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@ARTICLE{Spielmann:872939,
      author       = {Spielmann, Alina and Brack, Yannik and van Beek, Hugo and
                      Flachbart, Lion and Sundermeyer, Lea and Baumgart, Meike and
                      Bott, Michael},
      title        = {{NADPH} biosensor-based identification of an alcohol
                      dehydrogenase variant with improved catalytic properties
                      caused by a single charge reversal at the protein surface},
      journal      = {AMB express},
      volume       = {10},
      number       = {1},
      issn         = {2191-0855},
      address      = {Heidelberg},
      publisher    = {Springer},
      reportid     = {FZJ-2020-00399},
      pages        = {14},
      year         = {2020},
      note         = {Biotechnologie 1},
      abstract     = {Alcohol 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.},
      cin          = {IBG-1},
      ddc          = {570},
      cid          = {I:(DE-Juel1)IBG-1-20101118},
      pnm          = {581 - Biotechnology (POF3-581)},
      pid          = {G:(DE-HGF)POF3-581},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:31955268},
      UT           = {WOS:000514581800005},
      doi          = {10.1186/s13568-020-0946-7},
      url          = {https://juser.fz-juelich.de/record/872939},
}