| 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 |
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| 引用本文： | 崔 超,杨金晓,张春晓,王丽丽,陈小波,冯惠勇. 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[J]. 河北工业科技, 2011, 28(2): 94-99 |
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| 作者姓名： | 崔 超 杨金晓 张春晓 王丽丽 陈小波 冯惠勇 |
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| 作者单位： | 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 |
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| 摘 要： | 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...
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| 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 |
| 收稿时间： | 2010-11-09 |
| 修稿时间： | 2010-12-26 |
Cloning and expression of phenylacetyl-CoA ligase gene (phl) from penicillium chrysogenum in prokaryotic and properties of purified enzyme |
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| CUI Chao,YANG Jin-xiao,ZHANG Chun-xiao,WANG Li-li,CHEN Xiao-bo and FENG Hui-yong. Cloning and expression of phenylacetyl-CoA ligase gene (phl) from penicillium chrysogenum in prokaryotic and properties of purified enzyme[J]. Hebei Journal of Industrial Science & Technology, 2011, 28(2): 94-99 |
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| Authors: | CUI Chao YANG Jin-xiao ZHANG Chun-xiao WANG Li-li CHEN Xiao-bo FENG Hui-yong |
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| Affiliation: | College of Bioscience and Bioengineering, Hebei University of Science and Technology, Shijiazhuang Hebei 050018,China;Hebei Fisheries Research Institute, Qinhuangdao Hebei 066002,China;College of Bioscience and Bioengineering, Hebei University of Science and Technology, Shijiazhuang Hebei 050018,China;College of Bioscience and Bioengineering, Hebei University of Science and Technology, Shijiazhuang Hebei 050018,China;College of Bioscience and Bioengineering, Hebei University of Science and Technology, Shijiazhuang Hebei 050018,China;College of Bioscience and Bioengineering, Hebei University of Science and Technology, Shijiazhuang Hebei 050018,China |
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| Abstract: | 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. |
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| Keywords: | RT-PCR |
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