*3.9. Enzyme Assays*

#### 3.9.1. Substrate Specificity Determination

The endo-glucanase, cellobiohydrolase II, beta-glucosidase and cellobiohydrolase I activities were determined using the CMC-Na, 1, 4-β-<sup>d</sup>-cellopentaitol, *p*NPG and *p*NPC substrates, respectively. Standard assay conditions (50 mM sodium citrate bu ffer (pH 5.0) at 50 ◦C with continuous agitation at 45 rpm) were followed as previously described [26]. One unit (U) of enzyme activity is defined as the amount of enzyme releasing 1 μmol of glucose per minute under the specified assay conditions [6,7].

#### 3.9.2. Enzyme Cocktail Formulation

The experiments were carried out in quadruplicate at a paper sludge loading of 2% ( *w*/*v* DW) in 50 mM sodium citrate bu ffer (pH 5.0) in a 400 μL total volume (with 0.1 mg/mL BSA as a stabiliser) using 1.5 mL safe-lock Eppendorf tubes. Hydrolysis took place at 50 ◦C with mixing at 45 rpm for up to 48 h. Unless otherwise specified, enzyme loadings were maintained at 1.875 mg/g paper sludge with BGL at 10% of the total protein loading (0.1875 mg/g paper sludge). The aforementioned enzyme loading was used for the synergy studies as it assured greater than 5% glucan conversion yields of paper sludge. Binary and ternary combinations between the cellobiohydrolases; CBHI and CBHII, and the endo-glucanase, EGII, were formulated (Table 3).

The optimal combination for glucose release was selected as the "core cellulase cocktail" (Opt CelMix). The hydrolysis was terminated by boiling for 5 min at 100 ◦C to inactivate the enzymes. Hydrolysis controls included substrate (without the enzyme) and enzyme controls (without substrate). The samples were stored at 4 ◦C until analysed.

#### 3.9.3. E ffect of Cellulase Cocktail Loading on the Hydrolysis Yields of Paper Sludge

The hydrolysis of 2% ( *w*/*v* DM) paper sludge by the formulated cellulase enzyme cocktail was evaluated at a protein dosage range of 1.875–15 mg protein/g of paper sludge, following the procedure outlined in Section 3.9.2.

#### 3.9.4. E ffect of Paper Sludge Loading on Cellulase Cocktail Hydrolytic E fficiency

Hydrolysis experiments were carried out at 1%, 2%, 4%, 6%, 10%, 15% and 20% ( *w*/*v*) DM consistency using paper sludge at a cellulase cocktail loading of 15 mg/g of paper sludge to evaluate the effect of paper sludge loading on cellulase enzyme cocktail hydrolytic efficiency, following the procedure outlined in Section 3.9.2.


**Table 3.** Enzyme combinations (%) at a total protein dosage of 1.875 mg/g biomass for paper sludge hydrolysis synergy studies with BGL added subsequently at 10% of the total protein dosage.

3.9.5. Comparison of the Formulated Cellulase Cocktail to Commercial Enzyme Preparations

The hydrolysis of 2% (*w*/*<sup>v</sup>*, DM) paper sludge was conducted using the Opt CelMix, Celluclast® 1.5 L, Cellic CTec2® and Viscozyme® L at a protein loading of 7.5 mg protein/g of paper sludge, following the procedure outlined in Section 3.9.2. Due to the low level of β-glucosidase activity detected in Celluclast® 1.5 L and Viscozyme® L (data not shown), these enzyme cocktails were supplemented with the β-glucosidase preparation, Novozyme® 188 (Novozymes A/S) at 10% of the total protein dosage.
