D-Threonine is one of the important unnatural amino acids used as chiral building blocks in pharmaceutical drugs. Owing to the presence of two chiral centers, a synthetic protocol, either through chemocatalysis or biocatalysis, has not yet been available for one-step preparation of stereochemically pure D-threonine in terms of enantiomeric and diastereomeric excesses (i.e., both >99%). Here we demonstrate that facile production of D-threonine can be implemented using threonine deaminase (TD) via kinetic resolution of DL-threonine that can be readily prepared by conventional organic synthesis. TD catalyzes the dehydration/deamination of L-threonine, leading to generation of 2-oxobutyrate and ammonia. In contrast to mild substrate inhibition of the TD activity by L-threonine (i.e., apparent inhibition constant (K-1(app)) = 950 mM), e-threonine turned out to be a strong inhibitor (i.e., K-1(app) = 41 mM). In addition to the enzyme inhibitions by both enantiomers of threonine, cell lysis observed during small-scale kinetic resolutions of >= 1 M DL-threonine led us to carry out a preparative-scale reaction at 500 mM racemic substrate. The preparative-scale kinetic resolution in a 50 mL reaction mixture charged with 3 g DL-threonine and 3400 U whole cells was completed at 5 h with >99% ee of D-threonine. Product isolation by a cation-exchange chromatography led to white solid of D-threonine (1.36 g, 90.7% isolation yield). To explore whether our strategy could afford coproduction of another valuable unnatural amino acid, the pass-through solution from the cation-exchange column was further processed by a omega-transaminase (omega-TA) reaction where 2-oxobutyrate was converted to enantiopure homoalanine using isopropylamine as an amino donor. Addition of S- and R-selective omega-TA to the pass-through solution led to 93.2 and 90.9% reaction yield within 12 h with both >99% ee of the produced L- and D-homoalanine, respectively. (C) 2015 Elsevier B.V. All rights reserved.
