Cloning of the wide spectrum amidase gene from Brevibacterium sp. R312 by genetic complementation. Overexpression in Brevibacterium sp. and Escherichia coli.
The amiE gene of Brevibacterium sp. R312 encoding wide spectrum amidase was isolated by complementation of a Brevibacterium sp. mutant using a plasmid gene bank of chromosomal DNA. The amiE structural gene and its promoter were localized on a 1.8-kb fragment by subsequent subcloning and complementation studies. Another promoter localized in the pSR 1 fragment of the cloning vector was shown to be able to control amiE gene expression. In Brevibacterium sp., the investigation of amidase activities related to one copy of the gene suggested that the regulation of the amiE gene expression was under negative control. High expression levels have been obtained in Brevibacterium sp. and, after substitution of the amiE promoter by the tac promoter, in Escherichia coli.
Azza
S
S
Ecole Nationale Supérieure Agronomique de Montpellier, France.
Bigey
F
F
Arnaud
A
A
Galzy
P
P
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000235:genetics*; D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial / Q000235:genetics
D000581:Amidohydrolases; C019665:amidase