Identification, Cloning, and Characterization of a Novel Ketoreductase from the Cyanobacterium Synechococcus sp. Strain PCC 7942 |
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Authors: | Kathrin Hlsch Jan Havel Martin Haslbeck and Dirk Weuster-Botz |
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Institution: | Kathrin Hölsch, Jan Havel, Martin Haslbeck, and Dirk Weuster-Botz |
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Abstract: | A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44°C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 ± 2.15 U mg−1) and 2′,3′,4′,5′,6′-pentafluoroacetophenone (8.57 ± 0.49 U mg−1) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2′,3′,4′,5′,6′-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% S] versus 43.3% S]). |
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