An optimization by response surface methodology for the enhanced production of rMBSP from Pichia pastoris and study of its application

Background: Recombinant myofibril-bound serine proteinase (rMBSP) was successfully expressed in Pichia pastoris GS115 in our laboratory. However, low production of rMBSP in shake flask constraints further exploration of properties. 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 degrees 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 K-m of 2.89 +/- 0.09 mu M and a V-max of 14.20 +/- 0.12 nM center dot 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.