The commercial pectinase (polygalacturonase) was immobilized on celite through adsorption. There was 2.5% concentration of glutaraldehyde used as cross-linking agent and a binding time of 4 h was found to be the best time for its proper binding. Binding efficiency of enzyme to the matrix was 43.18%. The immobilized enzyme retained almost 50% of activity after third cycle of reuse. Free enzyme showed maximum activity with polygalacturonic acid (PGA, 0.9%) as substrate at 40C temperature, pH 5.5 and incubation time of 20 min while immobilized enzyme showed maximum activity with PGA (0.9%) as substrate at temperature of 45C, pH 5.5 and incubation time of 15 min. Both free and immobilized enzyme were inhibited by metal ions, thiols, phenolics and protein inhibitors. For clarification of 1.0 mL of pineapple juice, 20 mg of immobilized enzyme per mL of juice at 45C temperature and 1 h of holding time was optimized. PRACTICAL APPLICATIONSPectinases find commercial application in fruit juice, wine, oil, tea, coffee, textile and paper-making industries using a wide variety of carriers and methods. One of the vital applications is the clarification and depectinization of fruit juices. The raw fruit juice obtained after pressing is very turbid viscous and contains a significant amount of colloidal compounds, mainly pectin that causes cloudiness; therefore, clarification of fruit juices involves the removal of juice haze by enzyme hydrolysis with pectolytic enzymes. Although pectinases enhance the clarification of juices, immobilization of these enzymes proves to be beneficial for industrial use. Immobilization of pectinase on celite through adsorption is a simple, cheap and effective method. For the clarification of pineapple juice, excellent results were observed using immobilized polygalacturonase in comparison with free enzyme.

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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.