Purification and biochemical characterization of an exo-polygalacturonase from Aspergillus flavus MTCC 7589

Polygalacturonases represents an important member of pectin-degrading glycoside hydrolases of the family GH28 and is widely distributed among fungi, bacteria, yeasts, plants and some plant parasitic nematodes. Microbial polygalacturonases are widely used in food industry to clarify fruit and vegetable juice by degrading pectin. An exo- polygalacturonase from Aspergillus flavus MTCC 7589 was purified, biochemically characterized and its utility in clarification of orange juice was elucidated. The enzyme was purified by acetone precipitation (0–60%), CM- cellulose column chromatography and Gel filtration chromatography using Sephadex G-100 resulting in 8.24 fold purification with specific activity of 26.8 IU/mg protein and yield of 0.9%. A single band corresponding to a relative molecular mass of approximately 29.0 kDa was revealed by SDS-PAGE. The enzyme showed pH and temperature optima of 5 and 40 °C respectively. The K_m and kcat values of purified enzyme were 2.08 mg/mL and 96 s⁻¹ respectively. The metal ions Co²⁺, Cu²⁺ and K⁺ were found to stimulate PG activity while Ca²⁺, Ag⁺ and Hg²⁺ ions were inhibitory. The preliminary study revealed its potential in clarification of orange juice.