Engineering of novel Biocatalysts with Functionalities beyond Nature

  • Anastassja Gespers (Akal)

Student thesis: Doctoral Thesis

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.
Date of AwardJan 2019
Original languageEnglish (US)
Awarding Institution
  • Biological, Environmental Science and Engineering
SupervisorMagnus Rueping (Supervisor)

Keywords

  • Biocatalysis
  • DNA polymerase
  • Artificial Metalloensymes
  • protein engineering
  • Halo-thermophilic
  • Streptavidin-Biotin

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