一个新的L-半胱亚磺酸脱羧酶基因的分析和突变

发布时间：2018-05-31 16:24

本文选题：牛磺酸 + L-半胱亚磺酸脱羧酶　；参考：《微生物学报》2017年08期

【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。
[Abstract]:[objective] to complete the identification of a new L- cysteinesulfonic acid decarboxylase gene undec1A from a macro genomic library of alkaline contaminated soil. The enzyme properties were studied and modified by irrational design. [methods] Recombinant expression plasmid containing undec1A gene was constructed by using p ETBlue-2 as expression vector and transformed into host cell E.coli Tuner(DE3)p? The enzyme protein was isolated and purified by nickel affinity chromatography, and its biochemical characteristics were studied. Continuous error-prone PCR technique was used to modify Undec1A protein. [results] Bioinformatics analysis revealed that Undec1A protein had similar coenzyme binding sites and substrate recognition catalytic motifs with known L- cysteinesulfonic acid decarboxylase. The results of molecular docking showed that the amino acid residues Val237Asp239Asp266Ala268 and Lys298 determined the recognition and binding catalysis with the substrate L- cysteinesulfonic acid (L- cysteinesulfonic acid). Using L- cysteine sulfonic acid as substrate, the optimal action of recombinant Undec1A protein was 7.0 and the optimum temperature was 35 掳C, and the molecular dynamics constant K _ m was 1.557 卤0.015 mmol-1 路min ~ (-1) (n = 49.07 卤3.19) 渭 mol/(L 路min ~ (-1) 路min ~ (-1) ~ (-1) 路min ~ (-1) ~ (-1) ~ (-1) 路min ~ (-1) ~ (-1) 路min ~ (-1). The molecular transformation of parental enzyme was carried out by continuous error-prone PCR, and a mutant enzyme Undec1A-1180 with higher enzyme activity was isolated and screened. Under the optimized conditions, the specific activity of Undec1A-1180 was 5.62 times higher than that of the parent enzyme. [conclusion] this study provides a theoretical reference for the construction of taurine biosynthesis process, so it has important practical significance.
【作者单位】：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;
【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)~~
【分类号】：Q55;Q78

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