恶臭假单胞菌丙氨酸消旋酶基因的克隆、序列分析及表达  

Cloning,sequence analysis and expression of alanine racemase gene in Pseudomonas putida

作　　者：曹芹[1] 赵智[1] 张英姿[1] 王宇[1] 丁久元[1] 

机构地区：[1]中国科学院微生物研究所

出　　处：《微生物学报》2006年第1期80-84,共5页Acta Microbiologica Sinica

摘　　要：从恶臭假单胞菌(Pseudomonas putida)200的基因组出发,用PCR方法克隆到两个独立作用的丙氨酸消旋酶基因,称之为dadX和alr。DadX编码357个氨基酸长的多肽,计算分子量为38.82kDa,alr编码409个氨基酸长的多肽,计算分子量为44.182kDa。序列分析显示,DadX的氨基酸序列与Pseudomonas putidaKT2440,铜绿假单胞菌(Pseudomonas aeruginosa),鼠伤寒沙门氏菌(Salmonella typhimurium)和大肠杆菌(Escherichia coli)的DadX比较,相似性分别为96.64%、71.99%、44.88%和47.37%。Alr的氨基酸序列与Pseudomonas putidaKT2440比较,同源性为94.38%,而与铜绿假单胞菌(P.aeruginosa)、鼠伤寒沙门氏菌(S.typhimurium)和大肠杆菌(E.coli)的Alr比较,同源性均较低,分别为22.89%、25.72%和26.44%。在P.putida200的DadX和Alr氨基酸序列中部发现有对于酶活性至关重要的保守区域,如磷酸吡哆醛(PLP)结合位点。DadX和alr在大肠杆菌中得到表达,DadX丙氨酸消旋酶只对丙氨酸有消旋作用,而Alr丙氨酸消旋酶可以作用于丙氨酸和丝氨酸两种底物,且对丝氨酸特异性更高。Alr的表达不依赖于外源启动子,说明在其结构基因上游存在启动子结构。Two distinct alanine racemase genes from Pseudomonas putida 200 were cloned and sequenced. DadX encodes a peptide of 357 amino acids with a calculated molecular weight of 38.82kDa. The putative product of alr gene is a peptide of 409 amino acids with molecular weight of 44.182kDa. A homology comparison revealed identities of 96.64 %, 71.99%, 44.88% and 47.37% of the DadX alanine racemase to those from P. putida KT2440, Pseudomonas aeruginose, Salmonella typhimurium and Escherichia coli, respectively. The amino acids sequence deduced from alr gene showed the homologies of 94.38%, 22.89%, 25.72% and 26.44% to those from the microorganisms above, respectively. Two motifs believed essential to the enzyme activity are found both in DadX and Alr, such as pyridoxal-5＇- phosphate binding site. Both dadX and air were expressed in E. coli TG1. Neither alanine racemase activity or serine racemase activity was detected in the host strain. Only alanine racemase activity was found in E. coli TG1/pCTD. But both E. coli TG1/pCTA and TG1/pCBA exhibit activity toward L-alanine and L-serine. Transcription of air gene in E. coli is independent from extraneous promoter, a result confirmed by the significant enzyme activity observed in the E. coli TG1/pCBA, which indicates the presence of a possible promoter upstream the structure gene.

关 键 词：恶臭假单胞菌 丙氨酸消旋酶 基因克隆 表达 

分 类 号：Q78[生物学—分子生物学]