Department of Mocrobiology
MBiotec Group

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The Cellulose and Cellulosome Page

 



 

AN INTRODUCTION TO CELLULOSE HYDROLYSIS

 

 

SUMMARY

Our main research interest is the hydrolysis of cellulose because

1. cellulose is the most abundant carbohydrate in the biosphere
2. its hydrolysis plays a major role in the carbon cycle in the atmosphere
3. the hydrolysis of crystalline substrates like cellulose is an unsolved biochemical problem, and
4. the action of multienzyme complexes is a fascinating research subject.

Despite its simple chemical composition, cellulose exists in a number of crystalline and amorphous topologies. Its insolubility and heterogeneity makes native cellulose a recalcitrant substrate for enzymatic hydrolysis (despite its homogeneous chemical composition). Microorganisms meet this challenge with the aid of a multi-enzyme system. Aerobic bacteria produce numerous individual, extra-cellular enzymes with binding modules for different cellulose conformations. Specific enzymes act in synergy to elicit effective hydrolysis. In contrast, anaerobic bacteria possess a unique extracellular multienzyme complex, called cellulosome. Binding to a non-catalytic structural protein (scaffoldin) stimulates activity of the single components towards the crystalline substrate. (1) The most complex and best investigated cellulosome is that of the thermophilic bacterium Clostridium thermocellum. A scheme for the cellulosomes slowly emerges. Many crucial details of the cellulose hydrolysis are still to be uncovered - yet, a mechanistic model for the action of enzyme-complexes on the surface of insoluble substrates becomes apparent and application of enzymatic hydrolysis of cellulosic biomass can now be addressed.

Introduction to cellulose hydrolysis (1)

About half of the carbonaceous compounds in terrestrial biomass are cellulose, which is the most prominent single organic compound on earth. The net primary production of biomass was estimated to be 60 Gt yr-1 of carbon in terrestrial and 53 Gt yr-1 in marine ecosystems (1 Gt = 1012 kg) (Cox et al. 2000). Almost all of the biomass produced is mineralized again by enzymes which are provided by microorganisms. The polysaccharide hydrolysis thus is one of the most important enzymatic processes on earth, and cellulose synthesis and hydrolysis is a great part of the carbon cycle. Cellulose is a chemically homogeneous linear polymer of up to 10 000 D-glucose molecules, which are connected by ß-1,4-bonds. As each glucose residue is tilted by 180° towards its neighbors, the structural subunit of cellulose is cellobiose (structure of cellulose). The chemical uniformity provokes spontaneous crystallization of the cellulose molecules, the tightly packed microfibrils. Cellulose thus is a sturdy material ideally suited to insure the structural stability of land plants where it is a main component of the primary cell wall, especially in wood. Although crystalline cellulose is chemical homogeneous, no single enzyme is able to hydrolyze it, whereas soluble cellulose derivatives are easily degraded by a single endo-ß-1,4-glucanase. The extensive, level surface of the insoluble crystalline microfibrils is an unusual, resilient substrate for hydrolytic (soluble) enzymes. Enzyme mechanisms generally depend on single molecules fitting in their substrate pocket - with cellulose the substrate is much larger than the enzyme (a schematical drawing showing size relation). A model drawing shows the difference between enzymatic attack on a single and a multichain substrate (figure).

The crystalline material is hydrolyzed by a number of simultaneously present, interacting enzymes, or alternatively by a multienzyme complex. Only by cooperation with non-catalytic specific binding modules (the carbohydrate binding proteins or modules) the enzymes are able to disrupt the crystal surface at the solid-liquid interphase, to make single cellulose fibers accessible for hydrolysis. The investigation of the hydrolysis mechanisms of cellulases opens up a new way of looking at enzymatic activity: the dualism between mechanical and structural "preparation" of the insoluble (crystalline) substrate followed by the hydrolytic activity on a released molecule. The recently increased research in cellulases aims at the enzymatic mechanisms at the surface of the insoluble substrate. It also tries to solve the problems with direct conversion of biomass into valuable products by using isolated enzymes or cellulolytic microorganisms (Sheehan and Himmel 1999).

An artificial formulation of combined cellulases that works effectively in low concentration is a goal not yet accomplished. To be cost effective in a commercial process, it particularly would have to speed up the slowest step of the process, namely breaking up the crystal surface and releasing single cellulose molecules. Many reviews have been published in the recent years to describe the structure of cellulose (Heiner et al. 1995), the mode of action of enzymes on the crystalline cellulose surface (Teeri 1997; Boisset et al. 1999; Shoham et al. 1999), and the structure of cellulolytic multienzyme complexes (Beguin and Lemaire 1996; Bayer et al. 1998a, b). This article describes new advances in the understanding of the bacterial hydrolysis of crystalline cellulose with emphasis on the cellulosome.

Cellulose hydrolysis

Enzymatic cellulose hydrolysis generally is a slow and incomplete process. However, in relatively short time (up to 48 hrs) the microbial consortium in the bovine rumen hydrolyzes cellulose to 60 - 65 %, and the lower termites were even reported to assimilate wood cellulose to an extent greater than 90 % (Breznak and Brune 1994). In complex biological systems like a rotting tree or plant debris in soil, cellulose is decomposed in a time scale of months, although at lower temperature. Based on the intensive research on the cellulase system of the fungus T. reesei, it was accepted for some time as a general concept that cellulolysis is performed by the synergistic action of three types of enzymatic activities: endoglucanases or 1,4-ß-D-glucan 4-glucanohydrolases (EC 3.2.1.4) acting at random in the polymeric chain and producing new ends; exoglucanases which include both 1,4-ß-D-glucan glucanohydrolases (EC 3.2.1.74), liberating D-glucose from ß-glucan and cellodextrins, or 1,4-ß-D-glucan cellobiohydrolases (EC 3.2.1.91) that liberate D-cellobiose from ß-glucan in a processive manner; and ß-glucosidases or ß-D-glucoside glucohydrolases (EC 3.2.1.21), which release D-glucose units from soluble cellodextrins and a variety of glycosides. However this simplified model had to be modified in the light of new findings, as will be described below.The screening of cellobiohydrolase and endoglucanase clones is shown in a figure.All cellulases hydrolyze the ß-1,4-glucosidic bond between glucosyl moieties by a general acid catalysis requiring a proton donor and a nucleophile/base. They release the products either by overall retention or inversion of the anomeric configuration at C1. The protein sequences were classified in glycosyl hydrolase families (GHF) based on amino acid sequence similarities (Henrissat and Bairoch 1996; Coutinho and Henrissat 1999). The overall 3D-structure and the stereospecificity of hydrolysis are conserved within a family. More than 600 cellulase genes are known to date. A summary of the enzymic mechanisms involved was given by Schülein (2000).The enzymes have a complex molecular architecture comprising discrete modules: the catalytic domains are joined to non-catalytic modules, called carbohydrate binding modules (CBM), by linker regions (commonly proline-threonine-serine- or PTS-boxes). Other non-catalytic modules, partially of unknown function, may also be present. A recent nomenclature allows to describe enzymes composed of various modules unequivocally (Henrissat et al. 1998; Corpet et al. 1999).

Substrate binding modules are essential

Cellulases differ not only in the action mode (endo or exo), but also in the way they bind to the crystalline surface of the substrate. There are two sites in the enzymes which mediate binding: the active site of the catalytic domain and the separately folded and functionally independent carbohydrate binding module (CBM) which usually is attached through a PTS-box. This linker may act as a flexible arm, connecting the two functional parts of the protein, but leaving (limited) freedom for the catalytic domain to move around the binding module fixed to the substrate surface. The essential function of the CBM was shown for cellobiohydrolase CBHI from T. reesei, for which a detailed 3-dimensional model was constructed (Lee and Brown 1997). The catalytic domain without the CBM (the core enzyme) has a very limited overall-action on cellulose. The deletion of CBMs has no effect for activity on soluble substrates (like CMC or barley ß-glucan) where the possible sites of activity on the substrate are not limited. This behavior was confirmed by the characterization of genetically engineered deletion mutants (Tomme et al. 1995; Bolam et al. 1998).

The sequences of modules binding to various carbohydrates have been grouped in 26 families according to their amino acid sequence similarities (Coutinho and Henrissat 1999). A continually updated list is available at URL http://afmb.cnrs-mrs.fr/~pedro/CAZY/cbm_table.html. Members of each group have been investigated for their binding capacity for a number of polysaccharides: crystalline and amorphous cellulose, ß-1,3-glucan, xylan, starch, chitin and others (Tomme et al. 1998). Even within one family binding to different substrates is possible (Zverlov et al. 2001). Although CBMs bind to the cellulose with a high association constant and sometimes irreversibly, they show, in conjunction with a catalytic domain, surface diffusion and redistribute on the surface (Jervis et al. 1997; Carrard et al. 2000). This allows for erosion along the cellulose chains on the surface of the crystal (Väljamäe et al. 1998).

Although CBMs are important for the processivity of cellulases (Irwin et al. 1998), there is no hint for a driving force, neither by the CBM nor by the catalytic unit. Some CBMs, like that of the cellulosomal components (see below), exhibit a lower affinity for crystalline cellulose with a more general binding behavior for a broad range of sites on the cellulose crystal, and thus are especially useful in industrial applications. The CBMs of the soluble cellulases and the CBM IIIa of the cellulosomal scaffoldins generally have a higher affinity.

The hydrolytic strategy of the anaerobic bacteria

Some anaerobic microorganisms have developed a most economical way to circumvent at least a part of the above mentioned problems: the Syntrophomonadaceae (list of cellulolytic bacteria) combine more than one of the essential and synergistic catalytic domains with a CBM in one complex polypeptide. Such multifunctional cellulases of "Anaerocellum thermophilum" or Caldicellulosiruptor sp. contain modules of GHF 5, 9, 44 or 48 in varying pairs, also combined with mannanase and xylanase units (Zverlov et al. 1998; Gibbs et al. 2000). An intramolecular synergism can be assumed for these complex enzymes, promising enhanced activity and/or processivity even at low enzyme concentration. This kind of synergism was demonstrated with the cellulase system from C. stercorarium by combining a GHF 9 and 48 catalytic module with central CBMs (Riedel and Bronnenmeier 1998). An even more elaborate multienzyme system is realized in other anaerobic microorganisms, the clostridia: an extracellular multienzyme complex which is called cellulosome. It was discovered in the cellulolytic bacterium Clostridium thermocellum (Bayer et al. 1983). They are macromolecular machines specially designed for the hydrolysis of insoluble polysaccharides.

Cellulosomes are cell protuberances which tightly bind to crystalline cellulose (Lamed et al. 1987; Mayer et al. 1987). They mediate a close neighborhood between cell and substrate and thus minimize diffusion losses of hydrolytic products, which is thought to be a major advantage for attached cells. A cellulosome preparation contains a number of different proteins, most of them having enzymatic activity. However, attempts for mild denaturation, purification of single components and reconstitution were only partially successful (Beattie et al. 1994; Bhat et al. 1994; Choi and Ljungdahl 1996). Therefore the vast majority of investigations on the cellulosome were done by cloning and genetic engineering. Genetical and biochemical data revealed, that in all cellulosomes investigated so far the components of the multienzyme complex are strongly bound to each other by a duplicated, non-catalytic segment of 22 amino acid residues found to be conserved in all enzymes which are located in the cellulosome (Tokatlidis et al. 1991). This dockerin module binds specifically to the cohesin modules, located in a non-catalytic cellulosome component, for which the term "scaffoldin" was coined (cellulosome structure).

The catalytic components themselves are complex proteins consisting of catalytic and non-catalytic modules. Binding of the cellulosome to the crystalline substrate is mainly mediated by a very strongly binding CBM IIIa module of the scaffoldin. The production of the multienzyme-complex "cellulosome" may have a number of advantages for the effective hydrolysis of cellulose: 1. synergism is optimized by the correct ratio between the components, which is determined by the composition of the complex; 2. non-productive adsorption is avoided by the optimal spacing of components working together in synergistic fashion; 3. the competitiveness in binding to a limited number of binding sites is avoided by binding the whole complex to a single site through a strong binding domain with low specificity; 4. a stop of hydrolysis on depletion of one structural type of cellulose at the site of adsorption is avoided by the presence of other enzymes with different specificity. ___________________________________________________________________________

The cellulosome of Clostridium thermocellum

C. thermocellum is a moderately thermophilic bacterium (55 - 65 °C), repeatedly isolated from hot springs and wet, rotting biomass. It is highly specialized for growth on cellulose and cellodextrins as carbon and energy source. Cellobiose and soluble cellodextrins are taken up and hydrolyzed intracellularily by cellobiose- and cellodextrin-phosphorylase (Arai et al. 1994; Strobel et al. 1995; Tanaka et al. 1995). The extra-cellular cellulolytic complex of C. thermocellum differs between the species somewhat in size (from 2 to 6,5 MDa) and composition (Béguin and Lemaire 1996). In some strains the cellulosomes aggregate to larger supercomplexes, called polycellulosomes, with a molecular mass ranging up to 100 MDa. The protein pattern found in SDS-PAGE after complete denaturation by boiling in EDTA and SDS are identical for cellulosomes and polycellulosomes and show up to 50 components (= protein bands) depending on the strain and the growth condition. The single components of the cellulosomes are not present in the same amount; a few components dominate, some are underrepresented, indicating a heterogeneous composition for individual complexes (see table 2 for the major components). The composition of the complex varies with the carbon source (Bhat et al. 1993). The scaffoldin (Ct-CipA) holds the cellulosome complex together. It consists of nine cohesin domains, a cellulose binding domain CBM IIIa with broad binding specificity for different sites on crystalline cellulose, a hydrophilic X-domain and a modified dockerin domain (cellulosome structure).

Click here for a model of the cellulosome structure or for a list of the cellulosome components.

Despite overall similarity of the cohesin sequences of different species, the binding is highly specific. The four amino acid residues in the dockerin responsible for this specific protein-protein interaction have been determined, but additional residues seem to add more subtle secondary interactions modifying the biorecognition (Mechaly et al. 2000). Some components may bind preferentially to specific cohesin sites, whereas others may compete for the remaining sites. This would guarantee the presence of essential components in each individual cellulosome particle while other (minor) components are distributed more scarcely. Altogether 23 genes for cellulosomal components of C. thermocellum have been detected to date (table components). All components contain a dockerin repeat sequence. The presence of 10 components in the cellulosomal complex has been verified. CbhA, CelK, CelS and CelO are exoglucanases (cellobiohydrolases), and 9 genes code for endoglucanases (Guglielmi and Béguin 1998).

Surprisingly there are also cellulosomal genes for 1 lichenase (ß-1,3-1,4-endoglucanase), 1 chitinase, 1 mannanase and 5 xylanases. Xylanases XynY and XynZ also contain xylan-esterase modules to remove feruloyl-residues from native xylan (Blum et al. 2000). The attached xylan-binding modules play an important role in hemicellulose hydrolysis (Fernandes et al. 1999). These non-cellulolytic activities may assist in the degradation of biomass, where cellulose is embedded in a matrix of hemicellulose.

Interestingly the sequence of genes isolated from different strains of C. thermocellum did not differ except in single bases, irrespective of the point of isolation or the age of culture: Yellowstone National Park (strain ATCC 27405, 1948), Armenia (F-7, 1984) or Japan (F-1, 1994). The biogeographical distribution of C. thermocellum seems to be a rather recent event, at least within the northern hemisphere. Many questions remain to be solved, esp. towards the extracellular assembly of the cellulosome, the composition of the individual components and the synergism between the components and the CBM. Among the cellulosome forming bacteria C. thermocellum is the most thermophilic. It is unique in the arrangement of cellulosomal genes in the chromosome, and the size and complexity of its cellulosome. The efficiency of its cellulosome makes it a good candidate for commercial bioconversion.

Conclusion

Cellulose is an insoluble, crystalline substrate. Its sturdy structure poses serious problems on the enzymatic hydrolysis and requires the perfect interplay of at least four components: ¨ Carbohydrate binding modules (CBM): for the attachment and positioning of the catalytic components on the surface of the insoluble substrate; a role in the displacement of single molecules from the crystal was discussed; ¨ Endoglucanases (EC 3.2.1.4): to degrade soluble and amorphous regions of the cellulose, and to produce new ends by cutting into long cellulose-strands; ¨ Exoglucanases (EC 3.2.1.74) or cellobiohydrolases (EC 3.2.1.91): to hydrolyze processively from both ends and possibly also to displace cellulose molecules from the crystal surface; two types of cellobiohydrolases with specificity from the reducing and the non-reducing end are needed simultaneously; ¨Cellobiases and cellodextrinases, specific ß-glucosidases (EC 3.2.1.21): to degrade the resulting oligosaccharide products; in vivo, sugar transport systems and in some bacteria, as an energy-saving alternative to ß-glucosidases, cellobiose- and cellodextrin-phosphorylases (EC 2.4.1.29 and EC 2.4.1.49) are to be considered.

Clostridia and ruminococci have developed the cellulosome, an optimized multienzyme complex, which displays intra-molecular synergism by connecting all the necessary components in the correct ratio and order. It contains a non-catalytic scaffolding protein, which captures a number of hydrolytic components by specific protein-protein interaction. Major catalytic components of the cellulosome are two cellobiohydrolases with different direction of processivity (GHF 48 and 9). Endoglucanases also play an important role in hydrolysis. C. thermocellum, so far the most thermophilic cellulosome producer, has the largest cellulosome with the greatest complexity. Cellulosomes have many advantages for a biotechnological application in the hydrolysis of cellulosic biomass. By using gene technology they should have a considerable development potential.

The direct conversion of biomass into solvents is a promising perspective for targeted screening for new bacterial strains or directed strain development of existing cellulolytic clostridia. However, a commercial process is still far ahead. Understanding the cellulosome mechanism requires a lot more of basic research. This will improve our knowledge of new enzymatic mechanisms on solid surfaces and will help to rationally design a optimal enzymatic hydrolysis system for application in an economical process of biomass saccharification.

References

1. Schwarz, W.H. (2001). The cellulosome and cellulose degradation by anaerobic bacteria. Appl. Microbiol. Biotechnol. 56: 634-649.

The publication list of our group.

For all other citation see the review (1).