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 Award | Jan 2019 |
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Original language | English (US) |
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Awarding Institution | - Biological, Environmental Sciences and Engineering
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Supervisor | Magnus Rueping (Supervisor) |
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- Biocatalysis
- DNA polymerase
- Artificial Metalloensymes
- protein engineering
- Halo-thermophilic
- Streptavidin-Biotin