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    Engineering of novel Biocatalysts with Functionalities beyond Nature

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    Anastassja Gespers (Akal) Dissertation.pdf
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    10.25Mb
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    Description:
    Anastassja Gespers (Akal) Dissertation
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    Type
    Dissertation
    Authors
    Gespers (Akal), Anastassja cc
    Advisors
    Rueping, Magnus cc
    Committee members
    Arold, Stefan T. cc
    Hamdan, Samir cc
    Stingl, Ulrich cc
    Program
    Bioscience
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Date
    2019-01
    Embargo End Date
    2020-01-21
    Permanent link to this record
    http://hdl.handle.net/10754/630912
    
    Metadata
    Show full item record
    Access Restrictions
    At the time of archiving, the student author of this dissertation opted to temporarily restrict access to it. The full text of this dissertation became available to the public after the expiration of the embargo on 2020-01-21.
    Abstract
    Novel biocatalysts are highly demanded in the white biotechnology. Hence, the development of highly stable and enantioselective biocatalysts with novel functionalities is an ongoing research topic. Here, an osmium ligating single-site ArM was created based on the biotinstreptavidin technology for the dihydroxylation of olefins. For the creation of the artificial catalytic metal center in the streptavidin (SAV) cavity, efficient osmium tetroxide (OsO4) chelating biotin-ligands were created. The unspecific metal binding of the host scaffold was diminished through genetical and chemical modification of the host protein. The created single-site OsO4 chelating ArM was successfully applied in the asymmetric cyclopropanation, revealing a stable and tunable catalytic hybrid system for application. The structural analysis of protein-ligand complexes is essential for the advanced rational design and engineering of artificial metalloenzymes. In previous studies, a SAV-dirhodium ArM was created and successfully applied in the asymmetric cyclopropanation reaction. To improve the selectivity of the SAV-dirhodium complex, the structural location of the organometallic complex in the SAV cavity was targeted and small-angle x-ray scattering (SAXS) was used to obtain the structural information. The SAXS analysis revealed valuable information of the molecular state of the complexes; hence, the method proved to be useful for the structural analysis of protein-ligand interactions. The discovery of novel enzymes from nature is still the major source for improved biocatalysts. One of the most important enzymes used in the molecular biology are DNA polymerases in PCR reactions. The halothermophilic brine-pool 3 polymerase (BR3 Pol) from the Atlantis II Red Sea brine pool showed optimal activities at 55 °C and salt concentrations up to 0.5 M NaCl, and was stable at temperatures above 95 °C. The comparison with the hyperthermophilic KOD polymerase revealed the haloadaptation of BR3 Pol due to an increased negative electrostatic surface charge and an overall higher structural flexibility. Engineered chimeric KOD polymerases with swapped single BR3 Pol domains revealed increased salt tolerance in the PCR, showing increased structural flexibility and a local negative surface charge. The understanding of the BR3 Pol haloadaptation might enable the development of a DNA polymerase tailored for specific PCR reactions with increased salt concentrations.
    Citation
    Gespers (Akal), Anastassja. (2019). Engineering of novel Biocatalysts with Functionalities beyond Nature. <i>KAUST Research Repository</i>. https://doi.org/10.25781/KAUST-C89MZ
    DOI
    10.25781/KAUST-C89MZ
    ae974a485f413a2113503eed53cd6c53
    10.25781/KAUST-C89MZ
    Scopus Count
    Collections
    Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program; Dissertations

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