Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1.
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+).
Baek
Dae Heoun
DH
Biocatalysis Research Laboratory, National Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305-333, Korea.
Kwon
Seok-Joon
SJ
Hong
Seung-Pyo
SP
Kwak
Mi-Sun
MS
Lee
Mi-Hwa
MH
Song
Jae Jun
JJ
Lee
Seung-Goo
SG
Yoon
Ki-Hong
KH
Sung
Moon-Hee
MH
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
D000581:Amidohydrolases; C019665:amidase; D000409:Alanine