Chalcone isomerase from Citrus sinensis: purification and characterization
1994
Fouche, S.D. | Dubery, I.A.
A chalcone isomerase was purified to apparent homogeneity from developing fruit tissue of Citrus sinensis by gel filtration, hydrophobic interaction chromatography and anion exchange chromatography. A single protein band was observed on SDS-PAGE gels, corresponding to a Mr of 27 800. In addition, the Mr of the native protein was determined as 27 200 using analytical gel filtration. The enzyme exhibited micro-heterogeneity on native PAGE gels. Enzyme staining indicated the presence of four activity bands. Four protein bands were also observed on isoelectric focusing gels with pI values of 6.55, 6.38, 6.35 and 6.04. Charge-heterogeneity could not be ascribed to glycosylation as the enzyme was not found to be a glycoprotein. The enzyme catalysed isomerization was found to be optimal at pH 7.4; and an energy of activation of 31.9 kJ mol-1 was calculated. The enzyme cyclized only naringenin chalcone of all the aglycone substrates tested and a Km of 4 micromolar and Vmax of 67 mkat kg-1 were determined for this substrate. No activity was observed with chalcone glycosides. The turnover number was 1.87 sec-1 and the catalytic efficiency 4.62 X 10(5) M-1 sec-1. Competitive inhibition of enzyme activity was observed with naringenin (Ki = 180 micromolar), quercetin (Ki = 45 micromolar) and morin (Ki = 30 micromolar).
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