A novel amidohydrolase gene from Bacillus subtilis cloning: DNA-sequence analysis and map position of amhX

Abstract

The nucleotide sequence of a new Bacillus subtilis gene ( amhX) was determined that encodes a protein (AmhX) with strong sequence identity to amidohydrolases from both plant and bacterial species and a carboxypeptidase from the archaeon Sulfolobus sulfataricus. The amAJfgene encodes a hydrophilic polypeptide of 383 amino acids with a molecular mass of 41.5 kDa. The amhX gene was overexpressed in E. coli by using the T7 RNA polymerase/promoter system and the transcription initiation sites for the amhX mRNAs in B. subtilis were determined by primer extension analysis. Chromosomal amhX mutations were constructed by marker replacement and the amhX gene was positioned at 25 ° on the genetic and physical map of the B. subtilis chromosome.