Characterization of a Thermostable d -Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

Abstract

ABSTRACT A gene encoding a new thermostable d -stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards d -amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards l -amino acid amides, d -amino acid-containing peptides, and NH 2 -terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co 2+ and Mn 2+ . The k cat / K m for d -alaninamide was measured as 544.4 ± 5.5 mM −1 min −1 at 50°C with 1 mM Co 2+ .