Comparison of the ω-transaminases from different microorganisms and application to production of chiral amines

Microorganisms that are capable of (S)-enantioselective transamination of chiral amines were isolated from soil samples by selective enrichment using (S)-α-methylbenzylamine ((S)-α-MBA) as a sole nitrogen source. Among them, Klebsiella pneumoniae JS2F, Bacillus thuringiensis JS64, and Vibrio fluvialis JS17 showed good ω-transaminase (ω-TA) activities and the properties of the ω-TAs were investigated. The induction level of the enzyme was strongly dependent on the nitrogen source for the strains, except for V. fluvialis JS17. All the ω-TAs showed high enantioselectivity (E>50) toward (S)-α-MBA and broad amino donor specificities for arylic and aliphatic chiral amines. Besides pyruvate, aldehydes such as propionaldehyde and butyraldehyde showed good amino acceptor reactivities. All the ω-TAs showed substrate inhibition by (S)-α-MBA above 200 mM. Moreover, substrate inhibition by pyruvate above 10 mM was observed for ω-TA from V. fluvialis JS17. In the case of product inhibition, acetophenone showed much greater inhibitions than L-alanine for all ω-TAs. Comparison of the enzyme properties indicates that ω-transaminase from V. fluvialis JS17 is the best one for both kinetic resolution and asymmetric synthesis to produce enantiomerically pure chiral amines. Kinetic resolution of sec-butylamine (20 mM) was done under reduced pressure (150 Torr) to selectively remove an inhibitory product (2-butanone) using the enzyme from V. fluvialis JS17. Enantiomeric excess of (R)-sec-butylamine reached 94.7% after 12 h of reaction.