嗜热棉毛菌木糖苷酶Xyl43基因优化及其在毕赤酵母中高效表达  

Codon optimization of Thermomyces lanuginosus β-xylosidase Xyl43 and its overexpression in Pichia pastoris

作　　者：张云博[1,2] 白仁惠 王春迪[1] 张斐洋 岳春[4] 张震宇[1] 孙付保[1] 

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122 [2]南京林业大学江苏省生物质绿色燃料与化学品重点实验室,江苏南京210037 [3]河南天冠企业集团有限公司车用生物燃料技术国家重点实验室,河南南阳473000 [4]南阳理工学院生物与化学工程学院,河南南阳473004

出　　处：《微生物学通报》2017年第1期9-19,共11页Microbiology China

基　　金：国家自然科学基金项目(No.21176106);车用生物燃料技术国家重点实验室开放基金项目(No.KFKT2013010);中国博士后科学基金项目(No.2015M571666);江苏省生物质绿色燃料与化学品重点实验室资助项目(No.JSBGFC14006);河南省科技开放合作项目(No.162106000007)~~

摘　　要：【目的】通过外源表达手段构建重组毕赤酵母实现木糖苷酶的高效表达。【方法】基于毕赤酵母密码子偏好性优化嗜热棉毛菌β-木糖苷酶(Xyl43)基因密码子,将其导入毕赤酵母GS115中实现分泌表达,并对重组木糖苷酶酶学性质进行分析。通过单因素实验优化高产菌株的摇瓶发酵条件,并在5 L发酵罐中进行扩大培养。【结果】Xyl43基因优化后的序列中222个碱基发生改变,G+C含量由52.8%降低到44.6%,序列一致性为78.17%;将构建的表达载体p PIC9K-Opt Xyl43电击转入毕赤酵母中,利用平板初筛和摇瓶复筛获得一株高效表达重组菌(命名为P.pastoris GS115-Xyl43);其所产重组木糖苷酶大小为51.5 k D,动力学参数Km为2.93 mmol/L、Vmax为157.9μmol/(min·mg),最适反应温度55°C,最适p H 7.0,在p H 6.0-9.5条件下具有良好的稳定性;摇瓶优化结果表明:培养基初始p H 6.0、甲醇补加浓度1.0%、培养温度28°C、摇床转速250 r/min为最佳产酶条件,在此条件下发酵144 h胞外酶活达到42 U/m L(蛋白含量0.54 g/L);5 L发酵罐放大培养,发酵156 h(甲醇诱导96 h),木糖苷酶酶活为222.2 U/m L,蛋白含量2.36 g/L,较摇瓶提高了4.3倍。【结论】木糖苷酶在毕赤酵母中实现了高效表达,具有较好的工业化应用前景。[Objective] We overexpressed β-xylosidase with heterologous expression by constructing a recombinant Pichia pastoris. [Methods] According to the codon usage frequency of highly expressed genes in P. pastoris, the Thermomyces lanuginosus β-xylosidase（Xyl43） gene was optimized and expressed in P. pastoris GS115, followed by characterization of Xyl43. Then, single factor experiments were used to optimize the fermentation conditions, in a 5-L fermenter. [Results] The optimized Xyl43 gene changed greatly with 78.17% of the sequence homology, and the GC content reduced from 52.8% to 44.6% with 222 bases substituted. The optimized gene was transplanted with an expression vector p PIC9K-Opt Xyl43 into P. pastoris GS115 to produce transformants. Then, a high xylosidase activity secreting recombinant P. pastoris GS115-Xyl43 was selected from the transformants on G-418 resistant plates, followed by shake flask cultivation. Basic enzyme properties of the recombinant xylosidase was analyzed as below： the protein molecular weight 51.5 k D, the optimal reaction temperature 55 °C, the optimum p H 7.0, and kinetic parameters Km=2.93 mmol/L and Vmax=157.9 μmol/（min·mg）. β-xylosidase fermentation was optimized in shake flask as follows： methanol supply 1%（each 24 h）, shaking speed 250 r/min, incubation time 144 h, incubation temperature 28 °C and initial p H 6.0. Under the optimal condition, the extracellular enzyme activity reached 42 U/m L with a protein content of 0.54 g/L. Further, in a 5-L fermenter, P. pastoris GS115-Xyl43 achieved 222.2 U/m L of xylosidase at 156 h（methanol induction for 96 h）, with protein concentration at 2.36 g/L, which was 4.3 fold more than that in the shake-flask fermentation. [Conclusion] β-xylosidase can be expressed in P. pastoris GS115 with high level production and can be used as a candidate in various industrial applications.

关 键 词：嗜热棉毛菌 Β-木糖苷酶 密码子优化 发酵优化 毕赤酵母 

分 类 号：Q78[生物学—分子生物学] Q55