Kinetic resolution of α-methylbenzylamine with ω-transaminase screened from soil microorganisms: Application of a biphasic system to overcome product inhibition

Two microorganisms showing high ω-transaminase activity (Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64) were screened by the enrichment method using (S)-α-methylbenzylamine (α-MBA) as a sole nitrogen source. Optimal carbon and nitrogen sources for enzyme induction and the properties of ω-transaminases were investigated. ω-Transaminase from B. thuringiensis JS64 was highly enantioselective (E = 75.3) for (S)-enantiomer of α-MBA and showed remarkable stability. However, ω-transaminase showed severe product inhibition by acetophenone. An aqueous/organic two-phase system was introduced to overcome this problem. Through solvent screening, cyclohexanone and ethyl acetate were selected as the best organic phases. The acetophenone-extracting capacity of the solvent and the biocompatibility of the solvent to the cell were important determinants in the reaction rate at high concentrations of α-MBA. The reaction rate of ω-transamination was strongly influenced by the volume ratio of organic phase to aqueous phase as well as agitation speed in the biphasic mixture. Using the optimal volume ratio (V(org): V(aq) = 1:4) in the biphasic system with cyclohexanone, the reaction rate of ω-transaminase under vigorous mixing conditions increased ninefold compared with that in the monophasic aqueous system. At the same optimal conditions, using whole cells, 500 mM α-MBA could be resolved successfully to above 95% enantiomeric excess of (R)-α-MBA with ca. 51% conversion.