产黄青霉谷胱甘肽S-转移酶基因PcgstA的克隆与鉴定(英文)  

Molecular Cloning and Characterization of a Glutathione S-transferase Gene Repressed by Phenylacetic Acid From Penicillium chrysogenum

作　　者：王富强[1] 郑桂珍[2] 赵颖[2] 任志红[2] 贾茜[2] 贺建功[2] 于军[1] 

机构地区：[1]中国科学院北京基因组研究所 [2]华北制药集团新药研究开发有限责任公司,石家庄050015

出　　处：《生物化学与生物物理进展》2006年第12期1223-1229,1230,共8页Progress In Biochemistry and Biophysics

基　　金：国家科技攻关计划资助项目(2002BA7112A16)~~

摘　　要：从青霉素工业生产菌产黄青霉(Penicilliumchrysogenum)中首次克隆了一个谷胱甘肽S-转移酶(GST)基因,定名为PcgstA.该基因的开放阅读框长840bp,含有两个内含子,编码一个238氨基酸残基的蛋白质.其推断的氨基酸序列与一些已经鉴定的丝状真菌GST具有50%左右的序列一致性.PcgstA的完整编码区经RT-PCR扩增、验证,插入原核表达载体pET11a,转化大肠杆菌BL21(DE3)-RP菌株,表达得到重组PcGSTA蛋白.酶活测定证实,重组PcGSTA具有GST活性,其对底物CDNB(1-chloro-2,4-dinitrobenzene)的比活为(0.159±0.031)μmol/(min·mg).利用TaqMan探针法,对PcgstA的表达情况进行了比较.结果表明,在添加了侧链前体苯乙酸的青霉素生产培养基中,PcgstA的表达水平和在不含苯乙酸培养基中的表达相比明显下调,显示了该基因与苯乙酸代谢的关系.Glutathione S-transferase （GST） gene, PcgstA was cloned from the penicillin producing strain Penicillium chrysogenura, which is important for understanding the industrial fermentation process. PcgstA gene has an open-reading-frame of 840 bp in length, which is interrupted by two introns. The deduced amino acid sequence shows about 50% identity to several characterized filamentous fungi GSTs. The recombinant PcGSTA in Escherichia coli were overexpressed and purified. Enzymatic assays showed that the recombinant PcGSTA had a specific activity with 1-chloro-2, 4-dinitrobenzene of （0.159±0.031） μmol/（min·mg）. It was found that the expression level of PcgstA in the penicillin producing medium supplemented with phenylacetic acid, the side chain precursor of penicillin G, was significant down regulated than that in medium without phenylacetic acid. This result suggested that PcGST may be related to phenylacetic acid metabolism in the penicillin producing strain.

关 键 词：产黄青霉 谷胱甘肽S-转移酶 苯乙酸 

分 类 号：Q939[生物学—微生物学]