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

Author:
He, Fengping, Pan, Yonggui
Source:
Lebensmittel-Wissenschaft + [i.e. und] Technologie 2017 v.79 pp. 402-409
ISSN:
0023-6438
Subject:
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
Abstract:
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.
Agid:
5610071