Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimi β-1,4-Glucanases

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.     

Determination of the molecular states of the processive endocellulase Thermobifida fusca Cel9A during crystalline cellulose depolymerization

Detailed understanding of cell wall degrading enzymes is important for their modeling and industrial applications, including in the production of biofuels. Here we used Cel9A, a processive endocellulase from Thermobifida fusca, to demonstrate that cellulases that contain a catalytic domain (CD) attached to a cellulose binding module (CBM) by a flexible linker exist in three distinct molecular states. By measuring the ability of a soluble competitor to reduce Cel9A activity on an insoluble substrate, we show that the most common state of Cel9A is bound via its CBM, but with its CD unoccupied by the insoluble substrate. These findings are relevant for kinetic modeling and microscopy studies of modular glycoside hydrolases.     

Processive endoglucanase active in crystalline cellulose hydrolysis by the brown rot basidiomycete Gloeophyllum trabeum

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity--4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a beta-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.     

Crystal structure of the cellulase Cel9M enlightens structure/function relationships of the variable catalytic modules in glycoside hydrolases

Cellulases cleave the beta-1.4 glycosidic bond of cellulose. They have been characterized as endo or exo and processive or nonprocessive cellulases according to their action mode on the substrate. Different types of these cellulases may coexist in the same glycoside hydrolase family, which have been classified according to their sequence homology and catalytic mechanism. The bacterium C. celluloyticum produces a set of different cellulases who belong mostly to glycoside hydrolase families 5 and 9. As an adaptation of the organism to different macroscopic substrates organizations and to maximize its cooperative digestion, it is expected that cellulases of these families are active on the various macroscopic organizations of cellulose chains. The nonprocessive cellulase Cel9M is the shortest variant of family 9 cellulases (subgroup 9(C)) which contains only the catalytic module to interact with the substrate. The crystal structures of free native Cel9M and its complex with cellobiose have been solved to 1.8 and 2.0 A resolution, respectively. Other structurally known family 9 cellulases are the nonprocessive endo-cellulase Cel9D from C. thermocellum and the processive endo-cellulase Cel9A from T. fusca, from subgroups 9(B1) and 9(A), respectively, whose catalytic modules are fused to a second domain. These enzymes differ in their activity on substrates with specific macroscopic appearances. The comparison of the catalytic module of Cel9M with the two other known GH family 9 structures may give clues to explain its substrate profile and action mode.     

The processive endoglucanase EngZ is active in crystalline cellulose degradation as a cellulosomal subunit of Clostridium cellulovorans

Clostridium cellulovorans produces an efficient enzyme complex for the degradation of lignocellulosic biomass. In our previous study, we detected and identified protein spots that interacted with a fluorescently labeled cohesin biomarker via two-dimensional gel electrophoresis. One novel, putative cellulosomal protein (referred to as endoglucanase Z) contains a catalytic module from the glycosyl hydrolase family (GH9) and demonstrated higher levels of expression than other cellulosomal cellulases in Avicel-containing cultures. Purified EngZ had optimal activity at pH 7.0, 40°C, and the major hydrolysis product from the cellooligosaccharides was cellobiose. EngZ's specific activity toward crystalline cellulose (Avicel and acid-swollen cellulose) was 10-20-fold higher than other cellulosomal cellulase activities. A large percentage of the reducing ends that were produced by this enzyme from acid-swollen cellulose were released as soluble sugar. EngZ has the capability of reducing the viscosity of Avicel at an intermediate-level between exo- and endo-typing cellulases, suggesting that it is a processive endoglucanase. In conclusion, EngZ was highly expressed in cellulolytic systems and demonstrated processive endoglucanase activity, suggesting that it plays a major role in the hydrolysis of crystalline cellulose and acts as a cellulosomal enzyme in C. cellulovorans.     

Processive Endoglucanase Active in Crystalline Cellulose Hydrolysis by the Brown Rot Basidiomycete Gloeophyllum trabeum

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity—4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a β-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.     

Strategies to reduce end‐product inhibition in family 48 glycoside hydrolases

Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the product inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free‐energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. The theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity. Proteins 2016; 84:295–304. © 2016 Wiley Periodicals, Inc.     

Heterologous co-production of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> Cel9A with other cellulases in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The processive endoglucanase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in Saccharomyces cerevisiae. Recombinant Cel9A displayed activity on both soluble (carboxymethylcellulose) and insoluble (Avicel) cellulose substrates confirming its processive endoglucanase activity. High-performance anionic exchange chromatography analyses of soluble sugars released from Avicel revealed a cellobiose/glucose ratio of 2.5 ± 0.1. Growth by the recombinant strain on amorphous cellulose was possible due to the sufficient amount of glucose cleaved from the cellulose chain. This is the first confirmed report of S. cerevisiae growing on a cellulosic substrate as sole carbohydrate source while only expressing one recombinant gene. To improve the cellulolytic capability of S. cerevisiae and to investigate the level of synergy among cellulases produced by a recombinant host, the cel9A gene was co-expressed with four cellulase-coding genes of Trichoderma reesei: two endoglucanases cel5A (egII) and cel7B (egI), and two cellobiohydrolases cel6A (cbhII) and cel7A (cbhI). Synergy, especially between the Cel9A and the two cellobiohydrolases, resulted in a higher cellulolytic capability of the recombinant host.     

Product binding varies dramatically between processive and nonprocessive cellulase enzymes

Cellulases hydrolyze β-1,4 glycosidic linkages in cellulose, which are among the most prevalent and stable bonds in Nature. Cellulases comprise many glycoside hydrolase families and exist as processive or nonprocessive enzymes. Product inhibition negatively impacts cellulase action, but experimental measurements of product-binding constants vary significantly, and there is little consensus on the importance of this phenomenon. To provide molecular level insights into cellulase product inhibition, we examine the impact of product binding on processive and nonprocessive cellulases by calculating the binding free energy of cellobiose to the product sites of catalytic domains of processive and nonprocessive enzymes from glycoside hydrolase families 6 and 7. The results suggest that cellobiose binds to processive cellulases much more strongly than nonprocessive cellulases. We also predict that the presence of a cellodextrin bound in the reactant site of the catalytic domain, which is present during enzymatic catalysis, has no effect on product binding in nonprocessive cellulases, whereas it significantly increases product binding to processive cellulases. This difference in product binding correlates with hydrogen bonding between the substrate-side ligand and the cellobiose product in processive cellulase tunnels and the additional stabilization from the longer tunnel-forming loops. The hydrogen bonds between the substrate- and product-side ligands are disrupted by water in nonprocessive cellulase clefts, and the lack of long tunnel-forming loops results in lower affinity of the product ligand. These findings provide new insights into the large discrepancies reported for binding constants for cellulases and suggest that product inhibition will vary significantly based on the amount of productive binding for processive cellulases on cellulose.     

Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant Cellulomonas fimi β-1,4-Glucanases

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.Cellulose hydrolysis by aerobic fungi, such as Trichoderma reesei, is usually explained in terms of the synergistic activities of endo-β-1,4-glucanases and exocellobiohydrolases. Models that describe the attack of cellulose at susceptible regions by endoglucanases, followed by cellobiohydrolase attack at the newly formed chain ends, continue to form the basis of most discussions of enzymatic cellulose hydrolysis (2, 24).Although the occurrence of endoglucanases and cellobiohydrolases in fungi is firmly established, the extent to which the cellulase systems of aerobic bacteria resemble those from fungi was unclear until recently, because there was little evidence for the presence of cellobiohydrolases in bacteria. However, it now appears that at least some cellulolytic bacteria produce enzymes similar to the fungal cellobiohydrolases. For example, Cellulomonas fimi produces at least six β-1,4-glucanases, of which four (CenA, CenB, CenC, and CenD) are endoglucanases and two (CbhA and CbhB) appear to be cellobiohydrolases that are the functional equivalents of T. reesei CBHI and CBHII (6, 15, 21, 22). Similar cellobiohydrolases have been described for the actinomycete Thermomonospora fusca (9).

C. fimi cellobiohydrolases.

The preferential attack of cellulose at the ends of glucan chains by C. fimi cellobiohydrolases CbhA and CbhB is strongly suggested by hydrolysis experiments using cellooligosaccharides or carboxymethylcellulose (CMC) (14, 15, 21, 22). However, we lack direct evidence for exohydrolytic activity on cellulose itself. Accordingly, we have examined the activities of CbhA and CbhB on cellulose by measurement of molecular size distributions during hydrolysis. Analysis of CenA was also included to allow comparison of cellobiohydrolase and endoglucanase activities.

C. fimi CenC.

Previous studies have indicated that CenA attacks susceptible linkages in soluble CMC in a relatively nonprocessive manner (7, 14); i.e., the enzyme dissociates from the substrate after each hydrolytic event. While CenB and CenD attack CMC in a similar way (14, 23), C. fimi CenC seems to act in a more processive fashion (16, 23). Therefore, CenC activity was analyzed in order to determine if the enzyme behaves in a similarly processive manner on cellulose.

Cellulose substrates.

Previous determinations of molecular size distribution during hydrolysis have shown that the choice of substrate is an important consideration (10). In this study we used two forms of cellulose: bacterial microcrystalline cellulose (BMCC) and phosphoric acid-swollen cellulose (PASC). These celluloses were chosen in order to simplify the interpretation of data by avoiding complications due to low surface/volume ratios and substrate heterogeneity, which are associated with the use of substrates like cotton or pulp fibers (24). Both BMCC and PASC have a high surface/volume ratio (17). BMCC is a highly crystalline form of cellulose I prepared from cellulose produced by Acetobacter xylinum. PASC is produced by swelling microcrystalline cellulose in concentrated phosphoric acid; although often described as amorphous, it is probably a low-crystallinity form of cellulose II (1). Recent data suggest that cellulose I and cellulose II contain glucan chains arranged in parallel orientation (12).     

Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans

The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHΔCBM and EngYΔCBM devoid of the CBM lost activity toward all substrates including CMC. EngKΔCBM and EngMΔCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.     

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes.     

A minimal set of bacterial cellulases for consolidated bioprocessing of lignocellulose

Cost-effective release of fermentable sugars from non-food biomass through biomass pretreatment/enzymatic hydrolysis is still the largest obstacle to second-generation biorefineries. Therefore, the hydrolysis performance of 21 bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase (BsCel5), family 9 Clostridium phytofermentans processive endoglucanase (CpCel9), and family 48 C. phytofermentans cellobiohydrolase (CpCel48) was studied on partially ordered low-accessibility microcrystalline cellulose (Avicel) and disordered high-accessibility regenerated amorphous cellulose (RAC). Faster hydrolysis rates and higher digestibilities were obtained on RAC than on Avicel. The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was the most important for high cellulose digestibility regardless of substrate type. This study provides important information for the construction of a minimal set of bacterial cellulases for the consolidated bioprocessing bacteria, such as Bacillus subtilis, for converting lignocellulose to biocommodities in a single step.     

Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes

In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V _max and catalytic efficiency (k _cat/K _M) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.     

Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase

In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.     

CelI,a noncellulosomal family 9 enzyme from Clostridium thermocellum,is a processive endoglucanase that degrades crystalline cellulose

The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G. P. Hazlewood, K. Davidson, J. I. Laurie, N. S. Huskisson, and H. J. Gilbert, J. Gen. Microbiol. 139:307-316, 1993). We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein. The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b. The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70 degrees C, respectively, using carboxymethylcellulose as a substrate. Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase. A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates. A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined. This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates.     

Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting cellulolytic complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes.     

The noncellulosomal family 48 cellobiohydrolase from Clostridium phytofermentans ISDg: heterologous expression, characterization, and processivity

Family 48 glycoside hydrolases (cellobiohydrolases) are among the most important cellulase components for crystalline cellulose hydrolysis mediated by cellulolytic bacteria. Open reading frame (Cphy_3368) of Clostridium phytofermentans ISDg encodes a putative family 48 glycoside hydrolase (CpCel48) with a family 3 cellulose-binding module. CpCel48 was successfully expressed as two soluble intracellular forms with or without a C-terminal His-tag in Escherichia coli and as a secretory active form in Bacillus subtilis. It was found that calcium ion enhanced activity and thermostability of the enzyme. CpCel48 had high activities of 15.1 U μmol⁻¹ on Avicel and 35.9 U μmol⁻¹ on regenerated amorphous cellulose (RAC) with cellobiose as a main product and cellotriose and cellotetraose as by-products. By contrast, it had very weak activities on soluble cellulose derivatives (e.g., carboxymethyl cellulose (CMC)) and did not significantly decrease the viscosity of the CMC solution. Cellotetraose was the smallest oligosaccharide substrate for CpCel48. Since processivity is a key characteristic for cellobiohydrolases, the new initial false/right attack model was developed for estimation of processivity by considering the enzyme's substrate specificity, the crystalline structure of homologous Cel48 enzymes, and the configuration of cellulose chains. The processivities of CpCel48 on Avicel and RAC were estimated to be ∼3.5 and 6.0, respectively. Heterologous expression of secretory active cellobiohydrolase in B. subtilis is an important step for developing recombinant cellulolytic B. subtilis strains for low-cost production of advanced biofuels from cellulosic materials in a single step.     

Modeling the activity burst in the initial phase of cellulose hydrolysis by the processive cellobiohydrolase Cel7A

The hydrolysis of cellulose by processive cellulases, such as exocellulase TrCel7A from Trichoderma reesei, is typically characterized by an initial burst of high activity followed by a slowdown, often leading to incomplete hydrolysis of the substrate. The origins of these limitations to cellulose hydrolysis are not yet fully understood. Here, we propose a new model for the initial phase of cellulose hydrolysis by processive cellulases, incorporating a bound but inactive enzyme state. The model, based on ordinary differential equations, accurately reproduces the activity burst and the subsequent slowdown of the cellulose hydrolysis and describes the experimental data equally well or better than the previously suggested model. We also derive steady-state expressions that can be used to describe the pseudo-steady state reached after the initial activity burst. Importantly, we show that the new model predicts the existence of an optimal enzyme-substrate affinity at which the pseudo-steady state hydrolysis rate is maximized. The model further allows the calculation of glucose production rate from the first cut in the processive run and reproduces the second activity burst commonly observed upon new enzyme addition. These results are expected to be applicable also to other processive enzymes.     

Purification and characterization of five cellulases and one xylanase from Penicillium brasilianum IBT 20888

The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l⁻¹ cellulose and 10 g l⁻¹ xylan for 111 h and the resulting culture filtrate was used for protein purification. From the cultivation broth, five cellulases and one xylanase were purified. Hydrolysis studies revealed that two of the cellulases were acting as cellobiohydrolases by being active on only microcrystalline cellulose (Avicel). Three of the cellulases were active on both Avicel and carboxymethyl cellulose indicating endoglucanase activity. Two of these showed furthermore mannanase activity by being able to hydrolyze galactomannan (locust bean gum). Adsorption studies revealed that the smaller of the two enzymes was not able to bind to cellulose. Similarity in molecular mass, pI and hydrolytic properties suggested that these two enzymes were identical, but the smaller one was lacking the cellulose-binding domain or an essential part of it. The basic xylanase (pI>9) was only active towards xylan. Two of the purified cellulases with endoglucanase activity were partly sequenced and based on sequence homology with known enzymes they were classified as belonging to families 5 and 12 of the glycosyl hydrolases.