Abstract
We expanded the application of self-sufficient heterogeneous biocatalysts containing coimmobilized ω-transaminases and pyridoxal 5′-phosphate (PLP) to efficiently operate packed-bed reactors in continuous flow. Using a ω-transaminase from Halomonas elongata coimmobilized with PLP onto porous methacrylate-based carriers coated with polyethylenimine, we operated a packed-bed reactor continuously for up to 50 column volumes at 1.45 mL × min–1 in the enantioselective deamination of model amines (α-methylbenzyl amine), yielding >90% conversion in all cycles without exogenous addition of cofactor. In this work, we expanded the concept of self-sufficient heterogeneous biocatalysts to other ω-transaminases such as the ones from Chromobacterium violaceum and Pseudomonas fluorescens. We found that enzymes with lower affinities toward PLP present lower operational stabilities in flow, even when coimmobilizing PLP. Furthermore, ω-transaminases coimmobilized with PLP were successfully implemented for the continuous sy...
Highlights
The synthesis of optically pure amines is essential to manufacture a plethora of commodities, agrochemicals and pharmaceuticals.[1]
To expand the concept of self-sufficient heterogeneous biocatalysts to amine biotransformations using Pyridoxal ́-phosphate (PLP)-dependent wTAs, commercial porous methacrylate beads activated with epoxide groups were functionalized with cobalt-chelates and blocked with either hydroxylamine or ethanolamine (Figure 1), giving rise to carriers named as Pu-Co2+/hA and Pu-Co2+/eA, respectively (Table 1)
The deamination activity of immobilized HewTA towards S-α-methylbenzyl amine (S-MBA) was significantly lower when PLP was adsorbed onto Pu-Co2+/hA than when it was onto Pu-Co2+/eA (Figure S3), indicating that the cofactor was catalytically more available when the solid surface was activated with ethanolamine
Summary
The synthesis of optically pure amines is essential to manufacture a plethora of commodities, agrochemicals and pharmaceuticals.[1]. These self-sufficient heterogeneous biocatalysts have been successfully integrated into flow PBR to perform continuous transamination reactions in buffer environment and without exogenous supply of PLP. Enzyme immobilization 10 mL of enzyme solution (0.1 – 0.5 mg x mL-1) containing 0.1 mM of PLP in 50 mM sodium phosphate buffer at pH 8, were added to 1 g of carrier and incubated under orbital shaking for 4 hours (He-wTA and Cv-wTA), and for 1 hour (Pf-wTA) at room temperature.
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