现代食品科技2017，Vol.33Issue(9)：77-82,6.DOI:10.13982/j.mfst.1673-9078.2017.9.011
恶臭假单胞杆菌肌酐酶在大肠杆菌中的表达及其酶学特性分析
Expression of Creatininase from Pseudomonas putida in Escherichia coli and Its Enzymatic Characteristics Analysis
摘要
Abstract
Creatininase is the key enzyme used for creatinine enzymatic detection.The Cre gene from Pseudomonas putida was cloned into the prokaryotic expression vector pET-28a (+) and expressed in the soluble form by Escherichia coli BL21 (DE3) after induction of isopropyl β-D-1-thiogalactopyranoside.The expressed product was purified and isolated using heat treatment at 60℃,nickel-nitrilotriacetic acid affinity chromatography,and Sephadex G-200 gel filtration to yield recombinant creatininase.The recovery rate was 29.3％,and the specific activity was 489.17 U/mg,which was the highest level reported to date.The enzymatic characteristics of Cre were further analyzed,and the results showed that the optimum temperature was 60 ℃.Additionally,the recombinant Cre showed good thermal stability and could be stably stored at less than 50 ℃;the optimum pH value was 7.0,and the enzyme was relatively stable at pH 7.0～8.0.The activity of Cre could be inhibited by copper (Ⅱ),but significantly increased by manganese (Ⅱ),zinc (Ⅱ),and cobalt (Ⅱ).(Ethylenedinitrilo) tetraacetic acid(EDTA),sodium dodecyl sulfate(SDS),Tween-20,Tween-80,and Triton-100 had almost no effect on Cre activity,and sodium azide did not have any effect on Cre activity.When creatine was used as a substrate,the enzyme kinetic constant Km value of Cre was 47.38 mM.In summary,the expression of the soluble form of creatininase was successfully achieved in an E.coli system,and purification and analysis of the enzymatic characteristics of the creatininase were performed,establishing a theoretical foundation for the expression and potential industrial applications of creatininase.关键词
恶臭假单胞杆菌/肌酐酶/大肠杆菌/酶学特性Key words
Pseudomonas putida/creatininase/Escherichia coli/enzymatic characteristics引用本文复制引用
侯赣生,林影,梁书利..恶臭假单胞杆菌肌酐酶在大肠杆菌中的表达及其酶学特性分析[J].现代食品科技,2017,33(9):77-82,6.基金项目
国家自然科学青年基金资助项目(31400062) （31400062）
广州市创新创业领军人才百人计划项目(LCY201322) （LCY201322）
中央高校基本科研业务费项目(2017MS103) （2017MS103）
