红球菌g26中C17位脱氢酶的克隆表达及生物转化

目的对红球菌g26中的短链脱氢酶基因进行克隆表达并考察其能否将甾体化合物雄甾-4-烯-3.17-二酮(androst-4-ene-3,17-dione,AD) C17位羰基还原为羟基。方法通过检索数据库获得红球菌g26中的两个短链脱氢酶基因,应用PCR、重组表达载体的构建等生物技术在大肠杆菌中进行外源表达,破碎细胞后应用SDS-PAGE对可溶性蛋白进行分析,筛选出合适的目的蛋白诱导条件,进而进行酶的体外转化反应,应用薄层色谱分析、高效液相分析和质谱法鉴定产物结构。结果与结论红球菌g26中的短链脱氢酶可以转化甾体化合物AD为睾丸酮(TS)。红球菌g26内的短链脱氢酶参与了甾体化合物C17位羰基到羟基的还原反应。

Cloning,expression and biotransformation of the dehydrogenase in rhodococcus g26 at the C position

Objective To Clone and express the short-chain dehydrogenase gene in Rhodococcus sp.g26 and examine whether it can reduce the carbonyl group of the steroidal androst-4-ene-3.17-dione(AD) C17 to a hydroxyl group.Methods Two short-chain dehydrogenase genes in Rhodococcus g26 were obtained by searching the database.Exogenous expression was carried out in E.coli by biotechnology such as PCR and recombinant expression vector construction.After shattering the cells,SDS-PAGE was used to treat soluble proteins.The analysis was carried out to select appropriate conditions for inducing the target protein,and then the reaction of the enzyme in vitro conversion was carried out,and the product structure was identified by thin layer chromatography,high performance liquid phase analysis and mass spectrometry.Results and Conclusions The short-chain dehydrogenase in Rhodococcus g26 could transform the steroidal compound AD into testosterone(TS).The short-chain dehydrogenase genes in Rhodococcus g26 are involved in the reduction of the carbonyl group to the hydroxyl group at the C17 position of the steroid compound.