Aldo-keto reductases (AKRs) mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols due to their high chemo-, enantio-, and regio-selectivity. To date, the application of AKRs in the asymmetric synthesis of chiral alcohols has been limited, due to the challenges of cloning and purifying. In this work, the aldo-keto reductase (AKR3-2-9) from Bacillus sp. was obtained, purified and proved to be NADPH-dependent. It exhibits good bioactivity and stability at 37 °C, pH 6.0. AKR3-2-9 is catalytically active on 11 pairs of substrates such as 3-methylcyclohexanone and methyl pyruvate, among which it showed the highest catalytic activity for acetylacetone. In addition, AKR3-2-9 was able to be resistant to five common organic solvents such as methanol and ethanol, it retained high catalytic activity even in a reaction system containing 10% v/v organic solvent for 6 h, which indicates its broad substrate spectrum and exceptional organic solvent tolerance. Furthermore, its three-dimensional structure was constructed and catalytic-site-analysis of the enzyme was conducted. Notably, it was capable of catalyzing the reaction of the key intermediates of duloxetine. The extensive substrate spectrum and predominant organic solvents resistance makes AK3-2-9 a promising enzyme which can be potentially applied in medicine synthesis.

As one of the important research contents of synthetic biology, the construction of a regulatory system exhibits great potential in the synthesis of high value-added chemicals such as drug intermediates. In this work, a self-assembly coenzyme regeneration system, leucine dehydrogenase (LeuDH)–formate dehydrogenase (FDH) protein co-assembly system, was constructed by using the polypeptide, SpyTag/SpyCatcher. Then, it was demonstrated that the nonchromatographic inverse transition cycling purification method could purify intracellular coupling proteins and extracellular coupling proteins well. The conversion rate of the pure LeuDH–FDH protein assembly (FR-LR) was shown to be 1.6-fold and 32.3-fold higher than that of the free LeuDH–FDH system (LeuDH + FDH) and free LeuDH, respectively. This work has paved a new way of constructing a protein self-assembly system and engineering self-purification coenzyme regeneration system for the synthesis of chiral amino acids or chiral α-hydroxy acids.