CLONING AND CHARACTERIZATION OF AN AMIDASE GENE FROM RHODOCOCCUS SPECIES N-774 AND ITS EXPRESSION IN ESCHERICHIA-COLI

被引:64
作者
HASHIMOTO, Y [1 ]
NISHIYAMA, M [1 ]
IKEHATA, O [1 ]
HORINOUCHI, S [1 ]
BEPPU, T [1 ]
机构
[1] UNIV TOKYO,DEPT AGR CHEM,BUNKYO KU,TOKYO 113,JAPAN
关键词
AMIDASE GENE; CLONING; GENE EXPRESSION; (RHODOCOCCUS); (ESCHERICHIA-COLI);
D O I
10.1016/0167-4781(91)90058-T
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli. Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of M(r) 54 671. The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans. The 521-amino acid coding region was therefore expressed by use of the E. coli lac promoter in E. coli, and was found to direct a considerable amidase activity. This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide. These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase. Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner.
引用
收藏
页码:225 / 233
页数:9
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