Deconstruction by Enzymes 1:Cellulase

2007 Oct. 19

Speaker: Dr. Po-Huang Liang

CONTENT

INTRODUCTION

FUNDAMENTALS-Structure and Composition of Cellulosic Biomass-Cellulolytic Organisms-Cellulase Enzyme Systemes-Regulation of Cellulase Production

APPLICATION OF CELLULASES-Recombinant Cellulolytic Strategy-Methodology for Studying Cellulase Properties

CLOSING COMMENT

INTRODUCTION

Need for Alternative Energy Source

With the hike in oil price around the world in the 1970s andthe realization that the world’s oil supply is finite,

the quest for alternative fuels began in 1975.

The amount of solar energy received at the earth’s surface far exceedsthe amount of present human usage

2.5x1021 Btu/year >> 2.0x1017 Btu/yearThe amount of energy from sun is stored as carbon via photosynthesis,

which results in production of plant biomass having cellulose as the major component

Strategy of converting cellulose into fuel

Glucose

Biofuel

Biomass

Cellulase

FUNDAMENTALS

Structure and Composition of Cellulosic Biomass

In plants, the cell wall is constructed primarily from a carbohydrate polymer called cellulose, and the cell wall can therefore also function

as a carbohydrate store for the cell.

genomics.energy.gov/gallery/gtl/originals/420.jpg

Plant cell wall structure

Plants form two types of cell wall that differ in function and in composition:- primary walls surround growing and dividing plant cells. - secondary wall is much thicker and stronger than primary wall and accounts for most of the carbohydrate in biomass.

Middle lamella is a specialize region associated with the cell walls and isshared by neighboring cells.Plasmodesmata is the small passages penetrate the middle larmella as well ass the primary and secondary cell walls, providing pathways for transporting cytoplasmic molecules from one cellto another. tainano.com/.../image036.gif

Plant cell wall structure

Primary Cell WallsThe main chemical components of the primary plant cell wall include cellulose.In addition, the cell wall contains two groups of branched polysaccharides, thepectins and cross-linking glycans. Organized into a network with the cellulose microfibrils, the cross-linking glycans increase the tensile strength of the cellulose, whereas the coextensivenetwork of pectins provides the cell wall with the ability to resist compression.The middle lamella is rich in pectins.

pectin

cross-linkingglycan

cellulose microfibrils

middle lamella

primary cell wall

plasma membrane

micro.magnet.fsu.edu/cells/plamts/cellwall.html

Plant cell wall structure

Secondary Cell WallThe secondary cell walls contain cellulose, hemicellulose and pectin as well as the primary cell walls, albeit in different proportion. The cellulose fibrils are embedded in a network of hemicellulose and lignin to make a further strengthened form.Numerous technical challenges must be overcome to enable the efficient utilization of secondary walls for energy production.

middle lamellaprimary cell wall

cellulose

hemicellulose

lignin

plasma membrane

protein

www.ccrc.uga.edu/~mao/intro/ouline.htm

Structure of cellulose

Cellulose is a linear condensation polymer consisting of glucose subunit linked by -1, 4-glycosidic bonds.

Cellobiose Glucose

Coupling of adjacent cellulose molecules by the hydrogen bonds and van der Waal’s forces results in a parallel alignment and a crystallinestructure.

Approximately 30 individual crystalline cellulose molecules are packed into larger units called microfibrils, which are in turn assembled into the familiar cellulose fibers.

Microfibril

7-30nm

Lignin

Hemicellulose

Elementarycellulose fibril

The microfibril chain are oriented in parallel and form highly ordered,crystalline domains interspersed by more disordered, amorphous regions.

Structure of cellulose

Crystalline domain Crystalline domainAmorphous region

The native, crystalline form of cellulose has a structure designated as type I, which can be converted to other crystalline forms (II-IV) by various treatments.Cellulose I can be transformed into cellulose II by alkali treatment and intocellulose III by supercritical ammonia treatment. Cellulose IV could be prepared by heat treatment in glycerol aftertransformation into cellulose II or cellulose III but cannot be transformed directly from cellulose I.Native cellulose has two distinct crystallite form, I and I, which differ in theirintermolecular hydrogen bonding pattern.Form I is dominant in bacterial and algal cellulose and form I is dominant in higher plants.

Cellulolytic Organisms

In plants, cellulases hydrolyze their cell walls at various developmental stages (e.g., bean abscission, fruit ripening and abscission, and pedicelabscission).For microorganisms and animals, hydrolysis and utilization of cellulose by cellulsas can provide usable energy to them.

Microorganism- Bacteria: fermentative anaerobes (Clostridium, Ruminococcus, Fibrobacter) aerobic gram-positive (Cellulomonas, Thermobifida) aerobic gliding (Cytophaga, Sporocytophaga)

Ruminococcus Cellulomonas CytophagaClostridium

Microorganism- Protozoa: ciliate (Diplodinium, Eudiplodinium)

Protozoa of Diplodinium and Eidinium type attached to fodder molecules in rumen liquid(Dobicki et al., 2006)

Piromyces Anaeromyces Chaetomium TrichodermaPhanerochaete

- Fungi: monocentric (Neocllimastix, Piromyces, Caecomyces) policentric (Orpimomyces, Anaeromyces) Ascomycetes (Bulgaria, Chaetomium, Helotium) Basidiomycetes (Coriolus, Phanerochaete, Serpula) Deuteromyces (Aspergillus, Cladosporium, Penicillium, Trichoderma)

Animal- Arthropods: termite (Coptotermes, Nasutitermes, Neotermes, Reticulitermes) cockroach (Cryptocercus, Panesthia) beetle (Ergates)

Symbioses - higher termite-bacteria - lower termite-bacteria-protozoa - lower termite-fungus

(Ohkuma, 2003)

Fungus(Termitomyces)

Termite nest(Odontotermes)

Nasutitermes Cryptocercus Ergates

Animal- Molluscs snail (Achatina, Helix, Levantina)

bivalve (Mytilus, Xylophaga)

sea slug (Dolabella)

marine periwinkle (Littorina)

Cellulase Enzyme SystemsSince cellulose cannot get into the cells, cellulolytic enzymes are by necessity secreted into the medium or bound to the outside surface of cellulolytic organisms.

General Feature Microorganisms - cellulase systems including a multiplicity of enzyme components - cellulase systems exhibiting higher collective activity than the sum of the activities of individual enzymes - most cellulases containing both catalytic and carbohydrate- binding modules - cellulases often associated with each other and with the surface of cellulolytic microorganisms Termite - endogeneous cellulase excreted from the salivary glands or the mid-gut - cellulases of termite origin belonging to glycosyl hydrolase family 9 - cellulases containing a single catalytic domain and lacking the ancillary domains such as cellulose-binding domain - cellulase systems consisting of endogeneous cellulases from termite and cellulolytic enzymes of gut protists

Components of cellulase systems

Cellulases are distinguished from other glycoside hydrolases by their ability to hydrolyze -1, 4-glucosidic bind between glucosyl residues.Based on mode of catalytic action and on structural properties, threemajor types of enzymatic activities are found:

(i) Endo--1, 4-glucanase (E.C. 3.2.1.4) cleaves at internal amorphous site in the cellulose polysaccharide chain, generating oligosaccharides of various lengths and consequently new chain end

(ii) Exoglucanase (cellodextrinases E.C. 3.2.1.74 or cellobiohydrolase E.C. 3.2.1.91) acts in a processive manner on the reducing or nonreducing ends of cellulose polysaccharide chain, releasing either glucose or cellulobiose

(iii) -Glucosidase (E.C. 3.2.1.21) hydrolyzes soluble cellodextrins and cellobiose to glucose

Components of cellulase systems

Reaction mechanismsIt is generally assumed that the hydrolysis reaction catalyzed by cellulase proceeding via an acid-base mechanism involving tworesidues, one as a general acid catalyst and another as a nucleophile.

Asp-201 Asp-201

Glu-555 Glu-555

Catalytic mechanism of C. thermocellum endoglucanase CelD as a paradigm of -glucanase acting(Beguin and Aubert, 1992))

Microorganism cellulase systemMicroorganism have adapted different approaches to effectivelyhydrolyze cellulose. There are two different systems:

Noncomplexed cellulase systemsThe microorganisms, such as cellulolytic filamentous fungi and actinomycete bacteria, have the ability to penetrate cellulolytic substrate and produce ”free” cellulases, with or without cellulose- binding modules.

Crystalline CrystallineAmorphous

Endoglucanase -glucosidase

Exoglucanase

Exoglucanase

CellobioseGlucose

Cello-oligosaccharides

Nonreducimg end

Reducing end

(Lynd et al., 2002)

Microorganism cellulase system

Complexed cellulase systemsThe microrganisms lack the ability to effectively penetrate cellulosic material and perhaps exists under a condition in the present of competition from other microorganisms and with limited ATP availablefor cellulase synthesis.This could have led to the development of “complexed” cellulasesystems (called “cellulosome”).

Crystalline CrystallineAmorphous

Cellobiose

Endoglucanase(with dockerin)

Exoglucanase(with dockerin)

Exoglucanase(with dockerin)

Cohesin moiety

Carbohydrate-binding module (CBM)

Cellobiose/cellodextrin phosphorylase

Glucose

Cello-oligosaccharides

Bacterium cell wall

Scaffodin

(Lynd et al., 2002)

Microorganism cellulase system

Cellulose-binding domain (CBD)CBDs provide a specific means for linking enzymes or other proteins on cellulose.

These domains are usually located at the NH2 or COOHterminus of the enzymes and are often separated from the catalytic domains by glycosylated, Pro/Thr/Ser-rich linker segments.Hydrogen bond formation and van Waals interactions are the main driving forces for binding.

In proteins that possess hydrolytic activity the CBD concentrates itscatalytic domains on the surface of the insoluble cellulose substrate.In proteins that have no hydrolytic activity, CBD is part of a scaffolding subunit that organizes the catalytic subunits into a cohesive multienzyme complex know as a cellulosome.

Based on amino acid sequences, binding specificity and structures, CBD can be divided to 16 different families among 48 differentcarbohydrate-binding modules.

Microorganism cellulase system

Cellulose-binding domain (CBD)Most of the CBDs found belong to the four major families:

Family I II III IV

Typical size 33-40 90-108 130-172 125-170

Shape wedge elongated barrels barrels -

Binding

reversiblebinds to surface

of both amorphous and

crystalline cellulose, not to soluble cellulose

irreversiblebinds to surface

of both amorphous and

crystalline cellulose, not to soluble cellulose

irreversiblebinds to surface

of both amorphous and

crystalline cellulose, not to soluble cellulose

reversiblebinds to single molecules of amorphous cellulose or

soluble cellulose, not to crystalline

cellulose

CharacteristicsExclusively CBDs from fungal en

zymes

Two sub-families (IIa and IIb)

CBDs from bacterial enzymes

Two sub-families (IIIa and IIIb)

CBDs from bacterial enzymes

Also found in cellulososme

CBDs from bacterial enzyme

s

(Shoseyov and Warren, 1997)

Microorganism cellulase system

CellulosomeThe plant cell wall degrading enzymes in most anaerobic microorganisms associate into a supramolecular complex, termedthe “cellulosome” with a molecular mass higher than 2 MDa.The cellulosomes are associated with the cell surface and mediate cell attachment to the insoluble substrate and degrade it to soluble products which are then absorbed.

cellulosomes

Cell membrane

S-layer

anchoring proteins

cellulosomes

Cell

cellulose

(Bayer et al., 1998)

Microorganism cellulase system

CellulosomeThe principal component of the cellulosome is a scaffoldin subunit that contains cohesin modules and also frequently includes a carbohydrate-binding module.The enzymatic subunits of the cellulosome contain a complementary type of module, the dockerin domain, that is responsible forattachment to the cohesin modules of scaffoldin.

bacterial cell

N

N

CC

Type I Dockerin

Type II Dockerin

Type I Cohesin

Type II Cohesin

Scaffoldin

SLH Module

X Module Cellulose-binding domainCatalytic domain

(Demain et al., 2005)

Microorganism cellulase system

CellulosomeRecently, several different modular structures of scaffoldin have beendescribed in various anaerobic microognisms.

1 2 3 4 5 6 7 8 9 X II

CBD

Clostridium thermocellum CipA

X

X

CBD

1 2 3 4 5 6 7 8

C. celluloticum CipC

CBD

II54321 6 7 8 9 10 11

Bacteroides cellulosolvens ScaA

1 2 3 4 5 6 7 8 9

XX X X

CBD

C. cellulovorans CbpA

GH91 2 3 4 5 6 7 X II

CBD

Acetivibrio cellulolyticus CipV

X

CBD

1 2 3 4 5 6

C. josui CipJ

Microorganism cellulase system

CellulosomeThe cellulosome of most anaerobic bacteria is in essence a cell-surface component which need a cell-surface protein to mediate theits binding. The most studied specie, Clostridium thermocellum, has been identified four cell-surface proteins and one scaffoldin that are relativeto this binding.

Gene product

Description

SdbA Scaffoldin-dockerin binding component A

Orf2p Cell surface glycoprotein 2

OlpB Outer layer protein component B

OlpA Outer layer protein component A

SlpA S-layer glycoprotein A

SdbA

Orf2pOlpB

OlpA

Bacterial Cell

(Demain et al., 2005) (Bayer et al., 1998)

Termite cellulase system

The presence of gut protists is important to termite survival on a dietof cellulose as their energy and carbon source.Termites grind and crunch their ingested material, which may enhance digestion by increasing the amount of surface that can beaccessed by cellulolytic enzymes.Probably, the ingested cellulose can be partially degraded by the endoglucanase of termite origin, and the cellulose not hydrolyzed in the anterior portion of the gut then travels to the hindgut, where it can be endocytosed and fermented by the symbiotic microrganisms.

Cellulolyticmaterial

GringCrunch

Mid-gut

endogeneous cellulase

Ingested cellulose

Hindgut

Cellulosedecomposition

Protist

CO2 H2

CH4

AcetateAbsorbed by termite

Regulation of Cellulase Production

Carbon Source Regulation

Cellulose and Derived MetabolitesFor most microorganisms, cellulase synthesis generally requires thepresence of cellulose or its soluble metabolites.Cellobiose functions as an inducer is more complex because at highlevel it inhibits cellulase production.In case of fungi, cellulose induces cellulase synthesis in germinatingconidia, but not in mycelium.Sophorose (-1,2-glucobiose), that is formed via the transglycosylation of cellobiose by a -glucosidase, was identified as astrong inducer of cellulase formation in fungi.

Easily Metabolized SubstratesIn most moicroorganisms, cellulase synthesis is blocked in presenceof soluble substrate such as glucose.

Intracellular Molecules

Transcriptional FactorsACEI and ACEII were identified the ability to bind to the promoterregion of fungal cellulase gene thus can stimulate the expression ofcellulase gene.

Gene ClusterIn the case of anaerobic bacteria, several gene clusters have been found, suggesting the existence of operons as units of generegulation.

Negative controlFor clostridia, three protein, GlyR1, GlyR2, and GlyR3 were identified as regulatory proteins containing two major domains, a sugar-binding domain and a DNA-binding domain.These proteins inhibit the cellulase production by binding to thepromoter region of cellulase gene.

Intracellular Molecules

Carbon Catabolite RepressorCre1 or CreA was identified as an inhibitor for the transcription of cellulase gene of fungus.

ATP and cAMPIn the case of fungi, extracellular cellulase was repressed at intracellular ATP concentration at a high level and cAMP played a role in derepression of enzyme synthesis.

Enzyme Inhibitor

Nojirimycin and GluconolactoneThe inhibition of -glucosidase activity by these components mayprevent induction by celloluse.

CellulasesSome cellulase enzymes may play a role in the formation of the inducer for other enzymes.

In T. reesei, Fowler and Brown (1992) suggested that BGL1 may be partially responsible for formation of the inducer because the deletion of bgl1 gene resulted in decreased endoglucanase activities. Later, Seiboth et al. (1997) revealed that deletion of cbh2 andeg2 genes prevented the expression of other cellulase genes.

In R. flavefaciens, Doerner et al. (1992) reported that the celA and celC genes were expressed constitutively while expression of the celBand celD was induced by cellulose.

In C. thermocellum, Mishra (1991) revealed that transcription of several cel genes was induced sequentially when cellobiose concentration in the medium became limiting. Transcription of celAstarted first, followed by celD and celF, and finally celC.

Summery

Cellulose first undergoes limited hydrolysis by cellulasesconstitutively produced in low amounts.

The soluble hydrolysis products thus generate and cause inductionof cellulase synthesis.

Catabolite repression of cellulase genes occurs in the presence ofglucose and may be regulated by cAMP.

APPLICATION OF CELLULASES

Methodology for Studying Cellulase Properties

Substrate for cellulase activity assays

Substrate for cellulase activity assays can be divided into 2 categories, based on their solubility in water.

Substrate Detection a Enzymes

Soluble

Short chain (low DP)

Cellodextrins

Radio-labeled cellodextrins

Cellodextrin derivatives

-methylumbelliferyl –oligosaccharides

p-nitrophenol-oligosaccharides

Long chain cellulose derivatives

Carboxymethyl cellulose (CMC)

Hydroxyethyl cellulose (HEC)

Dyed CMC

RS, HPLC, TLC

TLC plus liquid scintillation

Fluorophore liberation, TLC

Chromophore liberation, TLC

RS, viscosity

RS, viscosity

Dye liberation

Endo,Exo, BG

Endo,Exo, BG

Endo,Exo, BG

Endo,Exo, BG

Endo

Endo

Endo

a RS, reducing sugars; TSS, total soluble sugars. (Zhang et al., 2006)

Substrate Detection a Enzymes

Insoluble

Crystalline cellulose

Microcrystalline cellulose (Avicel)

Cotton

Valonia cellulose

Bacterial cellulose

Amorphous cellulose

Phosphorous acid swollen cellulose (PASC)

Alkali-swollen cellulose (RAC)

Dyed cellulose

Fluorescent cellulose

Chromogenic and fluorephoric derivatives

Trinitrophenyl-carboxymethylcellulose

(TNP-CMC)

Fluram-cellulose

Practical cellulose-containing substrates

-cellulose

pertreated lignocellulosic biomass

RS, TSS, HPLC

RS, TSS, HPLC

RS, TSS, HPLC

RS, TSS, HPLC

RS, TSS, HPLC, TLC

RS, TSS, HPLC, TLC

Dye liberation

Fluorophore liberation

Chromophore liberation

Fluorophore liberation

HPLC, RS

HPLC, RS

Total, Endo, Exo

Total, Endo, Exo

Total, Endo, Exo

Total, Endo, Exo

Total, Endo, Exo

Total, Endo, Exo

Total, Endo

Total

Endo

Total, Endo

Total

Total

Substrate for cellulase activity assays

a RS, reducing sugars; TSS, total soluble sugars. (Zhang et al., 2006)

Cellulase activity assays

All existing cellulase activity assays can be divided into three types:

1) the accumulation of products after hydrolysis - reducing sugars - total sugars - glucose

2) the reduction in substrate quantity - gravimetry - chemical methods

3) the change in the physical properties of substrates - swollen factor - fiber strength - structure collapse - turbidity - viscosity

Cellulase activity assays – accumulation of hydrolysis products

Reducing sugarsdepend on the reduction of inorganic oxidants such as cupric ions (Cu2

+) or ferricyanide, which accepts electrons from the donating aldehyde groups of reducing cellulose chain ends.

Method Sample (ml)

Reagent (ml)

G amount

(g/sample)

Ref.

DNS Micro

DNS Macro

Nelson-Somogyi Micro

Nelson-Somogyi Macro

Nelson Semi-Micro

Ferricyanide-1

Ferricyanide-2

PAHBAH Micro

PAHBAH Macro

BCA

Modified BCA

1-3

0.5

1-5

2

2

1-3

1

0.5

0.01

0.5

1

3

3

2+2

2+2

2

1+5

0.25

1.5

3

0.5

1

20-600

100-2500

1-10

10-600

5-100

1-9

0.18-1.8

0.5-5

5-50

0.2-4.5

0.4-9

Miller, 1959

Ghose, 1987

Somogyi, 1952

Somogyi,1952

Nelson, 1944

Park & Johnson, 1949

Kidby & Davidson, 1973

Lever, 1972

Lever, 1972

Waffenschmidt & Janeicke, 1987

Zhang & Lynd, 2005

G: reducing sugar; DNS: dinitrosalicyclic acid; PAHBAH: 4-hydroxybenzoylhydrazine; BCA: 2,2’-bicinchroninate

The common colorimetric reducing sugar assays

Cellulase activity assays – accumulation of hydrolysis products

Total soluble sugarsPhenol or anthrone in the presence of sulfuric acid can be used for the quantitative colorimetric microdetermination of sugars and theirmethylderivatives, oligosaccharides, and polysaccharides, to give an orange-yellow or blue-green color.

Method Sample (ml)

Reagent (ml)

G amount

(g/sample)

Ref.

Phenol-H2SO4

Anthrone-H2SO4

1

1

1+5

1+5

5-100

5-100

Dubois et al., 1956;

Zhang & Lynd, 2005

Roe, 1955;

Viles & Silverman, 1949

G: reducing sugar

The common colorimetric total sugar assays

Cellulase activity assays – accumulation of hydrolysis products

Glucose Assay

Enzymatic Glucose Assaysdepend on the glucose oxidase-peroxide reaction for the determination of glucose concentrations by using coupled hexokinase (HK) and glucose-6-phosphate dehydrogenase(PGHD).

Method Sample (ml)

Reagent (ml)

G amount

(g/sample)

Ref.

Glucose-HK/PGHD kit

Glucose-HK/PGHD kit

0.01

0.5

1

0.5

2-50

2-50

Sigma kit

Zhang & Lynd, 2004

G: reducing sugar

The colorimetric enzymatic glucose assays

HPLCAfter post-hydrolysis conversion to glucose

Cellulase activity assays – loss of substrate

Gravimetryuses precipitation or volatilization method based on the determination of a substance of known composition that is chemically related to the sugar. The standard deviation of this method is strongly associatedwith sample weight.

Chemical Methodincludes the phenol-H2SO4 and the anthrone-H2SO4 method for residual cellulose, and HPLC quantitative saccharification for differentcarbohydrate components.

Cellulase activity assays – physical cellulose properties

Swollen Factormeasures by alkali uptake.

Structure Collapsemeasures the reduction in tensile strength of cellulotic fiber.

Turbiditymeasures a reduction in the absorbance of particle suspension duringthe hydrolysis process. Amorphous cellulose is recommended for this assay.

Viscositymeasures a reduction in substrate viscosity.Soluble cellulose derivatives are recommended for this assay.

Endoglucanase activity assays

Endoglucanases cleave intramolecular -1,4-glucosidic linkages randomly, and their activities can be measured based on a reductionin substrate viscosity and/or an increase in reducing ends determined by a reducing sugar assay. Because exoglucanases also increase the number of reducing ends, it is strongly recommended thatendoglucanase activities be measured by both methods. CMC, a soluble high DP (degree of polyerization) cellulose derivative, is oftenrecommended as a good substrate for endoglucanase activities.

Soluble oligosaccharides and their chromophore-substituted substrates, such as p-nitrophenyl glucosides and methylumbelliferyl--D-glucosides, are also to measure endoglucanase activities based on the release of chromophores or the formation of shorter oligosaccharide fragments, which are measuredby HPLC or TLC.

Endoglucanase activities can also be easily detected on agar plates by staining residual polysaccharides (CMC, cellulose) with various dyes, such as Congo red, because these dyes are adsorbed only bylong chains of polysaccharides.

Exoglucanase activity assays

Exoglucanases cleave the accessible ends of cellulose molecules to liberate glucose and cellobiose. During chromatographic fractionation of cellulase mitures, enzymes with little activity on soluble CMC, but showing relatively high activity on Avicel, are usually identified as exoglucanase. There is no substrates specific for exoglucanase withinthe cellulase mixtures.

Soluble oligosaccharides and their chromophore-substituted substrates, such as p-nitrophenyl--D-cellobioside, 4-methylumbelliferyl--D-lactoside, 4-methylumbelliferyl--D-aglycones and 4-methylumbelliferyl--D-glycosides, are also to measure exoglucanase activities, which can be differentiated from endoglucanase activities by the aid of their specific inhibitor such as cellobiose. But different exoglucanases have different activities onthese substrates.

glucosidase activity assays

-glucosidase hydrolyze soluble cellobiose and other cellodextrins in the aqueous phase. These enzymes are very amenable to a wide range of simple sensitive assay methods, based on colored or fluorescent products released from p-nitrophenyl -D-1,4-glucopyranoside, -naphthyl--D-glucopyranoside, 6-bromo-2-naphthyl--D-glucopyranoside, and 4-methylumbelliferyl--D-glucopyranoside.

Cellobiose, which is not hydrolyzed by endoglucanases and exoglucanases, is also used as a substrate for -glucosidase activityassays by determining the increase of reducing sugar.

Total cellulase activity assays

The total cellulase activity assays are always measured using insoluble substrates, including pure cellulosic substrates such as Whatman No. 1 filter paper, cotton fiber, microcrystalline cellulose, bacterial cellulose, algal cellulose; and cellulose-containing substrates such as dyed cellulose, -cellulose, and pretreatedlignocellulose.

The most common total cellulase activity assay is filter paper assay (FPA) which requires a fixed amount (2mg) of glucose released from a50-mg sample of Whatman No.1 filter paper. -cellulose and pretreated lignocellulose are often used to evaluate the digestibilityof a reconstituted cellulase mixture for a prolonged reaction.

Dyed celluloses and fluorescent-dyed celluloses are widely used fordetermining sugar inhibition for total cellulase.

Recombinant Cellulolytic Strategy

Fundamentals ofmicrobial cellulose

utilization

Underlying fundamental issue:Understand cellulose

hydrolysis at a microbialrather than an enzymatic level

Engineered strains able toutilize cellulose and produce

a desired product at high yield

Microbes with goodsubstrate utilization properties:

Cellulase production, utilizationof hydrolysis products

(e.g. thermophiles)

Microbes with goodproduct-producing properties:

High product yields, titers ect.(e.g. yeasts)

Native strategy:Metabolic engineering to

improve product yields, titer ect.

Recombinant strategy:Heterologous cellulase

expression

Strain characterizationand improvemetn

Applied objective:CBP-compatible strains

of use for industrialprocesses

(Lynd et al., 2005)

Strain-donor

The most studied cellulolytic organisms and their properties are listedas follow:

Specie Function

Acidothermus cellulolyticus

Clostridium thermocellum

Coptotermes formosanus

Erwinia chrysanthemi

Humicola grisea

Nasutitermes takasagoensis

Reticulitermes speratus

Thermoascus aurantiacus

Thermobifida fusca

Trichoderma reesei

thermostable cellulase

cellulosome

animal cellulase

high activity cellulase

thermostable cellulase

animal cellulase

animal cellulase

thermostable cellulase

cellulase system

cellulase system

Strain-host

The most studied host microorganisms and their functions are listedas follow:

Specie Function

Aspergillus oryzae

Clostridium cellulolyticum

Clostridium thermocellum

Escherichia Coli

Fusarium oxysporum

Klebsiella oxytoca

Neurospora crassa

Saccharomyces cerevisiae

Thermoanaerobacterium thermosaccarolyticum

Thermoanaerbacterium saccharolyticum

Trichoderma reesei

Zymomonas mobilis

overexpression

fermentation

fermentation

overexpression and

fermentation

fermentation

fermentation

fermentation

overexpression and

fermentation

fermentation

fermentation

expression

Fermentation

Cellulase improvementTwo methods are available for improving the properties of individualcellulase components:1) rational design and 2) directed evolution.

Rational designis the eariliest approach to protein engineering and requires detailedknowledge of protein structure.

(Zhang et al., 2006)

Cellulase improvement

Directed evolutionis independent of knowledge of enzyme structure and of interactions between enzyme and substrate, and is developing tools to correctly evaluate the performance of mutants generated by recombinant DNAtechniques.

(Zhang et al., 2006)

Cellulase improvement- cellulase structure

Cellulase improvement- cellulase structure

Figure7Stereo representation of environment of the catalytic acid/base residue Glu412 at pH 5.6 and 6.5. The carboxylate group of Glu412 at pH5.6 is in purple and at pH 6.5 is in green. The simulated-annealing omit map density for Glu412 is contouredat 0.75.

Cellulase improvement- cellulase structure

The cellulosome is a macromolecular machine, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose. The cellulsome complex is composed of numerous kinds of cellulases and related enzyme subunits, which are assembled into the complex by virtue of a unique type of scaffolding subunit (scaffoldin). Each of the cellulosomal subunits consists of a multiple set of modules, two classes of wich (dockerin domains on the enzymes and cohesin domains on scaffoldin) govern the incorporation of the enzymatic subunits into the cellulosome complex. Another scaffoldin module-the cellulose-binding domain-is responsible for binding to the substrate. Some cellulosomes appear to be tethered to the cell envelop via similarly intricate, multiple-domain anchoring proteins. The assemblage is organized into dymatic polycellulosomal organelles, which adorn the cell surface. The cellulosome dictates both the binding of the cell to the substrate and its extracellular decomposition to soluble sugars, which are thentaken up and assimilated by normal cellular processes.

Cellulase improvement- cellulase structure

FIG. 1. Schematic representation of cellulosome organization and attachment to the C. thermocellum cell surface. The scaffoldin protein of C. therocellum, shown in yellow, is composed primarily of nine copies of cohesin module, a Family-IIIa CBD and a type-II dockerin domain. The high-resolution crystal structures of the former tow domains have been solved and are shown in yellow in the insets. The cellulose-binding, planar aromatic strip of the CBD, and the putative dockerin-binding residues of the cohesin are highlighted in red. The schematic cellulosomal catalytic subunits are shown in shades of blue, green, and purple; the crystal structures of five of the known enzymes are shown in the large inset the top of the page. From left to right they are: endoglucanase A from C. cellulolyticum (PDB code 1EDG), cellobiohydrolase I from Trichroderma reesei (1CEL), endoglucanase E2 from Thermomonospora fusca (1TML), endoglucanase V from Humicola insolens (3ENG), and endoglucanase CelA from C. thermocellum (1CEM) These structures were chosen as representatives of the five classes of protein folds seen to date for the cellulosomal catalytic subunits, as designated at the top of each structure. The enxymes are shown bound to the scaffoldin protein via theirattached and highly conserved, type-I dockerin domains. A proposed structural model for the calcium-dependent dockerin domain, based on the EF-hand structure of troponin C (PDB code 5TNC), is shown modeled in green in the inset. The calcium ligands are shown as white spheres, and predicted positions of cohesin-recognition residues are color-coded red. The entire cellulosome, comprising the scaffoldin protein and the catalytic subunits, is bound to the cell surface (left) in either single or mutiple copies by interaction of its resident type-II dockerin with type-II cohesin domains of cell-surface anchoring proteins-SdbA, Orf2, and OlpB, shown in orange. Each of these components contains an SLH module, which anchors the parent protein and the attached scaffoldin, together with its complement of enzymes, to the cell surface. Another SLH-containing protein, OlpA, bears a type-I cohesin, which apparently serves to anchor a singlecellulase to the cell exterior.

Cellulase improvement- properties of cellulase

Cellulase improvement- properties of cellulase

Cellulase improvement- properties of cellulase

G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose; G6, cellohexaose

Cellulase improvement- properties of cellulase

Cellulase improvement- properties of cellulase

Cellulase improvement-increased specific activity

Cellulase improvement-increased specific activity

The level of saccharification of cellulose by T. reesei in the presence of recombinant H. grisea BGL4 was 1.4-2.2 times higher than in itsabsence.

Cellulase improvement-increased specific activity

Cellulase improvement-increased specific activity

Cellulase improvement-improved cellulase binding

In recent work (Fierobe, H.-P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaïch, A., Lamed, R., Shoham, Y., and Belaich, J.-P. (2002) J. Biol. Chem. 277, 49621–49630), we reported the self-assembly of a comprehensive set of defined "bifunctional" chimeric cellulosomes. Each complex contained the following: (i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates (microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes (from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on acomplex substrate.

Cellulase improvement-improved cellulase binding

Cellulase improvement-improved cellulase binding

Cellulase improvement-increased thermostability

Cellulase improvement-increased thermostability

Cellulase improvement-increased thermostability

Fig. 3. Thermal inactivation curve of the wild-type Clostridium thermocellum cellulase C (■, t1/2=3.28 min) and the disulfide+mutant (X, t1/2=9.85 min). t1/2 is the time while the enzyme loses 50% of its activity during incubation at 70℃. Incubations were performed in 20 mM tris buffer, pH 7.2 at 70 . The measurements were carried out at 60 ℃

in pH 6.0 succinate buffer, containing 0.2 mM ℃pNPC. The final proteinconcentration was 0.008 mg/ml.

Fig. 4. Thermal inactivation curve of the wild-type Clostridium thermocellum cellulase C (■, T1/2=68.0 ) and the disulfide+mutant (℃ X, T1/2=69.9 ℃). T1/2 is the time while the enzyme loses 50% of its activity during 10-min incubation Incubations were performed in 20 mM tris buffer, pH 7.2 for 10 min at increasing temperatures. The measurements were carried out at 60 in 100 mM succinat℃e buffer at pH 6.0, containing 0.2 mM pNPC. The final protein concentrationwas 0.008 mg/ml.

CLOSING COMMENT

After 20 years of research and development, application of cellulase system during production of bioethanol is becoming a reality. But reducing the cost of cellulase enzyme production is still a problem inthe establishment of commercial process. Genetic techniques are one solution to create new cellulase production systems with possibleimprovement of enzyme yield and activity. It will be interesting to see whether future approaches will be:

to identify new cellulases with more powerful ability to hydrolyze cellulose materials from cellulolytic organisms

to improve present cellulases properties such as thermostablity,specific activity, cellulose-binding ability or synergistic action

to establish a single microorganism strain or microbial systemwhich is able to utilize both cellulose and fermentable compound

CLOSING COMMENT

(Bayer et al., 2007)