Coupled Enzymatic Alcohol‐to‐Amine Conversion of Isosorbide using Engineered Transaminases and Dehydrogenases

Abstract

A matching dehydrogenase and transaminase pair was engineered with regard to substrate recognition, catalytic activity, and cofactor specificity with the final goal to convert the bicyclic dialcohol isosorbide into a diamine by a multistep biocatalytic process. Individual catalytic turnover rates as well as coupled conversion to the amine were investigated for the enzymes in analytical assays that used the substrate isosorbide and different intermediates along the multiple pathway reaction cascade, in particular, stereoisomers of hydroxy ketones, amino alcohols, and amino ketones. For parallel screening and evaluation of mutant enzymes with regard to catalytic activities and optimal reaction conditions, a robotic platform was established that comprised all steps from bacterial protein expression to the enzymatic assay. As a result, we present a three‐enzyme system composed of L. aquatica levodione reductase, an engineered P. denitrificans ω‐aminotransferase, and B. subtilis alanine dehydrogenase that catalyzes formation of the isosorbide monoamine with a yield of up to ≈7 % under our analytical assay conditions. After further optimization and adaptation to whole‐cell catalysis, this enzyme system may open a biotechnologically attractive route to a structurally rigid diamine for the production of biosynthetic polymer materials.

Engineering of alanine dehydrogenase from Bacillus subtilis for novel cofactor specificity

“…The triple biocatalytic reaction of isosorbide to its diamine diastereomers was performed with BasAlaDH(D196A/L197R) from this study together with RasADH and PDωAT(L417M), which were expressed and purified according to previously described procedures [22,24]. The 250 µL assay solution contained 25 mM Hepes/NaOH pH 8.3 with 300 mM isosorbide (Evonik Industries) as substrate, 2 mM NADP + sodium salt (Applichem) as hydride acceptor for the oxidation catalyzed by RasADH, 5 mM l-Ala and 0.3 mM PLP (Sigma, Munich, Germany) as (co)-substrates for PDωAT(L417M)-catalyzed transamination, as well as 100-300 mM NH 4 Ac as ammonium donor for the regeneration of the l-Ala cofactor via the BasAlaDH variant.…”

“…• C for 96 H, the reaction was stopped and products were quantified by FMOC/HPLC analytics of the mono-and diamino isosorbide derivatives as previously described [22].…”

“…Since the former reaction generates NADH from NAD + , whereas the latter converts the cosubstrate l-alanine into pyruvate, a third biocatalyst, BasAlaDH, was introduced [21] to recycle this cosubstrate and connect both transformations, also enabling the use of ammonia as nitrogen source. This strategy has been further applied to the amination of secondary alcohols, in particular the renewable platform chemical isosorbide (1,4:3,6-dianhydro-d-glucitol) [22,23].…”

“…(RasADH) is particularly suitable for the oxidation of the dicyclic chiral isosorbide substrate, and we determined the k cat and K M values for the forward and backward reactions [22,24]. However, the NADP + dependence of RasADH prevented efficient cofactor regeneration because of the NADH specificity of BasAlaDH.…”

“…Therefore, we describe here an endeavor to alter the cofactor specificity of BasAlaDH from NADH to NADPH using bioinformatic analysis and rational protein engineering. The engineered enzyme is applied in a coupled reaction with two other biocatalysts to convert isosorbide into dicyclic diamines which constitute promising intermediates for high-performance polymers [22].…”