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“Directed Evolution of a Subtilisin Carlsberg Varia nt
towards Improved Perhydrolytic Activity for Industr ial
Application”
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH
Aachen University zur Erlangung des akademischen Grades einer Doktorin der
Naturwissenschaften genehmigte Dissertation
vorgelegt von
Diplom -Biochemikerin
Dragana Despotovic
aus Sabac, Republic of Serbia
Berichter: Universitätsprofessor Dr. Ulrich Schwaneberg
Universitätsprofessor Dr. Martin Zacharias
Tag der mündlichen Prüfung: 12.04.2012
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
Table of Contents
i
Table of contents
Acknowledgments ........................................................................................................... 1
Abstract ........................................................................................................................... 3
Abbreviations .................................................................................................................. 4
Part I: General Introduction ............................................................................................. 5
1. Protein Engineering (directed evolution and rational design) ................................. 5
2. Perhydrolases and peroxycarboxylic acids .......................................................... 10
2.1 Enzyme promiscuity ..................................................................................... 12
2.2 Applications of the perhydrolase-catalyzed synthesis of peroxycarboxylic
acids ................................................................................................................... 13
3. Project objective ................................................................................................... 15
Part II: Assay Development for Detection of Peroxycarboxylic Acids ............................ 16
1. Introduction .......................................................................................................... 16
1.1 Aim of this work ............................................................................................ 16
1.2 Detection of peroxycarboxylic acids ............................................................. 16
1.3 Flow cytometry screening system ................................................................. 19
1.4 Enzyme surface display on Bacillus subtilis cells ......................................... 20
2. Materials .............................................................................................................. 22
2.1 Reagents ...................................................................................................... 22
2.2 Apparatus ..................................................................................................... 22
2.3 Bacterial strains ............................................................................................ 23
2.4 Plasmids ....................................................................................................... 23
2.5 Oligonucleotides ........................................................................................... 23
3. Methods ............................................................................................................... 24
3.1 Synthesis of 7-(4-aminophenoxy)-3-carboxy coumarin (APCC) ................... 24
3.2 Sample preparation and analysis of peroxyacetic acid (PAA) and 7-
hydroxycoumarin-3-carboxylic acid (HCC) ......................................................... 27
Table of Contents
ii
3.3 Expression of subtilisin Carlsberg ................................................................. 28
3.4 Determination of enzymatic activity .............................................................. 28
3.5 APCC assay performed in double emulsions ............................................... 29
3.6 Emulsification of Bacillus cells ...................................................................... 29
3.7 Fusion of CWBc domain to Perhydrolase ..................................................... 30
3.8 Proteolytic activity ......................................................................................... 30
4. Results ................................................................................................................. 31
4.1 APCC assay principle ................................................................................... 31
4.2 Effect of bromide concentration .................................................................... 32
4.3 Effect of APCC concentration ....................................................................... 33
4.4 Effect of hypobromite and peroxycarboxylic acid on fluorescence intensity of
HCC .................................................................................................................... 34
4.5 Sensitivity ..................................................................................................... 35
4.6 Peroxycarboxylic detection in the presence of hydrogen-peroxide ............... 36
4.7 Detection of enzymatic activity ..................................................................... 37
4.8 Future development – optimization for high-throughput screening ............... 40
4.9 Surface display of protease .......................................................................... 44
5. Discussion ........................................................................................................... 45
6. Conclusion ........................................................................................................... 46
Part III: Directed Evolution of Subtilisin Carlsberg for Improved Perhydrolytic Activity .. 47
1. Introduction .......................................................................................................... 47
1.1 Aim of this work ............................................................................................ 47
1.2 Proteases ..................................................................................................... 47
1.2.1 Serine proteases and subtilisins ..................................................... 50
1.2.2 Protein engineering in subtilisin proteases ...................................... 56
1.2.3 Subtilisin Carlsberg ......................................................................... 58
1.2.4 Promiscous activity of subtilisin Carlsberg and Thr59Ala/Leu217Trp
variant ...................................................................................................... 59
2. Materials .............................................................................................................. 63
Table of Contents
iii
2.1 Chemicals ..................................................................................................... 63
2.2 Bacterial strains ............................................................................................ 63
2.3 Plasmids ....................................................................................................... 64
2.4 Oligonucleotides ........................................................................................... 64
2.5 Cell culture media and cultivation ................................................................. 66
3. Methods ............................................................................................................... 67
3.1 Cloning ......................................................................................................... 67
3.2 Transformation of B. subtilis WB600 and DB104 .......................................... 67
3.3 epPCR library generation ............................................................................. 68
3.4 SeSaM library generation ............................................................................. 69
3.5 Site Saturation Mutagenesis of Perhydrolase ............................................... 71
3.6 Protein expression ........................................................................................ 71
3.7 Catalase inhibition ........................................................................................ 72
3.8 Screening for decreased proteolytic activity ................................................. 72
3.9 Screening for improved perhydrolytic activity ............................................... 72
3.10 Purification of serine proteases with affinity chromatography ..................... 73
3.11 Protein purification ...................................................................................... 74
3.12 Proteolytic activity with natural substrate – skim milk ................................. 75
3.13 Proteolytic activity – suc-AAPF-pNA ........................................................... 75
3.14 Perhydrolytic activity of the variants ........................................................... 75
3.15 Esterolytic activity of the variants ................................................................ 76
3.16 Modeling studies ......................................................................................... 77
4. Results ................................................................................................................. 78
4.1 Expression system ....................................................................................... 78
4.2 Perhydrolytic (APCC) assay optimization for microtiter plate screening ....... 79
4.3 Diversity generation ...................................................................................... 80
4.4 Screening ..................................................................................................... 82
4.5 Diversity generation and screening optimization .......................................... 83
4.6 Rational design approach ............................................................................. 88
Table of Contents
iv
4.6.1 SSM at positions included in catalysis and close to active site ....... 89
4.6.2 SSM of positions in the substrate binding pocket ........................... 91
4.7 Purification of selected variants .................................................................... 95
4.8 Characterization of the variants .................................................................... 99
4.8.1 Characterization with suc-AAPF-pNA as a substrate ...................... 99
4.8.2 Skim milk activity ........................................................................... 100
4.8.3 Perhydrolytic activity: kinetic parameters for hydrogen-peroxide and
methyl-propionate .................................................................................. 101
4.8.4 Perhydrolytic activity: kinetic parameters for methyl-butyrate and
methyl-pentanoate ................................................................................. 103
4.8.5 Esterolytic activity of the variants .................................................. 105
5. Discussion ......................................................................................................... 107
5.1 Suc-AAPF-pNA specificity .......................................................................... 110
5.2 Ester specificity ........................................................................................... 113
6. Conclusion ......................................................................................................... 120
Future prospects ......................................................................................................... 121
References .................................................................................................................. 122
Publication list ............................................................................................................. 129
Curriculum vitae .......................................................................................................... 130
Appendix ......................................................................................................................... A
Acknowledgements
1
Acknowledgments
During my PhD research at Jacobs University Bremen and RWTH Aachen
University many people influenced my scientific and personal development.
First, I would like to thank my supervisor Prof. Dr. Ulrich Schwaneberg for giving
me the opportunity to work in this group, as well for his guidance and support during my
PhD studies. I admire his scientific thoughts and his dedication to his work. He was an
excellent life and science teacher.
I would like to thank my PhD committee members Prof. Dr. Martin Zacharias and
Prof. Dr. Lothar Elling for accepting to be in my thesis committee. I appreciate their time
to read this thesis and Prof. Zacharias for making his trip from Munich to Aachen. I am
thankful to Prof. Zacharias for helping us with the modeling part of the project, hosting
our visit to Munich and Technische Universität München where we were introduced to
the group of great scientist and their interesting work.
I acknowledge BMBF for financial support and thank our collaborators Dr.
Hendrik Hellmuth, Dr. Timothy O’Connell, Nina Mussman and Inken Prueser at Henkel
AG & Co KGaA. It was a great pleasure to be part of their team even for at least a short
period. I learned a lot about science and collegiality. I also thank Prof. Dr. Karl-Heinz
Maurer for his support on this project; I truly enjoyed scientific discussions with him.
I thank my co-supervisors Dr. Radivoje Prodanovic and Dr. Ronny Martinez. My
gratitude to Ronny, who supported me from the beginning of my PhD. I learned a lot
from him, enjoyed our discussions and I am very thankful for his help during manuscript
writing and for careful reading of my thesis. It was also great pleasure to be part of HTS
subgroup together with my colleagues: Ronny Martinez, Ljubica Vojcic, Felix Jakob,
Christian Lehmann, Georgette Wirtz and Christian Pitzler.
I thank to my students Ranjan Shrestha and Jana Ognjenovic; I hope they
learned from me as much I learned from them. They were all a great help in the lab and
I really enjoyed working with them.
I am thankful to all members of Schwaneberg group for their support; it was a
great pleasure working and learning from them. Multicultural environment was a big
Acknowledgements
2
experience for me as a person and a scientist. I appreciate the help from Daniela,
Gisela, Wolfgang and Brigitta in lab and Marina, Monika and Anita’s for help with
administrative work.
I thank to my collegues and friends Dr. Milan Blanusa, Raluca Ostafe, Dr.
Hemanshu Mundhada and Dr. Amol Shivange for their support, scientific discussions,
advices and small talks which I enjoyed especially. I am also very thankful to Arsenovic
family who helped me a lot during my stay in Aachen, their house was a second home
to me.
At last, I would like to thank my friends and family: Jelena, Aleksandra, Milena,
Ana, aunt Julijana and cousin Danica for their love and support. Thank you for believing
in me, had time and patience to listen and provide advices. My gratitude and love goes
to my father, my mother and sister who always believed in me. My mother helped us to
achieve our goals and I wish we made her proud. She is the strongest person I know
and the best teacher I ever had. I am thankful to my father for his love and guidance; I
believe that he would be happy to see how much we are committed to our family and
work.
All this work would have not been possible without the financial support of the
German government through the Bundesministerium für Bildung und Forschung
(BMBF) and Henkel AG & Co KGaA.
Dragana Despotovic
Abstract
3
Abstract
Adapting biocatalysts to non-conventional substrates and environments is of high
academic and industrial interest. Directed evolution and rational design methods offer
opportunities to tailor protein properties by generating diversity on gene level and
screening for new variants on the protein level.
Subtilisin Carlsberg is one of the first described serine proteases from the
subtilisin family. Due to its stability under production conditions and broad specificity,
this enzyme has become industrially important, especially in the detergent industry.
Subtilisin Carlsberg also catalyzes ester perhydrolysis, generating peroxycarboxylic
acids, compounds well known as bleaching and disinfection agents.
The bottleneck for most experiments focused on finding and improving
peroxycarboxylic acid producing enzymes is the availability of a medium and
high‐throughput screening system. To overcome this obstacle, a sensitive, fluorescent
assay with detection limit below 1 µM of peroxyacetic acid and suitable for detection of
perhydrolytic activity without previous enzyme purification was developed. The assay
was optimized for screening in 96 and 384-well microtiter plate system, with the
possibility to further upscale throughput by using flow cytometry screening technology.
The screening platform was developed in this work to improve protease and likely other
hydrolases (e.g. lipases or esterases) having perhydrolytic activity or find new enzymes
with peroxycarboxylic acid production by screening of metagenome libraries.
Subtilisin Carlsberg was furthermore the first protease engineered through
directed evolution towards increased perhydrolytic activity which offers novel
opportunities for bleaching applications. The protease variant, T59A/G166L/L217W,
identified in the current work showed a 9.4-fold increased specificity constant of
perhydrolytic activity compared to the wild type. Position 166 is part of the substrate
binding pocket and changes in the side-chain volume at this position were found to
modulate substrate specificity of subtilisin Carlsberg.
Abbreviations
4
Abbreviations
ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
APCC 7-(4-aminophenoxy)-3-carboxy coumarin
B. subtilis Bacillus subtilis
CMC Carboxymethylcellulose
Da Dalton
DMSO Dimethyl-sulfoxide
DNA Deoxyribonucleic acid
E. coli Escherichia coli
epPCR Error prone PCR
HCC 7-hydroxycoumarin-3-carboxylic acid
HPLC High Pressure Liquid Chromatography
HTS High Throughput Screening
LB Luria-Bertani medium
MEGAWHOP Megaprimer PCR of Whole Plasmid
MTP Microtiter plate
OD Optical Density
PAA Peroxyacetic acid
PBS Phosphate buffered saline
PCR Polymerase Chain Reaction
PDB Protein Data Bank
pI Isoelectric point
PMSF Phenylmethylsulfonyl fluoride
SSM Site Saturation Mutagenesis
StEP Staggered Elongation Process
Suc-AAPF-pNA N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide
TCA Trichloroacetic acid
Tet Tetracycline
w/o/w Water-in-oil-in-water
WT Wild type
Part I: General Introduction
5
Part I: General Introduction
1. Protein Engineering (directed evolution and rati onal
design)
“There are two fundamental conditions for life. First, the living entity must be able
to self-replicate; second, the organism must be able to catalyze chemical reactions
efficiently and selectively.” (Lehninger, Nelson et al. 2005).
Enzymes, highly specialized proteins, are the most efficient catalysts having
efficiency often greater than that of synthetic or inorganic ones. Enzymes can perform
regio-selective and/or enantio-selective chemical transformations and accelerate
reaction rates by factors up to 1012, all under mild conditions. Although highly attractive
for chemical synthesis, enzymes are usually not well suited for bioindustrial applications
(Arnold 2001). Limitations include low catalysis of nonnatural substrates, low stability
(towards temperature, pH, and oxidative agents), poor activity in non-aqueous media,
requirements for expensive cofactors and of course, nature has not provided catalysts
for every reaction. The goal of protein engineering research is to alter nature catalysts
making them suitable for industrial technology. (Farinas, Bulter et al. 2001). The most
successful methods for optimization and redesign of biocatalysts are rational design and
directed evolution.
Rational design is the strategy of modifying biocatalysts that requires not only
knowledge of enzyme crystal structure and function, but also of the property which
should be improved. Many properties are well understood and in some of the cases
general rules might be applied, for example termostability can be improved by
introduction of disulfide bridges, ionic interactions and salt bridges that increase the
rigidity of the protein scaffold, reducing the possible conformations of the unfolded
protein (Korkegian, Black et al. 2005). On the other hand, there are cases in which
specific knowledge is necessary to change enzyme specificity or to increase catalytic
efficiency (Lassila, Keeffe et al. 2005). These general rules are derived from studying
the effect of certain mutations on
methods accelerate the generation of new biocatalysts
small and high-quality mutant libraries based on protein structure and/or interactions
protein-substrate (Reetz, Bocola et al. 2005
the structure and/or the molecular basis for a
enzymes.
Directed protein evolution
to create improvements within
presented in Figure 1.1, starting from diversity generation where mutations are
randomly introduced in the gene of interest
expression in appropriate host.
agar plate and/or microtiter plate assays.
usually used for next round of evolution until desired property
achieved.
Figure 1.1. General overview of a directed evolution experimentProdanovic et al. 2010).
Part I: General Introduction
6
the effect of certain mutations on the protein structure and/or activit
generation of new biocatalysts by the generation of
quality mutant libraries based on protein structure and/or interactions
Reetz, Bocola et al. 2005; Wong, Roccatano et al. 2007
the molecular basis for a desired function are not available for most
Directed protein evolution is an approach which requires an outside intelligence
improvements within species. Major steps in directed evolution experiment are
, starting from diversity generation where mutations are
ndomly introduced in the gene of interest (Step I) to library
in appropriate host. In Step II library is screened for improved variants
agar plate and/or microtiter plate assays. Identified variants are isolated (Step II
usually used for next round of evolution until desired property or level of improvement
General overview of a directed evolution experiment. Taken over from
t I: General Introduction
protein structure and/or activity. Rational design
the generation of relatively
quality mutant libraries based on protein structure and/or interactions
Wong, Roccatano et al. 2007). However,
not available for most
requires an outside intelligence
Major steps in directed evolution experiment are
, starting from diversity generation where mutations are
to library transformation and
for improved variants using
isolated (Step III) and
or level of improvement is
. Taken over from (Güven,
Part I: General Introduction
7
Among the main challenges in directed evolution campaigns are the generation
of unbiased diversity and development of screening systems which enable scanning
through a meaningful fraction of the generated sequence space (Wong, Zhurina et al.
2006). A large number of chemical and molecular biology methods are now available for
generating random mutation in gene sequences, site specific and recombination of
mutations. The most commonly used random mutagenesis techniques are the PCR
methods under low fidelity conditions (epPCR) (Cirino, Mayer et al. 2003). One of the
latest random mutagenesis methods is Sequence Saturation Mutagenesis (SeSaM),
which overcomes limitations caused by polymerase bias (transitions over
transversions), saturates every position in a protein and is able to introduce subsequent
mutations (Wong, Tee et al. 2004). The most used recombination methods in directed
evolution used in the last step to combine beneficial mutations from different variants
are DNA shuffling (Stemmer 1994) and Staggered Extension Process (StEP) (Zhao,
Giver et al. 1998). Which of these methods will be used for directed evolution
experiments usually depends on the property which is aimed to be improved and
screening capabilities. Engineering termostability usually demands higher number of
mutations and substitution with chemically different amino acids leading to SeSaM as
method of choice for diversity generation. While, for enantioselectivity changes to
similar amino acids are more appropriate with epPCR as a method of choice for
diversity generation (Reetz and Jaeger 1999; Arnold 2001).
A large variety of methods for the generation of genetic diversity is available,
although currently the bottleneck of directed evolution is the availability of screening
systems for a desired property. Screening technology should reflect as close as
possible the property of interest, with substrate identical or similar to the target
substrate. Furthermore, the assay should be optimized for medium and/or high
throughput screening and sensitive over the final application range of substrate
concentration, enabling identification of desired hits in the library (Aharoni, Griffiths et al.
2005).
The most common screening methods are solid phase screening assays and
microtiter plate assays. Solid phase screening systems can be performed directly on the
agar plate, on filter paper or on membrane. These types of methods have a high
Part I: General Introduction
8
throughput, of 104‐106. The crucial factor when using this screening technique is that
substrate conversion generates a visual signal, such as fluorescence or color, to identify
colonies expressing an enzyme with desirable properties. The drawback is that they
have low accuracy and that’s why they are usually used as a pre-screen method.
Microtiter plate based screenings are the most commonly applied method in identifying
desirable enzyme variants. Microtiter plate assay enables screening 96, 384 or 1536
clones at the same time and these assays are considered as medium throughput.
Single transformants are grown in microtiter plates and variants are usually assessed in
a second plate, while the original plate is stored as back-up. Supernatant or cell lysate
is used for detection of enzymatic activity and generated signal measured by
spectrophotometer or fluorometer. High-throughput screening (HTS) systems are
Fluorescent Activated Cell Sorter (FACS) and microfluidic devices which can routinely
screen 107 clones per hour (Farinas 2006). This system is based on detection of
fluorescent product generated by enzyme reaction and linked to the cell surface or
entrapped in double emulsion compartments/microbeads, enabling the link between
genotype and phenotype (Mastrobattista, Taly et al. 2005). FACS greatly accelerates
HTS, but this technique is still in development, allowing for now only selection between
positive and negative variants. Figure 1.2 summarizes screening strategies currently
used.
Part I: General Introduction
9
Figure 1.2. Overview of screening technologies. (A) Experimental steps from obtaining the gene library as PCR product to the actual screen. (B) explanation of symbols, (C) Cell growth/survival selection and agar plate screening, (D) microtiter plate screening, (E) cell as micro-reactor, (F) cell surface display, (G) cell-in-droplet, and (H) in vitro compartmentalization. Taken from (Leemhuis, Kelly et al. 2009).
2. Perhydrolases and per
Perhydrolases catalyze the
CO-O-OH) from carboxylic acids (or
had been known as metal free haloperoxidases which
triad and structure very similar to
catalyze hydrolysis of chemical bonds.
al. 2006).
Figure 1.3. Reaction scheme for enzyme catalyzed perhydrolysis
Perhydrolysis presumably t
which a carboxylic acid first reacts with the
enzyme intermediate, which
intermediate (TI) leading to release of free enzyme and per
(Hofmann, Tölzer et al. 1998
mechanism, Bugg suggested that acetic acid is directly attacked by hydrogen
without formation of acetyl-
is not covalently bound for protein
perhydrolytic reaction are presented at Figure
Part I: General Introduction
10
and per oxycarboxylic acids
catalyze the reversible formation of peroxycarboxylic acids
from carboxylic acids (or esters) and hydrogen peroxide
had been known as metal free haloperoxidases which contain a Ser
and structure very similar to serine hydrolyses-esterases
catalyze hydrolysis of chemical bonds. (Pelletier, Altenbuchner et al. 1995
Reaction scheme for enzyme catalyzed perhydrolysis.
Perhydrolysis presumably takes place with an ping-pong bi
which a carboxylic acid first reacts with the active site serine group to form an acyl
enzyme intermediate, which reacts with hydrogen peroxide forming second tetrahedral
leading to release of free enzyme and per
Hofmann, Tölzer et al. 1998). This ping-pong mechanism is not the only proposed
mechanism, Bugg suggested that acetic acid is directly attacked by hydrogen
-enzyme intermediate where second tetrahedral intermediate
t covalently bound for protein (Bugg 2004). Possible mechanism
rhydrolytic reaction are presented at Figure 1.4.
t I: General Introduction
reversible formation of peroxycarboxylic acids (R-
esters) and hydrogen peroxide, Figure 1.3. They
contain a Ser-His–Asp catalytic
and lipases which
Pelletier, Altenbuchner et al. 1995; Song, Ahn et
pong bi-bi mechanism in
active site serine group to form an acyl-
ing second tetrahedral
leading to release of free enzyme and peroxycarboxylic acid
pong mechanism is not the only proposed
mechanism, Bugg suggested that acetic acid is directly attacked by hydrogen-peroxide
nd tetrahedral intermediate
Possible mechanisms of the
Part I: General Introduction
11
Figure 1.4. Mechanisms for pehydrolysis of acetic acid catalyzed by P. fluorescens esterase (PFE), a) the ping-pong bi-bi mechanism which includes acetyl-enzyme intermediate, b) bi-bi mechanism proposed by Bugg. The numbering corresponds to the active site of PFE. Figure taken from (Yin, Bernhardt et al. 2010).
Although perhydrolases and hydrolases have similar structure and reaction
mechanism, it is not clear why hydrolases have much lower perhydrolytic activity than
hydrolytic and why perhydrolases have much lower hydrolytic activity compared to
Part I: General Introduction
12
perhydrolytic. For example, Pseudomonas fluorescens esterase (PFE) shows low
perhydrolytic activity and has similar tertiary structure with the perhydrolases and Ser-
His–Asp catalytic triad. Bernhardt et al. (Bernhardt, Hult et al. 2005) identified residues
around the catalytic center which are only conserved for pehydrolases by aligning the
sequences from six hydrolases and six perhydrolases. Residues close to the active site
of PFE were identified and exchanged. Using this approach, perhydrolytic activity of
PFE was increased 28-fold with only one substitution (Leu29Pro). The proposed
explanation of this change was that this amino acid substitution shifts the backbone of
the neighboring Trp which could form hydrogen bond with perhydroxy-group in the
second tetrahedral intermediate, stabilizing the state, Figure 1.3a. Specific stabilization
of the second tetrahedral intermediate for the perhydrolysis reaction is of key
importance for completion of the reaction.
2.1 Enzyme promiscuity
Currently, only seven bacterial perhydrolases have been cloned, sequenced and
characterized (Wiesner, van Pée et al. 1988; Bantleon, Altenbuchner et al. 1994;
Pelletier, Pfeifer et al. 1994; Burd, Yourkevich et al. 1995; Kirner, Krauss et al. 1996;
Song, Ahn et al. 2006). Thus, there is a need to expand the group of enzymes which
can be used as a perhydrolases. Esterases, lipases and some proteases beside their
natural hydrolytic activity also show perhydrolytic activity (Bornscheuer and Kazlauskas
2004; Lee, Vojcic et al. 2010). This ability of enzymes to catalyze different synthetic
reactions, which are more or less far from their natural role, is called catalytic
promiscuity. In many cases the origin of such promiscuity can be found in the evolution
of the enzymes from a common ancestor, which could have generated/maintained
different residual side-reactions.
Catalytic promiscuity has been classified in three groups: (1) condition
promiscuity which is the catalytic activity in various reaction conditions different from
natural ones, (2) substrate promiscuity which is shown by enzymes with broad substrate
specificity (3) catalytic promiscuity, exhibited by enzymes catalyzing distinctly different
chemical transformations with different transition states. Enzyme catalytic promiscuity
Part I: General Introduction
13
can be either accidental, a side reaction catalyzed by the wild-type enzyme; or induced,
a new reaction established by one or several mutations changing the reaction catalyzed
by the wild-type enzyme (Hult and Berglund 2007). Rational understanding of the
causes could allow the development of new and useful enzymatic reactions by using
“well-known” enzymes (Bornscheuer and Kazlauskas 2004; Hult and Berglund 2007).
Promiscuous enzymes are then protein of interest for directed evolution, and new
knowledge regarding their mechanisms has to be generated in order to make them
applicable in industrial processes.
In the case of enzymes with perhydrolytic activity, the switch from water to
hydrogen peroxide as the nucleophilic agent could be considered as a change in
substrate selectivity. However, some serine hydrolases do not exhibit perhydrolase
activity which suggests that the Ser–His–Asp catalytic triad is not the only determinant
for perhydrolase activity, also better stabilization of hydrolytic or perhydrolytic second
tetrahedral intermediate will favor one or another reaction (Bernhardt, Hult et al. 2005).
2.2 Applications of the perhydrolase-catalyzed synthesis of peroxycarboxylic
acids
The use of hydrogen peroxide as a bleaching agent (because of its oxidizing
power) is well known since a long time. Due to its instability, it was produced in situ by
using sodium perborate and high temperature. Thus, in the 1950s peroxycarboxylic
acids and peroxycarboxylic acids-generating bleaches were recognized for their
potential to provide efficient bleaching at lower washing temperatures (Stanley and
Gianfranco 1983). Medium chain alkyl peroxycarboxylic acids are very effective for the
bleaching system, they have a broad application in various industry branches such as
synthesis of epoxides (Zhao, Wu et al. 2011), alcohols oxidation (Cella, Kelley et al.
1975), nylon manufacturing (Dicosimo, Gavagan et al. 2008). As antimicrobial agents,
since they can damage virtually all types of macromolecules, which ultimately leads to
cell lysis and microbial death (Baldry 1983). Due to these properties, peroxycarboxylic
acids are widely used in agricultural industry, food establishments, and medical facilities
(Block 2001). However, neither hydrogen peroxide, nor the organic peroxycarboxylic
Part I: General Introduction
14
acids are stable over time in aqueous solution. Therefore, an in situ generation is almost
mandatory for an effective bleaching procedure. The combination of an ester-
hydrolyzing enzyme, an acyl-alkyl ester, and a hydrogen-peroxide/peroxide donor, is an
effective bleaching platform (Minning, Weiss et al. 1999). Since the process takes place
in aqueous environment, a close interaction between the peroxycarboxylic acid,
hydrogen-peroxide and the hydrolase, is expected. In such conditions the catalyst is
highly sensitive to denaturation, and consequently, it is important to find a biocatalyst
able to work in that environment (Carboni-Oerlemans, Domínguez de María et al. 2006).
Additionally, there is a competition between water and hydrogen-peroxide for the acyl–
enzyme complex of the hydrolase. Therefore, the ratio of perhydrolysis- to hydrolysis-
rate (P/H) in a reaction mixture determines the efficiency of the enzymatic bleaching
system. Hydrolases also perform the reverse reaction, and form hydrogen peroxide and
carboxylic acids from the peroxycarboxylic acids in aqueous solutions. Related to this,
most of the commercial hydrolases in aqueous media have low a P/H value which limits
their practical applications. Thus, there is a need to improve hydrolases to obtain
perhydrolases of industrial interest (Carboni-Oerlemans, Domínguez de María et al.
2006).
Part I: General Introduction
15
3. Project objective
The aim of this work is to generate new perhydrolytic enzymes by altering the
catalytic properties of a serine protease (subtilisin Carlsberg) from hydrolase to
perhydrolase by means of directed evolution. The work is divided in two parts: the first
part is the development of a medium throughput detection system for peroxycarboxylic
acids, and the second part is directed evolution of subtilisin Carlsberg. The aim of the
first part is to generate a sensitive, simple assay able to follow continuous enzymatic
production of peroxycarboxylic acids in expression media. This assay should be
optimized for medium and high throughput screening system for identification of
subtilisin Carlsberg variants with improved perhydrolytic activity.
The aim of the second part is to identify by the screening of mutagenesis
libraries, subtilisin Carlsberg variants with increased perhydrolytic activity for application
in washing and cosmetic industry using the screening system previously developed in
this work. The analysis of amino acid substitutions present in variants with increased
perhydrolytic/hydrolytic activity ratio should provide new insights in understanding the
mechanism of promiscuous enzymatic activity by the identification of common features
that could be extrapolated to other hydrolases for the generation of new perhydrolases.
Part II: Assay Development
16
Part II: Assay Development for Detection of Peroxy-
carboxylic Acids
1. Introduction
1.1 Aim of this work
The goal of this work was to develop a new detection system for
peroxycarboxylic acids with different length of alkyl chain, which is sensitive (detection
limit below 1 µM), simple and suitable for screening in microtiter plate (96 and 384-well
format) without previous enzyme purification.
1.2 Detection of peroxycarboxylic acids
Peroxycarboxylic acids (R-CO-O-O-H) are oxidants which are used in bleaching,
disinfection (Rüsch gen. Klaas, Steffens et al. 2002), synthesis of epoxides (Zhao, Wu
et al. 2011), alcohols oxidation (Cella, Kelley et al. 1975) and antimicrobial agents
(Block 2001).
In particular, peroxyacetic acid (PAA) and medium chain (C7–C12)
peroxycarboxylic acids, have been introduced in the detergent industry, in addition to
hydrogen peroxide, as bleaching agents (Akerman, Phillips et al. 2003). Despite their
multiple uses, peroxycarboxylic acids are not widespread in industry due to their
instability in aqueous solution (Yuan, Ni et al. 1997), and the explosive character of their
pure form (Swern 1949). As an alternative, peroxycarboxylic acids can be produced in
situ by hydrolysis of esters or amides in the presence of hydrogen peroxide directly into
their final application.
Current methods for determination and quantification of peroxycarboxylic acids
are summarized in the Table 2.1.
The first approach for detection of peroxycarboxylic acids includes analytical
methods based on two step titration (methods 1 and 2 in the table) (D'Ans and Frey
1913; Greenspan and MacKellar 1948), where hydrogen-peroxide titration is followed by
Part II: Assay Development
17
a iodometric determination of peroxycarboxylic acids. With some modifications (method
3) (Sully and Williams 1962) such is detection under low acidic condition in which the
rate of equilibration between peroxyacids and peroxide is negligible, these methods are
still widely used for PAA monitoring. Method 4 (Di Furia, Prato et al. 1984) is a gas-
liquid chromatography including pre-column derivatization, while methods 5 and 6 (Kirk,
Damhus et al. 1992; Effkemann, Pinkernell et al. 1998) are liquid chromatographic
(HPLC) methods with direct separation or post-column derivatization. The development
of chromatographic methods, significantly increased sensitivity; reaching a detection
limit below 1 µM (Kirk, Damhus et al. 1992). HPLC analysis is also used as a reference
technique to calibrate other methods for peroxycarboxylic acid determination.
Current colorimetric assays (methods 7-9) suitable for the screening of enzymes
catalyzing perhydrolytic reaction are based on the oxidation of azo-dyes (Minning,
Weiss et al. 1999), chromogens (aromatic amines, phenols) (Fischer, Arlt et al. 1990),
iodine (Davies and Deary 1988) or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS) in the presence of peroxycarboxylic acids (Pinkernell, Luke et al. 1997;
Harms and Karst 1999; Binder and Menger 2000). ABTS assay (method 9) is an end
point assay for determination of peroxycarboxylic acids, reported to be used in microtiter
plates (Pinkernell, Luke et al. 1997). Beside spectrophotometric assays, fluorescent
detection methods offer high sensitivity and simplicity. Method 10 (Jacks 2004) is the
only fluorescent assay reported for detection of hypohalites and peroxycarboxylic acids.
This method was validated by activity studies on purified and semi-purified heme and
non-heme haloperoxidase activities (Jacks 2005).
The fluorescence of 7-hydroxycoumarin in aqueous media is completely lost in 7-
aryloxycoumarins. O-dearylation of coumarins can be used as a trigger in detection of
highly reactive oxygen species, resulting in fluorescent compounds. Nonfluorescent 7-
arylcoumarins can also be O-dearylated by hypohalites, forming highly fluorescent 7-
hydroxycoumarin (Setsukinai, Urano et al. 2000).
Part II: Assay Development
18
Table 2.1. Current methods for determination and quantification of peroxycarboxylic acids. Methods labeled in gray could be optimized in a screening platform for directed evolution.
Reference Method type Principle Notes
(D'Ans and Frey 1913)
Titrimetric
H2O2 is titrated using K2MnO4, followed by iodometric determination of
peroxycarboxylic acids (iodide reacts with peroxycarboxylic acids forming
iodine,generated iodine is titrated with thiosulphate solution)
Detection limit: not reported, endpoint assay
Decomposition of peroxycarboxylic acid by Mn2+
(Greenspan and
MacKellar 1948)
Titrimetric H2O2 is titrated using ceric sulphate,
followed by iodometric determination of peroxycarboxylic acids
Detection limit: not reported, endpoint assay
(Sully and Williams
1962) Titrimetric
Direct iodometric determination of peroxycarboxylic acid and hydrogen peroxide in acidic conditions (pH 3-5)
based on greater reactivity of iodide with peroxycarboxylic acids than with H2O2
Detection limit: not reported, endpoint assay
(Di Furia, Prato et al.
1984)
Gas-liquid chromatographic
Peroxycarboxylic acids oxidize methyl p-tolylsulphide to methyl p-tolylsulphoxide, residual sulphide or produced sulphoxide
are determined by gas-liquid chromatography
Detection limit: not reported, endpoint assay
(Kirk, Damhus et al. 1992)
Liquid chromatography
Peroxycarboxylic acids are separated by reversed-phase chromatography followed
by amperometric detection
Detection limits of 0.1-0.6 µM; linear range of 0.05-5 mM.
(Effkemann, Pinkernell
et al. 1998)
Liquid chromatography with postcolumn
derivatization
Peroxycarboxylic acids with chain lengths from C2 to C12 are separated by HPLC on a
reversed-phase C18 column. After separation, peroxycarboxylic acids were
detected using ABTS method
Detection limit: 5 µM, linear range - 10-250 µM for C2 and
10-500 µM for C3 to C12 peroxycarboxylic acids,
endpoint assay
(Minning, Weiss et al.
1999) Colorimetric
Peroxycarboxylic acids oxidize methylsulfanyl-groups of azo-dye leading in
change of color from orange to yellow.
Detection limit: ~ 4 ppm (50 µM), detection range: 4-40 ppm
(50-500 µM) semi-quantitative agar plate method
(Davies and Deary 1988)
Spectrophoto-metric
Measurement of iodine produced in the reaction between peroxycarboxylic acid
and iodide at 352 nm. Linear range: 4.9-68.8 µM.
(Pinkernell, Luke et al.
1997)
Spectrophoto-metric
Peroxycarboxylic acids in the presence of iodide as a catalyst oxidize ABTS forming
green colored radical (pH 3.5)
Detection limit: 1 µM, linear range: 2.5-100 µM. End point assay
(Harms and Karst 1999)
Spectrophoto-metric
Flow injection analysis system for measuring PAA samples based on ABTS
method
Detection limit: 0.7 µM, linear range: 2-700 µM
(Binder and Menger 2000)
Spectrophoto-metric
H2O2 is decomposed by catalase and residual peroxycarboxylic acid determined under the action of peroxidase and ABTS
Detection limit: 45 µM, linear range: 45-550 µM
endpoint assay
(Jacks 2004)
Spectrofluoro-metric
Peroxycarboxylic acids oxidize bromide to hypobromite which reacts with kojic acid
forming fluorescent product. Linear range: 0-100 mM
Part II: Assay Development
19
Hydrolases (proteases, lipases or esterases) which are mainly used in
detergents, are reported to generate peroxycarboxylic acids as a result of a secondary
catalytic function in the presence of carboxylic esters and hydrogen peroxide (Björkling,
Frykman et al. 1992; Kirk, Christensen et al. 1994). Indentifying and improving enzymes
for production of peroxycarboxylic acids is a constant subject of interest in detergent
industry. Lipases (Bernhardt, Hult et al. 2005) and proteases (Wieland, Polanyi-Bald et
al. 2009) have been engineered in order to increase their perhydrolytic activity. Despite
significant advance, an efficient enzymatic system for in situ production of
peroxycarboxylic acids is yet to be reported. One of the main challenges is developing
an analytical method capable of monitoring peroxycarboxylic acid generation in the
presence of a large excess of hydrogen peroxide which at the same time is suitable for
high throughput screening in microtiter plates in order to identify new enzyme variants
either by directed evolution or from metagenome libraries.
In this work a novel, highly sensitive fluorescent assay (µM range) is described
as part of a high throughput screening platform for peroxycarboxylic acids producing
enzymes. This continuous assay relies on the detection of coumarin compounds. After
enzymatic reaction, the generated peroxycarboxylic acids oxidize bromide producing
hypobromite, which reacts with 7-(4’-aminophenoxy)-3-carboxy coumarin (APCC)
releasing the highly fluorescent 7-hydroxycoumarin-3-carboxylic acid (HCC) together
with iminocyclohexadienon.
Assay was validated by detection of perhydrolytic activity of protease subtilisin
Carlsberg and T58A/L217W variant expressed in B. subtilis WB600.
1.3 Flow cytometry screening system
One way of accelerating HTS is to use Fluorescence-Activated Cell Sorting
(FACS), which can sort >107 clones per hour. FACS is successfully used for selection of
proteins with high binding affinity such as antibodies. Additionally, FACS can be used
for catalysis, but only if certain requirements are fulfilled: diffusion of product out of the
cell has to be prevented, or to capture the fluorescent product on: the surface of the
cells, into microbeads or in double emulsion compartments, in order to keep the link
Part II: Assay Development
20
between phenotype and genotype (Mastrobattista, Taly et al. 2005). Conversion inside
water-in-oil-in-water (w/o/w) double emulsions enabled sorting the compartments by
FACS, and the isolation of living bacterial cells and their enzyme-coding genes. High
enzyme concentration in the reaction droplets (>104 enzyme molecules in <10
femtoliter) increases sensitivity of the method (Aharoni, Amitai et al. 2005).
Figure 2.1. Single-cell compartmentalization and selection by in vitro compartmentalization in w/o/w emulsions (1) A gene library is transformed into expression host, and (2) proteins are expressed in the cytoplasm, or on the surface of the cells, (3) single cells are compartmentalized in the aqueous droplets of a primary (w/o) emulsion, (4) and secondary w/o/w emulsion is formed by emulsification of the primary w/o emulsion, (5) compartments containing the fluorescent product are sorted by FACS. Taken from (Aharoni, Amitai et al. 2005).
1.4 Enzyme surface display on Bacillus subtilis cells
Regardless of the screening or selections system used, the linkage between the
genotype and the phenotype must be somehow maintained in order to isolate the
improved mutants. In vitro compartmentalization in double emulsions of the cells or in
vitro translation machinery in emulsions enables this link. Using in vitro translation
system for compartmentalization has many disadvantages such are: 1) presence of
protease inhibitors in the kit, which will influence on activity of subtilisin Carlsberg; 2)
product of perhydrolytic reaction is peracid, strong oxidative agent which can deaminate
nucleotide bases and mutate the sequence of the gene (a better variant has higher
Fluorescent
product
Part II: Assay Development
21
probability that gene will be damaged); 3) delivery of H2O2 through oil into the droplets;
4) very expensive commercial kits.
However, subtilisin Carlsberg is an extracellular protease and prior to
compartmentalization of the cells expressed protease is secreted in the medium, where
only small number of the molecules stays on the surface of the cells. Entrapping
protease on the cell wall would significantly increase the sensitivity of flow cytometry
screening system.
In Bacillus subtilis there are many proteins non-covalently bound on the cell
surface. Major cell wall-binding proteins are peptidoglycan hydrolases, some of which
consists of two domains: catalytic and cell wall-binding domain (CWB). A small cell wall-
binding domain (CWBc) of the Bacillus subtilis peptidoglycan hydrolase CwlC fused to
B. subtilis lipase B was able to be localized on the cell wall of B. subtilis (Kobayashi,
Fujii et al. 2002). Although there are several candidates as an anchoring protein, the C-
terminal domain of CwlC (CWBc) is one of the smallest cell wall-binding domains. CWBc
consists of only 79 amino acids with two direct repeats (each containing 36 amino
acids) and it will be fused to the C-terminus of the Perhydrolase gene.
Part II: Assay Development
22
2. Materials
2.1 Reagents
All chemicals were of analytical-reagent grade or higher quality and were
purchased from Fluka, Sigma-Aldrich (Steinheim, Germany) and AppliChem
(Darmstadt, Germany), except APCC which was synthesized. The peroxyacetic acid
(PAA) solution was purchased from Sigma-Aldrich with a labeled concentration of 32 %.
Subtilisin Carlsberg protease was purchased from Sigma-Aldrich. All enzymes were
purchased from New England Biolabs GmbH (Frankfurt, Germany) and Fermentas
GmbH (St. Leon-Rot, Germany).
TLC plates coated with silica gel 60 were from Merck (Haar, Germany). UV was
used as a detection system, TLC plates were exposed to UV light (366 nm and 254
nm).
PD-10 Desalting Column was from GE Healthcare, Munich, Germany.
Silica gel, used for chromatography, was type 60 and purchased from Roth
(Karlsruhe, Germany).
2.2 Apparatus
Rotary evaporator (Rotavap) was from Heidolp Instruments (Schwabach,
Germany). Infinite M1000 (Tecan Group AG, Zürich, Switzerland) and a flow cytometer
(CyFlow® space, Partec GmbH, Muenster, Germany) were used for the detection of
fluorescence. H+ NMR spectra were recorded using Bruker AMX 300 (Bruker BioSpin,
Germany). Thermal cycler (Mastercycler gradient; Eppendorf) and thin-wall PCR tubes
(Multi-ultra tubes; 0.2 ml; Carl Roth, Germany) were used in all PCRs. The PCR volume
was always 50 µl. The amount of DNA in cloning experiments was quantified using a
NanoDrop photometer (NanoDrop Technologies, Germany).
Part II: Assay Development
23
2.3 Bacterial strains
The bacterial strains used in this work were Bacillus subtilis DB104 (Yang,
Ferrari et al. 1984) and Bacillus subtilis WB600 (Wu, Lee et al. 1991).
2.4 Plasmids
Plasmids pBC-Car (pBC carrying the subtilisin Carlsberg gene) and pBC-Per
(pBC carrying the subtilisin Carlsberg double mutant T59A/L217W) were provided by
the collaborator Henkel AG & Co. KGaA (Düsseldorf, Germany).
Other plasmids used in this work are pHY300PLK (Takara BIO) and pHY300Per
produced in this work.
2.5 Oligonucleotides
The oligonucleotides used in this work are summarized in Table 2.2.
Table 2.2. List of oligonucleotides used in this work.
Name Sequence (5’ →3’) Description
CWBc_fp GCGCCTCGAGCTTAAAAAGACTTCCAGCTC Amplification of CwlC domain
CWBc_rp GCGCTCTAGACCGTCTGGAAACATGGTC Amplification of CwlC domain
pHY300Per_fp GCGCCTCGAGTTGAGCGGCAGCTTCG Amplification of vector with Perhydrolase
pHY300Per_rp GCGCTCTAGAAGCTTGGGCAAAGCGTTTTTCC Amplification of vector with Perhydrolase
Part II: Assay Development
24
3. Methods
3.1 Synthesis of 7-(4-aminophenoxy)-3-carboxy coumarin (APCC)
APCC was synthetized as described in literature (Khanna, Chang et al. 1991).
Step 1 - synthesis of 7-hydroxy-3-ethyl-carboxy-coumarin. 8.4 g of 2,4-
dihydroxybenzaldehyde was dissolved in 45 ml of anhydrous methanol. To the stirred
solution 10.5 g of diethylmalonate was added and the solution was brought to reflux
temperature (80°C). 0.45 g of morpholine and 0.15 g of acetic acid were added to 2 ml
of methanol and mixed until the precipitate was dissolved. That solution then was added
to a refluxed reaction mixture and refluxing continued during 3 h. After cooling, 200 ml
of methanol was added; at this point reaction product precipitated. The precipitate was
heated again until it was dissolved. The resulting solution was left to precipitate again.
Crystals were recovered by filtering, washed with 10-15 ml of cold methanol and dried.
4.2 g of yellow crystals was obtained. Chemical reaction is presented in Figure 2.2.
Figure 2.2. Synthesis of 7-hydroxy-3-ethyl-carboxy-coumarin.
Step 2 – 7-hydroxy-3-ethyl-carboxy-coumarin salt preparation. 1.5 g of 7-
hydroxy-3-ethyl-carboxy-coumarin was resuspended in 50 ml of toluene (it is not
soluble) and heated to 120°C until about 5 ml of to luene was evaporated, to be sure that
water is removed (about 30-60 min) with stirring. After cooling, 0.3 g of NaH was added
to the mixture (at this point gas was released). When gas releasing decreased (after 20
min), mixing and heating to 120°C continued until t oluene evaporated (about 1-2 h). The
obtained salt was dried in vacuum overnight. 1.7 g of orange salt was obtained.
Part II: Assay Development
25
Step 3 – Benzoilation of 7-hydroxy-3-ethyl-carboxy-coumarin salt. 1.7 g of
step 2 product was dissolved in 100 ml of dry dimethyl-formamide (DMF), (DMF was
dried with molecular sieves, 1-2 h). The mixture was heated to 120°C. During heating
(when 50°C was reached) 0.82 g of 1-fluoro-4-nitrob enzene in 3 ml of dry DMF was
added slowly adding the last portion when the temperature reached 120°C. The mixture
(black color) was mixed and heated for 2 h at 120°C and additionally 0.41 g of 1-fluoro-
4-nitrobenzene in 3 ml of DMF was added. After 4-6 h of heating at 120°C, mixture was
left to cool overnight and diluted to 300 ml with ice water (200 ml), a precipitate was
formed. Before ice was melted completely, the precipitate was filtrated washed with cold
water to remove DMF, and dried. After drying, the pellet was extracted twice with 100 ml
of dichloromethane. Water was removed with anhydrous Na-sulphate, dichlormethane
was evaporated using Rotavap and the remaining pellet was dried in vacuum. 0.7 g of
orange compound was obtained. Chemical reaction is presented in Figure 2.3.
Figure 2.3. Synthesis of 7-(4-nitrophenoxy)-3-ethyl-carboxy-coumarin.
0.7 g of product was dissolved in 2 ml of dichloromethane (DCM) and loaded into
a column with 30 g of silica gel. Column was washed with 150 ml of DCM and 7-(4-
nitrophenoxy)-3-ethyl-carboxy-coumarin was eluted using a 20:1 mixture of DCM and
ethyl-acetate. Fractions were analyzed on TLC using a 20:1 mixture of DCM and ethyl-
acetate as developing solution. 7-(4-nitrophenoxy)-3-ethyl-carboxy-coumarin was visible
under UV light at 366 nm. Fractions containing the product were pooled, solvent was
evaporated using Rotavap and product was dried. 0.28 g of white-yellow product was
obtained and structure was confirmed by H-NMR (Appendix, Figure A1).
Part II: Assay Development
26
Step 4 – reduction of 7-(4-nitrophenoxy)-3-ethyl-carboxy-coumarin. 0.28 g of
p-nitro-benzyl-ethyl-carboxy-coumarin was resuspended in 19 ml of pure ethanol and 1
ml of distilled water. To this suspension 50 µl of concentrated HCl was added and the
mixture was stirred at 100°C. During heating, a mix ture of 0.6 g of Fe and 0.06 g of
FeCl3x6H2O was added and the solution was stirred during 1h at 100°C. The reduction
was monitored by thin-layer chromatography (TLC) using DCM/ethyl-acetate (20:1) as a
mobile phase and UV for visualization. Product was less soluble in mobile phase than 7-
(4-nitrophenoxy)-3-ethyl-carboxy-coumarin. After cooling, the mixture was diluted with
40 ml of water and extracted four times, each time with 50 ml of diethyl ether. Ether
extracts were dried over anhydrous Na-sulphate, filtered and evaporated to dryness.
0.25 g of yellow amino compound was obtained. Chemical reaction is presented in
Figure 2.4.
Figure 2.4 . Reduction of 7-(4-nitrophenoxy)-3-ethyl-carboxy-coumarin to 7-(4-aminophenoxy)-3-ethyl-carboxy-coumarin.
Step 5 – deesterification of 7-(4-aminophenoxy)-3-ethyl-carboxy-coumarin.
0.25 g of 7-(4-aminophenoxy)-3-ethyl-carboxy-coumarin was dissolved in 30 ml of
dioxane and then 30 ml of 20 % (v/v) sulfuric acid in water was added. Mixture was
stirred at 80°C overnight. The mixture was cooled d own and neutralized using 1M
NaOH to pH 5.0 when the formation of precipitate was visible. Precipitate was filtered,
washed with water and dried. Yield was 140 mg of white-yellow 7-(4-aminophenoxy)-3-
carboxy coumarin (APCC). Structure of the product was confirmed by H NMR
(Appendix, Figure A2). Chemical reaction is presented in Figure 2.5.
Part II: Assay Development
27
Figure 2.5. Deesterification reaction of 7-(4-aminophenoxy)-3-ethyl-carboxy-coumarin.
3.2 Sample preparation and analysis of peroxyacetic acid (PAA) and 7-
hydroxycoumarin-3-carboxylic acid (HCC)
Standard solutions and samples were prepared in 96-well microplates
(polystyrene black flat 96-well no. 655076, Greiner Bio-One GmbH, Frickenhausen,
Germany). PAA was diluted to a concentration range from 0.0005 to 1 mM, the
reactions were performed in Na-phosphate buffer (100 mM, pH 7.5) containing 10 µl
PAA standard solution, varied concentration of NaBr (10, 50, 100, 200 or 400 mM) and
varied concentration of APCC (0.05, 0.25 or 0.5 mM) in a final volume of 100 µl. The
PAA calibration curve in presence of hydrogen peroxide contained: 10 µl of PAA
standard solution, NaBr (100 mM), APCC (0.5 mM) and varied concentration of H2O2 (1,
10, 30 or 100 mM). The reaction was measured at 30°C by monitoring the fluorescence
intensity (excitation/emission 382/445 nm, gain 100) using Infinite M100. Reported
values are the average of three measurements with average deviation from the mean
value after 10 min of reaction when fluorescent intensity is constant. The PAA solutions
were used within 1 h to prevent any disproportionate reactions.
Calibration curve in 384-well microplates (polystyrene black flat 384-well no.
781076, Greiner Bio-One GmbH, Frickenhausen, Germany) was performed in Na-
phosphate buffer (100 mM, pH 7.5) containing 4 µl PAA standard solution, 100 mM
NaBr and 0.5 mM APCC in a final volume of 40 µl.
Part II: Assay Development
28
Reactions for HCC standard curves were performed in Na-phosphate buffer (0.1
M, pH 7.5) containing 0.05-25 µM HCC, 200 mM NaBr, 100 mM H2O2 and 0.05-25 µM
PAA. The reaction was measured at 30°C by monitorin g the fluorescence intensity
(excitation/emission 382/445 nm, gain 100) using Infinite M100.
3.3 Expression of subtilisin Carlsberg
Expression of subtilisin Carlsberg and subtilisin Carlsberg double mutant
Thr59Ala/Leu217Trp (in further text addressed as Perhydrolase) was in B. subtilis
DB104 strain. Test tubes containing 4 ml of LB media supplemented with tetracycline
(15 µg/ml) were inoculated with a 1:250 dilution of overnight culture (B. subtilis DB104
harboring pBC-Car and pBC-Per) grown in LB media. After 24 hr of expression (250
rpm, 37°C) culture was centrifugated (Eppendorf 581 0R, 4°C, 3220 g, 20 min) and
supernatant of the cells was used for determination of enzymatic activity.
3.4 Determination of enzymatic activity
Before measurement of enzymatic activity, catalase was inhibited in the cell
culture supernatant. Inhibition reaction was performed with 100 mM 3-amino-1,2,4-
triazole and 30 mM H2O2 at RT for 2 hr.
After inhibition supernatant was diluted 5 times and used for determination of
perhydrolytic activity.
The assay was carried out in 96-well microplates (polystyrene black flat 96-well
no. 655076, Greiner Bio-One GmbH, Frickenhausen, Germany). Reactions were
performed in Na-phosphate buffer (100 mM, pH 7.5) containing 50 mM methyl-butyrate,
30 mM H2O2, 100 mM NaBr, 0.5 mM APCC and 10 µl of catalase inhibited cell culture
supernatant (commercial enzyme) in a final volume of 100 µl. The enzymatic activity
was measured at 30°C by monitoring the increase of fluorescence intensity using
microtiter plate reader Infinite M1000.
One unit of enzyme activity is defined as the amount of enzyme that produces 1
µmol of peroxycarboxylic acid per minute. Concentration of produced peroxycarboxylic
acid was determined from calibration curve for peroxyacetic acid.
Part II: Assay Development
29
3.5 APCC assay performed in double emulsions
For the preparation of water in oil in water (w/o/w) double emulsions protocol
(Miller, Bernath et al. 2006) was followed. The reaction mixture (W1) was prepared in
100 mM Na-phosphate buffer pH 9.0 by mixing 20 µl of purified Perhydrolase
(0.01mg/ml), 25 µl of 800 mM NaBr, 20 µl 313 mM of H2O2 and 1 µl of 50 mM APCC in
80 µl reaction volume. Samples were vortexed and kept in ice. Primary emulsion was
prepared by mixing 80 µl of reaction mixture and 80 µl of Oil solution (2.9 % (w/w) ABIL
EM90 in light mineral oil). Components were emulsified using Ultra Turax at 10000 rpm
during 5 minutes, on ice. Substrate (methyl-butyrate) was added directly in the primary
emulsion in a final concentration of 50 mM and 250 mM. After addition of methyl-
butyrate, primary emulsion was incubated at 37°C fo r 2 h. Secondary emulsion was
prepared by adding W2 solution (1.5 % (w/v) CMC and 2 % (w/v) Tween 20 in PBS).
Components were emulsified using Ultra Turax at 8000 rpm for 3 minutes, on ice.
Secondary emulsions were diluted 1:100 in 1x PBS and analyzed on CyFlow Space
flow cytometer (Partec). The fluorescence of the product was detected at 375/440 nm.
3.6 Emulsification of Bacillus cells
Cells expressing the Perhydrolase gene (pBC-Per and pHY300Per) and cells
with pHY300PLK vector were grown and expressed as described in Part II, Chapter 3.3.
Bacillus cultures were harvested using a bench centrifuge (Eppendorf centrifuge 5414D,
Eppendorf AG, Hamburg, Germany) at 8000 g for 3 min and rinsed three times in ice-
cold 1x PBS. Approximately 5*108 cells were resuspended in 1 ml Na-phosphate buffer
(100 mM, pH 9.0). Prior to emulsification, catalase is inhibited on the surface of the
cells. Bacillus subtilis cells were incubated in the presence of 100 mM 3-amino-1,2,4-
triazole and 60 mM H2O2 for 2 h at 800 rpm. Emulsified reaction mixture contained 50 µl
of cells (5*108 cells/ml), 20 µl of 600 mM H2O2, 10 µl of 800 mM NaBr and 1 µl of 50 mM
APCC. Reaction mixture is emulsified according to the protocol described in Part II,
Chapter 3.5.
Part II: Assay Development
30
3.7 Fusion of CWBc domain to Perhydrolase
Cloning of Perhydrolase in pHY300PLK is described in Part III, Chapter 3.1.
All gene cloning and DNA manipulation steps were carried out according to
standard molecular cloning protocols (Sambrook and Russell 2001). Preparation of E.
coli competent cells and transformation were carried out as standard heat shock
transformation (Inoue, Nojima et al. 1990).
To amplify the cell wall-binding domain region of the B. subtilis peptidoglycan
hydrolase gene CWBc from genomic DNA, the primers CWBc_fp (5’-gcgc-
CTCGAGCTTAAAAAGACTTCCAGCTC-3’, the underlined indicates XhoI restriction
site, the 3’ end corresponds to the position 545 bp downstream of the CwlC start codon)
and CWBc_rp (5’-gcgc-TCTAGACCGTCTGGAAACATGGTC-3’, the underlined
indicates XbaI site, the 3’ end corresponds to the position 856 downstream of the cwlC
start codon) were used. Perhydrolase with the vector backbone was amplified with
primers pHY300Per_fp (5’-gcgcCTCGAGTTGAGCGGCAGCTTCG-3’, underlined
indicates XhoI restriction site) and pHY300Per_rp (5’-gcgcTCTAGAAGCTTGGGCAAA-
GCGTTTTTCC-3’, underlined indicates XbaI restriction site). Amplified fragments were
digested with restriction enzymes, ligated and transformed in E. coli DH5α. Plasmid was
isolated and transformed in B. subtilis WB600.
3.8 Proteolytic activity
Proteolytic activity in the supernatant of the cells: 5 µl of the supernatant was
added to 95 µl of suc-AAPF-pNA solution (1.1 mM in reaction) in Na-phosphate buffer
(100 mM, pH 9.0). Proteolytic activity for samples was defined as the increase in
absorbance at 405 nm per second across 5 min of reaction.
Proteolytic activity on the surface of the cells: 1 ml of expression cultures (B.
subtilis WB600 pHYPer and B. subtilis WB600 pHYPer_ CWBc), were centrifugated at
8000 rpm for 5 min at RT, supernatant was discarded and cells washed 3 times with
PBS. After washing, cells were re-suspended in 1ml of Na-phosphate buffer (100 mM,
pH 9.0). 5 µl of the re-suspended cells was added to 95 µl of suc-AAPF-pNA solution
(1.1 mM in reaction) in Na-phosphate buffer (100 mM, pH 9.0).
Part II: Assay Development
31
4. Results
4.1 APCC assay principle
The main reaction on the assay is the oxidation of sodium bromide to
hypobromite by the presence of peroxycarboxylic acids, Figure 2.6. Subsequently, the
fluorogenic substrate 7-(4-amino-phenyl)-3-carboxy coumarin (APCC) is O-dearylated
by the generated hypobromite, releasing the fluorescent 7-hydroxycoumarin-3-
carboxylic acid (HCC) and the side product iminocyclohexadienon.
Figure 2.6. Schematic representation of APCC assay.
The excitation and emission spectra of fluorescent product 7-hydroxycoumarin-3-
carboxylic acid (HCC) has excitation and emission peaks at 382 and 445 nm,
respectively, Figure 2.7. The same spectrum was obtained by simulating the O-
dearylation reaction by the addition of sodium bromide and 7-(4-aminophenoxy)-3-
carboxy coumarin (APCC) to a peroxyacetic acid solution. Fluorescent intensity of HCC
increases towards alkaline pH with maximum values at pH 9. Because of this
characteristic, HCC is also used as pH indicator (Offenbacher, Wolfbeis et al. 1986).
APCC assay can be used for detection of peroxycarboxylic acids in the pH range 5-9.
Figure 2.7. 3D fluorescence spectra of acid (HCC).
In order to study theof each compound (bromide and APCC) was varied.
4.2 Effect of bromide concentration
Experimental data showed that p
dearylate APCC; and dearylation
where fluorescent product is gener
bromide (10 to 400 mM) with constant concentration of APCC (0.5 mM) in a calibration
curve for peroxyacetic acid was investigated,
bromide higher than 50 mM, a dramatic s
reaction was not observed, suggesting that the concentration of bromide is in excess in
this reaction setup. Hundred mM sodium bromide were finally selected as standard for
the APCC assay and for recording the ca
Part II: Assay Development
32
3D fluorescence spectra of the reaction product 7-hydroxycoumarin
In order to study the substrate effect on the sensitivity of the assay (bromide and APCC) was varied.
concentration
Experimental data showed that peroxycarboxylic acids cannot
dearylation only takes place in the presence of hypobromite
where fluorescent product is generated. The influence of different concentration of
bromide (10 to 400 mM) with constant concentration of APCC (0.5 mM) in a calibration
acid was investigated, Figure 2.8. With a concentration of
bromide higher than 50 mM, a dramatic shift on the calibration curves after 10 min
reaction was not observed, suggesting that the concentration of bromide is in excess in
Hundred mM sodium bromide were finally selected as standard for
the APCC assay and for recording the calibration curves.
Part II: Assay Development
hydroxycoumarin-3-carboxylic
sensitivity of the assay concentration
acids cannot directly O-
presence of hypobromite
The influence of different concentration of
bromide (10 to 400 mM) with constant concentration of APCC (0.5 mM) in a calibration
. With a concentration of
hift on the calibration curves after 10 min
reaction was not observed, suggesting that the concentration of bromide is in excess in
Hundred mM sodium bromide were finally selected as standard for
Figure 2.8. Calibration curves for 7.5) with different concentrations of NaBr and 0.5 mM APCC. Concentration range of PAA is 0.05-100 µM. The linearity of each calibration curve
4.3 Effect of APCC concentration
For the evaluation the influence
calibration curve for peroxyacetic
APCC substrate, (0.05-0.5 m
concentration of APCC in aqueous solutions
Figure 2.9. Calibration curves for 7.5) containing different concentrations PAA is 0.05-100 µM.
Part II: Assay Development
33
Calibration curves for peroxyacetic acid (PAA) in Na-phosphate buffer (0.1 M, pH 7.5) with different concentrations of NaBr and 0.5 mM APCC. Concentration range of PAA is
100 µM. The linearity of each calibration curve was with R2 > 0.992.
.3 Effect of APCC concentration
evaluation the influence of the secondary substrate
peroxyacetic acid was performed using different concentrations of
.5 mM) in presence of 100 mM bromide. The highest
aqueous solutions due to solubility limitation was 0.5 m
Calibration curves for peroxyacetic acid (PAA) in Na-phosphate buffer (0.1 M, pH ent concentrations of APCC and 100 mM NaBr. Concentration range of
Part II: Assay Development
phosphate buffer (0.1 M, pH 7.5) with different concentrations of NaBr and 0.5 mM APCC. Concentration range of PAA is
of the secondary substrate concentration, a
ifferent concentrations of
00 mM bromide. The highest
due to solubility limitation was 0.5 mM.
phosphate buffer (0.1 M, pH
00 mM NaBr. Concentration range of
Using 0.05 mM, APCC fluorescence intensit
assay is decreased to less than 50
4.4 Effect of hypobromit
of HCC
Peroxycarboxylic acids
substrates or products in APCC assay. Due to that
each reagent present in the assay on the fluorescent product
peroxyacetic acid, bromide and APCC,
calibration curve for HCC in the presence of
the reaction present in the same concentration as HCC)
together (forming hypobromite) these compounds
effect on HCC, Figure 2.10.
Figure 2.10. Calibration curves for HCC in NaHCC, ▲ HCC with 200 mM NaBr, 200 mM NaBr and peroxyacetic
Peroxyacetic acid in the 20 molar excess compared to HCC without sodium
bromide had no effect on fl
to 5 molar excess with sodium bromide (100 mM) showed no effect on HCC
fluorescence intensity, while
decreased the fluorescence intensit
Part II: Assay Development
34
APCC fluorescence intensity is higher, however sensitivity of the
assay is decreased to less than 50 µM PAA, after which linear range is lost, Figure 2.9
bromite and peroxycarboxylic acid on fluorescence intensity
eroxycarboxylic acids and hypohalites are bleaching agents, both are also
substrates or products in APCC assay. Due to that, it’s important to investigate effect of
the assay on the fluorescent product formed in
acid, bromide and APCC, 7-hydroxycoumarin-3-carboxylic acid
HCC in the presence of 200 mM bromide or peroxyacetic
he same concentration as HCC) showed that
together (forming hypobromite) these compounds don’t have a quenching or bleaching
.
Calibration curves for HCC in Na-phosphate buffer (0.1 M, pH 7.5) cont HCC with 200 mM NaBr, ▼ HCC with 200 mM NaBr and 100 mM H
peroxyacetic acid in the same concentration as HCC.
Peroxyacetic acid in the 20 molar excess compared to HCC without sodium
bromide had no effect on fluorescence intensity. Concentrations of peroxyacetic acid
to 5 molar excess with sodium bromide (100 mM) showed no effect on HCC
while further increasing concentration of PAA to 10 molar excess
uorescence intensity of HCC by 30 %, Figure 2.11.
Part II: Assay Development
y is higher, however sensitivity of the
er which linear range is lost, Figure 2.9.
acid on fluorescence intensity
and hypohalites are bleaching agents, both are also
it’s important to investigate effect of
formed in the reaction of
carboxylic acid (HCC). A
peroxyacetic acid (in
showed that independently and
a quenching or bleaching
phosphate buffer (0.1 M, pH 7.5) containing ■
HCC with 200 mM NaBr and 100 mM H2O2, ♦ HCC with
Peroxyacetic acid in the 20 molar excess compared to HCC without sodium
peroxyacetic acid up
to 5 molar excess with sodium bromide (100 mM) showed no effect on HCC
further increasing concentration of PAA to 10 molar excess
Part II: Assay Development
35
Figure 2.11. Fluorescence intensity of 1 µM HCC in the presence of sodium bromide (100 mM) and different molar excess of PAA (0, 1, 5, 10 and 20 µM PAA).
4.5 Sensitivity
The detection limit of the assay is 0.05 µM PAA and the linear range 0.05-100
µM PAA under optimal conditions (100 mM bromide and 0.5 mM APCC), Figure 2.12.
Linear range in 384-well microplates was from 0.25-100 µM PAA. It is also possible to
optimize the assay according to specific needs (i.e. enzymatic reactions) by decreasing
concentration of bromide and APCC or changing the reaction pH with little or no effect
on detection limit and linear range.
Part II: Assay Development
36
Figure 2.12. (A) Correlation of fluorescence (Y-axis) to peroxyacetic acid concentration (0.05-100 µM PAA, R2 value = 0.9954) in sodium phosphate buffer (100 mM, pH 7.5; supplemented with sodium bromide (100 mM) and APCC (0.5 mM)). (B) Enlarged Figure 3A to show the linearity of fluorescence to peroxyacetic acid concentration (0.05-2.5 µM PAA, R2 value = 0.9993) which are of high relevance for directed evolution experiments.
4.6 Peroxycarboxylic detection in the presence of hydrogen-peroxide
Peroxycarboxylic acids are mainly used in industry as bleaching agents, due to
their instability there is a need for in situ peroxycarboxylic acid generation. Current
enzymatic methods relay on the production of peroxycarboxylic methods in the
presence of an excess of hydrogen-peroxide. To be able to perform under these
conditions, and in order to be used as screening system for perhydrolytic enzymes,
APCC assay has to be reliable in the presence of hydrogen-peroxide.
Hydrogen-peroxide is a strong oxidizing agent with very similar characteristics to
peroxycarboxylic acids, due to this it is necessary to develop an assay which can detect
peroxycarboxylic acids in presence of excess of hydrogen-peroxide. Surprisingly, the
HCC product formation is reduced when hydrogen peroxide concentration is increased
for all investigated PAA concentrations (0.05
peroxide concentrations (1 mM, 10 mM, 30 mM and 100 mM) a linear trend is observed
between fluorescence and PAA concentration.
fluorescence intensity is also
2.13. Since hydrogen-peroxide doesn’t have a direct effect on the fluorescent intensity
of the product (HCC), it is not clear the reason for
with high excess of hydrogen
to oxidation of bromide to hypobromite by hydrogen
is much slower compared to reaction rate
Results showed that APCC
presence of 10000 times excess of hydrogen
fluorescent intensity.
Figure 2.13. Calibration curves for 7.5) containing different concentrations of Hof each calibration curve was with R
4.7 Detection of enzymatic activity
To determine whether APCC assay can be used for
perhydrolase activity, ester perhydrolysis catalyzed by s
variant subtilisin Carlsberg
Perhydrolase) was investigated
Part II: Assay Development
37
HCC product formation is reduced when hydrogen peroxide concentration is increased
for all investigated PAA concentrations (0.05-100 µM). Still, for all four hy
peroxide concentrations (1 mM, 10 mM, 30 mM and 100 mM) a linear trend is observed
between fluorescence and PAA concentration. In the presence of 100 mM H
fluorescence intensity is also 2.4-fold lower compared to signal without peroxide,
peroxide doesn’t have a direct effect on the fluorescent intensity
it is not clear the reason for decrease in fluorescence intensity
with high excess of hydrogen-peroxide. Also increased background
to oxidation of bromide to hypobromite by hydrogen-peroxide; however
is much slower compared to reaction rate against peroxycarboxylic acids.
ed that APCC assay can detect peroxycarboxylic acids in the
s excess of hydrogen-peroxide without influence
Calibration curves for peroxyacetic acid (PAA) in Na-phosphate buffer (0.1 M, pH 7.5) containing different concentrations of H2O2, 100 mM NaBr and 0.5 mM APCC. Theof each calibration curve was with R2 > 0.991.
.7 Detection of enzymatic activity
To determine whether APCC assay can be used for
ster perhydrolysis catalyzed by subtilisin Carlsberg
subtilisin Carlsberg Thr59Ala/Leu217Trp (in further text addressed as
investigated.
Part II: Assay Development
HCC product formation is reduced when hydrogen peroxide concentration is increased
or all four hydrogen
peroxide concentrations (1 mM, 10 mM, 30 mM and 100 mM) a linear trend is observed
In the presence of 100 mM H2O2
ed to signal without peroxide, Figure
peroxide doesn’t have a direct effect on the fluorescent intensity
decrease in fluorescence intensity
increased background was observed due
however this reaction rate
peroxycarboxylic acids.
assay can detect peroxycarboxylic acids in the
peroxide without influence in the
phosphate buffer (0.1 M, pH
00 mM NaBr and 0.5 mM APCC. The linearity
To determine whether APCC assay can be used for the detection of
ubtilisin Carlsberg and improved
Trp (in further text addressed as
At first, sensitivity of the assay and response to different amount of enzyme was
investigated. Figure 2.14 shows the response of the assay to different
commercial subtilisin Carlsberg p
Figure 2.14. Enzyme catalyzed perhyCarlsberg detected using APCC continuous assaybuffer (0.1M, pH 7.5) containing 50 mM methylmM APCC.
The difference in change of fluorescent signal per minute of reactions with
different enzyme concentration
assay responds at different enzyme concentration.
Although assay responds
the expression media can be changed due to interference of media components.
perhydrolytic activity was followe
expressing protease subtilisin Carlsberg (WT)
molecules from media such are salts by passing sample
column according to protocol provided by man
Part II: Assay Development
38
At first, sensitivity of the assay and response to different amount of enzyme was
shows the response of the assay to different
commercial subtilisin Carlsberg present in the reaction mixture.
Enzyme catalyzed perhydrolysis of methyl-butyrate by APCC continuous assay. Reaction mixture (40 µl) in Na
buffer (0.1M, pH 7.5) containing 50 mM methyl-butyrate, 30 mM H2O2, 1
ifference in change of fluorescent signal per minute of reactions with
concentration correlates with the enzyme dilution,
different enzyme concentration.
responds to different concentration of pure enzyme, sensitivity in
expression media can be changed due to interference of media components.
perhydrolytic activity was followed in the supernatant of B. subtilis
expressing protease subtilisin Carlsberg (WT) and in the sample after removing small
molecules from media such are salts by passing sample through
according to protocol provided by manufacturer, Figure 2.15
Part II: Assay Development
At first, sensitivity of the assay and response to different amount of enzyme was
shows the response of the assay to different concentrations of
butyrate by commercial subtilisin
0 µl) in Na-phosphate , 100 mM NaBr and 0.5
ifference in change of fluorescent signal per minute of reactions with
enzyme dilution, which proves that
to different concentration of pure enzyme, sensitivity in
expression media can be changed due to interference of media components. The
B. subtilis cell culture
and in the sample after removing small
through PD-10 desalting
, Figure 2.15.
Part II: Assay Development
39
Figure 2.15. Perhydrolytic activity in the supernatant of the cells and in the sample after desalting. Reaction mixture (40 µl) in Na-phosphate buffer (0.1M, pH 7.5) containing 50 mM methyl-butyrate, 30 mM H2O2, 100 mM NaBr and 0.5 mM APCC.
When perhydrolytic activity was measured directly in the supernatant of the cells,
a lag phase was visible in the first 10 minutes of reaction (without increase of
fluorescence intensity) suggesting interferences some of the media components,
probably by reacting with generated peroxycarboxylic acid. When additional
components were removed by exchanging media with buffer lag phase disappeared.
However, in the screening process, desalting every sample would be time consuming,
robust and increase standard deviation. Desalting process was substituted with the
dilution of supernatant which also decreased the influence of interfering compounds.
The optimal dilution, where lag phase disappears and fluorescence intensity is the
highest, was 5-fold. Figure 2.16 shows perhydrolysis of methyl-butyrate catalyzed by
subtilisin Carlsberg and Perhydrolase after catalase inhibition and dilution of the
supernatant. The enzymatic reaction was followed in the supernatant of B. subtilis cell
culture expressing protease obtaining an enzyme activity of 30 mU/ml for subtilisin
Carlsberg and 80 mU/ml for Perhydrolase. Purified Perhydrolase also showed 2.7-fold
higher perhydrolytic activity compared to pure subtilisin Carlsberg under the same
conditions. Perhydrolytic activity ratio between two variants is in agreement with
reported values (Wieland, Polanyi-Bald et al. 2009), which confirms the validity of the
APCC assay.
Part II: Assay Development
40
Figure 2.16. Enzyme catalyzed perhydrolysis of methyl-butyrate by subtilisin Carlsberg and Perhydrolase detected using APCC continuous assay. Reaction mixture (100 µl) in Na-phosphate buffer (0.1M, pH 7.5) containing 50 mM methyl-butyrate, 30 mM H2O2, 100 mM NaBr and 0.5 mM APCC.
4.8 Future development – optimization for high-throughput screening
High throughput screening methods developed until now have a throughput
between 103 and 106, meaning that a significant part of the diversity is lost due to
limitation of screening systems. Recently, methods based on flow cytometry and
fluorescent activated cell sorting (FACS) have been developed (Aharoni, Griffiths et al.
2005; Miller, Bernath et al. 2006), these methods can routinely sort >107 clones per
hour. The core of these methods is the activity detection assay, which needs to be
reproducible and adaptable to a flow cytometry-based screening.
The main requirements for such an assay in order to be used in double
emulsions are: (a) the presence of the charge on the detected molecule, since non-
charged molecules will diffuse through lipid layer leading to cross-contamination, and
(b) excitation wavelength has to be in the range of the laser employed by the flow
cytometry device. Since APCC and the product of the reaction (HCC) carry a positive
charge and fluorescent signal of HCC can be detected using our in-house flow
cytometer equipped with a cell sorter, APCC assay was tested for application in flow
cytometry based screening system.
The first approach was
double emulsions using the
water droplets containing the
with the delivery of ester substra
Perhydrolase was tested using
the water phase containing the rest of the reaction mixtur
Figure 2.17. Flow cytometry recordings of a blankcontaining Perhydrolase in double emulsions. FL3droplets. RN1 – gated region with fluorescence droplets. A) 50mM methylmethyl-butyrate was added in the prima
Results presented in Figure 2.17
reaction and droplets with high fluorescent signal.
intensity values were gated
the number of droplets in RN1 region wa
to control, while in the reaction with 250 mM methyl
higher for positive reaction
Part II: Assay Development
41
first approach was to detect the perhydrolytic activity of
using the flow cytometer. To prevent cross-contamination between
the reaction mixture, the perhydrolytic reaction
substrate through primary emulsions. Perhydrolytic
using different concentration of methyl-butyrate
water phase containing the rest of the reaction mixture.
Flow cytometry recordings of a blank reaction (without enzyme) and reaction containing Perhydrolase in double emulsions. FL3-DAPI represents blue fluorescence of
gated region with fluorescence droplets. A) 50mM methylbutyrate was added in the primary emulsions and incubated at 37°C for 2h.
Results presented in Figure 2.17 show a clear difference between
reaction and droplets with high fluorescent signal. Droplets with highest fluorescence
values were gated in RN1 region. In the reaction with 50 mM methyl
er of droplets in RN1 region was 7-fold higher for positive
n the reaction with 250 mM methyl-butyrate this number wa
reaction compared to control. According to this data
Part II: Assay Development
of purified enzyme in
contamination between
perhydrolytic reaction was started
Perhydrolytic activity of
butyrate, delivered in
reaction (without enzyme) and reaction DAPI represents blue fluorescence of
gated region with fluorescence droplets. A) 50mM methyl-butyrate, B) 250 mM C for 2h.
clear difference between the control
with highest fluorescence
reaction with 50 mM methyl-butyrate,
positive reaction compared
this number was 12-fold
According to this data, we could
Part II: Assay Development
42
conclude that APCC assay is suitable for the detection of perhydrolytic activity in double
emulsions.
Since this assay is suitable for flow cytometry screening, the next step is the
development of a whole-cell screening system using Bacillus cells as an expression
system. B. subtilis secretes subtilisin Carlsberg into the extracellular medium, whereas
~1000 molecules are always present at the surface of the cells (Power, Adams et al.
1986). We expected that after expression, washing and emulsification, 1000 molecules
of Perhydrolase are still on the surface of the cells and can catalyze perhydrolytic
reaction to give fluorescent signal strong enough for detection by flow cytometer. Cells
with empty pHY300PLK vector and cells carrying Perhydrolase gene in two different
vectors (pBC-Per and pHY300Per) were emulsified and activity (fluorescent intensity) in
the droplets was analyzed using flow cytometer device, Figure 2.18.
Figure 2.18. Flow cytometer recw/o/w double emulsions. A) B. subtilispHY300Per and C) B. subtilisthe released HCC product of perhydrolytic reaction.
The product of the p
expressing Perhydrolase in
compared to pHY300PLK vector.
2.18C, the number of fluorescent droplets wa
reaction, Figure 2.18A. This difference in signal between positive and negative reaction
is not significant and in the case of
variants a significant difference compared to
However, these results show that
expression host and APCC assay can be used for devel
screening system based on flow cytometry technology for dire
Perhydrolase.
Further optimization
concentration of Perhydrolase in
Part II: Assay Development
43
Flow cytometer recordings of fluorescence signals of B. subtilis B. subtilis WB600 harboring pHY300PLK, B)
B. subtilis WB600 harboring pBC-Per. FL3 represents blue fluorescence of sed HCC product of perhydrolytic reaction.
The product of the perhydrolyic reaction was detectable only in
expressing Perhydrolase in pBC vector, which has a higher level of expression
compared to pHY300PLK vector. In the RN1 region of that reaction mixture
number of fluorescent droplets was ~3.5-fold higher than in t
This difference in signal between positive and negative reaction
is not significant and in the case of a library which contains low percentage of positive
difference compared to the negative control cannot be achieved
these results show that an in vivo system which employs
expression host and APCC assay can be used for development of high throughput
screening system based on flow cytometry technology for dire
Further optimization of this system should consider: i)
concentration of Perhydrolase in the reaction droplets by attaching protein molecules for
Part II: Assay Development
B. subtilis cells entrapped in
WB600 harboring pHY300PLK, B) B. subtilis harboring Per. FL3 represents blue fluorescence of
only in B. subtilis cells
higher level of expression
at reaction mixture, Figure
fold higher than in the control
This difference in signal between positive and negative reaction
ntains low percentage of positive
cannot be achieved.
system which employs B. subtilis as an
opment of high throughput
screening system based on flow cytometry technology for directed evolution of
i) including a higher
hing protein molecules for
Part II: Assay Development
44
the surface of the cells (surface display), ii) higher expression of protease and iii)
constant delivery of hydrogen-peroxide through the emulsions since most of the
peroxide is destroyed by cells protective mechanism (catalases).
4.9 Surface display of protease
The cell wall binding (CWBc) domain fused to the C-terminus of Perhydrolase
should localize the protein on the surface of the Bacillus cells due to non-covalent
interactions between CWBc domain and structures on the cell wall. Increased number of
protease molecules on the Bacillus cells will increase the sensitivity of flow cytometry
screening system for detection of perhydrolytic activity in double emulsions.
CWBc domain was successfully fused to the C-terminus of Perhydrolase gene
and transformed in B. subtilis WB600. If the construct is functional, proteolytic activity at
the surface of the cells should be increased compared to cells which are expressing
Perhydrolase without this domain. In the Table 2.3 is presented activity in the
supernatant and on the surface of the cells after.
Table 2.3. Proteolytic activity of Perhydrolase and Perhydrolase_CWBc constructs in the supernatant and on the surface of the cells.
Expression time (h)
Supernatant (U/ml) Surface of the cells (U/ml)
Perhydrolase Perhydrolase_CWBc Perhydrolase Perhydrolase_CWBc
8 0.035 0.03 0.043 0.043
10 0.283 0.25 0.04 0.038
24 3.125 3.655 0.12 0.1
There was no difference in activity on the surface of the cells of Perhydrolase
and Perhydrolase_CWBc constructs. Similar level of activity was detected in the
supernatant of the cells, suggesting that Perhydrolase cleaves this small domain. SDS-
PAGE analysis of the supernatant confirmed the hypothesis that the small binding
Part II: Assay Development
45
domain is cleaved, since the band which corresponds to Perhydrolase without CWBc
domain was detected.
5. Discussion
The first fluorescent based screening system for detection of peroxycarboxylic
acids in microtiter plate was developed as part of a high throughput screening platform
for peroxycarboxylic acid producing enzymes. APCC allows a continuous detection of
peroxycarboxylic acids and relies on the detection of a generated fluorescent coumarin
compound (see Fig. 2.6). Main performance criteria for a high throughput screening
systems are: a low standard deviation, reproducibility, simplicity, high sensitivity and low
interference of media or reaction compounds on enzymatic activity. In order to
determine the sensitivity of the APCC assay, peroxyacetic acid concentrations were
quantified in the presence of varied concentrations of sodium bromide and APCC.
Additionally, the fluorescent detection product, HCC, was “treated” with bleaching
agents (hydrogen peroxide, peroxyacetic acid and hypobromite) to determine its
stability.
A main challenge of peroxycarboxylic acid detection systems for directed enzyme
evolution is the interference of cell lysate/supernatant components with the detection
system. Most peroxycarboxylic acid detection methods require enzyme purification
before peroxycarboxylic acid determination and only a few are downscaled to a
microtiter plate format, Table 2.1 (Pinkernell, Luke et al. 1997) and (Binder and Menger
2000)). The validated APCC screening system for directed perhydrolase evolution is the
first reported continuous assay which enables detection of perhydrolytic activity without
enzyme purification and which has been optimized for medium and high throughput
screening in microtiter plate format.
Part II: Assay Development
46
6. Conclusion
APCC assay is a spectrofluorometric assay for quantification of in situ
peroxycarboxylic acid production. This is the first reported continuous assay which
enables detection of perhydrolytic activity without previous purification of enzyme
optimized for medium and high throughput screening. The wide pH range (5-9) and
robustness (sodium bromide concentration, oxidative HCC stability) of the APCC
detection system offers a broad flexibility in terms of assay conditions. This simple,
highly sensitive assay is suitable for finding new or modifying existing enzymes with
perhydrolytic activity (lipases, esterases, proteases, nonheme haloperoxidases) to
develop efficient enzymatic bleaching system for industrial applications. APCC assay
enables detection of perhydrolytic activity in double emulsions, opening the possibility to
develop in vivo (cell host expression) and in vitro (cell free expression) high throughput
screening systems.
Part III: Directed Evolution of Subtilisin Carlsber g
47
Part III: Directed Evolution of Subtilisin Carlsberg for
Improved Perhydrolytic Activity
1. Introduction
1.1 Aim of this work
The aim of this work was to generate and identify variants of subtilisin Carlsberg
with increased perhydrolytic activity and simultaneously decreased proteolytic/hydrolytic
activity. In order to achieve this, random mutagenesis techniques (epPCR, SeSaM) and
rational design approach (Saturation Mutagenesis) were used employing high
throughput screening methods. Improved variants were characterized and structure-
function analysis was performed. Changed specificity from peptides toward ester
substrates and from water to hydrogen-peroxide will allow us to study enzymatic
promiscuity and gain more general knowledge about tailoring subtilisin catalysis in
direction of perhydrolases.
1.2 Proteases
Proteases (also named peptidases or proteinases) are enzymes that hydrolyze
peptide bonds in proteins and polypeptides. Different proteases have its specificity in
“choosing” which peptide bonds hydrolyze depending in neighboring amino acid
residues, but all proteases have in common that they can catalyze the hydrolysis of
ester bonds, since the chemical mechanism of hydrolysis of amide bonds and ester
bonds is similar (Hedstrom 2002).
Proteases are classified into six broad groups: serine, threonine, cysteine,
aspartate, glutamic acid proteases and metalloproteases (Barrett, Rawlings et al. 1998).
The mechanism used to cleave a peptide bond involves a nucleophilic attack to the
carbonyl group of the peptide bond by an amino acid residue (in serine, cysteine and
threonine proteases) or a water molecule (aspartic acid, metallo- and glutamic acid
proteases). This nucleophilic attack is induced by a catalytic triad, where a histidine
Part III: Directed Evolution of Subtilisin Carlsber g
48
residue is used to activate serine, cysteine or threonine as a nucleophile. Within each of
these groups, proteases are classified into families of related proteases. Alternatively,
proteases can be classified by the optimal pH in which they are active: acid, neutral and
alkaline proteases and peptide bond specificity: endopeptidase, exopeptidase
(carboxypeptidase and aminopeptidase) and amino acid specific peptidase (Barrett,
Rawlings et al. 1998).
Proteases were the first enzyme class to be discovered because of their
abundance in the digestive system. Additionally, they have a wide range of important
biological activities, as they are also involved in processing other proteins. For example,
the blood clothing cascade and activation of immune system involves serine proteases.
Metalloproteases and cysteine proteases are involved in the self destruction of cells
(apoptosis). Bacteria also secrete proteases to hydrolyze (digest) the peptide bonds in
proteins and therefore break the proteins down into their constituent monomers and use
them as nutrients. A secreted bacterial protease may also act as an exotoxin which
destroys extracellular structures (Fersht 1999).
Proteases find applications in various industrial sectors: medicine, pharmacology
and drug manufacture, hard surface cleaning formulations, contact lens cleaning
formulations, waste treatment, fermentation, animal feed additives, digestive
supplements, food and food processing applications (Kirk, Borchert et al. 2002). Today,
proteases are dominant in total enzyme sales and is expected that their dominance will
increase. Most of these proteases are microbial, since they can be produced in large
quantities in a short time. Microbial alkaline proteases hold the largest part of enzyme
market. These proteases are used in food industry, peptide synthesis, leather industry,
industrial and household waste management, photographic industry, medical usage, silk
degumming and the largest field is detergent industry (Gupta, Beg et al. 2002).
Commercial alkaline proteases are summarized in Table 3.1.
Part III: Directed Evolution of Subtilisin Carlsber g
49
Table 3.1. Commercial bacterial alkaline proteases, sources, applications and their industrial suppliers. n.s.- not specified, PE - protein engineered. Table taken from (Gupta, Beg et al. 2002)
Supplier Product trade name Microbial source Application
Novo Nordisk, Denmark
Alcalase Savinase Esperase
Biofeed pro Durazym
Novozyme 471MP Novozyme 243
Nue
B. licheniformis Bacillus sp.
B. lentus B. licheniformis
Bacillus sp. n.s
B. lentus Bacullus sp.
Detergent, silk degumming Detergent, textile
Detergent, food, silk degumming Feed
Detergent Photographic gelatin hydrolysis
Denture cleaners Leather
Genencor Inter-national, USA
Purafact Primatan
B. lentus Bacterial source
Detergent Leather
DSM Gist Brocades, The Netherlands
Subtilisin Maxacal
Maxatase
B. alcalophilus Bacillus sp. Bacillus sp.
Detergent Detergent Detergent
Solvay Enzymes, Germany
Opticlean Optimase Maxapem
HT-proteolytic
Protease
B. alcalophilus B. licheniformis PE variant of Bacillus sp. B. subtilis
B. licheniformis
Detergent Detergent Detergent
Alcohol, baking, brewing, feed,
Food, waste
Amano Enzyme Inc, Japan
Proleather Collagenase
Amano protease S
Bacillus sp. Clostridium sp.
Bacillus sp.
Food Technical
Food
Enzyme Development,
USA
Enzeco alkaline protease
Enzeco alkaline protease-L FG Enzeco high
alkaline protease
B. licheniformis
B. licheniformis
Bacillus sp.
Industrial
Food
Industrial
Nagase Biochemicals,
Japan
Bioprase concentrarte Ps. protease Ps. elastase
Cryst. protease Cryst. protease
Bioprase Bioprase SP-10
B. subtilis
P. aeruginosa P. aeruginosa B. subtilis (K2)
B. subtilis (bioteus) B. subtilis B. subtilis
Cosmetic, Pharmaceuticals
Research Research Research Research
Detergent, cleaning Food
Godo Shusei, Japan
Godo-Bap B. licheniformis Detergent, food
AB Enzymes, Germany
Corolase 7089 B. subtilis Food
Wuxi Synder Bioproducts,
China
Wuxi Bacillus sp. Detergent
Advance Biochemicals,
India
Protosol Bacillus sp. Detergent
Part III: Directed Evolution of Subtilisin Carlsber g
50
1.2.1 Serine proteases and subtilisins
Serine proteases are the most thoroughly studied class of proteases, and
perhaps in all of enzymology. Serine endo- and exo-peptidases widespread from
bacteria to mammals and have diverse functions, such as digestive and degradative
processes in blood clothing, immune system, tissue remodeling (Krem and Di Cera
2001).
The catalytic mechanism of serine proteases is based on nucleophilic attack of
the targeted peptide bond by a serine. Side chains of serine, histidine and aspartate
build the catalytic triad common to most serine proteases. Figure 3.1 present catalytic
mechanism of serine proteases on example of chymotripsin-like enzymes. The
deprotonated His 57 acts as a general base to subtract a proton from Ser 195,
enhancing its nucleophilicity as it attacks the electrophilic carbon of the amide or ester
link, forming the oxyanion tetrahedral intermediate. Asp 102 stabilizes the positive
charge on the His. His, as a general acid and base catalyst, stabilizes charges in the
transition state and provides a path for proton transfer, without which reactions would
have difficulty in proceeding. The first intermediate is the Michaelis complex. This is
followed by a tetrahedral intermediate in which a covalent bond is formed between the
substrate carbonyl carbon atom and serine O atom. This transition state is stabilized by
formation of hydrogen bonds with residues in the oxianion hole. A hydrogen atom is
transferred from histidine N atom to the leaving group of the substrate. When this
transfer is finished, the first product of the reaction is released and the acyl-enzyme
intermediate is formed. In the next step water comes as a second substrate, His
accepts a proton from the water and covalent bond between oxygen from water and the
carbon of acyl-enzyme intermediate is formed, this is the second tetrahedral
intermediate. At the end, the second tetrahedral intermediate collapses leading to the
release of the second product of the reaction and the regenerated active site of the
enzyme.
Part III: Directed Evolution of Subtilisin Carlsber g
51
Figure 3.1. Reaction mechanism of catalysis of serine proteases. Modified from (Garrett and Grisham 1999).
SSuubbssttrraattee
BBiinnddiinngg ooff
ssuubbssttrraattee
FFoorrmmaattiioonn ooff ccoovvaalleenntt EESS ccoommpplleexx
PPrroottoonn ddoonnaattiioonn bbyy
HHiiss5577
CC--HH bboonndd cclleeaavvaaggee
RReelleeaassee ooff aammiinnoo pprroodduucctt
NNuucclleeoopphhiilliicc aattttaacckk bbyy wwaatteerr
CCoollllaappssee ooff tteettrraahheeddrraall
iinntteerrmmeeddiiaattee
CCaarrbbooxxyyll pprroodduucctt rreelleeaassee
Part III: Directed Evolution of Subtilisin Carlsber g
52
Additional amino residues forming the oxyanion hole are involved in the
stabilization of tetrahedral intermediates. The negative oxygen ion of the intermediates
fits in the oxyanion hole, forming hydrogen bonds with the backbone of the residues in
the hole, Figure 3.2. This structure is favored, which means that activation energy of the
reaction is lowered and this binding is responsible for the catalytic efficiency of the
enzyme.
Figure 3.2. The “oxyanion hole” of chymotrypsin stabilizes the tetrahedral oxyanion transition states of chymotrypsin. Taken from (Garrett and Grisham 1999).
This type of catalysis is called ping-pong, where the first substrate binds to the
enzyme (peptide), then the first product is released (the N-terminus of peptide), the
second substrate binds (water) and at the end the final product is released (C-terminus
of peptide). The proof for this type of catalytic pathway is that three intermediates (the
Michaelis complex, tetrahedral intermediate and acyl-enzyme complex) have been
isolated and characterized by X-ray chystallography (Kraut 1977).
Many distinct families of serine proteases exist; they have been grouped into six
clans (Chemotrypsines, Subtilisin-like, Carboxipeptidase C, Alapeptidase E, Represor
Lexa-like, ATP-dependent serine peptidases) (Barrett and Rawlings 1995). The two
largest clans of serine proteases are the Trypsin-like and Subtilisin-like clans. Subtilisins
are a non-specific, extracellular proteases initially obtained from Bacillus subtilis. They
are physically and chemically well characterized enzymes. The catalytic triad of
Gly 193
Ser 195
The oxyanion hole
subtilisins consists of aspartic acid,
90 kDa, but most have a size of approximately 27 kDa
Subtilisins are produc
sequence) leads the protein
between the signal sequence and the N
folding of the subtilisin into its
1987). The pro-region also
membrane until it is cleaved off autocatalyt
Today, crystal structures of the most important representatives of this family are
available: Bacillus licheniformis
Bacillus amyloliquefaciens
alkaline protease Savinase
protease PB92 (van der Laan, Teplyakov et al
(Gros, Betzel et al. 1989),
Bacillus TA41 Subtilisin S41
structure of these enzyme
amino acids) has a globular shape and
active site is located in the larger
Figure 3.3. Three-dimensional modelsubtilisin Carlsberg (PDB code 1c3l).
Part III: Directed Evolution of Subtilisin Carlsber g
53
subtilisins consists of aspartic acid, histidine and serine, the size varies fro
ze of approximately 27 kDa (Maurer 2004
Subtilisins are produced as pre-proproteins, where the pre
sequence) leads the protein to the extracellular environment. The p
between the signal sequence and the N-terminal end of mature su
folding of the subtilisin into its enzymatically active conformation (Ikemura, Takagi et al.
also holds the pro-subtilisin molecule associated with the
membrane until it is cleaved off autocatalytically by subtilisin (Egnell and Flock 1992
rystal structures of the most important representatives of this family are
licheniformis subtilisin Carlsberg (Bode, Papamokos et al. 1986
subtilisin BPN’ (Wright, Alden et al. 19
alkaline protease Savinase (Betzel, Klupsch et al. 1992) , Bacillus alcalophilus
van der Laan, Teplyakov et al. 1992), Thermus vulgaris
, Thermus album proteinase K (Betzel, Pal et al. 1988
Subtilisin S41 (Almog, González et al. 2009). The secondary
enzymes is highly conserved. The central core of the protein
has a globular shape and consists of 7 β-sheet and nine
in the larger subdomain next to the central β-sheet, Figure 3.3
dimensional model of subtilisin Carlsberg derived from crystal structure of
subtilisin Carlsberg (PDB code 1c3l). Catalytic triad residues are Asp 32, His 64 and Ser 221.
Part III: Directed Evolution of Subtilisin Carlsber g
he size varies from 18 kDa to
Maurer 2004).
proteins, where the pre-sequence (signal
pro-region is located
terminal end of mature subtilisin, guiding the
Ikemura, Takagi et al.
subtilisin molecule associated with the
Egnell and Flock 1992).
rystal structures of the most important representatives of this family are
Bode, Papamokos et al. 1986),
Wright, Alden et al. 1969), Bacilus lentus
Bacillus alcalophilus alkaline
Thermus vulgaris thermitase
Betzel, Pal et al. 1988) and
The secondary and tertiary
entral core of the protein (194
sheet and nine α-helices, the
sheet, Figure 3.3.
derived from crystal structure of atalytic triad residues are Asp 32, His 64 and Ser 221.
Crystal structures of
involved in substrate binding
pockets from P6 to P3’. P is
residues extending from the cleaved bond toward
nomenclature for the substrate amino acid residues
toward COOH-terminus-leaving
corresponding binding sites on the enzyme. E
that the inhibitor binds in a surface channel of the enzyme interacting with
residues from P4 to P2’, Figure 3.4
Figure 3.4. Interaction of oligopeptide substrate with catalytic residues of serine proteasesurface interactions (A) and chemicalacid residues is P, where P1binding site. Part B taken from
Subtilisins are highly stable
a wide substrate specificity which makes them suitab
Additionally, as extracellular enzymes,
expression in Bacillus species enables secretion of enzymes in a short period of time
into the fermentation media
Although members of
continuously in many organisms
used in the detergent industry
Part III: Directed Evolution of Subtilisin Carlsber g
54
Crystal structures of the enzyme-inhibitor complex helped to identify residues
involved in substrate binding. More than nine amino acids residues form binding
P is the standard nomenclature for the substrate amino acid
residues extending from the cleaved bond toward NH2-terminus-acyl
nomenclature for the substrate amino acid residues extending from the cleaved bond
leaving-group side, S and S’ is the
corresponding binding sites on the enzyme. Every enzyme-inhibitor complex showed
inhibitor binds in a surface channel of the enzyme interacting with
Figure 3.4, (Perona and Craik 1995).
Interaction of oligopeptide substrate with catalytic residues of serine proteaseface interactions (A) and chemical interactions (B). Nomenclature for the substrate amino
acid residues is P, where P1-P1’ denotes the hydrolyzed bond, S is nomenclature for enzyme aken from (Perona and Craik 1995).
Subtilisins are highly stable in the presence of solvents and detergents
substrate specificity which makes them suitable for industrial application
extracellular enzymes, they are easily separated from the biomass
species enables secretion of enzymes in a short period of time
into the fermentation media (Maurer 2004).
embers of subtilase superfamily of serine proteases are
organisms, subtilisins from Bacillus species are
used in the detergent industry, Table 3.2. Since detergent industry demands high
Part III: Directed Evolution of Subtilisin Carlsber g
or complex helped to identify residues
residues form binding
nomenclature for the substrate amino acid
acyl-group side, P’ is
extending from the cleaved bond
the nomenclature for
inhibitor complex showed
inhibitor binds in a surface channel of the enzyme interacting with protein
Interaction of oligopeptide substrate with catalytic residues of serine protease:
Nomenclature for the substrate amino P1’ denotes the hydrolyzed bond, S is nomenclature for enzyme
in the presence of solvents and detergents and have
le for industrial application.
ily separated from the biomass and
species enables secretion of enzymes in a short period of time
proteases are identified
are still the only ones
Since detergent industry demands high
Part III: Directed Evolution of Subtilisin Carlsber g
55
amount of these enzymes, subtilisins are among the first produced using recombinant
strains. They were also among the first enzymes which were modified using protein
engineering techniques to satisfy needs of growing detergent industry for enzymes
efficient under different non-natural conditions (high or low temperature, presence of
denaturizing agents). Subtilisin BPN’ is the most engineered subtilisin protease and by
1996 substitutions at almost every position of mature subtilisin BPN’ have been
patented. This enzyme is today used as a model system for protein engineering of other
homologous subtilisins (Maurer 2004).
Table 3.2. Subtilisin variants used in detergent industry. Legend: aExclusive molecules for specific customer, bExlusive molecules for captive use, PE protein engineered, WT wild type. Taken from (Maurer 2004).
Trade mark Producer Origin WT/PE Production strain Synonim
Alcalase Novozymes B. licheniformis WT B. licheniformis subtilisin Carlsberg
FNAa Genencor B. amyloliquefaciens PE B. subtilis
Savinase Novozymes B. clausii WT B. clausii Subtilisin 309
Purafect Genencor B. lentus WT B. subtilis
KAPb Kao B. alkalophilus WT B. alkalophilus
Everlase Novozymes B. clausii PE B. clausii
Purafect OxP
Genencor B. lentus PE B. subtilis
FN4a Genencor B. lentus PE B. subtilis
BLAP Sb Henkel B. lentus PE B. licheniformis
BLAP Xb Henkel B. lentus PE B. licheniformis
Esperase Novozymes B. halodurans WT B. halodurans Subtilisin 147
Kannase Novozymes B. clausii PE B. clausii
Properase Genencor B. alkalophilus PB92 PE B. alkalophilus
Part III: Directed Evolution of Subtilisin Carlsber g
56
1.2.2 Protein engineering in subtilisin proteases
Almost every property of subtilisin has been altered by protein engineering
including its catalysis, substrate specificity, stability to oxidative, thermal and alkaline
inactivation and pH/rate profile. In this work, the main focus was on altered substrate
specificity of subtilisin and substrate-assisted catalysis.
Subtilisin BPN’ was, in most of the cases, subject of protein engineering
investigation. Amino acid substitution at positions Glu 156 and Gly 166 which form S1
site (P1 binding site) showed that charged substitutions increase the catalytic efficiency
toward complementary charged P1 substrates and decrease toward similarly charged
P1 substrates (Wells, Powers et al. 1987). Altering the electrostatic potential of the S1
site by introducing or removing Arg, Lys, Glu and Asp at positions 156 and 166 kcat/KM
1000-fold increases toward complementary charged substrates. This enzyme also
shows preference for the substrates with hydrophobic residues at position P1. To
investigate the effect of different hydrophobicity levels of S1 site, position Gly 166 was
substituted with 12 different amino acids. Results showed that an increase in the side-
chain volume at position 166 decrease the size of S1 gap leading to reductions in
kcat/KM toward large amino acids up to 5000-fold. The reason for reduced activity is due
to steric repulsion, which is more dominant than hydrophobic interactions. At the same
time catalytic efficiencies toward small P1 side chains were increased up to 10-fold in
these variants (Estell, Graycar et al. 1986). Results showed that specificity is easily
changed by replacing directly amino acids which are in contact with substrate. This
brought the idea of changing one protease into a related one by protein engineering
(Wells, Cunningham et al. 1987). Subtilisin Carlsberg and subtilisin BPN’ differ by 31 %
in sequence and by factor > 60 in catalytic efficiency toward different substrates.
Despite these differences in sequence and catalytic efficiency only 3 substitutions lie
within 7 Ǻ of the S1 pocket. Those substitutions are at positions 156, 217 and 169;
positions 156 and 217 are within 4 Ǻ of modeled substrate where position 156 is part of
S1 pocket, while position 217 is in the S1’ site. A third residue at position 169 is behind
the S1 pocket, 7 Ǻ from the substrate. In subtilisin BPN’ the corresponding amino acids
are Glu 156, Gly 169 and Tyr 217; these were replaced with analogous of subtilisin
Carlsberg Ser 156, Ala 169 and Leu 217. Substrate specificity of BPN’ triple mutant was
Part III: Directed Evolution of Subtilisin Carlsber g
57
tested with seven different substrates that vary in charge, size and hydrophobicity was
similar to wild type of subtilisin Carlsberg.
Subtilisin BPN’ can also function as a peptide ligase. This reaction happens
when a peptide carrying a free amino-terminal group can compete with water for the
nucleophilic attack on the acyl-enzyme intermediate. The substitution of the active site
Ser 221 by Cys improved ligase activity of BPN’ 1000-fold (Nakatsuka, Sasaki et al.
1987). The additional mutation Pro225Ala improved ligase activity additionally 10-fold
(Abrahmsen, Tom et al. 1991). Subtiligase, engineered variant of subtilisin BPN’, was
used to synthesize ribonuclease A and active-site variants of this enzyme by stepwise
ligation of six esterified peptide fragments (each 12 to 30 residues long) at yields
averaging 70 % per ligation (Jackson, Burnier et al. 1994).
Considerable specificity toward substrate residues depends from the interaction
between S4 and P4. The S4 site of subtilisin BPN’ and Bacillus lentus alkaline protease
(BLAP) is formed of two structural elements: residues 100-107 which are part of α-helix
in the small subdomain and residues 125-132 in a surface loop (Perona and Craik
1995). For example, introducing mutations at position Tyr 104 and Ile 107 in subtilisin
BPN’ changed specificity toward residues with large side chains at P4. Mutations
increased the size of the P4 site by replacing Ile 107 with smaller amino acids and to
disrupt the hydrogen bond between Tyr 104 and Ser 130 by introducing Phe at position
104. The largest improvement in specificity for P4-Phe relative to P4-Ala was 200-fold
for the variant which had Ile107Gly substitution (Rheinnecker, Baker et al. 1993).
Engineering of S4 site draw as a conclusion that both steric and hydrophobic effects
determinate the S4 specificity profile.
Substitutions within S1 and S4 site showed that only the local environment of
amino acids contacting substrate should be considered in designing substrate specificity
and polar and hydrophobic interactions enzyme-substrate are important since each of
them generates a different specificity.
Part III: Directed Evolution of Subtilisin Carlsber g
58
1.2.3 Subtilisin Carlsberg
Subtilisin Carlsberg is a serine protease produced by B. licheniformis. The size of
mature protein is approximately 27 kDa with a pH optimum 8-10. The original subtilisin
is described in 1954 (Guntelberg and Ottesen 1954) and later called subtilisin
Carlsberg. In 1968 subtilisin Carlsberg was sequenced, characterized and the
relationship with other proteinases determined (Smith, DeLange et al. 1968). Due to its
termostability and broad specificity, this enzyme has become industrially important. In
order to elucidate the evolutionary relationship between the subtilisins and simplify the
production of this enzyme, in 1985 the gene encoding subtilisin Carlsberg was isolated,
sequenced and cloned into Bacillus expression vectors (Jacobs, Eliasson et al. 1985).
Subtilisin Carlsberg consists of N-terminal signal peptide including a 29 residues
followed by a 76 residue highly charged pro-region and 274 residues of mature protein.
The signal sequence has similar properties to other signal sequences from Gram
positive bacteria, which includes a basic N-terminal segment followed by a stretch of
uncharged residues and cleavage site most frequently follows the (-3,-1) rule. Most
probably that cleavage of signal sequence takes place after the residues Ala-Ser-Ala.
Homology plot analysis revealed that C-terminus of the pro region is conserved, which
means that there is an evolutionary pressure to keep the structure of this region since is
most probably recognized by a proteolytic enzyme during maturation. For example, the
mature enzyme subtilisin Carlsberg is 70% homologous to BPN’ with identical catalytic
triad, while pro regions are less than 50 % homologous. Still, the secondary structure of
the whole pro region is very similar leading to conclusion that most probably tertiary
structure is also the same (Jacobs, Eliasson et al. 1985).
Maturation and release of subtilisins in an extracellular environment is an
autocatalytic process. Experimental data also confirmed this theory, when inactive
variants of B. amyloliquefaciens subtilisin were expressed in Bacillus protease deficient
strains, only preproenzyme was detected in the cell membrane, whereas the mature
form of the enzyme was absent in the media. Maturation can be carried out with
addition of active subtilisin during fermentation or co-expression together with the
inactive form (Power, Adams et al. 1986). However, when subtilisin Carlsberg variant
which has a deletion of amino acids 97-101 in the mature part of the enzyme was
Part III: Directed Evolution of Subtilisin Carlsber g
59
expressed in B. subtilis DB104 (protease deficient strain), the processing and secretion
of enzyme was blocked. Only the pre-proenzyme (molecular weight on SDS gel
corresponds to 42 kDa) was detected in the membrane fraction. When the deletion
variant was expressed in the protease-proficient Bacillus strain only slight increase in
proteolytic activity was detected which can be due to increased expression of the host
strain own proteases (Schulein, Kreft et al. 1991). Experimental data also showed that
the autocatalytic cleavage of the pro sequence and its recognition is independent of the
amino acid sequence around that site, suggesting that the cleavage specificity most
likely depends on the secondary structure of that region (Egnell and Flock 1992).
As of today, all the amino acid positions of subtilisin Carlsberg have been
modified either by site-directed mutagenesis based on rational design or by various
methods of random mutagenesis.
1.2.4 Promiscous activity of subtilisin Carlsberg and Thr59Ala/Leu217Trp variant
It is observed that subtilisin Carlsberg, besides proteolytic (hydrolytic) activity,
also shows perhydrolytic activity. As a result of random mutagenesis, it has been
discovered that modification of the subtilisin Carlsberg by point mutations can generate
perhydrolases with the potential to be used in bleaching systems. The variant
Thr59Ala/Leu217Trp (Perhydrolase) has an 8-fold decreased proteolytic activity and 3-
fold increased perhydrolytic activity compared to wild type subtilisin Carlsberg (Wieland,
Polanyi-Bald et al. 2009).
This results and the previously mentioned switching catalysis from esterase to
perhydrolase motivated to study the second tetrahedral intermediate during hydrolytic
and perhydrolytic reaction in order to understand how single mutation shifts activity
toward perhydrolysis (Lee, Vojcic et al. 2010). The initial structure used for Molecular
Dynamics (MD) simulation corresponded to the coordinates of the crystal structure of
wild type subtilisin Carlsberg, while mutations were introduced in silico. Thr59Ala
substitution is far from the catalytic Ser 221 so it was assumed that this mutation
doesn’t influence on activity of Carlsberg. Leu217Trp substitution is close to the active
site and the Trp 217 variant was used as a starting structure for MD simulations.
Results showed that Trp 217 is very important for the stabilization of the second
Part III: Directed Evolution of Subtilisin Carlsber g
60
tetrahedral intermediate (TI) of the perhydrolytic reaction. Trp 217 forms a hydrogen
bond with oxygen of the perhydroxyl group from the second TI, Figure 3.5, while in the
case of wild type there is no hydrogen bonding which can stabilize the second TI state
which can explain for lower perhydrolytic activity compared to Perhydrolase.
Figure 3.5. Hydrogen bonding between N-H in Trp217 and the hydroxyl oxygen of the 2nd tetrahedral intermediate (TI) of the hydrolysis (A) and perhydrolysis reaction (B). Taken over from (Lee, Vojcic et al. 2010).
MD simulation studies also showed differences between the hydrogen bonds
formed by the hydrogen attached to Nε of the catalytically important His 64 and the
second TI. In case of the hydrolysis reaction, this hydrogen interacts with the oxygen of
Ser 221 and any competing hydrogen bond may interfere with the catalysis. In the case
of wild type there were no interfering hydrogen bonds, but in case of the Perhydrolase
there was interfering hydrogen bonds which can be explanation for reduced hydrolytic
activity. During the perhydrolytic reaction, a similar effect was observed, just this time
the interfering hydrogen bond was present in a higher extent for wild type which could
explain the lower perhydrolytic activity compared to the mutant, Figure 3.6.
Part III: Directed Evolution of Subtilisin Carlsber g
61
Figure 3.6. Hydrogen bonding between His64 and oxygen atoms of the 2nd TI state of hydrolysis (A) and perhydrolysis (B). Hydrogen bonds are indicated with double arrows, where with dashed arrows are represented interfering hydrogen bonds. Taken over from (Lee, Vojcic et al. 2010).
We used Perhydrolase as a starting point for further directed evolution
experiments in order to increase perhydrolytic activity of subtilisin Carlsberg.
Table 3.3 shows the ratio of hydrolytic/perhydrolytic activity of enzymes present
in nature or engineered.
The values for the ratio of hydrolytic/perhydrolytic activity in the Table 3.3 are not
the representing the real ratio of activities since in most of the cases characterization of
hydrolytic and perhydrolytic activity was done with different substrates. For example,
hydrolytic activity of subtilisin Carlsberg and it’s variant Thr59Ala/Leu217Trp was
determined with tetrapeptide suc-AAPF-pNA as a substrate, while perhydrolytic activity
was determined with ester methyl-butyrate.
Part III: Directed Evolution of Subtilisin Carlsber g
62
Table 3.3. Perhydrolytic enzymes and ratio of their hydrolytic and perhydrolytic activites
Enzyme source Hydrolytic/perhydrolytic
activity Reference
P. fluorescens esterase mutant L29P
0.02 (Bernhardt, Hult et al. 2005)
T. maritime CE-7 0.2 (Dicosimo, Gavagan et al.
2008)
T. lettingae CE-7 0.3 (Dicosimo, Gavagan et al.
2008)
T. neapolitana CE-7 1.0 (Dicosimo, Gavagan et al.
2008)
C. parapsilosis polypeptide 1.5 (Dubreucq, Weiss et al. 2007)
P. mendocina lipase mutant Ser207
2.0 (Poulouse 1994)
P. putida lipase mutant Arg 127 2.0 (Poulose and Anderson
1992)
B. subtilis 31954 CE-7 4.5 (Dicosimo, Gavagan et al.
2008)
B. licheniformis subtilisin Carlsberg Thr59Ala/Leu217Trp
7.3 (Wieland, Polanyi-Bald et al.
2009)
Part III: Directed Evolution of Subtilisin Carlsber g
63
2. Materials
2.1 Chemicals
All chemicals used were of analytical reagent grade or higher quality and
purchased from Sigma-Aldrich (Taufkirchen, Germany), Applichem (Darmstadt,
Germany) or Carl Roth (Karlsruhe, Germany). All enzymes were purchased from New
England Biolabs (Frankfurt, Germany) or Fermentas GmbH (St. Leon-Rot, Germany).
The substrate for detection of proteolytic activity, Suc-AAPF-pNA, was purchased from
Sigma-Aldrich (Taufkirchen, Germany) and Bachem (Bubendorf, Switzerland). The
protease inhibitor PMSF was purchased from Sigma-Aldrich. The fluorogenic substrate
APCC was synthesized according to protocol described in Part II, Chapter 3.1.
2.2 Bacterial strains
The bacterial strains used in this work are listed in Table 3.4.
Table 3.4. Bacterial strains used in this work.
Strain Description References
E. coli DH5α
F'/endA1 hsdR17(rK-mK+) supE44
thi-1 recA1 gyrA (Nalr) relA1
D(laclZYA-argF)U169 deoR
(F80dlacD(lacZ)M15)
Strategene
B. subtilis DB104 nprE aprE (Yang, Ferrari et al. 1984)
B. subtilis WB600 nprE nprB aprE epr mpr bpr (Wu, Lee et al. 1991)
Part III: Directed Evolution of Subtilisin Carlsber g
64
2.3 Plasmids
The plasmids used and generated during this work are listed in Table 3.5.
Table 3.5. Plasmids used and generated in this work.
Plasmid Description References
pBC-Car pBC carrying subtilisin
Carlsberg Provided by Henkel AG & Co. KGaA, Düsseldorf, Germany
pBC-Per pBC carrying Perhydrolase Provided by Henkel AG & Co. KGaA, Düsseldorf, Germany
pHY300PLK Shuttle vector (E. coli, Bacillus
sp.), Apr, Tetr Takara BIO
pHY300Car pHY300PLK carrying subtilisin
Carlsberg This work
pHY300Per pHY300PLK carrying
Perhydrolase This work
2.4 Oligonucleotides
The oligonucleotides used in this work are summarized in Table 3.6.
Table 3.6. Oligonucleotides used in this work.
Name Sequence (5’ →3’) Description
Car_BamHI_fp GATGGATCCCCGGGACCTCTTTC Cloning SC and
T59A/L217W variant in pHY300PLK vector
Car_XbaI_rp TAGTCTAGATTATTGAGCGGCAGCTTCG Cloning SC and
T59A/L217W variant in pHY300PLK vector
pHY_shuttle_fp CAGATTTCGTGATGCTTGTCAGG Sequencing primer
pHY300PLK
pHY_shuttle_rp CGTTAAGGGATCAACTTTGGGAG Sequencing primer
pHY300PLK
ep_Per_mature_fp CATGTGGCCCATGCGCTAGCG epPCR library generation
Part III: Directed Evolution of Subtilisin Carlsber g
65
Name Sequence (5’ →3’) Description
pHYPer_pro_fw CGATGGCATTCAGCGATTCCGCTTC epPCR and SeSaM library
ep_Per_rp TGCCCAGCTTCTAGATTATTGAGCGG epPCR library generation
SeFpPer_pro_fp CACACTACCGCACTCCGTCGCGATGGCATTCAGCGATTCCGCTTC SeSaM library generation
pHYPer_rp GATTTCGTGATGCTTGTCAGGGGGC SeSaM library generation
SeRp_Per_rp GTGTGATGGCGTGAGGCAGCGATTTCGTGATGCTTGTCAGGGGGC SeSaM library generation
BioSeSaM_fp [Biotin]CACACTACCGCACTCCGTCG 5’ Biotin primer for SeSaM
BioSeSaM_rp [Biotin]GTGTGATGGCGTGAGGCAGC 5’ Biotin primer for SeSaM
SSM32_Per_fp GTAAAAGTAGCCGTCCTGNNNACAGGAATCCAAGCTTC SSM of Perhydrolase
SSM32_Per_rp GAAGCTTGGATTCCTGTNNNCAGGACGGCTACTTTTAC SSM of Perhydrolase
SSM33_Per_fp AAAGTAGCCGTCCTGGATNNKGGAATCCAAGCTTCTCAT SSM of Perhydrolase
SSM33_Per_rp ATGAGAAGCTTGGATTCCMNNATCCAGGACGGCTACTTT SSM of Perhydrolase
SSM68_Per_fp CGGACACGGCACACATNNKGCCGGTACAGTAGCTG SSM of Perhydrolase
SSM68_Per_rp CAGCTACTGTACCGGCMNNATGTGTGCCGTGTCCG SSM of Perhydrolase
SSM125_Per_fp GGATGTTATCAATATGNNKCTTGGGGGAGCATCAG SSM of Perhydrolase
SSM125_Per_rp CTGATGCTCCCCCAAGMNNCATATTGATAACATCC SSM of Perhydrolase
SSM127_Per_fp TATCAATATGAGCCTTNNKGGAGCATCAGGCTCGA SSM of Perhydrolase
SSM127_Per_rp TCGAGCCTGATGCTCCMNNAAGGCTCATATTGATA SSM of Perhydrolase
SSM155_Per_fp GTAGCTGCAGCAGGGNNNAGCGGATCTTCAGG SSM of Perhydrolase
SSM155_Per_rp CCTGAAGATCCGCTNNNCCCTGCTGCAGCTAC SSM of Perhydrolase
SSM156_Per_fp GTGTTTCCTGAAGATCCNNNGTTCCCTGCTGCAGCTAC SSM of Perhydrolase
SSM156_Per_rp GTAGCTGCAGCAGGGAACNNNGGATCTTCAGGAAACAC SSM of Perhydrolase
SSM166_Per_fp GAAACACGAATACAATTNNKTATCCTGCGAAATACGA SSM of Perhydrolase
SSM166_Per_rp TCGTATTTCGCAGGATAMNNAATTGTATTCGTGTTTC SSM of Perhydrolase
SSM169_Per_fp TACAATTGGCTATCCTNNKAAATACGATTCTGTCA SSM of Perhydrolase
SSM169_Per_rp TGACAGAATCGTATTTMNNAGGATAGCCAATTGTA SSM of Perhydrolase
SSM217_Per_fp GCCATTGACGTTCCGTTNNNTGTTGCATAAGTGTTCG SSM of Perhydrolase
SSM217_Per_rp CGAACACTTATGCAACANNNAACGGAACGTCAATGGC SSM of Perhydrolase
SSM218_Per_fp GAAGCCATTGACGTTCCNNNCCATGTTGCATAAGTG SSM of Perhydrolase
SSM218_Per_rp CACTTATGCAACATGGNNNGGAACGTCAATGGCTTC SSM of Perhydrolase
SSM219_Per_fp GGAGAAGCCATTGACGTNNNGTTCCATGTTGCATAAG SSM of Perhydrolase
SSM219_Per_rp CTTATGCAACATGGAACNNNACGTCAATGGCTTCTCC SSM of Perhydrolase
SSM222_Per_fp CATGGAACGGAACGTCANNKGCTTCTCCTCATGTAGC SSM of Perhydrolase
SSM222_Per_rp GCTACATGAGGAGAAGCMNNTGACGTTCCGTTCCATG SSM of Perhydrolase
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2.5 Cell culture media and cultivation
Cells were cultivated in Luria Broth (LB) medium supplemented with the
appropriate antibiotics using a shaking incubator (Multitron II, Infors GmbH, Einsbach,
Germany) at 37°C and 250 rpm for 24 h. Antibiotics used for cell culture are listed in
Table 3.7.
LB medium: 1 % (w/v) peptone from casein, 0.5 % (w/v) yeast extract, 1 % (w/v)
NaCl.
1.5 x LB medium buffered: 1.5 % (w/v) peptone from casein, 0.75 % (w/v) yeast
extract, 1 % (w/v) NaCl and 90 mM K-phosphate buffer pH 7.6.
LB skim milk (LBM) medium: LB medium plus 1 % (w/v) skim-milk. LB or LBM
agar plates contain 1.5 % (w/v) agar.
Table 3.7. Antibiotics used for cell culture in this work.
Antibiotic Stock (mg/ml) Solvent Working
concentration (µg/ml)
Ampicilin 100 water 100
Erythromycin 2.5 75 % ethanol 5
Lincomycin 2.5 water 5
Tetracycline 15 50 % ethanol 15
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3. Methods
3.1 Cloning
All gene cloning and DNA manipulation steps were carried out according to
standard molecular cloning protocols (Sambrook and Russell 2001). Preparation of E.
coli competent cells and transformation were carried out as standard heat shock
transformation (Inoue, Nojima et al. 1990). Subtilisin Carlsberg (Car) and subtilisin
Carlsberg Thr59Ala/Leu217Trp variant (Per) genes along with its pre-pro-sequence
including the Bacillus promoter were cloned into pHY300PLK shuttle vector (Takara Bio
Inc, Shiga, Japan) by using BamHI and XbaI restriction sites. The generated construct
was named pHY300Car and pHY300Per (6221 bp). Plasmids constructs were verified
by PCR, restriction enzyme digestion and sequence analysis. Sequence results were
analyzed by Vector NTI 9 (Invitrogen). Plasmid isolation, gel purification and PCR
purification kit were purchased from Macherey-Nagel (Düren, Germany).
3.2 Transformation of B. subtilis WB600 and DB104
For preparation of Bacillus competent cells and transformation two protocols
were used: modified (Matsuno, Ano et al. 1992) and protocol developed during work on
this project (Vojcic 2012).
Solutions for preparation of B. subtilis competent cells: T base (150 mM
ammonium sulfate, 800 mM potassium hydrogen phosphate (K2HPO4), 440 mM
potassium dihydrogen phosphate (KH2PO4) and 35 mM Na citrate); SpC media (10 ml T
base, 1 ml 50 % glucose, 1.5 ml 1.2 % MgSO4, 2.0 ml 10% yeast extract, 2.5 ml 1%
casamino acids in final volume 100 ml) and SpII media (10 ml T base, 1 ml 50 %
glucose, 7.0 ml 1.2 % MgSO4, 1.0 ml 10% yeast extract, 1.0 ml 1% casamino acids, 0.5
ml 100 mM CaCl2 in final volume 100 ml).
Freshly streaked cells were inoculated in 10 ml of SpC media overnight at 37°C,
250 rpm. Overnight culture was diluted in SpC media until OD600 was adjusted to 0.5.
Culture is grown for 3 hours 10 minutes in flask shaker (37°C, 200 rpm). Cell cultures
are diluted 1:1 with starvation medium (essential amino acid was added in final
Part III: Directed Evolution of Subtilisin Carlsber g
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concentration 10 µg/ml), and then incubated again for 2 h in flask shaker (37°C, 300
rpm). After incubation, cultures are maximally competent for about an hour.
Transformation of Bacillus competent cells: 4 µl of plasmid (DNA concentration:
>20 ng/ul) was mixed with 500 µl of competent cells in 2 ml Eppendorf tube, incubated
for 30 min at 37°C, 200 rpm. 300 µl of LB was added for the recovery of competent
cells. Culture was incubated for another 30 min at 37°C, 200 rpm. 250 µl of cells were
plated on LB agar plates containing selective antibiotic.
3.3 epPCR library generation
The random mutagenesis library was constructed using standard epPCR
(Cadwell and Joyce 1994) and megaprimer PCR of whole plasmid (MEGAWHOP)
(Miyazaki 2003) as described. The epPCR library was generated by PCR (95°C for 1
min, one cycle; 95°C for 30 s, 60°C for 30 s, 72°C for 75 s, 30 cycles; 72°C for 5 min,
one cycle) using dNTPs mix (0.2 mM), primers (ep_Per_mature_fp: 5´-
CATGTGGCCCATGCGCTAGCG-3´; ep_Per_rp: 5´-TGCCCAGCTTCTAGATTATTGA-
GCGG-3´; 0.5 µM each), plasmid DNA template (pHY300Per, 5 ng/50 µL), Taq
polymerase (5 U/50 µL) and MnCl2 (0.1 mM). epPCR library for Perhydrolase gene with
pro-sequence was generated according to same protocol, using ep_proPer_fp primer
(5’- CGA TGG CAT TCA GCG ATT CCG CTT C -3’). The MEGAWHOP PCR (72°C for
5 min, one cycle; 98°C for 1 min, one cycle; 98°C f or 30 s, 55°C for 45 s, 72°C for 3
min, 24 cycles; 72°C for 10 min, one cycle) was per formed in a final volume of 50 µL
using dNTPs mix (0.2 mM), megaprimer (purified epPCR amplification product, 500 ng),
plasmid DNA template (pHY300Per, 60 ng) and PfuS polymerase (1.5 U). The amplified
MEGAWHOP product was digested by DpnI (20 U) at 37°C overnight, purified using
Macherey-Nagel kit and transformed into E. coli strains. Plasmids were then pooled and
isolated using the Qiagen kit.
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3.4 SeSaM library generation
SeSaM library of Perhydrolase was generated based on the advanced protocol
(Mundhada, Marienhagen et al. 2011) which was modified to attain tunable mutational
load and transition/transversion bias.
Preliminary step: Generation of forward and reverse step 1 template
For the template-generation of of SeSaM-TvTv step 1-forward DNA-template, a
50 µl PCR-mixture contained: 1x Phusion HF Buffer; (New England Biolabs); 0.2 mM of
each dNTP; 12.5 pmol of each primer (SeFpPer_pro_fp and pHYPer_rp); 2.5 U of
Phusion polymerase (New England Biolabs) and 50 ng of pHY300Per plasmid as
template. PCR protocol: 98°C for 40 s (1 cycle); 98 °C for 10 s; 64°C for 30 s; 72°C for
40 s (20 cycles); 72°C for 3 min (1 cycle). The sam e PCR setup and protocol was
applied for the generation of the SeSaM-TvTv reverse template, only different primers
(pHYPer_pro_fw and SeRp_Per_rp) were used. After PCR, reactions were column-
purified using the Nucleospin kit from Macherey-Nagel to obtain pure PCR products.
Step 1: Generating a ssDNA fragment pool with random length distribution
The ‘G-Forward’ and ‘-Reverse’ library was constructed by linear amplification of
the pro-Perhydrolase gene in the presence of 0.07 mM of dGTPαS (35 %), 0.130 mM of
dGTP and 0.2 mM each of dATP, dTTP and dCTP. For forward library apart from the
nucleotides, 50 µl PCR mix (6 x 50 µl) contained: 1x buffer (Qiagen PCR), 20 pmol
primer BioSeSaM_fp, 100 ng of the step 1 forward template and 2.5 U Taq Polymerase.
Reverse ‘G’ library (6 x 50 µl) on the other hand contained 100 ng of reverse step 1
template and 20 pmol of primer BioSeSaM_rp, 1x buffer (Qiagen PCR) and 2.5 U Taq
Polymerase. On the similar lines, ‘A-Forward’ library 50 µl (6 x 50 µl) contained 0.07 mM
of dATPαS (35 %), 0.13 mM of dATP and 0.2 mM each of dGTP, dTTP and dCTP; 1x
buffer (Qiagen PCR), 20 pmol primer BioSeSaM_rp, 100 ng of the step 1 forward
template and 2.5 U Taq Polymerase. Whereas 50 µl ‘A-Reverse’ (6 x 50 µl) constituted
0.07 mM of dATPαS (35 %) and 0.130 mM of dATP; 0.2 mM each of dGTP, dTTP and
dCTP; 1x buffer (Qiagen PCR), 20 pmol primer BioSeSaM_rp, 100 ng of the step 1
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reverse template and 2.5 U Taq Polymerase. Common PCR protocol: 94°C for 2 min (1
cycle); 94°C for 30 s; 60°C for 30 s; 72°C for 35 s (25 cycles); 72°C for 3 min (1 cycle).
300 µl of PCR product was then column purified by Nucleospin kit and was eluted in 80
µl elution buffer. 10 µl of 10x Qiagen Taq buffer was added to provide the alkaline
condition followed by addition of 10 µl of cleavage solution (20 mM solution of Iodine in
99 % ethanol). The cleavage reaction was vortexed immediately after setup, and
incubated at 70°C for 1 h. Biotinylated-DNA fragmen ts were subsequently isolated
using, magnetic streptavidin beads. For better yield of single stranded DNA template
NTC buffer was used for binding on Nucleospin columns for all following steps of
SeSaM-TvTv.
Step 2: Enzymatic addition of dPTP αS to the DNA fragment pool
300 ng (2 x 150) each of ‘G’ Forward, ‘G’ reverse, ‘A’ ‘Forward’ and ‘A’ ‘Reverse’
step 1 DNA fragments were subjected for ‘tailing’ reaction with TdT. Fragments were
elongated at the 3´-OH groups by TdT-catalyzed incorporation of dPTPαS. The reaction
mixture contained: 1x NEB buffer 4 (New England Biolabs); 150 ng DNA fragments; 40
U TdT; 60 pmol dPTPαS and 0.05 mM CoCl2 in a total reaction volume of 25 µl (2 x 25
µl). After incubation at 37°C for 2 h and heat inac tivation for 20 min at 65°C, DNA
fragments were subsequently purified with the Nucleospin Kit.
Step 3: Full length gene synthesis
Full-length of pro-Perhydrolase genes were generated by combining ‘tailed’
‘forward’ and ‘reverse’ library fragments. The reaction mixture contained: 150 ng G and
A Forward and 150 ng of G and A Reverse step 2 fragments 0.25 mM of each dNTP; 1x
Super Taq buffer (MoBiTec) and 10 U 3D1 Polymerase in 100 µl (2 x 50 µl). PCR
Protocol: 94°C for 2 min (1 cycle); 94°C for 30 s; 51.5°C for 1 min; 72°C for 1 min (29
cycles); 72°C for 5 min (1 cycle). Fragments were s ubsequently purified using the
Nucleospin kit.
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Step 4: Degenerate base replacement
The universal bases were replaced by standard nucleotides by PCR-amplification
using primers pHYPer_pro_fw and pHYPer_rp. 100 µl of reaction contained 12.5 pmol
each of primer pHYPer_pro_fw and pHYPer_rp; 1x Qiagen Taq buffer; 5 U Taq
polymerase (Qiagen); 0.2 mM of each dNTP and 20 ng of purified step 3 PCR product.
PCR program: 94°C for 2 min (1 cycle); 94°C for 30 s; 60°C for 30 s; 72°C for 40 s (15
cycles); 72°C for 3 min (1 cycle). The resulting PC R product representing the final
mutant library was purified with the Nucleospin kit and cloned by MEGAWHOP in E. coli
cells.
3.5 Site Saturation Mutagenesis of Perhydrolase
Site Saturation Mutagenesis (SSM) of Perhydrolase was performed according to
the published method (Wang and Malcolm 2002). The oligonucleotides for SSMs of
Perhydrolase were listed as in Table 3.5. The procedure consists of two stages: In
stage one, two extension reactions are performed in separate tubes; one containing the
forward primer and the other containing the reverse primer. Subsequently, both
reactions were mixed and the stage two was carried out. For the mutagenic PCR (First
stage: 98°C for 30 s, one cycle; 98°C for 10 s, 55° C for 30 s, 72°C for 3 min, four
cycles. Second stage: 98°C for 30 s, one cycle; 98° C for 10 s, 55°C for 30 s, 72°C for 3
min, 14 cycles; 72°C for 3 min, one cycle), PfuS DNA polymerase (1 U), dNTP mix (0.2
mM), each primer (0.2 µM) together with plasmid template (10 ng) were used in 50 µL
reaction volume. Following the PCR, DpnI (20 U) was added, and the mixture was
incubated at 37°C overnight, purified using Machere y-Nagel kit, transformed into E. coli
strains, clones were pooled and plasmid isolated using Macherey-Nagel kit.
3.6 Protein expression
Plasmid library was transformed into B. subtilis WB600 and DB104 and plated
into LB agar plates containing 15 µg/ml tetracycline and 1% skim milk. Colonies
generating a clearance halo on skim milk were picked and resuspended in separate
wells of a 96-well flat bottom microtiter plate (Greiner, Frickenhausen, Germany)
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containing 150 µl of LB medium and 15 µg/ml tetracycline (master plates) and incubated
during 24 h at 37°C, 900 rpm and 70% humidity. Expr ession was carried out by
replicating the master plates into 96-well flat bottom microtiter plates (expression plates)
containing 200 µl of LB medium and 15 µg/ml tetracycline. Expression was carried out
during 24 h at 37°C, 900 rpm and 70% humidity. Afte r expression, the microtiter plates
were centrifuged at 4000 rpm during 20 min at 4°C a nd the supernatant was used for
activity testing.
3.7 Catalase inhibition
Since is known that Bacillus cells express catalase during stationary growth
phase (Naclerio, Baccigalupi et al. 1995) which was experimentally confirmed in this
work, the first step after cell centrifugation was the inhibition of catalase to prevent
degradation of hydrogen-peroxide, which is substrate in the perhydrolytic reaction.
Catalase was inhibited using 100 mM 3-amino-1,2,4-triazole (AT) and 30 mM hydrogen
peroxide at RT, 800 rpm (using microtiter plate shaker) for 2 h (Tephly, Mannering et al.
1961). After inhibition, the supernatant was diluted 5-fold and used for detection of
proteolytic and perhydrolytic activity.
3.8 Screening for decreased proteolytic activity
Screening was performed in 96-well microtiter plate by adding 5 µl catalase
inhibited supernatant from each clone to 95 µl of suc-AAPF-pNA solution (0.22 mM in
reaction) in 100 mM Na-phosphate buffer pH 7.5. Proteolytic activity for samples within
the same microtiter plate was defined as the increase in absorbance at 405 nm per
second across 5 min of reaction.
3.9 Screening for improved perhydrolytic activity
Screening was performed in 384-well plate by adding 4 µl catalase inhibited
supernatant to 36 µl of reaction mixture containing: 4 µl 313 mM hydrogen-peroxide, 5
µl 800 mM NaBr, 10 µl of 200 mM methyl-butyrate in buffer with 0.1 % SDS and 0.4 µl
APCC in DMSO, in 100 mM Na-phosphate buffer pH 7.5. Perhydrolytic activity was
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defined as increase in fluorescence (ex/em 360/465 nm) per second across 10 min of
reaction at 30°C.
3.10 Purification of serine proteases with affinity chromatography
The purification of proteases form supernatant can be carried out in a one-step
affinity chromatography with bacitracin as biospecific ligand. Bacitracin is a decapeptide
with antibiotic properties produced by Bacillus species. It is a competitive inhibitor for
serine proteases and binds in the active site via a D-glutamic acid. CNBr-activated
Sepharose couples preferentially at ε-lysine groups forming an iso-urea derivative,
provided that amino group on the ligand are in the unprotonated form and therefore
nucleophile. Thus bacitracin can be coupled directly to the support matrix. This method
enables purification of 96 samples at the same time by using 96-well plate column (2 ml,
Zinsser Analytic, Frankfurt, Germany) connected to a vacuum system. The protocol was
provided by the collaborator Henkel AG & Co. KGaA, Düsseldorf, Germany.
After washing CNBr-activated Sepharose 4B (Sigma-Aldrich) in 1 mM HCl,
bacitracin (100mg/g dry sepharose) was added to the soaked gel and incubated in 100
mM NaHCO3 , 500 mM NaCl while rotating (4°C, 14-15 h). The ge l was washed with
100 mM NaHCO3, 500 mM NaCl pH 10 first and then washed with destilled water. To
block remaining active groups of the sepharose, the washed gel was incubated with 1M
ethanolamine pH 8 while rotating (4°C, 2 h). Afterw ards, the gel was washed with 100
mM NH4-acetate, 10 mM CaCl2 pH 6.5 in steps using a membrane (regenerated
cellulose 0.45 µm) while applying vacuum to remove the washing solution. This gel can
be stored at 4oC. The gel was diluted 1:4 with 100 mM NH4-acetate; 10 mM CaCl2 pH
6.5 and 800 µl of this suspension was pipetted into each well of a Microelute plate and
used as Mini Columns (2 ml, Zinsser Analytic, Frankfurt, Germany).
The column matrix was regenerated by washing with 100 mM Na-acetate, 500
mM NaCl pH 4 and 100mM Tris/HCl, 500 mM NaCl pH 8. Afterwards column was
equilibrated with 100 mM NH4-acetate 10 mM CaCl2 pH 6.5 and 300 µl of the
supernatant was applied. Not bound protein was washed with 100 mM NH4-acetate pH
6.5 and 100 mM NH4-acetate, 50 mM NaCl pH 6.5. Protease was eluted with 100 mM
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74
NH4-acetate, 1 M NaCl pH 6.5, 25 % isopropanol. The matrix was washed and stored in
100 mM Na-acetate pH 4.
Total protein concentration of the final samples was determined using the Micro
BCA assay according to the protocol provided by manufacturer.
3.11 Protein purification
Pehydrolase and its variants were expressed in B. subtilis DB104 strain at 37°C
for 60 h in 250 ml of LB media. Cell pellet was separated from supernatant by
centrifugation at 8000 g for 30 min at 4°C. The resulting supernatant was filtered using a
#30 glass fiber filter (Schleicher & Schuell Microscience, Dassel, Germany) and again
by using a 0.2 µm Polyethersulfone (PES) membrane filter (Sartorius, Göttingen,
Germany). The supernatant was concentrated to 10 ml using a Millipore ultra-filtration
stirred cell with a 5 kDa cut off regenerated cellulose ultra-filtration membrane (Millipore,
Billerica, MA, USA). The concentrated supernatant was diluted 10 times with 10 mM
HEPES buffer pH 7.0, re-concentrated to 10 ml and loaded into a Toyopearl Super Q
650c (TOSOH) anion exchange chromatography column (25 ml) previously equilibrated
with 10 mM HEPES pH 7.0 buffer. The flow rate and pressure were managed by an
ÄKTA prime system (Amersham, GE Healthcare Europe, Freiburg, Germany) using the
Unicorn software package. The perhydrolase did not bind to this column at pH 7.0 and
was collected on the buffer front fractions. Pulled fractions are concentrated to 2 ml and
diluted 5 times with distilled water to decrease conductivity (< 1.5 mS/cm) and loaded
into a Toyopearl Super SP-650c (TOSOH) cation exchange chromatography column
(20 ml) previously equilibrated with 20 mM HEPES pH 7.0 and Perhydrolase was
binding for negatively charged matrix. Elution was achieved with 4 % 20 mM HEPES,
1 M NaCl pH 7.0. The collected fractions were analyzed by SDS-PAGE.
The optimized protocol was used for purification of all variants. Total protein
concentration was determined using the BCA assay, and purity was assessed by
running Experion Pro260 Analysis Kit (Bio-Rad, Munich, Germany) to confirm validity of
protocol. The fractions with the highest purity were pooled together, concentrated in
0.1 M Na-phosphate buffer pH 7.5 up to volume of 500 µl and stored at -80°C.
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3.12 Proteolytic activity with natural substrate – skim milk
Proteolytic activity of the variants was determined using skim milk as a substrate.
The reaction was started by adding 10 µl of enzyme (3.66 µM, final 0.18 µM) to 190 µl
of skim milk in 100 mM TRIS/HCl buffer pH 8.6 at 30°C and kinetic the reaction was
followed by the decrease in absorbance at 650 nm across 10 min of reaction. Change in
absorbance per minute (slope of the reaction) was compared between the variants.
3.13 Proteolytic activity – suc-AAPF-pNA
Kinetic constants for proteolytic activity were determined using suc-AAPF-pNA as
substrate. The amount of released product was determined from Lambert-Beer’s law
using extinction coefficient of pNA ε410 = 8480 M-1 cm-1 provided by manufacturer.
The reaction was started by adding 5 µl (0.37 µM, final 18.3 nM, concentration of
M2 variant was 9.2 µM, final 0.46 µM) of enzyme to 95 µl of suc-AAPF-pNA reaction
mix in 100 mM Na-phosphate buffer pH 7.5 preheated at 30°C. The change in
absorbance was followed every 15 s during 5 min of reaction at 30°C. Kinetic
parameters (KM, kcat) were calculated from initial rates (Vmax) at different concentrations
of substrate (the range from 0.05 to 3 mM).
3.14 Perhydrolytic activity of the variants
Specificity of subtilisin Carlsberg and its variants for three different ester
substrates and hydrogen-peroxide was determined through perhydrolytic activity of the
enzyme with these substrates.
Kinetic parameters (KM, kcat) were determined for methyl-propionate, methyl-
butyrate and methyl-pentanoate in presence of 100 mM hydrogen-peroxide. The
reaction was started by adding 10 µl of enzyme (1.50 µM, final 0.15 µM) to 90 µl of
reaction mixture preheated at 30°C. The reaction mi xture contains: 12.5 µl of 0.8 M
NaBr, 10 µl of 1 M H2O2, 1 µl of 0.05 M APCC and ester substrates of different
concentration in 100 mM Na-phosphate buffer pH 7.5. Concentration range of: methyl-
propionate ranged from 10 to 600 mM; methyl butyrate from 10 to 200 mM and methyl-
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76
pentanoate from 5 to 90 mM in reaction. Kinetic parameters (KM, kcat) for hydrogen-
peroxide were determined in the presence of 500 mM methyl-propionate. The reaction
started by adding 10 µl of enzyme (1.50 µM, final 0.15 µM) to 90 µl of reaction mixture
preheated at 30°C. Reaction mixture contains: 12.5 µl of 0.8 M NaBr, 1 µl of 0.05 M
APCC and H2O2 in different concentration in 0.1 M Na-phosphate buffer pH 7.5.
Concentration range of H2O2 was from 1 to 60 mM.
The amount of produced peroxycarboxylic acid per minute was calculated from a
peroxyacetic acid calibration curve. The change in fluorescence was followed every 15
s during 5 min of reaction at 30°C. Kinetic parameters (KM, kcat) were calculated from
initial rates (V0) at different concentrations of substrate.
3.15 Esterolytic activity of the variants
Subtilisin Carlsberg catalyzes hydrolysis of methyl esters generating methanol
and carboxylic acid as a product of the reaction. Amount of generated carboxylic acids
was determinate by HPLC.
Esterolytic reaction: 5 µl of enzyme (concentration 1 mg/ml) was added to 195 µl
of methyl-butyrate (reaction concentration 200 mM) in 100 mM Na-phosphate buffer pH
7.5. Reaction was carried at 30°C for 10 min in mic rotiter plate shaker at 650 rpm.
Reaction was stopped by addition of 1 µl of phosphoric acid. Samples were analyzed by
reversed-phase HPLC.
The HPLC instrument was from Beckman Coulter. Data acquisition was
performed using Beckman Coulter software. The column (Macherey-Nagel, Düren,
Germany) was reversed-phase C18 Nucleosil 100-5 (50 x 4.6 mm). The injection volume
for all measurements was 50 µl, column was heated at 50°C and the UV detection
wavelength was 210 nm. The following gradient of acetonitrile and 20 mM KH2PO4 pH
3.0 was selected to achieve separation: 0-0.2 min 25 % acetonitrile, 0.2-5.8 min applied
gradient from 25 to 100 % acetonitrile, 5.8-9.3 min 100 % acetonitrile and 9.3-16 min
25 % acetonitrile. The flow rate of the mobile phase was 1 ml/min. Concentration of
butyric acid was determined from calibration curve generated with standard solutions.
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Standard solutions contained butyric acid with concentration range from 0.25 to 25 mM
and 200 mM methyl-butyrate, area below peak for butyric acid was calculated.
3.16 Modeling studies
Analyses are based on the crystal structure of subtilisin Carlsberg (PDB code
1uy6) obtained from RCSB Protein Data Bank. Program Yasara Structure
(http://www.yasara.org/) was used for docking studies and generating the images of the
molecular structures.
Amino acid numeration in the thesis is according to numeration in PDB file (PDB
code 1uy6), where all amino acids starting from position 56 are shifted by one position
(missing position 56).
4. Results
In this work Directed Evolution was performed
subtilisin Carlsberg Thr59A
perhydrolytic activity and decreased proteolytic activity.
4.1 Expression system
Carlsberg and Perhydrolase
shuttle vector pHY300PLK obtaining pHY300Car
3.7. The presence of insert in
Constructs were transformed in expression strains
and expression was confirmed on skim milk agar plates by formation of halos
subtilis colonies. Proteolytic acti
detected in liquid culture.
Figure 3.7 . pHY300PLK shuttle vector containing Carlsberg(pHY300Per) genes for expression and dive
Part III: Directed Evolution of Subtilisin Carlsber g
78
In this work Directed Evolution was performed to generate and identify a
59Ala/Leu217Trp (Perhydrolase) variant having increased
perhydrolytic activity and decreased proteolytic activity.
Carlsberg and Perhydrolase genes including the promoter
shuttle vector pHY300PLK obtaining pHY300Car and pHY300Per,
resence of insert in the shuttle vector was confirmed by sequencing analysis.
Constructs were transformed in expression strains Bacillus subtilis
expression was confirmed on skim milk agar plates by formation of halos
Proteolytic activity with artificial substrate suc-AAPF
. pHY300PLK shuttle vector containing Carlsberg (pHY300Car)genes for expression and diversity library generation.
Part III: Directed Evolution of Subtilisin Carlsber g
to generate and identify a
variant having increased
promoter were cloned into a
respectively, Figure
shuttle vector was confirmed by sequencing analysis.
DB104 and WB600,
expression was confirmed on skim milk agar plates by formation of halos around B.
AAPF-pNA was also
(pHY300Car) and Perhydrolase
Part III: Directed Evolution of Subtilisin Carlsber g
79
4.2 Perhydrolytic (APCC) assay optimization for microtiter plate screening
Screening of random mutagenesis libraries for increased perhydrolytic activity of
Perhydrolase was performed using APCC assay in 384-well microtiter plates,
measuring the increase in fluorescence at ex/em 360/465 nm over time at 30°C. Methyl-
butyrate was chosen as substrate, since Perhydrolase showed the highest performance
with this ester whereas background activity (autoperhydrolysis of methyl-butyrate) was
not detectable under screening conditions. Perhydrolytic reaction is presented in Figure
3.8.
Figure 3.8. Enzyme catalyzed perhydrolysis of methyl-butyrate.
Subtilisin Carlsberg is an alkaline protease with pH optimum 8-10, however,
peroxycarboxylic acids are more stable at a lower pH, where the fluorescent product
(HCC) also shows higher fluorescence intensity. The screening was performed at pH
7.5 in 100 mM phosphate buffer. At this pH, the proteolytic activity of Perhydrolase
measured with the artificial substrate suc-AAPF-pNA was 1.5-fold decreased compared
to the activity at pH 9.0. On the other hand, fluorescent intensity generated in
perhydrolytic reaction was 1.5-fold higher at pH 7.5. Standard deviation of APCC assay,
calculated for 94 clones under these conditions, was approximately 14 %, as shown in
Figure 3.9.
Part III: Directed Evolution of Subtilisin Carlsber g
80
Figure 3.9. Validation of APCC assay for screening in MTP. The assay was performed in 100 mM Na-phosphate buffer pH 7.5 with 50 mM methyl-butyrate, 31.3 mM H2O2, 100 mM NaBr, 0.9 mM SDS and 0.5 mM APCC in 40 µl reaction volume using supernatant of B. subtilis WB600 cells after catalase inhibition containing Perhydrolase. The coefficient of variation was calculated for the change of relative fluorescence intensity (RFU) per second.
4.3 Diversity generation
Diversity was generated using error prone PCR (epPCR). Different
concentrations of MnCl2 (0.05 to 0.2 mM) were used in epPCR library generation to
adjust mutation frequency at which the ratio of active to inactive clones is 50 %. Only
the mature Perhydrolase was used following described protocol, PCR product was
cloned into pHY300Per using MEGAWHOP and transformed into competent E. coli
DH5α cells, Figure 3.10.
Part III: Directed Evolution of Subtilisin Carlsber g
81
Figure 3.10. Library generation and cloning, epPCR product has size 0.8 kb (line 1) and MEGAWHOP product has 6.2 kb size (line 2).
Transformants were pooled together; plasmid DNA was isolated and transformed
into the expression host B. subtilis WB600 cells. Active/inactive ratio was determined
according to proteolytic activity on skim milk agar plates and perhydrolytic activity in
MTP, Table 3.8.
Table 3.8. Percentage of inactive clones (inactive ratio) with different concentration of MnCl2.
MnCl 2 concentration (mM)
Inactive ratio (%) on skim milk Inactive ratio (%) for perhydrolytic activity
0.05 17 24
0.1 35 60
0.15 62 80
0.2 70 85
A concentration of 0.1 mM MnCl2 was chosen for library generation, since
libraries with similar mutational load have often been used in directed evolution
improving enzymes by gradual changes.
Part III: Directed Evolution of Subtilisin Carlsber g
82
4.4 Screening
Approximately 2250 B. subtilis WB600 clones were picked into 96-well flat
bottom MTP containing 150 µl of 1.5 x LB buffered medium, after 24 h colonies were
replicated in 96-well flat bottom MTP containing 200 µl of 1.5 x LB buffered medium and
expression was carried out for 24 h, at 37°C, shaki ng 800 rpm and 70 % humidity.
Plates were centrifuged for 20 min at 4000 rpm and 4°C. After centrifugation, catalase
activity was inhibited by adding 2 µl of 2.5 M 3-amino-1,2,4-triazole and 1 µl of 3.13 M
hydrogen-peroxide to 47 µl of supernatant and incubated for 2 h at RT with shaking at
800 rpm. 4 µl of catalase inhibited supernatant from each well was transferred in 384-
well black, flat MTP for activity assay at 30°C.
Figure 3.11 shows activity profile of epPCR library, where in descendent order, is
presented ratio of active clones versus Perhydrolase.
Figure 3.11. MTP screening profile of active clones from error prone library plotted in descendent order. Activity ratio was calculated versus wild type.
After screening, 15 clones which showed activity at least 1.3-fold higher than
Perhydrolase were rescreened and besides perhydrolytic activity; proteolytic activity
was determined. In the rescreening, 4 clones showed all three activities higher than
Perhydrolase; plasmid DNA was isolated from the variants and again transformed in B.
subtilis WB600. After re-transformation activity was at the level of Perhydrolase,
suggesting that increased activity measured in the screening step was due to higher
protein expression. Additionally, one clone showed 3 silent mutations and another
Part III: Directed Evolution of Subtilisin Carlsber g
83
showed a change Ser252Asn which is at the surface of the protein and distant from the
active site.
4.5 Diversity generation and screening optimization
Since after screening more than 2000 clones, there was no improved variant
mutational load of the libraries was confirmed by sequencing random clones. SeSaM
library covering the pro- and mature sequence of Perhydrolase and epPCR libraries
were generated following described protocols, resulting PCR products were cloned in
pHY300Per using MEGAWHOP and transformed into E. coli DH5α cells. Mutational
load of SeSaM and 0.15 mM MnCl2 epPCR library was evaluated by sequencing 3
random clones from each library, Table 3.9.
Table 3.9. Sequencing results for 3 clones from Perhydrolase epPCR library with 0.15 mM MnCl2 and 3 clones from Perhydrolase SeSaM library.
Clone Position (prepro -Perhydrolase) Mutation Type
ep library_1
431 A→G Transition 443 A→T Transversion 483 A→C Transversion 564 T→C Transition 613 A→G Transition 642 A→G Transition 857 C→A Transversion 863 G→C Transversion 891 A→G Transition 970 A→G Transition 975 A→G Transition
1012 A→G Transition ep library_2 none none None
ep library_3
422 A→G Transition 546 A→G Transition 798 G→A Transition 870 A→G Transition 929 A→T Transversion
1013 T→C Transition 1090 T→A Transversion
SeSaM library_1 none none None
SeSaM library_2 152 C→T Transition 518 T→A Transversion 841 T→C Transition
Part III: Directed Evolution of Subtilisin Carlsber g
84
Clone Position (prepro -Perhydrolase) Mutation Type
SeSaM library_3 345 C→T Transition 843 T→C Transition 948 C→A Transversion
Analysis of sequencing data showed that the average number of mutations per
gene was 6.3 in epPCR library and 2 in SeSaM library.
To rule out expression mutants, proteolytic activity of Perhydrolase variants was
measured in addition to perhydrolytic activity and the perhydrolytic/proteolytic activity
ratio was calculated for each variant. Proteolytic activity was measured using the
tetrapeptide suc-AAPF-pNA. Although skim milk would reflect the real macromolecular
substrate for proteases, it was not used for screening due to inability to detect activity in
cell supernatant. Additionally, at pH 7.5 skim milk has an increased autohydrolysis,
making it difficult to get accurate measurements.
Screening conditions for perhydrolytic activity were the same as described in Part
III, Chapter 4.4. The proteolytic assay was performed using 0.22 mM suc-AAPF-pNA in
Na-phosphate buffer pH 7.5 using 5 µl of catalase inhibited supernatant containing
Perhydrolase. Standard deviation of the ratio perhydrolytic/proteolytic activity calculated
for 94 clones under these conditions was approximately 13 %.
In the second round, 3350 B. subtilis WB600 colonies were re-picked on fresh
LBtet skim milk agar plates. After 16 h incubation at 37°C colonies producing halos on
skim milk were inoculated into 96-well flat bottom MTP containing 150 µl of 1.5 x LB
buffered medium, after 24 h colonies were replicated in 96-well flat bottom MTP
containing 200 µl of 1.5 x LB buffered medium and expression was carried out for 24 h,
at 37°C, shaking 800 rpm and 70 % humidity. Perhydr olytic/proteolytic activity ratio
versus Perhydrolase ratio of active clones in epPCR (0.1 and 0.15 mM MnCl2) library is
summarized in Figure 3.12.
Part III: Directed Evolution of Subtilisin Carlsber g
85
Figure 3.12. Ratio perhydrolytic/proteolytic activity versus Perhydrolase ratio of active clones from error prone library plotted in descendent order.
All clones from epPCR and SeSaM libraries with higher ratio of
perhydrolytic/proteolytic activity and clones with just increased perhydrolytic activity
were rescreened. In the rescreening only the clones which had both activities increased
were found, most likely due to higher expression or substitution which leads to improved
both activities. Plasmids were isolated from 6 variants and re-transformed into
competent B. subtilis WB600 cells to avoid variants with increased expression due to
host. Two clones from each variant were re-screened by measuring proteolytic and
perhydrolytic activity while expression level was analyzed by SDS-PAGE. Proteolytic
and perhydrolytic activity of the variants versus activity of Perhydrolase (E1 to E6) and
SDS analysis are presented in Figure 3.13.
Figure 3.13. Perhydrolytic (Perhydrolase activity and expression in means empty vector, Per is for Perhydrolase.for SDS analysis, 250 µl of the supernatant was TCA precipitated and loaded on the gelmature form of Perhydrolase variants
After re-transformation
however, SDS-PAGE analysis showed
variants, while decreased total protein content in the supernatant of E6 variant can be
due to sample handling during TCA precipitation.
revealed that there was no mutation in the Perhydrolase gene, suggesting that
increased expression could be
Part III: Directed Evolution of Subtilisin Carlsber g
86
Perhydrolytic (blue)/proteolytic (red) activity of the variants compared to and expression in the supernatants of the cells from selected
means empty vector, Per is for Perhydrolase. Proteins in the supernatant were TCA precipitated SDS analysis, 250 µl of the supernatant was TCA precipitated and loaded on the gel
form of Perhydrolase variants (27 kDa) is labeled in red square.
transformation, four variants (E3, E4, E5 and E6) had increased activity,
analysis showed an increased expression level for E3, E4 and E5
variants, while decreased total protein content in the supernatant of E6 variant can be
sample handling during TCA precipitation. DNA sequencing of E4 variant
was no mutation in the Perhydrolase gene, suggesting that
could be due to a mutation in the vector backbone during
Part III: Directed Evolution of Subtilisin Carlsber g
of the variants compared to
from selected variants. EV Proteins in the supernatant were TCA precipitated
SDS analysis, 250 µl of the supernatant was TCA precipitated and loaded on the gel, and
four variants (E3, E4, E5 and E6) had increased activity,
evel for E3, E4 and E5
variants, while decreased total protein content in the supernatant of E6 variant can be
DNA sequencing of E4 variant
was no mutation in the Perhydrolase gene, suggesting that the
vector backbone during
Part III: Directed Evolution of Subtilisin Carlsber g
87
MEGAWHOP amplification. E4 variant (in further text addressed as pHY300PerE4) was
used as a parent in the next round of diversity generation.
Screening conditions were optimized for B. subtilis DB104 pHY300PerE4 and
standard deviation calculated for perhydrolytic and proteolytic activity. Expression host
was changed from WB600 to DB104 due to higher transformation efficiency of DB104
strain with new method described in Chapter 3.2 of Part III. Expression conditions were
the same as for WB600 and carried out in 96-well microtiter plate for 24 h at 37°C, 900
rpm and 70 % humidity. Supernatant was separated from the biomass by centrifugation
and catalase inhibited, after inhibition supernatant was diluted 5 times to decrease the
influence of interfering compounds from media. The coefficient of variation for
perhydrolytic activity calculated for the change of relative fluorescence intensity (RFU)
per second (slope of the reaction) was 11 %. Standard deviation of proteolytic activity
calculated for the slope of the reaction was 9 % and standard deviation of ratio
perhydrolytic/proteolytic reaction slopes was 11 %, Figure 3.14.
Figure 3.14. MTP screening validation of proteolytic and perhydrolytic activity. On the graph is presented ratio of perhydrolytic/proteolytic reaction slopes plotted in descendent order. Perhydrolytic reaction was carried out in 100 mM Na-phosphate buffer pH 7.5 in the presence of 31.3 mM H2O2, 100 mM methyl-butyrate, 100 mM NaBr, 0.9 mM SDS and 0.5 mM APCC in 40 µl reaction mixture. Proteolytic activity was carried out in 100 mM Na-phosphate buffer pH 7.5 in the presence of 0.22 mM suc-AAPF-pNA in 100 µl reaction mixture.
4.6 Rational design approach
Overall, 6000 clones
improved ratio perhydrolytic/proteolytic activity
obtained. Considering the limitations of medium thr
approach was employed to reduce sequence space and screening efforts. Furthermore
the motivation for this work was to define
pockets (S1, S3 and S4) of
catalytic triad in order to improve perhydrolytic a
are divided into 3 groups: 1) positions dire
155), 2) positions close to the active site (T
Gly 219 and Met 222) and 3) positions forming binding pockets (Gl
166 and Ala 169), Figure 3.15
Figure 3.15. Saturated position in Perhydrolase surrounding active site and forming substrate binding pocket. Active site residuesand Ser 221 – yellow), orange (group 2), purple – amino acids in substrate binding pockets (gr
Part III: Directed Evolution of Subtilisin Carlsber g
88
4.6 Rational design approach
6000 clones were screened for improved perhydrolytic activity
ratio perhydrolytic/proteolytic activity and only expression mutant
. Considering the limitations of medium throughput screening system a rational
approach was employed to reduce sequence space and screening efforts. Furthermore
ion for this work was to define important residues in subs
) of the protein and interaction of the ester substrates with
catalytic triad in order to improve perhydrolytic activity of the protein. Saturated position
are divided into 3 groups: 1) positions directly involved in catalysis (Asp
s close to the active site (Thr 33, Val 68, Ser 125, Trp
222) and 3) positions forming binding pockets (Gl
169), Figure 3.15.
Saturated position in Perhydrolase surrounding active site and forming substrate nding pocket. Active site residues with surface presentation (Asp 32 – red, His
w), orange – oxyanion hole (Asn 155), blue – amino acids around active site amino acids in substrate binding pockets (group 3).
Part III: Directed Evolution of Subtilisin Carlsber g
screened for improved perhydrolytic activity or
and only expression mutants were
oughput screening system a rational
approach was employed to reduce sequence space and screening efforts. Furthermore
important residues in substrate binding
of the ester substrates with
ctivity of the protein. Saturated position
ctly involved in catalysis (Asp 32 and Asn
125, Trp 217, Asn 218,
222) and 3) positions forming binding pockets (Gly 127, Ser 156, Gly
Saturated position in Perhydrolase surrounding active site and forming substrate
red, His 64 – light green amino acids around active site
Part III: Directed Evolution of Subtilisin Carlsber g
89
4.6.1 SSM at positions included in catalysis and close to active site
Proteolytic activity of subtilisin Carlsberg is essential for maturation and enzyme
expression in extracellular environment. However, the goal of this project was to
decrease proteolytic (hydrolytic) and increase promiscuous, perhydrolytic activity.
Motivation was to decrease the self-digestion of the enzyme, and instead of ester
hydrolysis to favor the perhydrolytic reaction and production of peroxycarboxylic acids.
Since both reactions are catalyzed by the same active site, it is very likely that residues
involved in the interaction with ester substrate and the peptide chain are the same, as
well as residues involved in stabilization of hydrolytic and perhydrolytic tetrahedral
intermediate. This fact significantly limits diversity, since variants with improved
perhydrolytic activity can be lost due to inability to self-activate and express in the
supernatant.
Saturation of the positions in the catalytic triad (Asp 32) and oxyanion hole
(Asn155) was performed to study relationship maturation-expression and enzyme
processing by addition of active subtilisin Carlsberg in expression media.
Site Saturation libraries were generated in pHY300PerE4 vector and expressed
in B. subtilis DB104, 400 clones was re-picked on fresh LBtet skim milk agar plates. The
oligonucleotides used for SSM are listed in Table 3.5. Active ratio on skim milk was
15 % in both libraries; ~60 active clones and 20 inactive were inoculated in microtiter
plate for expression. Screening results showed that all active variants have ratio of
activities in the range of standard deviation. SDS analysis of the supernatant some of
the inactive variants showed that the mature form of subtilisin Carlsberg was not
present. Maturation carried by extracellular protease was tested; where two inactive
clones from each library were expressed with and without commercial subtilisin
Carlsberg in media. After 24 h of expression perhydrolytic and proteolytic activity was
tested in the supernatant and 100 µl of the supernatant after TCA precipitation was
analyzed by SDS-PAGE, Figure 3.16.
Figure 3.16. SDS analysis of maturation process with addition of subtilisin Carlsberg (1 µg/ml) in expression media. Supernatant fractions without (are shown for: EV - empty vector, P1 and P2 clones, N3 and N4 – Asp 155 negative clones. Mature protein is labeled with
On the SDS-PAGE
with and without subtilisin Carlsberg, except in the case N2, but since whole profile was
different cannot be concluded that activation was present. It is also possible that amount
of mature protein generated by extr
SDS-PAGE. As a conclusion
was not possible or amount and activity of enzyme was below detection limit under
screening conditions.
Motivated with results were substitution close to active site improved ratio of
perhydrolytic/hydrolytic activity by increasing stabilization of tetrahedral intermediate
seven position surrounding act
each position and none of the variants had improved perhydrolytic activity or improved
ratio perhydrolytic/proteolytic activity. Active ratio for each saturation li
summarized in Table 3.10.
Part III: Directed Evolution of Subtilisin Carlsber g
90
SDS analysis of maturation process with addition of subtilisin Carlsberg (1 µg/ml) in expression media. Supernatant fractions without (-) and with (+) subtilisin Carlsberg in media
empty vector, P1 and P2 - positive clones, N1 and N2 155 negative clones. Mature protein is labeled with
analysis there was no difference in the supernatant profile
with and without subtilisin Carlsberg, except in the case N2, but since whole profile was
different cannot be concluded that activation was present. It is also possible that amount
generated by extracellular activation was below detection limit on
As a conclusion, activation with addition of wild type in expression media
or amount and activity of enzyme was below detection limit under
sults were substitution close to active site improved ratio of
perhydrolytic/hydrolytic activity by increasing stabilization of tetrahedral intermediate
seven position surrounding active site were saturated. 400 clones were
one of the variants had improved perhydrolytic activity or improved
ratio perhydrolytic/proteolytic activity. Active ratio for each saturation li
Part III: Directed Evolution of Subtilisin Carlsber g
SDS analysis of maturation process with addition of subtilisin Carlsberg (1 µg/ml)
with (+) subtilisin Carlsberg in media ositive clones, N1 and N2 – Asn 32 negative
155 negative clones. Mature protein is labeled with the red box.
o difference in the supernatant profile
with and without subtilisin Carlsberg, except in the case N2, but since whole profile was
different cannot be concluded that activation was present. It is also possible that amount
s below detection limit on
, activation with addition of wild type in expression media
or amount and activity of enzyme was below detection limit under
sults were substitution close to active site improved ratio of
perhydrolytic/hydrolytic activity by increasing stabilization of tetrahedral intermediate
400 clones were screened for
one of the variants had improved perhydrolytic activity or improved
ratio perhydrolytic/proteolytic activity. Active ratio for each saturation library is
Part III: Directed Evolution of Subtilisin Carlsber g
91
Table 3.10. Activity profile of Saturation Mutagenesis libraries on skim milk agar and in microtiter plate.
Position Active ratio for proteolytic activity (%)
Active ratio for perhydrolytic activity (%)
Thr 33 50 18
Val 68 40 14
Ser 125 40 12
Trp 217 95 43
Asn 218 65 41
Gly 219 7 7
Met 222 72 26
Modifications of the active site and substrate binding pocket of subtilisin
Carlsberg lead to a decrease of the proteolytic activity, which also had, as a
consequence, decreased perhydrolytic activity below detection limit.
4.6.2 SSM of positions in the substrate binding pocket
To further investigate amino acid substitutions that could affect the activity of
subtilisin Carlsberg, positions in S1 binding pocket (Ser 156 and Gly 166), which are in
direct contact with the first residue of the peptide substrate (phenylalanine of suc-AAPF-
pNA substrate) and with small substrates such as ester compounds, were saturated.
Although it was reported that position 156 has significant influence in
determination of substrate specificity (Wells, Powers et al. 1987), (Wells, Cunningham
et al. 1987), screening results of Saturation Mutagenesis library showed that out of 364
screened clones 88 % had perhydrolytic activity, suggesting that this position is not
affecting binding of small substrates such as esters.
When SSM was performed at position Gly 166, out of 184 screened clones 93 %
active variants were identified, and 90 clones had increased ratio
perhydrolytic/proteolytic activity. Screening results are summarized in Figure 3.17.
Part III: Directed Evolution of Subtilisin Carlsber g
92
Figure 3.17. SSM library profile described by comparison perhydrolytic/proteolytic activity ratio of the variants versus Perhydrolase.
Seven variants with the highest ratio were further analyzed. Variants were re-
screened and expression level of the best variants was analyzed by Experion system,
Figure 3.18. Re-screening data for perhydrolytic and proteolytic activity compared to
Perhydrolase are summarized in Table 3.11.
Table 3.11. Re-screening results (assay conditions for perhydrolytic activity - 31.3 mM H2O2, 100 mM methylbutyrate, 100 mM NaBr, 0.5 mM APCC in 100 mM Na – phosphate buffer pH 7.5 with 0.9 mM SDS; assay conditions for proteolytic activity – 0.22 mM AAPF-pNA in 0.1 M Na-phosphate buffer pH 7.5). M- mutant, P - Perhydrolase
Variant Proteolytic activity M/P
Perhydrolytic activity M/P
Ratio perhydrolytic/proteol
ytic activity M/P
Amino acid substitution
P1B7 0.003 1.3 > 100 Gly→Ile
P1B12 0.152 2.8 18.4 Gly→Leu
P1C2 0.734 1.5 2.0 Gly→Phe
P1C10 0.260 1.6 6.2 Gly→Val
P1D12 0.008 1.2 > 100 Gly→Ile
P1F1 0.006 1.5 > 100 Gly→Ile
P1H2 0.192 3.0 15.6 Gly→Leu
Figure 3.18. Expression analysis of Perhydrolase and G166 variants byµl of supernatant after 24 h of expression was TCA precipitated and loaded on SDS gel. empty vector, 2 – Perhydrolase, 3 Perhydrolase, 9 – P1C2, 10 –
Selected variants were purified using bacitracin affinity chromatography
described in Chapter 3.10, Part II to confirm data
screening conditions. Bacitracin, decapeptide with antibiotic properties, is a competitive
inhibitor for serine proteases and binds in the active site via a D
ligand is coupled for CNBr Sepharose
plate column. 1.5 ml of expression culture
fractions were washed with 100 mM NH
mM NaCl pH 6.5. Protease was eluted from column with 100 mM
NaCl pH 6.5, 25 % isopro
interactions and isopropanol
substrate. Concentration of the variants was in µg/ml range (50
perhydrolytic and proteolytic
Part III: Directed Evolution of Subtilisin Carlsber g
93
pression analysis of Perhydrolase and G166 variants by
µl of supernatant after 24 h of expression was TCA precipitated and loaded on SDS gel. Perhydrolase, 3 – P1B7, 4 – P1B12, 5 – P1D12, 6 –
– P1C10. Black arrow indicates mature subtilisin Carlsberg.
variants were purified using bacitracin affinity chromatography
described in Chapter 3.10, Part II to confirm data using purified
Bacitracin, decapeptide with antibiotic properties, is a competitive
inhibitor for serine proteases and binds in the active site via a D
coupled for CNBr Sepharose to prepare an affinity matrix and loaded in 96
1.5 ml of expression culture was applied on the column; non
fractions were washed with 100 mM NH4-acetate pH 6.5 and 100 mM NH
mM NaCl pH 6.5. Protease was eluted from column with 100 mM
isopropanol, where high salt concentration
isopropanol disturbs hydrophobic interactions between protein and
oncentration of the variants was in µg/ml range (50-100 µg/ml).
perhydrolytic and proteolytic activity of the variants is presented in Table 3.12
Part III: Directed Evolution of Subtilisin Carlsber g
Experion system. 100 µl of supernatant after 24 h of expression was TCA precipitated and loaded on SDS gel. 1 –
P1F1, 7 – P1H2, 8 – Black arrow indicates mature subtilisin Carlsberg.
variants were purified using bacitracin affinity chromatography
using purified samples under
Bacitracin, decapeptide with antibiotic properties, is a competitive
inhibitor for serine proteases and binds in the active site via a D-glutamic acid. This
affinity matrix and loaded in 96-well
was applied on the column; non-bound
acetate pH 6.5 and 100 mM NH4-acetate, 50
mM NaCl pH 6.5. Protease was eluted from column with 100 mM NH4-acetate, 1 M
affects electrostatic
hydrophobic interactions between protein and
100 µg/ml). Specific
iants is presented in Table 3.12.
Part III: Directed Evolution of Subtilisin Carlsber g
94
Table 3.12. Specific activities of the variants. Perhydrolytic activity of the variants in the presence of 100 mM methyl-butyrate and 31.3 mM hydrogen-peroxide and proteolytic activity of the variants with 0.22 mM suc-AAPF-pNA in 100 mM Na-phosphate pH 7.5 buffer.
Variant Perhydrolytic activity (U/mg) Proteolytic activity (U/mg)
Perhydrolase 2.0 60.0
P1B7 2.2 0.9
P1B12 4.1 8.6
P1C2 2.4 31.1
P1C10 2.1 10.5
Results using purified enzyme confirmed that variant P1B7 (Gly166Leu) has the
highest performance when methyl-butyrate was used as substrate, while proteolytic
activity with suc-AAPF-pNA was 7-fold decreased compared to Perhydrolase. However,
the Mutant/Perhydrolase activity ratio was slightly different compared to screening
results, which can be due to process of purification where bacitracin (ligand in affinity
chromatography) could be eluted simultaneously with protease and since it is an
inhibitor it will decrease the measured activity.
Single amino acid substitution in the S1 binding pocket changed the
perhydrolytic/proteolytic ratio more than 100-fold. Further modification of the pocket
included Saturation Mutagenesis at positions Gly 127/Ala 169, and Gly 127/Gly 166. Gly
127 as a part of S3 pocket is opposite to Gly 166 and Ala 169, substituting Gly/Gly and
Gly/Ala with more robust amino acids would additionally decrease the size of S1 pocket
and ‘’push’’ the ester substrate toward the active site. However, screening data showed
that 85 % of the variants were inactive, suggesting that position 127 is important for
autoprocessing of the enzyme. Identified variants with increased perhydrolytic activity
had substitutions only at Gly 166. Beside Gly166Leu and Gly166Ile, an additional
substitution Gly166Tyr was found. This variant showed 2-fold increased perhydrolytic
activity and 3-fold decreased proteolytic activity.
Protein purification on large scale (250 ml expression media) was necessary to
determine kinetic parameters and compare the variants with wild type subtilisin
Carlsberg. Additionally, variants Thr59Ala and Leu217Trp were generated and purified
Part III: Directed Evolution of Subtilisin Carlsber g
95
in order to understand the influence of each position on proteolytic and perhydrolytic
activity.
4.7 Purification of selected variants
The calculated isoelectric point (pI) of the parent is 7.1 whereas reported values
are 6.7 (Vitale and Gamulin 1975) and 9.4 (Ottesen and Spector 1960). If pI of subtilisin
Carlsberg is around 7.0, protein at pH 7.8 should bind for anion exchange column,
however results showed that at this pH subtilisin Carlsberg does not bind to the anion or
cation exchange column. Binding to the anion exchange column was achieved at pH
8.6, while at pH 7.0 subtilisin Carlsberg binds to the cation exchange column, indicating
that pI value is around 7.8.
Purification of the variants was optimized at pH 7.0, where in the first step anion
exchange column was used to remove protein impurities from the cell supernatant.
Subtilisin Carlsberg did not bind to this column, while most of the impurities remained in
the column. Protease fractions were pooled together and loaded on the cation
exchange column. The detailed purification protocol is described in Chapter 3.11 of Part
III.
Figure 3.19 shows the purification profile of subtilisin Carlsberg. Panel A shows
the resulting fractions and SDS-PAGE analysis after the concentrated cell culture
supernatant was loaded into the anion exchange column. The blue line on the
chromatogram represents the protein content in each fraction related to the absorbance
of the sample at 280 nm. The black arrow points the fractions exhibiting proteolytic
activity, corresponding to subtilisin Carlsberg. After increasing the ionic strength of the
running buffer, the remaining protein content retained in the column was eluted. The
fractions with proteolytic activity were collected, concentrated and loaded into the cation
exchange column.
Panel B shows the result of the cation exchange chromatography; subtilisin
Carlsberg was retained in the column while other proteins present in the sample passed
through. These fractions did not show proteolytic activity. The retained protein was
eluted by increasing ionic strength and collected as a single protein peak.
Part III: Directed Evolution of Subtilisin Carlsber g
96
The proteolytic activity of the eluted fractions was confirmed and the positive
fractions were collected, pooled together, desalted and reconcentrated. The protein
purity was confirmed by the presence of a single band at 28 kDa corresponding to
subtilisin Carlsberg, using the Experion Pro260 Analysis Kit, Figure 3.20. Before
Experion analysis protein content of pure samples was inhibited with PMSF to avoid
degradation during sample preparation. Obtained purity of the variants was above 90 %.
Figure 3.19. Two step ion exchange chromatography purification of subtilisin Carlsberg. (ml of concentrated supernatant was loaded into an anion exchange column (Toy650c), active fractions (3, 4, 5, 6, 7, 8) weexchange chromatography Toyopearl SPconcentrated.
Part III: Directed Evolution of Subtilisin Carlsber g
97
Two step ion exchange chromatography purification of subtilisin Carlsberg. (rated supernatant was loaded into an anion exchange column (Toy
e fractions (3, 4, 5, 6, 7, 8) were pooled together, concentrated and loaded on cation exchange chromatography Toyopearl SP-650c (B) fractions 16, 17, 18 and 19 were p
Part III: Directed Evolution of Subtilisin Carlsber g
Two step ion exchange chromatography purification of subtilisin Carlsberg. (A) 10 rated supernatant was loaded into an anion exchange column (Toyopearl SuperQ-
re pooled together, concentrated and loaded on cation fractions 16, 17, 18 and 19 were pooled and
Figure 3.20 . Experion analysis of purified variants. 1 3 – Leu217Trp, 4 – Perhydrolase, 5Carlsberg.
Total protein concent
standard for calibration curve. Concentration of the each variant was calculated
according to the Experion analysis respect to the total protein concentration, val
summarized in Table 3.13.
Table 3.13. Calculated specific subtilisin Carlsberg concentration for each variant after purification.
Variant
Subtilisin Carlsberg
Thr59Ala variant
Leu217Trp variant
Perhydrolase
Thr59Ala/Gly166Leu/Leu217Trp (
Thr59Ala/Gly166Ile/Leu217Trp (
Thr59Ala/Gly166Tyr/Leu217Trp (
Part III: Directed Evolution of Subtilisin Carlsber g
98
nalysis of purified variants. 1 – subtilisin Carlsberg, 2
Perhydrolase, 5 - M1, 6 - M2, 7 - M3. Black arrow indicates mature subtilisin
concentration was determined using the BCA kit, using BSA as a
standard for calibration curve. Concentration of the each variant was calculated
according to the Experion analysis respect to the total protein concentration, val
Calculated specific subtilisin Carlsberg concentration for each variant after
Variant Specific concentration (mg/ml)
Subtilisin Carlsberg (wild type) 1.0
Thr59Ala variant 0.6
Leu217Trp variant 0.6
Perhydrolase 0.6
6Leu/Leu217Trp (M1) 1.9
Thr59Ala/Gly166Ile/Leu217Trp (M2) 4.5
Thr59Ala/Gly166Tyr/Leu217Trp (M3) 1.6
Part III: Directed Evolution of Subtilisin Carlsber g
subtilisin Carlsberg, 2 - Thr59Ala variant, Black arrow indicates mature subtilisin
BCA kit, using BSA as a
standard for calibration curve. Concentration of the each variant was calculated
according to the Experion analysis respect to the total protein concentration, values are
Calculated specific subtilisin Carlsberg concentration for each variant after
Specific concentration (mg/ml)
4.8 Characterization of the variants
Kinetic parameters
activity using suc-AAPF-pNA as a substra
different ester substrates (methyl
Variants Thr59Ala and Leu217Trp were
showed that substitution Thr59Ala does no
4.8.1 Characterization with suc
Although suc-AAPF-
most widely used substrate for characterization of proteolytic activity.
kinetic parameters for subtilisin Carlsberg and four variants are summarized in Figure
3.21 and Table 3.14.
Enzyme
Wild type
Perhydrolase
M1
M2
M3
Figure 3.21/Table 3.14. Kinetic parameters of the selected variants for proteolytic activity using suc-AAPF-pNA as a substrate.
Part III: Directed Evolution of Subtilisin Carlsber g
99
4.8 Characterization of the variants
inetic parameters of the selected variants were determined for proteolytic
pNA as a substrate and for perhydrolytic activity with three
different ester substrates (methyl-propionate, methyl-butyrate and methyl
Variants Thr59Ala and Leu217Trp were excluded from characterization, since r
substitution Thr59Ala does not have any effect on activity
4.8.1 Characterization with suc-AAPF-pNA as a substrate
-pNA does not represent “real” protease
most widely used substrate for characterization of proteolytic activity.
tic parameters for subtilisin Carlsberg and four variants are summarized in Figure
KM (mM) kcat (min -1) k
0.59 ± 0.06 10353 ± 342
0.15 ± 0.02 1988 ± 43
0.42 ± 0.03 908 ± 21
0.54 ± 0.04 104 ± 2
0.2 ± 0.02 1370 ± 27
Kinetic parameters of the selected variants for proteolytic activity using substrate.
Part III: Directed Evolution of Subtilisin Carlsber g
were determined for proteolytic
te and for perhydrolytic activity with three
butyrate and methyl-pentanoate).
excluded from characterization, since results
t have any effect on activity.
protease substrate it is the
most widely used substrate for characterization of proteolytic activity. The calculated
tic parameters for subtilisin Carlsberg and four variants are summarized in Figure
kcat /KM (mM-1min -1*103)
17.5
13.2
2.2
0.2
6.8
Kinetic parameters of the selected variants for proteolytic activity using
As already described, Perhydrolase has
surprisingly KM value is also decreased
Perhydrolase only 1.3-times compared to WT
and M3 variants is lower compared to Perhydrolase which is in acco
screening results. Additionaly, M3 variant has
M1 and M2 variants have increased K
The best variant is M2 with
87.5-fold decreased specificity constant (
4.8.2 Skim milk activity
Proteolytic activity of the variants was tested
the activity towards complex
Figure 3.22. Skim milk activityabsorbance per minute at 650 nm
As expected, difference in activity between
obtained by characterization with suc
decreased proteolytic activity compared to WT and M2 variant
than Perhydrolase and 4.6-
Part III: Directed Evolution of Subtilisin Carlsber g
100
described, Perhydrolase has a decreased kcat co
value is also decreased, decreasing specificity constant
times compared to WT. The catalytic efficiency (k
and M3 variants is lower compared to Perhydrolase which is in acco
screening results. Additionaly, M3 variant has similar KM value to
M1 and M2 variants have increased KM, but still slightly lower than in the case of WT.
The best variant is M2 with 99.5-fold decreased catalytic efficiency compared to WT and
fold decreased specificity constant (kcat/KM).
Proteolytic activity of the variants was tested using skim milk i
complex substrate.
Skim milk activity of the selected variants. Activity is represented as decrease in at 650 nm caused by 5 ng/ml of enzyme with 2 % skim milk
As expected, difference in activity between the variants is different from data
rization with suc-AAPF-pNA. Perhydrolase has only 2
eolytic activity compared to WT and M2 variant has 1.7
-lower activity compared to WT, while activity with
Part III: Directed Evolution of Subtilisin Carlsber g
compared to WT, but
specificity constant (kcat/KM) of
The catalytic efficiency (kcat) of M1, M2
and M3 variants is lower compared to Perhydrolase which is in accordance with the
Perhydrolase, while
, but still slightly lower than in the case of WT.
cy compared to WT and
skim milk in order to compare
of the selected variants. Activity is represented as decrease in
ng/ml of enzyme with 2 % skim milk at pH 8.6.
variants is different from data
Perhydrolase has only 2.8-fold
has 1.7-fold lower activity
activity with suc-AAPF-
Part III: Directed Evolution of Subtilisin Carlsber g
101
pNA is 87.5-fold lower. In this case, M1 is the best variant having 5-times lower activity
than WT.
4.8.3 Perhydrolytic activity: kinetic parameters for hydrogen-peroxide and methyl-
propionate
Amount of produced peroxycarboxylic acid in perhydrolytic reaction was
calculated from a calibration curve for peroxyacetic acid generated in the presence of
hydrogen-peroxide and an ester substrate.The relationship between concentration of
peroxyacetic acid in the reaction mixture and intensity of fluorescent signal is presented
in Figure 3.23.
Figure 3.23. Calibration curve for peroxyacetic acid
Kinetic constants for perhydrolysis of methyl-propionate were obtained by varying
each substrate independently. The fixed methyl-propionate concentration was 500 mM,
which is also the highest solubility of this substrate and fixed concentration of hydrogen-
peroxide was 100 mM. Kinetic constants are presented in Figure 3.23 and Table 3.15.
Enzyme Varied substrate
Wild type Hydrogen
Methyl-propio
Perhydrolase Hydrogen
Methyl-propionate
M1 Hydrogen
Methyl-propionate
M2 Hydrogen
Methyl-propionate
M3 Hydrogen
Methyl-propionate
Figure 3.23/Table 3.15. Kinetic parameters of the propionate.
As expected from the
2.4-fold increased kcat value,
and M3 variants have slightly i
fold decreased KM values for methyl
peroxide to Perhydrolase. The best vari
constant (kcat/KM) compared to
comparison with WT. The values of k
were completely saturated with both constant substrates. The K
propionate was high for all variants so the
did not completely saturate the enzyme.
Part III: Directed Evolution of Subtilisin Carlsber g
102
Varied substrate KM (mM) kcat (min -1
Hydrogen-peroxide
propionate
41.0 ± 8.3
478.2 ± 55.7
66.7 ± 7.0
67.8 ± 4.3
Hydrogen-peroxide
propionate
16.4 ± 2.4
566.2 ± 75.3
115.8 ± 6.3
164.6 ± 12.7
Hydrogen-peroxide
propionate
18.8 ± 2.5
265.1 ± 27.1
173.3 ± 9.1
182.4 ± 8.0
Hydrogen-peroxide
propionate
20.9 ± 0.9
255.2 ± 27.7
183.8 ± 3.4
204.0 ± 9.3
Hydrogen-peroxide
propionate
18.7 ± 2.0
349.4 ± 42.8
209.8 ± 8.6
238.4 ± 14.1
Kinetic parameters of the selected variants for perhydrolysis
the reported data (Lee, Vojcic et al. 2010), Perhydrolase has
value, and decreased KM value for hydrogen
and M3 variants have slightly increased kcat values compared to Perhydrolase, but
values for methyl-propionate and similar KM values for hy
. The best variant, M3, shows 1.6-fold increased
compared to Perhydrolase and 7-fold increased
he values of kcat would be the same in both cases, if the enzyme
were completely saturated with both constant substrates. The K
propionate was high for all variants so the methyl-propionate concentration of 500 mM
did not completely saturate the enzyme.
Part III: Directed Evolution of Subtilisin Carlsber g
1) kcat/KM (mM-1min -1)
7.0
4.3
1.6
0.14
6.3
12.7
7.1
0.29
9.1
8.0
9.2
0.69
3.4
9.3
8.8
0.80
8.6
14.1
11.2
0.68
iants for perhydrolysis of methyl-
, Perhydrolase has a
ydrogen-peroxide. M1, M2
values compared to Perhydrolase, but ~2-
values for hydrogen-
fold increased specificity
fold increased specificity in
would be the same in both cases, if the enzyme
were completely saturated with both constant substrates. The KM value for methyl-
propionate concentration of 500 mM
4.8.4 Perhydrolytic activity: kinetic parameters for
pentanoate
Since position Gly 166 is part of S1 binding pocket, different substitutions at
position can change enzyme specificity toward different
perhydrolytic activity were determined
which was substrate of choice for screening and already mentioned methyl
variants are also characterized with methyl
and methyl-pentanoate are present
Enzyme Substrate
Wild type Methyl-butyrate
Methyl-pentanoate
Perhydrolase Methyl-butyrate
Methyl-pentanoate
M1 Methyl-butyrate
Methyl-pentanoate
M2 Methyl-butyrate
Methyl-pentanoate
M3 Methyl-butyrate
Methyl-pentanoate
Figure 3.24/Table 3.16. Kinetic parameters omethyl-butyrate and methyl-pentanoate
Part III: Directed Evolution of Subtilisin Carlsber g
103
Perhydrolytic activity: kinetic parameters for methyl-butyrate
166 is part of S1 binding pocket, different substitutions at
can change enzyme specificity toward different substrates.
perhydrolytic activity were determined for three ester substrates. Beside methyl
which was substrate of choice for screening and already mentioned methyl
variants are also characterized with methyl-pentanoate. Kinetic data for methyl
pentanoate are presented in Figure 3.24 and Table 3.16
Substrate KM (mM) kcat (min
butyrate
pentanoate
117.7 ± 26.8
35.2 ± 4.2
124.6 ± 13.7
69.5 ± 3.6
butyrate
pentanoate
122.6 ± 36.9
38.3 ± 5.1
258.3 ± 38.1
170.8 ± 12
butyrate
pentanoate
64.7 ± 12.5
33.3 ± 5.5
676.0 ± 49.4
382.7 ± 27.2
butyrate
pentanoate
93.1 ± 10.4
29.2 ± 3.4
605.5 ± 29.7
378.8 ± 18.2
butyrate
pentanoate
88.8 ± 20.1
26.3 ± 4.6
525.8 ± 51.2
182.3 ± 12.7
Kinetic parameters of the selected variants for perhydrolysispentanoate.
Part III: Directed Evolution of Subtilisin Carlsber g
butyrate and methyl-
166 is part of S1 binding pocket, different substitutions at that
Kinetic constants for
Beside methyl-butyrate
which was substrate of choice for screening and already mentioned methyl-propionate,
pentanoate. Kinetic data for methyl-butyrate
ed in Figure 3.24 and Table 3.16.
(min -1) kcat/KM
(mM-1min -1)
13.7
3.6
1.1
2.0
38.1
12.9
2.1
5.7
49.4
27.2
10.4
11.5
29.7
18.2
6.5
13.0
51.2
12.7
5.9
6.9
ected variants for perhydrolysis activity of
Perhydrolase has a ~2.5
ester substrates and similar K
perhydrolytic reaction variants M1, M2 and M3 have increased k
2.0-fold, respectively and decreased K
the highest affinity for methyl
compared to WT.
In the case of methyl
M2 variant have ~2.2-fold increased k
Perhydrolase. KM values are up to 1.5
substrate M2 variant has the highest specificity constant which is 11.8
compared to WT.
Due to decreased KM
length of the acyl chain, Figure 3.25
Figure 3.25: Effect of acyl chain length on the catalytic efficiency of the variants.
Part III: Directed Evolution of Subtilisin Carlsber g
104
has a ~2.5-fold increased kcat for perhydrolytic activity
ester substrates and similar KM to WT. With methyl-butyrate as a substrate fo
drolytic reaction variants M1, M2 and M3 have increased kcat
fold, respectively and decreased KM values compared to Perhydrolase.
the highest affinity for methyl-butyrate is M1 with specificity constant
In the case of methyl-pentanoate as a substrate in perhydrolytic reaction M1 and
fold increased kcat, while kcat of M3 variant is
values are up to 1.5-fold decreased. With methyl
substrate M2 variant has the highest specificity constant which is 11.8
M, the catalytic efficiency of all the variants increases
h of the acyl chain, Figure 3.25.
chain length on the catalytic efficiency of the variants.
Part III: Directed Evolution of Subtilisin Carlsber g
for perhydrolytic activity with all three
butyrate as a substrate for
cat values 2.6, 2.3 and
values compared to Perhydrolase. Variant with
specificity constant 9.5-fold increased
pentanoate as a substrate in perhydrolytic reaction M1 and
of M3 variant is similar to kcat of
ethyl-pentanoate as a
substrate M2 variant has the highest specificity constant which is 11.8-fold higher
fficiency of all the variants increases with the
chain length on the catalytic efficiency of the variants.
Part III: Directed Evolution of Subtilisin Carlsber g
105
4.8.5 Esterolytic activity of the variants
Substrate (methyl-butyrate) and product (butyric acid) in the esterolytic reaction
were separately passed through a reversed-phase C18 column under described
conditions (Chapter 3.15 of Part III) to determine retention time for each component.
After identification of each component on chromatogram, a calibration curve for butyric
acid was generated. Relationship between concentration of butyric acid and peak area
on the chromatogram is presented in Figure 3.26.
Figure 3.26. Calibration curve for butyric acid.
Esterolytic activity of the variants was carried with 200 mM methyl-butyrate at
30°C for 10 min. The reaction was stopped by decrea sing the pH value to 3 with
addition of phosphoric acid. Samples were analyzed by reversed-phase
chromatography and amount of produced butyric acid in esterolytic reaction was
calculated from calibration curve generated in the presence of methyl-butyrate. Specific
activity (U/mg) was calculated for all variants, where 1 U presents amount of enzyme
which generates 1 µmol of product per minute.
Part III: Directed Evolution of Subtilisin Carlsber g
106
Table 3.17. Esterolytic activity of the variants with 200 mM methyl-butyrate in 100 mM Na-phosphate buffer pH 7.5. Product was separated from substrate by HPLC on a reversed-phase C18 column with acetonitrile/K-phosphate buffer (20 mM, pH 3.0) gradient elution and detected at 210 nm.
Variant Esterolytic activity (U/mg)
Wild type 0.96 ± 0.02
Perhydrolase 0.63 ± 0.06
M1 1.72 ± 0.08
M2 1.06 ± 0.02
M3 1.46 ± 0.02
Results for esterolytic activity also confirmed that Perhydrolase has decreased
specificity for water compared to WT, since has lower esterolytic activity with methyl-
butyrate, while perhydrolytic activity with the same substrate is increased. This data
additionally confirms Molecular Dynamics simulation results (Lee, Vojcic et al. 2010)
which suggested a better stabilization of the perhydrolytic tetrahedral intermediate over
hydrolytic intermediate in Perhydrolase variant compared to WT. Variants M1, M2 and
M3 besides increased perhydrolytic activity also have an increased esterolytic activity
compared to Perhydrolase, leading to the conclusion that a position 166, as a part of S1
binding pocket, interacts with both peptide and ester substrates.
Part III: Directed Evolution of Subtilisin Carlsber g
107
5. Discussion
Subtilisin proteases are among the first engineered enzymes and until today
most of the amino acids have been exchanged to increase activity, termostability, alter
specificity and pH profile or to understand the function of each position in the active site
and substrate binding pocket. However, there has not been a study about esterolytic
and perhydrolytic activity of this proteases or protein engineering experiments which
alter their ester specificity. Wieland at al. (Wieland, Polanyi-Bald et al. 2009) for the first
time reported that one of the serine proteases, subtilisin Carlsberg, besides it’s natural
role catalyzes the reaction of ester perhydrolysis. They also performed directed
evolution experiments obtaining variant (Thr59Ala/Leu217Trp) which has 2.7-fold
increased perhydrolytic activity with methyl-butyrate and 8-fold decreased proteolytic
activity with suc-AAPF-pNA. We continued to engineer variant Thr59Ala/Leu217Trp in
order to further increase perhydrolytic activity.
Despite subtilisins having broad substrate specificity, they can differ significantly
from each other in catalytic efficiency against selected substrates. Specificity is the
result of chemical binding forces which includes hydrogen bonding, electrostatic
interactions, hydrophobic and steric effects (Fersht 1999). Strength and specificity of the
enzyme-substrate binding also determines the kinetic parameters of the reaction such
are kcat, KM and the catalytic constant (kcat/KM). KM is defined by the binding energy of
unaltered substrate for the enzyme, while kcat is determined by activation energy
(energy difference between ES and ES‡). In the reaction of peptide bond hydrolysis, the
limiting step is formation of the first transition state, which determines kcat of the
reaction, while limiting step in the reaction of ester hydrolysis is degradation of acyl-
enzyme intermediate and second product formation (Wells and Estell 1988), Figure
3.27.
Part III: Directed Evolution of Subtilisin Carlsber g
108
Figure 3.27: The rate-limiting acylation step (which defines kcat) for hydrolysis of peptide bonds by subtilisin BPN’. Taken over from (Wells and Estell 1988).
Position Gly 166 is conserved in five known subtilisins: subtilisin BPN’ (Wells,
Ferrari et al. 1983), subtilisin Carlsberg (Guntelberg and Ottesen 1954), subtilisin DY
(Nedkov, Oberthür et al. 1985), subtilisin Amylosacchariticus (Kurihara, Markland et al.
1972), subtilisin E (Wong, Price et al. 1984). Still, engineering of this position produced
variants with changed substrate specificity and same or similar catalytic efficiency
(Estell, Graycar et al. 1986). Gly 166 is located at the bottom of S1 substrate binding
cleft, Figure 3.28. This pocket is a hydrophobic cleft which can accept any amino acid at
P1 position of the substrate, providing broad substrate specificity for subtilisin
Carlsberg. We identified variants with substitutions at position 166 which not only have
changed specificity towards suc-AAPF-pNA, but also for different ester substrates with
increasing size and hydrophobicity. Proteolytic activity of these variants using the
artificial substrate suc-AAPF-pNA and a complex substrate was decreased, while
perhydrolytic and hydrolytic activities with ester substrate were increased.
Figure 3.28: Model of the substrate, sucactive site of subtilisin Carlsberg
Part III: Directed Evolution of Subtilisin Carlsber g
109
substrate, suc-AAPF-pNA (element representation),active site of subtilisin Carlsberg (gray).
Part III: Directed Evolution of Subtilisin Carlsber g
t representation), bound to the
5.1 Suc-AAPF-pNA specificity
To understand the nature of substrate binding pocket of subtilisin
interactions with suc-AAPF
Figure 3.29: Kinetic parameters of the variantswhite bars, KM 10-5(M) – gray bars and k
Even though kcat of Perhydrolase is 5.2 times less compared to WT,
efficiency did not change significantly, since K
already proposed by Lee at al.
destabilization of second tetrahedral intermediate by introducing substitution
Leu217Trp. Additionally, the
pocket where introduction of “bulkier”
nitroanilide group of the substrate to
By introducing Leu, Ile and Tyr at
S1 pocket are changed, suggesting that specificity is determined by steric and
hydrophobic effects. The nature of p
AAPF-pNA) binding in the S1 pocket is analyz
parameters. Increasing the hydrophobicity of S1 binding pocket by amino acid
substitution at position 166 may increase binding of hydrophobic P1 substrates.
Part III: Directed Evolution of Subtilisin Carlsber g
110
pNA specificity
To understand the nature of substrate binding pocket of subtilisin
AAPF-pNA, the observed kinetic parameters were
Kinetic parameters of the variants for hydrolysis of suc-AAPFgray bars and kcat/KM*105(s-1*M-1) – black bars.
of Perhydrolase is 5.2 times less compared to WT,
change significantly, since KM decreased 4-fold, Figure 3.29
Lee at al. (Lee, Vojcic et al. 2010), kcat is decreased due to a
econd tetrahedral intermediate by introducing substitution
the decreased kcat value might be due to a
pocket where introduction of “bulkier” tryptophan could prevent
nilide group of the substrate to the S1’ pocket.
By introducing Leu, Ile and Tyr at position 166, the size and hydrophobicity of the
S1 pocket are changed, suggesting that specificity is determined by steric and
ophobic effects. The nature of phenylalanine (Phe is at position P1 in the suc
pNA) binding in the S1 pocket is analyzed according to the obtained kinetic
parameters. Increasing the hydrophobicity of S1 binding pocket by amino acid
substitution at position 166 may increase binding of hydrophobic P1 substrates.
Part III: Directed Evolution of Subtilisin Carlsber g
To understand the nature of substrate binding pocket of subtilisin Carlsberg and
kinetic parameters were analyzed.
AAPF-pNA: kcat (1/s) –
of Perhydrolase is 5.2 times less compared to WT, the catalytic
fold, Figure 3.29. As
is decreased due to a
econd tetrahedral intermediate by introducing substitution
might be due to a changes in S1’ the
could prevent binding of the p-
position 166, the size and hydrophobicity of the
S1 pocket are changed, suggesting that specificity is determined by steric and
henylalanine (Phe is at position P1 in the suc-
ed according to the obtained kinetic
parameters. Increasing the hydrophobicity of S1 binding pocket by amino acid
substitution at position 166 may increase binding of hydrophobic P1 substrates.
Part III: Directed Evolution of Subtilisin Carlsber g
111
However, since hydrophobicity is proportional to its surface area (Rose, Geselowitz et
al. 1985), increasing the hydrophobicity of the S1 pocket may sterically hinder binding of
larger substrates.
Catalytic efficiency of M3 variant (Gly→Tyr) is 2-fold decreased, M1 (Gly→Leu)
has a 6-fold decreased kcat/KM, while M2 (Gly→Ile) has a 65-fold decreased efficiency
compared to Perhydrolase (Gly 166). Although, M3 has introduced a Tyr at position
166, which has a large residue, the efficiency is not as affected as when Leu and Ile
were introduced. This result suggests that the steric effect of Tyr is lower, which can be
due to the orientation of the residue in the S1 pocket. Figure 3.30 shows interaction of
suc-AAPF-pNA bound to the active site residue Ser 221 with amino acid at position 166.
The phenol ring of Tyr residue is orientated towards the solvent, while Leu and Ile
residues remain in the pocket, further decreasing the S1 pocket size. Decreased pocket
size will increase steric repulsion between the Phe residue of substrate and Ile 166,
possibly explaining why M2 (Gly→Ile) is less efficient when large aromatic substrates
are at position P1.
Figure 3.30: Interaction of sucdifferent variants (Perhydrolase, M1, M2 and M3). The closest interactionsposition 166 and phenol ring was observed in M2 variant (G166I).
Substitution of Gly 166
the variants when a complex substrate
Part III: Directed Evolution of Subtilisin Carlsber g
112
Interaction of suc-AAPF-pNA bound to Ser 221 with amino acid at positiondifferent variants (Perhydrolase, M1, M2 and M3). The closest interactions
166 and phenol ring was observed in M2 variant (G166I).
Substitution of Gly 166 narrows substrate specificity, explain
complex substrate such skim milk is used, Figure 3.22.
Part III: Directed Evolution of Subtilisin Carlsber g
1 with amino acid at position 166 in
different variants (Perhydrolase, M1, M2 and M3). The closest interactions between residue at
explaining lower activity of
, Figure 3.22.
Part III: Directed Evolution of Subtilisin Carlsber g
113
5.2 Ester specificity
Perhydrolytic activity of the variants was determined using three ester substrates
which differ in the length of acyl chain (from propyl to pentanoyl group). As the size of
the acyl chain increases, the KM value of each variants decreases, suggesting a
stronger substrate binding to the hydrophobic S1 pocket as substrate hydrophobicity
increases. Each additional methylene group theoretically changes the Gibbs free energy
by 2.84 KJ/mol (Fersht 1999). Docking results of each substrate showed increase in
binding energy with increase of substrate acyl chain, which indicates stronger binding of
the substrate to the enzyme. Table 3.18 summarizes binding energies of substrate
conformations for each substrate to all the variants of subtilisin Carlsberg.
Table 3.18. Binding energies of the substrates (only the score with the highest energies are included, A and B) to each variant of subtilisin Carlsberg. In bolded are presented binding energies of substrate in the position which enables conversion (productive binding of the substrate).
Binding energy (kcal/mol) Variant Methyl -propionate Methyl -butyrate Methyl -pentanoate
A B A B A B
Wild type 3.34 3.21 3.7 3.58 4.09 3.98
Perhydrolase 3.36 3.21 3.77 3.61 4.22 3.99
M1 3.27 3.68 4.23
M2 3.34 3.78 4.11
M3 3.27 3.68 3.6 4.25 3.84
Next, we analyzed the effect of each mutation on the catalytic efficiency of each
variant using the same substrate.
Methyl-propionate: the increased hydrophobicity (Tyr→Leu/Ile) and decreased
size of S1 pocket decrease KM value of the variants from 1.6 to 2-fold. Catalytic
constant (kcat) of the variants is increased up to 1.4-fold (for M3) compared to parent.
Methyl-propionate binds in the S1 pocket of Perhydrolase in two conformations
(productive and nonproductive), Figure 3.31. Non-productive conformation (A) has
stronger binding energy than productive (B), meaning that at saturation less than half of
the substrate is bound productively, see Table 3.18.
Figure 3.31. Docking result of methylbinds in two conformation: A –
Introduction of Leu, Ile and Tyr in the S1 pocket instead of Gly
resulted in only one possible
is the productive conformation, Figure 3.32. This result also explains
since at saturation all the substrate
Part III: Directed Evolution of Subtilisin Carlsber g
114
stronger binding energy than productive (B), meaning that at saturation less than half of
the substrate is bound productively, see Table 3.18.
Docking result of methyl-propionate in the active site of Perhydrolase. Substrate – non-productive binding and B – productive binding.
Introduction of Leu, Ile and Tyr in the S1 pocket instead of Gly
d in only one possible binding mode of methyl-propionate in the active site, which
productive conformation, Figure 3.32. This result also explains
t saturation all the substrate should be bound productively.
Part III: Directed Evolution of Subtilisin Carlsber g
stronger binding energy than productive (B), meaning that at saturation less than half of
propionate in the active site of Perhydrolase. Substrate
productive binding.
Introduction of Leu, Ile and Tyr in the S1 pocket instead of Gly at position 166
propionate in the active site, which
productive conformation, Figure 3.32. This result also explains the increased kcat,
Figure 3.32: Docking result of methylvariant (Ile 166) and M3 variant
Methyl-butyrate: KM
case of M1, while kcat is significantly increas
increased kcat of M1, M2 and M3 variant, respectively
propionate, methyl-butyrate
(productive and non-productive), Figure 3.33
has stronger binding energy than productive
Part III: Directed Evolution of Subtilisin Carlsber g
115
Docking result of methyl-propionate in the active site of M1 variant M3 variant (Tyr 166).
values of the variants were decreased,
is significantly increased for all the variants (
of M1, M2 and M3 variant, respectively). As in the case of methyl
butyrate also shows two binding conformations in the S1 pocket
productive), Figure 3.33. Again, non-productive conformation (A)
has stronger binding energy than productive conformation (B), see Table 3.18.
Part III: Directed Evolution of Subtilisin Carlsber g
M1 variant (Leu 166), M2
values of the variants were decreased, up to 1.8-fold in the
ed for all the variants (2.6-, 2.3- and 2-fold
As in the case of methyl-
binding conformations in the S1 pocket
productive conformation (A)
(B), see Table 3.18.
Figure 3.33. Docking result of methylbinds in two conformation: A –
Docking results show that m
one conformation that favors
and M2). In the case of M3 variant, methyl
productive (A) and non-productive
conformations of the substrate are
When substrate binding is productive
site, inside the S1 pocket, while in the case of non
oriented towards the solvent, having
(M3(A) and M3(B)). The presence of the non
decreased kcat value for M3 variant compared to M1.
Part III: Directed Evolution of Subtilisin Carlsber g
116
king result of methyl-butyrate in the active site of Perhydrolase. Substrate – nonproductive binding and B – productive binding.
Docking results show that methyl-butyrate binds for M1 and M2 variant only in
one conformation that favors catalysis, resulting in a increased kcat
of M3 variant, methyl-butyrate docks in two
productive (B), Figure 3.34 (M3(A) and M3(B)
substrate are possible due to ineraction of Tyr 166 with substrate.
is productive Tyr 166 is more oriented in the direction of active
site, inside the S1 pocket, while in the case of non-productive binding Tyr 166 is
solvent, having less influence on the size of S1 pocket, Figure 3.34
resence of the non-productive conformation explains slightly
for M3 variant compared to M1.
Part III: Directed Evolution of Subtilisin Carlsber g
in the active site of Perhydrolase. Substrate
productive binding.
for M1 and M2 variant only in
cat, Figure 3.34, (M1
ocks in two conformations,
M3(A) and M3(B)). Two different
Tyr 166 with substrate.
ented in the direction of active
productive binding Tyr 166 is
less influence on the size of S1 pocket, Figure 3.34
ive conformation explains slightly
Figure 3.34: Docking result of methylvariant (Ile 166) and M3 variant (Tyr 166).conformation for M3 variant andconformation for M3 variant. Conformation A has higher binding energy
Methyl-pentanoate: K
hydrophobicity of the substrate, where further increase in the hydrophobicity of the
pocket does not influence significantly on the binding energy of the substrate. Again, M1
and M2 show higher catalytic constant values, which
to parent, while M3 variant has the same k
methyl-pentanoate also binds in two conformations for parent and M3 variant, which
explains similar kcat values
M3(A) and M3(B)). Docking results
is bound only in the productive conformation, Figure 3.35
Part III: Directed Evolution of Subtilisin Carlsber g
117
king result of methyl-butyrate in the active site of M1 variant (Leu 166), M2 variant (Ile 166) and M3 variant (Tyr 166). A present methyl-butyrate bou
or M3 variant and B present methyl-butyrate bound in . Conformation A has higher binding energy than conformation
KM values of the variants are very similar, most
hydrophobicity of the substrate, where further increase in the hydrophobicity of the
pocket does not influence significantly on the binding energy of the substrate. Again, M1
and M2 show higher catalytic constant values, which are 2.2-fold in
M3 variant has the same kcat as parent. Docking results showed that
pentanoate also binds in two conformations for parent and M3 variant, which
values between these two variants, Figure 3.35
Docking results for M1 and M2 variant show that
productive conformation, Figure 3.35 (M1 and
Part III: Directed Evolution of Subtilisin Carlsber g
M1 variant (Leu 166), M2
butyrate bound in productive butyrate bound in nonproductive
than conformation B.
values of the variants are very similar, mostly defined by
hydrophobicity of the substrate, where further increase in the hydrophobicity of the
pocket does not influence significantly on the binding energy of the substrate. Again, M1
fold increased compared
. Docking results showed that
pentanoate also binds in two conformations for parent and M3 variant, which
, Figure 3.35 (Per(A), Per (B),
variant show that methyl-pentanoate
and M2). At saturation
concentration of substrate,
catalysis, resulting in a kcat
Figure 3.35: Docking result of methyl(Leu 166), M2 variant (Ile 166) and M3 variant (Tyr 16present methyl-pentanoate conformationbinding energy than conformation B.
Part III: Directed Evolution of Subtilisin Carlsber g
118
all the molecules are bound in the conforma
value more than 2-fold increased compared to parent.
king result of methyl-pentanoate in the active site of Perhydrolase, (Leu 166), M2 variant (Ile 166) and M3 variant (Tyr 166). A (productive) and B (nonproductive)
pentanoate conformation bound in the S1 pocket. Conformation A has higher binding energy than conformation B.
Part III: Directed Evolution of Subtilisin Carlsber g
the conformation which favors
compared to parent.
Perhydrolase, M1 variant
A (productive) and B (nonproductive) bound in the S1 pocket. Conformation A has higher
Part III: Directed Evolution of Subtilisin Carlsber g
119
Decreased size of the S1 pocket allows binding of the ester substrate
preferentially in the productive conformation which leads to catalysis. Leu, Ile and Tyr at
position 166 “push” the ester substrate toward active site, affecting the susceptibility of
the scissile ester bond to the nucleophilic attack by the hydroxyl group of the catalytic
Ser 221.
Part III: Directed Evolution of Subtilisin Carlsber g
120
6. Conclusion
In summary, an expression system for subtilisin Carlsberg in B. subtilis WB600
and B. subtilis DB104 was developed in microtiter plates suitable for perhydrolytic and
proteolytic activity screening. APCC assay was optimized for detection of perhydrolytic
activity in the supernatant of Bacillus cells and used for final characterization of the
variants.
Screening site saturation libraries generated at positions near the active site and
in the substrate binding pockets identified position 166 as residue that determines
substrate specificity of subtilisin Carlsberg. An increase in the side-chain volume at
position 166 decreased the size of S1 gap leading to a reduction in kcat/KM value
towards large amino acids at position P1. At the same time, catalytic efficiencies
towards ester substrates were increased up to 10-fold in these variants. Results showed
that the specificity of subtilisin Carlsberg could be changed by replacing directly amino
acids that are in contact with the substrate, which brings the idea of changing protease
into esterase and perhydrolase.
Important substitutions for increasing specificity toward hydrogen-peroxide
instead water were not identified by screening random mutagenesis libraries and SSM
libraries at positions identified by rational design. Same catalytic center for hydrolytic
and perhydrolytic activity, similar structure of hydrogen-peroxide with water leads to
similar structure of perhydrolytic and hydrolytic tetrahedral intermediate, suggesting that
the same residues in subtilisin Carlsberg are included in interactions with substrates
and intermediates. This limits the possibilities to find residues that will favor one type
reaction over the other. Additionally, diversity is lost since variants without hydrolytic
activity can’t autocatalytically process into mature form, although it is possible that once
the pro-sequence is cleaved they still can have perhydrolytic activity.
Future prospects
121
Future prospects
Results of the directed evolution and modeling studies gave insight how to
engineer protease into esterase and perhydrolase preserving the level of proteolytic
activity necessary for protease maturation.
However, interesting questions come from the results and discussion of this
work:
1. Is it possible to make better perhydrolase if we avoid prescreening step
caused by automaturation?
2. How much of diversity is lost during this self caused prescreening?
3. Can serine protease be converted into effective perhydrolase?
4. Why only some subtilisins show perhydrolytic activity?
5. Will engineering of pro-sequence induce conformational changes that alter
substrate specificity and catalytic activity?
To tackle some of these questions it is necessary to develop an expression
system without autocatalytic processing. Development of in vitro translation systems of
the mature protease form in the presence of pro-sequence to guide folding or wild type
protease to activate variants which can not process combined with high throughput
screening system might provide a platform to address this questions.
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122
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Publications
129
Publication list
1. Wook Lee, Ljubica Vojcic, Dragana Despotovic , Radivoje Prodanovic, Karl-
Heinz Maurer, Ulrich Schwaneberg, Martin Zacharias (2010): "Rationalizing
perhydrolase activity of aryl-esterase and subtilisin Carlsberg mutants by molecular
dynamics simulations of the second tetrahedral intermediate state." Theoretical
Chemistry Accounts 125(3-6): 375-386.
2. Dragana Despotovic , Ljubica Vojcic, Radivoje Prodanovic, Ronny Martinez,
Karl-Heinz Maurer, Ulrich Schwaneberg (2012): “Fluorescent assay for directed
evolution of perhydrolases.” Journal of Biomolecular Screening. Accepted
3. Ljubica Vojcic, Dragana Despotovic , Ronny Martinez, Karl-Heinz Maurer, Ulrich
Schwaneberg (2012). “An efficient transformation method for Bacillus subtilis DB104.”
Applied Microbiology and Biotechnology. Accepted
4. Ljubica Vojcic, Dragana Despotovic , Karl-Heinz Maurer, Ronny Martinez, Ulrich
Schwaneberg (2012). “Directed evolution of a double mutant subtilisin Carlsberg,
Thr59Ala/Leu217Trp towards increased oxidative stability”. Under preparation
5. Dragana Despotovic , Ljubica Vojcic, Milan Blanusa, Karl-Heinz Maurer, Ronny
Martinez, Ulrich Schwaneberg (2012). Switching substrate specificity of a subtilisin
Carlsberg mutant towards ester perhydrolysis. Under preparation
Curriculum vitae
130
Curriculum vitae
Personal data
Name: Dragana Despotovic
Gender: Female
Date of birth: 24th February, 1983
Place of birth: Sabac, Republic of Serbia
Marital Status: Single
Nationality: Serbian
Education
2009 - present PhD fellow of Biotechnology, RWTH Aachen University, Aachen,
Germany
2008 – 2009 PhD fellow of Engineering and Science, Jacobs University Bremen,
Bremen, Germany
2002 – 2008 Diploma in Biochemistry, Faculty of Chemistry, University of
Belgrade, Belgrade, Serbia
1998 – 2002 High school Gimnazija “Vera Blagojevic”, Sabac, Serbia
1991 – 1998 Elementary school, Vladimirci, Serbia
Appendix
A
Appendix
Figure A1. H-NMR spectra of 7-(4-nitrophenoxy)-3-ethyl-carboxy-coumarin
Appendix
B
Figure A2. H-NMR spectra of 7-(4-aminophenoxy)-3-carboxy-coumarin