Enzymatic preparation of optically pure t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate by a novel alcohol dehydrogenase discovered from Klebsiella oxytoca |
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| Institution: | 1. Centre for Environmental Management of Degraded Ecosystems, University of Delhi, Delhi 110007, India;2. Malaria Research Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India;1. State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing 100029, People’s Republic of China;2. Department of Chemistry, Biochemistry, Philipps-University Marburg, Hans-Meerwein-Strasse 4 and LOEWE-Centre for Synthetic Microbiology, D-35032 Marburg, Germany;1. Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China;2. Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041, China;3. University of the Chinese Academy of Sciences, Beijing 100049, China;1. Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China;2. Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041, China;3. University of Chinese Academy of Sciences, Beijing 100049, China;1. Department of Chemistry, Norwegian University of Science and Technology (NTNU), NO-7491 Trondheim, Norway;2. Department of Material Science and Engineering, Norwegian University of Science and Technology (NTNU), NO-7491 Trondheim, Norway |
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| Abstract: | Alcohol dehydrogenases can catalyze the inter-conversion of aldehydes and alcohols. The t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate is a key chiral intermediate in the synthesis of statin-type drugs such as Crestor (rosuvastatin calcium) and Lipitor (atorvastatin). Herein, a novel alcohol dehydrogenase (named as KleADH) discovered from Klebsiella oxytoca by a genome mining method was cloned and characterized. The KleADH was functionally overexpressed in Escherichia coli Rosetta (DE3) and the whole cell biocatalyst was able to convert t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate to t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate with more than 99% diastereomeric excess (de) and 99% conversion in 24 h without adding any expensive cofactors. Several factors influencing the whole cell catalyst activity such as temperature, pH, the effects of metal ions and organic solvent were determined. The optimum enzyme activity was achieved at 30 °C and pH 7.0 and it was shown that 1 mM Fe3+ can increase the enzyme activity by 1.2 times. N-hexane/water and n-heptane/water biphasic systems can also increase the activity of KleADH. Substrate specificity studies showed that KleADH also exhibited notable activity towards several aryl ketones with high stereoselectivity. Our investigation on this novel alcohol dehydrogenase KleADH reveals a promising biocatalyst for producing chiral alcohols for preparation of valuable pharmaceuticals. |
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| Keywords: | Alcohol dehydrogenase Whole-cell biocatalyst Rosuvastatin Atorvastatin |
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