产黄青霉苯乙酰CoA连接酶基因phl的克隆表达及其纯化酶的性质分析  

Cloning and expression of phenylacetyl-CoA ligase gene (phl) from penicillium chrysogenum in prokaryotic and properties of purified enzyme

作　　者：崔超 杨金晓[2] 张春晓[1] 王丽丽[1] 陈小波[1] 冯惠勇[1] 

机构地区：[1]河北科技大学生物科学与工程学院,河北石家庄050018 [2]河北省水产研究所,河北秦皇岛066002

出　　处：《河北工业科技》2011年第2期94-99,共6页Hebei Journal of Industrial Science and Technology

摘　　要：苯乙酸与CoA反应,生成苯乙酰CoA,利用RT-PCR方法成功地从产黄青霉中扩增得到1737 bp不含内含子的phl基因,并将其克隆到pET30a原核表达载体,在大肠杆菌BL21（DE3）中低温诱导表达。经SDS-PAGE检测分析,获得与预期大小（62.1 kDa）一致的蛋白表达特异条带。通过Ni-NTA亲和层析柱纯化了重组酶,经检测苯乙酸的吸收,表明纯化酶对苯乙酸和CoA有明显的催化活性,酶活为1 680 U/mL。研究表明,纯化酶PCL最适宜温度为30℃,有一定的热稳定性,最适宜pH值为8.0,pH值为7.0~8.5时酶比较稳定。The phenylacetic acid（PAA） and CoA to produce phenylacetyl-CoA.The 1 737 bp intronless phl was amplified from penicillium chrysogenum by RT-PCR.Using pET30a as vector and escherichia coli BL21（DE3） as host,phl was cloned and overexpressed in low culture temperature.Through analysis and detecting by SDS-PAGE,the protein bands of about 62.1 kDa expected molecular mass of the phl was visualized clearly.The recombinant protein was purified by Ni-NTA affinity chromatography,and possessed high activities with PAA and CoA as the substrates.The activity of PCL was 1 680 U/mL.The study shows that the optimum temperature for enzyme PCL is 30 ℃,where a certain degree of thermal stability is maintained,and the optimum pH of PCL is 8.0,while the range for stable enzyme is from pH 7.0 to pH 8.5.

关 键 词：苯乙酰CoA连接酶基因(phl) 基因克隆 表达 纯化 产黄青霉 RT-PCR 

分 类 号：Q786[生物学—分子生物学]