芽孢杆菌环氧化物水解酶的生信分析、异源表达及催化活性研究  

The Bioinformatic Analysis,Heterologous Expression and Catalytic Activity of Epoxide Hydrolase from Bacillus sp.

作　　者：吴燕红 陈妮 徐婧然 郝昳昕 彭仁[1] WU Yanhong;CHEN Ni;XU Jinran;HAO Yixin;PENG Ren(College of Life Science,Jiangxi Normal University,Nanchang Jiangxi 330022,China)

机构地区：[1]江西师范大学生命科学学院,江西南昌330022

出　　处：《江西师范大学学报（自然科学版）》2024年第3期240-246,共7页Journal of Jiangxi Normal University(Natural Science Edition)

基　　金：国家自然科学基金(31560018)资助项目.

摘　　要：环氧化物水解酶是一类能催化环氧化物水解生成相应邻位二醇的酶,环氧化物水解酶不需辅酶与金属离子,具有底物专一性,在药物中间体的制备方面具有广泛的利用价值.在Bacillus sp.V3-13中的环氧化物水解酶含有383个氨基酸残基,理论分子量为44119.61 Da,理论等电点为5.33,不稳定系数为39.25,总平均亲水性为-0.493,属于稳定的亲水蛋白.该酶无信号肽、二硫键、跨膜区,含有EHN(pfam06441)和MenH(COG0596)保守结构域.该文利用pET-28a(+)作为载体、E.coli BL21(DE3)作为宿主,对该酶在30℃、0.4 mmol·L^(-1) IPTG、诱导10 h的条件下成功进行了表达.经镍柱亲和层析纯化后,获得了电泳纯的重组环氧化物水解酶.重组环氧化物水解酶在20 min内对苄基缩水甘油醚、3-(1-萘氧基)-1,2-环氧丙烷和氧化苯乙烯的水解率分别为14.40%、11.05%和8.17%.Epoxide hydrolases are a class of enzymes that can catalyze the hydrolysis of epoxides to produce corresponding ortho-diols.The reactions catalyzed by epoxide hydrolases do not require coenzymes and metal ions with substrate specificity.It has a wide application values in the preparation of pharmaceutical intermediates.Epoxide hydrolases from Bacillus sp.V3-13 contains 383 amino residues with a theoretical molecular weight of 44119.61 Da and an isoelectric point of 5.33.It also has an instability index of 39.25 and a grand average hydropathicity of-0.493.Therefore,it is stable and hydrophilic.The enzyme has no signal peptide,disulfide bonds and two conservative domains named EHN(pfam06441)and MenH(COG0596).Using pET-28a(+)as vector and E.coli BL21(DE3)as host,the enzyme is successfully expressed at 30℃,0.4 mmol·L^(-1) IPTG and the induction time of 10 h.Recombiant EH with electrophoretical purity is obtained after purification by nickel column affinity chromato-graphy.The hydrolysis rates ofbenzyl glycidyl ether,3-(1-naphthoxy)-1,2-propylene oxide and styrene oxide catalyzed by the recombinant EH within 20 min is 14.40%,11.05%and 8.17%,respectively.

关 键 词：环氧化物水解酶 表达与纯化 环氧化物 生信分析 

分 类 号：Q89[生物学]