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Purification and characterization of chalcone isomerase from fresh-cut Chinese water-chestnut

He, Fengping, Pan, Yonggui
Lebensmittel-Wissenschaft + [i.e. und] Technologie 2017 v.79 pp. 402-409
EDTA (chelating agent), ammonium sulfate, ascorbic acid, beta-mercaptoethanol, calcium, chalcone, chalcone isomerase, citric acid, copper, enzyme activity, high performance liquid chromatography, iron, molecular weight, naringenin, octoxynol, pH, polyacrylamide gel electrophoresis, polysorbates, proteins, sodium, sodium dodecyl sulfate, temperature, zinc
Chalcone isomerase (CHI) from fresh-cut Chinese water-chestnut was isolated and purified by precipitation with ammonium sulfate (35%–80%), sequential chromatography using DEAE-cellulose (DE-52) and Sephadex™ G-100. The purified CHI was obtained with a 12.35-fold purity, a yield of 6.86% and a specific activity of 125 U/mg·proteins. The molecular weight was estimated to approximately 14.4 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The spectral and HPLC analysis demonstrated that the end enzymatic product was naringenin. Further analysis based on enzymatic characterization indicated the optimal temperature was 45 °C, as well as pH was 7.5. The CHI activity was significantly stimulated by Ca2+ and Cu2+, but inhibited by Zn2+, Fe2+ and Na+. A significant active effect of DTT, Triton X-100 and PVP on the enzyme activity was observed, while a negative correlation was found between the CHI activity and the chemical compounds of β-mercaptoethanol, SDS and Tween 80. In addition, EDTA, ascorbic acid, sodium ascorbate and citric acid presented no significant (p > 0.05) influence on CHI activity. Finally, the Km (2.91 mM) and Vmax (45.25 U/mL) for naringenin chalcone were obtained from the Lineweaver-Burk plot. Therefore, the properties of CHI could contribute to controlling the yellowing of fresh-cut CWC.