摘要
PCR扩增了苜蓿根瘤菌乙酰辅酶A合成酶编码基因 (acsA1) ,克隆到连接 -非依赖型载体pET30LIC ;在E .coliBL2 1(DE3)pLysS中得到了有效表达 ,表达需IPTG的诱导 ,诱导 3h达到酶活高峰 .采用His·Bind柱层析对ACS进行了纯化 ,纯化的酶蛋白经SDS-PAGE呈单一浓带 ,分子量约 72 0 0 0 ,具较高的酶活 ,是无细胞提取液的 12 .7倍 .酶动力学分析显示 ,Vmax、Km分别为 (4 13.6± 11.7)mmolL-1和 (5 .8± 0 .6 )mmolL-1.图 4表 2参
s Acetyl-CoA synthetase encoding gene acsA1 of Sinorhizobium meliloti was amplified by PCR, then cloned into ligation-independent vector pET30 LIC, and over-expressed in E. coli BL21(DE3)pLysS. The expression of acsA1 needed IPTG induction and the ACS specific activity reached the highest point when the culture induced with IPTG for 3 hours. ACS was successfully purified through His·Bind chromatography and the purified enzyme protein gave a single band (M r=72 000 Da) on SDS-PAGE gel. The activity of purified ACS was 12.7 times of cell-free extract activity. Kinetic analysis indicated that the V max and K m of ACS was (413.6±11.7) mmol L -1 and (5.8±0.6) mmol L -1, respectively. Fig 4, Tab 2, Ref 8
出处
《应用与环境生物学报》
CAS
CSCD
2004年第1期113-115,共3页
Chinese Journal of Applied and Environmental Biology
