Isolation, Identification of the Antimicrobial Protein on Staphyloccus aureus and Optimization of Fermentation Process from Marine Bacillus subtilis UBMR1027
Abstract：It was all known that Staphylococcus aureus was an infectious pathogen to human and animal. In order to study the protein of inhibitory activities to S. aureus, the protein from Bacillus subtilis UMBR1027 was extracted by ammonium sulfate precipitation, and separated by Sephadex LH-20 column chromatography as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and was also identified by ABI 4800 plus MALDI-TOF. Furthermore, the conditions of fermentation were optimized by the response surface method to increase the quantity of the bioactive protein from B. subtilis UMBR1027. The bacteriostasis activity against S. aureus was assessed by the filter paper diffusion method. The 3 key factors including the pH value of the buffer, the ionic strength in the solution and temperature of culture were evaluated respectively to optimize the conditions of fermentation by design Box-Behnken experiments. The results showed that the alkaline serine protease, subtilisin, endo-β-1,3-1,4 glucanase and alaph amylase were identified in the bioactive protein from B. subtilis UMBR1027. The optimization experiments were achieved by systematically adjusting the pH of the buffer, the ionic strength in the solution and temperature. The optimal culture conditions were the medium pH 6.95, salinity 10.38% and the temperature 34.95 ℃, respectively. The selected experiment under the culture conditions were that pH was 6.95, salinity was 10.38% and the temperature was 35 ℃, and the bacteriostatic circle diameter was 20.4 mm which was close to the predicted values. This paper identified antibactial protein from B. subtilis UMBR1027 and optimized ferment process by the response surface method. The results will provide theoretical basis to discover new natural drug molecules from marine.
[1]袁萍, 王晓, 吴根鹏, 等.金黄色葡萄球菌α-溶血素及其突变体的原核表达和免疫学活性[J].中国生物制品学杂志, 2014, 27(11):1400-1411[2]李毅.金黄色葡萄球菌及其肠毒素研究进展[J].中国卫生检验杂志, 2004, 14(4):392-395[3]杨桂柳, 杨立芳, 姜明国, 等.北仑河红树林内生放线菌分离、鉴定及活性研究[J].农业生物技术学报, 2015, 23(7):894-904[4]Carlet J.Ten tips on how to win the war against resistance to antibiotics[J].Intensive Care Medicine, 2015, 41(5):1-3[5]姜明国, 甘光华, 杨立芳, 等.广西红树林根际土壤放线菌的原位培养分离及其活性筛选[J].海洋学报, 2015, 37(2):55-64[6]Shan H, Hong-qiang W, Bin W, et al.Response Surface Methodology optimization of fermentation conditions for rapid and efficient accumulation of Macrolactin A by Marine Bacillus amyloliquefaciens ESB-2[J].Molecules, 2012, 18(1):408-417[7]Zhang X, Zhou J, Fu W, et al.Response Surface Methodology used for statistical optimization of jiean-peptide production by Bacillus subtilis[J].Electronic Journal of Biotechnology, 2010, 13(4):2548-2552[8]Xiao-xi C, Xiao-kun L, She-zeng L.Interval of high fermentation yield of Bacillus amyloliquefaciens determined by Response Surface Method[J].Guizhou Agricultural Sciences, 2010, 38(5):110-111[9]Carlet J.Ten tips on how to win the war against resistance to antibiotics[J].Intensive Care Medicine, 2015, 41(5):1-3[10]陈亮, 毛晶晶, 宇光海, 等.响应面法优化Bacillus subtilis抗菌脂肽发酵工艺条件[J].食品科技, 2015, 40(4):17-21[11]王幸, 吴文惠, 陈志华, 等.海洋微生物次生代谢产物的结构特征和生物活性的研究进展[J].Chinese Journal of Natural Medicines, 2010, 8(04):309-320[12]史翠娟, 闫培生, 赵瑞希, 等.海洋微生物酶研究进展[J].生物技术进展, 2015, 5(3):185-190[13]尹伊, 屈建航, 李海峰, 等.耐酸耐高温α-淀粉酶及其菌种选育研究进展[J].粮油食品科技, 2015, 23(5):101-105[14]王淑军, 吕明生, 秦松, 等.热球菌高温α-淀粉酶基因克隆、表达及性质研究[J].海洋学报, 2011, 33(3):158-164[15]李玉环, 张岩, 时威.枯草芽孢杆菌碱性蛋白酶分离纯化及其性质研究[J].食品工业科技, 2012, 33(19):187-190[16]张向武, 郑江, 伏建峰, 等.金黄色葡萄球菌细胞壁可溶性肽聚糖的分离纯化和鉴定[J].中国临床药理学与治疗学, 2007, 3(12):321-323
