Abstract: Central composite design(CCD)and response surface methodology(RSM)were used to optimize the expression conditions of bovine pancreatic carboxypeptidase A. The results of single-factor test showed that the optimal conditions as following：the inducible expression time was 96 hours, the methanol content was 0.5% every 24 h, the initial induction pH4, and OD₆₀₀ before induction expression was 7. Based on the above result, CCD was used to design the tests, and RSM to have regression analysis. The optimal parameters for the expression were obtained as：methanol content was 0.5% every 24 hours, the induction pH 5.90, OD₆₀₀ before inducible expression was 6.54, the expression level after 96 h reached 0.325 mg/mL. Compared with the theoretical value(0.326 mg/mL), the small relative error was 0.38%. Also, the expression of the target protein was confirmed by Western blot. Conclusively, using RSM for optimizing the expression condition of bovine pancreatic carboxypeptidase A was accurate and reliable, and conducive to seek the optimal condition, thus this lays a foundation for guiding the high-density fermentation production of bovine pancreatic carboxypeptidase A in fermenter.

Key words: bovine pancreatic carboxypeptidase A, Pichia pastoris, response surface method