A new alkaline proteinase with pI 2.8 from alkalophilicBacillus sp.

Abstract

A new serine alkaline proteinase (ALPase II) was purified from the culture broth of an alkalophilicBacillus sp. NKS-21. The molecular weight of ALPase II was estimated to be 32,000 by SDS-polyacrylamide gel electrophoresis. The enzyme had a very low isoelectric point (pI), which was determined to be 2.8. An optimum pH of this enzyme was 10.2. The specific activity was 0.28 katal/kg of protein for milk casein, 0.34 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) and 8.5 katal/kg for succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA). The substrate specificity of the alkaline proteinase was studied with the synthetic fluorogenic and chromogenic substrates. It was most favorable for the enzyme that the P1 site of the substrate might be hydrophobic and bulky amino residue (Phe or Tyr). When the substrate contained four amino residues, the proteinase efficiently expressed its activity. The alkaline proteinase had higher specificity than those of the bacterial serine proteinases, subtilisins Carlsberg and BPN′, and lower specificity than that of serine alkaline proteinase with pI 8.2 (ALPase I) obtained from the same bacteria NKS-21. ALPase II did not react with the anti-ALPase I antiserum.