Expression, Purification and Catalytic Turnover of Mn-Quercetin Dioxygenase on Various Substrates
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Gray, Donald II
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Genetically transformed E. coli cell line was cultured to express Mn-QDO. The product was collected for use in the catalytic turnover rate studies conducted, as well as for future EPR analysis. One ~250 μL purified and quantified batch of QDO was obtained, and determined to have a QDO concentration of ~229 μM. Another unpurified batch, which showed high catalytic activity, was used for preliminary catalytic activity. The activity was studied on the native substrate quercetin and was shown to have an aerobic catalytic turnover rate, or oxygenase activity, of 3.5 x 10-4 Au/s. Nitroxygenase activity is measured using a similar analysis of an iosolectronic anaerobic reaction using HNO. The nitroxygenase activity on quercetin was 2.9 x 10-4 Au/s. The oxygenase and nitroxygenase activity on another flavonol, myricetin was determined to be 1.1 x 10-4 Au/s and 4.3 x 10-4 Au/s, respectively. An activity study toward HOPTO in buffers of pH from 5 to 8 was performed. The lower pHs seemed to correspond to the fastest rates of activity.