Enzyme assay

The PG assay was performed by reaction of alginate beads with 0.5 mg of protein and 0.5% (w/v) polygalacturonic acid solution as substrate in buffer 25 mM Tris–HCl pH 7. PG activity was measured by the colorimetric method with 3′,5′-dinitrosalicylic (DNS) acid reagent using d-galacturonic acid as standard for the quantification of reducing sugars released during reaction at 40 °C. Complete hydrolysis involved the release of 5 mg/mL of d-galacturonic acid, so the relation of this value to the reducing sugars produced by enzymatic reactions indicated substrate conversion.

Viscosity reduction was measured with a rheometer (AR-G2/TA Instruments) at 24 °C and 300/s shear rate. The Km and Vmax parameters of Michaelis–Menten kinetics were determined using substrate solutions at concentrations of 0.1, 0.25, 0.50, 1.00 and 1.50% (w/v). Periodically, product conversion was measured, and the results were fitted to the Lineweaver–Burk linearization plot.