Cellulase assays
Cellulase enzymes show activity during the ripening of some fruits, where their effects on cell walls results in softening of the fruit. In cases of programmed cell death, such as the formation of aerenchyma (large air spaces in the cortex of plants in flooded soils), and in the abscission zones of leaves and fruits, cellulases are once again very active, breaking down the cellulose walls of the dead cells. Two possible protocols are described here, a gel diffusion assay method and a viscosity reduction assay method.
Gel diffusion method
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A fruit extract is placed in a well in some agar containing carboxymethylcellulose (CMC), and, as cellulase molecules diffuse outwards from the well, they destroy cellulose molecules. The more concentrated the enzymes in the well, the larger the zone of cellulose destruction. |
- Prepare an agar gel containing 1.7% agar and 0.5% CMC (carboxymethylcellulose). Pour this gel into petri dishes and allow it to set.
- After the gel has set, use a narrow cork-borer to punch small cylinders in the gel. Then, using a mounted needle, remove each of these cylinders to create a series of similar sized wells in the agar. Four or more wells can be put in a single dish, provided they are spaced apart.
- Place similar volumes of extracts of fruits in the each of the wells. In one well, place some distilled water, as a control. Incubate the dishes for at least 24 hours at 30 °C.
- After the incubation period is finished, use tap water to rinse out the contents of the wells, and then flood the dishes with Congo red solution for 15 minutes. Then rinse the dishes with 1 M NaCl solution for at least 10-15 minutes.
Wells containing cellulase should have a clear zone around them, and the diameter of the zone gives a measure of the cellulase activity in that well.
Viscosity reduction method
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The technique is based on the action of cellulase enzymes which shorten the lengths of cellulose molecules in a viscous solution of wallpaper paste and cause it to become less viscous (runnier). |
- Make up a 2% (w/v) wallpaper paste solution, sufficient to provide 25 cm3 for each sample to be tested.
- Place 25 cm3 of the paste in a boiling tube and add 2 to 5 cm3 of fruit extract. Mix thoroughly.
- Then pour the mixture into the barrel of a syringe, held in a retort stand, pointing downwards into a small beaker. Note the time taken for all the mixture to drain through the syringe nozzle into the beaker.
- Incubate the mixture in a water-bath at 30°C, checking the change in viscosity about every 30 minutes.
The more active the enzyme, the greater the reduction in viscosity, and so the shorter the drainage times.
作者：
2008-2-3