Cloning and characterization of an oxiranedicarboxylate hydrolase from Labrys sp. WH-1  

双头菌WH-1环氧乙烷二酸水解酶的克隆和酶学性质的研究（英文）

作　　者：Wen-na BAO Zi-sheng LUO Shi-wang LIU Yuan-feng WU Pei-lian WEI Gong-nian XIAO Yong LIU 

机构地区：[1]College of Biosystems Engineering and Food Science,Zhejiang University,Hangzhou 310058,China [2]School of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Hangzhou 310023,China [3]Zhejiang Provincial Key Laboratory for Chemical and Biological Processing Technology of Farm Products,Hangzhou 310023,China

出　　处：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2019年第12期995-1002,共8页浙江大学学报（英文版）B辑（生物医学与生物技术）

基　　金：Project supported by the Zhejiang Provincial Natural Science Foundation of China(No.LQ19C200001);the Education Department of Zhejiang Province Scientific Research Project(No.Y201737925);the Zhejiang Provincial Key Laboratory for Chemical and Biological Processing Technology of Farm Products(No.2016KF0048);the Key Laboratory of Bioorganic Synthesis of Zhejiang Province(No.20170110),China

摘　　要：Objective:This study aimed to clone and characterize the oxiranedicarboxylate hydrolase(ORCH) from Labrys sp.WH-1.Methods:Purification by column chromatography,characterization of enzymatic properties,gene cloning by protein terminal sequencing and polymerase chain reaction(PCR),and sequence analysis by secondary structure prediction and multiple sequence alignment were performed.Results:The ORCH from Labrys sp.WH-1 was purified 26-fold with a yield of 12.7%.It is a monomer with an isoelectric point(pl) of 8.57 and molecular mass of 30.2 kDa.It was stable up to 55℃with temperature at which the activity of the enzyme decreased by 50% in 15 min(T5015) of 61℃and the half-life at 50℃(t1/2,50℃) of 51 min and was also stable from pH 4 to 10,with maximum activity at 55℃and pH 8.5.It is a metal-independent enzyme and strongly inhibited by Cu2+,Ag+,and anionic surfactants.Its kinetic parameters(Km,kcat,and kcat/Km) were 18.7 mmol/L,222.3 s-1,and 11.9 mmol/(L s),respectively.The ORCH gene,which contained an open reading frame(ORF) of 825 bp encoding 274 amino acid residues,was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain.Conclusions The catalytic efficiency and thermal stability of the ORCH from Labrys sp.WH-1 were the best among the reported ORCHs,and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.目的:克隆双头菌WH-1环氧乙烷二酸水解酶(ORCH)的基因并研究其酶学性质。创新点:首次获得双头菌ORCH的基因,且该酶催化效率高,热稳定性好。方法:柱层析纯化ORCH后,进行酶学性质研究;通过蛋白末端测序和PCR获得其基因序列;通过二级结构预测和多序列比对进行ORCH序列分析。结论:来源于双头菌WH-1的ORCH是迄今报道的催化效率和热稳定性最好的ORCH,为L(+)-2,3-二羟基丁二酸的生产提供了新的催化剂。

关 键 词：Oxiranedicarboxylate hydrolase(ORCH) L(+)-2 3-Dihydrobutanedioic acid CHARACTERIZATION CLONING 

分 类 号：Q556[生物学—生物化学]