Pectinase Assay

Method: One unit liberates 1 umole of D-galacturonic acid from polygalacturonic acid per minute at 37°C, pH 5.0.

Reagents

  • 0.1 M Citric Acid/Phosphate Buffer (Assay Buffer), pH 5.0
  • Color Reagent A: Dissolve 40g anhydrous Na2CO3 in 600 ml reagent grade H2O. Add 16 gm glycine and stir until dissolved. Add 0.450 gm CuSO4 5H2O, stir until dissolved. Bring to 1 liter with reagent grade H2O.
  • Color Reagent B: Dissolve 1.2 g neocuprine - HCl in 1 liter reagent grade H2O. Store at 4°C in a brown bottle.
  • D-galacturonic acid standard, 1mg/ml
  • 0.5% Polygalacturonic Acid Substrate:
  • Heat 500 ml Assay Buffer on a hot plate. While heating and stirring, slowly add 2.5 gm polygalacturonic acid. Heat and stir until dissolved. It will be slightly viscous and opaque. Do not allow solution to boil. Cool and bring back up to volume if necessary. Store at 4°C.

Enzyme

Make up fresh daily. Keep solutions on ice until used. Make serial dilutions to 0.01 mg/ml in assay buffer.

Procedure

Prepare three tubes, each containing 1 ml of buffer and 6 ml of substrate (Reagent Blanks). Prepare three tubes, each containing 6 ml of substrate and 1 ml of enzyme sample at 0.1 mg/ml (Test). Prepare three tubes, each containing 6 ml of buffer and 1 ml of enzyme sample at 0.1 mg/ml (Sample Blanks).

Incubate tubes in 37°C water bath for 60 minutes ± 1 minute. Aliquot the standard curve tubes, in triplicate, using 10 μg to 125 μg of D-galacturonic acid standard.

After the assay tubes have incubated for 60 minutes, place rack immediately into ice water to stop reaction. Aliquot from each tube 100 μl into another set of tubes (also on ice). To each reaction tube and standard tube add 2 ml of Color Reagent A and 2 ml of Color Reagent B. Mix well by inversion. Place rack into boiling water bath for 13 minutes ±1 minute. Cool tubes, then add 2ml H2O. Mix well by inversion. Read absorbance at A450 against a water blank using disposable cuvettes. Do not use a flowcell; the orange color adheres to glass. Calculate mean absorbances for each set of triplicates.

Plot a standard curve:

equation

Calculate rate of change. The curve should be linear up to 100 ug D-galacturonic acid, if not, repeat curve with fresh reagents.

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