黑曲霉葡萄糖氧化酶基因在毕赤酵母中的表达及产酶条件的优化  

Expression of Glucose Oxidase Gene from Aspergillus niger in Pichia pastoris and Optimization of Enzyme Production Conditions

作　　者：廖兆民 蔡俊 林建国 杜馨 王常高 LIAO Zhao-min;CAI Jun;LIN Jian-guo;DU Xin;WANG Chang-gao(School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068;Hubei Key Laboratory of Industrial Microbiology,School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068;Key Laboratory of Fermentation Engineering(Ministry of Education),School of Food and Biological Engineering,Hubei University of Technology,Wuhan 430068)

机构地区：[1]湖北工业大学生物工程与食品学院,武汉430068 [2]湖北工业大学生物工程与食品学院工业微生物湖北省重点实验室,武汉430068 [3]湖北工业大学生物工程与食品学院发酵工程教育部重点实验室,武汉430068

出　　处：《生物技术通报》2021年第6期97-107,共11页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31401807)。

摘　　要：从黑曲霉中克隆葡萄糖氧化酶(glucose oxidase,E.C.1.1.3.4,GOD)基因,使其在毕赤酵母GS115中高效表达,为葡萄糖氧化酶的工业化生产提供理论依据。通过设计简并引物,扩增GOD,将该基因连接到质粒pPICZαA上并在毕赤酵母中表达,依据摇瓶水平优化最佳诱导产酶条件,在30 L发酵罐中进行发酵放大试验,共表达His4,采用DO-STAT的甲醇流加策略进行高密度发酵。结果表明,重组毕赤酵母GS115/pPICZαA-GOD在摇瓶水平经0.5%甲醇诱导96 h,发酵液上清GOD酶活达到32.25 U/mL。经优化得到最佳发酵条件为:在30℃、pH 6.0、250 r/min条件下,发酵液体积1%的甲醇诱导96 h,GOD酶活达到50.1 U/mL,相比初始发酵,酶活提高了55.3%。经30 L发酵罐放大试验,GOD酶活达到307.52 U/mL,提高了5.1倍。共表达His4使细胞湿重提高了72.5%,GOD酶活达到了461 U/mL,相比初始重组酵母提高了50.2%。SDS-PAGE分析发现GOD分子量约90 kD。重组GOD能在毕赤酵母中高效表达,且表达产物纯度高,杂蛋白少,有利于纯化。Glucose oxidase gene(GOD)was cloned from Aspergillus niger and highly expressed in Pichia pastoris GS115.The GOD gene was amplified by designing degenerate primers,and the gene was connected to the plasmid pPICZαA and expressed in P.pastoris.The optimized induction conditions for enzyme production were optimized according to the shaking flask level.The fermentation scale-up experiment was carried out in a 30 L bioreactor.The His4 was co-expressed and the methanol feed strategy of DO-STAT was used for highdensity fermentation.The results showed that the GOD enzyme activity in the supernatant of the fermentation broth reached 32.25 U/mL after the recombinant P.pastoris GS115/pPICZ-GOD was induced by 0.5%methanol at the shake flask level for 96 h.The optimal fermentation conditions were obtained as follows:under the condition of 30℃,pH 6.0 and 250 r/min,the activity of GOD enzyme induced by 1.0%methanol for 96 h reached 50.1 U/mL,which was 55.3%higher than that of the initial fermentation.Through the scale-up experiment in a 30 L bioreactor,the GOD enzyme activity reached 307.52 U/mL,which increased by 5.1 times.Co-expression of the His4 increased the wet cell weight by 72.5%and the GOD enzyme activity reached 461 U/mL,which was an increase of 50.2%compared to the original recombinant yeast.SDS-PAGE analysis showed that the molecular weight of GOD was about 90 kD.Recombinant GOD can be highly expressed in P.pastoris with high purity and few miscellaneous proteins,which is beneficial to purification.

关 键 词：毕赤酵母 葡萄糖氧化酶 异源表达 产酶优化 

分 类 号：TQ925.4[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]