University of Natural Resources and Life Sciences, Vienna (BOKU) - Research portal
Gewählte Master / Diploma Thesis:
Amal Chandra Manidas
(2013):
Investigations of substrate specificity and physiological relevance of chalcone 3-hydroxylase using genetically modified organism.
Master / Diploma Thesis - Abteilung Pflanzenbau,
BOKU-Universität für Bodenkultur,
pp 80.
UB BOKU
obvsg
- Abstract:
- Chalcone 3-hydroxylase (CH3H) is a cytochrome P450 dependent monooxygenase which catalyzes the introduction of hydroxyl group in position 3 (B-ring) of chalcones. Two heterologous expression systems, one with yeast cells (Strep-Tag) and one with bacterial cells E. coli (pGEX-6P-1 with GST tag) were used to test functional activity, thereby confirming identity of the cloned sequence as CH3H. P450 reductase also was heterlogously expressed in E.coli support the CH3H functional activity. Functional activity of recombinant CH3H was checked with isoliquiritigenin and naringenin. The results showed that recombinant CH3H was not functionally active from the heterologous expression with E.coli but was active in Strep-Tag system with both substrates. The conversion rate with isoliquiritingenin (ISO) was 88.95% while with naringenin (NAR) was 93.06%. Four mutations were performed to test the relevance of 4 specific sites on isoliquiritigenin acceptance. Functional activity studies with recombinant CH3H and mutants showed that all mutants were functionally active but conversion rates were generally lower compared to the recombinant parent protein. During the characterization of recombinant Dahlia CH3H, highest activity was observed at a pH of 6.5 (with isoliquiritigenin) and 6.25 (with naringenin) and a temperature of 20°C. The reaction was linear with time until 20 minutes and amount of protein was 15 μg with both substrates. Michaelis–Menten kinetics for recombinant Dahlia CH3H indicated that CH3H prefers isoliquritingenin over naringenin
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