One pot simultaneous preparation of both enantiomer of ß-amino alcohol and vicinal diol via cascade biocatalysis.

OBJECTIVES: To investigate the efficiency of a new cascade biocatalysis system for the conversion of R, S-ß-amino alcohols to enantiopure vicinal diol and ß-amino alcohol. RESULTS: An efficient cascade biocatalysis was achieved by combination of a transaminase, a carbonyl reductase and a cofactor regeneration system. An ee value of > 99% for 2-amino-2-phenylethanol and 1-phenyl-1, 2-ethanediol were simultaneously obtained with 50% conversion from R, S-2-amino-2-phenylethanol. The generality of the cascade biocatalysis was further demonstrated with the whole-cell approaches to convert 10-60 mM R, S-ß-amino alcohol to (R)- and (S)-diol and (R)- and (S)-ß-amino alcohol in 90-99% ee with 50-52% conversion. Preparative biotransformation was demonstrated at a 50 ml scale with mixed recombinant cells to give both (R)- and (S)-2-amino-2-phenylethanol and (R)- and (S)-1-phenyl-1, 2-ethanediol in > 99% ee and 40-42% isolated yield from racemic 2-amino-2-phenylethanol. CONCLUSIONS: This cascade biocatalysis system provides a new practical method for the simultaneous synthesis of optically pure vicinal diol and an ß-amino alcohol.

A high-throughput microtiter plate assay for the discovery of active and enantioselective amino alcohol-specific transaminases.

Chiral vicinal amino alcohols are important chiral building blocks and intermediates in the pharmaceutical industry. The transaminase (TAm) catalyzed kinetic resolution of racemic amino alcohols provides a straightforward approach to access these important compounds. This study describes the development of a novel microtiter plate assay to screen vicinal amino alcohol-specific TAms using a tetrazolium red-based colorimetric assay to monitor the rate of α-hydroxy ketone formation at 510 nm. This approach is the first to determine the Michaelis-Menten parameters for a recombinant TAm (PpbauA) from Pseudomonas putida NBRC14164. The corresponding Vmax and KM values for both enantiomers of 2-amino-1-propanol and 2-amino-1-butanol were obtained, and the calculated kinetic E-factors of PpbauA toward 2-amino-1-propanol and 2-amino-1-butanol are 3 (S) and 6 (R), respectively. The method is sensitive and exhibits low level background coloration. Moreover, this method can be used to detect transaminase activity and enantioselectivity toward amino alcohols in a high-throughput format. Additionally, this simple method is compatible with the most widely used (R)- and (S)-selective transaminases and may be a broadly applicable tool for screening transaminases from a transaminase mutant library.

Characterization of Four New Distinct ω-Transaminases from Pseudomonas putida NBRC 14164 for Kinetic Resolution of Racemic Amines and Amino Alcohols.

Four uncharacterized ω-transaminases (ωTAs) from Pseudomonas putida NBRC 14164 have been identified and cloned from the pool of fully sequenced genomes. The genes were functionally expressed in Escherichia coli BL21, and the enzymes were purified and characterized. Four TAs showed highly (S)-selective ωTA activity and converted (S)-α-methylbenzylamine and pyruvate to acetophenone and L-Ala. The maximum activity of cloned enzymes was in the pH range of 8.0-8.5 (Pp36420), 8.5-9.5 (Pp21050), 9.0-9.5 (PpspuC), and 9.5-10.5 (PpbauA), and the optimal temperatures were at 35 °C (Pp36420, Pp21050, and PpspuC) and 50 °C (PpbauA), respectively, with K M of 161.3 mM (Pp21050), 136.7 mM (PpbauA), 398.5 mM (Pp36420), and 130.9 mM (PpspuC) and yielding a catalytic efficiency k cat/K M of 0.015, 0.003, 0.012, and 0.023 mM-1 s-1. Several racemic amines and amino alcohols were resolved by the cloned ωTAs; perfect conversions (48-50 %) were obtained by at least one enzyme, and the residual substrates were left with 97-99 % ee. Kinetic resolution of racemic phenylglycinol was done with PpspuC in a 100-mL scale. Enaniomeric excess of (S)-phenylglycinol reached 99 % with 45 % isolated yield. The high enantioselectivity and large substrate spectra of the cloned PpTAs showed an attractive potency for biotechnology application in production of chiral amines and amino alcohols.

Cloning and characterization of two distinct water-forming NADH oxidases from Lactobacillus pentosus for the regeneration of NAD.

Two uncharacterized nicotinamide adenine dinucleotide (NADH) oxidases (named as LpNox1, LpNox2) from Lactobacillus pentosus ATCC 8041 were cloned and overexpressed in Escherichia coli BL21 (DE3). The sequence analysis revealed that the two enzymes are water-forming Noxs with 64 % and 52 % identity to LbNox from Lactobacillus brevis DSM 20054. The optimal pH and temperature of the purified LpNox1 and LpNox2 were 7.0 and 8.0 and 35 and 40 °C, respectively, with K M of 99.0 µM (LpNox1) and 27.6 µM (LpNox2), and yielding catalytic efficiency k cat/K M of 1.0 and 0.2 µM(-1) s(-1), respectively. Heat inactivation studies revealed that the two enzymes are relatively instable. The application of LpNox1 for the regeneration of NAD(+) was demonstrated by coupling with a glycerol dehydrogenase-catalyzed oxidation of glycerol to 1,3-dihydroxyacetone. The characteristics of the LpNox1 could prove to be of interest in industrial application such as NAD(+) regeneration in dehydrogenase-catalyzed oxidations.