Statistical Optimization of the Production of NaCl-Tolerant Proteases by a Moderate Halophile, Virgibacillus sp. SK37

Sornchai Sinsuwan1, Anuvat Jangchud2, Sureelak Rodtong3, Sittirak Roytrakul4 and Jirawat Yongsawatdigul1*

1School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon
  Ratchasima 30000, Thailand
2Department of Product Development, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand
3School of Microbiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
4National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand


Article history: 
Received November 29, 2014
Accepted March 9, 2015


Key words
response surface methodology, protease, Virgibacillus sp., moderate halophile, fish sauce

The objectives of this study are to optimize the conditions for providing high yield of NaCl-tolerant extracellular protease from Virgibacillus sp. 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 500–1000 m/z compared with the medium containing 0 % NaCl, indicating the higher proteolytic activity of the high-salt medium. The Virgibacillus sp. 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.




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