Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design |
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| Authors: | Ronnie Machielsen Loren L Looger John Raedts Sjoerd Dijkhuizen Werner Hummel Hans‐Georg Hennemann Thomas Daussmann John van der Oost |
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| Institution: | 1. Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands;2. Howard Hughes Janelia Farm Research Campus, Ashburn, USA;3. Institute of Molecular Enzyme Technology, Heinrich Heine University Dusseldorf, Research Centre Julich, Julich, Germany;4. Julich Chiral Solutions GmbH, Julich, Germany |
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| Abstract: | The R‐specific alcohol dehydrogenase from Lactobacillus brevis (Lb‐ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure‐based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb‐ADH. Mutations were introduced into Lb‐ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb‐ADH mutants may prove useful for the green synthesis of pharmaceutical precursors. |
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| Keywords: | Alcohol dehydrogenase Cofactor engineering Computational protein design Lactobacillus brevis |
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