一个新的L-半胱亚磺酸脱羧酶基因的分析和突变  

Protein engineering by random mutagenesis and analysis of a metagenome-derived cysteine sulfinate decarboxylase

作　　者：邓洁[1] 吴巧芬 高华[1] 徐悦[1] 欧倩[1] 武波[1] 蒋承建[1] Jie Deng Qiaofen Wu Hua Gao Yue Xu Qian Ou Bo Wu Chengjian Jiang(State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi Province, Chin)

机构地区：[1]亚热带农业生物资源保护与利用国家重点实验室,广西大学生命科学与技术学院,广西南宁530004

出　　处：《微生物学报》2017年第8期1283-1292,共10页Acta Microbiologica Sinica

基　　金：国家自然科学基金(21262003);2017年度广西高等教育创优计划教学相关项目-优势特色专业项目(优质本科专业)~~

摘　　要：【目的】完成一个来源于碱性污染土壤宏基因组文库的新的L-半胱亚磺酸脱羧酶基因undec1A的鉴定,研究其酶学性质并利用非理性设计技术对其进行分子改造。【方法】以p ETBlue-2为表达载体构建包含undec1A基因的重组表达质粒,转化至宿主细胞E.coli Tuner(DE3)p Lac?中构建重组表达克隆,采用镍亲和层析完成了酶蛋白的分离纯化,完成其生化特征研究,利用连续易错PCR技术对Undec1A蛋白进行分子改造。【结果】生物信息学分析结果揭示Undec1A蛋白与已知的L-半胱亚磺酸脱羧酶存在类似的辅酶结合位点和底物识别催化基序等。分子对接结果显示氨基酸残基Val237、Asp239、Asp266、Ile267、Ala268和Lys298等决定了与底物分子L-半胱亚磺酸的识别和结合催化。以L-半胱亚磺酸作为底物,重组Undec1A蛋白的最适作用p H为7.0,最适作用温度为35°C;分子动力学常数K_m为(1.557±0.015)mmol/L,V_(max)为(49.07±3.19)μmol/(L·min),k_(cat)为(45.80±1.32)/min。利用连续易错PCR技术完成了亲本酶的分子改造,分离筛选到了一个酶活力更高的突变酶Undec1A-1180。在优化条件下,Undec1A-1180的比活力较亲本酶提高了约5.62倍。【结论】本研究为构建牛磺酸的生物合成工艺提供了理论参考,因而具有重要的实践意义。[Objective] The aim of this study was to identify a novel L-cysteine sulfinate decarboxylase from alkaline polluted soil metagenome and to use non-rational design method to improve the enzyme. [Methods] L-cysteine sulfinate decarboxylase gene undec1A was cloned into pETBlue-2 vector and expressed in Escherichia coli Tuner （DE3） pLacI. The recombinant Undec1A protein was purified to homogeneity. The original Undec1A protein was characterized and a mutagenesis library was constructed with sequential error-prone PCR method, then an interesting variant was identified. [Results] Multiple sequence alignment analysis showed that Undec1A protein shared the similar pyridoxal 5＇-phosphate binding sites and the substrate recognition motif with the other known L-cysteine sulfinate decarboxylases. Molecular docking results indicated that amino acid residues Val237, Asp239, Asp266, Ile267, Ala268 and Lys298 contributed to the decarboxylation of L-cysteine sulfinate acid. Recombinant Undec1A protein had an apparent Km of （1.557±0.015） mmol/L, a Vmax of （49.07±3.19） μmol/（L·min）, and a kcat of （45.80 ±1.32）/min at the optimal reaction conditions of 35℃ and pH 7.0 when using L-cysteine sulfinate acid as the substrate. Furthermore, the protein engineering approach of random mutagenesis via sequential error-prone PCR was applied on the original Undec1A protein. Compared with the original Undec1A protein, the best variant of Undec1A-1180 in the random mutagenesis library, exhibited 5.62-folds at the optimal reaction conditions of 35℃ and pH 7.0. [Conclusion] These results are the first step towards a better understanding of the properties of Undec1A protein. Protein engineering with error-prone PCR paves the way toward the metagenome-derived genes for biotechnological applications.

关 键 词：牛磺酸 L-半胱亚磺酸脱羧酶 宏基因组文库 连续易错PCR技术 突变酶 

分 类 号：Q55[生物学—生物化学]