Use of enrichment culture for directed evolution of the Vibrio fluvialis JS17 omega-transaminase, which is resistant to product inhibition by aliphatic ketones.
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.
Yun
Hyungdon
H
School of Chemical Engineering and Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
Hwang
Bum-Yeol
BY
Lee
Jae-Hun
JH
Kim
Byung-Gee
BG
D000588:Amines / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D002074:Butanones / Q000494:pharmacology*; D003470:Culture Media; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D007700:Kinetics; D013379:Substrate Specificity; D000637:Transaminases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*
D000588:Amines; D002074:Butanones; D003470:Culture Media; C005222:methylethyl ketone; D000637:Transaminases