C: Food Chemistry Evidence of Cell-Associated Proteinases from Virgibacillus sp. SK33 Isolated from Fish Sauce Fermentation Sornchai Sinsuwan, Sureelak Rodtong, and Jirawat Yongsawatdigul 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. Keywords: cell-associated proteinases, NaCl-stable enzyme, Virgibacillus sp. Practical Application: 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. Introduction Production of fish sauce involves the addition of salt to un- eviscerated fish at a ratio of approximately 1:3. Fermentation process relies mainly on proteolytic activity of endogenous pro- teinases in digestive tract of fish as well as proteinases secreted by halophilic/halotolerant bacteria naturally presented in solar salt and fish (Beddows 1998). Studies of proteinase from halophilic bacteria isolated from fish sauce fermentation have been exten- sively focused on extracellular enzymes that completely dissociate from cell surface (Hiraga and others 2005; Namwong and oth- ers 2006; Sinsuwan and others 2007, 2008b, 2010a, b). Another important class of proteinase is a cell-associated proteinase, which has rarely been characterized in halophilic/halotolerant bacteria. Cell-associated proteinases have been mainly explored in lactic acid bacteria used as a starter culture for various fermented milk products. These enzymes play an important role in the initial stage of casein degradation, providing peptides and amino acids for translocation into the cell (Bockelmann 1995). Lactic acid bacte- ria convert these amino acids to alcohols, aldehydes, acids, which MS 20101053 Submitted 9/18/2010, Accepted 12/28/2010. Authors Sinsuwan and Yongsawatdigul are with Food Protein Research Unit, School of Food Technology, Suranaree Univ. of Technology, Nakhon Ratchasima 30000, Thailand. Author Rodtong is with School of Microbiology, Suranaree Univ. of Technology, Nakhon Ratchasima 30000, Thailand. Direct inquiries to Jirawat Yongsawatdigul (E-mail: ji- rawat@sut.ac.th) significantly contribute to flavor characteristic of fermented dairy products (van Kranenburg and others 2002). Cell-associated pro- teinases from halotolerant/halophilic bacteria might play as vital a role as extracellular counterparts in hydrolyzing proteins during fish sauce fermentation. Cell-associated proteinases are defined as the enzyme anchoring the C-terminus region to cell envelop and adhering to cell surface. Cell envelop consists of cell membrane and cell wall whose major components are amphipatic lipids and peptidoglycan, respectively (McKane and Kandel 1985). Deletion of C-terminus from the proteinase rendered liberation of the enzyme into a soluble form without reducing the enzyme activity and substrate specificity (Kok and others 1988; Haandrikman and others 1989; Haandrik- man and others 1991; Bruinenberg and others 2000; Germond and others 2003). Parts of polypeptide chain embedding in an integral part of lipid bilayer are nonpolar amino acids, allowing hydropho- bic interactions between the enzyme and lipid. On the other hand, parts of the enzyme associated with peripheral membrane are rich in polar amino acids, and form electrostatic interactions or hy- drogen bonds with phosphate group of lipid bilayer or integral membrane protein (Palmer 1991). Proline-, glycine-, threonine- and serine-rich region, and lysine-rich region on C-terminus of the cell-associated proteinase were capable to bind peptidoglycan and teichoic acids of cell wall, respectively (Piard and others 1997; Rathsam and Jacques 1998; Germond and others 2003). Typically, membrane-associated proteins can be extracted and isolated using salts, sonication, chelating agents, mild alkaline C  2011 Institute of Food Technologists R  doi: 10.1111/j.1750-3841.2011.02058.x Vol. 76, Nr. 3, 2011  Journal of Food Science C413 Further reproduction without permission is prohibited