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Use of enrichment culture for directed evolution of the Vibrio fluvialis JS17 omega-transaminase, which is resistant to product inhibition by aliphatic ketones.

ABSTRACT: 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.

SUBMITTER: Yun H

PROVIDER: S-EPMC1183280 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Use of enrichment culture for directed evolution of the Vibrio fluvialis JS17 omega-transaminase, which is resistant to product inhibition by aliphatic ketones.

Yun Hyungdon H Hwang Bum-Yeol BY Lee Jae-Hun JH Kim Byung-Gee BG

Applied and environmental microbiology 20050801 8

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 mut ...[more]

PMID: 16085806

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