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In this article we will discuss about the tests for estimation of cellulase in plants.
Principle:
Endo-β-1, 4 glucanase acts on carboxymethyl cellulose (CMC) and hydrolyses the β-1, 4 glycosidic bonds in a random manner. As a result, the viscosity of CMC solution is reduced. This is measured in an Ostwald viscometer.
Materials:
a. Citrate-phosphate Buffer 0.1 M (pH 6.0)
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b. Carboxymethyl Cellulose 1%
Dissolve 1 g carboxymethyl cellulose in 100 ml Sodium Citrate Buffer 0.1 M (pH 5.0)
c. Di-nitro-salicylic Acid (DNS) Reagent (Refer carbohydrate section)
d. 40% Rochelle salt solution (Potassium sodium tartrate).
Test Procedure:
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1. 0.45 ml of 1% CMC solution is pipetted out at a temperature of 55°C and 0.05 ml of enzyme extract.
2. The mixture is incubated at 55°C for 15 min.
3. Immediately after removing the enzyme substrate mixture from the bath add 0.5 ml DNS reagent.
4. The mixture is heated in a boiling water-bath for 5 min.
5. While the tubes are warm, add 1.0 ml potassium sodium tartrate solution.
6. Cool to room temperature.
7. Water is added to make 5 ml volume.
8. The absorbance is measured at 540 nm.
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9. A standard graph is prepared with glucose in the concentration range 50 µg to 1000 µg/ml.
Calculation:
Express the enzyme activity as the mg glucose released per min per mg protein.
