ACTAUNIVERSITATISUPSALIENSISUPPSALA2006

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 185

Hydrolytic and OxidativeMechanisms Involved in CelluloseDegradation

ANU NUTT

ISSN 1651-6214ISBN 91-554-6571-4urn:nbn:se:uu:diva-6888

To my family

List of Papers

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I Nutt, A., Sild, V., Pettersson, G. and Johansson, G. (1998) Pro-gress curves. A mean for functional classification of cellulases. European Journal of Biochemistry, 258, 200-206.

II Väljamäe, P., Sild, V., Nutt, A., Pettersson, G. and Johansson, G. (1999) Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and en-doglucanase I. European Journal of Biochemistry, 266, 327-334.

III Henriksson, G., Nutt, A., Henriksson, H., Pettersson, B., Ståhl-berg, J., Johansson, G. and Pettersson, G. (1999) Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium cellulase. European Journal of Biochemistry, 259, 88-95.

IV Nutt, A., Salumets, A., Henriksson, G., Sild, V. and Johansson, G. (1997) Conversion of O2 species by cellobiose dehydrogenase (cellobiose oxidase) and glucose oxidase - a comparison. Bio-technology Letters, 19, 379-383.

V Nutt, A., Nilsson, M., Väljamäe, P., Ståhlberg, J., Isaksson, R. and Johansson, G. o-nitrophenyl cellobioside as an active site probe for family 7 cellobiohydrolases. Manuscript.

Papers I-III were reproduced with kind permission of Blackwell Publishing. Paper IV was reproduced with kind permission of Springer Science and Business Media. The typographical corrections were approved by the pub-lisher and the Editor-in-Chief of the Journal.

Contents

Introduction.....................................................................................................9Cellulose.....................................................................................................9

Chemical structure .................................................................................9Crystalline structure.............................................................................10

Other components of plant cell wall.........................................................11Cellulases .................................................................................................12

Biological degradation of cellulose .....................................................13Classification of cellulases ..................................................................13Fungal cellulases..................................................................................16Specific aspects of cellulase kinetics ...................................................17Hypocrea jecorina cellulases...............................................................20Phanerochaete chrysosporium cellulases............................................23Phanerochaete chrysosporium cellobiose dehydrogenase ..................25

Present investigation .....................................................................................27Aims of the present study.........................................................................27Action of cellulases on end-labelled cellulose (Paper I) ..........................28Influence of acid pre-treatment of bacterial cellulose on mode of synergy between Cel7A (CBH I) and Cel7B (EG I) (Paper II)................29Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporiumcellulase (Paper III) ..................................................................................31Conversion of oxygen species by cellobiose dehydrogenase (Paper IV).33o-nitrophenyl cellobioside as an active site probe for family 7 cellobiohydrolases (Paper V) ...................................................................36Conclusions ..............................................................................................38

Summary in Swedish ....................................................................................39

Acknowledgements.......................................................................................41

References.....................................................................................................43

Abbreviations

BC bacterial cellulose BMCC bacterial microcrystalline cellulose CBD cellulose binding domain CBH Cellobiohydrolase CBM carbohydrate binding module CD catalytic domain CDH cellobiose dehydrogenase CMC carboxymethyl cellulose DP degree of polymerisation EG Endoglucanase GH glycosyl hydrolase GOX glucose oxidase kcat catalytic constant Ki inhibition constant KD dissociation constant KM Michaelis-Menten constant MeUmb (Glc)2 methylumbelliferyl cellobioside oNPC o-nitrophenyl cellobioside pNPC p-nitrophenyl cellobioside pNPL p-nitrophenyl lactoside

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Introduction

Cellulose Cellulose is the most abundant biopolymer on earth. It is the main structural component of plant cell walls, constituting up to 50% of the mass in trees. Apart from vascular plants, cellulose is also produced by most groups of algae, the slime mold Dictyostelium, a number of bacterial species and by tunicates. Despite the simple chemical structure, the physical properties of cellulose, such as the crystalline state, degree of crystallinity and molecular weight are highly variable.

Chemical structure Cellulose is a linear polymer composed of D-glucose residues joined by -1,4-glucosidic bonds. The cellulose molecule forms a straight, almost fully extended chain, where glucose residues are rotated 180° relative to each other along the main axis, which means that the repetitive unit is the glucose dimer, cellobiose, rather than glucose (Fig. 1). Although glucose is a highly water-soluble molecule, the solubility of cellodextrines decreases rapidly with the degree of polymerisation, cellohexaose already being only slightly soluble. Each chain is stabilised by intrachain hydrogen bonds formed be-tween the pyranose ring oxygen in one residue and the hydrogen of the OH-group on C3 in the next residue (O5...H-O3’) and between the hydroxyls on C2 and C6 in the next residue (O2-H...O6’) [Gardner and Blackwell, 1974].

Figure 1. Molecular structure of the cellulose. Reproduced from [Hildén and Jo-hansson, 2004] with kind permission from the publisher.

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Crystalline structure Cellulose, as all carbohydrates, has both hydrophilic (from the HO-groups) and hydrophobic (from the HC-groups) character [Sundari et al., 1991]. Strong inter- and intramolecular O H···O bonds retain the chains straight and stacked in a sheet-like structure. Individual chains co-crystallise together shortly after biosynthesis into highly crystalline microfibrils held together by hydrogen bonds, hydrophobic interactions and van der Waals forces [Brown, Jr. and Saxena, 2000].

The shape of the cellulose microfibril, where the chains are running in parallel, is determined by the geometry of the cellulose synthase complex and by the local environment [Doblin et al., 2002]. In plants, the unit mi-crofibrils are about 3 nm wide and contain around 36 cellulose chains and are often packed into larger, 20-100 nm microfibril bundles in the secondary cell wall. Interestingly, in certain algae, the microfibril width has been re-ported to be up to 20 nm [Jarvis, 2003]. Bacterial cellulose (BC) synthesised by the bacterium Acetobacter xylinum is a long ribbon with a diameter of about 40-60 nm, consisting of microcrystals with a width of about 3.0 x 6.8 nm [White and Brown, Jr., 1981].

Figure 2. A schematic diagram representing the differences between the monoclinic and triclinic forms of cellulose I. Each rectangle represents a single glucose unit. In the monoclinic form, the cellobiose units stagger with a shift of a quarter of the c axis period, whereas the triclinic form exhibits a diagonal shift of the same amount. Two spacings and angles are given, the first referring to the (100) face and the sec-ond to the (010) face of the triclinic crystal. Reproduced with the permission from [Baker et al., 1997].

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In nature, most cellulose is produced as crystalline and is defined as cellu-lose I. It is composed of two distinct crystalline forms (triclinic cellulose Iand monoclinic cellulose I , which differ from each other in their crystal packing, molecular conformation and intermolecular hydrogen bonding pat-terns (Fig. 2) [Atalla and Vanderhart, 1984; Sugiyama et al., 1991; Heiner et al., 1995]. These differences may influence the physical properties of the cellulose [Nishiyama et al., 2003]. The ratio of the two phases depends on the origin of the cellulose. The I form is dominant in cellulose produced by primitive organisms, such as the bacterium Acetobacter xylinum and the alga Valonia macrophysa, whereas the I form dominates in cellulose produced by higher plants [Atalla and Vanderhart, 1984]. The I form is more stable than the I form, which has been reported to be more susceptible to enzy-matic hydrolysis [Hayashi et al., 1997].

The size of an exposed cellobiose unit on the cellulose surface is ca 1x0.5 nm. Native cellulose also contains less ordered, amorphous or paracrystalline regions. Amorphous cellulose has not been studied widely, but it is thought to be held together by hydrogen bonds between the C2, C3 and C6 hydroxyl groups [Kondo and Sawatari, 1996].

Other components of plant cell wall In the plant cell wall, the cellulose microfibrils are embedded into a matrix, cross-linked mainly by hemicellulose and pectin (Fig. 3). Wood cells contain also lignin, a non-polysaccharide polymer. In wood, hemicellulose and lig-nin comprise 20 to 25 and 5 to 30% of the plant dry weight [Sjöström, 1993].

HemicelluloseHemicellulose is used as a common name for a large number of different carbohydrate heteropolymers, of which xylans and glucomannans are the main components. It is a heterogeneous mixture of different polysaccharides and the composition varies depending on the plant type. In contrast to cellu-lose, which itself is crystalline, strong and resistant to hydrolysis, hemicellu-lose is a highly branched and amorphous structure with little inherent strength. Apart from glucose, it may contain mannose, xylose, arabinose, rhamnose and L-fucose.

LigninLignin is a highly branched random polymer of coniferyl, sinapyl and p-coumaryl alcohols generated by radical polymerisation. In wood, lignin is bound covalently to the side groups of different hemicelluloses by ester- or ether bonds and forms a matrix surrounding the cellulose microfibril.

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PectinPectin is an important part of a fruit cell wall, but it is present in all plant cell walls. Pectin is composed of “smooth regions”, consisting of -1,4 linked galacturonic acid residues and “hairy regions”, consisting mainly of rham-nogalacturonan I, II and xylogalactouronan.

Figure 3. The structure of wood. Adapted with modifications from [Harrington, 1998].

Cellulases Cellulases are O-glycosyl hydrolases (GHs) that hydrolyse -1,4-glucosidic bonds in cellulose. Cellulose degradation is brought about mainly by bacte-ria, fungi and protozoa, but the production of cellulases is documented also in plants and in a number of invertebrate taxa that includes insects, crusta-ceans, annelids, molluscs, mussels and nematodes [Watanabe and Tokuda, 2001; Davison and Blaxter, 2005].

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Biological degradation of cellulose In wood, crystalline cellulose microfibrils are tightly packed in a complex network of hemicellulose constituents and lignin. Most cellulolytic micro-organisms produce, in addition to cellulases that hydrolyse the -1,4-glucosidic bonds, a number of other cell-wall-degrading enzymes, e.g. ligni-nases, xylanases, pectinases, etc. Only a few micro-organisms produce a complete set of enzymes capable of degrading native cellulose efficiently. Aerobic and anaerobic micro-organisms use different strategies to feed on cellulose. Whereas aerobes generally secrete a set of individual cellulases, some anaerobes have evolved a multi-enzyme complex- cellulosome which is associated with the cell surface of the micro-organism, reviewed recently by Bayer et al. [Bayer et al., 2004].

The cellulolytic enzyme systems in fungi can be divided into two groups. The white-rot fungi, such as Phanerochaete chrysosporium and soft-rot fungi, such as Hypocrea jecorina (formerly known as Trichoderma reesei)and Penicillum pinophilum have complete cellulolytic enzyme systems ca-pable of the breakdown of crystalline cellulose to glucose. They consist of several secreted enzymes acting at the ends (exoglucanases) or in the middle (endoglucanases) of the cellulose chains. The released cellobiose is hydro-lysed to glucose by -glucosidases. The second group of fungi reportedly degrade cellulose by means of oxidative components together with endoglu-canases, but lack the strict cellobiohydrolases. A representative of this mechanism is the cellulolytic system of the brown-rot fungus Postia pla-centa [Kleman-Leyer et al., 1992].

Classification of cellulases Cellulases can be classified by different means, according to their substrate specificities, reaction mechanisms or structural similarities.

Functional classification Cellulases have traditionally been classified into two distinct classes: cello-biohydrolase (1,4- -D-glucan cellobiohydrolase, EC 3.2.1.91) and endoglu-canase (1,4- -D-glucan glucanohydrolase, EC 3.2.1.4), based on their activ-ity toward a wide range of substrates. This is rather difficult, since the en-zymes have overlapping specificities toward substrates which themselves are poorly defined.

By definition, cellobiohydrolases release cellobiose from the non-reducing ends of the cellulose chain, but the experimental evidence for this assumption is obscure. Enzyme kinetics on soluble oligosaccharides and structural data on enzyme-oligosaccharide complexes show that some cello-biohydrolases may have opposite chain-end preferences [Barr et al., 1996; Divne et al., 1998; Koivula et al., 1998]. Cellobiohydrolases are thought to

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work processively, that is, one enzyme molecule can release several cello-biose units from the cellulose chain without leaving the substrate. Cellobio-hydrolases show little or no activity on substituted celluloses, such as CMC, but microcrystalline cellulose with relatively low DP is relatively rapidly degraded [Beguin and Aubert, 1994].

Endoglucanases cut cellulose chains at random positions in less crystal-line regions, creating new chain ends. Extreme endoglucanases, often called CM-cellulases (carboxymethyl-cellulases) have little activity towards crys-talline cellulose, but hydrolyse readily CMC, acid-swollen cellulose and even barley -glucan in a random fashion, resulting in a rapid fall in the degree of polymerisation [Kleman-Leyer et al., 1994].

The classification of cellulases as purely endoglucanases or exogluca-nases is not absolute and is an over-simplification, since several studies indi-cate that several cellobiohydrolases can attack also the internal glucosidic bonds of the cellulose chain [Ståhlberg et al., 1993; Armand et al., 1997; Boisset et al., 2000]. Also, several endoglucanases have been shown to hy-drolyse cellulose processively, which is a common property of cellobiohy-drolases [Reverbel-Leroy et al., 1997; Gilad et al., 2003; Cohen et al., 2005; Zverlov et al., 2005]. As a result, cellulases seem to have a more or less con-tinuous spectrum of properties ranging from virtually random endogluca-nases to highly processive strict cellobiohydrolases [Teeri, 1997; Hildén and Johansson, 2004].

Hydrolytic mechanism In glycosyl hydrolases, enzymatic hydrolysis of the glycosidic bond usually takes place via general acid/base catalysis, which requires two critical resi-dues: a proton donor (HA) and a nucleophile/base (B-). This catalytic activity is provided by two aspartic- or glutamic acid residues.

Two different mechanisms can be distinguished- retaining and inverting mechanisms. In both cases, the acid-base (HA) protonates the leaving glyco-sidic oxygen with the concomitant formation of a partial positive charge on the C1 carbon.

In the inverting mechanism, the base (B-) deprotonates a water molecule, which then attacks the C1 carbon of the glucose ring in an Sn2 type dis-placement reaction, resulting in inversion of the configuration at the ano-meric carbon C1.

In the retaining mechanism, a glycosidic bond is hydrolysed via two sin-gle displacement steps. First, the nucleophile (B-) attacks directly the C1 carbon, resulting in a covalent intermediate between the enzyme and the substrate, the first product is released. In the second step, the acid-base acti-vates a water molecule by abstracting a proton from it, promoting an attack on the C1 carbon.

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OR

A

B-

HOOH

A

B-

HO

OR

A

B-

HO

A-

B

H

HROHO

A-

B

OO

HHO

ROH

A-

BH

O

HO

5.5 Å

10 Å

A

B

Figure 4. The two major mechanisms of enzymatic hydrolysis of the glycosidic bond as first proposed by Koshland [Koshland D.E., 1953]. (A) The retaining mechanism. (B) The inverting mechanism.

Recently, a fundamentally different glycosidase mechanism has been un-veiled for NAD+- and divalent metal ion-dependent GH4 glycosidases whereby hydride abstraction at C3 generates a ketone, followed by deproto-nation of C2 accompanied by acid-catalysed elimination of the glycosidic oxygen and formation of a 1,2-unsaturated intermediate. This - -unsaturated species undergoes a base-catalysed attack by water to generate a 3-keto derivative, which is then reduced by NADH to complete the reaction cycle [Lodge et al., 2003; Rajan et al., 2004; Varrot et al., 2005].

Glycoside hydrolase families The glycoside hydrolases can be classified into structurally related families based on similarities in the distribution of hydrophobic amino acids in their sequences [Henrissat, 1991]. Up to date, 106 families have been distin-guished and the continuously updated information is available on Carbohy-drate Active Enzymes Database server (http://www.cazy.org/CAZY) [Coutinho and Henrissat, 1999]. Cellulolytic enzymes are grouped into at least 14 families. The family classification reflects the structural features of the enzymes and the evolution of glycoside hydrolases. Some families con-tain enzymes with different substrate specificities. For example, families 5, 6, 7, 8, 9 and 48 contain both cellobiohydrolases and endoglucanases. Fam-ily 7 contains only fungal hydrolases, whereas family 8 contains only bacte-rial hydrolases. Some families are evolutionally deeply rooted, containing cellulases from bacteria, fungi, and plants. Also, cellulases from different families are found in the same organism. So far, the hydrolysis mechanism

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seems to be conserved among the members of a given glycosyl hydrolase family.

Fungal cellulases Cellulases face the difficult problem of working on a solid substrate. Most of the fungal cellulases share a common molecular organisation where a large catalytic domain (CD) is connected by a highly glycosylated linker-peptide to a small carbohydrate-binding module (CBM). Upon limited proteolysis with papain the enzymes can, in many cases, be cleaved easily into the two functional domains [Tomme et al., 1988].

Figure 5. A general sketch of a fungal cellulase. The three hexagons in the CBM indicate the aromatic residues responsible for interaction with the hydrophobic face of every second pyranose ring. The grey area in the figure represents the loops cov-ering the substrate-binding sites. Reprinted with permission from [Hildén and Jo-hansson, 2004].

The active site of a cellulase consists of multiple binding sites for glucose units, which enhances the probability for the enzyme to remain bound to the substrate after a catalytic cycle and thereby work processively [Divne et al., 1994]. These binding subsites are labelled, according to convention, from -nto +n, with -n at the nonreducing end and +n at the reducing end. The cleav-age occurs between the -1 and +1 subsites [Davies et al., 1997].

Generally, cellobiohydrolases have a tunnel-shaped active site, whereas the active site for endoglucanases is more open, forming a cleft or groove, allowing the enzyme to bind to the middle of the substrate chain and cleave it. Since some cellobiohydrolases also can perform these internal cuts, the loops closing the tunnel must be flexible to allow a cellulose chain to enter the active site.

Most fungal cellulases contain, in addition to the CD, a carbohydrate binding module (CBM), more specifically called cellulose-binding domain (CBD). The CBDs are believed to play an important role in cellulose hy-drolysis. Although these domains do not affect the activity of cellulases to-

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ward soluble and amorphous substrates, they significantly enhance the ca-pacity of the enzymes to hydrolyse crystalline cellulose. Currently, the CaZy classification lists 45 families of characterised CBMs based on amino acid sequence similarity (see http://www.cazy.org/CAZY/), but this number will most probably increase [Davies et al., 2005]. These families have been re-viewed recently by Boraston et al., [Boraston et al., 2004]. All fungal CBDs belong to the family I containing 35 to 40 amino acids and show strong se-quence similarity with an overall amino acid identity of 60%, some residues being completely conserved and some displaying conservative substitutions [Gilkes et al., 1991].

The first structure of a fungal CBD was determined by nuclear magnetic resonance [Kraulis et al., 1989]. The CBDs of the fungal cellulases have a wedge-shaped fold containing a basic structure of a distorted -sheet of three short antiparallel strands. One face of the wedge is planar and contains three conserved aromatic amino acids separated by a distance corresponding to the length of the repeating unit in cellulose, cellobiose [Tomme et al., 1995]. This interaction is often supplemented by polar residues forming hydrogen bonds [Tormo et al., 1996]. The other surface is rougher and less hydrophilic in character.

Specific aspects of cellulase kinetics The enzymatic degradation of solid cellulose is a complicated process which takes place at a solid-liquid phase boundary where the enzymes are the mo-bile components. Several properties of the substrate influence the kinetics of enzymatic hydrolysis of cellulose: the crystallinity and probably also the type of the cellulose crystals, the degree of polymerisation, the distribution of the molecular weight, the accessible surface for the enzymes and the mi-crostructure of the cellulose surface [Zhang and Lynd, 2004]. To study the individual effects of these parameters on the enzymatic hydrolysis is a com-plicated task, because within any given cellulose sample there is a great de-gree of variability. Recently, bacterial cellulose produced by Acetobacter xylinum has become a widely used substrate for cellulose studies. The ad-vantages of using bacterial cellulose as a substrate are that it consists of pure cellulose, is relatively well-defined and is available in never-dried form.

The studies of enzymatic attack of cellulose have focused primarily on the release of reducing sugars from insoluble cellulose or soluble cellulose de-rivatives. Relatively little is known about the effect of the individual en-zymes on the macromolecular structure of insoluble cellulose substrates.

The rate of enzymatic hydrolysis of the cellulosic materials always de-creases rather quickly. Generally, enzymatic cellulose degradation is charac-terised by a rapid initial phase followed by a slow secondary phase that may last until all substrate is consumed. This has been explained most often by the rapid hydrolysis of the readily accessible fraction of cellulose, strong

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product inhibition and slow inactivation of absorbed enzyme molecules [Converse et al., 1988]. The erosion of the cellulose surface by the cellulases has been proposed as one of the rate retardation factors [Väljamäe et al., 1998].

It has been shown that the surface area of cellulose which is accessible to cellulase enzymes is the most important factor in determining initial rates of hydrolysis [Thompson et al., 1992; Helle et al., 1993]. The efficiency of cellulose hydrolysis by an individual enzyme is dependent on the degree of polymerisation and crystallinity of the substrate. Generally, cellobiohy-drolases are relatively more active towards highly crystalline substrates with relatively low DP, such as BMCC; endoglucanases have only very limited action on crystalline substrates, but hydrolyse readily amorphous cellulose [Henrissat et al., 1985].

The interplay of the cellulose domains on crystalline cellulose degradation, the role of the CBD The adsorption of cellulase to cellulose is a prerequisite step for hydrolysis. The overall binding efficiency of the cellulases to the cellulose is enhanced by the presence of the CBM, and this correlates clearly with higher activity towards insoluble cellulose [Tomme et al., 1988; Gilkes et al., 1988; Reini-kainen et al., 1992; Ståhlberg et al., 1993; Reinikainen et al., 1995]. At the same time, the strong binding via CBD to the cellulose surface can lead to a population of nonproductively bound enzymes [Ståhlberg et al., 1991]. In addition to anchoring the enzyme molecules to the cellulose surfaces, the disruption of cellulose microfibrils by family II CBDs has been reported [Din et al., 1991]. Simultaneous addition of separated CBD and catalytic domain resulted in synergy between these domains in the hydrolysis of cot-ton cellulose [Din et al., 1994]. Similar results have not so far been obtained with the CBDs from other families and even with family II CBDs the effect was seen only using cotton as substrate and not on microcrystalline cellulose [Esteghlalian et al., 2001].

It is probable that different CBDs bind to different regions on the cellu-lose surface. CBDs can promote the enzyme activity towards different re-gions on the cellulose surface, thereby determining the substrate specificity [Carrard et al., 2000]. Lately, CBMs from families 1 and 3 were shown to bind preferentially to the obtuse corners of Valonia cellulose microcrystals, which expose the hydrophobic phase [Lehtio et al., 2003].

Many cellulases, like other enzymes acting on polymeric substrates, have been thought to work processively, i.e. they can perform several hydrolytic events without dissociating from the substrate. Generally, the kcat value for oligosaccharide hydrolysis increases together with the DP of the substrate. In 1983, Lee et al. found that the cellulase catalysis does not significantly affect the DP of a solid cellulose substrate. These authors proposed that cellulose chains are peeled off progressively from the fibrils by the cellulase enzymes,

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since the DP remains constant during the course of hydrolysis. [Lee et al., 1983]. Similar findings have been described for H. jecorina cellobiohy-drolase Cel6A acting on cotton fibers [Kleman-Leyer et al., 1996]. The thin-ning of cellulose microfibrils by the action of cellobiohydrolases has been observed several times [Chanzy and Henrissat, 1985; Boisset et al., 2000; Lee et al., 2000]. The processivity hypothesis is supported by the structure of the CD of cellulases, which contain multiple binding sites for the glycosyl units. The cellulose chains are held together in microcrystals by van der Waals interactions and hydrogen bonds. In order to separate a cellulose chain from the cellulose crystal, a cellulase molecule has to overcome an energy barrier in breaking the interaction between cellulose chains, which may slow down the rate of hydrolysis [Skopec et al., 2003]. Since the active site of a cellobiohydrolase often is a long tunnel, the cellulose chain is en-closed and held in the active site of the enzyme by numerous interactions, which makes the enzyme less likely to dissociate after each hydrolytic step and thus compete more efficiently with the interactions driving the cellulose chain back onto the cellulose crystal [von Ossowski et al., 2003]. Deletion in the loops covering the active site of cellobiohydrolase Cel7A resulted in loss of activity on crystalline cellulose, whereas the activity on amorphous cellu-lose and soluble substrates remained the same or increased [von Ossowski et al., 2003]. In the case of processive endoglucanases, the active-site covering loop undergoes a large “loop-flip” conformational change to enclose the active site upon substrate binding [Davies et al., 1995; Varrot et al., 2000]. Movements in the loops have been shown also for GH6 family cellobiohy-drolases, and probably facilitate substrate gliding into the tunnel to allow occasional endo type of cleavages [Zou et al., 1999; Varrot et al., 1999]. The efficient hydrolysis of cellulose needs interplay between those two domains. Experiments with H. jecorina Cel7A mutants with deletions in the hinge region connecting the CD and CBD have shown that sufficient distance be-tween CD and CBD is needed in the cellulases for efficient hydrolysis of crystalline cellulose [Srisodsuk et al., 1993]. Recently, Mulakala and Reilly presented a model based on the interaction energies and forces on cello-oligosaccharides computationally docked to CD and CBD, where CBD wedges itself under a free chain end on the crystalline cellulose surface and feeds it to the CD active site tunnel [Mulakala and Reilly, 2005]. The energy for cellulose structure disruption comes ultimately from the chemical energy of glycosidic bond breakage [Sinnott, 1998].

Synergism of cellulases Effective degradation of crystalline cellulose requires cooperation between different types of cellulases. This cooperation, resulting in higher total activ-ity, is called synergism. The synergy factor (SFp) is defined as the ratio of the activity of combined enzymatic action to the sum of the activities of in-dividual components. Two classes of synergism between cellulases have

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been described: the cooperation between endoglucanases and cellobiohy-drolases (endo-exo synergism) and that between two cellobiohydrolases (exo-exo synergism). Generally, synergism between endo- and exoenzymes is highest on semicrystalline cellulose of high DP, lower on amorphous cel-lulose and non-existent on soluble cellulose derivatives [Henrissat et al., 1985; Nidetzky et al., 1993; Samejima et al., 1997]. The molecular basis of synergism is not yet completely understood, largely because the modes of action of the individual enzymes are not clear. The synergism has been found to be dependent on the relative proportions of the enzyme components [Henrissat et al., 1985] and also on the degree of saturation of the substrate with the enzymes, decreasing at higher enzyme concentration [Woodward et al., 1988a; Woodward et al., 1988b].

It is generally assumed that the mechanism of endo-exo synergism can be discussed in terms of sequential action where by the random endoglucanase initiates attack and the new chain ends generated are then hydrolysed by the endwise-acting cellobiohydrolase. Furthermore, -glucosidases can work in synergy with cellulases by removing the cellobiose produced.

Hypocrea jecorina cellulases The filamentous soft-rot fungus Hypocrea jecorina (formerly known as Trichoderma reesei) is one of the most studied cellulolytic micro-organisms. It degrades plant litter in its natural environment, in the soil. H. jecorinaproduces a complete cellulolytic enzyme system and is capable of very effi-cient degradation of crystalline cellulose.

Table 1. Hypocrea jecorina (Trichoderma reesei) cellulases.

Enzyme Old name Molecular weight (kDa)

Isoelectric point (pI)

Position of the CBM

Cel7A CBH I 57 3.9 C Cel6A CBH II 53 5.9 N Cel7B EG I 55 4.5 C Cel5A EG II 50 5.5 N Cel12A EG III 25 7.5 - Cel61A EG IV 55 C Cel45A EG V 36 2.9 C

Hitherto, two cellobiohydrolases (Cel7A and Cel6A), five endoglucanases (Cel7B, Cel5A, Cel12A, Cel61A and Cel45A) and two -glucosidases have been isolated from H. jecorina culture medium. In addition, transcription analysis and genome sequencing have additionally identified three putative endoglucanases belonging to the families GH5, GH61 and GH74 and five putative -glucosidases (one belonging in family GH 1 and four in family GH3) [Foreman et al., 2003]. Each H. jecorina cellulase is expressed from a single gene, and a simple on-off co-regulation results in constant ratios

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among the major enzymes, regardless of the growth conditions [Ilmen et al., 1997]. All isolated H. jecorina cellulases except Cel12A have a multidomain structure based on a catalytic domain and a cellulose-binding domain. Both domains bind to cellulose, but the affinity of the CD is, in most cases, much lower than that of the CBD [Ståhlberg et al., 1991].

Three-dimensional structures of the catalytic modules of the cellobiohy-drolases Cel7A, Cel6A and for the endoglucanases Cel7B and Cel12A have been solved [Rouvinen et al., 1990; Divne et al., 1994; Kleywegt et al., 1997; Sandgren et al., 2001]. The overall shape of Cel7A and Cel6A has been determined by low-resolution small-angle X-ray scattering analysis (SAXS). Both enzymes were shown to have similar structures with a large ellipsoidal head and an elongated cylindrical tail with the average dimen-sions 4.5x18 nm for Cel7A and 4.5x21.5 nm for Cel6A [Abuja et al., 1988; Abuja et al., 1989].

Cel7ACel7A (CBH I) is the major cellulase produced by H. jecorina. It comprises about 45-50% of the total cellulolytic protein of H. jecorina and hydrolyses crystalline cellulose, liberating cellobiose as the main product [Fägerstam and Pettersson, 1980]. The crystal structure of the CD revealed a -sandwichstructure with a 50Å-long substrate-binding tunnel formed by the inner -sheets and the extensive loops covering the active site [Divne et al., 1994; Divne et al., 1998]. Ten glycosyl-unit binding subsites have been identified, 3 of these at the product side. Four tryptophan residues form a glycosyl-binding platform in sites –7, –4, –2 and +1 in the tunnel of Cel7A. Cello-biose has its highest affinity towards the +1,+2 subsites of the enzyme, the experimentally determined value for Kd, based on competitive binding or inhibition experiments using various chromogenic or fluorogenic substrates (p-nitrophenyl glycosides and methylumbelliferyl glycosides) being about 20 µM [Claeyssens et al., 1989; van Tilbeurgh et al., 1989; Henriksson et al., 1999b]. The action of Cel7A on cellulose is much less sensitive to inhibition by cellobiose, with an apparent Ki around 1.5 mM [Gruno et al., 2004].

Cel7A, like the other GHs belonging to family 7, hydrolyse the -1,4 glu-cosidic bond of cellulose with retention of the anomeric carbon configura-tion [Knowles et al., 1988; Claeyssens et al., 1990]. Site-directed mutagene-sis confirmed that Glu217 acts as the proton donor and Glu212 as the nu-cleophile in a double-displacement mechanism, whereas Asp214 is likely to be involved in maintenance of the appropriate pKa values of the other cata-lytic residues [Divne et al., 1994; Ståhlberg et al., 1996; Kleywegt et al., 1997]. It has been shown that Cel7A hydrolyses soluble oligosaccharides from the reducing end [Biely et al., 1993; Barr et al., 1996]. The kcat values for Cel7A have been shown to increase with the DP of the substrate, having values of 4.0 s-1 for cellotetraose and 9.5 s-1 for cellohexaose, whereas KMvalues decrease from 7 µM to 3 µM [Nidetzky and Claeyssens, 1994]. Struc-

22

tural and kinetic data, taken together, indicate strongly that Cel7A is a highly processive enzyme. Recently, the processivity values for Cel7A acting on cellulosic substrates labelled at the reducing end with anthranilic acid were determined and found to be 88±10 for bacterial cellulose, 42±10 for bacterial microcrystalline cellulose and 34±2.0 for endoglucanase-pretreated bacterial cellulose, respectively [Kipper et al., 2005].

Cel6ACel6A (CBH II) is another cellobiohydrolase produced by H. jecorina, con-stituting approximately 20% of the secreted protein. The crystal structure of the Cel6A catalytic domain was the first cellulase structure solved [Rouvinen et al., 1990]. Cel6A CD is an / barrel protein, similar to triose phosphate isomerase (TIM) with the exception that it contains seven instead of eight -strands. The active site forms a 20 Å long tunnel with four glyco-syl unit binding subsites. Two additional subsites close to the tunnel entrance have been identified [Koivula et al., 1998]. A tryptophan residue (Trp 272) at the +4 subsite is critical in the crystalline cellulose degradation by Cel6A. Its mutation leads to an overall decrease in activity by at least an order of magnitude. Compared to H. jecorina Cel7A, the active-site tunnel of Cel6A is shorter and more open. The tunnel-covering loops can undergo move-ments, resulting in the closing or opening of the tunnel [Zou et al., 1999; Varrot et al., 1999]. This is apparently the reason for the observed endoactiv-ity and lower processivity of Cel6A.

Cel6A hydrolyses the glucosidic bond with inversion of the configuration at the anomeric carbon by a single displacement mechanism [Knowles et al., 1988; Claeyssens et al., 1990]. The bond cleavage takes place from the non-reducing end of the substrate. Two catalytically important aspartate residues have been identified, of which Asp 221 acts as proton donor and Asp 175 stabilises the positively-charged transition state [Koivula et al., 2002].

EndoglucanasesCel7B (EG I) is the main endoglucanase of H. jecorina, accounting for 5-10% of the total cellulase [Bhikhabhai et al., 1984] and shows 45% sequence homology to Cel7A [Penttilä et al., 1986]. Cel7B also shows a very similar fold. However, four loops covering the tunnel in Cel7A are partially deleted in Cel7B, resulting in an open-groove-shaped active site [Kleywegt et al., 1997]. Cel7B cleaves the polymeric substrates in random fashion and pos-sesses transglycosylation activity [Claeyssens et al., 1990; Biely et al., 1991].

Another endoglucanase, Cel5A (EG II) is produced by H. jecorina in comparable amounts [Saloheimo et al., 1988]. Cel5A hydrolyses the gluco-sidic bond via the double-displacement mechanism and has slightly lower activity than Cel7B on substituted celluloses and -glucan [Penttilä et al., 1987]. Its three-dimensional structure is not yet established, but the kinetic

23

studies of oligosaccharide hydrolysis by Cel5A suggest that the active site of Cel5A consists of five glucosyl binding subsites [Macarron et al., 1993; Biely et al., 1993].

Cel12A (EG III) is a small, 25 kDa endoglucanase that does not have a CBD. It can hydrolyse, in addition to cellulose, also -1,3-1,4 glucan, xy-loglucan and xylan [Hayn et al., 1993; Karlsson et al., 2002]. The crystal structure revealed that Cel12A consists of two largely anti-parallel -sheets which pack on top of each other. The substrate-binding cleft is approxi-mately 35 Å long, 8 Å wide and 15 Å deep with at least six sugar-binding subsites, from –4 to +2 [Sandgren et al., 2001; Sandgren et al., 2005]. Cel12A hydrolyses the glucosidic bonds in cellulose using the double-displacement mechanism with Glu 116 as nucleophile and Glu 200 as gen-eral acid/base [Okada et al., 2000].

The expression of Cel61A (EG IV) is induced together with the other cel-lulases in H. jecorina [Saloheimo et al., 1997]. However, the specific en-doglucanase activity of Cel61A (EG IV) is several orders of magnitude lower than that of Cel7B toward both cello-oligosaccharides and amorphous and substituted celluloses, and its role remains obscure [Karlsson et al., 2001].

Cel45A (EG V) seems to have quite unique hydrolytic properties. It does not hydrolyse cellotriose, cellotetraose and cellopentaose and has lower ac-tivity toward cellulosic substrates than do other H. jecorina endoglucanases [Karlsson et al., 2002]. The main product of Cel45A (EG V) cellulose hy-drolysis is cellotetraose, with significant amounts of cellotriose and cel-lopentaose. It hydrolyses readily glucomannan, being able to cleave a glyco-sidic bond between glucose and a mannose unit, which indicates that Cel45A is a glucomannanase rather than a strict endoglucanase [Karlsson et al., 2002].

Phanerochaete chrysosporium cellulases The white-rot basidomycete, Phanerochaete chrysosporium, employs an array of extracellular enzymes capable of completely degrading the major polymers of wood: cellulose, hemicellulose and lignin. P. chrysosporiumexhibits a system of synergistically-acting cellulases homologous to H. je-corina [Uzcategui et al., 1991a; Uzcategui et al., 1991c]. Sequencing of the whole P. chrysosporium genome revealed at least 40 putative endogluca-nase-encoding genes (in families GH5, GH9, GH12, GH61 and GH74), six genes encoding GH7 cellobiohydrolases and one gene for a cellobiohy-drolase belonging to the GH6 family [Martinez et al., 2004]. The data con-cerning cellobiohydrolases are in accordance with previous findings [Sims et al., 1988; Covert et al., 1992a; Tempelaars et al., 1994].

Hitherto, three cellobiohydrolases: Cel7D (CBH 58), Cel7C (CBH 62) and Cel6A (CBH 50), and three endoglucanases: Cel5A (EG 38), Cel5B (EG

24

44) and Cel12A (EG 28) have been isolated and characterised from P. chry-sosporium [Uzcategui et al., 1991a; Uzcategui et al., 1991c; Henriksson et al., 1999a]. The fourth previously characterised endoglucanase, EG 36, is probably a truncated isoform of EG 38 lacking a cellulose-binding module [Uzcategui et al., 1991a]. Recently, from cellulose-grown medium of P. chrysosporium, two peptides with previously identified cDNAs matching to cel7e and cel7f were identified for the first time [Wymelenberg et al., 2005]. In addition, several peptides matching putative endoglucanases from GH families 12, 45, 61 and 74 were identified [Wymelenberg et al., 2005].

Table 2. Phanerochaete chrysosporium cellulases

Enzyme Old name Molecular weight (kDa)

Isoelectric point (pI)

Position of the CBM

Cel7D CBH 58 58 3.8 C Cel7C CBH 62 62 4.9 C Cel6A CBH 50 50 4.9 N Cel5B EG 44 44 4.3 N Cel5A EG 38 38 5.6-5.7 N Cel12A EG 28 28 5.2 -

In contrast to H. jecorina, where the expression of cellulases is co-regulated, the cellulase genes of P. chrysosporium are differentially transcribed, de-pending on the substrate and the stage of degradation. During growth on cellulose powder, the highest expression is observed for Cel7D and Cel6A, with lower levels of Cel7C and GH5 endoglucanases [Uzcategui et al., 1991b; Vanden Wymelenberg et al., 1993]. Using aspen wood chips as the growth medium, transcripts of cel6A, cel7C and cel7E dominate [Vallim et al., 1998]. During growth on minimal medium containing glucose, only cel7A and cel7B transcripts were seen [Covert et al., 1992b].

Cel7DCel7D is the major secreted cellulase in the cultures grown on cellulose powder as a carbon source [Szabo et al., 1996]. It has 55% amino acid se-quence identity to H. jecorina Cel7A. The main architecture of the Cel7D catalytic module, including most aspects of substrate-binding- and catalytic machinery, resembles the structure of Cel7A, the main differences being deletions and other changes in the loops covering the substrate-binding tun-nel, which makes the tunnel more open without any direct contacts between one side and the other [Munoz et al., 2001]. In total, 11 substrate binding subsites have been identified, three of them at the product side. Cel7D has higher activity than H. jecorina Cel7A toward both soluble and insoluble substrates [von Ossowski et al., 2003].

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Phanerochaete chrysosporium cellobiose dehydrogenase Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by many lignocellulose-degrading soft-rot, white rot and brown-rot fungi, in-cluding P. chrysosporium [Westermark and Eriksson, 1974; Schmidhalter and Canevascini, 1993; Roy et al., 1996; Fang et al., 1998; Schou et al., 1998; Temp and Eggert, 1999]. The enzyme is produced at relatively high levels (0.5% of the secreted protein) when cellulose is the main carbon source [Szabo et al., 1996]. CDH oxidises cellobiose, lactose and longer cello-oligosaccharides to the corresponding lactones using a wide spectrum of electron acceptors, including quinones, phenoxyradicals, Fe3+, Cu2+ and molecular oxygen. CDH exhibits strong discrimination against glucose, as indicated by the 87,000-fold larger specificity constant (kcat/KM) for cello-biose compared to glucose [Henriksson et al., 1998]. This feature has been used to construct amperometric biosensors for the measurement of cello-biose, lactose and cellooligosaccharides [Nordling et al., 1993].

CDH is a monomeric enzyme with molecular weight of 90 kDa consisting of two distinct domains: a flavin domain, which contains FAD and a cyto-chrome b type heme-carrying domain. These two domains are connected via a 25-residue peptide linker which is susceptible to cleavage by papain [Henriksson et al., 1991]. The overall shape of CDH is reported to be “cigar shaped” with a length of 180 Å and a maximal width of about 50 Å [Lehner et al., 1996]. The crystal structures of both domains have been solved [Hallberg et al., 2000; Hallberg et al., 2002]. The flavin domain is peanut-shaped and consists of two structurally distinct subdomains; one that binds the FAD cofactor and one that binds the substrate, cellobiose. The interface between these two subdomains forms a 12 Å-long funnel-shaped tunnel that leads down to the active site. Two glycosyl-binding subsites were identified; the innermost glycosyl-binding subsite (C) adjacent to the flavin ring and the binding subsite (B) close to the tunnel entrance. The architecture of sugar-binding subsites supports prevhious findings [Henriksson et al., 1998] that the specificity of the CDH is determined mostly by the configuration of the C2 carbon in the C subsite, whereas in the B subsite, configurations at C2, C3 and possibly also C6 carbons appear to be important. The tight binding of the substrate at the B subsite partly explains the observed strong glucose discrimination.

To date, CDH is the only known extracellular flavocytochrome. It is still unclear how the electrons are transferred between these two domains, but according the crystal structures, both domains where the active site is located display a high degree of surface complementarity allowing the cofactors to communicate over a distance relevant for inter-domain electron transfer [Hallberg et al., 2002].

CDH can transfer electrons both to one- and two-electron acceptors. The reduction rate of two-electron acceptors is virtually unaffected by the loss of

26

the heme domain [Samejima and Eriksson, 1992; Henriksson et al., 1993]. For one-electron acceptors, both the sink model, where the heme domain acts as electron sink, enhancing the rate of reduction of one-electron accep-tors, and the electron chain model, where the heme directly reduces the elec-tron acceptor after obtaining the electrons from FADH2, have been dis-cussed. Recent results indicate that both electrons from two-electron-reduced CDH are transferred to one-electron acceptor cytochrome c via the heme [Igarashi et al., 2005]. However, this does not have to be the case for all one-electron acceptors.

CDH can produce both hydrogen peroxide and superoxide as primary re-duction products of molecular oxygen [Morpeth, 1985; Kremer and Wood, 1992a] and can also degrade hydrogen peroxide under the same conditions [Henriksson et al., 1993].

CDH binds to cellulose with an estimated binding constant in the submi-cromolar range. The cellulose binding is probably of hydrophobic nature and the putative binding site is located on the flavin domain [Henriksson et al., 1997]. However, in the crystal structure there is no obvious substructure or surface patch that can be assigned as the cellulose-binding site [Hallberg et al., 2002].

The physiological role of CDH in wood degradation has not been estab-lished unambiguously. Several roles for CDH in wood degradation have been proposed and these hypotheses have been reviewed by Henriksson et al. [Henriksson et al., 2000]. Some examples include preventing the repoly-merisation of lignin by reducing phenoxyl radicals produced by lignolytic enzymes; participating in lignin degradation by supporting manganese per-oxidase, relieving product inhibition of cellulases by oxidating cellobiose- the main hydrolysis product cellulases, etc. One of the suggested functions of the CDH is involved in generating reactive oxygen species, such as super-oxide and hydroxyl radicals. Kremer and Wood proposed that the Fe3+ -reducing activity of CDH may be important for the production of hydroxyl radicals [Kremer and Wood, 1992b]. Reactive oxygen species may acceler-ate the depolymerisation of cellulose by attacking its crystalline structure, thereby making it more accessible for hydrolytic enzymes.

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Present investigation

Aims of the present study This thesis has focused on the characterisation of the dynamics in the enzy-matic degradation of cellulose microfibrils, including both the action of the individual enzymes and their synergistic interplay. More specifically, the following tasks were addressed:

To determine the end-preference of cellobiohydrolases on their natural substrate - crystalline cellulose.

To investigate synergistic mechanisms between endo- and exo-acting cellulases with specific focus on its dependence on the nature of the substrate.

To evaluate the conversion of oxygen species by cellobiose dehydro-genase.

In addition, a new endoglucanase from Phanerochaete chrysosporium was isolated and characterised. The interactions between GH7 family cellobiohy-drolases and o-nitrophenyl cellobioside were investigated and employed for indirect binding studies.

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Action of cellulases on end-labelled cellulose (Paper I) The aim of paper I was to determine the end-preference of cellobiohy-drolases acting on crystalline cellulose.

It was assumed for a long time that cellobiohydrolases hydrolyse cellu-lose starting from the non-reducing end of the cellulose chain. An increasing amount of both structural and kinetic data using soluble substrates show indeed strong evidence that cellobiohydrolases have different end-preferences. However, the behaviour of the enzymes on crystalline cellulose is not only active-site mediated, but may be influenced by the binding to the bulk cellulose, especially if a binding domain is involved.

To study the end-preference of cellulases we used reducing-end-labelled bacterial microcrystalline cellulose (BMCC) as substrate. We followed the release of the labelled end group in relation to the total reaction course (total fraction of cellulose solubilised). Conclusions were drawn with the help of computer-simulated progress curves.

Figure 6. Some examples of the simulated progress curves. The first example shows a progress curve for a strict exoenzyme that hydrolyses the substrate from the la-beled end. The second example shows the progress curve for a highly processive strict exoenzyme hydrolyzing the substrate from the non-labelled end.

The computer simulations were carried out to mimic the actual experimental situation. The following parameters, which may influence the shape of the progress curve, were taken into account: end-preference of the enzyme,

29

probability for endo/exoactivity, processivity towards labelled ends, proces-sivity towards non-labelled end and enzyme concentration (non-productively bound enzymes can disturb productively bound ones). Some examples of the simulated progress curves are shown in Figure 6. In short, one can conclude that the initial slope of the progress curve over the 1:1 line indicates that the enzyme prefers the labelled end. The slope is also dependent on the enzyme processivity. An exo-acting enzyme with end-preference for the labelled end and low processivity performs only a few hydrolytic events before dissociat-ing from the substrate, and the resulting progress curve for such an enzyme is strongly convex with a high initial slope. If the processivity of an enzyme is higher than the average DP of the substrate, a productively bound enzyme will hydrolyse whole cellulose chains without dissociating from the sub-strate, releasing one labelled end group per hydrolysed cellulose chain. In this case, the resulting progress curve is a straight line.

Experimental progress curves showed clearly distinguishable patterns for GH6 and GH7 family cellobiohydrolases. The progress curves for Cel7A and Cel7D were virtually identical, showing a strong preference for the la-belled end, whereas the progress curves for Cel6 enzymes were close to the 1:1 line, showing no obvious end-preference. These data can be explained by combined endo/exo activity. In order to clarify the end-preference of Cel6 enzymes, a substrate labelled at both ends is needed.

Influence of acid pre-treatment of bacterial cellulose on mode of synergy between Cel7A (CBH I) and Cel7B (EG I) (Paper II) In this study, the separate, sequential and simultaneous actions of Cel7A (CBH I) and Cel7B (EG I) were investigated using well-defined substrates derived from bacterial cellulose.

The high synergy between cellobiohydrolases and endoglucanases is common among cellulolytic enzymes. According to the conventional model, endoglucanases create new chain ends at more amorphous substrate areas upon which cellobiohydrolases can start to hydrolyse the cellulose proces-sively. The aim of our study was to evaluate the synergistic mechanism be-tween Cel7A and Cel7B on substrates with different physical properties, crystallinity and DP.

The bacterial cellulose was hydrolysed in boiling 1 M hydrochloric acid, the samples were withdrawn at certain time points, neutralised and washed. From these samples, the crystallinity index and DP were determined and these samples were used as substrate.

Some characteristic properties of different cellulose samples are shown in Table 3.

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Table 3. Parameters of cellulose samples. BC, appearance common to bacterial cellulose (large fibrous bundles); BMCC, appearance common to bacterial micro-crystalline cellulose (suspension with a milky consistency)

Property Sample

BC A25 A40 BMCC Time in 1M HCl, min 0 25 40 760 Weight loss by HCl, % 0 0.26 1.0 5.2 Crystallinity index, % 87.9 90.3 91.3 92.4 DP, glucose unit 2620 212 151 114 Appearance BC BC BC BMCC Relative activity Cel7A, % 37 69 100 66 Cel7B, % 95 53 57 100 Cel7A/Cel7B, % 100 56 50 33 Maximum synergy factor 7.8 4.1 2.3 1.7

Maximum effect of pretreatment with Cel7B

2.12 - 1.09 -

The most dramatic changes during cellulose hydrolysis by acid occurred within the first 25 minutes of the acid treatment, when the DP decreased more than ten-fold and the synergy by a factor of two.

Similarly to the results obtained by Samejima et al. [Samejima et al., 1997], the synergism between endo- and exoglucanases was highest on un-treated bacterial cellulose and decreased during the course of the acid hy-drolysis. The high synergy on BC is in accordance with the classical endo/exo synergy model, where the endoglucanases create new starting points for cellobiohydrolases.

Interestingly, the relative activities of Cel7A on different cellulose sam-ples did not reflect the changes in cellulose DP, as would be expected if the number of chain ends were the limiting factor for activity. Cel7A did not have maximal activity on the cellulose sample of lowest DP (the sample with the highest concentration of end-groups). Also, if the number of chain ends were the main limiting factor, one should expect much lower activity for Cel7A on BC than on BMCC, but the activities of Cel7A differed only by a factor of two on these substrates.

The activity of endoglucanase Cel7B was found to be almost equal on BC and BMCC. However, since we compared the release of solubilised sugar and not the concentration of the end-groups on the cellulose, the amount of released soluble sugar does not necessarily reflect the total hydrolytic activ-ity of an endoenzyme.

Pre-treatment of cellulose with Cel7B increased the activity of Cel7A on BC about two-fold, whereas the effect of EG pre-treatment on BMCC was neglible. Therefore, the synergy observed between EG and CBH on BMCC cannot be explained by the sequential attack of EG and CBH. We propose a

31

new, more interactive mechanism of synergy between EG and CBH on BMCC, whereby EG “polishes” the cellulose surface from small cellulose chains, supporting in this way the processive action of CBH.

Endoglucanase 28 (Cel12A), a new Phanerochaetechrysosporium cellulase (Paper III) Paper III describes the isolation and characterisation of a new small en-doglucanase from Phanerochaete chrysosporium.

Protein purification Phanerochaete chrysosporium strain K3 was cultivated and the culture fil-trate was precipitated with ammonium sulphate as described by Szabo et al. [Szabo et al., 1996]. After dissolving and desalting the sample, the initial separation of the proteins on DEAE-Sepharose was performed as described by Uzcatequi et al. [Uzcategui et al., 1991b]. Material corresponding to pool B was collected and after adjusting the pH to 5 and adding ammonium sul-phate to 2 M concentration, the protein was applied to a Phenyl Sepharose®

CL-4B column equilibrated with 0.5 M ammonium sulphate in 0.1 M am-monium acetate buffer, pH 5. Proteins were eluted by two linear gradients, first from 0.5 M ammonium sulphate in 0.1 M ammonium acetate, pH 5, to 0.1 M ammonium acetate buffer, pH 5, followed by a second gradient from 0.1 M ammonium acetate buffer, pH 5 to the same buffer containing 50% ethylene glycol. EG activity was eluted in the first gradient, corresponding to Cel5A (EG 38). In the second gradient, two protein peaks were isolated cor-responding to Cel7C (CBH 62) and Cel6A (CBH 50). The latter of these coincided with the second peak of EG activity. This last peak was collected, concentrated and applied to a Biogel P100 column equilibrated with 0.05 M ammonium acetate buffer, pH 5. Two major peaks were obtained, the first corresponding to Cel6A and the second to an enzyme with high activity to-wards CMC.

Protein characterisation The purified enzyme showed a single band on SDS/PAGE with an estimated molecular weight of 28 kDa and an isoelectric point at pH 5.2. Deglycosyla-tion analysis together with amino acid analysis showed that this protein con-tains 1-2 N-glycosylation sites. Peptide mapping after cleavage with the V8 protease showed a pattern clearly distinguishable from the previously char-acterised P. chrysosporium endoglucanases Cel5A (EG 38) and Cel5B (EG 44) (Fig.7). A sequence obtained from a peptide showed strong homology with endoglucanases belonging to the GH12 family.

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Figure 7. Peptide mapping of P. chrysosporium endoglucanases on SDS/PAGE using V8 protease. The peptide pattern of EG28 is totally different from the other endoglucanases, indicating that this is enzyme is not a fragment of either of the others.

The Cel12A protein sequence translated from P. chrysosporium cel12a gene (protein data bank access code: AAU12276) [Vanden Wymelenberg et al., 1993] shows about 40% homology with H. jecorina Cel12A. The peptide isolated and sequenced in protein mapping belongs apparently to the -helixof the protein located near to the C-terminus. The catalytically important amino acids seem to be conserved also in P. chrysosporium Cel12A.

Kinetic properties Cel12A did not bind to crystalline cellulose to any detectable extent. In con-trast to many other cellulases, pNPL was not a substrate for this enzyme. The comparison of the catalytic constants on pNPC as substrate are shown in Table 4.

Table 4. Kinetic constants on p-nitrophenyl cellobioside.

Enzyme kcat, min-1 KM, mM kcat/KM, min-1/mM-1

Cel12A 1.61 12.6 0.13 Cel7B 87.4 3.46 2.5 * 103

Cel7A 0.146 97.4 * 10-3 1.6

Cel12A showed high activity towards CMC and amorphous cellulose, with lower activity on xylan and glucan. Interestingly, Cel12A did not produce soluble sugars using Avicel (a microcrystalline cellulose derived from wood sample) as substrate. Cel12A showed synergy both with Cel7A and Cel6A from H. jecorina using filter paper and Avicel as substrate, but not with en-doglucanases.

33

Cel12A swells efficiently filter paper and disperses the filter paper struc-ture, releasing short fibres. This phenomenon led us to propose a physiologi-cal function for Cel12A enzymes in cellulose degradation. We speculate that Cel12A has an important function in an early stage of cellulose degradation by degrading amorphous material located in the narrow regions between or on the surface of the microfibrils that are sterically inaccessible to the larger two-domain cellulases, leading to the swelling and separation of microfibrils.

Figure 8. Suggested cellulolytic strategy in P. chrysosporium. 1) Crystalline mi-crofibrils are held together by more amorphous material. 2) Cel12A diffuses into pores and nicks the amorphous chains, thereby releasing the microfibrils. 3) “Classi-cal” endoglucanases now have access to the substrate and can create nicks. 4) Cello-biohydrolases degrade the crystalline cellulose processively.

Conversion of oxygen species by cellobiose dehydrogenase (Paper IV) Cellobiose dehydrogenase produces hydrogen peroxide in the presence of an electron donor and molecular oxygen. Typically, during the reaction course, cellobiose and molecular oxygen are consumed in equimolar amounts, whereas the level of hydrogen peroxide produced is significally lower and seems to reach pseudo-steady state conditions. It has been shown that CDH can degrade hydrogen peroxide under the same conditions [Henriksson et al., 1993].

The aim of this paper was to find out what actually happens to the hydro-gen peroxide produced by CDH and into which species it is converted. We compared CDH to a well-characterised enzyme – glucose oxidase (GOX).

34

This enzyme catalyses the oxidation of glucose into gluconolactone by re-ducing molecular oxygen to hydrogen peroxide:

GOX produced H2O2 in equimolar amounts to the cellobiose and O2 con-sumed (Fig. 9B), whereas the level of hydrogen peroxide produced by CDH was much lower than would be expected from the basic stochiometry (Fig. 9A).

Figure 9. Balance of saccharide oxidation by CDH (A) or glucose oxidase (B). On A, empty circles show the molar amount of cellobiose consumed at the given time point, filled triangles show the consumed O2 and filled rectangles show hydrogen peroxide formation. On B, empty circles show consumed glucose, filled triangles consumed O2 and filled squares formed hydrogen peroxide.

Catalase catalyses the decomposition of hydrogen peroxide into water and O2 according the following scheme:

If all of the hydrogen peroxide produced by CDH is decomposed either by catalase or by CDH into water and dioxygen, the total balance between the cellobiose and oxygen consumption rates should be 2:1.

Adding catalase to the reaction mixture indeed caused the ratio between the consumption rates of cellobiose and O2 to become 2:1, which supports the idea that hydrogen peroxide is the primary product of the oxygen reduc-tion by CDH.

35

CDH is known to reduce ferric ions:

These divalent ferrous ions are known to decompose hydrogen peroxide into a hydroxyl radical and a hydroxyl ion in Fenton’s reaction according the following scheme:

The hydroxyl radicals produced are known to react with cellulose, causing its depolymerisation. Kremer and Wood [Kremer and Wood, 1992b] have proposed the following reaction scheme, where HROH is a part of a saccha-ride:

Since the solutions inevitably contain traces of ferric ions, we repeated the experiments using 1 mM desferrioxamine mesylate to inactivate any traces of ferric ions. Under these conditions, hydrogen peroxide was formed stoichiometrically from cellobiose and O2.

The reduction rate of Fe3+ ions by CDH is much higher than for O2 and if the Fe2+ ions produced by CDH are oxidised back by hydrogen peroxide, this makes the reduction of Fe3+ ions by CDH even more kinetically favourable.

Taking all results together, we can conclude that hydrogen peroxide is a primary product of cellobiose oxidation by CDH under aerobic conditions and that hydrogen peroxide is not decomposed further when traces of ferrous ions are eliminated from the system. The inevitable traces of metal ions pre-sent in standard test conditions and in nature seem to be sufficient for an enhanced Fenton reaction, where CDH produces both components needed- reduced metal ions and hydrogen peroxide.

36

o-nitrophenyl cellobioside as an active site probe for family 7 cellobiohydrolases (Paper V) Nitrophenyl glycosides are widely used chromogenic substrates for glycosyl hydrolases, since they contain good leaving groups with favourable spectral properties, which make the reaction easy to monitor. In many cases, how-ever, the kcat observed has been orders of magnitude lower than that observed for the cleavage of oligosaccharides. For cellulase studies, mostly p-nitro-phenyl cellobioside and lactoside have been used as model substrates. Inter-estingly, many of the GH12 family endoglucanases can readily hydrolyse oNPC, some of them being more than 40 times active on oPNC as compared to pNPC [Sandgren et al., 2005]. In this study, interactions between o-nitro-phenyl cellobioside (oNPC) and GH7 family cellobiohydrolases were inves-tigated.

Table 5. Comparison of kinetic constants of Cel7A and Cel7D on oNPC and pNPC.

Enzyme / substrate kcat, s-1 KM, µM kcat /KM, s-1M-1

Cel7A / oNPC (66±15)*10-6 7.0±4.5 9.5 Cel7A / pNPC 0.0026±0.0001 26±3 100 Cel7D / oNPC 0.015±0.002 3200±100 4.6 Cel7D / pNPC 0.046±0.0021 1300±160 35

For both Cel7A and Cel7D, the observed rate constants for oNPC are lower than those for pNPC, as shown in Table 5. Moreover, oNPC was found to be an inhibitor of both enzymes. Since the active site of the cellu-lases consists of multiple binding sites for sugar units, it is likely that these model substrates can bind to the active site of the enzyme in more than one mode, some of these being non-productive. In certain cases where some binding modes may coexist, cooperativity can occur for productive or non-productive binding. This may result in devations from the standard Micha-elis-Menten kinetics, which has been observed for certain cellobiohydrolase-model substrate combinations (data not published). The modelling data show that apart from the competition between productive and non-productive binding modes, there may also be some sterical constraints imposed by the o-nitro group that give a real decrease in the kcat.We found that oNPC quenches the natural fluorescence of Cel7A and Cel7D. The fluorescence quenching increased upon addition of the oNPC and followed a Langmuir isotherm. The fluorescence of the Cel7A could be recovered by adding cellobiose. The dissociation constant for cellobiose determined from competitive binding experiments was in fair agreement with that obtained earlier [Claeyssens et al., 1989; Henriksson et al., 1999b], suggesting that oNPC binds to the active site of the enzyme with a strong preference for the +1, +2 subsites for which cellobiose is known to have

37

highest affinity [Divne et al., 1998] The fluorescence of Cel7D did not re-cover upon adding up to 1 mM cellobiose, suggesting that oNPC may bind possibly also closer to the entrance of the cellulose binding tunnel.

The competitive binding approach was used to study the binding of the cellobiose to the catalytically inactive Cel7A mutants. The catalytic group Cel7A mutants D214N, E212Q and E217Q all bind oNPC with an affinity very similar to that observed for the wild type enzyme (Table 6), strongly suggesting that it binds at the same position. The dissociation constants thus determined for cellobiose were in the same range as that for the wild type but generally somewhat lower. A possible explanation for the difference observed could be that the lower charge density found in the active site of the mutants is favourable for the binding of a neutral ligand, such as cello-biose, especially since the carboxylate-amide substitution allows hydrogen bond interactions to be retained.

Table 6. Binding constants for o-nitrophenyl cellobioside and cellobiose for Cel7D, Cel7A and its catalytically inactive mutants.

Enzyme Kd for oNPC, µM Kd for cellobiose, µM

Cel7A wt 7.4±0.4 23±4 D214N 7.1±0.7 8.9±1.1E212Q 4.7±0.4 8.1±0.3E217Q 3.9±0.4 13.5±3Cel7D 110±10 -

38

Conclusions

GH7 family cellobiohydrolases Cel7A and Cel7D degrade crystalline cellu-lose processively with a strong preference for the reducing end.

There is a common pattern of hydrolysis for GH7 family cellobiohydrolases and another clearly distinguishable pattern for GH6 family cellobiohy-drolases.

Synergistic action between cellobiohydrolases and endoglucanases cannot be explained using only the classical endo-exo model. A new concept whereby endoglucanase “polishes” the cellulose surface is proposed.

A new endoglucanase, Cel12A from P. chrysosporium, was isolated and characterised. This enzyme may have a role in disintegrating larger struc-tures.

Hydrogen peroxide produced by CDH is decomposed via an enhanced Fen-ton’s reaction.

o-nitrophenyl cellobioside has a potential as an active site probe for GH7 family cellobiohydrolases.

39

Summary in Swedish

Cellulosa är den dominerande kolhydraten som produceras av växter, och kan i träd utgöra över 50% av vikten. Kemiskt utgörs cellulosa, liksom stär-kelse, av långa kedjor av hopkopplade glukos-(druvsocker)molekyler. Skill-naden mellan cellulosa och stärkelse bygger väsentligen på att glukosenhe-terna är hopkopplade på olika sätt. Den nedbrytning av cellulosabaserad biomassa som åstadkommes av mikroorganismer är följaktligen en mycket viktig faktor i det totala kolkretsloppet i naturen.

I anslutning till de hela tiden stigande oljepriserna har ett ökande antal länder uppmärksammat problemet med fossila bränslen, och intresset för att hitta alternativa förnyelsebara energikällor som kan ersätta olja är större än någonsin. Sveriges regering har beslutat att skapa förutsättningar för att bryta Sveriges beroende av fossila bränslen till år 2020. Biologiskt styrda proces-ser förefaller lovande för energiomsättning, i synnerhet då för omvandling av lignocellulosamaterial till bekvämare bränslen. Därmed är en fördjupad för-ståelse av mekanismerna för enzymatisk cellulosanedbrytning av högsta betydelse och kommer att bidra till förbättrade processer för storskalig och miljövänlig biologisk nedbrytning av cellulosabaserad biomassa, vilket ännu är alltför kostsamt för att kunna konkurrera med fossila bränslen under rim-liga villkor.

I den här avhandlingen ligger fokus på cellulosanedbrytande enzymer från två mögelsvampar, nämligen Hypocrea jecorina, en rötsvamp som lever i tropisk förna och där bryter ner cellulosarester samt Phanerochaete chrysos-porium, en tränedbrytande s.k.vitrötesvamp som har isolerats från flishögar och är kapabel att bryta ned alla komponenter i ved. Studien har omfattat såväl undersökningar av separata enzymer som deras förmåga att samverka synergistiskt, dvs att effekten när enzymerna arbetar tillsammans blir större än summan av deras individuella effekter.

I det första delarbetet har metoder utvecklats för att avgöra från vilken ände av cellulosakedjorna som enzymerna fördrar att arbeta och sedan an-vänts för att funktionellt karakterisera ett antal viktiga enzymer. Här kunde vi avgöra att de hos svampar ofta förekommande enzymerna från struktur-familj 7 startade nedbrytningen av cellulosakedjorna från den s.k. reduceran-de änden och sedan kunde frisätta ett stort antal lösliga sockermolekyler utan att lämna kedjan, vilket kallas processiv funktion.

I nästa arbete studerades speciellt samverkan mellan enzymer med delvis olika sätt att attackera cellulosakedjorna. Ett klassiskt sätt för samverkan är

40

här den så kallade endo-exosynergismen, där en typ av enzym har förmågan att klippa en lång kedja internt till ett fåtal kortare kedjor, som sedan stegvis bryts ned av enzymer som endast verkar från ändarna och därmed gynnas av när flera fria kedjeändar skapas. Våra resultat tydde dock på att samverkan även följde andra principer, till exempel att ett enzym "städar bort" korta rester av kedjor, vilket därmed gör andra kedjor mera tillgängliga.

En annan aspekt på samverkan av enzymer belyses av ett nytt endogluka-nas med ovanligt små dimensioner, som isolerades från P. chrysosporiumoch studerades funktionellt. Dess släktskap med andra kända enzymer fast-slogs också. Detta enzym var jämförelsevis ineffektivt i sin förmåga att fri-sätta lösligt socker från cellulosa, men visade stor förmåga att luckra upp strukturer i t.ex filterpapper. Vi tolkade detta som att enzymet har som sin främsta uppgift att försvaga kontakterna mellan mikrofibrerna i naturliga cellulosastrukturer så att andra enzymer lättare kommer åt att genomföra en fullständig nedbrytning. En sådan uppluckrande roll skulle gynnas av att enzymet är litet, och lättare kan ta sig in i en från början kompakt struktur, något som är svårt/omöjligt för de flesta andra cellulosanedbrytande enzy-mer.

Hos P. chrysosporium och många andra vitrötesvampar hittar man förut-om de enzymer som bryter ner cellulosakedjorna, också ett enzym som kal-las cellobiosdehydrogenas vilket i stället styr elektronöverföringar, främst från sockret cellobios till ett brett spektrum av mottagarmolekyler. Intressant nog har man inte lyckats fastställa den grundläggande biologiska rollen för detta enzym. En föreslagen roll var att enzymet skulle producera väteperox-id, vilket motsades av andra resultat som antydde att sådan inte kunde pro-ducerades. Vi kunde, bland annat med hjälp av syrgaselektrod, påvisa att enzymet verkligen kunde producera väteperoxid, men att det också, under realistiska förhållanden med spårmängder av järnjoner närvarande, också hade förmågan att förbruka väteperoxid, vilket kunde förklara de tidigare motstridiga resultaten.

Ett avslutande arbete är inriktat på en del speciella mätresultat som kan fås när enzymer med uppgift att bryta ned långa kedjemolekyler studeras med hjälp av små, lösliga modellsubstanser. Resultaten kompliceras av att dessa substanser ofta kan bindas till enzymet på flera sätt än det som ligger till grund för en reaktion. Många gånger kan olika bindningslägen leda till ömsesidig utestängning, dvs bara ett av bindningssätten är möjligt, medan andra bindningssätt kan vara samtidiga. Vi har med olika metoder studerat sådana effekter, men också visat att en substans, orto-nitrofenylcellobiosid, som blott mycket långsamt bryts ned av enzymet, vid bindning förändrar enzymets förmåga att fluorescera, så att den också kan användas som en "reportermolekyl" för att studera bindning av andra molekyler som inte ger några lätt observerbara effekter, men som konkurrerar med reportermoleky-len om att bindas på samma ställe.

41

Acknowledgements

This study was carried out at the Department of Biochemistry and Organic Chemistry, Uppsala University. This work was supported by the Swedish Research Council for Engineering Sciences, the Swedish Natural Science Research Council, The Royal Academy of Sciences, the Swedish Institute, the Swedish Pulp and Paper Research Institute and Wood Ultrastructure Research Center (WURC).

I would like to express my sincere gratitude to all the people who have sup-ported me throughout this long journey.

My very special gratitude belongs to Gunnar Johansson, my supervisor, for Your constant support and encouragement (not mentioning Your natural talent to generate creative ideas) during all these years, especially for allow-ing me to compromise between science and family.

Thank You, all my co-supervisors: Göran Pettersson, The grand father of the “cellulasgruppen”, for making

our lab a warm and friendly place, and of course, for sharing Your enormous knowledge about cellulases and nature in general;

Late Veljo Sild, for teaching me to look into the fascinating world of cel-lulases in a unique way, being a friend, teacher and supervisor in one person;

Jerry Ståhlberg, for being always so positive and helpful. Special thanks for Your contribution to the pending manuscript;

I would like to thank also: Professor Bengt Mannervik, for always finding time for discussions. Priit Väljamäe, for Your critical mind, discussions and comments. My closest co-authors, Andres Salumets, Gunnar Henriksson, Hongbin

Henriksson, Bert Pettersson, Roland Isaksson, Mikael Nilsson for fruitful collaboration.

David Eaker for linguistic revisions of my manuscripts and this thesis. All of the people at our department for creating nice and friendly envi-

ronment. Special thanks to Per-Axel for finding solutions to ALL technical problems in Your smart way. I am deeply impressed of Your skills. The people at the amino-acid lab for Your excellent work (as always, indeed). Lilian, Your assistance at the course lab is indispensable. Gun and

42

Brigitta T., it has been always a pleasure to assist in teaching at Your courses. All other PhD students at the department for all fun and support. Present and former members of our group, especially Jing, my room-mate, for Your friendship and all stories about Chinese culture; Lisa and Caroline, for pleasant time together after moving into new lab; Lars, Istvan and Gabriel, for truly great company.

My fellow-Estonians in Uppsala, especially 2xAnneli, Mart, Sirje, 2xTanel, Taavo, Teet and Triin. My friends here and there. Special thanks goes to Linda for keeping things together during my stays in Estonia.

My mother and father, for the foundation. My sister and brother, for al-ways being there for me.

Finally, my dearest: Toomas, for all Your support and love and my children, Tuuli and Siim, who added a whole new dimension to my life.

43

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