Development of reproducible assays for polygalacturonase and pectinase

• Developed previously missing reproducible assays for polygalacturonase/pectinase.
• Optimal assay reaction time is 30 min to provide consistent results.
• 5 g/L is chosen substrate concentration to avoid substrate inhibition or limitation.
• Optimal enzyme activity ranges established for obtaining reproducible assay results.
• Substrate preparation and storage methods were studied.

Polygalacturonase and pectinase activities reported in the literature were measured by several different procedures. These procedures do not give comparable results, partly owing to the complexity of the substrates involved. This work was aimed at developing consistent and efficient assays for polygalacturonase and pectinase activities, using polygalacturonic acid and citrus pectin, respectively, as the substrate. Different enzyme mixtures produced by Aspergillus niger and Trichoderma reesei with different inducing carbon sources were used for the method development. A series of experiments were conducted to evaluate the incubation time, substrate concentration, and enzyme dilution. Accordingly, for both assays the recommended (optimal) hydrolysis time is 30 min and substrate concentration is 5 g/L. For polygalacturonase, the sample should be adjusted to have 0.3–0.8 U/mL polygalacturonase activity, because in this range the assay outcomes were consistent (independent of dilution factors). Such a range did not exist for the pectinase assay. The recommended procedure is to assay the sample at multiple (at least 2) dilution factors and determine, by linear interpolation, the dilution factor that would release reducing sugar equivalent to 0.4 g/L d-galacturonic acid, and then calculate the activity of the sample accordingly (dilution factor × 0.687 U/mL). Validation experiments showed consistent results using these assays. Effects of substrate preparation methods were also examined.