title: Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1.
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 degrees 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 degrees C with 1 mM Co(2+).
authors.affiliation: Biocatalysis Research Laboratory, National Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305-333, Korea.
authors.forename: Dae Heoun
authors.initials: DH
authors.lastname: Baek
authors.forename: Seok-Joon
authors.initials: SJ
authors.lastname: Kwon
authors.forename: Seung-Pyo
authors.initials: SP
authors.lastname: Hong
authors.forename: Mi-Sun
authors.initials: MS
authors.lastname: Kwak
authors.forename: Mi-Hwa
authors.initials: MH
authors.lastname: Lee
authors.forename: Jae Jun
authors.initials: JJ
authors.lastname: Song
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
authors.forename: Ki-Hong
authors.initials: KH
authors.lastname: Yoon
authors.forename: Moon-Hee
authors.initials: MH
authors.lastname: Sung
mesh_terms: D000409:Alanine / Q000378:metabolism*; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism
chemical_list: D000581:Amidohydrolases; C019665:amidase; D000409:Alanine