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

Abstract

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 °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 μM and a V_max 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.