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

- The purified chalcone isomerase (CHI) from fresh-cut CWC was obtained through sequential chromatography.
- The molecular weight of CHI was estimated with SDS-PAGE to approximately 14.4 kDa.
- The spectral and HPLC analysis demonstrated that the end enzymatic product of purified CHI was naringenin.
- The characterization of purified CHI was studied.
- The Km (2.91 mmol/L) and Vmax (45.25 U/mL) of purified CHI for naringenin chalcone were extracted.

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.