Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 185
Hydrolytic and Oxidative
Mechanisms Involved in Cellulose
Degradation
ANU NUTT
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2006
ISSN 1651-6214
ISBN 91-554-6571-4
urn:nbn:se:uu:diva-6888
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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) Progress 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 endoglucanase I. European Journal of Biochemistry, 266, 327-334.
III
Henriksson, G., Nutt, A., Henriksson, H., Pettersson, B., Ståhlberg, 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. Biotechnology 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 publisher and the Editor-in-Chief of the Journal.
Contents
Introduction.....................................................................................................9
Cellulose.....................................................................................................9
Chemical structure .................................................................................9
Crystalline structure.............................................................................10
Other components of plant cell wall.........................................................11
Cellulases .................................................................................................12
Biological degradation of cellulose .....................................................13
Classification of cellulases ..................................................................13
Fungal cellulases..................................................................................16
Specific aspects of cellulase kinetics ...................................................17
Hypocrea jecorina cellulases...............................................................20
Phanerochaete chrysosporium cellulases............................................23
Phanerochaete chrysosporium cellobiose dehydrogenase ..................25
Present investigation .....................................................................................27
Aims of the present study.........................................................................27
Action of cellulases on end-labelled cellulose (Paper I) ..........................28
Influence of acid pre-treatment of bacterial cellulose on mode of
synergy between Cel7A (CBH I) and Cel7B (EG I) (Paper II)................29
Endoglucanase 28 (Cel12A), a new Phanerochaete chrysosporium
cellulase (Paper III) ..................................................................................31
Conversion of oxygen species by cellobiose dehydrogenase (Paper IV).33
o-nitrophenyl cellobioside as an active site probe for family 7
cellobiohydrolases (Paper V) ...................................................................36
Conclusions ..............................................................................................38
Summary in Swedish ....................................................................................39
Acknowledgements.......................................................................................41
References.....................................................................................................43
Abbreviations
BC
BMCC
CBD
CBH
CBM
CD
CDH
CMC
DP
EG
GH
GOX
kcat
Ki
KD
KM
MeUmbȕ(Glc)2
oNPC
pNPC
pNPL
bacterial cellulose
bacterial microcrystalline cellulose
cellulose binding domain
Cellobiohydrolase
carbohydrate binding module
catalytic domain
cellobiose dehydrogenase
carboxymethyl cellulose
degree of polymerisation
Endoglucanase
glycosyl hydrolase
glucose oxidase
catalytic constant
inhibition constant
dissociation constant
Michaelis-Menten constant
methylumbelliferyl cellobioside
o-nitrophenyl cellobioside
p-nitrophenyl cellobioside
p-nitrophenyl lactoside
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 E1,4-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 between the pyranose ring oxygen in one residue and the hydrogen of the OHgroup 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 Johansson, 2004] with kind permission from the publisher.
9
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 OíH···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 microfibrils 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 reported 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 second to the (010) face of the triclinic crystal. Reproduced with the permission from
[Baker et al., 1997].
10
In nature, most cellulose is produced as crystalline and is defined as cellulose I. It is composed of two distinct crystalline forms (triclinic cellulose IĮ
and monoclinic cellulose Iȕ, which differ from each other in their crystal
packing, molecular conformation and intermolecular hydrogen bonding patterns (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 IĮ form is dominant in cellulose produced by
primitive organisms, such as the bacterium Acetobacter xylinum and the alga
Valonia macrophysa, whereas the Iȕ form dominates in cellulose produced
by higher plants [Atalla and Vanderhart, 1984]. The Iȕ form is more stable
than the IĮ form, which has been reported to be more susceptible to enzymatic 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 lignin comprise 20 to 25 and 5 to 30% of the plant dry weight [Sjöström, 1993].
Hemicellulose
Hemicellulose 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 cellulose, which itself is crystalline, strong and resistant to hydrolysis, hemicellulose is a highly branched and amorphous structure with little inherent
strength. Apart from glucose, it may contain mannose, xylose, arabinose,
rhamnose and L-fucose.
Lignin
Lignin is a highly branched random polymer of coniferyl, sinapyl and pcoumaryl 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.
11
Pectin
Pectin 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 Į-1,4 linked
galacturonic acid residues and “hairy regions”, consisting mainly of rhamnogalacturonan 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 ȕ-1,4-glucosidic
bonds in cellulose. Cellulose degradation is brought about mainly by bacteria, fungi and protozoa, but the production of cellulases is documented also
in plants and in a number of invertebrate taxa that includes insects, crustaceans, annelids, molluscs, mussels and nematodes [Watanabe and Tokuda,
2001; Davison and Blaxter, 2005].
12
Biological degradation of cellulose
In wood, crystalline cellulose microfibrils are tightly packed in a complex
network of hemicellulose constituents and lignin. Most cellulolytic microorganisms produce, in addition to cellulases that hydrolyse the E-1,4glucosidic bonds, a number of other cell-wall-degrading enzymes, e.g. ligninases, 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 capable 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 hydrolysed to glucose by ȕ-glucosidases. The second group of fungi reportedly
degrade cellulose by means of oxidative components together with endoglucanases, but lack the strict cellobiohydrolases. A representative of this
mechanism is the cellulolytic system of the brown-rot fungus Postia placenta [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: cellobiohydrolase (1,4-E-D-glucan cellobiohydrolase, EC 3.2.1.91) and endoglucanase (1,4-E-D-glucan glucanohydrolase, EC 3.2.1.4), based on their activity toward a wide range of substrates. This is rather difficult, since the enzymes have overlapping specificities toward substrates which themselves are
poorly defined.
By definition, cellobiohydrolases release cellobiose from the nonreducing 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 cellobiohydrolases may have opposite chain-end preferences [Barr et al., 1996;
Divne et al., 1998; Koivula et al., 1998]. Cellobiohydrolases are thought to
13
work processively, that is, one enzyme molecule can release several cellobiose units from the cellulose chain without leaving the substrate. Cellobiohydrolases 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 crystalline regions, creating new chain ends. Extreme endoglucanases, often called
CM-cellulases (carboxymethyl-cellulases) have little activity towards crystalline cellulose, but hydrolyse readily CMC, acid-swollen cellulose and
even barley E-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 exoglucanases is not absolute and is an over-simplification, since several studies indicate 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 hydrolyse cellulose processively, which is a common property of cellobiohydrolases [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 continuous spectrum of properties ranging from virtually random endoglucanases 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 residues: 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 glycosidic 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 displacement reaction, resulting in inversion of the configuration at the anomeric carbon C1.
In the retaining mechanism, a glycosidic bond is hydrolysed via two single 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 activates a water molecule by abstracting a proton from it, promoting an attack
on the C1 carbon.
14
A
A-
A
H
O
O
O
R
B-
B
B
B
H
ROH
R
O
O
H
B-
O
OH
H
5.5 Å
B-
O
O
H
H
A-
A
O
A
H
O
ROH
O
A-
10 Å
H
BH
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 unveiled for NAD+- and divalent metal ion-dependent GH4 glycosidases
whereby hydride abstraction at C3 generates a ketone, followed by deprotonation of C2 accompanied by acid-catalysed elimination of the glycosidic
oxygen and formation of a 1,2-unsaturated intermediate. This Į-ȕunsaturated 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 distinguished and the continuously updated information is available on Carbohydrate 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 contain enzymes with different substrate specificities. For example, families 5,
6, 7, 8, 9 and 48 contain both cellobiohydrolases and endoglucanases. Family 7 contains only fungal hydrolases, whereas family 8 contains only bacterial 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
15
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 covering the substrate-binding sites. Reprinted with permission from [Hildén and Johansson, 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 -n
to +n, with -n at the nonreducing end and +n at the reducing end. The cleavage 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 hydrolysis. Although these domains do not affect the activity of cellulases to16
ward soluble and amorphous substrates, they significantly enhance the capacity 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 reviewed 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 sequence 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 ȕ-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 mobile 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 microstructure of the cellulose surface [Zhang and Lynd, 2004]. To study the
individual effects of these parameters on the enzymatic hydrolysis is a complicated task, because within any given cellulose sample there is a great degree of variability. Recently, bacterial cellulose produced by Acetobacter
xylinum has become a widely used substrate for cellulose studies. The advantages 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 derivatives. Relatively little is known about the effect of the individual enzymes on the macromolecular structure of insoluble cellulose substrates.
The rate of enzymatic hydrolysis of the cellulosic materials always decreases rather quickly. Generally, enzymatic cellulose degradation is characterised 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
17
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, cellobiohydrolases 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; Reinikainen 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 cotton 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 cellulose surface. CBDs can promote the enzyme activity towards different regions 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,
18
since the DP remains constant during the course of hydrolysis. [Lee et al.,
1983]. Similar findings have been described for H. jecorina cellobiohydrolase Cel6A acting on cotton fibers [Kleman-Leyer et al., 1996]. The thinning 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 enclosed 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 cellulose 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 cellobiohydrolases, 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 between 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 cellooligosaccharides 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 activity, 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 individual components. Two classes of synergism between cellulases have
19
been described: the cooperation between endoglucanases and cellobiohydrolases (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 cellulose 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, E-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. jecorina
produces a complete cellulolytic enzyme system and is capable of very efficient 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
Cel6A
Cel7B
Cel5A
Cel12A
Cel61A
Cel45A
CBH I
CBH II
EG I
EG II
EG III
EG IV
EG V
57
53
55
50
25
55
36
3.9
5.9
4.5
5.5
7.5
C
N
C
N
C
C
2.9
Hitherto, two cellobiohydrolases (Cel7A and Cel6A), five endoglucanases
(Cel7B, Cel5A, Cel12A, Cel61A and Cel45A) and two ȕ-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 ȕ-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
20
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 cellobiohydrolases 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 dimensions 4.5x18 nm for Cel7A and 4.5x21.5 nm for Cel6A [Abuja et al., 1988;
Abuja et al., 1989].
Cel7A
Cel7A (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 ȕ-sandwich
structure with a 50Å-long substrate-binding tunnel formed by the inner ȕsheets 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 glycosylbinding platform in sites –7, –4, –2 and +1 in the tunnel of Cel7A. Cellobiose 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 ȕ-1,4 glucosidic bond of cellulose with retention of the anomeric carbon configuration [Knowles et al., 1988; Claeyssens et al., 1990]. Site-directed mutagenesis confirmed that Glu217 acts as the proton donor and Glu212 as the nucleophile in a double-displacement mechanism, whereas Asp214 is likely to
be involved in maintenance of the appropriate pKa values of the other catalytic 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 KM
values decrease from 7 µM to 3 µM [Nidetzky and Claeyssens, 1994]. Struc21
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].
Cel6A
Cel6A (CBH II) is another cellobiohydrolase produced by H. jecorina, constituting 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 Į/ȕ barrel protein, similar to triose
phosphate isomerase (TIM) with the exception that it contains seven instead
of eight ȕ-strands. The active site forms a 20 Å long tunnel with four glycosyl 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 movements, 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 endoactivity 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 nonreducing 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].
Endoglucanases
Cel7B (EG I) is the main endoglucanase of H. jecorina, accounting for 510% 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 possesses 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 glucosidic bond via the double-displacement mechanism and has slightly lower
activity than Cel7B on substituted celluloses and ȕ-glucan [Penttilä et al.,
1987]. Its three-dimensional structure is not yet established, but the kinetic
22
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 ȕ-1,3-1,4 glucan, xyloglucan and xylan [Hayn et al., 1993; Karlsson et al., 2002]. The crystal
structure revealed that Cel12A consists of two largely anti-parallel ȕ-sheets
which pack on top of each other. The substrate-binding cleft is approximately 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 doubledisplacement mechanism with Glu 116 as nucleophile and Glu 200 as general acid/base [Okada et al., 2000].
The expression of Cel61A (EG IV) is induced together with the other cellulases in H. jecorina [Saloheimo et al., 1997]. However, the specific endoglucanase 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 activity toward cellulosic substrates than do other H. jecorina endoglucanases
[Karlsson et al., 2002]. The main product of Cel45A (EG V) cellulose hydrolysis is cellotetraose, with significant amounts of cellotriose and cellopentaose. It hydrolyses readily glucomannan, being able to cleave a glycosidic 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. chrysosporium
exhibits a system of synergistically-acting cellulases homologous to H. jecorina [Uzcategui et al., 1991a; Uzcategui et al., 1991c]. Sequencing of the
whole P. chrysosporium genome revealed at least 40 putative endoglucanase-encoding genes (in families GH5, GH9, GH12, GH61 and GH74), six
genes encoding GH7 cellobiohydrolases and one gene for a cellobiohydrolase belonging to the GH6 family [Martinez et al., 2004]. The data concerning 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
23
44) and Cel12A (EG 28) have been isolated and characterised from P. chrysosporium [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
Cel7C
Cel6A
Cel5B
Cel5A
Cel12A
CBH 58
CBH 62
CBH 50
EG 44
EG 38
EG 28
58
62
50
44
38
28
3.8
4.9
4.9
4.3
5.6-5.7
5.2
C
C
N
N
N
-
In contrast to H. jecorina, where the expression of cellulases is co-regulated,
the cellulase genes of P. chrysosporium are differentially transcribed, depending 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].
Cel7D
Cel7D 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 sequence 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 tunnel, 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].
24
Phanerochaete chrysosporium cellobiose dehydrogenase
Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by
many lignocellulose-degrading soft-rot, white rot and brown-rot fungi, including 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 cellobiose compared to glucose [Henriksson et al., 1998]. This feature has been
used to construct amperometric biosensors for the measurement of cellobiose, 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 cytochrome 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 peanutshaped 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 sugarbinding 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
25
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 acceptors, and the electron chain model, where the heme directly reduces the electron acceptor after obtaining the electrons from FADH2, have been discussed. 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 oneelectron acceptors.
CDH can produce both hydrogen peroxide and superoxide as primary reduction 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 submicromolar 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 established 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 repolymerisation of lignin by reducing phenoxyl radicals produced by lignolytic
enzymes; participating in lignin degradation by supporting manganese peroxidase, relieving product inhibition of cellulases by oxidating cellobiosethe main hydrolysis product cellulases, etc. One of the suggested functions
of the CDH is involved in generating reactive oxygen species, such as superoxide 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 accelerate the depolymerisation of cellulose by attacking its crystalline structure,
thereby making it more accessible for hydrolytic enzymes.
26
Present investigation
Aims of the present study
This thesis has focused on the characterisation of the dynamics in the enzymatic degradation of cellulose microfibrils, including both the action of the
individual enzymes and their synergistic interplay. More specifically, the
following tasks were addressed:
x To determine the end-preference of cellobiohydrolases on their natural
substrate - crystalline cellulose.
x To investigate synergistic mechanisms between endo- and exo-acting
cellulases with specific focus on its dependence on the nature of the
substrate.
x To evaluate the conversion of oxygen species by cellobiose dehydrogenase.
In addition, a new endoglucanase from Phanerochaete chrysosporium was
isolated and characterised. The interactions between GH7 family cellobiohydrolases and o-nitrophenyl cellobioside were investigated and employed for
indirect binding studies.
27
Action of cellulases on end-labelled cellulose (Paper I)
The aim of paper I was to determine the end-preference of cellobiohydrolases acting on crystalline cellulose.
It was assumed for a long time that cellobiohydrolases hydrolyse cellulose 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 endpreferences. 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 labeled 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,
28
probability for endo/exoactivity, processivity towards labelled ends, processivity 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 dissociating 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 substrate, 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 labelled 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 processively. The aim of our study was to evaluate the synergistic mechanism between 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.
29
Table 3. Parameters of cellulose samples. BC, appearance common to bacterial
cellulose (large fibrous bundles); BMCC, appearance common to bacterial microcrystalline cellulose (suspension with a milky consistency)
Property
Time in 1M HCl, min
Weight loss by HCl, %
Crystallinity index, %
DP, glucose unit
Appearance
Relative activity
Cel7A, %
Cel7B, %
Cel7A/Cel7B, %
Maximum synergy
factor
Maximum effect of
pretreatment with
Cel7B
Sample
BC
0
0
87.9
2620
BC
A25
25
0.26
90.3
212
BC
A40
40
1.0
91.3
151
BC
BMCC
760
5.2
92.4
114
BMCC
37
95
100
69
53
56
100
57
50
66
100
33
7.8
4.1
2.3
1.7
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 untreated bacterial cellulose and decreased during the course of the acid hydrolysis. 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 samples 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 activity 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
30
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 Phanerochaete
chrysosporium cellulase (Paper III)
Paper III describes the isolation and characterisation of a new small endoglucanase from Phanerochaete chrysosporium.
Protein purification
Phanerochaete chrysosporium strain K3 was cultivated and the culture filtrate 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 sulphate 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 ammonium 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 corresponding 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 towards 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. Deglycosylation analysis together with amino acid analysis showed that this protein contains 1-2 N-glycosylation sites. Peptide mapping after cleavage with the V8
protease showed a pattern clearly distinguishable from the previously characterised 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.
31
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 Į-helix
of 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 contrast 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
Cel7B
Cel7A
1.61
87.4
0.146
12.6
3.46
97.4 * 10-3
0.13
2.5 * 103
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 endoglucanases.
32
Cel12A swells efficiently filter paper and disperses the filter paper structure, releasing short fibres. This phenomenon led us to propose a physiological 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 microfibrils are held together by more amorphous material. 2) Cel12A diffuses into
pores and nicks the amorphous chains, thereby releasing the microfibrils. 3) “Classical” endoglucanases now have access to the substrate and can create nicks. 4) Cellobiohydrolases 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 hydrogen peroxide produced by CDH and into which species it is converted. We
compared CDH to a well-characterised enzyme – glucose oxidase (GOX).
33
This enzyme catalyses the oxidation of glucose into gluconolactone by reducing molecular oxygen to hydrogen peroxide:
GOX produced H2O2 in equimolar amounts to the cellobiose and O2 consumed (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 reduction by CDH.
34
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 saccharide:
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 present in standard test conditions and in nature seem to be sufficient for an
enhanced Fenton reaction, where CDH produces both components neededreduced metal ions and hydrogen peroxide.
35
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, however, the kcat observed has been orders of magnitude lower than that observed
for the cleavage of oligosaccharides. For cellulase studies, mostly p-nitrophenyl cellobioside and lactoside have been used as model substrates. Interestingly, 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-nitrophenyl cellobioside (oNPC) and GH7 family cellobiohydrolases were investigated.
Table 5. Comparison of kinetic constants of Cel7A and Cel7D on oNPC and
pNPC.
Enzyme / substrate
Cel7A / oNPC
Cel7A / pNPC
Cel7D / oNPC
Cel7D / pNPC
kcat, s-1
-6
(66±15)*10
0.0026±0.0001
0.015±0.002
0.046±0.0021
KM, µM
kcat /KM, s-1M-1
7.0±4.5
26±3
3200±100
1300±160
9.5
100
4.6
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 cellulases 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 nonproductive binding. This may result in devations from the standard Michaelis-Menten kinetics, which has been observed for certain cellobiohydrolasemodel 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
36
highest affinity [Divne et al., 1998] The fluorescence of Cel7D did not recover 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 cellobiose, 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
D214N
E212Q
E217Q
Cel7D
7.4±0.4
7.1±0.7
4.7±0.4
3.9±0.4
110±10
23±4
8.9±1.1
8.1±0.3
13.5±3
-
37
Conclusions
GH7 family cellobiohydrolases Cel7A and Cel7D degrade crystalline cellulose 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 cellobiohydrolases.
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 structures.
Hydrogen peroxide produced by CDH is decomposed via an enhanced Fenton’s reaction.
o-nitrophenyl cellobioside has a potential as an active site probe for GH7
family cellobiohydrolases.
38
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 stärkelse, av långa kedjor av hopkopplade glukos-(druvsocker)molekyler. Skillnaden mellan cellulosa och stärkelse bygger väsentligen på att glukosenheterna 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 processer förefaller lovande för energiomsättning, i synnerhet då för omvandling av
lignocellulosamaterial till bekvämare bränslen. Därmed är en fördjupad förståelse 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 rimliga 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 chrysosporium, 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 använts för att funktionellt karakterisera ett antal viktiga enzymer. Här kunde
vi avgöra att de hos svampar ofta förekommande enzymerna från strukturfamilj 7 startade nedbrytningen av cellulosakedjorna från den s.k. reducerande 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
39
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 endoglukanas med ovanligt små dimensioner, som isolerades från P. chrysosporium
och studerades funktionellt. Dess släktskap med andra kända enzymer fastslogs också. Detta enzym var jämförelsevis ineffektivt i sin förmåga att frisätta 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 enzymer.
Hos P. chrysosporium och många andra vitrötesvampar hittar man förutom de enzymer som bryter ner cellulosakedjorna, också ett enzym som kallas 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 väteperoxid, vilket motsades av andra resultat som antydde att sådan inte kunde producerades. 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 reportermolekylen om att bindas på samma ställe.
40
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 supported 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 allowing 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 cellulases 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 environment. 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
41
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 always 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.
42
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