An alkaline proteinase of an alkalophilicBacillus sp.
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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.
KeywordsAngiotensin Serine Proteinase Amino Acid Composition Alkaline Proteinase Casein Hydrolysis
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