Characterization and Catalytic-Site-Analysis of an Aldo-Keto Reductase with Excellent Solvent Tolerance

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) fromBacillussp. 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.Keywords:aldo-keto reductase;enzyme assay;organic solvents;chiral alcohol;substrate spectrum