title: Evidence of cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation.
abstract: Cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation were extracted and characterized. Proteinases were effectively released when washed cells were incubated in 0.3 mg/mL lysozyme in 50 mM Tris-maleate (pH 7) at 37 °C for 2 h. Major cell-associated proteinases exhibited molecular mass of 17, 32, and 65 kDa, but only a 32-kDa proteinase showed strong amidolytic activity toward Suc-Ala-Ala-Pro-Phe-AMC. Activity of all cell-associated proteinases was completely inhibited by phenylmethanesulfonyl fluoride, indicating a characteristic of serine proteinase. In addition, a 65-kDa serine proteinase was also inhibited by ethylenediaminetetraacetic acid, implying a metal-dependent characteristic. Optimum activity toward a synthetic peptide substrate was at 50 °C and pH 8 and 11. Proteinases with molecular mass of 17 and 32 kDa exhibited caseinolytic activity at 25% NaCl and activity based on a synthetic peptide substrate increased with NaCl concentrations up to 25%, suggesting their role in hydrolyzing proteins at high salt concentrations. This is the first report of liberated cell-associated proteinases from a moderate halophile, Virgibacillus sp.
  
  
  CONCLUSIONS
  The cell-associated proteinases could be extracted from Virgibacillus sp. SK 33 using lysozyme. The extracted enzyme could be applied to hydrolyze food proteins at NaCl content as high as 25%. In addition, this study demonstrated that not only extracellular but also cell-associated proteinases are key factors contributing to protein-degrading ability at high salt environment of Virgibacillus sp. SK 33.
authors.affiliation: Food Protein Research Unit, School of Food Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Sornchai
authors.initials: S
authors.lastname: Sinsuwan
authors.forename: Sureelak
authors.initials: S
authors.lastname: Rodtong
authors.forename: Jirawat
authors.initials: J
authors.lastname: Yongsawatdigul
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D003212:Condiments / Q000382:microbiology*; D003374:Coumarins / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D005285:Fermentation; D005396:Fish Products / Q000382:microbiology*; D006863:Hydrogen-Ion Concentration; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D009994:Osmolar Concentration; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D057057:Serine Proteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013785:Thailand; D058679:Virgibacillus / Q000201:enzymology* / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D003374:Coumarins; D009842:Oligopeptides; D011480:Protease Inhibitors; D012965:Sodium Chloride; D010664:Phenylmethylsulfonyl Fluoride; C064159:succinylalanylalanyl-prolyl-phenylalanine-4-methylcoumaryl-7-amide; D004492:Edetic Acid; D045726:Metalloproteases; D010447:Peptide Hydrolases; D057057:Serine Proteases