PURIFICATION AND CHARACTERIZATION OF ALKALINE ENDO-1,4-BETA-GLUCANASES FROM ALKALOPHILIC BACILLUS SP KSM-635

Two carboxymethylcellulases (CMCase, 1,4-β-D-glucan glucanohydrolase, EC 3.2.1.4), designated E-H and E-L, were purified to homogeneity from a culture filtrate of the alkalophilic Bacillus sp. KSM-635, by chromatography on DEAE-Toyopearl 650S and gel filtration on Bio-Gel A-0.5m. The purified CMCases both contained approximately 2-3% (w/w) glucosamine. Molecular masses deduced from SDS-PAGE were 130 kDa for E-H and 103 kDa for E-L. The pH optima of the enzymes were both about 9.5, and their optimum temperatures were around 40°C. Activities of both enzymes were inhibited by Hg2+, Cu2+, Fe2+ and Fe3+, but sulphydryl inhibitors, such as N-ethylmaleimide, monoiodoacetate and 4-chloromercuribenzoate, had either no effect or a slightly inhibitory effect. N-Bromosuccinimide was strongly inhibitory, suggesting that a tryptophan residue is essential for the activity of the CMCases from Bacillus. In addition, the activities of both E-H and E-L were stimulated by Co2+, and they required Mg2+, Ca2+, Mn2+ or Co2+ for stabilization. Both enzymes efficiently hydrolysed carboxymethylcellulose (β-1,4-linkage) and lichenan (β-1,3; 1,4-linkage), but crystalline cellulosic substrates, curdlan (β-1,3-linkage), laminarin (β-1,3; 1,6-linkage) and 4-nitrophenyl-β-D-glucopyranoside were hydrolysed very little, if at all. 4-Nitrophenyl-β-D-cellobioside was hydrolysed by both enzymes to liberate 4-nitrophenol, and their hydrolysis rates were higher at neutral pH than at alkaline pH.