Shigehito Adachi

Purification and properties of an alkaline protease from alkalophilic Bacillus sp. KSM-K16

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°C and at pH 12.3 in 50 mM phosphate/NaOH buffer. The activity was inhibited by phenylmethylsulfonyl flouride and chymostatin. The enzyme was very stable in long-term incubation with liquid detergents at 40°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 cyrstals.

Enhanced Production of Extracellular Enzymes by Mutants of Bacillus That Have Acquired Resistance to Vancomycin and Ristocetin

A new mutagenic method for increasing the productivity of extracellular enzymes, such as cellulases, proteases, and amylases, has been developed, using vancomycin-and ristocetin-resistance as indicators. Among the mutants of Bacillus that were resistant to these atibiotics, strains with improved productivity of the extracellular enzymes were found at high frequency. The use of these antibiotics seems to provide a very effective method for the improvement of industrially important strains of Bacillus, regardless of the specific strains used and the particular extracellular enzymes produced by them.