Sinsuwan Sornchai, Jangchud Anuvat, Rodtong Sureelak, Roytrakul Sittirak, Yongsawatdigul Jirawat
School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Department of Product Development, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand.
Food Technol Biotechnol. 2015 Jun;53(2):136-145.
The objectives of this study are to optimize the conditions for providing high yield of NaCl-tolerant extracellular protease from 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 compared with the medium containing 0% NaCl, indicating the higher proteolytic activity of the high-salt medium. The 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.
本研究的目的是基于鱼基培养基优化从sp. SK37中高产耐盐胞外蛋白酶的条件，并研究关键因素（干鳀鱼、酵母提取物和NaCl的质量体积比以及培养基的初始pH值）对蛋白酶分泌模式的影响。基于预测响应模型，优化后的培养基含有1.81%的干鳀鱼、0.33%的酵母提取物和1.25%的NaCl，pH值为7.8。在这些条件下，与仅含有1.2%干鳀鱼（pH值为7时含5% NaCl）的肉汤相比，蛋白酶产量提高了5.3倍。三次回归充分描述了蛋白酶的产生。使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）在合成底物（Suc-Ala-Ala-Pro-Phe-AMC）上测定蛋白酶活性。在有NaCl存在的情况下，分泌出分子量为19、34、35和44 kDa的蛋白酶，而在无NaCl的情况下，检测到的主要蛋白酶分子量为22和42 kDa。此外，当培养基的初始pH值为6时，未检测到分泌的蛋白酶。与含0% NaCl的培养基相比，用10% NaCl培养的培养基的肽质量指纹图谱显示，分子量在500-1000之间的低质量肽丰度更高，表明高盐培养基的蛋白水解活性更高。sp. SK37蛋白酶在有NaCl存在时对Lys、Arg和Tyr有明显偏好，在无NaCl时对Lys和Arg有明显偏好。
