Production and characterization of NaCl-activated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation

Abstract

Virgibacillus sp. SK33 newly isolated from one-month-old Thai fish sauce was studied for its proteinase production. Neopeptone broth (NEO), halobacterium broth (HL) and HL without either yeast extract (HL-Y), peptone (HL-P) or casamino acid (HL-C) were found to be suitable for proteinase production, whereas fish broth (FB) and skim milk salts broth (SKS) appeared to suppress proteinase production. Moreover, yeast extract, peptone and casamino acid equally stimulated proteinase production. The optimal enzyme production for Virgibacillus sp. SK33 was at 5% NaCl, 40 °C. Maximum proteinase production was achieved at 36 h and maximum cell growth was obtained at 72 h in the modified HL supplemented with only yeast extract (Ym). Extracellular proteinase of Virgibacillus sp. SK33 exhibited optimum activity at 50 °C and pH 8, 10 and 11. Crude proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating the presence of serine alkaline proteinase. The enzymes preferentially cleaved Suc-Ala-Ala-Pro-Phe-AMC, a substrate for subtilisin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) activity staining showed molecular weights of 56, 46, 42, 32, 25 and 19 kDa. All proteinases exhibited caseinolytic activity at 25% NaCl. Proteolytic activity towards synthetic substrate increased about 4 times in the presence of 25% NaCl, indicating the characteristic of NaCl-activated proteinase. In addition, crude proteinase from Virgibacillus sp. SK33 showed higher proteolytic activity towards anchovy than did commercial proteinases at 25% NaCl, demonstrating its potential for protein hydrolysis at high salt content.