Characterization of a Bifunctional Cellulase and Its Structural Gene

Abstract

Bacillus sp. D04 secreted a bifunctional cellulase that had a molecular weight of 35,000. This cellulase degraded Cm-cellulose, cellotetraose, cellopentaose, p-ni-trophenyl-D-cellobioside, and avicel PH101. Based on the high performance liquid chromatography analysis of the degradation products, this cellulase randomly cleaved internal-1,4-glycosidic bonds in cellotetraose and cellopentaose as an endoglucanase. It also hydro-lyzed the aglycosidic bond in p-nitrophenyl-D-cellobio-side and cleaved avicel to cellobiose as an exoglucanase. Cellobiose competitively inhibited the p-nitrophenyl-D-cellobioside degrading activity but not Cm-cellulose degrading activity. Ten mM p-chloromercuribenzoate inhibited p-nitrophenyl-D-cellobioside degrading activity completely, but Cm-cellulose degrading activity incompletely. Cm-cellulose increased p-nitrophenyl-D-cellobioside degrading activity, and vice versa, whereas methylumbelliferyl-D-cellobiose strongly inhibited p-nitrophenyl-D-cellobioside degrading activity. The cellulase gene (cel gene), 1461 base pairs, of Bacillus sp. D04 was cloned. The nucleotide sequence of the cel gene was highly homologous to those of Bacillus subtilis DLG and B. subtilis BSE616. The cel gene was overexpressed in Escherichia coli, and its product was purified. The substrate specificity and substrate competition pattern of the purified recombinant cellulase were the same as those of the purified cellulase from Bacillus sp. D04. These results suggest that a single polypeptide cellulase had both endo-and exoglucanase activities and each activity exists in a separate site. Cellulose is an unbranched glucose polymer composed of an anhydro-1,4-glucose units linked by a-1,4-D-glycosidic bond. Cellulolytic enzymes degrade cellulose by cleaving this glycosidic bond. Cellulases can be classified into three types: endoglucanases (1,4-Dglucan 4-glucohydrolase, EC 3.2.1.4), exoglucanases (-1,4-D-glucan cellobiohydrolase), and-gluco-sidases (-D-glucoside glucohydrolase, EC 3.2.1.21). Endoglu-canases randomly hydrolyze internal-1,4-glycosidic bonds in cellulose. As a result, the polymer rapidly decreases in length, but the concentration of the reducing sugar increases slowly (1). Exoglucanases hydrolyze cellulose by removing the cellobi-ose unit from the nonreducing end of cellulose; the reducing sugars are rapidly increased, but the polymer length changes little (1-3).-Glucosidases cleave cellobiose and oligosaccha-rides to glucose (1). Therefore, crystalline cellulose is efficiently hydrolyzed by the synergistic action of all three types of cellulases. Cellulosic substrates hydrolyzed by only one type of cellulase are catagorized as follows. Acid-swollen cellulose, Cm-cellulose, cellulose azure, and trinitrophenyl Cm-cellulose are hydrolyzed by endoglucanases (1). MUC 1 (4) and pNPC (5) are used as substrates for the determination of exoglucanase activity, and MUG (4) and pNPG (5) are cleaved by-glucosidases. Filter paper and avicel are efficiently hydrolyzed by the synergistic action of endo-and exoglucanases, but not by either one alone (6). Some organisms (for example, Trichoderma sp.) (6-9, 11) produce all three types of cellulases and efficiently degrade cellulose by their synergistic effect. A cellulolytic hydrolase with a considerable level of endo-, exoglucanase, and xylanase activity has been described (3, 12-14). For example, Saul reported a cellulase gene (cel B) of Caldocellum saccharolyticum with a Cm-cellulose-degrading domain in the C-terminal region and an MUC degrading domain in the N-terminal region (14). A polysaccharide hydrolase of the rumen fungus Neocallima-trix patriciarum has a multifunctional catalytic domain with high endoglucanase, cellobiohydrolase, and xylanase activities (12, 13). Extensive recent studies on proteins (such as cellulase, pro-tease, and amylase) secreted by Bacillus species (15) have shown that the following Bacillus species produce cellulases: Bacillus cereus (16), Bacillus licheniformis (17), Bacillus sub-tilis (18), and Bacillus polymyxa (19). Because these strains did not produce all three types of cellulase, they did not extensively hydrolyze crystalline cellulose. We have investigated another strain of this species, Bacillus sp. D04, having the ability to degrade crystalline cellulose. We have determined that the cellulase of Bacillus sp. D04 differed from that of other Bacillus species in several respects. In particular it has both endo-and exoglucanase activity. It degraded Cm-cellulose, cellotetraose, and cellopentaose as an endoglucanase and cleaved aglycosidic bonds in pNPC as an exoglucanase. It also cleaved avicel to cellobiose. Substrate competition assays showed that the cel-lulase of Bacillus sp. D04 had separate sites for endo-and exoglucanase activity.