Pectinase activity assay

Pectinase activity production was assessed using the 3,5-dinitrosalicylic acid (DNS) method ([29] #1193; [9] #1173), and the absorbance was measured at 540 nm with a UV-VIS spectrophotometer (Cary 50 UV-Vis, Varian, Inc., North America). Cell-free supernatant (20 μl) was mixed with 1 mL pectin (0.2%, galacturonic acid≥74.0%, Shanghai Macklin Biochemical Co., Ltd.) as the substrate using NaOH-glycine buffer (50 mmol/L, pH 9.0) and incubated at 40 °C for 30 min. DNS solution (1 mL; Beijing Leagene Biotechnology Co., Ltd., China) was added, and the reaction mixture was boiled for 5 min. The absorbance of the cooled reaction mixture was read at 540 nm, while the supernatant was boiled for 5 min to inactivate the pectinase.

A standard curve was established using D-galacturonic acid as the reducing sugar. The final concentrations of the solutions in each tube with 5 mL DNS solution were 0.008, 0.016, 0.024, 0.032, 0.040, and 0.048 mg/mL D-galacturonic acid (Beijing Leagene Biotechnology Co., Ltd.). The tubes were boiled for 5 min and cooled. A blank sample was prepared with distilled water instead of D-galacturonic acid. One unit (U) of polygalacturonase activity was defined as the amount of enzyme that generated 1 μmol galacturonic acid per min under the assay conditions. Pectinase activity was calculated using the following formula:

where ΔA is the absorbance of samples subtracted from control absorbance, N is the dilution factor, K is the slope of the standard curve, T is 30 min, and M is the molecular weight of D-galacturonic acid (M = 194.14 Da).