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

摘要

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 degreesC within a broad pH range from 6.5 to 10.0, and its maximum activity was measured at pH 9.5 and 70degreesC. The enzyme activity increased about five-fold with the addition of Co2+, yet was strongly inhibited by Hg2+, 2-mercaptoethanol, dithiothreitol, and ethylenediaminetetracetic acid.