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Applied and Environmental Microbiology, March 2007, p. 1772-1782, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.02119-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Cloning and Characterization of a Novel ß-Transaminase from Mesorhizobium sp. Strain LUK: a New Biocatalyst for the Synthesis of Enantiomerically Pure ß-Amino Acids

Juhan Kim, Dohyun Kyung, Hyungdon Yun, Byung-Kwan Cho, Joo-Hyun Seo, Minho Cha, and Byung-Gee Kim^*

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

Received 7 September 2006/ Accepted 20 January 2007

A novel ß-transaminase gene was cloned from Mesorhizobium sp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the ß-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The ß-transaminase showed higher activities toward D-ß-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The ß-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and -ketoglutarate/oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of ß-aminocarboxylic acids in the active site is reversed relative to that of -amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the ß-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic -keto acids such as -ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the -carboxylate group of the -amino acids and -keto acids. The ß-transaminase was used for the asymmetric synthesis of enantiomerically pure ß-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor.

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* Corresponding author. Mailing address: School of Chemical Engineering, Seoul National University, Seoul 151-742, Korea. Phone: 82-2-880-6774. Fax: 82-2-874-1206. E-mail: byungkim{at}snu.ac.kr.

Published ahead of print on 26 January 2007.

Present address: UCB 216, Cooperative Institute for Research in Environmental Sciences, CIRES 318, University of Colorado, Boulder, CO 80302.

Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

Present address: Department of Bioengineering, University of California—San Diego, 9500 Gilman Dr., La Jolla, CA 92093.

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Applied and Environmental Microbiology, March 2007, p. 1772-1782, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.02119-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.