Efficient synthesis of an antiviral drug intermediate using overexpressed short‐chain dehydrogenase and cross‐linked enzyme aggregates stabilization

Abstract

BACKGROUND (2R,3S)‐N‐tert‐Butoxycarbonyl‐3‐amino‐1‐chloro‐2‐hydroxy‐4‐phenylbutane (1b) is key for the synthesis of the antiviral drug atazanavir. It can be obtained via the stereoselective bioreduction of (3S)‐3‐(N‐Boc‐amino)‐1‐chloro‐4‐phenyl‐butanone (1a) with short‐chain dehydrogenase/reductase (SDR) (EC 1.1.1.1). To develop a practical and cost‐effective biocatalytic approach with high stability and reusability, an SDR mutant (muSDR) from Novosphingobium aromaticivorans was overexpressed in Escherichia coli cell and immobilized by cross‐linked enzyme aggregates with enhanced stability and high efficiency. RESULTS muSDR gene was successfully cloned and overexpressed in Escherichia coli and reduced 1a to 1b with high stereoselectivity. Four resin‐bound muSDR and cross‐linked muSDR aggregates (muSDR‐CLEAs) were prepared and compared. With addition of Tween‐80, the muSDR‐CLEAs demonstrated much higher activity recovery than resin‐bound muSDR. Compared with free enzyme, muSDR‐CLEAs demonstrated enhanced thermal, pH, and storage stabilities and could obtain 1b with high substrate concentration, high de, and complete conversion. The reusability of CLEAs was also determined as no significant activity loss was observed after seven batches of reactions. The space time yield of the bioreduction using muSDR‐CLEAs was 226.6 g∙L−1∙day−1. CONCLUSION Overexpressed muSDR was efficiently immobilized, and it demonstrated superior stability and reusability. The recombinant muSDR and muSDR‐CLEAs are practical potential biocatalysts for industrial production of the atazanavir precursor. © 2020 Society of Chemical Industry (SCI)

New Strategy for Effective Biosynthesis of Chiral Aryl Alcohols: Co-Cultivation Microbe with Natural Deep-Eutectic Solvent

“…Based on the performance evaluation of various surfactants addition in the CFPO reduction process (Figure S3a), Tween 80 manifested as the most favorable co-solvent to enhance ( S )-CFPL synthesis efficiency. As an applicable co-solvent in biocatalysis, the proportion of Tween 80 in the reaction system showed a great discrepancy for different biocatalysts in previous reports. − To determine its optimal concentration in CFPO reduction, the impact of varied Tween 80 content on ( S )-CFPL yield was examined in detail. As shown in Figure S3b, the catalytic yields showed a bell shape in the range of Tween 80 volume fractions from 0–50.0% (w/v) and reached 95.1% yield at 5.0% (w/v) concentration under 50 mM substrate concentration, which is 138.4% higher than that in pure buffer solution.…”