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.     

Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically

The genome of Clostridium thermocellum contains a number of genes for polysaccharide degradation-associated proteins that are not cellulosome bound. The list includes beta-glucanases, glycosidases, chitinases, amylases and a xylanase. One of these 'soluble'-enzyme genes codes for a second glycosyl hydrolase (GH)48 cellulase, Cel48Y, which was expressed in Escherichia coli and biochemically characterized. It is a cellobiohydrolyse with activity on native cellulose such as microcrystalline and bacterial cellulose, and low activity on carboxymethylcellulose. It is about 100 times as active on amorphic cellulose and mixed-linkage barley beta-glucan compared with cellulase Cel9I. The enzyme Cel48Y shows a distinct synergism of 2.1 times with the noncellulosomal processive endoglucanase Cel9I on highly crystalline bacterial cellulose at a 17-fold excess of Cel48Y over Cel9I. These data show that C. thermocellum has, besides the cellulosome, the genes for a second cellulase system for the hydrolysis of crystalline cellulose that is not particle bound.     

A comparative analysis of two cDNA clones of the cellulase gene family from anaerobic fungus Piromyces rhizinflata

Cellulase family and some other glycosyl hydrolases of anaerobic fungi inhabiting the digestive tract of ruminants are believed to form an enzyme complex called cellulosome. Study of the individual component of cellulosome may shed light on understanding the organization of this complex and its functional mechanism. We have analysed the primary sequences of two cellulase clones, cel5B and cel6A, isolated from the cDNA library of ruminal fungus, Piromyces rhizinflata strain 2301. The deduced amino acid sequences of the catalytic domain of Cel5B, encoded by cel5B, showed homology with the subfamily 4 of the family 5 (subfamily 5(4)) of glycosyl hydrolases, while cel6A encoded Cel6A belonged to family 6 of glycosyl hydrolases. Phylogenetic tree analysis suggested that the genes of subfamily 5(4) glycosyl hydrolases of P. rhizinflata might have been acquired from rumen bacteria. Cel5B and Cel6A were modular enzymes consisting of a catalytic domain and dockerin domain(s), but not a cellulose binding domain. The occurrence of dockerin domains indicated that both enzymes were cellulosome components. The catalytic domain of the Cel5B (Cel5B') and Cel6A (Cel6A') recombinant proteins were purified. The optimal activity conditions with carboxymethyl cellulose (CMC) as the substrate were pH 6.0 and 50 degrees C for Cel5B', and pH 6.0 and 37-45 degrees C for Cel6A'. Both Cel5B' and Cel6A' exhibited activity against CMC, barley beta-glucan, Lichenan, and oat spelt xylan. Cel5B' could also hydrolyse p-nitrophenyl-beta-d-cellobioside, Avicel and filter paper while Cel6A' did not show any activity on these substrates. It is apparent that Cel6A' acted as an endoglucanase and Cel5B' possessed both endoglucanase and exoglucanase activities. No synergic effect was observed for these recombinant enzymes in vitro on Avicel and CMC.     

Cellobiose: A true inducer of cellulosome in different strains of Clostridium thermocellum

Abstract Growth and production of cellulosome by three strains (YS, LQRI and NCIB 10682) of Clostridium thermocellum were compared using Avicel (microcrystalline cellulose) and cellobiose as carbon sources. All three strains grew faster on cellobiose than on Avicel and produced 0.71–0.74 IU of endoglucanase/ml compared to 0.88–1.18 IU/ml on Avicel. Also, the cellulase produced by these strains in the presence of 0.2–1% cellobiose and Avicel, when compared on the basis of equal units of endoglucanase (0.5 IU), degraded cotton almost completely. SDS-PAGE further confirmed the production of cellulosome by all three strains when grown on cellobiose and Avicel. Thus, the cellobiose, like Avicel, acts as a true inducer of cellulosome in C. thermocellum .     

Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum

The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C. cellulolyticum.     

Degradation of microcrystalline cellulose: synergism between different endoglucanases of Cellulomonas sp. ATCC 21399

Three immunologically and enzymatically distinct endoglucanases of Cellulomonas sp. ATCC 21399 were purified previously. Endoglucanase A and endoglucanase B acted synergistically on microcrystalline cellulose (Avicel), whereas no synergistic action was observed between endoglucanase B or endoglucanase C. Only endoglucanase A was capable of hydrolyzing Avicel when acting alone and this enzyme resulted in "short fiber formation" when acting on Avicel. The end product of hydrolysis of acid swollen Avicel produced by the three endoglucanases was in all cases dominated by cellobiose and showed lower content of glucose and cellotriose. Higher cellodextrins appeared as transient end products. The results indicate that the function of endoglucanase A in the cellulase system of Cellulomonas might be very similar to the function of the cellobiohydrolases of Trichoderma reesei.     

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.     

Exo-mode of action of cellobiohydrolase Cel48C from Paenibacillus sp. BP-23. A unique type of cellulase among Bacillales.

Sequence analysis of a Paenibacillus sp. BP-23 recombinant clone coding for a previously described endoglucanase revealed the presence of an additional truncated ORF with homology to family 48 glycosyl hydrolases. The corresponding 3509-bp DNA fragment was isolated after gene walking and cloned in Escherichia coli Xl1-Blue for expression and purification. The encoded enzyme, a cellulase of 1091 amino acids with a deduced molecular mass of 118 kDa and a pI of 4.85, displayed a multidomain organization bearing a canonical family 48 catalytic domain, a bacterial type 3a cellulose-binding module, and a putative fibronectin-III domain. The cloned cellulase, unique among Bacillales and designated Cel48C, was purified through affinity chromatography using its ability to bind Avicel. Maximum activity was achieved at 45 degrees C and pH 6.0 on acid-swollen cellulose, bacterial microcrystalline cellulose, Avicel and cellodextrins, whereas no activity was found on carboxy methyl cellulose, cellobiose, cellotriose, pNP-glycosides or 4-methylumbeliferyl alpha-d-glucoside. Cellobiose was the major product of cellulose hydrolysis, identifying Cel48C as a processive cellobiohydrolase. Although no chromogenic activity was detected from pNP-glycosides, TLC analysis revealed the release of p-nitrophenyl-glycosides and cellodextrins from these substrates, suggesting that Cel48C acts from the reducing ends of the sugar chain. Presence of such a cellobiohydrolase in Paenibacillus sp. BP-23 would contribute to widen up its range of action on natural cellulosic substrates.     

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.     

Production and Characteristics of Avicel-Disintegrating Endoglucanase from a Protease-Negative Humicola grisea var. thermoidea Mutant

Mutational experiments were performed to decrease the protease productivity of Humicola grisea var. thermoidea YH-78 using UV light and N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, no. 140, exhibited higher endoglucanase activity than the parent strain in mold bran culture at 50°C for 4 days. The culture extract rapidly disintegrated filter paper but produced a small amount of reducing sugar. About 30% of total endoglucanase activity in the extract was adsorbed onto Avicel. The electrophoretically homogeneous preparation of Avicel-adsorbable endoglucanase (molecular weight, 128,000) showed intensive filter-paper-disintegrating activity but did not release reducing sugar. The preparation also exhibited a highly synergistic effect with the cellulase preparation from Trichoderma reesei in the hydrolysis of microcrystalline cellulose. This endoglucanase was observed via scanning electron microscopy to disintegrate Avicel fibrils layer by layer from the surface, yielding thin sections with exposed chain ends. A mutant, no. 191, producing higher protease activity and an Avicel-unadsorbable, Avicel-nondisintegrating endoglucanase was isolated. The purified enzyme (molecular weight, 63,000) showed no disintegrating activity on filter paper and Avicel and a less synergistic effect with the T. reesei cellulase in hydrolyzing microcrystalline cellulose than did the former enzyme. Endoglucanase was therefore divided into two types, Avicel disintegrating and Avicel nondisintegrating.     

The brown-rot basidiomycete Fomitopsis palustris has the endo-glucanases capable of degrading microcrystalline cellulose

Two endoglucanases with processive cellulase activities, produced from Fomitopsis palustris grown on 2% microcrystalline cellulose (Avicel), were purified to homogeneity by anion-exchange and gel filtration column chromatography systems. SDS-PAGE analysis indicated that the molecular masses of the purified enzymes were 47 kDa and 35 kDa, respectively. The amino acid sequence analysis of the 47-kDa protein (EG47) showed a sequence similarity with fungal glycoside hydrolase family 5 endoglucanase from the white-rot fungus Phanerochaete chrysosporium. N-terminal and internal amino acid sequences of the 35-kDa protein (EG35), however, had no homology with any other glycosylhydrolases, although the enzyme had high specific activity against carboxymethyl cellulose, which is a typical substrate for endoglucanases. The initial rate of Avicel hydrolysis by EG35 was relatively fast for 48 h, and the amount of soluble reducing sugar released after 96 h was 100 microg/ml. Although EG47 also hydrolyzed Avicel, the hydrolysis rate was lower than that of EG35. Thin layer chromatography analysis of the hydrolysis products released from Avicel indicated that the main product was cellobiose, suggesting that the brown-rot fungus possesses processive EGs capable of degrading crystalline cellulose.     

Functional analysis of a gene encoding endoglucanase that belongs to glycosyl hydrolase family 12 from the brown-rot basidiomycete Fomitopsis palustris

The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and beta- glucosidases. A gene encoding endoglucanase, designated as cel12, was cloned from total RNA prepared from F. palustris grown at the expense of Avicel. The gene encoding Cel12 has an open reading frame of 732 bp, encoding a putative protein of 244 amino acid residues with a putative signal peptide residing at the first 18 amino acid residues of the N-terminus of the protein. Sequence analysis of Cel12 identified three consensus regions, which are highly conserved among fungal cellulases belonging to GH family 12. However, a cellulose-binding domain was not found in Cel12, like other GH family 12 fungal cellulases. Northern blot analysis showed a dramatic increase of cel12 mRNA levels in F. palustris cells cultivated on Avicel from the early to late stages of growth and the maintenance of a high level of expression in the late stage, suggesting that Cel12 takes a significant part in endoglucanase activity throughout the growth of F. palustris. Adventitious expression of cel12 in the yeast Pichia pastoris successfully produced the recombinant protein that exhibited endoglucanase activity with carboxymethyl cellulose, but not with crystalline cellulose, suggesting that the enzyme is not a processive endoglucanase unlike two other endoglucanases previously identified in F. palustris.     

Hydrolysis of cellulose by a mixture of Trichoderma reesei cellobiohydrolase and Aspergillus niger endoglucanase

Two endoglucanase-containing fractions were separated from Aspergillus niger cellulase by gel filtration and fast protein liquid chromatofocusing (FPLC). They possessed no ability to bind to or hydrolyze insoluble microcrystalline cellulose (Avicel) but were active toward soluble carboxymethylcellulose. No synergism was observed between Trichoderma reesei cellobiohydrolase I and either endoglucanase from A. niger. These findings may indicate that the role of the endoglucanase component of cellulase in insoluble microcrystalline cellulose hydrolysis is dependent upon its ability to be adsorbed upon the substrate.     

Characterization of a metagenome-derived halotolerant cellulase

Metagenomes of uncultured microorganisms represent a sheer unlimited resource for discovery of novel biocatalysts. Here, we report on the biochemical characterisation of a novel, soil metagenome-derived cellulase (endoglucanase), Cel5A. The deduced amino acid sequence of Cel5A was similar to a family 5, single domain cellulase with no distinct cellulose binding domain from Cellvibrio mixtus. The 1092bp ORF encoding Cel5A was overexpressed in Escherichia coli and the corresponding 42.1 kDa protein purified using three-step chromatography. The recombinant Cel5A protein was highly active against soluble cellulose substrates containing beta-1,4 linkages, such as lichenan and barley beta-glucan, and not active against insoluble cellulose. Glucose was not among the initial hydrolysis products, indicating an endo mode of action. Cel5A displayed a wide range of pH activity with a maximum at pH 6.5 and at least 60% activity at pH 5.5 and 9.0. The enzyme was highly stable at 40 degrees C for up to 11 days, and retained 86-87% activity after incubation with 3M NaCl, 3M RbCl or 4M KCl for 20 h. Cel5A was also active in the presence of diverse divalent cations, detergents and EDTA. This highly stable, salt and pH tolerant cellulase is an ideal candidate for industrial applications.     

Molecular Cloning of Novel Cellulase Genes <Emphasis Type="Italic">cel</Emphasis>9A and <Emphasis Type="Italic">cel</Emphasis>12A from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> GXN151 and Synergism of Their Encoded Polypeptides

A thermophilic bacterial strain GXN151 which could degrade Avicel efficiently was isolated and identified as Bacillus licheniformis. A genomic library of GXN151 was constructed and two novel endoglucanase genes designated cel9A and cel12A were isolated by screening the library on carboxylmethyl cellulase indicator plates. The analysis of amino acid sequences deduced from the genes indicated that Cel9A consisted of a catalytic domain belonging to glycosyl hydrolase family 9, a linker domain, and a carbohydrate binding module family 3 from N-terminal to C-terminal; Cel12A had only one catalytic domain belonging to glycosyl hydrolase family 12. The combinations of Cel9A and Cel12A produced by the recombinant E. coli exhibited synergistic action against substrates of carboxylmethyl cellulose as well as Avicel.     

Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with a cell surface-engineered yeast strain

Enzymatic hydrolysis of cellulosic material is an essential step in the bioethanol production process. However, complete cellulose hydrolysis by cellulase is difficult due to the irreversible adsorption of cellulase onto cellulose. Thus, part of the cellulose remains in crystalline form after hydrolysis. In this study, after 96-h hydrolysis of Avicel crystalline cellulose, 47.1 % of the cellulase was adsorbed on the cellulose surface with 10.8 % crystalline cellulose remaining. In simultaneous saccharification and fermentation of 100 g/L Avicel with 1.0 filter paper unit/mL cellulase, a wild-type yeast strain produced 44.7 g/L ethanol after 96 h. The yield of ethanol was 79.7 % of the theoretical yield. On the other hand, a recombinant yeast strain displaying various cellulases, such as β-glucosidase, cellobiohydrolase, and endoglucanase, produced 48.9 g/L ethanol, which corresponds to 87.3 % of the theoretical yield. Higher ethanol production appears to be attributable to higher efficiency of cellulase displayed on the cell surface. These results suggest that cellulases displayed on the yeast cell surface improve hydrolysis of Avicel crystalline cellulose. Indeed, after the 96-h simultaneous saccharification and fermentation using the cellulase-displaying yeast, the amount of residual cellulose was 1.5 g/L, one quarter of the cellulose remaining using the wild-type strain, a result of the alleviation of irreversible adsorption of cellulases on the crystalline cellulose.     

Effect of cellulase mole fraction and cellulose recalcitrance on synergism in cellulose hydrolysis and binding

Elucidating the molecular mechanisms that govern synergism is important for the rational engineering of cellulase mixtures. Our goal was to observe how varying the loading molar ratio of cellulases in a binary mixture and the recalcitrance of the cellulose to enzymatic degradation influenced the degree of synergistic effect (DSE) and degree of synergistic binding (DSB). The effect of cellulose recalcitrance was studied using a bacterial microcrystalline cellulose (BMCC), which was exhaustively hydrolyzed by a catalytic domain of Cel5A, an endocellulase. The remaining prehydrolyzed BMCC (PHBMCC) was used to represent a recalcitrant form of cellulose. DSE was observed to be sensitive to loading molar ratio. However, on the more recalcitrant cellulose, synergism decreased. Furthermore, the results from this study reveal that when an exocellulase (Cel6B) is mixed with either an endocellulase (Cel5A) or a processive endocellulase (Cel9A) and reacted with BMCC, synergism is observed in both hydrolysis and binding. This study also revealed that when a "classical" endocellulase (Cel5A) and a processive endocellulase (Cel9A) are mixed and reacted with BMCC, only limited synergism is observed in reducing sugar production; however, binding is clearly increased by the presence of the Cel5A.     

Optimization of cellulase mixture for efficient hydrolysis of steam-exploded corn stover by statistically designed experiments

To improve the enzymatic hydrolytic efficiency and reduce production cost, a statistically designed experimental approach was used to optimize the composition of cellulase mixture so as to maximize the amount of glucose produced from steam-exploded corn stover (SECS). Using seven purified enzymes (cellobiohydrolases, Cel7A, Cel6A, Cel6B; endoglucanases, Cel7B, Cel12A, Cel61A; and beta-glucosidase) from Trichoderma viride T 100-14 mutant strain, a multi-enzyme mixture was constituted after screening and optimization. The final optimal composition (mol%) of the multi-enzyme mixture was Cel7A (19.8%), Cel6A (37.5%), Cel6B (4.7%), Cel7B (17.7%), Cel12A (15.2%), Cel61A (2.3%) and beta-glucosidase (2.8%). The subsequent verification experiments followed by glucose assay together with scanning electron microscopy (SEM) observation confirmed the validity of the models. The multi-enzyme mixture displayed a high performance in converting the cellulosic substrate (SECS). The amount of glucose produced (15.5mg/ml) was 2.1 times as that of the crude cellulase preparation. The results indicated that the optimized cellulase mixture is an available and efficient paradigm for the hydrolysis of lignocellulosic substrate. The enhanced cellulolytic activity displayed by the constructed cellulase mixture could be used as an effective tool for producing bioethanol efficiently from cellulose.     

Effects of agitation on enzymatic saccharification of cellulose

Summary Crystalline cellulose Avicel has been hydrolyzed byTrichoderma viride cellulase (Meicelase CEPB) under vaned agitation conditions and the effect of agitation on the adsorption of cellulase on cellulose has been studied. Agitation was found to enhance the hydrolysis pf crystalline cellulose; possibly the agitation enhances the adsorption of exoglucanase to shift the adsorption balance of exoglucanase and endoglucanase to a direction favorable for their synergistic action on the surface of cellulose.