The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans:: molecular cloning, nucleotide sequence, and expression in Escherichia coli
The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans:: molecular cloning, nucleotide sequence, and expression in Escherichia coli
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DOI：
10.1007/s004380050802
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发表时间：
1998-08-01
期刊：
影响因子：
--
通讯作者：
Cohen, JD
中科院分区：
文献类型：
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作者：
Chou, JC;Mulbry, WW;Cohen, JD
利用基于n端氨基酸序列的杂交探针，从凝集肠杆菌菌株GK12中分离到含有吲哚-3-乙酰-天冬氨酸（iaaspH）水解酶基因（iaaspH）的5.5 kb DNA片段。测定了该片段2.4 kb区域的DNA序列，发现了一个1311个核苷酸的ORF，足以编码45 kda的IAA-asp水解酶。将含有iaaspH的1.5 kb DNA片段亚克隆到大肠杆菌表达质粒pTTQ8中，得到质粒pJCC2。含有pJCC2重组质粒的iptg诱导的大肠杆菌培养物的IAA-asp水解酶水平比聚集大肠杆菌提取物高5 ~ 10倍。同源性研究表明，IAA-asp水解酶与多种氨基水解酶相似。此外，IAA-asp水解酶与大肠杆菌的一种推定的耐热羧肽酶的序列同源性为70%。
A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli.