Methods

         Four types of wastepaper (office paper, newsprint, cardboard, paper towel) were used along with filter paper (containing almost 100% cellulose) as a positive control. Cellulases from Trichoderma viride, Trichoderma reesei, and Aspergillus niger (Sigma-Aldrich) were tested with or without the addition of cellobiase from Aspergillus niger (Novozyme® 188) for reducing sugar production. 

        In the first experiment to survey the digestibility of wastepaper with cellulases from T. viride, T. reesei, and A. niger, 100mg of cut wastepaper or filter paper was digested in 5ml of 50 mM sodium acetate buffer (pH4.8) which contained cellulases at 2 unit/ml concentration in a 37ºC incubator for 24 hrs. The total reducing sugars produced were determined with the Dinitrosalicylic Acid  Assay (DNS assay) with glucose as standard.

       The later experiments with cellulases and wastepaper were done as follows.

Equipment required:

Three shakers with temperature settings at 25°C, 37°C and 50°C

Reagents required:

50 mM sodium acetate buffer, pH 4.8

An enzyme mixture containing the cellulase Celluclast® 1.5L (Sigma-Aldrich) and the cellobiase Novazyme®188 in 50 mM sodium acetate buffer (pH 4.8).

 

Procedure:

  1. Add 0.5 g of 0.5 cm x 0.5 cm cut wastepaper or filter paper into each 50 ml flask.
  2. Add 24.8 ml 50 mM sodium acetate buffer (pH 4.8) to each flask.
  3. Add 0.2 ml enzyme mixture to each flask and cover the flask with foil.
  4. Place flasks in shakers with presetting temperatures (25°C, 37°C and 50°C) at 150 rpm
  5. An aliquot (200 ul) of the reactions is taken at 2, 4, 8, 12, 24, and 48 hours for DNS assay.

          The Dinitrosalicylic Acid  Assay (DNS assay) was employed to determine the total amount of reducing sugars in the reactions. Additionally, thin-layer chromatography (TLC) was preformed to identify and semi-quantify the glucose and cellobiose produced in the reactions.  

      

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