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Applied and Environmental Microbiology, April 2004, p. 2529-2534, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2529-2534.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

{omega}-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of ß-Amino Acids and Amines

Hyungdon Yun, Seongyop Lim, Byung-Kwan Cho, and Byung-Gee Kim*

Institute for Molecular Biology and Genetics and School of Chemical Engineering, Seoul National University, Seoul 151-742, Korea

Received 8 July 2003/ Accepted 17 January 2004

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed {omega}-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic ß-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-ß-amino-n-butyric acid (L-ß-ABA). The enzyme converts various ß-amino acids and amines to the corresponding ß-keto acids and ketones by using pyruvate as an amine acceptor. The apparent Km and Vmax for L-ß-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-ß-ABA, the apparent Km and Vmax for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-ß-ABA, producing optically pure D-ß-ABA (99% enantiomeric excess) with 53% conversion.

* Corresponding author. Mailing address: Laboratory of Biotechnology and Biomaterials, Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea. Phone: 82-2-880-6774. Fax: 82-2-874-1206. E-mail: byungkim{at}snu.ac.kr.

Applied and Environmental Microbiology, April 2004, p. 2529-2534, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2529-2534.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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