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Isolation, Purification, and Characterization of a Polygalacturonase Produced in Penicillium solitum-Decayed ‘Golden Delicious’ Apple Fruit

First, third, fourth, and sixth authors: Food Quality Laboratory, United States Department of Agriculture--Agricultural Research Service (USDA-ARS), BARC-West, Beltsville, MD; second author: Institute of Phytomedicine, Faculty of Agriculture, University of Belgrade, Serbia; and fifth author: Appalachian Fruit Research Station, USDA-ARS, Kearneysville, WV.

Polygalacturonase (PG) was extracted and purified from decayed ‘Golden Delicious’ apple fruit inoculated with Penicillium solitum. Ammonium sulfate, gel filtration, and cation exchange chromatography were used to purify the enzyme. Both chromatographic methods revealed a single peak corresponding to PG activity. The purified PG most likely originates from the fungus because PG activity from healthy and wounded apple tissue was undetectable. Analysis of cation exchange-purified material using sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single 50-kDa band. The enzyme was active over a broad pH range (3 to 7), with optimal activity between pH 4 and 5. PG was highly active at 20 and 37°C but was also detectable at 2, 50, and 75°C. Divalent cations affected PG enzyme activity; Mg and Fe increased, whereas Ca and Mn reduced activity in vitro. Thin-layer chromatographic separation of hydrolysis products and data from a PG plate activity assay based on staining with ruthenium red showed that the enzyme exhibits both exo and endo activity. Purified PG incubated with intact apple fruit tissue in vitro caused a 30% reduction in mass after 48 h, suggesting a role in P. solitum-mediated decay of apple fruit.

Additional keywords:maceration, postharvest decay.