Isolation, purification and characterization of extracellular pectinase produced by Valsa mali

Abstract:

[Objective] The purpose of this study was to separate and purify the pectinase of Valsa mali, and explore the corresponding enzymatic properties. [Methods] Valsa mali was cultured in MS medium containing 0.5% starch, and maintained for different days. The pectinase was extracted using ammonium sulphate gradient fractionation and purified by Sephacryl S-100 gel fitration and DEAE-Sepharose Fast Flow ion exchange chromatography. The purity was examined by SDS-PAGE. The properties of rhamnose galacturonic acid enzyme were studied using biochemical techniques. [Results] The result showed that the activity of the pectinase was highest after 10 days’ fermentation. The pectinase was identified to be rhamnose galacturonic acid enzyme. The properties including the molecular mass, the isoelectric point, the optimal temperature and pH were 58.83 kD, 6.03, 40 °C and 3.5, respectively. The rhamnose galacturonic acid enzyme was stable when its pH varied from 2.0 to 5.5. Ca2+, Li+, Co2+ could active the rhamnose galacturonic acid enzyme, while the metal ions of K+, Fe2+, Pb2+, Zn2+, Cu2+, Mn2+, Ni+ could inhibit it, especially Ba2+ and Mg2+. Besides, the kinetic constants was also detected, and the Km and Vm values were 3.600 g/L and 0.162 7 g/(L·min), respectively. [Conclusion] The rhamnose galacturonic acid enzyme was isolated from the fermentation liquor of Valsa mali, and enzymatic properties were explored. The results will lay the foundation for the preparation of antibody to pectinase and studies of cytochemistry.

Key words:Valsa mali, Pectinase, Separation and purification, Enzymatic property