Kinetic Resolution of Racemic Amines to Enantiopure (S)-amines by a Biocatalytic Cascade Employing Amine Dehydrogenase and Alanine Dehydrogenase

Abstract

Amine dehydrogenases (AmDHs) efficiently catalyze the NAD(P)H-dependent asymmetric reductive amination of prochiral carbonyl substrates with high enantioselectivity. AmDH-catalyzed oxidative deamination can also be used for the kinetic resolution of racemic amines to obtain enantiopure amines. In the present study, kinetic resolution was carried out using a coupled-enzyme cascade consisting of AmDH and alanine dehydrogenase (AlaDH). AlaDH efficiently catalyzed the conversion of pyruvate to alanine, thus recycling the nicotinamide cofactors and driving the reaction forward. The ee values obtained for the kinetic resolution of 25 and 50 mM rac-α-methylbenzylamine using the purified enzymatic systems were only 54 and 43%, respectively. The use of whole-cells apparently reduced the substrate/product inhibition, and the use of only 30 and 40 mgDCW/mL of whole-cells co-expressing AmDH and AlaDH efficiently resolved 100 mM of rac-2-aminoheptane and rac-α-methylbenzylamine into the corresponding enantiopure (S)-amines. Furthermore, the applicability of the reaction protocol demonstrated herein was also successfully tested for the efficient kinetic resolution of wide range of racemic amines.

Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin‐derived substrates

“…The introduction of AlaDH from B . subtilis for the regeneration of l ‐alanine from pyruvate could assist in shifting the equilibrium, as previously shown for E. coli (Wu et al ., 2016 ; Wu et al ., 2017a ; Patil et al ., 2019 ). l ‐alanine regeneration was investigated by cloning the AlaDH gene in the plasmid pJH004 and inserting it in GN442ΔPP_2426.…”

“…Thus, strategies for surpassing this typically involves using an excess amount of amine donors (such as l ‐alanine or isopropylamine) and/or coupled reactions to remove the co‐product or to recycle the amine donor (Wu et al ., 2016 ; Wu et al ., 2017a ; Zhou et al ., 2018 ). Whole‐cell transamination (Weber et al ., 2014a ; Weber et al ., 2017 ; Patil et al ., 2019 ; Molnár et al ., 2019 ) offers several advantages over in vitro because it supplies PLP and amine donor, recycles cofactors, removes co‐product, as well as simplifies upstream preparation of the biocatalyst, which altogether result in significant process improvement (Tufvesson, Lima‐Ramos, Jensen, et al ., 2011 ; Tufvesson, Lima‐Ramos, Nordblad, et al ., 2011 ). On the other hand, whole‐cell systems are often sensitive to adverse process conditions, such as high titres of inhibitory substrates and products.…”

An Integrated Cofactor/Co‐Product Recycling Cascade for the Biosynthesis of Nylon Monomers from Cycloalkylamines

“…The reactions catalyzed by CHMO and LA in the present cascade are practically irreversible. The deaminated products are detrimental in transamination reactions, because they impede the reaction equilibrium and also inhibit the enzymes, thereby necessitating instant removal to shift the equilibrium towards product formation [2d–h] …”

Kinetic Resolution of Racemic Primary Amines Using Geobacillus stearothermophilus Amine Dehydrogenase Variant

“…Similarly, cFL1‐AmDH performed the reductive amination of respective ketones 1 – 4 b yielding poor‐to‐moderate conversions after 48 h, and low activity was observed with Es‐LeuDH (AmDH) for the reductive amination of 1 b and 4 b . In the oxidative deamination direction (KR), only a few α‐methylbenzylamines could be resolved, but at the expense of a large amount of whole cells biocatalysts, and using a less atom‐efficient pyruvate/AlaDH system for NAD + recycling . Herein, we used the LE‐AmDH‐v1/NOx system to access pharmaceutically relevant, optically active amines in S ‐configuration starting from the respective racemic mixtures.…”