A novel d-stereoselective amino acid amidase from Brevibacterium iodinum: Gene cloning, expression and characterization

Abstract

The gene encoding d-stereoselective amino acid amide amidohydrolase ( d-amino acid amidase, named DaaA Bi) was cloned from a chromosomal DNA library of Brevibacterium iodinum TPU 5850 and sequenced. The gene, daaA Bi , encoded a protein composed of 266 amino acids with a M r of 30035. The deduced amino-acid sequence of the daaA Bi gene did not exhibit any similarity with any other previously reported d-amino acid amidases, but did show similarity with hypothetical class A β-lactamases. DaaA Bi protein was produced in Escherichia coli, purified to electrophoretic homogeneity, and characterized. The purified enzyme was about 290,000 based on gel filtration chromatography and about 30,000 based on SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is active as a decamer with identical subunits. DaaA Bi showed maximum activity at pH 7.2 and 35 °C. It exhibited strict d-stereoselective hydrolyzing activity towards a broad range of d-amino acid amides including d-methioninamide, d-lysinamide, d-glutaminamide, and d-phenylalaninamide, while l-amino acid amides, peptides composed of l- or d-amino acids, and β-lactam compounds could not serve as substrates for the enzyme. Almost complete hydrolysis of d-phenylalaninamide with highly strict d-stereoselectivity was achieved in 3 h from 180 mM of dl-phenylalaninamide using the purified DaaA Bi enzyme.