新型深海来源蛋白酯酶E4的纯化、结晶和X射线衍射分析

Purification,Crystallization and X-ray Diffraction Analysis of a Novel Marine Esterase E4

酯酶和脂酶是水解短链酯类和长链甘油三酯的两类水解酶,在制药和食品工业酯(脂)键的合成和分解过程中发挥着关键的作用.我们前期已鉴定深海细菌Croceicoccus marinus E4A9T来源的新型蛋白酯酶E4,该酶属于酯酶SGNH亚家族,具有潜在的水解溶血磷脂键的作用,但其催化机制尚不清楚,因此我们对蛋白E4进行了纯化、结晶和初步的X射线衍射分析.E4在大肠杆菌中高效表达,动态光散射(DLS)分析显示其主要以四体的形态均匀分布在溶液中.我们对E4进行了晶体初筛和优化,最终优化培养出棒状晶体且衍射分辨率达2.2.X射线衍射结果表明E4属于P43212空间群,晶胞参数为a=88.863,b=88.863,c=318.914,α=90°,β=90°,γ=90°.此外,酶学活力测定结果表明,它能够水解具有不同酰基链长度的酯类(C2～C10),尤其倾向于水解己酸酯(C6)和辛酸酯(C8).

Esterases and lipases hydrolyze short-chain esters and long-chain triglycerides respectively,and play key roles in the synthesis and decomposition of ester bonds in pharmaceutical and food industries.We previously identified an esterase E4 from a marine bacteriumCroceicoccus marinus E4A9 T.E4is a new member of the SGNH subfamily and may act as a catalyst in hydrolyzing lysophospholipid bonds.However,the structure and catalytic mechanism of E4 remain unknown.Here,we report the purification,crystallization and preliminary X-ray diffraction analysis of the esterase E4.The high purity E4 was obtained by in vitro expression in E.coli.Dynamic light scattering(DLS)analysis showed that E4 protein forms a tetramer and is distributed homogeneously in solution.The E4 protein was crystallized in rod shape and diffracted to a 2.2resolution.The result of X-ray diffraction analysis showed that E4 belongs to a space group P43212,with unit-cell parameters a=88.863,b=88.863,c =318.914,α=90°,β=90°,γ=90°.In addition,E4 hydrolyzed p-nitrophenyl esters with different acyl chain lengths(C2-C10)and shows preference for caproate(C6)and caprylate(C8).