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

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Title
New thermostable D-methionine amidase from Brevibacillus borstelensis BCS-1 and its application for D-phenylalanine production
Author(s)
Dae Heoun Baek; Jae Jun SongSeung Goo Lee; Seok Joon Kwon; Y Asano; Moon Hee Sung
Bibliographic Citation
Enzyme and Microbial Technology, vol. 32, no. 1, pp. 131-139
Publication Year
2003
Abstract
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 199kDa by gel permeation chromatography and 30kDa 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-1s-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.
Keyword
Brevibacillus borstelensis BCS-1D-Phenylalanine productionThermophilic bacteriumThermostable D-methionine amidase
ISSN
0141-0229
Publisher
Elsevier
DOI
http://dx.doi.org/10.1016/S0141-0229(02)00268-5
Type
Article
Appears in Collections:
Jeonbuk Branch Institute > Microbial Biotechnology Research Center > 1. Journal Articles
Division of Biomaterials Research > Synthetic Biology and Bioengineering Research Center > 1. Journal Articles
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