Abstract
Background(S)-(−)-N,N-Dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine (DHTP) is a key intermediate for the preparation of (S)-duloxetine, an important antidepressant drug. However, so far, the catalytic efficiency of (S)-DHTP synthesis by asymmetric bioreduction is yet limited. The present study aims to develop an efficient system for synthesis of (S)-DHTP by bioreduction.ResultsVarious recombinant carbonyl reductases were evaluated for asymmetric reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP) to produce (S)-DHTP. The NADPH-dependent carbonyl reductase CR2 was identified as the suitable candidate, giving (S)-DHTP in absolute configuration. Then the fusion protein involving CR2 and glucose dehydrogenase (CR2-L-GDH) was constructed to further improve cofactor regeneration and resulted catalytic efficiency of the enzymatic reduction. By studying the effects of reaction conditions involving cofactor regeneration, suitable catalytic system was achieved for CR2-L-GDH catalyzing (S)-DHTP synthesis. Consequently, (S)-DHTP (>99.9% e.e.) with yield of 97.66% was obtained from 20 g L−1 DKTP within 8-h reaction, employing 40 g L−1 glucose and 0.1 mmol L−1 NADP+ to drive the cofactor regeneration, resulting in the space–time yield of 2.44 g L−1 h−1.ConclusionOptically pure (S)-DHTP with improved yield was obtained by fusion enzyme CR2-L-GDH. Fusion enzyme-mediated biocatalytic system would be promising to enhance reaction efficiency of enzyme-coupled system for preparation of optically active alcohols.
Highlights
Active alcohols are valuable and promising chiral building blocks imposed in the production ofSun et al Bioresour
For the reaction system involving the crude enzymes of CR2 and Glucose dehydrogenase (GDH), the desired product (S)-DHTP with optical purity over 99.9% e.e. and yield of 90.43% was obtained from 10 g L−1 DKTP at 30 °C and pH 8.0, in the presence of 120 g L−1 glucose
Using the cell-free extract of recombinant E. coli BL21(DE3)/ pET-28a-cr2-l-gdh as the catalyst, with DKTP of 20 g L−1, optically pure (S)-DHTP (>99.9% e.e.) was obtained at the yield of 83.26% at 30 °C and pH 8.0 (TEA buffer). These results indicated that it would be much potential to improve the reaction efficiency under increased substrate concentration of 20 g L−1 by the fusion enzyme CR2-L-GDH
Summary
Various recombinant carbonyl reductases were evaluated for asymmetric reduction of N,N-dimethyl-3-keto3-(2-thienyl)-1-propanamine (DKTP) to produce (S)-DHTP. The NADPH-dependent carbonyl reductase CR2 was identified as the suitable candidate, giving (S)-DHTP in absolute configuration. The fusion protein involving CR2 and glucose dehydrogenase (CR2-L-GDH) was constructed to further improve cofactor regeneration and resulted catalytic efficiency of the enzymatic reduction. By studying the effects of reaction conditions involving cofactor regeneration, suitable catalytic system was achieved for CR2-L-GDH catalyzing (S)-DHTP synthesis. (S)-DHTP (>99.9% e.e.) with yield of 97.66% was obtained from 20 g L−1 DKTP within 8-h reaction, employing 40 g L−1 glucose and 0.1 mmol L−1 NADP+ to drive the cofactor regeneration, resulting in the space–time yield of 2.44 g L−1 h−1
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