新颖顺式环氧琥珀酸水解酶及其催化机制和热稳定性研究

cis-Epoxysuccinate hydrolase can transform cis-epoxysuccinate to tartaric acid and the stereospecificity of the product is determined by the enzyme. We currently isolate a novel strain from soil which can transform cis-epoxysuccinate to L(+)-tartaric acid and study on its cis-epoxysuccinate hydrolase from primary levels. The enzyme owns higher specific activity and higher temperature stability. Howerver, its molecular weight and pI show significant differences from other reported cis-epoxysuccinate hydrolases, indicating that it is a novel cis-epoxysuccinate hydrolase. This study is to obtain its cDNA sequence through purification of enzyme, N terminal amino acid sequencing, construction of gene library and Southern hybridization，to probe its catalytic mechanism through multiple sequence alignment, homology modelling, molecular docking, site-directed mutagenesis, isotopic labeling experiment and LC/MS analysis of the proteolytic digest, and to propose its thermal stability through topology structure, secondary structure, disulfide bond, amino acid composition, ionic bond, hydrogen bond, cation-π bond, helix propensity and helix stability. This study is not only has theoretical significance for basic enzymology, but also provides application value.

顺式环氧琥珀酸水解酶能将顺式环氧琥珀酸转化为酒石酸，产物酒石酸的立体特异性取决于酶学性质。本课题组最近从土壤中分离到一株能转化顺式环氧琥珀酸为L(+)-酒石酸的新菌株，并对该菌的顺式环氧琥珀酸水解酶进行了初步研究。该酶具有比活力高和热稳定性好的优势，但表观分子量、等电点等数据表明，它与现有报道的顺式环氧琥珀酸水解酶有显著差别，是一种新颖的顺式环氧琥珀酸水解酶。本研究拟通过分离纯化、N端测序、基因文库构建和Southern杂交等获得该酶的基因序列，然后通过多序列比对、同源建模、分子对接、定点突变、同位素标记、多肽片段液质联用等揭示其催化机制，最后通过拓扑结构、二级结构、二硫键、氨基酸组成、离子键、氢键、cation-π键、螺旋倾向性和螺旋稳定性探讨其热稳定性机制。本研究不仅对基础酶学具有重要的理论意义，还具有重要的应用价值。

顺式环氧琥珀酸水解酶（CESH）能将顺式环氧琥珀酸转化为酒石酸，产物酒石酸的立体特异性取决于酶学性质。本课题组从土壤中分离到一株能转化顺式环氧琥珀酸为L(+)-酒石酸的新菌株，经形态、生理生化性质和16S rDNA序列鉴定为双头菌属，并将菌株命名为Labrys sp. BK-8。野生菌经培养条件优化后，10L罐中细胞酶活达3597 U/g。通过Phenyl-Sepharose疏水层析、DEAE- Sepharose离子层析、Sephadex G100分子筛层析法纯化得到目的蛋白。经基因组分析后得到Labrys sp. BK-8 CESH酶的基因序列，由825 bp核苷酸编码的274个氨基酸组成，分子量约30.2 kDa，pI值约为8.4。Labrys sp. BK-8 CESH酶的最适pH8.5，最适温度55℃，pH稳定范围3.5-10.0，小于45℃时温度稳定性好。多序列比对发现，Labrys sp. BK-8 CESH酶属于卤酸脱卤酶超家族成员，与已报道的红球菌的CESH酶序列相似性只有37.5%。同源建模结果显示，CESH酶具有典型的α/β-桶状折叠结构。D48、T52、R85、N165、K195、Y201、A219、H221、D224这9个氨基酸保守位点进行定点突变，酶活显著降低，不足野生菌酶活的2%，说明它们是重要的催化位点。同位素标记的单转化和多转化实验证明，该酶是通过酶-底物酯中间体的两步法催化进行，并的推测其D48-H221-D224的催化三联体反应机制。通过二级结构预测、二硫键测定、氨基酸组成分析，比较该CESH酶与已报道的CESH酶的差别，发现该酶中带电荷氨基酸（Arg、Lys、His、Asp、Glu）和芳香族氨基酸（Phe、Tyr和Trp）的含量较高，它们在离子键和cation-π键形成中有重要作用，是蛋白热稳定的原因之一。定点突变实验表明，Phe10位点在酶的活性和热稳定性中有重要作用，并获得了高活性和高热稳定性的突变菌株F10Q，其比活力较野生性CESH酶提高230%，具有良好的工业化应用前景。