联合策略优化葡萄糖氧化酶的酵母表达  

High-level expression of glucose oxidase in Pichia pastoris optimized by a combined strategy

作　　者：王玥 董聪 罗同阳 王庆庆 高庆华 王云鹏 唐兆宏 耿革霞 WANG Yue;DONG Cong;LUO Tongyang;WANG Qingqing;GAO Qinghua;WANG Yunpeng;TANG Zhaohong;GENG Gexia(Hebei Research Institute of Microbiology Co.,Ltd.,Baoding Hebei 071051,China;Baoding Xianerkang Biological Engineering Co.,Ltd.,Baoding Hebei 071051,China;Hebei Pharmaceutical Professional Inspector Crops,Shijiazhuang Hebei 050024,China)

机构地区：[1]河北省微生物研究所有限公司,河北保定071051 [2]保定鲜尔康生物工程有限责任公司,河北保定071051 [3]河北省药品职业化检查员总队,河北石家庄050024

出　　处：《河北省科学院学报》2023年第4期1-9,共9页Journal of The Hebei Academy of Sciences

基　　金：河北省科学院高层次人才培养与资助项目(2023G23,2022G13)。

摘　　要：葡萄糖氧化酶(GOD)应用广泛,随着市场对葡萄糖氧化酶的需求逐渐增大,挖掘性能高、稳定性好的葡萄糖氧化酶成为研究热点。为提高GOD在毕赤酵母中的表达,本文以黑曲霉(Aspergillus niger)为出发菌株,首先分别构建了重组表达载体pMD-AOX-GOD和多拷贝表达载体pPICZαA-GOD_((n(n=1-5))),通过高浓度G418、博来霉素抗性筛选平板,得到1株高表达GOD的重组菌。在此基础上分别构建了HAC1、PDI两种分子伴侣1~4拷贝共表达重组菌株,结果表明,共表达1拷贝PDI的重组菌株GOD酶活最高,比出发菌株提高123%。RT-qPCR检测结果符合预期。Glucose oxidase is widely used and in great demand in the market,and mining glucose oxidase with high performance and good stability has become a research hotspot.In order to improve the gene expression efficiency of GOD in Pichia pastoris,the recombinant expression vector pMD-AOX-GOD and the multi-copy expression vector pPICZαA-GOD_(n(n=1-5))are constructed with Aspergillus niger as the starting strain.One recombinant strain with high expression of GOD is obtained by G418 and zeocin resistance screening.On this basis,1~4 copies of HAC1 and PDI molecular chaperones are constructed to express recombinant strains respectively.The results show that the recombinant strain with 1 copy of PDI has the highest GOD activity,which is 123% higher than that of the original strain.Besides,the results of RT-qPCR are in line with expectations.

关 键 词：葡萄糖氧化酶 毕赤酵母 多拷贝 组合优化 分子伴侣 

分 类 号：Q815[生物学—生物工程]