Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans

Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.     

Synergistic interaction of Clostridium cellulovorans cellulosomal cellulases and HbpA

Clostridium cellulovorans, an anaerobic bacterium, produces a small nonenzymatic protein called HbpA, which has a surface layer homology domain and a type I cohesin domain similar to those found in the cellulosomal scaffolding protein CbpA. In this study, we demonstrated that HbpA could bind to cell wall fragments from C. cellulovorans and insoluble polysaccharides and form a complex with cellulosomal cellulases endoglucanase B (EngB) and endoglucanase L (EngL). Synergistic degradative action of the cellulosomal cellulase and HbpA complexes was demonstrated on acid-swollen cellulose, Avicel, and corn fiber. We propose that HbpA functions to bind dockerin-containing cellulosomal enzymes to the cell surface and complements the activity of cellulosomes.     

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.     

Cellulosome and noncellulosomal cellulases of Clostridium cellulovorans

This paper reviews the properties of the cellulosome and noncellulosome cellulases produced by Clostridium cellulovorans, an anaerobic, mesophilic, spore-forming microorganism that produces copious amounts of cellulase. The three major subunits of the cellulosome, CbpA, exoglucanase S (ExgS), and P100, are described, as well as the properties of the functional domains of CbpA. The properties of two noncellulosomal endoglucanases, EngD and EngF, are compared. The functions of the cellulose-binding domain (CBD) of CbpA indicate its potential uses in biotechnology. Received: November 18, 1997 / Accepted: November 26, 1997     

Cohesin-dockerin interactions of cellulosomal subunits of Clostridium cellulovorans

The cellulosome of Clostridium cellulovorans consists of three major subunits: CbpA, EngE, and ExgS. The C. cellulovorans scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits. Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity. Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes. Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues. Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA. By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6. The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared. Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1. Significantly, the abilities of the two fusion proteins to bind CbpA were similar. The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes. In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin. These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA.     

Regulation of expression of cellulosomal cellulase and hemicellulase genes in Clostridium cellulovorans

The regulation of expression of the genes encoding the cellulases and hemicellulases of Clostridium cellulovorans was studied at the mRNA level with cells grown under various culture conditions. A basic pattern of gene expression and of relative expression levels was obtained from cells grown in media containing poly-, di- or monomeric sugars. The cellulase (cbpA and engE) and hemicellulase (xynA) genes were coordinately expressed in medium containing cellobiose or cellulose. Growth in the presence of cellulose, xylan, and pectin gave rise to abundant expression of most genes (cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and/or pelA) studied. Moderate expression of cbpA, engH, manA, engE, and xynA was observed when cellobiose or fructose was used as the carbon source. Low levels of mRNA from cbpA, manA, engE, and xynA were observed with cells grown in lactose, mannose, and locust bean gum, and very little or no expression of cbpA, engH, manA, engE, and xynA was detected in glucose-, galactose-, maltose-, and sucrose-grown cells. The cbpA-exgS and engE genes were most frequently expressed under all conditions studied, whereas expression of xynA and pelA was more specifically induced at higher levels in xylan- or pectin-containing medium, respectively. Expression of the genes (cbpA, hbpA, manA, engM, and engE) was not observed in the presence of most soluble di- or monosaccharides such as glucose. These results support the hypotheses that there is coordinate expression of some cellulases and hemicellulases, that a catabolite repression type of mechanism regulates cellulase expression in rapidly growing cells, and that the presence of hemicelluloses has an effect on cellulose utilization by the cell.     

Characterization of two noncellulosomal subunits,ArfA and BgaA,from Clostridium cellulovorans that cooperate with the cellulosome in plant cell wall degradation

Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases. To characterize the alpha-L-arabinosidases that are involved in hemicellulose degradation, we screened the C. cellulovorans genomic library for clones with alpha-L-arabinofuranosidase or alpha-L-arabinopyranosidase activity, and two clones utilizing different substrates were isolated. The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci. The amino acid sequences for ArfA and BgaA were related to alpha-L-arabinofuranosidase and beta-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively. Recombinant ArfA (rArfA) had high activity for p-nitrophenyl alpha-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl alpha-L-arabinopyranoside. On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl alpha-L-arabinopyranoside but also p-nitrophenyl beta-D-galactopyranoside. However, when the affinities of rBgaA for p-nitrophenyl alpha-L-arabinopyranoside and p-nitrophenyl beta-D-galactopyranoside were compared, the K(m) values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for alpha-L-arabinopyranose residues than for beta-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 beta-galactosidase. Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction. When rArfA and rBgaA were incubated with beta-1,4-xylanase A (XynA), a cellulosomal enzyme from C. cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone. These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits.     

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.     

The engL gene cluster of Clostridium cellulovorans contains a gene for cellulosomal manA

A five-gene cluster around the gene in Clostridium cellulovorans that encodes endoglucanase EngL, which is involved in plant cell wall degradation, has been cloned and sequenced. As a result, a mannanase gene, manA, has been found downstream of engL. The manA gene consists of an open reading frame with 1,275 nucleotides encoding a protein with 425 amino acids and a molecular weight of 47, 156. ManA has a signal peptide followed by a duplicated sequence (DS, or dockerin) at its N terminus and a catalytic domain which belongs to family 5 of the glycosyl hydrolases and shows high sequence similarity with fungal mannanases, such as Agaricus bisporus Cel4 (17.3% identity), Aspergillus aculeatus Man1 (23.7% identity), and Trichoderma reesei Man1 (22.7% identity). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal amino acid sequence analyses of the purified recombinant ManA (rManA) indicated that the N-terminal region of the rManA contained a DS and was truncated in Escherichia coli cells. Furthermore, Western blot analysis indicated that ManA is one of the cellulosomal subunits. ManA production is repressed by cellobiose.     

Putative role of cellulosomal protease inhibitors in Clostridium cellulovorans based on gene expression and measurement of activities

This study is the first to demonstrate the activity of putative cellulosomal protease/peptidase inhibitors (named cyspins) of Clostridium cellulovorans, using the Saccharomyces cerevisiae display system. Cyspins exhibited inhibitory activities against several representative plant proteases. This suggests that these inhibitors protect their microbe and cellulosome from external attack by plant proteases.     

Degradation of corn fiber by Clostridium cellulovorans cellulases and hemicellulases and contribution of scaffolding protein CbpA

Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.     

Structural basis for the substrate specificity of the feruloyl esterase domain of the cellulosomal xylanase Z from Clostridium thermocellum

Feruloyl esterases function in the cleavage of ferulic acid's bonds to arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose. This work presents the crystal structure of FAE_XynZ, the domain of Clostridium thermocellum's cellulosomal xylanase Z that displays feruloyl esterase activity. The structure was obtained via multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives and refined against X-ray diffraction data of up to 1.75 A resolution. The R-value of the final model was 0.187 (R(free) = 0.21). FAE_XynZ displays an eight-stranded alpha/beta-fold with the characteristic "catalytic triad" at the heart of the active site. To define the substrate specificity determinants of the enzyme, the crystal structures of FAE_XynZ and the inactive FAE_XynZ(S172A) mutant were determined in complexes with the feruloyl-arabinoxylans FAXX and FAX(3), respectively. In the complex crystals, the ferulic acid moieties are clearly recognizable and allowed identification of the hydrophobic binding pocket. The carbohydrate part of both substrates is not visible in either structure. The location of the putative carbohydrate binding-pocket was inferred based on the location and orientation of the adjacent ferulic acid molecule. Five of the six residues lining the pocket were found to be conserved in FAE A from Orpinomyces sp., which further supports the proposed role of these amino acids.     

Site-directed mutagenesis and expression of the soluble form of the family IIIa cellulose binding domain from the cellulosomal scaffolding protein of Clostridium cellulovorans

The planar and anchoring residues of the family IIIa cellulose binding domain (CBD) from the cellulosomal scaffolding protein of Clostridium cellulovorans were investigated by site-directed mutagenesis and cellulose binding studies. By fusion with maltose binding protein, the family IIIa recombinant wild-type and mutant CBDs from C. cellulovorans were expressed as soluble forms. Cellulose binding tests of the mutant CBDs indicated that the planar strip residues played a major role in cellulose binding and that the anchoring residues played only a minor role.     

Enzymatic degradation of cell wall and related plant polysaccharides

Polysaccharides such as starch, cellulose and other glucans, pectins, xylans, mannans, and fructans are present as major structural and storage materials in plants. These constituents may be degraded and modified by endogenous enzymes during plant growth and development. In plant pathogenesis by microorganisms, extracellular enzymes secreted by infected strains play a major role in plant tissue degradation and invasion of the host. Many of these polysaccharide-degrading enzymes are also produced by microorganisms widely used in industrial enzyme production. Most commerical enzyme preparations contain an array of secondary activities in addition to the one or two principal components which have standardized activities. In the processing of unpurified carbohydrate materials such as cereals, fruits, and tubers, these secondary enzyme activities offer major potential for improving process efficiency. Use of more defined combinations of industrial polysaccharases should allow final control of existing enzyme processes and should also lead to the development of novel enzymatic applications.     

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

An endoglucanase gene, engB, from Clostridium cellulovorans, previously cloned into pUC19, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichia coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosome-binding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.