The whole cells of Candida parapsilosis ATCC 7330 (Cp), a versatile biocatalyst, harbors (S) and (R)-specific enzymes that carry out various oxidation-reduction reactions generating a plethora of industrially important intermediates. The next logical progression is to isolate these stereospecific enzymes. The focus of this work is to purify an (S) specific carbonyl reductase (SRED) and to understand the bases of its substrate selectivity and stereospecificity at the molecular level. SRED was purified and characterized. This enzyme is a tetramer with subunit molecular weight of 30 kDa with optimum pH and temperature of 5.0 and 25 °C, respectively. Its substrate selectivity is α-ketoesters > ketones > aldehydes. SRED asymmetrically reduces α-ketoesters to (S)-hydroxyesters (ee 70%) in presence of both NADPH and NADH, while gives (S)-alcohols (ee >99%) from ketones, with NADPH only. SRED displays dual kinetic behavior i.e. hyperbolic and sigmoidal for the reduction of ketones and α-ketoesters, respectively. This was followed by an extensive study employing quantum mechanics/molecular mechanics (QM/MM) analysis of the modelled SRED-NADPH with two representative substrates, 2-hydroxyacetophenone (ketone) and ethyl-2-oxo-4-phenylbutanoate (α-ketoester). The energy profiles thus obtained along the reduction paths of these substrates complement the experimental substrate selectivity and stereospecificity. The intrinsic reaction coordinates (IRC) suggesting different reduction mechanisms for these substrates having a possible link with the distinct kinetic behavior of the substrates, at the atomic level, is presented. In addition, the distinct infrared absorption bands for the hydroxyl and carbonyl groups make a strong case for using Fourier transform infrared spectroscopy (FTIR) for quantifying Cp mediated redox reactions, usually done by the elaborate and sometimes time consuming gas chromatography (GC) and/or high performance liquid chromatography (HPLC). Thus, the current work also includes developing a quick FTIR based assay to monitor Cp mediated oxidations.

Publications:

1. Sneha Sudhakara and Anju Chadha (2015) A fourier transform infrared spectroscopy (FTIR) based assay for Candida parapsilosis ATCC 7330 mediated oxidation of aryl alcohols, Journal of Biotechnology, 209, 102-107.

2. Sneha Sudhakara and Anju Chadha (2017) A carbonyl reductase from Candida parapsilosis ATCC 7330: substrate selectivity and enantiospecificity, Organic and Biomolecular Chemistry, 15, 4165-4171.