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    Measurement of Cellulase Activity

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    This document outlines the procedure for measuring cellulase activity using a filter paper assay. It describes preparing a sodium citrate buffer at pH 4.8 to dissolve the cellulase enzyme. Filter paper, cotton, or sodium carboxymethyl cellulose are used as substrates. The substrate and enzyme are incubated at 50°C for 60 minutes, then stopped with DNS reagent. Absorbance is measured at 540nm after boiling and dilution, with glucose standards used to calculate activity units.

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    Measurement of Cellulase Activity

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    [email protected]
    76 views2 pages
    This document outlines the procedure for measuring cellulase activity using a filter paper assay. It describes preparing a sodium citrate buffer at pH 4.8 to dissolve the cellulase enzyme. Filter paper, cotton, or sodium carboxymethyl cellulose are used as substrates. The substrate and enzyme are incubated at 50°C for 60 minutes, then stopped with DNS reagent. Absorbance is measured at 540nm after boiling and dilution, with glucose standards used to calculate activity units.

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    Measurement of Cellulase activity

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    This document outlines the procedure for measuring cellulase activity using a filter paper assay. It describes preparing a sodium citrate buffer at pH 4.8 to dissolve the cellulase enzyme. Filter paper, cotton, or sodium carboxymethyl cellulose are used as substrates. The substrate and enzyme are incubated at 50°C for 60 minutes, then stopped with DNS reagent. Absorbance is measured at 540nm after boiling and dilution, with glucose standards used to calculate activity units.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    76 views2 pages

    Measurement of Cellulase Activity

    Uploaded by

    [email protected]
    This document outlines the procedure for measuring cellulase activity using a filter paper assay. It describes preparing a sodium citrate buffer at pH 4.8 to dissolve the cellulase enzyme. Filter paper, cotton, or sodium carboxymethyl cellulose are used as substrates. The substrate and enzyme are incubated at 50°C for 60 minutes, then stopped with DNS reagent. Absorbance is measured at 540nm after boiling and dilution, with glucose standards used to calculate activity units.

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    © All Rights Reserved
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    Measurement of Cellulase activity (Filter paper assay)

    Preparation of sodium citrate buffer (0.05 M, pH 4.8)

    Preparation of Stock sodium citrate buffer solution (10 ml, 1M)

    Tri-sodium citrate dihydrate  2.94 g

    water  7.50 mL

    Add NaOH until pH equals 4.3

    Dilute to 10 ml and check pH.

    If necessary, add NaOH until the pH is 4.5.

    Then the 1 M stock citrate buffer stock is diluted with water to 0.05 M citrate buffer the pH
    should be 4.8. After diluting the citrate buffer check and adjust the pH if necessary, to pH 4.8

    Enzyme preparation

    Dissolve 40 mg of enzyme in 10 ml 0.05 M citrate buffer

    Store at 4 °C

    Substrate

    Filter paper -> 50 mg

    Cotton -> 50 mg

    Sodium carboxymethyl cellulose -> 50 mg

    Add substrate to the test tubes.

    Add 1.0 mL 0.05 M citrate buffer, pH 4.8 to the tube; the buffer should saturate the substrate

    Equilibrate tubes with buffer and substrate to 50 °C.

    Add 0.5 mL enzyme

    Incubate at 50°C for exactly 60 min.

    At the end of the incubation period, remove each assay tube from the 50 °C bath and stop the enzyme
    reaction by immediately adding 3.0 mL DNS reagent and mixing. *
    Blank and controls:

    Reagent blank: 1.5 mL citrate buffer.

    Enzyme control: 1.0 mL citrate buffer + 0.5 mL enzyme

    Substrate control: 1.5 mL citrate buffer + filter-paper strip.

    Glucose standards:

    A working stock solution of anhydrous glucose (10 mg/mL) should be made up.

    Dilutions are made from the working stock in the following manner:

    1.0 mL + 0.5 mL buffer => 6.7 mg/mL

    1.0 mL + 1 mL buffer => 5 mg/mL

    1.0 mL + 2 mL buffer => 3.3 mg/mL

    1.0 mL + 4 mL buffer => 2 mg/mL

    Glucose standard tubes should be prepared by adding 0.5 mL of each of the above glucose dilutions to
    1.0 mL of citrate buffer test tube.

    Blanks, controls and glucose standards should be incubated at 50°C along with the enzyme assay tubes,
    and then "stopped" at the end of 60 minutes by addition of 3.0 mL of DNS reagent.

    _________________________________

    *Now, boil all tubes for exactly 5.0 minutes in a boiling water bath containing sufficient water to cover
    the portions of the tubes occupied by the reaction mixture plus reagent.

    All samples, controls, blanks, and glucose standards should be boiled together. After boiling, transfer to
    a cold ice-water bath.

    Dilute all tubes (assays, blanks, standards and controls) in water (0.20 mL of color-developed reaction
    mixture plus 2.5 mL of water in a spectrophotometer cuvette. Use the pipettor to mix.

    Determine color formation by measuring absorbance against the reagent blank at 540 nm.

    Reference:

    Ghose, T. K. (1987). Measurement of cellulase activities. Pure and applied Chemistry, 59(2), 257-268.

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