| 李 婕,余 磊,赵晓雅,郑桂兰,王洪钟.简单节杆菌3-甾酮-△1 -脱氢酶基因的克隆表达及定点突变研究[J].现代生物医学进展英文版,2017,17(25):4801-4806. |
| 简单节杆菌3-甾酮-△1 -脱氢酶基因的克隆表达及定点突变研究 |
| The Clone, Expression and Site-directed Mutagenesis of 3-Ketosteroid-Delta(1)-Dehydrogenase from Arthrobacter Simplex |
| Received:March 05, 2017 Revised:March 29, 2017 |
| DOI：10.13241/j.cnki.pmb.2017.25.001 |
| 中文关键词: 3-甾酮-△1-脱氢酶 简单节杆菌 同源建模 定点突变 |
| 英文关键词: 3-Ketosteroid-delta(1)-dehydrogenase Arthrobacter simplex Homologous modeling Site-directed mutagenesis |
| 基金项目:国家自然科学基金项目（21476124） |
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| 中文摘要: |
| 摘要 目的：将来源于简单节杆菌的3-甾酮-△1-脱氢酶（3-ketosteroid-Delta(1)-dehydrogenase，KSDD）在大肠杆菌中进行表达，获得具有活性的脱氢酶；利用计算机预测KSDD的三级结构，并通过定点突变确定酶的关键位点以期优化脱氢酶的活性及性质。方法：克隆简单节杆菌编码KSDD的基因ksdd构建原核表达载体，以Escherichia coli BL21(DE3)为表达宿主构建重组菌并诱导表达，HPLC法检测重组酶催化4-AD脱氢的转化率；通过SWISS-MODEL同源建模分析KSDD结构，对预测的催化关键位点氨基酸残基进行定点突变并研究突变后重组酶的活性变化。结果：成功构建了表达脱氢酶KSDD的重组菌E. coli pET-22-ksdd，21 ℃下诱导表达后，重组酶对4-AD的转化率为27%；通过SWISS-MODEL同源建模预测出脱氢酶结构并对4个关键位点进行定点突变设计，获得突变子Y120R、Y320L、Y488F和G492Y。突变子Y120R和Y488F失活，证明其为酶的活性位点；突变子Y320L的转化率与野生型基本一致，但37 ℃反应条件下稳定性有所提高；突变子G492Y对4-AD的转化率是野生型的1.2倍，37 ℃条件下稳定性有所提高，是突变后氨基酸位点疏水性增加和周围静电作用改变所导致。结论：目前对简单节杆菌3-甾酮-△1-脱氢酶结构分析及催化机理相关的研究较少，本研究验证了KSDD的活性位点，优化了酶的稳定性，为进一步对酶的性质进行定向改造打下了基础。 |
| 英文摘要: |
| ABSTRACT Objective: In this study, a prokaryotic expression of the 3-ketosteroid-Delta(1)-dehydrogenase (KSDD) which came from Arthrobacter simplex was built. Moreover, in order to investigate the catalytic mechanism of KSDD and improve its stability, the structure of KSDD was predicted by computer and the critical sites were confirmed by site-directed mutations. Methods: The recombi- nant plasmid was constructed by eukaryotic expression vector pET-22b and the recombinant strain was constructed and expressed in Es- cherichia coli BL21(DE3). High-performance liquid chromatography was used to determine the transformation rate of 4-AD to ADD. The KSDD structure and key sites were predicted by SWISS-MODEL. Site-directed mutations for the amino acid residues of key sites were constructed and activities of the mutations were detected. Results: The recombinant strain E. coli pET-22-ksdd was successfully constructed. It was induced to express the dehydrogenase by IPTG and the conversion rate of 4-AD to ADD was 27% at 21℃. The struc- ture of 3-ketosteroid-Delta(1)-dehydrogenase and the four key sites was analyzed by SWISS-MODEL. Four mutants, Y120R, Y320L, Y488F and G492Y were constructed. Mutants Y120R and Y488F were inactivated, so they were proved to be the key active sites of KSDD. The conversion rate of mutant Y320L was consistent with that of wild type, but the stability at 37 ℃ was improved. The conversion rate of mutant G492Y was 1.2 times of the wild type and the stability has been improved at 37 ℃. Conclusion: At present, there are few studies about the structure and catalytic mechanism of dehydrogenase. The active sites of the enzyme were verified by this study, which laid the foundation for the further study of the properties of the enzyme KSDD. |
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