title: Statistical Optimization of the Production of NaCl-Tolerant Proteases by a Moderate Halophile,Virgibacillussp. SK37.
abstract: The objectives of this study are to optimize the conditions for providing high yield of NaCl-tolerant extracellular protease fromVirgibacillussp. SK37 based on a fish-based medium and to investigate the effects of the key factors (mass per volume ratios of dried anchovy, yeast extract and NaCl, and initial pH of the medium) on the secretion pattern of proteases. Based on the predicted response model, the optimized medium contained 1.81% of dried anchovy, 0.33% of yeast extract and 1.25% of NaCl at pH=7.8. Under these conditions, a 5.3-fold increase in protease production was achieved, compared with the broth containing only 1.2% of dried anchovy (5% of NaCl at pH=7). The cubic regression adequately described the protease production. Protease activity was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on the synthetic substrate (Suc-Ala-Ala-Pro-Phe-AMC). Proteases of molecular masses of 19, 34, 35 and 44 kDa were secreted in the presence of NaCl, whereas those of 22 and 42 kDa were the main proteases detected in the absence of NaCl. In addition, no secreted proteases were detected when initial pH of the medium was pH=6. The peptide mass fingerprint of the medium cultured with 10% NaCl showed a higher abundance of peptides with lower mass of 500-1000m/zcompared with the medium containing 0% NaCl, indicating the higher proteolytic activity of the high-salt medium. TheVirgibacillussp. SK37 proteases showed a marked preference towards Lys, Arg and Tyr in the presence of NaCl and towards Lys and Arg in the absence of NaCl.
authors.affiliation: School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Sornchai
authors.initials: S
authors.lastname: Sinsuwan
authors.affiliation: Department of Product Development, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Anuvat
authors.initials: A
authors.lastname: Jangchud
authors.affiliation: School of Microbiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Sureelak
authors.initials: S
authors.lastname: Rodtong
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand.
authors.forename: Sittirak
authors.initials: S
authors.lastname: Roytrakul
authors.affiliation: School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
authors.forename: Jirawat
authors.initials: J
authors.lastname: Yongsawatdigul
keywords: Virgibacillus sp.; fish sauce; moderate halophile; protease; response surface methodology