Enzymatic conversion

of plant biomass

Henrik Stålbrand

Department of Biochemistry

Lund University

Sweden

henrik.stalbrand@biochemistry.lu.se

www.lubiofuels.org

OUTLINE

1. Intro/Basics including basic enzyme knowledge and enzyme classes

2. Cellulases with general aspects for enzymatic plant cell wall conversion

3. Hemicellulases with current research & enzyme development

4. Bioinformatics: CAZy database of carbohydrate active enzymes

BIOREFINERY CONCEPTS

Cellulose, Hemicellulose

, Pectin, Lignin

THE PLANT CELL WALL

Cellulose

THE PLANT CELL WALL

Half life of cellulose decomposition: ”several millions of years”

In absence of enzymes, pH 7. D Wilson Curr. Opinion Microb., 14, 259 (2011)

Glycoside

Hydrolases (GHs)

Cellulases: Endo-glucanase

Exo-glucanase (CBH)

b-Glucosidase

Cellulose

Oligomers Sugars

ENZYMES:

Degradation:

24 h – a year

6 9

10 - 10

times faster

(a very rough estimate)

ENZYMES INCREASE THE RATE OF DEGRADATION

6 9

Cellulose degradation 10 - 10 times faster!

(Rough estimate)

ENZYMES INCREASE THE REACTION RATE

Glycoside

Hydrolases (GHs) Cellulase:

Endo-glucanase

Exo-glucanase (CBH)

b-Glucosidase

Cellulose

Oligomers Sugars

Enzyme

Active Site

Topologies:

Pocket Cleft Tunnel

Biofuels &

green chemicals

S P

S

P

G

Reaction coordinate

(”Reactant conformation”)

DG

Transition state

DG# =

Activation

energy

Cellulose

Sugars

ENZYMES LOWER THE ACTIVATION ENERGY

S P

S

P

G

Reaction coordinate

(”Reactant conformation”)

DG

Transition state

DG# =

Activation

energy

E

DG# (enz)

ENZYMES LOWER THE ACTIVATION EBERGY

Cellulose

Sugars

The first step: the enzyme (E) binds to the substrate (S) forming a ES complex

P=product

S

E

k1 k2 (kcat)

E+S ES E+P

k-1 k-2

Many enzyme reactions

can be described by

classical Michaelis-Menten (MM)

Kinetic model from1913

Basic introduction:

Berg et al ”Biochemistry” 7th ed

(2011) Freeman

THE ENZYME BINDS THE SUBSTRATE

ENZYMES HAVE A DEFINED SURFACE (ACTIVE SITE) with amino acids binding and interacting with the substrate

Enzymes are folded

proteins (=Polypeptides)

with a defined 3D structure

Amino acids interact

With the substrate

in the active site

Active site 1 nm

amino N terminal

C carboxy

terminal

Endoglucanase

BaCel5A from

Bacillus

agaradhaerens ( Varrot et al 2000,

JMB, 297, 819)

Gene

(DNA)

Protein

ADSORPTION TO INSOLUBLE SUBSTRATE BY ADDITIONAL (NON-CATALYTIC) BINDING SITE

Carbohydrate binding module (CBM) Catalytic module (CM) Review of cellulose-affinity CBMs:

Linder and Teeri, J Biotech, 57, 15 (1997)

General review: Boraston et al 2004

Specificity and affinity directed by several binding sites:

Picture: Divne et al

Glycosidic

bonds between Glc

units are cleaved

by hydrolysis

A water molecule

Gets activated in

the catalytic

process

Cellulose

Sugars

Cellulase Trichoderma

Cellobiohydrolase

(Cbh)

Processive

Glycoside

Hydrolases (GHs)

Cellulases: Endo-glucanase

Exo-glucanase (CBH)

b-Glucosidase

Cellulose

Oligomers Sugars

Enzyme

Active Site

Topologies:

Pocket Cleft Tunnel

Biofuels &

green chemicals

Glycoside

Hydrolases (GHs) Redox enzymes Cellulases:

Endo-glucanase

Exo-glucanase (CBH) Cellobiose dehydrogenase

b-Glucosidase Glucoseoxidase

GH61´s?

Hemicellualses:

b-Mannanase

b-Mannosidase

a-Galactosidase Galactose oxidase

Xylanase

XGH, XET (Lignin: laccase, lignin peroxidase)

Carbohydrate Polysacch. lyases (PLs)

Ester Hydrolases (CEs) Pectin deg. enzymes (> 15)

Acetylesterase

Pectin methyl esterase

Cellulose, Hemicellulose

Lignin, Pectin

Oligomers Sugars

New materials, polymers &

green products

Enzyme

Active Site

Topologies:

Pocket Cleft Tunnel

Biofuels &

green chemicals

POLYSACCHARIDE DEGRADATION

Hydrolases

a

b

b

11 338

Reviewed Gilbert et al 2008

Carbohydrate Esterases (CEs)

Glycoside Hydrolases (GHs)

Polysaccharide Lyases (PLs)

H2O

H2O

Lyases

CARBOHYDRATE OXIDOREDUCTASES

Example: Galactose oxidase

(EC 1.1.3.9)

Gal

Man-Man-Man-Man-Man-Man-Man-Man-Man*

Gal Gal

Galactose

Cupper enzyme, conserved Tyr

Whittaker et al Arch Bioch Biophys

433, 227

Guar galactomannan

oxidation:

Hartmans et al, ACS Symp 864, 360

Enzymklasser.

Tabell 8.8 sid 237

1. Cellobiose dehydogenase, Glucose

oxidase ,Lignin peroxidase , Laccase etc

3. Cellulases (endoglucanase,

cellobiohydrolase), Xylanases, Mannanases

Beta-glucosidase, alpha-galactosidase etc etc

4. Pectin lyases erc

OUTLINE

1. Intro/Basics including basic enzyme knowledge and enzyme classes

2. Cellulases with general aspects for enzymatic plant cell wall conversion

3. Hemicellulases with current research & enzyme development

4. Bioinformatics: CAZy database of carbohydrate active enzymes

CELLULOSE DEGRADATION

BY GLYCOSIDE HYDROLASES (GHs)

MAJOR CELLULASES

EG

CBH

Typical operational pH´s:

3.5 – 6 (fungal) 5 – 7.5 (bacterial) > 7.5 - 9 (extremophiles) Shanmughapriya et al 2010 Kumar et al 2008

Typical operational temperatures:

37-50 C (bacterial, fungal), > 65-70 C (extremophiles) Lee et al 2010 EMT 46 206 Approx. values

ACTIVE SITE TOPOLOGIES

Pocket Cleft Tunnel

Figure by: E. Sabini, PhD thesis York Univ

Endo-activity Exo-activities

Examples:Cel7B (EGI) Cel7A (CBHII)

Loop deletion:

Meinke et al 1995

JBC 270 4383

Small substrates

Exo-activity

EG CBHICBHII

Crystalline cellulose Amorphous celluloseEndohydrolysis:

Endoglucanases (EGs)

Exohydrolysis:

Cellobiohydrolases (CBHs)

Beta-glucosidase (BGs)

(hydrolyses cellobiose)

BG

EG - CBH synergy values 2-3 Steamed spruce: Karlsson et al 1999 Appl Bioch Biot 82 243

red Non-red

Glucose

Cellulose

Hydrolysis by

Cellulases (overview)

CELLULASES ACT IN SYNERGY Review Lynd et al (2002) Microb Mol Biol Rev 66 506, Hilden and Johansson 2002

CELLULOSE DEGRADING ENZYMES

Trichoderma reesei (aerobic fungus)

• More than 10 “free” cellulases are secreted

Trichoderma reesei (aerobic fungus)

EG´s (Endo-beta-1,4-glucanase) EC 3.2.1.4

CBH´s (cellulose-beta-1,4-cellobiase) EC3.2.1.91

Old Name New Name Enzyme in culture (%) Mw (kDa) C

B

M

CBHI Cel7A 50 59-68 +

CBHII Cel6A 20 50-58 +

EGI Cel7B 10 50-55 +

EGII Cel5A 10 48 +

EGIII Cel12A low 25 -

• Anaerobic bacteria (eg Clostridia) have cellulosomes, cell attached

multi-enzymes complexes (up to 30 or more enzymes).

ADSORPTION TO INSOLUBLE SUBSTRATE BY ADDITIONAL (NON-CATALYTIC) BINDING SITE (CBM)

Carbohydrate binding module (CBM) Catalytic module (CM) Review of cellulose-affinity CBMs:

Linder and Teeri, J Biotech, 57, 15 (1997)

General review: Boraston et al 2004

Specificity and affinity directed by several binding sites:

Picture: Divne et al

Glycosidic

bonds between Glc

units are cleaved

by hydrolysis

A water molecule

Gets activated in

the catalytic

process

Cellulose

Sugars

Cellulase Trichoderma

Cellobiohydrolase

(Cbh)

Processive K Igarashi et al 2011

AFM (to be publsihed)

Deletion of CBM:

5-fold decrease of activity

Hägglund et al

2003 J Biotech 101 37

ADSORPTION TO INSOLUBLE SUBSTRATE

IS CRUCIAL

•Adsorption equilibrium reached > 30 – 90 min Lynd et al 2002 Microb Mol Biol Rev 66 506

•Correlation with rate of hydrolysis

•Carbohydrate binding modules (CBM´s) important

(Review Linder &Teeri et al 1997, Boraston et al 2004)

•Problem: unproductive irreversible binding (eg lignin) Aid:Surfactant addition (PEG) Börjesson et al 2007 Investigated: Lignin removal

Stalbrand, H. et al. 1998. Appl. Environ. Microbiol. 64(7):2374-2379

CRYSTALIN CELLULOSE DEGRADED BY EROSION

AMORPHOUS CELLULOSE MORE ACCESSIBLE

EARLY OBSERVATIONS ON CELLULOSE

HYDROLYSIS BY ENZYMES

Reese et al J Bact 59 485 (1950) (Trichoderma)

Ryu & Mandels EMT 2 91 (1980) (Trichoderma)

C1 = substrate disruption factor

CX= hydrolases

Trichoderma

Lignocellulose Cellulose

Reese et al J Bact 59 485 (1950)

Ryu & Mandels EMT 2 91 (1980)

C1 = substrate disruption factor

CX= hydrolases

G Vaaje-Kolstad et al. Science 2010;330:219-222

C1 may be an oxidative

enzyme. Homologue

active against chitin

Enzymes in GH familiy 61 may be active

toward cellulose

EARLY OBSERVATIONS ON CELLULOSE

HYDROLYSIS BY ENZYMES

New observations on degrad

of recalitrant polysaccharides

HEMICELLULOSE CONVERSION

Glycoside Hydrolases (GHs)

Hemicellulases

Cellulases

HEMICELLULOSE CONVERSION

Glycoside Hydrolases (GHs)

Hemicellulases:

Xylanases

Cellulases

Synergy (6-fold) Patricia Orozco et al

submitted 2011

GALACTOGLUCOMANNAN (GGM)

-the major soft-wood hemicellulose

-Plant cell wall polysaccharide, associates with cellulose

-Major renawable resource, ~25% of wood dr wght

Gal Acetyl

Man-Man-Glc-Man-Man-Man-Glc-Man-Man-Man-Man*

Acetyl Gal Galactoglucomannan (GGM)

Xylan

Mannan

GALACTOGLUCOMANNAN (GGM)

-the major soft-wood hemicellulose

--Major renawable resource, ~25% of wood dr wght

-Numerous developing and potential applications:

Polymeric: films, emulsions, composites, bioplastics

Oligomeric: food additives, conjugates: surfactants

-Modification possible with specific enzymes: properties altered

Gal Acetyl

Man-Man-Glc-Man-Man-Man-Glc-Man-Man-Man-Man*

Acetyl Gal Galactoglucomannan (GGM)

Oxygen barrier

for food packaging?

Hydrogels for drug

delivery? (Roos et al

2008 Biomacrom 9 2104)

HOW CAN WE OBTAIN GGM?

Gal Acetyl

Man-Man-Glc-Man-Man-Man-Glc-Man-Man-Man-Man*

Acetyl Gal Galactoglucomannan (GGM)

1. Hot water or steam extraction of soft-wood chips

Extraction and structure determination of spruce GGM :

Man (1); Glc (0.3); Gal (0.1); Ac (0.3): DP 100-200

Lundqvist et al. Carb. Polymers, 48,281 (2002); Lundqvist et al Carb Polymers, 51, 203 (2003)

Could be combined with bioethanol process

2. Recovery from existing industrial side streams

Recovery from forestry: technical mechanical pulping (TMP)

e.g. Willfor et al (2003) TAPPI, 2, 27 & Andersson et al Appl Bioch Bioeng 136-140 971 (2007) Biological properties: Ebringerova et al (2008) Int J Biol Macromol 42 1-5

COST EVALUATION: 670 Euro/tonne (Persson et al 2006)

Process economy potentially better with GGM byproducts

GALACTOMANNAN (GGM) can be modified with hydrolases

Gal Acetyl

Man-Man-Glc-Man-Man-Man-Glc-Man-Man* (b-1,4)

Acetyl (C2,C3) Gal (a-1,6)

Glycoside hydrolases: Exo- a-galactosidase, Endo- b-mannanase

Clan D Clan A, Families 5, 26, 113

a-galactosidase Acetyl-esterase b-mannanase

b-MANNANASES HAVE ACTIVE SITE CLEFTS

* Reducing end

3D: Sabini et al 2000

Acta Cryst D 56 3-13

Donor binding (-) Acceptor binding (+)

+1

-1

-2

+2

3D fr lit: Sabini et al,2000, Act Cryst D, 56, 3

TrMan5A from

Trichoderma reesei

M

M 2

M 3

M 4

M 5 M 6

St M4 1 2 Tr

M

M 2

M 3

M 4

M 5 M 6

*)First report from litt: Harjunpää et al 1995 & 1999

Eur J Bioch 234 278 ; FEBS lett 443 149

Products: Rosengren , Stalbrand (submitted 2011)

TLC

Relative rates

(M6 build up)

1 0.15 0.2

Origin:

Cellulomonas fimi

(Soil bacterium)

Mytilus edulis

(Blue mussel,

a marine mollusc)

Trichoderma reesei *

(Soil filamentous fungus)

*) Aka Hypocrea jecorina

Enzyme:

CfMan26A

MeMan5A

TrMan5A

b-Mannanses of Glycoside Hydrolase

Clan A (GHA), families 5&26 Related (b/a)8- TIM-barrels

Le Nours et al 2005

Biochem 44 12703

Larsson et al

2006, JMB,357,1500

Sabini et al

2000, Act Cryst, 57, 3

b-MANNANASES USE A RETAINING

TWO-STEP MECHANISM

O O H

O O H O R

O H

R´

B-

A H

O O H

O H O R

O H

B

A-

O O H

O H O R

O H

B

A-

H

O H

O O H

O H O H O R

O H A H

B-

Nucleophile: Glu

+ R´-OH

Covalent

glycosyl-enzyme

intermediate

Acid/base: Glu

b-MANNASES CAN DISPLAY

TRANSGLYCOSYLATION ACTIVITY

O O H

O O H O R

O H

R´

B-

A H

O O H

O H O R

O H

B

A-

O O H

O H O R

O H

B

A-

R´´

O H

O O H

O -R´´ O H O R

O H A H

B-

+ R´-OH

Acid/base: Glu

Nucleophile: Glu Covalent

glycosyl-enzyme

intermediate

Donor Acceptor

R´´-OH = eg saccharide or alcohol acceptor

New glycoside/

glycoconjugate

334.0 409.2 484.4 559.6 634.8 710.0

Mass (m/z)

6593.4

0

10

20

30

40

50

60

70

80

90

100

% I

nte

nsit

y

(M4 + Na)

(M3 + Na)

(M2 + Na)

(M2-O-C2H5 + Na) (M3-O-C2H5)

365.0

724

527.1

062

393.1

004

689.1

381

555.1

341

374.9

698

381.0

439

544.9

439

360.9

814

TRANSGLYCOSYLATION

sugars and non-sugars acceptors

Trichoderma reesei b-mannanase (TrMan5A) incubated with M4 (Mannotetraose)

M4 M4 + EtOH

DP2

DP3

Conjugate DP2

Substrate, DP4

Conjugate DP3

Anna Rosengren

et al Biocat Biotransf

Accepted 2011

Man4-OH + HO-Et Man4-O-Et

MALDI-TOF MS

(DP=degree of polymerisation)

3297 20 mM

0

20

40

60

80

100

120

140

160

180

0 10 20 30 40 50 60

Time (min)

De

te

cto

r r

es

po

nc

e (

nC

)

3

7

2

4

5 6 8 9

Dionex HPAEC

M2 M3

M4

M5

M6

M7

M8

Origin:

Cellulomonas fimi

(Soil bacterium)

Mytilus edulis

(Blue mussel,

a marine mollusc)

Trichoderma reesei * (Soil filamentous fungus)

*) Aka Hypocrea jecorina

Enzyme:

CfMan26A

MeMan5A

TrMan5A

DIFFERENCES IN TRANSGLYCOSYLATION. WHY?

Le Nours et al 2005

Biochem 44 12703

Larsson et al

2006, JMB,357,1500

Sabini et al

2000, Act Cryst, 57, 3

Related (b/a)8- TIM-barrels

PROTEIN ENGINEERING OF CfMan26A: SHIFT OF SUBSTRATE BINDING AND TRANSGLYCOSYLATION INTRODUCED

3D: Le Nours et al (2005), Biochemistry, 44, 12707

+1

-3

-1 -2 -3 -4

CfMan26A Ala Phe

CfMan26-DM Arg Ala

CjMan26A* Arg Ala

*) Cellvibrio japonicus Man26A

Hogg et al JBC 276 31176 (2000)

-3: Phe 325 Ala

-2: Ala 323Arg

Omid Hekmat, Anna Rosengren et al

Biochemistry, 49, 4884, (2010)

CfMan26-DM (A323R, F325A) mimicks CjMan26A

Creation of a double mutant (DM):

PROTEIN ENGINEERING OF CfMan26A: SHIFT OF SUBSTRATE BINDING AND TRANSGLYCOSYLATION INTRODUCED

WT DM

82

18

31

61

8

33

58

13

88

12

85

11

4

ND

24

ND

11

52

ND

ND

4

-3 -2 -1 +1 +2

OO

OH

OHOH

OHO

O

OHOH

OH

OH

O

OO

OH

OHOH

non-reducing end internal reducing end

44

56

16

8

5

38

38

O Hekmat A Rosengren et al

Biochemistry, 49,

4884, (2010)

pH 6.0, 1 mM M3, 0.5 mM M4

Dominant binding mode for M4 changed

from -3 to +1 (WT) to -2 to +2 (DM)

Wild-

Type

(WT)

(%)

Double-

Mutant

(DM)

(%)

+1

-1

-2

+2

-4

-1

-2

-3

+1

+2 +1

-1

-2 -3

CfMan26A TrMan5A

MeMan5A

Subsite binding *

-3 -2 -1 +1 +2

TrMan5A - + + + + transglyc.

CfMan26A + ( +) + + - MeMan5A + + + + + transglyc.

Larsson et al 2006,

J Mol Biol, 357, 1500

Le Nours et al 2005,

Biochemistry, 44, 12707

3D:Sabini et al 2000,

Acta Cryst D, 56, 3

(from litt.)

*) 3D overlay analysis and complex structures

+2: Trp?/Trp

+2: Arg/Trp

PROTEIN ENGINEERING OF TrMAN5A

.... H-bonds

Mannobiose

R171 (in +2) (mutated to Lysine:

R171K)

Acid Catalyst:

Glu169

Stacking hydrofob:

Trp114

3D fr litt.

Sabini et al

2000, Act Cr.

57, 3

TrMan5A R171 is semi-conserved in family 5 Trichoderma IFAWELGNEPRCNG---------CSTDVIVQWATSVSQYVKSLDSNHLVTLGDEGL

Aspergillus IFAWELANEPRCQG---------CDTSVLYNWISDTSKYIKSLDSKHLVTIGDEGF

Agaricus VMAWELANEPRCKGSTG-TTSGTCTTTTVTNWAKEMSAFIKTIDSNHLVAIGDEGF

Geobacillus IMAWELANEPRNDSD--------PTGDTLVRWADEMSTYIKSIDPHHLVAVGDEGF

Orpinomyces IFSWQLANEARCNNGPHGLPVKNCNTDTITKWMDEIATFIHQEDPNHLVSSGIEGI

Mytilus LGGWDIMNEPEGEIKP-------GESSSEPCFDTRHLSGSGAGWAGHLYSAQEIGR

*:: ** ** *

Anna Rosengren et al

Biocat Biotransformation

Accepted 2011

PROTEIN ENGINEERING: pH OPTIMUM SHIFT

R171 (in +2) (mutated to Lysine:

R171K)

kcat

pH

3.5 4 5 6 7

300

200

100 WILD TYPE

PROTEIN ENGINEERING

ALTER OPTIMAL pH and TEMP, STABILITY

ALTERED FUNCTION(S) / SPECIFICITY

INCREASE ACTIVITY (eg LOWERED INHIBITION)

DECREASE UNPRODUCTIVE BINDING

INCREASE PRODUCTIVITY / SECRETION

CELLULASES AND HEMICELLULASES

THE CAZy DATABASE

www.cazy.org

Glycoside hydrolases: 115 families

classfied in 13 clans (A-M)