Cloning of a wide-spectrum amidase from Bacillus stearothermophilus BR388 in Escherichia coli and marked enhancement of amidase expression using directed evolution☆

Abstract

A 1.6-kb DraI– HindIII DNA fragment from Bacillus stearothermophilus BR388 chromosomal DNA encoding a wide-spectrum amidase was cloned into Escherichia coli DH5α. With acrylamide substrate, the amidase showed maximum activity at 55°C, pH 7.0, and 0.12-M substrate, and demonstrated significant activity in 1-M acrylamide. A mutant prepared by PCR-based random mutagenesis of a 1.65 kb segment of B. stearothermophilus BR388 chromosomal DNA containing the amidase gene had two adenine bases replaced with guanine, resulting in a single primary structure alteration of His26 into Arg. This mutant demonstrated a 23-fold increase in amidase activity compared to wild–type, which is attributed to increased amidase gene transcription.