Purification of Pectin Lyase fromByssochlamys fulva: Its Application in Wine Fermentation

Abstract

Pectin lyase from Byssochlamys fulva was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. The purified pectin lyase exhibited single band of molecular mass, 29 kDa, as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme had an optimum activity at pH 4.5, reaction temperature at 25C and incubation time at 60 min. It was activated by Ca2+, Mg2+, sodium arsenate, L-cysteine, ascorbic acid and β-mercaptoethanol, and significantly inhibited by Zn2+, Mn2+, Fe3+, sodium dodecyl sulfate (SDS), mercuric chloride, cinnamic acid, chlorogenic acid, p-coumaric acid, ferulic acid and salicylic acid. It had specificity toward polygalacturonic acid (0.1% w/v). Purified enzyme was used for apple juice fermentation and clarification of wine. Fermentation was carried out for 30 days. The amount of carbohydrates decreased from 160 to 142.28 μg/mL; the amount of phenolics increased from 0.42 to 1.02 mg/mL and ethanol from 6.6 to 9.01%, respectively, when juice was treated with pectin lyase. Practical Applications Pectinases play a significant role in many industrial applications such as in fruit juice, wine fermentation, tea and coffee fermentation, waste water treatment, textile and paper-making industries. In winemaking, pectin lyase preparations, often in combination with cellulase and hemicellulase, are used to increase the degradation of skin cell walls in order to obtain an increased pressing yield and improved clarification processes, extraction of color and aroma precursors during maturation. Pectinases can be used to prevent filter clogging prior to bottling. Purification of enzymes is very important to understand their affinity for particular substrates. Most of the commercial applications of enzymes do not always need their homogeneous preparation; a certain degree of purity is required. For the extraction and clarification of apple wine, significant results were observed using purified pectin lyase in comparison with both positive (inactive enzyme was added) and negative (no external enzyme was added) control.