Thermostable alkaline cellulase (endo-1,4-beta-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val- Lys-Arg. The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of 8.6-9.0 and displayed maximum activity at 45 degrees C. The alkaline enzyme was stable up to 50 degrees C and more than 30% of the original activity was detectable after heating at 100 degrees C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan (beta-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H3PO4-swollen cellulose, NaOH-swollen cellulose, curdlan (beta-1,3-linkage), laminarin (beta-1,3;1,6-linkage), and xylan were barely hydrolyzed at all.

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Alkaline protease (EC 3.4.21.14) activity, suitable for use in detergents, was detected in the alkaline culture medium of Bacillus sp. KSM-K16, which was originally isolated from soil. The enzyme, designated M protease, was purified to homogeneity from the culture broth by column chromatographies. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg- Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-Leu-Thr-Gly. The molecular mass of the protease was 28 kDa, and its isoelectric point was close to pH 10.6. Maximum activity toward casein was observed at 55 degrees C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl fluoride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40 degrees C. The enzyme cleaved the oxidized insulin B chain initially at Leu15-Tyr16 and efficiently at ten more sites. Among various oligopeptidyl p-nitro-anilides (pNA) tested, N-succinyl-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed by M protease. M protease was precipitated in (NH4)2SO4-saturated acetate buffer (pH 5.0) as plank-like crystals.

Alkaliphilic Bacillus sp. strain KSM-S237 (a relative of Bacillus pseudofirmus) produces a thermostable, alkaline endo-1,4-beta-glucanase (Egl). The entire gene for the enzyme harbored a 2,472-bp open reading frame (ORF) encoding 824 amino acids, including a 30-aminoacid signal peptide. The deduced amino acid sequence of the mature enzyme (794 amino acids, 88,284 Da) showed very high similarity to those of family 5 mesophilic, alkaline Egls from some alkaliphilic bacilli. The enzyme had a region similar to a novel cellulose binding domain proposed for an Egl (EngF) from Clostridium cellulovorans. Expression of the Bacillus Egl gene in Bacillus subtilis resulted in high carboxymethy cellulase activity (2.0 g/l) in the culture broth, concomitant with the appearance of a protein band on an SDS gel at 86 kDa. Site-directed mutagenesis delineated the importance of Arg111, His151, Glu190, His262, Tyr264, and Glu305 in catalysis and/or substrate binding of the enzyme.

Two carboxymethylcellulases (CMCase, 1,4-P-D-glucan glucanohydrolase, EC 3.2.1.4), designated E-H and E-L, were purified to homogeneity from a culture mtrate of the alkalophilic Bacillus sp. KSM-635, by chromatography on DEAE-Toyopearl65OS 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 103kDa for E-L. The pH optima of the enzymes were both about 9.5, and their optimum temperatures were around 40 OC. Activities of both enzymes were inhibited by Hg2+, Cu2+, Fez+ 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 carboxymethylcelldose (p-1 &linkage) and lichenan (p-1,3; 1,4linkage), but crystalline cellulosic substrates, curdlan @1,3-linkage), laminarin (p-1,3; 1,6-linkage) and 4-Ntrophenyl-/?-~-glucopyranoside were hydrolysed very little, if at all. 4-Nitrophenyl-P-D-cellobioside was hydrolysed by both enzymes to liberate 4-Ntrophenol, and their hydrolysis rates were higher at neutral pH than at alkaline pH.