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Applied and Environmental Microbiology, August 2005, p. 4220-4224, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4220-4224.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Enrichment Culture for Directed Evolution of the Vibrio fluvialis JS17 {omega}-Transaminase, Which Is Resistant to Product Inhibition by Aliphatic Ketones

Hyungdon Yun, Bum-Yeol Hwang, Jae-Hun Lee, and Byung-Gee Kim*

School of Chemical Engineering and Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea

Received 7 June 2004/ Accepted 26 February 2005

A novel high-throughput screening method that overcame product inhibition was used to isolate a mutant {omega}-transaminase from Vibrio fluvialis JS17. An enzyme library was generated using error-prone PCR mutagenesis and then enriched on minimal medium containing 2-aminoheptane as the sole nitrogen source and 2-butanone as an inhibitory ketone. An identified mutant enzyme, {omega}-TAmla, showed significantly reduced product inhibition by aliphatic ketone. The product inhibition constants of the mutant with 2-butanone and 2-heptanone were 6- and 4.5-fold higher than those of the wild type, respectively. Using {omega}-TAmla (50 U/ml) overexpressed in Escherichia coli BL21, 150 mM 2-aminoheptane was successfully resolved to (R)-2-aminoheptane (enantiomeric excess, >99%) with 53% conversion with an enantioselectivity of >100.

* Corresponding author. Mailing address: School of Chemical Engineering and Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea. Phone: 82-2-880-6774. Fax: 82-2-873-6020. E-mail: byungkim{at}snu.ac.kr.

Applied and Environmental Microbiology, August 2005, p. 4220-4224, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4220-4224.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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