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Applied and Environmental Microbiology, February 2003, p. 980-986, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.980-986.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization of a Thermostable D-Stereospecific Alanine Amidase from Brevibacillus borstelensis BCS-1

Dae Heoun Baek,1 Seok-Joon Kwon,1 Seung-Pyo Hong,2 Mi-Sun Kwak,1 Mi-Hwa Lee,1 Jae Jun Song,1 Seung-Goo Lee,1 Ki-Hong Yoon,3 and Moon-Hee Sung1,2*

Biocatalysis Research Laboratory (National Research Laboratory), Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305-333,1 Bioleaders Corporation, Yuseong, Daejeon 305-600,2 Division of Food Life Engineering, Woosong University, Daejeon 300-718, Korea3

Received 21 May 2002/ Accepted 4 August 2002

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 NH2-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85°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 Co2+ and Mn2+. The kcat/Km for D-alaninamide was measured as 544.4 ± 5.5 mM-1 min-1 at 50°C with 1 mM Co2+.

* Corresponding author. Mailing address: Biocatalysis Research Laboratory, Laboratory of Function Microbial, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Oun-dong, Yusong-gu, Daejeon 305-333, Korea. Phone: 82-42-860-4372. Fax: 82-42-860-4595. E-mail: smoonhee{at}mail.kribb.re.kr.

Applied and Environmental Microbiology, February 2003, p. 980-986, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.980-986.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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