Degradation Of Crystalline Cellulose Research Articles

Multiresolution Molecular Dynamics Simulations of Crystalline Nanofibrils

We introduce a multiresolution computational approach for the study of crystalline nanofibrils.Our multiresolution approach integrates fully-atomistic and coarse-grained levels of detail and it's particularly suited for the study of structural transitions between crystalline allomorphs. First, fully-atomistic simulations are used to gain a detailed understanding of the main structural differences between the crystalline phases under consideration. Second, we introduce a new coarse-grained, off-lattice model for the crystalline fibrils whose relevant degrees of freedom have been identified from the analysis of our fully-atomistic simulations. Both the structural transition and the relative thermal stability of the two allomorphs are studied at the coarse-grained level by means of Replica exchange molecular dynamics. The structural transition is analyzed within the framework of the Ginzburg-Landau formalism. As an example application of our method we consider two different allomorphs of crystalline cellulose nanofibrils, namely cellulose I-beta (the naturally--occurring form of cellulose) and cellulose III(I) (obtained from cellulose I-beta via ammonia pretreatment). Recent experiments show that the enzymatic degradation rate increases 2-5 times in cellulose III(I) respect to cellulose I-beta. Understanding the factors that regulate enzyme degradation of crystalline cellulose is a major challenge in the context of biofuels production from cellulosic biomass. Our multiresolution computational approach sheds new light on how the main structural and thermodynamic differences between these two cellulose crystalline forms affect their different enzyme activity rates.

Demonstration of the importance for cellulose hydrolysis of CelS, the most abundant cellulosomal cellulase in Clostridium thermocellum

Anaerobic and aerobic bacteria use different mechanisms to degrade cellulose, with many aerobic microorganisms secreting a synergistic set of individual enzymes that includes multiple cellulases, most of which contain a carbohydrate-binding module (CBM) in addition to their catalytic domain (free enzyme mechanism). Anaerobic microorganisms usually produce a similar set of enzymes, but they are present in large multienzyme complexes (>1 million molecular weight) called cellulosomes (cellulosomal mechanism) (1). Most of the cellulosomal enzymes do not contain a CBM, but the scaffoldin protein in the cellulosome does contain a CBM. At this time, there are still a number of unanswered questions about how cellulosomes are able to degrade cellulose so well, despite their large size, which should limit their access to the cellulose surface, much of which is present in narrow pores (2). One suggestion is that enzyme proximity on the cellulosomes increases the rate of cellulose degradation, but it would seem that enzyme proximity would be most effective if enzymes were incorporated in a specific order in cellulosomes. All of the different cohesin domains in the scaffoldin protein have equal affinity for all of the docerin domains present on the cellulosomal enzymes so that it is thought that the enzymes are randomly incorporated into cellulosomes, but this has not been proven. Synergism between cellulases that have different sites of attack (i.e., endocellulases and exocellulases) is important for crystalline cellulose degradation by both mechanisms, because the specific activity of a synergistic mixture can be up to 15 times that of the most active individual cellulase (3).

The genes encoding glycoside hydrolase family 6 and 7 cellulases from the brown-rot fungus Coniophora puteana

Four genes encoding glycoside hydrolase (GH) family 6 and 7 cellulases (cel6A, cel6B, cel7A, and cel7B) were obtained from the brown-rot fungus Coniophora puteana by genomic polymerase chain reaction (PCR) using consensus degenerate hybrid oligonucleotide primers (CODEHOPs) designated from the amino acid sequence of cellobiohydrolases (CBHs) from white-rot fungi. The nucleotide sequences of four genes showed high homology with basidiomycetes CBHs, suggesting the fi rst cloning of the genes encoding Cel6 and Cel7 from brown-rot fungi. PCR using CODEHOP pairs at the catalytic domain successfully amplifi ed both cel6A and cel6B, whereas only cel6A fragment was obtained using the primers including the carbohydrate-binding modules (CBMs), suggesting lack of CBM in Cel6B. Moreover, both cel7A and cel7B were amplified by the PCR using CODEHOP pairs at the catalytic domain, but not by those including CBM, suggesting the absence of Cel7 with CBM in the fungus. From these results, three of four cellulases from C. puteana may not carry CBM, which has an important role for the degradation of crystalline cellulose.

Quantitative transcriptional analysis of the genes encoding glycoside hydrolase family 7 cellulase isozymes in the basidiomycetePhanerochaete chrysosporium

Cellulolytic fungi generally secrete a cellulase mixture consisting mainly of glycoside hydrolase family 7 cellulases (Cel7s) during degradation of crystalline cellulose. Although several Cel7s have been investigated so far, the marked similarity in their amino acid and nucleotide sequences makes independent quantitative analysis difficult. Here, we present a real-time PCR method for the detection and quantification of Cel7 genes (cel7A-F/G) in the basidiomycete Phanerochaete chrysosporium using PCR primer sets designed based on the 3' untranslated region sequences. It was confirmed by agarose gel electrophoresis, sequencing, and dissociation curve analysis of the PCR products that each cel7 transcript was specifically amplified by the corresponding primers. We applied this real-time reverse-transcription PCR method using the presented primer sets to evaluate quantitatively the expression changes of cel7 genes in P. chrysosporium under conditions of carbon catabolite derepression.

Probing the role of N-linked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis

The filamentous fungi Trichoderma reesei and Penicillium funiculosum produce highly effective enzyme mixtures that degrade the cellulose and hemicellulose components of plant cell walls. Many fungal species produce a glycoside hydrolase family 7 (Cel7A) cellobiohydrolase, a class of enzymes that catalytically process from the reducing end of cellulose. A direct amino acid comparison of these two enzymes shows that they not only have high amino acid homology, but also contain analogous N-linked glycosylation sites on the catalytic domain. We have previously shown (Jeoh et al. in Biotechnol Biofuels, 1:10, 2008) that expression of T. reesei cellobiohydrolase I in a commonly used industrial expression host, Aspergillus niger var. awamori, results in an increase in the amount of N-linked glycosylation of the enzyme, which negatively affects crystalline cellulose degradation activity as well as thermal stability. This complementary study examines the significance of individual N-linked glycans on the surface of the catalytic domain of Cel7A cellobiohydrolases from T. reesei and P. funiculosum by genetically adding or removing N-linked glycosylation motifs using site directed mutagenesis. Modified enzymes, expressed in A. niger var. awamori, were tested for activity and thermal stability. It was concluded that N-linked glycans in peptide loops that form part of the active site tunnel have the greatest impact on both thermal stability and enzymatic activity on crystalline cellulose for both the T. reesei and P. funiculosum Cel7A enzymes. Specifically, for the Cel7A T. reesei enzyme expressed in A. niger var. awamori, removal of the N384 glycosylation site yields a mutant with 70% greater activity after 120 h compared to the heterologously expressed wild type T. reesei enzyme. In addition, similar activity improvements were found to be associated with the addition of a new glycosylation motif at N194 in P. funiculosum. This mutant also exhibits 70% greater activity after 120 h compared to the wild type P. funiculosum enzyme expressed in A. niger var. awamori. Overall, this study demonstrates that “tuning” enzyme glycosylation for expression from heterologous expression hosts is essential for generating engineered enzymes with optimal stability and activity.

Exploring The Dynamic Actions Of Cellulolytic Enzymes In A Heterogeneous System With Micro-cantilever Technology

The cellulolytic enzyme degradation process suffers from low efficiency and high cost because of the low activity of cellulases against their natural substrates cellulose, which is insoluble and crystalline in its native form. As a result, the degradation of crystalline cellulose becomes the rate-limiting step in the overall scheme of biomass conversion to ethanol. To address this problem, the development of a highly efficient and cost-effective cellulase has become one of the top priorities of the Advance Energy Initiative. Such an effort requires a thorough understanding of the mechanisms of cellulolytic enzyme actions including interactions between the cellulases and their native substrates. The current technologies such as ellipsometry and quartz crystal microbalance have not been able to provide with the level of sensitivity and resolution required for detailed characterization of the cellulolytic enzyme actions, especially the interaction between cellulase and glucan chains of cellulose, and the impact of such interaction on overall cellulose structure. To define cellulase actions in such a heterogeneous system, we focus on investigating the initial interaction between cellulase and its native substrate, crystalline cellulose by taking advantage of emerging micro-cantilever technology. We have constructed a micro-cantilever with a cellulose coating which allows us to detect the actions of cellulolytic enzymes in real time.

Carbohydrate metabolism inFibrobacter succinogenes: What NMR tells us

Fibrobacter succinogenes is a major rumen fibrolytic bacterium found in high numbers when ruminants are fed cellulose-rich diets. It produces a very efficient fibrolytic system comprising numerous enzymes, but the organization and catalytic features of this system remain unclear. We were using nuclear magnetic resonance (NMR) to study carbohydrate metabolism by Fibrobacter spp. well before the term ‘metabolomics’ was coined. We first analyzed in detail the NMR spectra arising from resting cells of F. succinogenes provided with glucose, cellobiose or cellulose as a substrate. We were able to show carbohydrate cycling and reversibility of some steps. The use of 1D and 2D 1 H and 13 C NMR showed the synthesis by the cells of unexpected oligosaccharides: maltodextrins, maltodextrin-1-phosphate, and other glucose derivatives. We also showed that maltodextrins and maltodextrins-1-phosphate were synthesized and excreted out of the cell, and could originate from exogenous sugars or endogenous glycogen. According to the substrate and the physiological state of the bacterium (resting or growing cells), metabolic deviations toward the synthesis of these oligosaccharides were observed. We have also used liquid- and solid-state NMR to monitor wheat straw degradation by F. succinogenes . The originality of this NMR approach was to investigate the activity of an entire fibrolytic system on an intact complex substrate (wheat straw). No preferential degradation of amorphous versus crystalline cellulose was observed, nor of cellulose versus hemicelluloses. We showed for the first time a sequential activity of some enzymes of F. succinogenes S85 on wheat straw. In conclusion, our studies illustrate the utility of various NMR approaches to study and better understand sugar and polysaccharide metabolism in rumen bacteria. Key words: ruminants, NMR, Fibrobacter succinogenes, polysaccharide metabolism, cellulose

Mutations in the scaffoldin gene, cipA, of Clostridium thermocellum with impaired cellulosome formation and cellulose hydrolysis: insertions of a new transposable element, IS1447, and implications for cellulase synergism on crystalline cellulose.

Mutants of Clostridium thermocellum that had lost the ability to adhere to microcrystalline cellulose were isolated. Six of them that showed diminished ability to depolymerize crystalline cellulose were selected. Size exclusion chromatography of the proteins from the culture supernatant revealed the loss of the supramolecular enzyme complex, the cellulosome. However, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis resulted in extracellular protein patterns comparable to those of isolated cellulosomes, except for a missing CipA band. Sequencing of the six mutant cipA genes revealed a new insertion (IS) element, IS1447, belonging to the IS3 family. It was inserted into the cipA reading frame in four different locations: cohesin module 1, two different positions in the carbohydrate binding module, and cohesin module 3. The IS sequences were identical and consisted of a transposase gene and the inverted repeats IRR and IRS. The insertion resulted in an obviously nonspecific duplication of 3 base pairs within the target sequence. This lack of specificity allows transposition without the need of a defined target DNA sequence. Eighteen copies of IS1447 were identified in the genomic sequence of C. thermocellum ATCC 27405. At least one of them can be activated for transposition. Compared to the wild type, the mutant culture supernatant, with a completely defective CipA protein, showed equal specific hydrolytic activity against soluble beta-glucan but a 15-fold reduction in specific activity with crystalline cellulose. These results identify a genetic basis for the synergistic effect of complex formation on crystalline-cellulose degradation.

A Tomato Endo-β-1,4-glucanase, SlCel9C1, Represents a Distinct Subclass with a New Family of Carbohydrate Binding Modules (CBM49)

A critical structural feature of many microbial endo-beta-1,4-glucanases (EGases, or cellulases) is a carbohydrate binding module (CBM), which is required for effective crystalline cellulose degradation. However, CBMs are absent from plant EGases that have been biochemically characterized to date, and accordingly, plant EGases are not generally thought to have the capacity to degrade crystalline cellulose. We report the biochemical characterization of a tomato EGase, Solanum lycopersicum Cel8 (SlCel9C1), with a distinct C-terminal noncatalytic module that represents a previously uncharacterized family of CBMs. In vitro binding studies demonstrated that this module indeed binds to crystalline cellulose and can similarly bind as part of a recombinant chimeric fusion protein containing an EGase catalytic domain from the bacterium Thermobifida fusca. Site-directed mutagenesis studies show that tryptophans 559 and 573 play a role in crystalline cellulose binding. The SlCel9C1 CBM, which represents a new CBM family (CBM49), is a defining feature of a new structural subclass (Class C) of plant EGases, with members present throughout the plant kingdom. In addition, the SlCel9C1 catalytic domain was shown to hydrolyze artificial cellulosic polymers, cellulose oligosaccharides, and a variety of plant cell wall polysaccharides.

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.

Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose

An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.

Relationship between production of hydroxyl radicals and degradation of wood, crystalline cellulose, and a lignin-related compound or accumulation of oxalic acid in cultures of brown-rot fungi

The degradation of wood, filter paper cellulose, and a lignin-substructure model, was measured in cultures of seven fungi usually regarded as brown-rot fungi. Hydroxyl radical production and the accumulation of oxalic acid in the cultures were also measured. Four of the fungi, Gloeophyllum trabeum, Tyromyces palustris, Laetiporus sulphureus, and Postia placenta, were typical brown-rot fungi, in that they preferentially degraded and eliminated the polysaccharides in wood and produced large amounts of hydroxyl radical. The rates of hydroxyl radical generation in cultures of the four fungi were directly proportional to the degradation rates of wood, cellulose, and the lignin-related compound, and inversely proportional to the amount of oxalic acid in the cultures. Two of the fungi, Daedalea dickinsii and Lentinus lepideus, did not degrade any of the substrates significantly and produced very little hydroxyl radical. Coniophora puteana had the highest rate of cellulose degradation, but did not degrade wood or the lignin model significantly and produced only negligible amounts of hydroxyl radical. These results indicate that brown-rot fungi produce large amounts of hydroxyl radical for the degradation of wood and crystalline cellulose.

Alternative Splicing Produces Two Endoglucanases with One or Two Carbohydrate-Binding Modules in Mucor circinelloides

We previously cloned three endoglucanase genes, rce1, rce2, and rce3, that were isolated from Rhizopus oryzae as the first cellulase genes from a member of the subdivision Zygomycota. In this study, two cDNAs homologous to the rce1 gene, designated the mce1 and mce2 cDNAs, were cloned from Mucor circinelloides, a member of the subdivision Zygomycota. The mce1 cDNA encoded an endoglucanase (family 45 glycoside hydrolase) having one carbohydrate-binding module (CBM), designated MCE1, and the mce2 cDNA encoded the same endoglucanase having two tandem repeated CBMs, designated MCE2. The two cDNAs contained the same sequences but with a 147-bp insertion. The corresponding genomic mce gene consisted of four exons. The mce1 cDNA was created from exons 1, 3, and 4, and the mce2 cDNA was created from exons 1, 2, 3, and 4. These results indicate that the mce1 and mce2 cDNAs were created from one genomic mce gene by alternative splicing. MCE1 and MCE2, purified to apparent homogeneity from the culture supernatant of M. circinelloides, had molecular masses of 43 and 47 kDa, respectively. The carboxymethyl cellulase specific activity of MCE2 was almost the same as that of MCE1, whereas the Avicelase specific activity of MCE2 was two times higher than that of MCE1. Furthermore, MCE2, whose two tandem CBMs might be more effective for degradation of crystalline cellulose than one CBM, was secreted only at an early culture stage when crystalline cellulose was abundant.

Influence of particle size and pH on anaerobic degradation of cellulose by ruminal microbes

Abstract Batch experiments were performed to investigate the influence of cellulose particle size and pH on the anaerobic degradation of crystalline cellulose by ruminal microbes. At a particle size of 50 μm there was a higher hydrolysis and acidogenesis rate, and a reduced degradation time, than for 100-μm particles. Reduction in cellulose particle size resulted in decreased methane production, but an increase of soluble products. Cellulose degradation increased with pH from pH 6.0 to 7.5, whereas at pH⩽5.5 there was no degradation. The inhibitory effect of low pH (⩽5.5) on ruminal microbes was not completely remedied even when the pH of the medium was adjusted to a neutral range. In an anaerobic cellulosic waste degrading system inoculated with ruminal microbes the fermentation system should therefore be maintained above pH 6.0. In all cases, volatile fatty acids were the major water-soluble products of cellulose degradation; acetate and propionate accounted for more than 90% of the volatile fatty acid total.

Expression, purification and structural characterization of the scaffoldin hydrophilic X-module from the cellulosome of Clostridium thermocellum

The cellulosome is a membrane-bound, extracellular multi-subunit complex responsible for the degradation of crystalline cellulose by a number of organisms including anaerobic bacteria and fungi. The hydrophilic X-module (CipA-X) from the modular scaffoldin subunit of Clostridium thermocellum cellulosome has been proposed to play various roles in cellulosomal function, including thermal and structural stability. Towards elucidating the function of CipA-X using structural and biophysical studies, the region comprising residues 1692–1785 from the C. thermocellum CipA cDNA encoding CipA-X was cloned into a pET21b expression vector. When expressed in Escherichia coli, the C-terminal His-tagged protein accumulated in the insoluble fraction. Cell fractionation experiments showed that the recombinant protein was localized to inclusion bodies. Refolding and purification involved denaturation of the whole cell lysate by addition of urea, followed by a nickel-Sepharose chromatography step and dialysis into native conditions (25 mM Tris–HCl, pH 7.4, 50 mM NaCl, and 10 mM EDTA). A final gel filtration step purified the protein to homogeneity, yielding 40 mg/L. The two-dimensional 1H– 15N correlation spectrum of uniformly 15N-labelled CipA-X showed the characteristics of a well-folded protein comprising significant β-structure, which is in agreement with the circular dichroism data.

Anaerobic degradation of cellulose by rumen microorganisms at various pH values

Batch experiments were performed to investigate the anaerobic degradation of crystalline cellulose of 10 g l −1 by rumen microorganisms at pH from 4.8 to 7.3. The degradation efficiency increased with pH and the highest value of about 78% was achieved at pH 6.8 and 7.3. Acetate and propionate were the major aqueous products at all pH values. With increasing pH, the molar percentage of acetate decreased, whereas that of propionate increased. A modified Gompertz equation was able to adequately model the fermentation of cellulose by rumen microorganisms. The results from this study might provide useful information for the application of rumen cultures for conversion of cellulosic wastes into value-added products.

Cellulolysis is severely affected in Clostridium cellulolyticum strain cipCMut1.

Progress towards understanding the molecular basis of cellulolysis by Clostridium cellulolyticm was obtained through the study of the first cellulolysis defective mutant strain, namely cipCMut1. In this mutant, a 2 659 bp insertion element, disrupts the cipC gene at the sequence encoding the seventh cohesin of the scaffoldin CipC. cipC is the first gene in a large 'cel' gene cluster, encoding several enzymatic subunits of the cellulosomes, including the processive cellulase Cel48F, which is the major component. Physiological and biochemical studies showed that the mutant strain was affected in cellulosome synthesis and severely impaired in its ability to degrade crystalline cellulose. It produced small amounts of a truncated CipC protein (P120), which had functional cohesin domains and assembled complexes which did not contain any of the enzymes encoded by genes of the 'cel' cluster. The mutant cellulolytic system was mainly composed of three proteins designated P98, P105 and P125. Their N-termini did not match any of the known cellulase sequences from C. cellulolyticum. A large amount of entire CipC produced in the cipCMut1 strain by trans-complementation with plasmid pSOScipC did not restore the cellulolytic phenotype, in spite of the assembly of a larger amount of complexes. The complexes produced in the mutant and complemented strains contained at least 12 different dockerin-containing proteins encoded by genes located outside of the 'cel' cluster. The disturbances observed in the mutant and trans-complemented strains were the result of a strong polar effect resulting from the cipC gene disruption. In conclusion, this study provided genetic evidence that the cellulases encoded by the genes located in the 'cel' cluster are essential for the building of cellulosomes efficient in crystalline cellulose degradation.

Integration of computer modeling and initial studies of site-directed mutagenesis to improve cellulase activity on Cel9A from Thermobifida fusca.

Cellulases are a complex group of enzymes that are fundamental for the degradation of amorphous and crystalline cellulose in lignocellulosic material. Unfortunately, cellulases have a low catalytic efficiency on their substrates when compared to similar enzymes such as amylases, which has led to a strong interest in improving their activities. Thermobifida fusca secretes six cellulose degrading enzymes: two exo- and three endocellulases and an endo/exocellulase Cel9A (formerly called E4). Cel9A shows unique properties because of its endo- and exocellulase characteristics, strong activity on crystalline cellulose, and good synergistic properties. Therefore, it is an excellent target for mutagenesis techniques to improve crystalline cellulose degradation. In this article, we describe research conducted to improve Cel9A catalytic efficiency using a rational design and computer modeling. A computer model of Cel9A was created using the program CHARMM plus its PDB structure and a cellohexose molecule attached to the catalytic site as a starting model. Initially molecular graphics and energy minimization were used to extend the cellulose chain to 18 glucose residues spanning the catalytic domain and cellulose-binding domain (CBD). The interaction between this cellulose chain and conserved CBD residues was determined in the model, and mutations likely to improve the binding properties of the CBD were selected. Site-directed mutations were carried out using the pET vector pET26b, Escherichia coli DH5-alpha, and the QuickChange mutagenesis method. E. coli BL21-DE3 was used for protein production and expression. The purified proteins were assayed for enzymatic activity on filter paper, swollen cellulose, bacterial microcrystalline cellulose, and carboxymethylcellulose (CMC). Mutation of the conserved residue F476 to Y476 gave a 40% improved activity in assays with soluble and amorphous cellulose such as CMC and swollen cellulose.

Engineering the Exo-loop of Trichoderma reesei Cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Δ(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k cat reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k cat and K M and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc 2/(Glc 3+Glc 1) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.

Cellulose Hydrolysis - The Role of Monocomponent Cellulases in Crystalline Cellulose Degradation

Changes in the molecular structure of cellulose during hydrolysis with four recombinant β-1,4-glycanases from the cellulolytic bacterium Cellulomonas fimi were assessed and compared in an attempt to elucidate the mechanism of crystalline cellulose degradation. It was apparent that the two endoglucanases, Cel6A and Cel5A, degraded sigmacell cellulose differently; Cel5A liberated more soluble sugars (cellobiose and cellotriose) and significantly altered the molecular weight distribution, while Cel6A had a limited effect on the polymer size and liberated primarily cellobiose and glucose. Additionally, both endoglucanases slightly increased the crystallinity of cellulose. In contrast, the cellobiohydrolases, Cel6B and Cel48A, had no effect on cellulose molecular weight and liberated only cellobiose and cellotriose. However, Cel48A was shown to be effective at reducing the crystallinity of the cellulosic substrate, while Cel6B increased the crystallinity index. Synergistic hydrolysis using combinations of the different enzymes showed that, although the cellulose was extensively hydrolysed, the molecular structure of the substrate was similar to the original material. This phenomenon suggests that the actions of individual monocomponent enzymes are offset by the concurrent modification by the complementing enzymes during synergistic hydrolysis.