[关键词]
[摘要]
肌酐酶(Creatininase)是肌酐酶法检测的一个关键酶,将恶臭假单胞菌(Pseudomonas putida)肌酐酶基因(Cre)克隆至原核表达载体pET-28a(+),通过IPTG的诱导,实现了肌酐酶Cre在大肠杆菌(Escherichia coli)BL21(DE3)的可溶性表达。表达产物经60 ℃加热处理、Ni-NTA亲和层析和Sephadex G-200分子筛层析,分离纯化出重组肌酐酶,回收率为29.3%,其比酶活达到489.17 U/mg,是已有文献中报道中的最高水平。进一步进行酶学特性分析,结果表明其最适反应温度为60 ℃,50 ℃以下可以稳定保存,具有良好的热稳定性;最适pH值为7.0,在pH 7.0~8.0条件下比较稳定;Cu2+会抑制肌酐酶的活性;Mn2+、Zn2+和Co2+对肌酐酶有明显的激活作用;EDTA、SDS、Tween-20、Tween-80和Trinton-100几乎对酶活力没影响;NaN3不影响酶的活力。以肌酸为底物时,酶动力学常数Km值为47.38 mmol/L。本研究在大肠杆菌系统成功实现肌酐酶的可溶性表达,进行分离纯化并测定其酶学性质,为肌酐酶的表达和潜在工业化应用提供理论基础。
[Key word]
[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.
[中图分类号]
[基金项目]
国家自然科学青年基金资助项目(31400062);广州市创新创业领军人才百人计划项目(LCY201322);中央高校基本科研业务费项目(2017MS103)