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

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pETBlue-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） pLacI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pH为7.0，最适作用温度为35℃；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] 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 K_m of (1.557±0.015) mmol/L, a V_max of (49.07±3.19) μmol/(L·min), and a k_cat 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.