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000872939 1001_ $$0P:(DE-Juel1)166526$$aSpielmann, Alina$$b0
000872939 245__ $$aNADPH biosensor-based identification of an alcohol dehydrogenase variant with improved catalytic properties caused by a single charge reversal at the protein surface
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000872939 520__ $$aAlcohol dehydrogenases (ADHs) are used in reductive biotransformations for the production of valuable chiral alcohols. In this study, we used a high-throughput screening approach based on the NADPH biosensor pSenSox and fluorescence-activated cell sorting (FACS) to search for variants of the NADPH-dependent ADH of Lactobacillus brevis (LbADH) with improved activity for the reduction of 2,5-hexanedione to (2R,5R)-hexanediol. In a library of approx. 1.4 × 106 clones created by random mutagenesis we identified the variant LbADHK71E. Kinetic analysis of the purified enzyme revealed that LbADHK71E had a ~ 16% lowered KM value and a 17% higher Vmax for 2,5-hexanedione compared to the wild-type LbADH. Higher activities were also observed for the alternative substrates acetophenone, acetylpyridine, 2-hexanone, 4-hydroxy-2-butanone, and methyl acetoacetate. K71 is solvent-exposed on the surface of LbADH and not located within or close to the active site. Therefore, K71 is not an obvious target for rational protein engineering. The study demonstrates that high-throughput screening using the NADPH biosensor pSenSox represents a powerful method to find unexpected beneficial mutations in NADPH-dependent alcohol dehydrogenases that can be favorable in industrial biotransformations.
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000872939 7001_ $$0P:(DE-Juel1)171836$$aBrack, Yannik$$b1
000872939 7001_ $$0P:(DE-Juel1)167342$$avan Beek, Hugo$$b2
000872939 7001_ $$0P:(DE-Juel1)162145$$aFlachbart, Lion$$b3
000872939 7001_ $$0P:(DE-Juel1)178698$$aSundermeyer, Lea$$b4
000872939 7001_ $$0P:(DE-Juel1)145489$$aBaumgart, Meike$$b5
000872939 7001_ $$0P:(DE-Juel1)128943$$aBott, Michael$$b6$$eCorresponding author
000872939 773__ $$0PERI:(DE-600)2621432-5$$a10.1186/s13568-020-0946-7$$gVol. 10, no. 1, p. 14$$n1$$p14$$tAMB express$$v10$$x2191-0855$$y2020
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