Methods: A 5-L high cell density fermentation was performed and the fermentation medium was optimized. Response surface methodology (RSM) was used to optimize the culture condition through modeling three selected parameter.
Results: Under the optimized culture medium (LBSM, 1% yeast powder and 1% peptone) and culture conditions (induction pH 5.5, temperature 29 °C, time 40 h), the yield of rMBSP was 420 mg/L in a 5-L fermenter, which was a 6-fold increase over thar, expressed in flask cultivation. The desired enzyme was purified by two-step, which yielded a 33.7% recovery of a product that had over 85% purity. The activity of purified rMBSP was significantly inhibited by Ca2+, Mg2+, SDS, guanidine hydrochloeide, acetone, isopropanol, chloroform, n-hexane and n-heptane. Enzymatic analysis revealed a Km of 2.89 ± 0.09 μM and a Vmax of 14.20 ± 0.12 nM•min-1 for rMBSP. LC-MS/MS analysis demonstrated the specific cleavage of bovine serum albumin by rMPSP.
Conclusion: These findings suggest that rMPSP has potential as a valuable enzyme for protein science research.
方法:进行5-L高细胞密度发酵并优化发酵培养基。响应面法(RSM)用于通过建模三个选定的参数来优化培养条件。
结果:在优化的培养基(LBSM,1%酵母粉和1%蛋白胨)和培养条件(诱导pH5.5,温度29°C,时间40小时),在5升发酵罐中,rMBSP的产量为420毫克/升,比塔尔增加了6倍,在烧瓶培养中表达。通过两步纯化所需的酶,其产生具有超过85%纯度的产品的33.7%回收率。纯化的rMBSP的活性被Ca2+显著抑制,Mg2+,SDS,盐酸胍,丙酮,异丙醇,氯仿,正己烷和正庚烷。酶分析显示rMBSP的Km为2.89±0.09μM,Vmax为14.20±0.12nM•min-1。LC-MS/MS分析表明rMPSP对牛血清白蛋白的特异性裂解。
结论:这些发现表明rMPSP具有作为蛋白质科学研究有价值的酶的潜力。