IMMOBILIZATION OF ENZYMES ONTO NANOPOROUS.pdf

IMMOBILIZATION OF ENZYMES ONTO NANOPOROUS

MATERIALS AND APPLICATION AS IMMOBILIZED

BIOCATALYST

MALIK JAMAL J

(B.Tech., University of Madras)

A THESIS SUBMITTED

FOR THE DEGREE OF MASTER OF ENGINEERING

DEPARTMENT OF CHEMICAL AND BIOMOLECULAR

ENGINEERING

NATIONAL UNIVERSITY OF SINGAPORE

2007

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Acknowledgements

At the outset, I thank almighty for providing me the strength and courage to

complete this thesis successfully. I wish to record my deep sense of gratitude to my

research guide, Assoc. Prof. Sibudjing Kawi and Assoc. Prof. Kus Hidajat, Department

of Chemical and Biomolecular Engineering, National University of Singapore for their

constant support and encouragement during the course of my research work. Their

guidance and suggestion was invaluable and went a long way towards the completion of

this thesis. My thanks are duly acknowledged to the department and the University for

their Support in the form of Graduate Student Tutorship.

A very special thanks goes to Song Shiwei for getting me familiarized with all

sophisticated characterization techniques. My heartfelt thanks to my friends Yang Jun,

Sun Gebiao, Yong Siek Ting, Li Peng and Dr.M.Selvaraj for their untiring and continued

support during my thesis work. Their timely help and friendship shall always be

remembered.

My heartfelt thanks to Mdm Jamie Siew, Mdm Chow Pek, Ms Novel Chew, Ms

Sylvia Wan, Ms Li Feng Mei and other staffs in the department who have gone out of

their way to help me with all the lab facilities and making me feel at home during my

days of M.Eng Studies. The cooperation I received from other faculty members is

gratefully acknowledged.

This thesis could not have been completed without the endless love and blessing

from my parents, family members and friends for their constant encouragement and

support to go ahead, especially during difficult times.

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Table of Contents

1 Introduction……………………………………………………………………………1

1.1 Biocatalysis...................................................................................................................1

1.2 Immobilization of Enzymes…………………………………………………………..3

1.3 Research Objectives…………………………………………………………………..5

1.4 Organization of Thesis………………………………………………………………..6

2 Literature Review……………………………………………………………………...7

2.1 Introduction…………………………………………………………………………...7

2.2 Nonaqueous Enzymology…………………………………………………………….7

2.2.1 Factors Affecting the Activity of Enzymes in Organic Media…………………..8

2.2.2 Enzyme Activation in Organic Media………………………………………….12

2.2.3 Enzyme Activation by Immobilization……………………………………………15

2.2.3.1 Enzyme Immobilization in Polymers………………………………………...15

2.2.3.2 Enzyme Immobilization in Inorganic Materials……………………………...17

2.3 Enzyme Immobilization in Mesoporous Silicate for Use in Aqueous Media……….19

2.4 Conclusions………………………………………………………………………….21

3 Synthesis of Mesoporous Silicate……………………………………………………22

3.1 Introduction………………………………………………………………………….22

3.2 Experimental Methods………………………………………………………………23

3.2.1 Materials………………………………………………………………………..23

3.2.2 Synthesis of Pure SBA-15……………………………………………………...23

3.2.3 Synthesis of Thiol Functionalized SBA-15…………………………………….23

3.2.4 Synthesis of Pure SBA-15 with Rod-like Morphology………………………...24

3.2.5 Characterization of Materials…………………………………………………..24

3.3 Results and Discussion………………………………………………………………25

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3.3.1 Characterization of Pure and Thiol Functionalized SBA-15…………………...25

3.3.2 Characterization of SBA-15 with Rod-Like Morphology……………………...28

3.4 Conclusions………………………………………………………………………….30

4 Immobilization of α-chymotrypsin into Mesoporous Silicate and Its

Activity in Aqueous or Organic Media……………………………………………….31

4.1 Introduction………………………………………………………………………….31

4.2 Materials and Methods………………………………………………………………33

4.2.1 Materials………………………………………………………………………..33

4.2.2 Model Reaction for Activity Studies in Aqueous and Organic Media…………33

4.2.3 Enzyme Immobilization for Activity Studies in Aqueous or Organic Media….35

4.2.4 Thermal Stability and Leaching Studies in Aqueous Media…………………...37


4.3.1 Immobilization of α-chymotrypsin into Mesoporous Silicate and Commercial Silica Gel……………………………………………………………….38 4.3.2 Activity of Native and Immobilized α-chymotrypsin in Aqueous Media……...39

4.3.3 Thermal Stability of Native and Immobilized α-chymotrypsin in Aqueous Media……………………………………………………………………40 4.3.4 Leaching of α-chymotrypsin form Pure SBA-15 and Thiol Functionalized SBA-15………………………………………………………………41

4.3.5 Activity of Immobilized α-chymotrypsin in Organic Media…………………..42

4.4 Conclusions………………………………………………………………………….44

5 Effects of Enzyme Loading and Thermodynamic Water Activity………………..45

5.1 Introduction………………………………………………………………………….45

5.2 Materials and Methods………………………………………………………………46

5.2.1 Materials………………………………………………………………………..46

5.2.2 Activity of Immobilized α-chymotrypsin in Acetonitrile and Tetrahydrofuran…………………………………………………….46 5.2.3 Immobilization of α-chymotrypsin……………………………………………..47

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5.3.1 Effect of Enzyme Loading on Activity in Acetonitrile and Tetrahydrofuran….50

5.3.2 Effect of Thermodynamic Water Activity……………………………………...53

5.4 Conclusions………………………………………………………………………….55

6 Conclusions and Future Research…………………………………………………..56

6.1 Conclusions………………………………………………………………………….56

6.2 Future Research……………………………………………………………………...58

References………………………………………………………………………………59

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Abstract

Mesoporous silicate (purely siliceous SBA-15, thiol functionalized SBA-15 and rod-like

SBA-15) have been utilized as hosts for the immobilization of α-chymotrypsin and the

activities of the immobilized enzymes in aqueous and organic media have been

investigated. The activity of α-chymotrypsin in aqueous media decreased upon

immobilization in pure and thiol functionalized SBA-15 but immobilized α-

chymotrypsin showed enhanced thermal stability at high temperature (70oC) compared to

native α-chymotrypsin. Interestingly, α-chymotrypsin immobilized in pure and thiol

functionalized SBA-15 showed minimal leaching from the support in aqueous buffer due

to the strong electrostatic interaction between positively charged enzyme and negatively

charged mesoporous silicate. α-chymotrypsin immobilized in pure SBA-15 and thiol

functionalized SBA-15 showed higher activity in organic media compared to α-

chymotrypsin immobilized in the commercial silica gel at the thermodynamic water

activity of 0.22. It is postulated that the higher activity of α-chymotrypsin immobilized in

mesoporous silicate in organic media as compared with that on commercial silica gel is

due to the smaller particle size of mesoporous silicate, which reduces the internal mass

transfer limitation and hence increases the activity of the immobilized enzyme.

Furthermore, α-chymotrypsin loading of 20 wt % in rod-like SBA-15 showed higher

catalytic activity compared to other enzyme loading amount as well as α-chymotrypsin

immobilized onto pure SBA-15 and commercial silica gel at the thermodynamic water

activity of 0.22. The optimum thermodynamic water activity of α-chymotrypsin

immobilized in rod like SBA-15 was found to be 0.55 in either acetonitrile or

tetrahydrofuran.

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List of Tables

3.1 Textural parameters of pure SBA-15, thiol functionalized SBA-15, rod-like SBA-15 and commercial silica gel…………………..………………………………29

4.1 Activity of immobilized α -chymotrypsin in dry octane, tetrahydrofuran or acetonitrile at thermodynamic water activity of 0.22………..….…………………...42

5.1 Water content required to attain selected water activities in 1-propanol, acetonitrile and tetrahydrofuran (Halling, 2002)………………….………………..47

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List of Figures

3.1 X-ray diffraction spectra of pure SBA-15, rod-like SBA-15 and thiol functionalized SBA-15…………………..…………………………………………..27 3.2 N2 adsorption/desorption isotherm of the pure SBA-15 and thiol functionalized SBA-15………………………………..……………………………………………..28 3.3 Pore size distribution curve of Pure and thiol functionalized SBA-15……………...29

3.4 N2 adsorption/desorption isotherm of the rod-like SBA-15…………………………30

3.5 Pore size distribution curve of rod-like SBA-15…………………………………….30

3.6 FESEM images of Pure SBA-15 and Rod Like SBA-15……………………………31

4.1 Reaction scheme for the hydrolysis of N-benzoyl-L-tyrosine ethyl ester…………...33

4.2 Reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester…………………….……………………………………………………...34 4.3 Procedure for the immobilization of α-chymotrypsin for carrying out the reaction in dry octane, acetonitrile or tetrahydrofuran at thermodynamic water activity of 0.22……………………………………………………………………………….37 4.4 Thermal stability of native and immobilized α-chymotrypsin in aqueous media…...41

4.5 Leaching of α-chymotrypsin from pure and thiol functionalized SBA-15………….42

5.1 Reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester in acetonitrile and tetrahydrofuran……………………..………………..47 5.2 Procedure for the immobilization of α-chymotrypsin for carrying out the reaction in either acetonitrile or tetrahydrofuran……..……………………………..49 5.3 Effect of enzyme loading on the activity of α-chymotrypsin immobilized in pure SBA-15 and rod-like SBA-15 in either acetonitrile or tetrahydrofuran at aw of 0.22………..…………………………………………………………………...50 5.4 Effect of enzyme loading on the activity of α-chymotrypsin immobilized in commercial silica gel in either acetonitrile or tetrahydrofuran at aw of 0.22………………………………………………………………………..…...51 5.5 Schematic representation of the effect of enzyme loading in porous support………52

5.6 Effect of thermodynamic water activity on the activity of α-chymotrypsin immobilized in rod-like SBA-15 in either acetonitrile or tetrahydrofuran…..……...53

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List of Abbreviation

Ǻ angstrom

○C Degree Centigrade

FESEM Field Emission Scanning Electron Microscopy

XRD X-Ray Diffraction

HPLC High Performance Liquid Chromatography

aw Thermodynamic Water Activity

Pure SBA-15 Unfunctionalized SBA-15

SH-SBA-15 Thiol functionalized SBA-15

Rod-like SBA-15 SBA-15 with rod like morphology

TEOS Tetraethylorthosilicate

MPTMS (3-mercaptopropyl) trimethoxy silane

HCl Hydrochloric Acid

KCl Potassium Chloride

v Volume

wt Weight

APEE N-acetyl-L-phenylalanine ethyl ester

BTEE N-benzoyl-L-tyrosine ethyl ester

CaCl2 Calcium Chloride

Chapter 1 Introduction

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Chapter 1

Introduction

1.1 Biocatalysis

Biocatalysis is an important tool for the production of pharmaceutical and fine chemicals

because of its high selectivity and specificity, mild reaction temperature, neutral or

almost neutral pH. Moreover, it offers less pollution, hazardous and energy consumption

compared to conventional metal based catalyst. Biocatalysis can also provide new routes

for synthesis of high value compounds that cannot be produced by conventional metal

based catalyst. The total number of biotransformation process carried out on an industrial

scale increased tremendously during the past decade (Straathof et al., 2002). It is

expected that there will be a huge growth on the application of enzymes in large scale

production of fine chemicals (Schmid et al., 2002).

If the reaction proceeds slowly under given temperature, an increase in

temperature often leads to denaturation of enzymes. Moreover, enzymes are expensive

and it often needs to be recovered and reused for economic viability. The soluble nature

of enzymes in aqueous media requires the use of laborious techniques to separate

enzyme from reaction mixture. However, these limitations can be overcome by

immobilizing the enzymes.

Immobilization of enzymes onto the solid support offers the most inexpensive

way of removing enzymes from reaction media. Furthermore, it imparts some special

features to biocatalyst such as improved operational and thermal stability, as well as


2

stability against solvent induced denaturation. Even though it provides the advantages

mentioned above, the major disadvantages upon immobilization are the loss of enzyme

activity, diffusional limitation and increased cost. Despite of these disadvantages,

immobilized enzymes are often used in pharmaceutical, food and chemical industries

(KatchalskiKatzir, 1993).

The most economical and preferred way to carry out biotransformation is to use

aqueous media. However, most of the organic compounds are not soluble in water

thereby requiring the use of organic solvent as the reaction medium. The use of organic

solvent is not recommended for synthesis due to environmental concern, however it still

serves as the best choice compared to the reaction performed in water due to the

following reasons (Dordick, 1989):

� Increased solubility of substrate since most of the organic compounds are sparingly soluble in water

� Prevents undesirable side reaction e.g. hydrolysis

� Shifts thermodynamic equilibrium to favor synthesis over hydrolysis e.g. ester synthesis

� Easier recovery of enzymes from organic media due to its insolubility

� Enhances thermal stability

� Eliminates microbial contamination

� Alters the specificity of enzyme

Enzymes in organic media possess very low activity due to diffusional limitation.

However, this limitation can be overcome by immobilizing the enzymes onto solid

support. Various factors which can affect the activity of enzymes in organic media are


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discussed in Chapter 2. It should be noted that, in the case of aqueous media, enzyme is

immobilized in order to recycle the enzyme, but in the case of organic media enzyme is

immobilized in order to suppress the diffusional limitation which leads to increased

activity.

1.2 Immobilization of Enzymes

Immobilization is the process of arresting the mobility of enzyme. Numerous methods

are available in the literature for immobilization of enzymes and each method has its own

efficiency and complexity. Various methods used till date for immobilization of enzymes

can be divided into two main categories namely, covalent method and non-covalent

method. Covalent method involves the formation of covalent linkage between amino

acid side chain residues of protein with functional group on the support. The latter

utilizes week forces such as ionic, hydrogen and van der Walls interaction. Enzyme

entrapment in the polymer matrix, sol-gel encapsulation, adsorption of enzymes on the

surface and porous materials are some of the examples of non-covalent method of

immobilization. Selection of a particular method depends on the requirements to be met

and the efficiency of the selected method depends on the nature of the enzyme and

supports. In this study, enzyme is adsorbed onto porous support which is segregated

under non-covalent method of immobilization. In some cases, the adsorption of enzymes

onto solid support shows severe leaching due to weak interaction between the support

and enzyme. In those cases, covalent method of immobilization is preferred over non-

covalent methods.


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The supports available for the immobilization of enzymes range from simple

synthetic polymers such as polypropylene, polyamide, polystyrene to inorganic materials

such as zeolites, controlled pore glass, sol-gel encapsulation and silica gel. Each of these

materials has its own advantages and disadvantages. Inorganic materials are preferred

over polymeric materials because of its high pore volume and high surface area. One of

the most widely used methods for immobilizing enzymes is encapsulation inside sol-gel

silica. However, due to small pore sizes and partially closed pore structures, most of the

enzymes immobilized by this procedure show much lower activity compared to the

native enzymes. The invention of ordered mesoporous silicate attracted much attention

as a host for enzyme immobilization due to its tunable large pore size (25 – 300 Å), very

high surface area (~ 1200 m2 g−1), narrow pore size distribution and easier chemical

surface modification.

Porous materials are classified by International Union of Pure and Applied

Chemistry (IUPAC) into three classes such as microporous (pore size < 20 Å),

mesoporous (pore size 20 - 500 Å), and macroporous materials (pore size >500 Å)

(Taguchi and Schuth, 2005). Zeolite is a well known member of the microporous

materials but its application as a host for immobilization of enzymes is limited due to its

smaller pore size (2 - 10 Å) compared to molecular dimension of the enzymes. This

limitation can be overcome by utilizing mesoporous silicate which has the pore size

larger than the molecular dimension of enzyme (Yiu and Wright, 2005).

In 1990s, researchers at Mobil Corporation invented the first family of highly

ordered mesoporous molecular sieves M41S called MCM-41 where long chain cationic

surfactant was used as the template or pore forming agents during the hydrothermal


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solgel synthesis. This material possesses narrow pore size distribution in the range of

26 – 40 Å (Kresge et al., 1992). The early studies on the use of this material were

focused on immobilizing organometallic compounds and very few studies were carried

out in utilizing MCM-41 as a host for the immobilization of enzymes. Moreover, this

material possesses low hydrothermal stability due to thin pore walls.

In 1998, Stucky and coworkers invented another mesoporous material called

SBA-15 with non-ionic amphiphilic triblock copolymer micelles as template under

acidic reaction conditions (Zhao et al., 1998). The specific surface area and pore volumes

are somewhat smaller than that of MCM-41 but it possess thicker pore walls. Therefore

the mechanical and hydrothermal stability of these materials are found to be better. The

pore diameters can be varied between 50 – 300 Å, which is quite large enough to

accommodate macromolecules. The functionalization of SBA-15 with organic moieties

in the framework was also developed, attracting enzymologist to use this material as a

host for the immobilization of enzymes.

1.3 Research Objectives

The objective of this work is to use unfunctionalized and thiol functionalized SBA-15 as

a host for the immobilization of enzymes and to find out the activity of the immobilized

enzymes in aqueous and organic media. α-chymotrypsin is chosen as the model enzyme

for this study. Since the native enzyme possesses very low thermal stability in aqueous

media, it is of great interest to investigate whether the enzyme immobilized in

mesoporous silicate has better thermal stability compared to native enzyme. Organic

solvent is the most preferred media for biotransformation. Hence, we are interested in


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finding out the efficiency of enzyme immobilization in mesoporous silicate for

application in organic media. We also studied the effect of particle of size and α-

chymotrypsin loading on the activity in polar organic solvents. Furthermore, it is our

interest to investigate whether the particle size and enzyme loading have any effect on

the activity of enzyme because in most of studies reported in the literature, very little

attention had been paid to this parameter. Moreover, enzyme hydration is one of the

critical parameter affecting the activity of enzyme; it is also of interest to investigate how

the activity of immobilized enzyme changes with enzyme hydration.

1.4 Organization of Thesis

The rest of this thesis is divided into five chapters. Chapter 2 gives a brief introduction to

non-aqueous enzymology, various factors affecting the activity of enzymes in organic

media and the methods to increase the activity of enzymes in organic media. In Chapter

3, preparation and characterization of pure SBA-15, thiol functionalized SBA-15 and

SBA-15 with rod like morphology are described. Chapter 4 is the most important chapter

in this thesis as it describes the immobilization of α-chymotrypsin into pure SBA-15 and

thiol functionalized SBA-15 and its activity studies in aqueous or organic media. In

Chapter 5, the effects of enzyme loading and thermodynamic water activity on the

reaction rate in polar organic solvents are also discussed. Conclusions and future scope

of the work are presented in Chapter 6.

Chapter 2 Literature Review

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Chapter 2

Literature Review

2.1 Introduction

This literature review gives the brief introduction to non-aqueous enzymology, various

reasons for the low activity of enzyme in organic media as well as the ways to increase

the activity of enzymes in organic media. Enzyme immobilization is one of the methods

of increasing the activity of enzymes in organic media, which is the main objective of

this work; hence, it is discussed in details. At first, immobilization of enzymes onto

polymers for use in organic media is given followed by the enzyme immobilization in

controlled pore glass, sol-gel and mesoporous silicate. Finally, enzyme immobilization in

mesoporous silicate for use in aqueous media is also discussed.

2.2 Nonaqueous Enzymology

Biocatalysis in organic media emerged in the nineteenth century. Scientists in the early

days recognized the insolubility of organic compounds in water and tried to replace it

with organic solvents. At first water miscible organic solvents such as ethanol or acetone

were added to the bulk water medium. As long as high water content was retained, the

enzymes were found to be catalytically active. The next phase of non-aqueous

enzymology was to use the biphasic mixture in which aqueous solution of enzyme was


8

emulsified in a water immiscible organic solvent e.g. isooctane or heptane. In this

system, the substrate present in the organic phase diffused into the aqueous media

underwent reaction and the product diffused back to the organic media (Klibanov, 1986;

Krishna, 2002). The seminal work of Zaks and Klibanov (Zaks and Klibanov, 1998a,

1998b) led to the next stage of development to employ the enzymes in nearly non-

aqueous solvents. They found that the enzymes were not only catalytically active in dry

organic solvents but also possess very distinct properties such as very high thermal

stability, altered regio- enantio- and stereo selectivity compared to pure aqueous and

aqueous-organic solvent mixtures (Klibanov, 2001; Krishna, 2002; Castro and

Knubovets, 2003). However, the activity of enzymes in dry organic media was very low

compared to its activity in aqueous media (Klibanov, 2001). Michels et al. (1997)

reported that the kcat/Km (turnover frequency/specific binding constant) of the

transesterification of N-acetyl-L-phenylalanine ethyl ester in hexane and its hydrolysis in

water were 0.43 and 2800 M−1s−1 respectively. The very low activity of enzymes in

organic media was a main problem and various solutions for improving the activity of

enzymes in organic media had been suggested which ranged from simple addition of

salts during the lyophilization (Khmelnitsky et al., 1994) to the covalent incorporation of

enzymes into the polymer named ”Biocatalytic Plastics” (Wang et al., 1997).

2.2.1 Factors Affecting the Activity of Enzymes in Organic Media

Several attempts had been made to elucidate the various reasons for the decrease in the

activity of enzyme upon switching from aqueous to organic media. Dependence of

enzyme activity on its three dimensional conformation is well known. The question,

which first comes to our mind, is whether the enzyme maintains its native fold upon


9

placing in organic media. X-ray crystal structure of α-chymotrypsin soaked in hexane

was found to be identical to the aqueous solution structure, which indicated that the

conformational changes did not, occurred upon placing the α-chymotrypsin in organic

media (Yennawar et al., 1994). Schmitke et al. (1997) studied the X-ray crystal structure

of another widely studied enzyme subtilisin carlsberg soaked in acetonitrile or dioxane

and compared it with aqueous solution structure which also confirmed that the

conformational changes did not occurred upon soaking the crystal in organic media.

Moreover, active site structure also found to be similar in all the media studied.

Molecular dynamics simulation of enzymes in organic solvents also provided the

evidence that the enzyme maintained its native fold in organic solvents (Soares et al.,

2003; Yang et al., 2004). All these studies eliminated the dogma that the diminished

activity of enzymes upon switching from aqueous to organic media was not due to

conformational changes in enzymes.

Fourier transform infrared (FTIR) spectroscopy is the most powerful technique

for studying the secondary structure of lyophilized enzyme powders, enzymes

suspensions in organic solvents and enzyme adsorbed onto solid surfaces (Griebenow et

al., 1999). FTIR spectroscopic studies on several lyophilized enzyme powder had shown

that lyophilization can cause severe structural damage to the enzyme but reversible upon

dissolving in aqueous media (Griebenow and Klibanov, 1995). However, the secondary

structure of the lyophilized enzyme powder suspended in several organic solvents

remained unchanged between the solvents and to the lyophilized enzyme powder

(Griebenow and Klibanov, 1997). Recently, solid-state nuclear magnetic resonance

spectroscopic study of lyophilized papain also confirmed that enzyme underwent

conformational changes during lyophilization and irreversible upon placing in organic


10

media (Matsubara et al., 2006). Thus, lyophilization induced conformational changes in

the enzymes which was irreversible upon placing in organic media was suggested as one

of the reason for the drastic decrease in the activity of enzymes upon switching from

aqueous to organic media. Various methods of preventing the lyophilization induced

structural changes in enzymes are discussed in Section 2.2.2.

Enzymes found to be precipitate upon placing in organic media (with an

exception to dimethyl sulfoxide, formamide) in contrast to the aqueous media in which

the enzyme dissolves (Chin et al., 1994). Zaks and Klibanov, (1998b) reported that the

decrease in the activity of enzymes in organic media was not due to the diffusional

limitation because the increase in the shaking rate from 160 to 300 rpm or ultrasonication

which reduced the particle size from 270 to 5 µm did not increase the activity of enzyme

in organic media. Rees and Halling (2001) studied the acetylation of lyophilized and

immobilized myoglobin powder in organic media by electron spray-mass spectrometry.

The rate of acetylation was higher on immobilized myoglobin compared to lyophilized

myoglobin powder. They concluded that, the adsorption of myoglobin onto the solid

surfaces spread the myoglobin over the large surface area and thereby eliminates the

mass transfer limitation that resulted in higher affinity for acetylation. However,

lyophilized myoglobin power had suffered severe mass transfer limitation, which

hindered the acetylation process. Persson et al. (2002a) showed that the specific activity

of lipase immobilized in polypropylene was 770 times higher compared to lyophilized

lipase powder and they interpreted that the higher activity of lipase upon immobilization

was due to reduced mass transfer limitation. All these results clearly showed the

advantages of employing immobilized enzymes in organic media, which was the main

aim of this work.


11

From the origin of non-aqueous enzymology, attention had been paid to the

availability of water around the enzyme molecule. Water in close proximity to the

protein surface is fundamental to protein folding, stability, recognition, and activity

(Phillips and Pettitt, 1995). Moreover, water also acts as a molecular lubricant, which

facilitates the conformational flexibility necessary for the enzyme to perform catalysis

(Klibanov, 1997). Hence, the completely dehydrated enzyme remains inactive due to the

reduced flexibility and it is always necessary to have a minimum amount of water

molecule in the enzyme in order to have a flexibility and thereby the activity. However,

the minimum number of water molecules necessary for the enzyme to possess the

catalytic activity found to be dependent on enzyme. For example, α -chymotrypsin

suspended in octane possess catalytic activity with only 50 molecules of water per

enzyme molecule but polyphenol oxidase requires about 3.5 ×107 molecules of water per

enzyme molecule to possess the activity in chloroform (Dordick, 1989). It was found that

the catalytic activity of the enzymes in organic media depend on the water molecules

associated with an enzyme and did not depend on the water content in the system (Zaks

and Klibanov, 1998a). In order to quantify the amount of water associated with an

enzyme molecule, thermodynamic water activity was suggested as the useful parameter

(Halling, 1994) because if the water equilibrated between the various phases present,

they would all come to the same water activity. The enzyme molecule will tend to

equilibrate in this way; hence, the quantity of water associated with the enzyme molecule

will reflect the system water activity (Halling, 2004).

Apart from decrease in the activity of enzyme upon transition from aqueous to

organic media, it is interesting to note that the activity changes over 10000 fold within

the organic media itself and the activity is higher in nonpolar solvents compared to polar


12

solvents (Zaks and Klibanov, 1998b). Removal of essential water molecules from the

enzyme surface (water striping) by polar solvents was suggested as one of the reason for

the low activity of enzymes in polar solvents. Because, when the enzyme was assayed in

dry polar solvent, the solvent can strip the essential water molecule present in the

enzyme surface and thereby lock the enzyme conformation, which led to decreases

enzyme flexibility and activity. In the case of nonpolar solvent, water stripping will be

minimum due to its low solubility in water, which results in low dehydration of enzymes,

hence high activity (Zaks and Klibanov, 1998b; Wangikar et al., 1997; Klibanov, 2001).

The water stripping by polar solvents can be clearly visualized in the molecular

dynamics simulation by Yang et al. (2004). However, the water stripping in polar

solvents could be eliminated by carrying out the reaction at constant thermodynamic

water activity. This is because, in a non-controlled system (phases with uneven

thermodynamic water activity or water content), water molecules tend to transfer

between various phases until they all reach the same water activity. However, if the

reaction is initiated at constant thermodynamic water activity, then the transfer of water

molecules between the phases will not take place and thereby the enzyme dehydration

will be prevented (Partridge et al., 1998a; Halling, 2002).

2.2.2 Enzyme Activation in Organic Media

There are several methods reported in the literature in order to improve the activity of

enzymes in organic media. Some methods prevent the conformational changes in

enzyme during lyophilization and there by increases the activity, and other methods are

specific and do not involve the lyophilization. The enzymes in organic media show pH

memory i.e. its activity in organic media depends on the pH to which it was finally


13

exposed and it was found to be maximum when it is lyophilized from the optimum pH

for the catalytic activity in aqueous media. For example, transesterification activity of

subtilisin Carlsberg increased to about 75 times when it was lyophilized from the

optimum pH compared to subtilisin Carlsberg as received from the company (Zaks and

Klibanov, 1998b). Hence, the enzyme as received from the company serves as the poor

control for comparing the activity of enzymes in organic media and it is a good practice

to obtain enzyme from optimum pH for use in organic media. Through out this literature

survey, lyophilized enzyme refers to relyophilized enzyme from optimum pH unless

otherwise stated.

Khmelnitsky et al. (1994) found that the lyophilization of subtilisin Carlsberg in

the presence of 98 wt % potassium chloride enhanced the activity to about 4000 fold in

hexane compared to lyophilized enzyme powder. By optimizing the freeze-drying time,

water and salt content, the transesterification activity of subtilisin Carlsberg in hexane

increased to about 20000 fold relative to lyophilized enzyme powder (Ru et al., 1999).

The prevention of lyophilization induced conformational changes and the role of salt

matrix as the immobilization support suggested as the reason for the increased activity of

enzyme upon addition of salt during lyophilization (Griebenow and Klibanov, 1997).

However, Laszlo and Compton, (2001) showed that α-chymotrypsin lyophilized in the

presence of potassium chloride possessed negligible activity in acetonitrile and they

concluded that the salt activation was effective only in nonpolar organic solvents.

Not only salt but also addition of crown ether during lyophilization increases the

activity of enzymes in organic media. Engbersen et al. (1996) found that the addition of

crown ethers increases the transesterification activity of α-chymotrypsin to about 640


14

fold relative to the lyophilized α-chymotrypsin. Unen et al. (2002) reported the crown

ether activation of enzymes was due to the macrocyclic interaction of crown ethers with

enzyme (lysine ammonium groups) which reduced the formation of inter and

intramolecular salt bridges and hence enabled the enzyme to refold into more active

conformation as well as preservation of enzymes during lyophilization. Tremblay et al.

(2005) found that the crown ether modified peptide shows higher activity compared to

native crown ether.

Several other methods such as addition of sorbitol (Debulis and Klibanov, 1993),

methyl cyclodextrin (Santos et al., 1999; Griebenow et al., 1999; Montanez et al., 2002),

urea (Guo and Clark, 2001) and Poly (ethylene glycol) (Debulis and Klibanov, 1993;

Kwon et al., 1999 (1999); Castillo et al., 2006) during lyophilization had also been

reported to increase the activity of enzymes in organic media.

Cross-linked enzyme crystal (CLEC) serves as the most robust biocatalyst in

either aqueous or organic media. Although there were some successful applications of

CLEC in organic media (Khalaf et al., 1996 ; Wang et al. (1997)), immobilization of

subtilisin carlsberg onto silica gel followed by rinsing with propanol (Patridge et al.,

1998b) and lyophilization of subtilisin carlsberg in presence of methyl cyclodextrin

shows higher activity in organic solvents compared to subtilisin CLEC. Moreover, CLEC

is not available for some enzyme such as chymotrypsin, papin and the advantages of

using CLEC compared to other methods of enzyme preparation for use in organic media

remains unclear.

Cross-linked enzyme aggregates (CLEA) (Cao et al., 2000), protein-coated micro

crystal (PCMC) (Kreiner et al., 2001), enzyme precipitated and rinsed with propanol


15

(Roy and Gupta, 2004), three-phase partitioning (Roy et al., 2004) were some of the

other methods available in the literature which increases the enzyme activity in organic

solvents compared to lyophilized enzyme powders.

2.2.3 Enzyme Activation by Immobilization

Immobilized enzyme employed in organic media from the origin of non-aqueous

enzymology (Dordick, 1989). Organic and inorganic materials were used as an

immobilization support for use in organic media. In all cases, the immobilized enzymes

possess higher activity in organic media compared to lyophilized enzyme powder due to

very low diffusional limitation. In this section, enzyme immobilization in polymer was

discussed first followed by enzyme immobilization in controlled pore glass, sol-gel and

mesoporous silicate for application in organic media.

2.2.3.1 Enzyme Immobilization in Polymers

Barros et al. (1998) showed that the α-chymotrypsin immobilized in polyamide not only

showed low activity for peptide synthesis but also possessed very low enzyme loading

(5-10 mg g−1 of support) compared to controlled pore glass (100 mg g−1 of support).

Persson et al. (2002a) reported that lipase adsorbed onto polypropylene possessed higher

activity compared to lyophilized lipase. Jia et al. (2002) immobilized α -chymotrypsin in

polystyrene nanofiber, which showed higher activity in organic media compared to

native α-chymotrypsin, but they did not state whether the native α-chymotrypsin used

was relyophilized or used as received from Sigma. Not only native polymers,

functionalized polymers have also been used for the immobilization of enzymes for use


16

in organic media which showed higher activity compared to lyophilized enzyme powder

(Markvicheva et al., 2005; Bacheva et al., 2005).

Apart from the enzyme adsorption onto polymers, covalent incorporation of

enzymes into the polymeric materials was also carried out to improve the activity of

enzymes in organic media (Ito et al., 1993; Yang et al., 1995a; Yang et al., 1995b). Wang

et al. (1997) incorporated acryloyl chloride modified α-chymotrypsin into the various

polymers such as poly vinylalcohol, poly (methyl methacrylate), poly (styrene) and

studied its activity in the transesterification of N-acetyl-L-phenylalanine ethyl ester with

propanol in hexane and toluene. They observed not only less diffusional limitation even

at the enzyme loading of 9.6 wt % but also high activity. Kim et al. (2001) used flash

devolatilization to incorporate α-chymotrypsin into the polymer and subsequent cross-

linking resulted in the enzyme with the high stability and activity relative to lyophilized

α-chymotrypsin. However, they observed severe leaching of enzyme while cross-linking

and the final enzyme loading was only 2 wt %. Enzymes incorporated in room-

temperature vulcanizable silicone composite had also showed high activity and stability

when the reaction was carried out in non-polar solvents saturated with buffer (Gill et al.,

1999; Ragheb et al., 2003).

Although, the covalent incorporation of enzymes into the polymeric support has

showed higher activity in organic media, it is not clear in what way it is a more

advantageous to use this method than simple enzyme adsorption which also has showed

higher activity in organic media (Halling, 2002)


17

2.2.3.2 Enzyme Immobilization in Inorganic Materials

Most of the studies in the immobilization of enzymes into inorganic materials use

celite, controlled pore glass, sol-gel glass, and silica gel. Barros et al. (1998) showed that

the activity and enzyme loading of α-chymotrypsin immobilized in controlled pore glass

was higher compare to polyamide and celite. However, lipase adsorbed onto

polypropylene shows higher activity compared to celite (Persson et al., 2000). Reetz et

al. (1996) showed that the lipase immobilized in sol-gel possessed 157 fold higher

activity in isooctane compared to lyophilized enzyme powder. Unen et al. (2001)

immobilized α-chymotrypsin, subtilisin Carlsberg and trypsin into sol-gel glass, which

also showed higher activity in non-polar solvents at the thermodynamic water activity of

0.7 compared to lyophilized enzyme powder. However, Persson et al., (2002b) found that

lipase adsorbed onto polypropylene powder showed higher activity (400 fold) compared

to crude lipase but sol-gel entrapment was also showed 340 fold higher activity

compared to crude lipase. However, the other form of enzyme preparation such as salt

and crown ether activation showed lower activity compared to lipase adsorbed in

polypropylene but higher than the lyophilized lipase.

Mesoporous silicate is a unique material for the immobilization of enzymes due

to its large specific surface area, tunable pore size, narrow pore size distribution, and

easier chemical modification. However, to the best of our knowledge very few studies

were carried out in the application of enzyme immobilized in mesoporous silicate and its

use in organic media.


18

Takahashi et al. (2000) was the first who evaluated the activity of enzyme in

organic media upon immobilization in mesoporous silicate. They immobilized

horseradish peroxidase in FSM-16, MCM-41 and SBA-15 of different pore size and

compared its activity with horseradish peroxidase immobilized in commercial silica gel

and lyophilized horseradish peroxidase as received from company. The adsorption of

horseradish peroxidase were found to be high when the material pore size was larger then

the molecular dimension of horseradish peroxidase, FSM-16 and MCM-41 were found to

possess highest enzyme loading of 183 and 147 mg g-1 respectively. The activities of

horseradish peroxidase immobilized in FSM-16 and MCM-41 were higher when pore

size of the support just matches with the molecular dimension of the horseradish

peroxidase. However, SBA-15 and silica gel possess not only possess low enzyme

loading (24 and 49 mg g−1 respectively) but also low activities. Since the activity of the

immobilized enzyme depends on the enzyme loading (Wehtje et al., 1993; Day and

Legge (1995); Barros et al., 1998), it is not clear to us that the low activity of

horseradish peroxidase immobilized in silica gel and SBA-15 was due to the low enzyme

loading or intrinsic nature of the interaction between enzyme and support. Moreover, the

reaction was not performed at constant thermodynamic water activity, which also can

lead to misleading conclusion about the effect of support.

Deere et al. (2003) reported the catalytic activity of cytochrome c in organic

media upon immobilization in commercial kieselgel silica and MCM-41 of pore size 28

and 45 Ǻ respectively. The activity was higher in methanol, ethanol and formamide

(containing 2.5% v/v water) but lower in 1-methoxy 2-propanol compared to lyophilized

cytochrome C powder. Wang et al., (2001) found that immobilization of α-chymotrypsin


19

onto mesoporous silicate showed higher activity compared to lyophilized enzyme

powder.

As the enzyme hydration is the critical parameter affecting the activity in organic

media to about 100 fold (Klibanov, 1997), none of these studies carried out the reaction

at constant thermodynamic water activity which is of paramount importance in order to

observed the true nature of support (Adlercretuz, 1991). Moreover, none of the studies in

mesoporous silicate had paid attention to the internal mass transfer limitation commonly

occurs in porous support which can also greatly affect the performance of the enzyme.

This inspired us to study the importance of mesoporous silicate a host for the

immobilization of enzymes for catalysis in organic media.

2.3 Enzyme Immobilization in Mesoporous Silicate for Use in

Aqueous Media

Application of mesoporous silicate as a host for the immobilization of enzymes

commence in 1996. Initial attempt on the immobilization of enzymes onto mesoporous

silicate showed that the loading efficiency of the enzyme depend on its molecular size as

well as solution pH and the immobilized enzyme was also possessed high storage

stability compared to native enzyme (Diaz and Balkus, 1996). He et al. (2000) showed

that the incorporation of aluminium in the framework of MCM-41 not only enhanced the

activity of penicillin acylase but also prevented enzyme leaching from the support. Lei et

al. (2002) found that the activity of organophosphorus hydrolase immobilized on

carboxylic acid functionalized SBA-15 showed higher activity and enzyme loading

compared to amine functionalized SBA-15. They rationalized that positive charge on

amine functionalized SBA-15 and in the enzyme caused repulsion in between them

which leads to lower enzyme loading, however the negative charge on acid


20

functionalized SBA-15 with positive charge on enzyme increased the interaction between

support and enzyme, which led to higher enzyme loading. The study on the effect of pore

size on the activity of trypsin by Yiu et al. (2001b) showed the activity increases with

increase in pore size, however the trypsin adsorbed in unfunctionalized mesoporous

silicate shows severe leaching. The same group latter found that the surface

functionalization can not only prevents the enzyme leaching but also influences the

enzyme activity (Yiu et al., 2001). From these studies, we can infer that the presence of

functional group in the framework of mesoporous silicate not only prevents the enzyme

leaching but also induces conformational changes in enzyme and thereby affecting the

activity. Pandya et al. (2005) found that α- amylase immobilized in MCM-41 and SBA-

15 in which the enzyme adsorbed only on the outer surface showed low specific activity

compared to meso-cellular foams (MCF) in which the enzyme seemed to be immobilized

inside the pores and exhibit high specific activity.

It is also interesting to note that horseradish peroxidase immobilized in

mesoporous silicate possesses high thermal stability compared to native enzyme when

the support pore size matches with the molecular dimension of enzyme (Takahashi et al.,

2000). Ravindra et al., 2004 found that the melting point of ribonuclease A increased

dramatically (∆Tm ~ 30○C) compared to native enzyme upon immobilization in

mesoporous silicate. They concluded that the increase in thermal stability of ribonuclease

A upon immobilization was not only due to excluded volume effect but may also due to

an increased strength of the protein in the narrow pore channel. This study confirmed

that enzyme immobilization in mesoporous silicate not only provides the easier way to

recover enzymes from aqueous media but also increases the thermal stability

dramatically. Fan et al. (2003) invented that the rate of immobilization was found to be


21

varied depending on the morphology of mesoporous silicate; however, activity was not

reported in their study. To the best of our knowledge, none of the studies had considered

the effect of particle size on the activity of enzyme.

2.4 Conclusions

Mesoporous silicate was found to be the excellent host for the immobilization of proteins

but little attention had been paid in the use of mesoporous material as a host for the

immobilization of enzymes for application in organic solvents. Although some have

reported, the activities of enzymes in organic media upon, none of the studies considered

the enzyme hydration, which can greatly affect the activity of enzymes in organic media.

In this study, we utilized mesoporous silicate as a host for the immobilization of enzymes

and found out its activity in aqueous and organic media taking enzyme hydration into

account.

Chapter 3 Synthesis of Mesoporous Silicate

22

Chapter 3

Synthesis of Mesoporous Silicate

3.1 Introduction

Porous materials have been used as adsorbents and catalyst supports for many decades

(Corma, 1997). Most of them are based on zeolites which possess pore sizes in the range

of 2- 10 Ǻ. However, they are not suitable for the immobilization of enzymes due to their

smaller pore sizes as the molecular dimension of enzymes are several angstroms larger

than the pore size of zeolites. Hence, zeolites receive less attention as supports for the

immobilization of enzymes. The discovery of highly ordered mesoporous silica materials

(M41S) has opened the way to use inorganic material as a support for the immobilization

of enzymes due to its tunable larger pore size (40-300 Å), narrow pore size distribution,

high specific surface area (~ 1000 m2 g-1 ), highly ordered pore structure and easier

chemical surface modification.

This chapter describes the preparation of mesoporous silicate unfunctionalized

SBA-15 (pure SBA-15), thiol functionalized SBA-15 (SH-SBA-15) as well as pure

SBA-15 with rod-like morphology (rod-like SBA-15). The prepared material was well

characterized by various techniques. α-chymotrypsin immobilized onto commercial silica

gel was used as a control for comparing the activity of α-chymotrypsin immobilized onto

mesoporous silicate in organic media (Chapter 4 and 5). Hence, surface area, pore


23

volume and particle size of commercial silica gel obtained from Sigma were given in this

chapter.

3.2 Experimental Methods

3.2.1 Materials

Silica source tetraethylorthosilicate (TEOS) (98 %) and poly (ethylene glycol)-block-

poly (propylene glycol)-block-poly (ethylene glycol) (Pluronic P123) were purchased

from Aldrich. Hydrochloric acid (HCl) (37%) and potassium chloride (KCl) were

purchased from Merck. (3-mercaptopropyl)trimethoxy silane (MPTMS) (95%) was

purchased from Alfa Aesar. Commercial silica gel was purchased from Sigma.

3.2.2 Synthesis of Pure SBA-15

Pure SBA-15 was prepared according to the procedure reported in the literature (Zhao et

al., 1998). The molar composition of the gel was 1 mole TEOS: 0.017 mole pluronic

P123:2.9 mole HCl: 202.6 mole water. In a brief protocol, about 11.4 g of hydrochloric

acid was added to the solution of 4 g of pluronic P123 mixed with 138 g of water. Then

8.4 g of TEOS was added and the resulting solution was stirred for about 24 hours at

40○C. The slurry was then transferred to polypropylene bottle and heated at 100○C for

about 48 hours. The solid was collected by filtration and dried at 60○C. Surfactant was

removed by calcination at 550○C for about 8 hours.

3.2.3 Synthesis of Thiol Functionalized SBA-15

Synthesis of thiol functionalized SBA-15 was carried out by the method similar to the

synthesis of pure SBA-15 except that MPTMS was introduced along with TEOS at


24

MPTMS/TEOS ratio of 95:5 mol %. The surfactant was removed by refluxing with

ethanol for about 24 hours.

3.2.4 Synthesis of Pure SBA-15 with Rod-like Morphology

Pure SBA-15 with rod-like morphology was prepared according to the procedure

reported in the literature (Yu et al., 2002). The molar composition of the gel was 1mole

TEOS: 0.02 mole pluronic P123: 1.5 mole KCl: 6 mole HCl: 166 mole water. In a brief

protocol, 2.3 g of pluronic P123 and 2.2 g of potassium chloride was dissolved in 59.8 g

of water containing 4.4 g of hydrochloric acid at 38±1○C. Then 4.2 g of TEOS was added

and the resulting solution was stirred vigorously for about 8 min and kept at the same

temperature under static condition for 24 hours. The slurry was then maintained at 100○C

for 24 hours. The solid was collected by filtration and dried at room temperature.

Surfactant was removed by calcination at 550○C for about 8 hours.

3.2.5 Characterization of Materials

X-ray diffraction (XRD) is a well established method for the identification of ordered

phases. This is because the wavelength of X-rays is comparable to the size of the atoms.

Hence it is ideally suitable for probing the structural arrangement of atoms and

molecules in a wide range of materials. XRD patterns of the prepared materials were

obtained using Shimadzu XRD-6000 with CuKα radiation. The X-ray tube was operated

at 40 kV and 30 mA and a continuous 2θ scan was performed. Since the walls of

mesoporous materials are amorphous and only the arrangement of the pores induces the

regularity in the solid, hence the reflections in powder XRD were only obtained in the 2θ

range between 0.5 to 10○.


25

The specific surface area, pore diameter and pore volume of the pure SBA-15,

SH-SBA-15 and pure SBA-15 with rod-like morphology were analyzed by N2

adsorption/ desorption measurements using Quantachrome Autosorb-1 at 77 K. Before

subjecting to the analysis, about 50 - 100 mg of sample was pelletized and degassed at

170○C for pure SBA-15 and SBA-15 with rod-like morphology for about 6 hours. SH-

SBA-15 was degassed at 80○C for about 6 hours. The specific surface area of the

samples were calculated in the relative pressure (P/P0) range of = 0.05 - 0.35. Pore size

distribution was obtained from the adsorption branch of the isotherm using Barrett-

Joyner-Halenda method. The total pore volume was determined from the adsorption

branch of the N2 isotherm at the relative pressure of P/P0 = 0.95.

Particle size distributions were measured using Coulter particle size analyzer.

Sulfur content in thiol-functionalized SBA-15 was determined from Perkin-Elmer Series

II CHNS Analyzer 2400. Field-emission scanning electron microscopy (FESEM) images

of pure SBA-15 and rod-like SBA-15 were obtained using JEOL JSM-6700F

microscope.

3.3 Results and Discussion

3.3.1 Characterization of Pure and Thiol Functionalized SBA- 15

X-ray diffraction pattern of pure SBA-15 and SH-SBA-15 were given in Figure 3.1. The

spectra clearly shows three well resolved peaks which can be assigned to (100), (110)

and (200) reflections associated with 2D hexagonal space group (P6mm) consistent with


26

the spectra reported in Zhao et al., (1998). The lower intensity of (110) and (200)

reflections in the spectra of SH-SBA-15 indicates the decrease in the ordered structure of

this sample due to the addition of 5 mol % functionalization agent (MPTMS) along with

the silica source. The decrease in the ordered structure upon functionalization is also

observed by Hodgkins et al., (2005).

0

5000

10000

15000

20000

25000

30000

35000

0 2 4 6 8

100

100

100

200

Pure SBA-15

Rod-like SBA-15

SH-SBA-15

110

Inte

nsit

y (

Arb

itary

Un

its)

2θ (Degree)

Figure 3.1: X-ray diffraction spectra of pure SBA-15, rod-like SBA-15 and thiol functionalized SBA-15

Figure 3.2 shows the N2 adsorption/desorption isotherms of the pure and thiol

functionalized SBA-15. Pure SBA-15 exhibits the typical type IV adsorption isotherms

where the volume of nitrogen adsorbed increases with increasing relative pressure with

sharp rise in adsorption due to capillary condensation in the mesopores. However, SH-

SBA-15 sample shows less-well defined capillary condensation step and lower porosity

due to the introduction of functionalization agent along with the silica source.


27

0

200

400

600

800

1000

1200

0 0.2 0.4 0.6 0.8 1

Relative Pressure (P/Po)

Vo

lum

e (

cc

/g)

Pure SBA-15

SH-SBA-15

Figure 3.2: N2 adsorption/desorption isotherm of the pure SBA-15 and thiol functionalized SBA-15

Figure 3.3 show the pore size distribution of pure SBA-15 and SH-SBA-15 which

indicate the synthesized material possess narrow pore size distribution which is the

hallmark of mesoporous silicate. The pore diameter, specific surface area, pore volume

and particle size of pure SBA-15 and SH-SBA-15 were given in Table 3.1. The decrease

in specific surface area, pore diameter and pore volume of SH-SBA-15 can be attributed

to the addition of 5 mol % MPTMS in the initial synthesis mixture. Even though, the

surface area and pore volume of pure SBA-15 differ from SH-SBA-15, the particle size

of pure SBA-15 and SH-SBA-15 were 22 and 20 µm respectively. Particle size of the

support mentioned in Table 3.1 refers to the length of the particle.

Table 3.1: Textural parameters of pure SBA-15, thiol functionalized SBA-15, rod-like SBA-15 and commercial silica gel

Materials Pore Diameter

(Ǻ)

Surface Area

(m2 g

-1)

Pore Volume

(cm3 g

-1)

Particle Size

(µm)

Pure SBA-15 83 790 1.09 22 SH-SBA-15 67 660 0.84 20 Commercial Silica Gel 56 470 0.73 64 Rod-like SBA-15 75 690 0.96 2.0


28

0.00E+00

2.00E-02

4.00E-02

6.00E-02

8.00E-02

1.00E-01

0 50 100 150 200 250

Pore Diameter (Å)

Ad

so

rpti

on

Dv (

d)

(cm

3/Ǻ

/g)

Pure SBA-15

SH-SBA-15

Figure 3.3: Pore size distribution curve of Pure and thiol functionalized SBA-15

CHNS elemental analysis of SH-SBA-15 sample shows that the carbon,

hydrogen, and sulfur content were 15.58, 3.38, 2.12 % respectively, confirming the

presence of sulfur in the SH-SBA-15.

3.3.2 Characterization of SBA-15 with Rod-Like Morphology

Figure 3.1 shows the XRD spectra of rod-like SBA-15 consisting of three well

resolved peak which can be assigned to (100), (110) and (200) reflections of the 2D

hexagonal space group (P6mm) which are similar to those of pure SBA-15. Figure 3.4

and 3.5 shows the N2 adsorption/desorption isotherms and pore size distribution curve of

rod-like SBA-15 which are identical to that of pure SBA-15. The surface area, pore

volume and pore size of rod-like SBA-15 given in 3.1 were closer to pure SBA-15 but

differ only in particle size. The particle size (length of the rod) of rod like SBA-15 is

2 µm which is 10 times smaller compared to pure SBA-15 and SH-SBA-15. This smaller

particle size of rod-like SBA-15 is of paramount importance in our investigation on the


29

effect of particle size on the catalytic activity of immobilized α-chymotrypsin in organic

media.

0

100

200

300

400

500

600

700

0 0.2 0.4 0.6 0.8 1

Relative Pressure (P/Po)

Vo

lum

e (

cc/g

)

Rod Like SBA-15

Figure 3.4: N2 adsorption/desorption isotherm of the rod-like SBA-15

0.00E+00

5.00E-03

1.00E-02

1.50E-02

2.00E-02

2.50E-02

3.00E-02

3.50E-02

0 50 100 150 200 250Pore Diameter (Å)

Ad

so

rpti

on

Dv(d

) (c

m3/Å

/g)

Rod Like SBA-15

Figure 3.5: Pore size distribution curve of rod-like SBA-15

The FESEM images shown in the Figure 3.6 also confirm that rod-like SBA-15

possesses discrete rod-like morphology with uniform length (1-2 µm) while pure SBA-


30

15 has a fibrous macrostructure with the length extending to 20 µm by stacking and

coupling of rod-like SBA-15 (Yu et al., 2002; Fan et al., 2003).

Pure SBA-15 Rod-like SBA-15

Figure 3.6: FESEM images of Pure SBA-15 and Rod-like SBA-15

3.4 Conclusions

The synthesized mesoporous silicates pure SBA-15 and SBA-15 with rod-like

morphology were found to possess highly ordered structure consistent with the published

results in the literature. The less ordered pore structure of thiol-functionalized SBA-15

was attributed to the presence of 5 mol % MPTMS in the initial synthesis mixture. Rod-

like SBA-15 possesses smaller particle size compared to pure SBA-15 and SH-SBA-15.

The smaller particle size of rod-like SBA-15 is used in our study as reported in Chapter 5

to investigate the effect of particle size on the activity of immobilized α-chymotrypsin in

acetonitrile or tetrahydrofuran.

Chapter 4 Activity Studies in Aqueous and Organic Media

31

Chapter 4

Immobilization of α-chymotrypsin into Mesoporous

Silicate and its Activity in Aqueous or Organic Media

4.1 Introduction

Biocatalysis in nonaqueous media has grown rapidly since 1980s. The use of organic

solvent is not recommended for synthesis due to environmental concern; however, it

serves as the best choice compared to the reaction performed in water due to the

increased solubility of substrate in organic solvents (Klibanov, 2001). Lyophilized

enzymes in organic media possess very low activity, which hinders its potential

application (Michels et al., 1997). Diffusional limitation experienced by lyophilized

enzyme powder is suggested as one of the reason for low activity of enzymes in organic

media (Rees and Halling, 2001). However, these limitations can be overcome by

immobilizing enzymes onto solid support, which spreads the enzyme over large surface

area and thereby increases the active site accessibility.

Mesoporous silicate such as SBA-15 has emerged to be an excellent carrier for

immobilization of enzymes due to its high specific surface area, tunable large pore size

and narrow pore size distribution. Although there are several studies on the

immobilization of enzymes in mesoporous silicate and its activity studies in aqueous

media (discussed in section 2.3), to the best of our knowledge there are only few reports

(Takahashi et al., 2000; Deere et al., 2003; Goradia et al., 2006) which have evaluated

the activity of enzymes in organic media upon immobilization in mesoporous silicate.


32

Enzyme hydration is one of the critical parameter affecting the activity of

enzymes in organic media to about 100 fold (Klibanov, 1997), Adlercretuz (1991) had

shown that the true nature of the support can only be revealed upon carrying out the

reaction at constant thermodynamic water activity (aw). Since water adsorption depends

on the nature of the support, if one uses uncontrolled support (not at constant aw) in

organic media, the support may adsorb the water available for the enzyme and thereby

changes the enzyme hydration, which in turn can affect reaction rate and led to

misleading conclusion about the effect of support. Therefore, the reaction needs to be

carried out at constant thermodynamic water activity when comparing immobilized

enzymes in organic media.

This chapter describes the immobilization of α-chymotrypsin onto

unfunctionalized SBA-15 (pure SBA-15) and thiol functionalized SBA-15 (SH-SBA-15).

The activities of α-chymotrypsin immobilized in pure SBA-15 and SH-SBA-15 in

aqueous or organic media are studied and compared with native α-chymotrypsin and α-

chymotrypsin immobilized in commercial silica gel. The thermodynamic water activity

of the immobilized α-chymotrypsin is fixed to 0.22 by rinsing it with 1-propanol

containing 1.60 % water (Patridge et al., 1998b). As the reaction is carried out at constant

thermodynamic water activity, the experimental results should reflect the true nature of

support.


33

4.2 Materials and Methods

4.2.1 Materials

α-chymotrypsin (EC 3.4.21.1) from bovine pancreas, N-benzoyl-L-tyrosine ethyl ester

(BTEE), N-acetyl-L-phenylalanine ethyl ester (APEE), N-acetyl-L-phenylalanine and

commercial silica gel were all purchased from Sigma. Tris(hydroxymethyl)

aminomethane was purchased from Aldrich. 1-propanol was purchased from Merck. All

the organic solvents used in this study were dried overnight by 3 Å molecular sieves

before use. The mesoporous silicates used in this work were prepared as described in

Chapter 3.

4.2.2 Model Reaction for Activity Studies in Aqueous and Organic

Media

Hydrolysis of 2 mM BTEE in 0.01 M tris buffer (pH = 7.5) containing 0.1 M

NaCl, 1 mM CaCl2 and 30 % v/v ethanol was chosen as a model reaction for studying

the enzyme activity in aqueous media. The reaction scheme for the hydrolysis of N-

benzoyl-L-tyrosine ethyl ester by native and immobilized α-chymotrypsin is given in

figure 4.1.

Figure 4.1 Reaction scheme for the hydrolysis of N-benzoyl-L-tyrosine ethyl ester

Tris buffer pH 7.5 with 0.1M NaCl and 1mM CaCl2

30 wt % v/v Ethanol, 23°C

NH

O

OH

OH

O

HNH

O

OH

O

O

H

N-Benzoyl-L-tyrosine Ethanol

+ CH3-CH2-OH

N-Benzoyl-L-tyrosine ethyl ester


34

The hydrolysis reaction was followed by an auto titrator Titralab TIM 854 from

Radiometer Analytical, France at room temperature equipped with magnetic stirrer

(500rpm). The activity was calculated by measuring the volume of 0.02 M sodium

hydroxide required for maintaining the pH of the reaction mixture at 7.5 for about 10

minutes is calculated using the formula given below.

Transesterification of APEE with 1 M 1-propanol in dry octane, tetrahydrofuran

or acetonitrile at the thermodynamic water activity of 0.22 was chosen as the model

reaction for finding the activity of immobilized α-chymotrypsin in organic media. The

reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester by

immobilized α-chymotrypsin is given in figure 4.2.

+ CH3-CH2-OH

Figure 4.2 Reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester

N-acetyl-L-phenylalanine ethyl ester

NH

O

O

OH

NH

O

O

OH

+ CH3-CH2-CH2-OH

Acetonitrile or Octane or Tetrahydrofuran

Water as required, 23°C

N-acetyl-L-phenylalanine propyl ester Ethanol

1-propanol

((AAmmoouunntt.. ooff NNaaOOHH aaddddeedd ppeerr mmiinnuuttee xx NNoorrmmaalliittyy ooff NNaaOOHH xx 11000000)) SSppeecciiffiicc AAccttiivviittyy ((UUnniittss//mmgg ooff eennzzyymmee)) == ---------------------------------------------------------------------------------------------------------- mmgg ooff eennzzyymmee


35

The reaction was carried out in 5 ml scale at room temperature (23○C). At first,

APEE was dissolved in dry 1-propanol and then mixed with dry octane, acetonitrile or

tetrahydrofuran at the thermodynamic water activity of 0.22, followed by the addition of

immobilized α-chymotrypsin. The resulting suspension was shaken at 260rpm using

water bath shaker. At regular interval, 100 µl of the sample was withdrawn and the rate

of formation of propyl ester (product) was analyzed by Shimazdu HPLC system

equipped with Inertsil ODS-3 column and UV-detector operated at the wavelength of

254 nm. The column was eluted isocratically with mobile phase composition of 55 %

water containing 0.1 % v/v trifluoroacetic acid and 45 % acetonitrile at the flow rate of 1

ml min−1. All the experiments were repeated at least twice and the error was found to be

less than 15 %. The activity was calculated on the basis of the formation of propyl ester

by assuming that the extinction coefficients of the product and substrate (APEE) were

equal (Wang et al., 2001) using the formula given below.

4.2.3 Enzyme Immobilization for Activity Studies in Aqueous or

Organic Media

Enzyme immobilization for activity studies in aqueous media was carried out as

follows. At first, α-chymotrypsin (4 mg ml-1) was dissolved in 25 mM tris buffer (pH =

7.8) containing 10 mM CaCl2. Then 25 mg of support was added to 1 ml of

chymotrypsin solution and the mixture was shaken at 260 rpm at room temperature

(23○C) for about 4 hours. The amount of α-chymotrypsin loaded onto the support was

obtained by measuring the concentration of supernatant collected by centrifugation. The

protein concentration was determined using Bradford assay (Bradford, 1976). The solid

collected by centrifugation was dried overnight in vacuum dryer at room temperature.

µµmmoollee ooff pprroodduucctt ffoorrmmeedd ppeerr mmiinnuuttee SSppeecciiffiicc AAccttiivviittyy == ---------------------------------------------------------------------------------------------------------------- ((µµmmoollee mmiinn--11..mmgg ooff eennzzyymmee--11)) mmgg ooff eennzzyymmee


36

Enzyme immobilization for activity studies in organic media was carried out

according to the procedure reported in the literature (Patridge et al., 1998b). Figure 4.3

shows the schematic representation of the preparation of immobilized α-chymotrypsin

for activity studies in dry octane, tetrahydrofuran or acetonitrile at thermodynamic water

activity of 0.22. Thermodynamic water activity was fixed to 0.22 in 1-propanol,

acetonitrile and tetrahydrofuran by adding 1.60, 1.00 and 0.45 % water into dry 1-

propanol, acetonitrile and tetrahydrofuran (Halling, 2002). At first, α-chymotrypsin (4

mg ml-1) was dissolved in 25 mM tris buffer (pH = 7.8) containing 10 mM CaCl2. Then

25 mg of support was added to 1 ml of α-chymotrypsin solution and the mixture was

shaken at 260 rpm at room temperature (23○C) for about 4 hours (Step 1). The amount of

α-chymotrypsin loaded onto the support was obtained by measuring the concentration of

supernatant collected by centrifugation. The protein concentration was determined using

Bradford assay (Bradford, 1976) (Step 2). The solid collected by centrifugation was

rinsed with 1-propanol containing 1.60 % water which led α-chymotrypsin immobilized

onto support (either pure SBA-15 or SH-SBA-15 or commercial silica gel) with

thermodynamic water activity of 0.22 which of paramount importance for determining

the true nature of support (step 3). Then it is rinsed with solvent used in the reaction

mixture and assayed without further delay (Step 4).


37

Figure 4.3: Procedure for the immobilization of α-chymotrypsin for carrying out the reaction in dry octane, acetonitrile or tetrahydrofuran at thermodynamic water activity of 0.22

The first three steps for the preparation of immobilized α-chymotrypsin for

activity studies in organic media were same regardless of the solvent used in the reaction

mixture. For example, to find the activity of immobilized α- chymotrypsin in dry octane,

after step 3, the immobilized α-chymotrypsin was rinsed with dry octane and quickly

transferred to dry octane which contains substrates APEE and 1-propanol (step 4). Then

the initial rate of transesterification was followed by HPLC described in section 4.1.

4.2.4 Thermal Stability and Leaching Studies in Aqueous Media

The thermal stability of both native and immobilized α-chymotrypsin was

determined by placing the free enzyme (native α-chymotrypsin) and immobilized

Mixing 1 ml of α-chymotrypsin (4 mg ml-1) with 25 mg of support and shaken for 4 hours

Centrifuge to separate immobilized α-chymotrypsin

Rinsing immobilized α-chymotrypsin with 1-propanol containing 1.60 % water

Addition of Immobilized α-chymotrypsin into 2.5 mM N-acetyl-L- phenylalanine ethyl ester and

1M 1-propanol solution mixed in dry Octane or Tetrahydrofuran containing 0.45 % water or

Acetonitrile containing 1.00 % water

Step 1

Step 2

Step 3

Step 4


38

enzyme (immobilized α-chymotrypsin in either pure SBA-15 or SH-SBA-15) in tris

buffer (pH 7.8, containing 10 mM CaCl2) in a water bath maintained at (70○C) under

static condition. Samples were removed at the regular interval and allowed to cool down

to room temperature and the residual hydrolytic activity was determined as described

above. The initial activity of native and immobilized enzymes before subjected to

thermal exposure was taken as 100 %. To study the leaching of α-chymotrypsin from the

support, about 100 mg of immobilized enzyme was suspended in a tris buffer (pH 7.8,

containing 10 mM CaCl2) solution and placed in a water bath shaker shaken at 260 rpm

at room temperature. The amount of enzyme leaching from the support was determined

by removing 1 ml of solution and the α-chymotrypsin concentration in the solution was

determined by Bradford assay (Bradford, 1976).


4.3.1 Immobilization of α-chymotrypsin into Mesoporous Silicate and

Commercial silica Gel

α-chymotrypsin is a class of serine protease, which hydrolyzes ester bonds in addition to

peptide bonds. Its molecular dimension and isoelectric point are 50 Å × 40 Å × 40 Å and

8.1 respectively (Zoungrana et al., 1997). Since the pore size of all the support (given in

Chapter 3) utilized in this study is higher than the molecular dimension of α-

chymotrypsin, it is expected that the enzyme can easily diffuse into the pores. It is well

known that the enzymes in organic media shows pH memory i.e. the activity of enzymes

in organic media depends on the pH to which it is finally exposed and it is found to be

maximum when it is brought from the optimum pH for catalytic activity in aqueous

media (Zaks and Klibanov, 1998b). Since the optimum pH for the catalytic activity of α-


39

chymotrypsin has been found to be 7.8 (Patridge et al., 1998b), hence this pH has been

selected in this study for the immobilization of α-chymotrypsin into the mesoporous

silicate SBA-15.

Several studies (Barros et al., 1998; Persson et al., 2000; Persson et al., 2002a)

have shown that the activity of immobilized enzyme depends on the amount of enzyme

loaded onto the support; hence, the α-chymotrypsin loading in all the support (pure SBA-

15, SH-SBA-15 and commercial silica gel) is fixed to 8 % only. Because the commercial

silica gel has lower surface area (470 m2 g-1) than mesoporous silicate (660-790 m2 g-1),

hence it is expected that commercial silica gel has a lower enzyme loading capacity

compared to mesoporous silicate. Patridge et al., (1998b) used only 8 wt % loading of

chymotrypsin in silica gel and found that the immobilized α- chymotrypsin showed

better activity compared to the lyophilized enzyme powder. Since the silica gel used in

Patridge et al. (1998b) work has surface area, pore volume and pore size more close to

the commercial silica gel used in our study, hence only 8 wt % enzyme loading is used

our study for comparison purpose.

4.3.2 Activity of Native and Immobilized α-chymotrypsin in Aqueous

Media

Upon immobilization in pure SBA-15 and SH-SBA-15, α-chymotrypsin retains only

about 3.35 and 3.44 % of its original activity at 23°C, indicating that there were

significant changes in α-chymotrypsin conformation upon adsorption in the mesopores of

SBA-15. This is not surprising as the enzyme activity depends on its three dimensional

conformation and perturbation in the three-dimensional conformation often leads to

changes in enzyme activity (Clark, 1993). Upon adsorption, the catalytic triad where the

substrate molecule will bind may orient in a different conformation, there by limiting the


40

substrate binding and decreasing the enzyme activity (Clark and Bailey, 2002). The

decrease in the activity of α-chymotrypsin upon immobilization is not only attributed to

the changes in the active site conformation but also due to diffusional limitation often

encountered in immobilized enzyme. The decrease in activity of α-chymotrypsin upon

immobilization is also observed in other immobilization carriers such as Ludox silica and

Teflon (Zoungrana et al., 1997) and recently single walled carbon nanotube (Karajanagi

et al., 2004). Lee et al. (2005) have also shown that the Michaelis constant (Km) was

increased to about 7 fold and turnover number (kcat) declined to about 37 fold upon

immobilization of α-chymotrypsin into hierarchically ordered mesocellular mesoporous

silica materials (HMMS) relative to the native α-chymotrypsin. All these observation

support our conclusion that both conformational changes and mass transfer limitation are

the main factors responsible for the decrease of activity of α-chymotrypsin upon

immobilization in mesoporous silicate.

4.3.3 Thermal Stability of Native and Immobilized α-chymotrypsin in

Aqueous Media

The slight increase of temperature in an enzymatic reaction generally leads to the

increase in reaction rate, at relatively high temperature (> 55○C), most of the enzyme

shows very low thermal stability, which hinders its potential application. This has

prompted us to study the thermal stability of α-chymotrypsin immobilized in mesoporous

silicate at 70○C. It can be seen from Figure 4.4 that α-chymotrypsin immobilized in pure

SBA-15 and SH-SBA-15 losses only about 40 % of the original activity after exposing it

to 70○C for about 3 hours while the native α-chymotrypsin losses all its activity ~ 98 %

within 20 minutes. This increased thermal stability of α-chymotrypsin upon

immobilization may be due to the effect of excluded volume and strong hydration of


41

protein upon immobilization in the narrow pore channel, which stabilizes the enzyme

against unfolding and thereby increases thermal stability (Ravindra et al., 2004).

0

20

40

60

80

100

120

0 30 60 90 120 150 180 210Time in Minutes

Re

sid

ua

l Ac

tiv

ity

(%

)

SH-SBA-15

Pure-SBA-15

Native α-chymotrypsin

Figure 4.4: Thermal stability of native and immobilized α-chymotrypsin in aqueous media

4.3.4 Leaching of α-chymotrypsin form Pure SBA-15 and Thiol

Functionalized SBA-15

Figure 4.5 shows the leaching of α-chymotrypsin immobilized in pure SBA-15 and SH-

SBA-15 at 23°C. α-chymotrypsin immobilized in mesoporous silicate shows only about

18 % of leaching from the support even after 12 hours. Adsorption of protein onto the

support is believed to be stabilized by electrostatic interaction. The isoelectric point of α-

chymotrypsin and SBA-15 has been reported to be 8.1 and 2.0, respectively (Zhao et al.,

1998; Zoungrana et al., 1997). At pH value of 7.8, which is the pH value used in the

leaching studies, α-chymotrypsin is slightly positively charged and silica surface is

negatively charged. Hence, the lower leaching of α-chymotrypsin from the support can

be attributed to the strong electrostatic interaction between α-chymotrypsin and

mesoporous silicate.


42

70

75

80

85

90

95

100

105

0 120 240 360 480 600 720

Time in Minutes

% o

f C

hym

otr

yp

sin

in

Su

pp

ort

SH-SBA-15

Pure-SBA-15

Figure 4.5: Leaching of α-chymotrypsin from pure and thiol functionalized SBA-15

4.3.5 Activity of Immobilized α-chymotrypsin in Organic Media

Table 4.1 shows the activities of α-chymotrypsin immobilized in pure SBA-15, SH-

SBA-15 and commercial silica gel in dry octane and acetonitrile or tetrahydrofuran with

thermodynamic water activity of 0.22 at 23°C. In the all the media taken in this study, α-

chymotrypsin immobilized in mesoporous silicate (pure SBA-15 and SH-SBA-15) shows

higher activity compared to α-chymotrypsin immobilized in commercial silica gel.

Table 4.1: Activity of immobilized α-chymotrypsin in dry octane, tetrahydrofuran or acetonitrile at thermodynamic water activity of 0.22

Activity (nanomole min-1

mg of enzyme-1

) Supports

Octane Tetrahydrofuran Acetonitrile

SH-SBA-15 212 9.5 2.75 Pure SBA-15 89 6.5 2.0

Commercial Silica gel 75 3.8 0.6

The reason for the higher activity of α-chymotrypsin upon immobilization is

mesoporous silicate (pure SBA-15 and SH-SBA-15) and the difference in activity

between the solvents is rationalized as follows.


43

Factors that can affect the activity of immobilized enzyme in organic media is

suggested to be related to enzyme hydration, substrate solvation and mass transfer

limitation. Since our reaction was carried out at constant thermodynamic water activity

of 0.22, the difference in activity due to enzyme hydration/dehydration (water stripping

in polar solvents) was eliminated (Halling, 2002). The difference in activity between the

solvents is suggested to be attributed to the difference in the solubility of substrate

(APEE) in the solvents employed. Because more polar solvents, such as acetonitrile and

tetrahydrofuran has better solvation for the substrate (APEE) compared to octane, hence

APEE is less available to the immobilized enzyme in polar solvents, resulting in lower

activity of the immobilized enzyme in polar solvents than in octane (Schmitke et al.,

1996; Partridge et al., 1998a). The higher activity observed in nonpolar solvents

compared to polar solvents is also in agreement with the results reported for immobilized

α-chymotrypsin (Suzawa et al., 1995).

The higher activity of α-chymotrypsin upon immobilization in mesoporous

silicate compared to commercial silica gel can be explained based on mass transfer

limitation, which commonly occurs in porous supports. The distance that has to be

traveled by substrate and product inside the pore depends on the particle size of the

support. In the case of support with larger particle size, the substrate and product have to

travel longer distance, thereby limiting the activity of the immobilized enzyme by

internal mass transfer (Barros et al., 1998). Thus the higher activity of α-chymotrypsin

immobilized in mesoporous silicate is due to the smaller particle size of mesoporous

silicate, which results in reduced internal mass transfer and thereby led to higher activity.

The increase in reaction rate upon decrease in particle size of the support is also observed

in the immobilization of lipase in controlled pore glass (Bosley and Clayton, 1994). The


44

slightly higher activity of α-chymotrypsin immobilized in SH-SBA-15 compared to pure

SBA-15 may be due to the difference in the mode of binding of α-chymotrypsin in SH-

SBA-15 compared to pure SBA-15.

4.4 Conclusions

The activity of α-chymotrypsin in aqueous and organic media has been investigated upon

immobilization in pure and SH-SBA-15. The activity of α-chymotrypsin in aqueous

media decreases upon immobilization, but the immobilized enzyme shows better thermal

stability, which is believed to be of practical importance for relatively high temperature

applications. Immobilized α-chymotrypsin in mesoporous silicate also shows less

leaching from the support. α-chymotrypsin immobilized in SBA-15 also shows higher

activity in organic media compared to enzyme immobilized in commercial silica gel,

possibly attributed to the smaller particle size of SBA-15, which reduces the internal

mass transfer limitation. However, α-chymotrypsin immobilized in SH-SBA-15 shows

only minor increase in activity as compared to pure SBA-15.

Chapter 5 Effect of Enzyme Loading and Thermodynamic Water Activity

45

Chapter 5

Effect of Enzyme Loading and Thermodynamic Water

Activity

5.1 Introduction

This chapter describes our results on the effect of enzyme loading and thermodynamic

water activity on the activities of α-chymotrypsin immobilized in pure SBA-15, SBA-15

with rod-like morphology (rod like SBA-15), and commercial silica gel. The reaction

media taken for this study were acetonitrile or tetrahydrofuran. SBA-15 with rod like

morphology which has the surface area, pore diameter and pore volume closer to pure

SBA-15 but differ only in the particle size (given in Chapter 3) has been used in this

study in order to observe the effect of particle size on the reaction rate. The use of rod-

like SBA-15 in this study is also to re-confirm our conclusion observed in Chapter 4 that

the higher activity of α-chymotrypsin immobilized in mesoporous silicate was due to the

reduced internal mass transfer limitation because of the smaller particle size of

mesoporous silicate (pure SBA-15 and thiol functionalized SBA-15) compared to that of

commercial silica gel. At first, the effect of α-chymotrypsin loading on the activities in

either acetonitrile or tetrahydrofuran was studied. The combination of support and

loading of enzyme that showed the highest activity was chosen to study the effect of

thermodynamic water activity on the reaction rate of immobilized α-chymotrypsin in

either acetonitrile or tetrahydrofuran.


46

5.2 Materials and Methods

5.2.1 Materials

N-acetyl-L-phenylalaine ethyl ester (APEE), N-acetyl-L-phenylalaine, α-chymotrypsin

from bovine pancreas, commercial silica gel was purchased from Sigma. 1-propanol was

purchased from Merck. Acetonitrile and tetrahydrofuran were purchased from Tedia. All

the organic solvents used in this work were dried overnight using 3 Å molecular sieve

before use. Pure SBA-15 and rod-like SBA-15 were prepared as described in the Chapter

3.

5.2.2 Activity of Immobilized α-chymotrypsin in Acetonitrile and

Tetrahydrofuran

The activities of the immobilized α-chymotrypsin were measured in acetonitrile or

tetrahydrofuran through the initial rate of transesterification of 2.5 mM APEE with 1 M

1-propanol. The reaction was carried out in 5 ml scale at room temperature (23○C). At

first, APEE was dissolved in dry 1-propanol and then mixed with acetonitrile or

tetrahydrofuran containing water (the water content was varied according to the

thermodynamic water activity in the reaction mixture as shown in Table 5.1), followed

by the addition of immobilized α-chymotrypsin. The resulting suspension was shaken at

260 rpm using water bath shaker. At regular interval, 100 µl of the sample was

withdrawn and the rate of formation of N-acetyl-L-phenylalanine propyl ester (product)

was analyzed by Shimazdu HPLC system equipped with Inertsil ODS-3 column and

UVdetector operated at the wavelength of 254 nm. The column was eluted isocratically

with mobile phase composition of 55 % water containing 0.1 % v/v trifluoroacetic acid

and 45 % acetonitrile at the flow rate of 1 ml min-1. All the experiments were repeated at

least twice and the error was found to be less than 15 %. The activity was calculated on


47

the basis of the formation of propyl ester by assuming that the extinction coefficients of

the product and substrate (APEE) were equal (Wang et al., 2001) which is given below.

The reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester

by immobilized α-chymotrypsin is given in figure 5.1.

+ CH3-CH2-OH

Figure 5.1 Reaction scheme for the transesterification of N-acetyl-L-phenylalanine ethyl ester in acetonitrile and tetrahydrofuran

5.2.3 Immobilization of α-chymotrypsin

The immobilization of α-chymotrypsin onto pure SBA-15, SBA-15 with rod-like

morphology and commercial silica gel was carried out as follows. At first, α-

chymotrypsin (4 to 9 mg ml-1) was dissolved in 25 mM tris buffer (pH = 7.8) containing

N-acetyl-L-phenylalanine ethyl ester

NH

O

O

OH

NH

O

O

OH

+ CH3-CH2-CH2-OH

Acetonitrile or Tetrahydrofuran

Water as required, 23°C

N-acetyl-L-phenylalanine propyl ester Ethanol

1-propanol

µµmmoollee ooff pprroodduucctt ffoorrmmeedd ppeerr mmiinnuuttee SSppeecciiffiicc AAccttiivviittyy == ---------------------------------------------------------------------------------------------------------------- ((µµmmoollee mmiinn--11..mmgg ooff eennzzyymmee--11)) mmgg ooff eennzzyymmee


48

10 mM CaCl2. Then 15 mg of support was mixed with 1 ml of α- chymotrypsin solution

and shaken at 260 rpm using water bath shaker (23○C) for about 4 hours and then

centrifuged to collect the immobilized α-chymotrypsin. The loading amount of α-

chymotrypsin into the support was obtained by measuring the concentration of

supernatant collected by centrifugation. The α-chymotrypsin concentration in the

supernatant was determined using Bradford assay (Bradford, 1976). The solid collected

by centrifugation was rinsed with 1-propanol containing water (the water content was

varied according to the thermodynamic water activity in the reaction mixture as shown in

Table 5.1) for about 3 times, finally rinsed with the solvent used in the reaction mixture

(acetonitrile or tetrahydrofuran) and assayed without further delay. The α-chymotrypsin

loading into the support was controlled by varying the enzyme concentration from 4 to 9

mg ml-1 and the amount of support was fixed to 15 mg.

Table 5.1: Water content required to attain selected water activities in 1-propanol, acetonitrile and tetrahydrofuran (Halling, 2002)

Water Content v/v % Water Activity

(aw) 1-propanol Acetonitrile Tetrahydrofuran

0.22 1.60 1.00 0.45 0.44 3.40 2.70 1.20 0.55 4.70 4.50 1.60 0.76 9.80 13.00 3.60

Figure 5.2 shows schematic representation of the procedure adopted to

immobilize α-chymotrypsin for carrying out the activity studies in either acetonitrile or

tetrahydrofuran as the reaction media. For example, to determine the activity of

immobilized α-chymotrypsin at the enzyme loading of 8 % in acetonitrile at the

thermodynamic water activity of 0.22, the procedure adopted was follows. 1 ml of α-

chymotrypsin solution (4 mg ml-1) was mixed with 15 mg of support and shaken at room

temperature for about 4 hours and then centrifuged. The supernatant was removed


49

carefully and then the solid was rinsed with 1-propanol containing 1.60 % water (aw =

0.22). Then the immobilized α- chymotrypsin was transferred to acetonitrile solution

containing 1.00 v/v % water (aw = 0.22) and substrates. The product formation was

determined by HPLC as described in the above paragraph. The α-chymotrypsin loading

onto the support was varied by changing the solution concentration in step 1. The water

content in 1-propanol at step 3 as well as in reaction medium (acetonitrile and

tetrahydrofuran) at step 4 was varied according to the thermodynamic water activity

required in the reaction mixture. The amount of water content to be added into the dry

organic solvent to achieve the required thermodynamic water activity is given in Table

5.1.

Figure 5.2: Procedure for the immobilization of α-chymotrypsin for carrying out the reaction in either acetonitrile or tetrahydrofuran

Mixing 1 ml of α-chymotrypsina with 15 mg of support and shaken for 4 hours

Centrifuge to separate immobilized α-chymotrypsin

Rinsing Immobilized α-chymotrypsin with 1-propanol containing waterb

Addition of Immobilized α-chymotrypsin into 2.5 mM N-acetyl-L-phenylalanine ethyl ester and 1M 1-propanol solution mixed in tetrahydrofuran

or acetonitrile containing waterb

Step 1

Step 2

Step 3

Step 4

a Concentration varied from 4 to 9 mg ml-1 to vary the enzyme loading

b Water content varied according to thermodynamic water activity required


50


5.3.1 Effect of Enzyme Loading on Activity in Acetonitrile and

Tetrahydrofuran

Figure 5.3 and 5.4 show the effect of α-chymotrypsin loading on the activities in

acetonitrile or tetrahydrofuran with thermodynamic water activity of 0.22 at 23°C. Since

the reaction is carried out at constant thermodynamic water (aw) of 0.22, the difference in

activity between the supports cannot be attributed to the enzyme hydration, but due to the

intrinsic nature of the support. It is seen that the activity of immobilized α-chymotrypsin

increases with increase of enzyme loading up to the certain limit (20 wt % in mesoporous

silicate and 13 wt % in commercial silica gel) and then decreases irrespective of nature

of the support, which indicates that this phenomenon is common for all the support

utilized in this study.

0

5

10

15

20

25

30

50 100 150 200 250 300

Enzyme Loading (mg/g of support)

Acti

vit

y

(nan

om

ole

/min

.mg

of

en

zym

e)

Rod-like SBA-15 Pure SBA-15

Rod-like SBA-15 Pure-SBA-15Tetrahydrofuran

Acetonitrile

Figure 5.3: Effect of enzyme loading on the activity of α-chymotrypsin immobilized in pure SBA-15 and rod-like SBA-15 in either acetonitrile or tetrahydrofuran at aw of 0.22


51

0

2

4

6

8

10

70 80 90 100 110 120 130 140 150 160 170

Enzyme Loading (mg/g of support)

Acti

vit

y

(nan

om

ole

/min

.mg

of

en

zym

e) Acetonitrile Tetrahydrofuran

Figure 5.4: Effect of enzyme loading on the activity of α-chymotrypsin immobilized in commercial silica gel in either acetonitrile or tetrahydrofuran at aw of 0.22

Figure 5.5 shows the schematic representation of the effect of enzyme loading on

changes in the microenvironment of the immobilized enzyme. At low enzyme loading,

the enzyme molecule has a higher tendency to unfold/spread on the support surface which

results in lowering the catalytic activity. As the loading increases, enzyme unfolding at

the support surface gets restricted by adjacent enzyme molecules which lad to increases in

the catalytic activity of immobilized enzyme. However, as the loading increases further,

the problem of mass transfer limitation arise which causes the drop in the catalytic

activity of immobilized enzyme. Hence, increase in the activity of immobilized α-

chymotrypsin upon increase in enzyme loading in all the support can be attributed to the

suppression of enzyme unfolding at the support surface by enzyme-enzyme interaction.

However, the decrease in activity at higher loading can be attributed to mass transfer

limitation which is quite common on porous support.


52

Figure 5.5: Schematic representation of the effect of enzyme loading in porous supports

It is seen from Figure 5.3 that mesoporous silicate not only possess high enzyme

loading capacity but also shows high enzyme activity compared to commercial silica gel

(Figure 5.4). At loading amount we tested, α-chymotrypsin immobilized in mesoporous

silicate shows higher catalytic activity compared to commercial silica gel which can be

attributed to the smaller particle size of mesoporous silicate compared to commercial

silica gel which reduces the internal mass transfer limitation and thereby increases the

activity. The enzyme loading at which α-chymotrypsin immobilized in mesoporous

silicate shows higher catalytic activity is around 20 wt %, which is higher compared to

commercial silica gel in which the maximum activity can be attained at only 13 wt %

enzyme loading. The higher loading capacity of mesoporous silicate compared to

commercial silica gel can be attributed to higher surface area and pore volume of

mesoporous silicate compared to commercial silica gel (given in Chapter 3). The

increase in enzyme activity with an increase in enzyme loading is also observed in many

Enzyme Molecule

Pore Opening

Solid Support

Low Enzyme Loading

Increasing Enzyme Loading

High Enzyme Loading


53

studies (Zoungrana et al., 1997, Barros et al., 1998 and Persson et al., 2000). It is

interesting to note that the enzyme immobilized in rod-like SBA-15 shows higher

activity compared to pure SBA-15 which has the same physico-chemical properties with

an exception to particle size. The higher activity of α-chymotrypsin immobilized in rod-

like SBA-15 can be attributed to the smaller particle size (2 µm) compared to pure-SBA-

15 (22 µm) which greatly reduces the internal mass transfer limitation and leads to higher

activity. These results also support our conclusion in Chapter 4 that the internal mass

transfer limitation was responsible for increased activity of α-chymotrypsin upon

immobilization in mesoporous silicate compared to commercial silica gel.

5.3.2 Effect of Thermodynamic Water Activity

Figure 5.6 shows the effect of thermodynamic water activity on the reaction rate either in

acetonitrile or tetrahydrofuran as the reaction medium at 23°C. Rod-like SBA-15 with an

enzyme loading of 20 wt % has been selected for determining the effect of

thermodynamic water activity due to its higher activity compared to other supports and

enzyme loading.

0

10

20

30

40

50

60

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

Thermodynamic Water Activity (aw)

Acti

vit

y

(nan

om

ole

/min

.mg

of

en

zym

e)

Acetonitrile Tetrahydrofuran

Figure 5.6: Effect of thermodynamic water activity on the activity of α-chymotrypsin immobilized in rod-like SBA-15 in either acetonitrile or tetrahydrofuran


54

The activity increases with increasing thermodynamic water activity up to about 0.55 and

then decreases upon further increase in thermodynamic water activity. There are a

number of possible explanations on this effect of thermodynamic water activity on the

reaction rate. One possible reason is that, enzyme hydration which can greatly affect the

flexibility of the enzyme molecule necessary for the catalysis. At lower water activity,

there will be less number of water molecules present on the enzyme surface which

reduces the enzyme flexibility and thereby the activity. However, as the thermodynamic

water activity increases, the number of water molecules on the enzyme surface increases,

thus increasing the flexibility and thereby the activity (Partridge et al., 1998a). However,

enzyme activity decreases above the thermodynamic water activity of 0.55 may be due to

the competitive inhibition by water molecule. At higher thermodynamic water activity,

the water content in the system is higher which can compete well with the propanol,

which results in the favor of hydrolysis instead of synthesis. In other words, the

equilibrium has been shifted thermodynamically to hydrolysis instead of synthesis. The

other possible reason may be the conformational changes. It should be noted that the

enzyme denatures in aqueous-organic mixture but not in pure organic solvents due to the

availability of molecular lubricant (water) in the former which can aid in the unfolding of

enzyme. Hence the higher water content at higher thermodynamic water activity can also

aid in the unfolding of enzyme which will results in diminishing the activity. The higher

activity of α-chymotrypsin immobilized in mesoporous silicate in tetrahydrofuran

compared to acetonitrile can be attributed to the difference in substrate solvation

(Schmitke et al., 1996). This is because acetonitrile has higher solvation capability

compared to tetrahydrofuran, thus affecting the substrate binding and reducing the

activity of the immobilized enzyme.


55

5.4 Conclusions

α-chymotrypsin immobilized in rod-like SBA-15 shows higher activity compared to

pure-SBA-15 and commercial silica gel, which can be attributed to the smaller particle

size, which reduces the internal mass transfer limitation and increases the activity. The

activity was found to be increased with increase in thermodynamic water activity due to

increased flexibility of the enzyme and the optimum thermodynamic water activity was

found to be 0.55 in both tetrahydrofuran and acetonitrile.

Chapter 6 Conclusions and Future Research

56

Chapter 6

Conclusions and Future Research

6.1 Conclusions

Mesoporous silicate was found to be an excellent carrier for the immobilization of

enzymes for application in aqueous and organic media. The activity of α-chymotrypsin

decreased upon immobilization as compared to the native α-chymotrypsin possibly due

to the active site orientation and diffusional limitation. However, thermal stability studies

carried out at 70○C in aqueous buffer showed that the immobilized α-chymotrypsin

losses only 40 % of original activity after 3 hours of exposure but the native α-

chymotrypsin losses almost all of its activity within 20 minutes. Interestingly, the

immobilized α-chymotrypsin also showed low leaching from the support in aqueous

buffer due to strong electrostatic interaction between positively charged enzyme and

negatively charged support. The combination of enhanced thermal stability and low

leaching of α- chymotrypsin immobilized in mesoporous silicate may find application in

industry even though the immobilized enzyme possess low activity.

α-chymotrypsin immobilized in mesoporous silicate (pure SBA-15 and SH-SBA-

15) possess higher activity in octane, tetrahydrofuran and acetonitrile compared to α-

chymotrypsin immobilized in commercial silica gel, possibly due to the smaller particle

size of mesoporous silicate compared to commercial silica gel which reduces the internal


57

mass transfer limitation and thereby increases the activity. Since the reaction was carried

out at constant thermodynamic water activity of 0.22, the difference in activity between

the three supports cannot be attributed to the difference in enzyme hydration. The higher

activity of α-chymotrypsin immobilized in thiol functionalized SBA-15 may be due to

the difference in the mode of binding compared to pure SBA-15.

The study on the effect of α-chymotrypsin loading on the activity of immobilized

enzyme in pure SBA-15, rod-like SBA-15 and commercial silica gel shows that the

activity increases with increase in α-chymotrypsin loading up to certain level and then

decreases. The enzyme loading at which the catalytic activity maximum was found to be

around 20 wt % in rod-SBA-15 and pure SBA-15 which can be attributed to the high

pore volume and surface area of mesoporous silicate compared to commercial silica gel.

The activity of α-chymotrypsin immobilized in rod-like SBA-15 in acetonitrile and

tetrahydrofuran was found to be higher compared to other support which can be

attributed to the reduced internal mass transfer limitation in rod-like SBA-15 due to its

smaller particle size (2 µm) compared to pure SBA-15 (22 µm) and commercial silica gel

(64 µm). The study on the effect of thermodynamic water activity on the reaction rate of

α-chymotrypsin immobilized in rod-like SBA-15 shows that the activity increases with

increase in thermodynamic water activity and optimum attained up to 0.55 in acetonitrile

and tetrahydrofuran and the catalytic activity of the immobilized α-chymotrypsin reached

a maximum before it decreased upon further increase in thermodynamic water activity.


58

6.2 Future Research

The future work on this study involves the effect of various functional groups on the

activity of α-chymotrypsin in aqueous and organic media. Since, the enzyme loading can

be influenced by solution pH at the time of immobilization, the effect of functional group

together with initial solution pH and enzyme loading can give more information on the

interaction between mesoporous silicate and α-chymotrypsin. The study on the effect of

thermodynamic water activity on the reaction rate of α-chymotrypsin immobilized in

functionalized mesoporous silicate in non-polar organic solvents can provide information

on the effect of functional group on the activity in nonpolar organic solvents.

References

59

References

Adlercretuz, P. (1991), ‘On the importance of the support material for enzymatic synthesis in organic media support effects at controlled water activity’, European Journal of Biochemistry 199, 609-614. Bacheva, A. V., Belyaeva, A. V., Lysogorskaya, E. N., Oksenoit, E. S., Lozinsky, V. I. and Filippova, I. Y. (2005), ‘Biocatalytic properties of native and immobilized subtilisin 72 in aqueous-organic and low water media’, Journal of Molecular Catalysis B: Enzymatic 32, 253-260. Barros, R. J., Wehtje, E. and Adlercreutz, P. (1998), ‘Mass transfer studies on immobilized α-chymotrypsin biocatalyst prepared by deposition for use in organic medium’, Biotechnology and Bioengineering 59, 364-373. Bosley, J. A. and Clayton, J. C. (1994), ‘Blueprint for a lipase support: use of hydrophobic controlled pore glasses as model systems’, Biotechnology and Bioengineering 43, 934-938. Bradford, M. M. (1976), ‘A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding’, Analytical Biochemistry 72, 248-254. Cao, L., Rantwijk, F. V. and Sheldon, R. A. (2000), ‘Cross-linked enzyme aggregates: A simple and effective method for the immobilization of penicillin acylase’, Organic Letters 2, 1361-1364. Castillo, B., Mendez, J., Al-Azzam, W., Barletta, G. and Griebenow, K. (2006), ‘On the relationship between the activity and structure of peg-α-chymotrypsin conjugates in organic solvents’, Biotechnology and Bioengineering 94, 565-574. Castro, G. R. and Knubovets, T. (2003), ‘Homogeneous biocatalysis in organic solvents and water-organic mixtures’, Critical Reviews in Biotechnology 23, 195-231. Chin, J. T., Wheeler, S. L. and Klibanov, A. M. (1994), ‘On protein solubility in organic solvents’, Biotechnology and Bioengineering 44, 140-145. Clark, D. S. (1993), ‘Can immobilization be exploited to modify enzyme activity?’, Trends in Biotechnology 12, 439-443. Clark, D. S. and Bailey, J. E. (2002), ‘Structure-function relationships in immobilized α-chymotrypsin catalysis’, Biotechnology and Bioengineering 79, 539-549. Corma, A. (1997), ‘From microporous to mesoporous molecular sieve materials and their use in catalysis’, Chemical Reviews 97, 2373-2419. Day, S. H. and Legge, R. L. (1995), ‘Immobilization of tyrosinase for use in nonaqueous media: Enzyme deactivation phenomena’, Biotechnology Techniques 9, 471-476.

References

60

Debulis, K. and Klibanov, A. M. (1993), ‘Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants’, Biotechnology and Bioengineering 41, 566-571. Deere, J., Magner, E., Wall, G. and Hodnett, B. K. (2003), ‘Oxidation of ABTS by silicate-immobilized cytochrome C in nonaqueous solutions’, Biotechnology Progress 19, 1238-1243. D´ıaz, J. F. and Balkus, K. J. (1996), ‘Enzyme immobilization in MCM-41 molecular sieve’, Journal of Molecular Catalysis B : Enzymatic 2, 115-126. Dordick, J. S. (1989), ‘Enzymatic catalysis in monophasic organic solvents’, Enzymes and Microbial Technology 11, 194-211. Engbersen, J. F. J., Broos, J., Verboom, W. and Reinhoudt, D. N. (1996), ‘Effect of crown ethers and small amount of cosolvent on the activity and enantioselectivity of α-chymotrypsin in organic solvents’, Pure and Applied Chemistry 68, 2171- 2178. Fan, J., Lei, J., Wang, L., Yu, C., Tu, B. and Zhao, D. (2003), ‘Rapid and high capacity immobilization of enzymes based on mesoporous silicas with controlled morphologies’, Chemical Communication, 2140-2141. Gill, I., Pastor, E. and Ballesteros, A. (1999), ‘Lipase-silicone biocomposites: Efficient and versatile immobilized biocatalysts’, Journal of the American Chemical Society 121, 9488-9496. Goradia, D., Cooney, J., Hodnett, B. K. and Magner, E. (2006), ‘Characteristics of a mesoporous silicate immobilized trypsin bioreactor in organic media’, Biotechnology Progress 22, 1125-1131. Griebenow, K. and Klibanov, A. M. (1995), ‘Lyophilization-induced reversible changes in the secondary structure of proteins’, Proceedings of the National Academy of Science of the United States of America 92, 10969-10976. Griebenow, K. and Klibanov, A. M. (1997), ‘Can conformational changes be responsible for solvent and excipient effects o the catalytic behavior of subtilisin Carlsberg in organic solvents?’, Biotechnology and Bioengineering 53, 351-362. Griebenow, K., Santos, A. M. and Carrasquillo, K. G. (1999), ‘Secondary structure of proteins in the amorphous dehydrated state probed by ftir spectroscopy’, The internet Journal of Vibrational Spectroscopy 3(1), [http://www.ijvs.com]. Guo, Y. and Clark, D. S. (2001), ‘Activation of enzymes for nonaqueous Biocatalysis by denaturing concentrations of urea’, Biochimica et Biophysica Acta 1546, 406-411. Halling, P. J. (1994), ‘Thermodynamic predictions for biocatalysis in nonconventional media:theory, tests, and recommendations for experimental design and analysis’, Enzymes and Microbial Technology 16, 178-206.

References

61

Halling, P. J. (2002), Enzyme Catalysis in Organic Synthesis, Vol. 1, Wiley-VCH Verlag GmBH, chapter 8, Enzymatic conversion in organic and other low water media, 259-285. Halling, P. J. (2004), ‘What can we learn by studying enzymes in non-aqueous media?’, Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 359, 1287-1297. He, J., Li, X., Evans, D. G., Duan, X. and Li, C. (2000), ‘A new support for the immobilization of penicillin acylase’, Journal of Molecular Catalysis B : Enzymatic 11, 45-53. Hodgkins, R. P., Garcia-Bennett, A. E. and Wright, P. A. (2005), ‘Structure and morphology of propylthiol-functionalised mesoporous silicas templated by nonionic triblock opolymers’, Microporous and Mesoporous Materials 79, 241-252. Ito, Y., Fujii, H. and Imanishi, Y. (1993), ‘Catalytic peptide synthesis by trypsin modified with polystyrene in chloroform’, Biotechnology Progress 9, 128-130. Jia, H., Zhu, G., Vugrinovich, B., Kataphinan, W., Reneker, D. H. and Wang, P. (2002), ‘Enzyme-carrying polymeric nanofibers prepared via electrospinning for use as unique biocatalysts’, Biotechnology Progress 18, 1027-1032. Karajanagi, S. S., Vertegel, A. A., Kane, R. S. and Dordick, J. S. (2004), ‘Structure and function of enzymes adsorbed onto single walled carbon nanotubes’, Langmuir 20, 11594-11599. KatchalskiKatzir, E. (1993), ‘Immobilized enzymes-learning from past successes and failures’, Trends in Biotechnology 11, 471-478. Khalaf, N., Govardhan, C. P., Lalonde, J. J., Persichetti, R. A., Wang, Y. F. and Margolin, A. L. (1996), ‘Cross-linked enzyme crystals as highly active catalysts in organic solvents’, Journal of the American Chemical Society 118, 5494-5495. Khmelnitsky, Y. L., Welch, S. H., Clark, D. S. and Dordick, J. S. (1994), ‘Salts dramatically enhance activity of enzymes suspended in organic solvents’, Journal of the American Chemical Society 116, 2647-2648. Kim, J., Kosto, I. T. J., Manimala, J. C., Nauman, E. B. and Dordick, J. S. (2001), ‘Preparation of enzyme in polymer composites with high activity and stability’, AIChE Journal 41, 240-244. Klibanov, A. M. (1986), ‘Enzymes that work in organic solvents’, Chemtech 16, 354-359. Klibanov, A. M. (1997), ‘Why are enzymes less active in organic solvents than in water?’, Trends in Biotechnology 15, 97-101. Klibanov, A. M. (2001), ‘Improving enzymes by using them in organic solvents’, Nature 409, 241-246.

References

62

Kreiner, M., Moore, B. D. and Parker, M. C. (2001), ‘Enzyme-coated micro-crystals: a 1-step method for high activity biocatalyst preparation’, Chemical Communication, 1096-1097. Kresge, C. T., Leonowicz, M. E., Roth, W. J., Vartuli, J. C. and Beck, J. S. (1992), ‘Ordered mesoporous molecular sieves synthesized by a liquid crystal template mechanism’, Nature 359, 710-712. Krishna, S. H. (2002), ‘Developments and trends in enzyme catalysis in nonconventional media’, Biotechnology Advances 20, 239-267. Kwon, O. H., Imanishi, Y. and Ito, Y. (1999), ‘Catalytic activity and conformation of chemically modified subtilisin Carlsberg in organic media’, Biotechnology and Bioengineering 66, 265-270. Lee, J., Kim, J., Kim, J., Jia, H., Kim, M., Kwak, J., Jin, S., Dohnalkova, A., Park, H., Chang, H. N., Wang, P., Grate, J. W. and Hyeon, T. (2005), ‘Simple synthesis of hierarchically ordered mesocellular mesoporous silica materials hosting crosslinked enzyme aggregates’, Small 1, 744-753. Lei, C., Shin, Y., Liu, J. and Ackerman, E. J. (2002), ‘Entrapping enzyme in a functionalized nanoporous support’, Journal of the American Chemical Society 124, 11242-11243. Markvicheva, E. A., Lozinsky, V. I., Plieva, F. M., Kochetkov, K. A., Rumsh, L. D., Zubov, V. P., Maity, J., Kumar, R., Parmar, V. S. and Belokon, Y. N. (2005), ‘Gel-immobilized enzymes as promising biocatalysts: Results from indo-russian collaborative studies’, Pure and Applied Chemistry 77, 227-236. Matsubara, T., Fujita, R., Sugiyama, S. and Kawashiro, K. (2006), ‘Stability of proteases in organic solvents: structural identification by solid-state NMR of lyophilized papin before and after 1-propanol treatment and the corresponding enzymatic activities’, Biotechnology and Bioengineering 93, 928-933. Michels, P. C., Dordick, J. S. and Clark, D. S. (1997), ‘Dipole formation and solvent electrostriction in subtilisin catalysis’, Journal of the American Chemical Society 119, 9331-9335. Montanez, I., Alvira, E., Macias, M., Ferrer, A., Fonseca, M., Rodriguez, J., Gonzalez, A. and Barletta, G. (2002), ‘Enzyme activation in organic solvents: co-lyophilization of Subtilisin Carlsberg with methyl cyclodextrin renders an enzyme catalyst more active than the cross-linked enzyme crystals’, Biotechnology and Bioengineering 78, 53-59. Pandya, P. H., Jasra, R. V., Newalkar, B. L. and Bhatt, P. N. (2005), ‘Studies on the activity and stability of immobilized a-amylase in ordered mesoporous silica’, Microporous and Mesoporous Materials 77, 67-77. Partridge, J., Dennison, P. R., Moore, B. D. and Halling, P. J. (1998a), ‘Activity and mobility of subtilisin in low water organic media: hydration is more important than solvent dielectric’, Biochimica et Biophysica Acta 1386, 79-89.

References

63

Patridge, J., Halling, P. J. and Moore, B. D. (1998b), ‘Practical route to high activity enzyme preparation for synthesis in organic media’, Chemical Communication, 841-842. Persson, M., Wehtje, E. and Adlercreutz, P. (2002a), ‘Factors governing the activity of lyophilized and immobilized lipase preparations in organic solvents’, ChemBioChem 3, 566-571. Persson, M., Mladenoska, I., Wehtje, E. and Adlercreutz, P. (2002b), ‘Preparation of lipases for use in organic solvents’, Enzymes and Microbial Technology 31, 833-841. Persson, M., Wehtje, E. and Adlercreutz, P. (2000), ‘Immobilization of lipases by adsorption and deposition: high protein loading gives lower water activity optimum’, Biotechnology Letters 22, 1571-1575. Phillips, G. J. and Pettitt, B. M. (1995), ‘Structure and dynamics of water around myoglobin’, Protein Science 4, 149-158. Ragheb, A., Brook, M. A. and Hrynyk, M. (2003), ‘Highly activated, silicone entrapped, lipase’, Chemical Communication, 2314-2315. Ravindra, R., Zhao, S., Gies, H. and Winter, R. (2004), ‘Protein encapsulation in mesoporous silicate: The effects of confinement on protein stability, hydration, and volumetric properties’, Journal of the American Chemical Society 126, 12224-12225. Rees, G. D. and Halling, P. J. (2001), ‘Chemical modification probes accessibility to organic phase: Proteins on surfaces are more exposed than in lyophilized powders’, Enzymes and Microbial Technology 28, 282-292. Reetz, M. T., Zonta, A., Simpelkamp, J. and Konen, W. (1996), ‘In situ fixation of lipase containing hydrophobic sol-gel materials on sintered glass-highly efficient heterogeneous biocatalysts’, Chemical Communication, 1397-1398. Roy, I. and Gupta, M. N. (2004), ‘Preparation of highly active α-chymotrypsin for catalysis in organic media’, Bioorganic and Medicinal Chemistry Letters 14, 2191-2193. Roy, I., Sharma, A. and Gupta, M. N. (2004), ‘Obtaining higher transesterification rates with Subtilisin Carlsberg in nonaqueous media’, Bioorganic and Medicinal Chemistry Letters 14, 887-889. Ru, M. T., Dordick, J. S., Reimer, J. A. and Clark, D. S. (1999), ‘Optimizing the salt induced activation of enzymes in organic solvents: effect of lyophilization time and water content’, Biotechnology and Bioengineering 63, 233-241. Santos, A. M., Montanez-clemente, I., Barlett, G. and Griebenow, K. (1999), ‘Activation of serine protease Subtilisin Carlsberg in organic solvents: combined effect of methyl cyclodextrin and water’, Biotechnology Letters 21, 1113-1118. Schmid, A., Hollmann, A. F., Park, J. B. and B¨uhler, B. (2002), ‘The use of enzymes in the chemical industry in Europe’, Current Opinion in Biotechnology 13, 359-366.

References

64

Schmitke, J. L., Wescott, C. R. and Klibanov, A. M. (1996), ‘The mechanistic dissection of the plunge in enzymatic activity upon transition from water to anhydrous solvents’, Journal of the American Chemical Society 118, 3360-3365. Schmitke, J., Stern, L. J. and Klibanov, A. M. (1997), ‘The crystal structure of Subtilisin Carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile’, Proceedings of the National Academy of Science of the United States of America 94, 4250-4255. Soares, C. M., Teixeira, V. H. and Baptista, A. M. (2003), ‘Protein structure and dynamics in nonaqueous solvents: Insights from molecular dynamics simulation studies’, Biophysical Journal 84, 1628-1641. Straathof, A. J., Panke, S. and Schmid, A. (2002), ‘The production of fine chemicals by biotransformations’, Current Opinion in Biotechnology 13, 548-556. Suzawa, V. M., Khmelnitsky, Y. L., Giarto, L., Dordick, J. S. and Clark, D. S. (1995), ‘Suspended and immobilized chymotrypsin in organic media: structure function relationship revealed by electron spin resonance spectroscopy’, Journal of the American Chemical Society 117, 8435-8440. Taguchi, T. and Schuth, F. (2005), ‘Ordered mesoporous materials in catalysis’, Microporous and Mesoporous Materials 77, 1-45. Takahashi, H., Li, B., Sasaki, T., Miyazaki, C., Kajino, T. and Inagaki, S. (2000), ‘Catalytic activity in organic solvents and stability of immobilized enzymes depend on the pore size and surface characteristics of mesoporous silica’, Chemistry of Materials 12, 3301-3305. Tremblay, M., Cote, S. and Voyer, N. (2005), ‘Enhanced activity of α-chymotrypsin in organic media using designed molecular staples’, Tetrahedron 61, 6824-6828. Unen, D. J. V., Engbersen, J. F. J. and Reinhoudt, D. N. (2002), ‘why do crown ethers activate enzymes in organic solvents?’, Biotechnology and Bioengineering 77, 248–255. Unen, D. V., Engbersen, J. F. J. and Reinhoudt, D. N. (2001), ‘Sol-gel immobilization of serine proteases for application in organic solvents’, Biotechnology and Bioengineering 75, 154-158. Wang, P., Dai, S., Waezsada, S. D., Tsao, A. Y. and Davison, B. H. (2001), ‘Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis’, Biotechnology and Bioengineering 74, 249-255. Wang, P., sergeeva, M. V., Lim, L. and Dordick, J. S. (1997), ‘Biocatalytic plastics as active and stable materials for biotransformation’, Nature Biotechnology 15, 789-793. Wangikar, P. P., Michels, P. C., Clark, D. S. and Dordick, J. S. (1997), ‘Structure and function of subtilisin BPN solubilized in organic solvents’, Journal of the American Chemical Society 119, 70-76.

References

65

Yang, L., Dordick, J. S. and Garde, S. (2004), ‘Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity’, Biophysical Journal 87, 812-821. Yang, Z., Mesiano, A. J., Venkatasubramanian, S., Gross, S. H., Harris, J. M. and Russell, A. J. (1995b), ‘Activity and stability of enzymes incorporated into acrylic polymers’, Journal of the American Chemical Society 117, 4843-4850. Yang, Z., Williams, D. and Russell, A. J. (1995a), ‘Synthesis of protein-containing polymers in organic-solvents’, Biotechnology and Bioengineering 45, 10-17. Yennawar, N. H., Yennawar, H. P. and Farber, G. K. (1994), ‘X-ray crystal structure of chymotrypsin in hexane’, Biochemistry 33, 7326-7336. Yiu, H. H. P. and Wright, P. A. (2005), ‘Enzymes supported on ordered mesoporous solids: A special case of an inorganic-organic hybrid’, Journal of Materials Chemistry 15, 3690-3700. Yiu, H. H. P., Wright, P. A. and Botting, N. P. (2001), ‘Enzyme immobilization using SBA-15 mesoporous molecular sieves with functionalized surfaces’, Journal of Molecular Catalysis B:Enzymatic 15, 81-92. Yu, C., Fan, J., Tian, B., Zhao, D. and Stucky, G. D. (2002), ‘High-yield synthesis of periodic mesoporous silica rods and their replication to mesoporous carbon rods’, Advanced Materials 14, 1742-1745. Zaks, A. and Klibanov, A. M. (1988a), ‘The effect of water on enzyme action in organic media’, The Journal of Biological Chemistry 263, 8017-8021. Zaks, A. and Klibanov, A. M. (1988b), ‘Enzymatic catalysis in nonaqueous solvents’, The Journal of Biological Chemistry 263, 3194-3201. Zhao, D., Feng, J., Huo, Q., Melosh, N., Fredrickson, G. H., Chmelka, B. F. and Stucky, G. D. (1998), ‘Triblock copolymer syntheses of mesoporous silica with periodic 50 to 300 angstrom pores’, Science 279, 548-552. Zoungrana, T., Findenegg, G. H. and Norde, W. (1997), ‘Structure, stability and activity of adsorbed enzymes’, Journal of Colloids and Interface Science 190, 437- 448.

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