摘要
采用Central Composite Design(CCD)实验设计及响应面分析相结合的方法,对毕赤酵母表达牛胰脏羧肽酶A的表达条件进行优化,旨在获得牛胰脏羧肽酶A的最佳表达条件。单因素实验结果显示,诱导时间为96 h时,最佳甲醇含量为0.5%、最佳p H值为4、诱导时最佳菌体OD600值为7。在此基础上,利用CCD实验设计及响应面分析法进行回归分析,获得最优表达参数为:甲醇含量0.5%、诱导pH为5.90、接种前种子培养基OD600为6.54。在优化后的条件下进行验证实验,得到牛胰脏羧肽酶A的表达量为0.325 mg/m L,与理论值(0.326 mg/m L)相比,相对误差较小,为0.38%。同时,对表达产物进行了Western Blot鉴定。研究表明,采用响应面法分析优化牛胰脏羧肽酶A的表达条件准确可靠,有助于找出最佳条件,为指导其在发酵罐中进行高密度发酵生产牛胰脏羧肽酶A奠定基础。
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 p H4,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 p H 5.90,OD600 before inducible expression was 6.54,the expression level after 96 h reached 0.325 mg/m L. Compared with the theoretical value（0.326 mg/m L）,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.
出处
《生物技术通报》
CAS
CSCD
北大核心
2016年第3期190-197,共8页
Biotechnology Bulletin
基金
国家高技术研究发展计划("863"计划)(2012AA101609-7)
关键词
牛胰脏羧肽酶A
毕赤酵母
响应面
bovine pancreatic carboxypeptidase A
Pichia pastoris
response surface method
