一种高R-选择性水解R,S-甲霜灵的酯酶基因的克隆表达及表征

Cloning, expression and characterization of a gene encoding the esterase hydrolyzing R,S-metalaxyl high R-enantioselectively

摘要:

[目的] 将一种可以高选择性水解R-甲霜灵的新脂酶基因，在大肠杆菌中进行克隆和表达，并研究重组脂酶的性质。[方法] 根据已知的目标酯酶N端10个氨基酸序列，在已测序的Albibacters sp. zjut528基因组中找到相匹配的一个酯酶基因，它全长969 bp，编码322个氨基酸，将该基因命名为RMest。通过引物扩增得到该基因的DNA片段，将它与表达载体pET-28a（+）连接后，转化大肠杆菌BL21Gold（DE3），构建重组菌，IPTG诱导表达该酯酶，并用Ni²⁺亲和层析介质进行纯化。[结果] 在重组菌RMest-pET-28a（+）-E.coli BL21 Gold（DE3）中成功表达了重组酯酶RMesterase，大小约为46 kDa。用胞内重组酶液催化水解R，S-甲霜灵，底物浓度10 g/L，反应6 h，底物转化率为49.8%，产物（甲霜灵酸）的ee_p为99.3%，对底物的对映体R-构型具有专一（选择）性。该酶最适温度和pH分别为40℃和pH 9.0。该酶的活性受到产物甲醇的抑制。通过Blast+在Uniprot KB数据库中搜寻与酯酶RMest同源的蛋白，采用邻近法构建该酶的蛋白系统发育树，结果显示它与某些Lysophospholipase、AB hydrolase-1 domain-containing protein和Esterase的同源性最高，但是与它们均存在较大的进化距离，表明该酶是一种相对独立进化的新酯酶。[结论] 在大肠杆菌中成功克隆和表达了一种新的脂酶基因RMest，重组酯酶RMesterase可以高手性选择性水解R，S-甲霜灵生成R-甲霜灵酸。

Abstract:

[Objective] A gene encoding the esterase to hydrolyze R, S-metalaxyl high enantioseclectively to R-metalaxyl acid was cloned and expressed in E. coli. The properties of the recombinant esterase were studied. [Methods] Based on known N-terminal 10 amino acid sequence of the target esterase, a candidate esterase gene matching with it was found in the sequenced Albibacters sp. zjut528 genome. It had a length of 969 bp encoding 322 amino acids and was named RMest. The RMest DNA segment was amplified by PCR, ligated with pET-28a(+), transformed into E. coli BL21Gold(DE3) to construct the recombinant strain. The recombinant esterase was expressed by induction with IPTG and purified with Ni²⁺ resin. [Results] The recombinant RMesterase was expressed successfully in RMest-pET-28a(+)-E. coli BL21 Gold (DE3). Its size was about 46 kDa in SDS-PAGE. Hydrolysis of 10 g/L of R,S-metalaxyl for 6 h catalyzed by intracellular crude enzyme from the recombinant strain, the substrate conversion rate reached 49.8% with product (metalaxyl acid) ee_p of 99.3%. The main product was R-metalaxyl acid. The optimal temperature and pH of RMestrase was 40℃ and 9.0 respectively. The activity of RMesterase was inhibited by methanol, the other product of metalaxyl hydrolysis. After aligning the amino acid sequence of RMest with other proteins in Uniprot KB database by Blast+, the neighboring-joining phylogenetic tree was constructed. It shows that the esterase RMest had a long evolution distance with homologous Lysophospholipases, AB hydrolase-1 domain-containing proteins and other esterases. That indicates that the esterase RMest was a new one that evolved relative independently. [Conclusion] The esterase gene RMest was successfully cloned and expressed in RMest-pET-28a(+)-E. coli BL21 Gold (DE3). The recombinant RMesterase could hydrolyze R, S-metalaxyl high R-enantioselectively to produce R-metalaxyl acid.