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 coil 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.%笨乙酸与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不舍内含子的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(62.1kDa) -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.
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