Determination of Optimal Assay Conditions for Olygalacturonase

Chen, Wei-Da, Brian Roy, and Mary Lou Caspers

The long term goal of this project is to determine if a correlation exits between the activity of polygalacturonase (PGase) and the quality of coffee produced in small plantations in Nicaragua. PGase, also known as pectinase, catalyzes the hydrolysis of α1-4 galactosidic bonds in pectin, a complex polysaccharide found in fruit including coffee beans. PGase is assayed by the release of galacturonic acid from polygalacturonic acid in 0.05 M sodium acetate buffer pH 5.5. Galacturonic acid reacts with 3,5 dinitrosalicyclic acid (DSN) reagent (1% DSN, 0.2% phenol, 0.05% sodium sulfite and 1% sodium hydroxide) to produce a colored product which absorbs visible light at 575 nm. The amount of galacturonic acid produced by PGase can be quantitated by comparison to a standard curve of known concentrations of galacturonic acid. Preliminary results indicate that increasing the amount of PGase from 5 to 22 mUnits produces increasing amounts of galacturonic acid. A mUnit is defined as the amount of PGase required to produce 1 nmole of galacturonic acid per min at 37 oC. Furthermore, PGase activity produces increasing amounts of galacturonic acid as the assay time increases up to 20 minutes. Finally, PGase exhibits first-order kinetics as the concentration of substrate polygalacturonic acid increases up to 3.75 mg/mL. Once the exact conditions to optimize PGase activity have been ascertained, assay of coffee mucilage samples will begin.