title: Purification and characterization of a salt-activated and organic solvent-stable heterotrimer proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce.
abstract: 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 approximately 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 degrees 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 Gln4, Cys7, Glu13, Ala14, Leu15,17, Tyr16,26, Arg22, Phe24,25, and Lys29. Dominant cleavage sites of the enzyme were Tyr16-Leu17 and Phe25-Tyr26, 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.
authors: School of Food Technology, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
authors: Sornchai
authors: S
authors: Sinsuwan
authors: Sureelak
authors: S
authors: Rodtong
authors: Jirawat
authors: J
authors: Yongsawatdigul
mesh_terms: D000818:Animals; D001406:Bacillaceae / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D004795:Enzyme Stability; D005396:Fish Products / Q000382:microbiology*; D005399:Fishes; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D012965:Sodium Chloride / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D012965:Sodium Chloride; D010447:Peptide Hydrolases