耐辐射奇球菌类胡萝卜素C3',4'-脱氢酶底物特异性

Substrate specificity of carotenoid 3',4'-desaturase from Deinococcus radiodurans

为了鉴定耐辐射奇球菌类胡萝卜素C3',4'-脱氢酶(DR2250)催化底物的特异性,利用PCR方法将dr2250基因的克隆到载体pUC19上,形成了重组载体pUC-CRTD。利用质粒共转化方法将不同组合的质粒pACCRT-EBIEu、pRK-CRTC和pUC-CRTD转化到大肠杆菌中,筛选阳性克隆并提取其色素进行产物分析。结果表明,DR2250修饰的底物具有选择性,它不能以未经过羟基化修饰的直链类胡萝卜素为底物,而能在C1(1')羟基修饰的羟基化类胡萝卜素的基础上进行C3',4'-脱氢反应。

To examine the substrate specificity of carotenoid 3＇,4＇-desaturase （DR2250） from Deinococcus radiodurans, we amplified the dr2250 gene by using PCR methods. The PCR products were digested by Hind III-BamH I and ligated into the vector pUC19, yielding recombinant vector pUC-CRTD. We analyzed the carotenoids of E. coli transformants containing pACCRT-EBIEu and （or） pRK-CRTC and （or） pUC-CRTD. Our results demonstrated that DR2250 had substrate specificity on the carotenoids with hydroxyl group at C1 （1＇）.