Determination of the Michaelis-Menten constant

The K_m and V_max values of the enzyme were assessed by measuring the reaction velocity at different substrate concentration and the citrus pectin was used as a substrate. A stock solution of citrus pectin with a concentration of 10 mg/mL was firstly prepared in citrate buffer (pH 4.5). The stock was subsequently diluted by a volume of citrate buffer to make a serial dilution with the final concentration of citrus pectin in between 2 mg/mL to 10 mg/mL. The pectinase activity was determined using the previously standard assay. The relationship between velocity (enzyme activity) and substrate concentration was plotted using GraphPad Prism 8 software. The Michaelis-Menten constant values (K_m and V_max) were calculated using non-regression linear.