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@PHDTHESIS{Okrob:20792,
      author       = {Okrob, Danial},
      title        = {{O}ptimierung der {H}ydroxynitril-{L}yase aus {A}rabidopsis
                      thaliana für die enantio-selektive {S}ynthese von
                      ({R})-{C}yanhydrinen: {E}ntwicklung und {E}tablierung
                      geeigneter {R}eaktionsparameter und molekulare
                      {S}tabilisierung durch rationales {E}nzymdesign},
      volume       = {53},
      school       = {Universität Düsseldorf},
      type         = {Dr. (Univ.)},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zenralbibliothek, Verlag},
      reportid     = {PreJuSER-20792},
      isbn         = {978-3-89336-782-5},
      series       = {Schriften des Forschungszentrums Jülich. Reihe Gesundheit
                      / Health},
      pages        = {XV, 135 S.},
      year         = {2012},
      note         = {Record converted from JUWEL: 18.07.2013; Universität
                      Düsseldorf, Diss., 2012},
      abstract     = {In this work the potential of the hydroxynitrile lyase from
                      $\textit{Arabidopsis thaliana}$ (AtHNL) for the
                      enantioselective synthesis of industrially important
                      cyanohydrins was demonstrated by two different principles:
                      reaction- and protein engineering. The enantiomeric excess
                      of this enzymatic reaction is strongly compromised by a
                      non-selective side-reaction resulting in racemic
                      cyanohydrins and thus lowering the enantiomeric excess of
                      the biotransformation. This non-selective product formation
                      is influenced by pH, temperature and the water content of
                      the reaction medium. For industrial applications aqueous or
                      aqueous-organic reaction systems are used where the racemic
                      product formation is suppressed by lowering the pH below pH
                      5 and running the process at $\le$ 10 °C. However, both
                      approaches are not feasible with AtHNL, since the enzyme is
                      rapidly inactivated below pH 5. In order to enable the use
                      of AtHNL for the enantioselective synthesis of industrial
                      important cyanohydrins two strategies were developed in this
                      work. The first strategy concerned the suppression of the
                      racemic product formation by reaction engineering. Thereby,
                      a micro-aqueous reaction medium (buffer-saturated
                      mono-phasic methyl $\textit{tert}$. butyl ether) was used to
                      suppress the undesired side reaction, which resulted in a
                      good enzymatic activity and high enantioselectivities for
                      several industrially important cyanohydrins. Efficient
                      cyanohydrin syntheses were reached in the established
                      reaction system by application of whole cells and the use of
                      immobilized enzyme (e.g., enzyme adsorption at celite
                      particles, encapsulation in solgel, cross-linking of enzyme
                      aggregates), espectively. Best results were obtained using
                      whole recombinant $\textit{E. coli}$ cells as well as
                      celite-adsorbed isolated AtHNL. For the synthesis of (R)-
                      mandelonitrile both preparations showed comparable catalytic
                      efficiencies, process-and storage stabilities, resulting in
                      a maximal productivity of ~1.6 g (R)-mandelonitrile per g
                      wet cell mass (after 6-8 recycling steps). The second
                      strategy was the development of a stabilized AtHNL variant
                      which is stable under acidic conditions and thus applicable
                      in industrially established aqueous-organic two-phase
                      reaction systems. Based on the highly similar HNL from
                      $\textit{Manihot esculenta}$ (MeHNL), which is stable under
                      these conditions, the protein [...]},
      cin          = {IBT-2},
      ddc          = {500},
      cid          = {I:(DE-Juel1)VDB56},
      pnm          = {Biotechnologie},
      pid          = {G:(DE-Juel1)FUEK410},
      typ          = {PUB:(DE-HGF)11 / PUB:(DE-HGF)3},
      url          = {https://juser.fz-juelich.de/record/20792},
}