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.

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In cellulosomes produced by Clostridium spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex. Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a "designer" cellulosome, or a recombinant cellulosome with a specific function. We report the preparation of Clostridium cellulovorans recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units. The full-length EngB containing the dockerin domain was expressed by Bacillus subtilis WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with Escherichia coli. The assembly of recombinant EngB with the miniCbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis.Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome) containing a variety of cellulolytic subunits attached to the nonenzymatic component termed CbpA (2). All cellulosomal enzymatic subunits contain a twice-repeated sequence, usually at their C termini, called the dockerin domain. These dockerin domains are considered to bind to the hydrophobic domains termed cohesins (19,26), which are repeated nine times in CbpA (14). So far, we have cloned and sequenced eight cellulosomal cellulase genes from C. cellulovorans (engB [3] [17], and exgS [9]). In addition, the genes for cellulosomal mannanase (manA) (15) and pectate lyase (pelA) (17) have also been cloned and sequenced.In order to understand the hydrolytic mechanism of the cellulosome and to use cellulosomes for industrial purposes, it is very important to make recombinant cellulosomes, or "designer" cellulosomes, with specific functions. To prepare recombinant cellulosomes, it is necessary to express the cellulosomal enzyme subunits that retain their dockerin domains. Escherichia coli is a very efficient host for expression of heterologous genes. However, during the expression of cellulosomal subunits from C. cellulovorans by E. coli, we found that almost all recombinant enzyme proteins were partially degraded between the catalytic and dockerin domains by the proteases of E. coli. Thus, this problem prevented us from preparing recombinant cellulosomes.As a host for heterologous expression, Bacillus subtilis has many attractive features (21). These features include the ability to secrete extracellular proteins, ease of genetic manipulation, and fast growth. However, one major problem is the presence of high levels of extracellular proteases. Aminov et al. (1) tried to express the celE gene, encoding one of the cellulosomal subunits from Clostridium thermocellum, ...

Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases. To characterize the ␣-L-arabinosidases that are involved in hemicellulose degradation, we screened the C. cellulovorans genomic library for clones with ␣-L-arabinofuranosidase or ␣-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 ␣-L-arabinofuranosidase and ␤-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively. Recombinant ArfA (rArfA) had high activity for p-nitrophenyl ␣-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl ␣-L-arabinopyranoside. On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl ␣-L-arabinopyranoside but also p-nitrophenyl ␤-D-galactopyranoside. However, when the affinities of rBgaA for p-nitrophenyl ␣-L-arabinopyranoside and p-nitrophenyl ␤-D-galactopyranoside were compared, the K m values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for ␣-L-arabinopyranose residues than for ␤-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 ␤-galactosidase. Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction. When rArfA and rBgaA were incubated with ␤-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.Clostridium cellulovorans ATCC 35296 (25), an anaerobic, mesophilic, and spore-forming bacterium, produces a large extracellular polysaccharolytic multicomponent complex called the cellulosome, in which several cellulases are tightly bound to a scaffolding protein called CbpA (7). C. cellulovorans utilizes not only cellulose but also hemicelluloses, such as xylan, pectin, and several other carbon sources, for growth (7,15,20). Our laboratory has characterized the gene for the scaffolding protein CbpA as well as several genes necessary for the degradation of crystalline cellulose and plant cell wall polysaccharides (7,15,20,26,27).Plant cell walls consist of a composite structure containing a complex mixture of polysaccharides such as cellulose, hemicellulose (xylan and galactomannan), pectic substances (galacturonan and arabinogalactan), and other polysaccharides (e.g., type II arabinogalactan and fucoxyloglucan) (1). L-Arabinose is a common component of several of these polysaccharides, and the arabinose residues are widely distributed in plant cell walls, where...