Purification and Characterization of a Salt-Activated and Organic Solvent-Stable Heterotrimer Proteinase from Virgibacillus sp. SK33 Isolated from Thai Fish Sauce

摘要

A NaCl-activated proteinase produced by Virgibacillus sp. SK33 was purified to homogeneity using phenyl-Sepharose and Sephadex G-75 with a yield of 12% and purification of 2.6-fold. A single protein was detected at ～32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, three subunits with molecular weights of 27,858, 33,918, and 35,368 Da were obtained from MALDI-TOF mass spectra, implying that the enzyme was a heterotrimer. The isoelectric point of the proteinase was 5.4. Optimum catalytic activity was at 55°C and pH 7.5. The enzyme showed serine characteristics as it was completely inhibited by phenylmethanesulfonyl fluoride. The purified proteinase showed broad specificity toward oxidized insulin B including Gln~4, Cys~7, Glu~(13), Ala~(14), Leu~(15,17), Tyr~(16,26) Arg~(22), Phe~(24,25), and Lys~(29). Dominant cleavage sites of the enzyme were Tyr~(16)-Leu~(17) and Phe~(25)-Tyr~(26), indicating that it preferably hydrolyzed aromatic amino acids located on the P1 site. Among various substrates studied, the enzyme hydrolyzed anchovy protein to the greatest extent at 4 M NaCl. Activity increased with either CaCl2 or NaCl concentration with the maximum 2-fold increase at either 50 mM CaCl2 or 4 M NaCl. The enzyme was also highly stable up to 500 mM CaCl2 or 4 M NaCl. The proteinase showed high stability in various organic solvents (25%, v/v) including dimethylsulfoxide, methanol, acetonitrile, and ethanol. Results of peptide mass fingerprint and de novo peptide sequencing showed that the purified proteinase is a novel proteinase. The proteinase from Virgibacillus sp. SK33 could have a potential application in high ionic strength environments and aqueous-organic solvent systems.