TY  - THES
AB  - Broadening the scope of biocatalysts could be achieved by giving new functionality to non-functional proteins or by improving enzymatic features such as stability and catalytic activity. To develop new biocatalysts for application in organic synthesis, three enzymes were introduced in this study.<br />
The metalloprotein MoWSto from Azotobacter. Vinelandii was chosen for organic synthesis due to its large metal loading in the enzyme cavity. The tungstate amounts in MoWSto was measured as 20 ions per protein molecule after purifications. Chemical synthetic condition of acetophenone from (R,S)-1-phenylalcohol was modified for the biocatalytic oxidation. Subsequently, the Metalloprotein was used for the oxidation in combination with 3-5% hydrogen peroxide and phase transfer catalyst at 50 °C. The maximum conversion using cell crude extract was 12.4% in 13 days at 50 °C with 0.14 mM tungstate in MoWSto, indicating that oxidation proceeded slowly in the presence of metalloenzyme. However, the repeated experiments with tungstate salt and crude extracts showed lower conversion; furthermore, protein degradation was accelerated in the reaction solution. On the other hand, the moderative kinase activity was observed from MoWSto that suggests the possibility for utilization in the production of metabolic intermediates.
For organic synthesis using biocatalysts in the pharmaceutical industry, OCR was a perfect example of engineering of an enzyme to increase catalytic specificity. The substrate specificity of a new type of carbonyl reductase OCR toward pitavastatin intermediates was improved by error-prone PCR. After directed evolution of OCR, The productivity was dramatically enhanced up to 70% from 15% toward 10 g·L-1 DOXE and up to 95% from 60% toward 2.6 g·L-1 MOLE using OCR mutant D54V. To enhance the mixing condition, a segmented flow reactor was applied. OCR successfully converted 2,2,2-trifluoroacetophenone to the corresponding alcohol, showing approximately 70% conversion in 2 h in the presence of MTBE as an organic solvent. The segmented flow reactor improved phase separation to a great extent and showed the same conversion quality per each residence time.<br />
Finally, aldoxime dehydratase isolated from Rhodococcus sp. YH3-3 was constructed to the recombinant enzyme in E. coli. The optimal condition for overexpression was observed using overexpression vector pET28b containing C-terminus histidine tag and BL21(DE3) as a host cell. Directed evolution of OxdYH3-3 was conducted to improve the substrate loading up to 100 mM. OxdYH3-3 mutant N266S showed four and three times higher activities toward 50 and 100 mM E-pyridine-3-aldoxime, respectively. Also, the mutant exhibited 4 and 5 times higher activities towards 2-furfuryl aldoxime compared to OxdYH3-3 WT. Since 2-furfuryl aldoxime has high potential as fine chemicals, the increment of substrate loading implies the utilization of OxdYH3-3 mutants in the food ingredient industry or pharmaceuticals.
DA  - 2020
LA  - eng
PY  - 2020
TI  - Development of Biocatalysts for Organic Synthesis by Protein Engineering based on Metal Replacement and Directed Evolution
UR  - https://nbn-resolving.org/urn:nbn:de:0070-pub-29437298
Y2  - 2024-11-22T02:22:14
ER  -