New thermostable d-methionine amidase from Brevibacillus borstelensis BCS-1 and its application for d-phenylalanine production

A new thermostable d-methionine amidase was found in a cell-free extract of Brevibacillus borstelensis BCS-1. After five steps of purification, the specific activity increased approximately 207-fold and the purity was more than 98%. The molecular weight of the enzyme was estimated to be 199 kDa by gel permeation chromatography and 30 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, which indicates that the thermostable d-methionine amidase was a homo-hexamer consisting of a single subunit. The purified enzyme was stable up to 65 ◦C within a broad pH range from 6.5 to 10.0, and its maximum activity was measured at pH 9.5 and 70 ◦C. The enzyme activity increased about five-fold with the addition of Co2+, yet was strongly inhibited by Hg2+, 2-mercaptoethanol, dithiothreitol, and ethylenediaminetetracetic acid. The thermostable d-methionine amidase exhibited a high amidase activity and d-stereospecificity toward d-amino acid amides and esters, yet did not hydrolyze d-peptides. The catalytic efficiencies (kcat/Km, mM−1 s−1) of the enzyme for d-methioninamide and d-alaninamide were 3086 and 21.5, respectively, and the enantiomeric excess (ee) and enantiomeric ratio of d-phenylalanine produced from dl-phenylalaninamide were 97.1 and 196%, respectively. © 2002 Elsevier Science Inc. All rights reserved.