An alkaline proteinase of an alkalophilicBacillus sp.

An alkaline serine proteinase was purfied from the culture broth of an alkalophilicBacillus sp. NKS-21. The molecular weight was estimated to be 22,000 by a gel filtration method and 31,000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 8.2. The amino acid composition and CD spectrum were determined. The alkaline proteinase had a pH optimum at 10–11 for milk casein digestion. The specific activity of the alkaline proteinase was 0.35 katal/kg of protein at pH 10.0 for milk casein hydrolysis.The substrate specificity of the alkaline proteinase was studied by using the oxidized, insulin B-chain and angiotensin. An initial cleavage site was observed at Leu¹⁵-Tyr¹⁶, secondary site at Leu¹¹-Val¹², and additional sites at Gln⁴-His⁵, Tyr²⁶-Thr²⁷, and Asn³-Gln⁴ in the oxidized insulin B-chain at pH 10.0. In comparison with the subtilisins Carlsberg and Novo, the alkaline proteinase fromBacillus sp. showed a unique specificity toward the oxidized insulin B-chain. Hydrolysis of angiotensin at pH 10.0 with the alkaline proteinase was observed at Tyr⁴-Ile⁵. The proteinase has aK _m of 0.1 mM andk _cat of 3.3 s^–1 with angiotensin as substrate.