*2.2. Characterization of Optimal Reaction Temperature and pH of the Crude Glucoamylase Extract*

Optimal pH and reaction temperature of the crude GA extract were determined. The results are shown in Figure 2. In order to determine the optimal reaction pH of the crude GA extract, assays were carried out at various pHs from 3.5 to 7.5 and the results are indicated in Figure 2A. The maximal enzyme activity was obtained at pH 5.5, indicating it was the optimal pH for starch hydrolysis. Fungal GAs from *Aspergillus* strains are usually active at acidic pH, their enzyme activities vary from pH 3.5 to 7 depending on the strains and amino acid sequences (isoform) [3]. Similarly, GA assay was conducted at different temperatures as indicated in Figure 2B from 40–75 °C with 5 °C increment at pH 5.5 in order to determine the optimal reaction temperature for the crude GA extract and assuming that the pH factor is independent of the temperature factor. A significant increase in enzyme activity was observed as temperature increases from 40 to 55 °C. Maximal GA activity was observed from 55 to 65 °C suggesting the range for optimal reaction temperature of the crude GA extract and in accordance with optimal reaction temperatures in the range of 50 to 60 °C usually found for GAs from *Aspergillus* [3]. Further increase in reaction temperature greatly reduced the enzyme activity most likely due to enzyme denaturation.

**Figure 2.** Effect of (**A**) pH at 55 °C and (**B**) temperature at pH 5.5 on crude GA extract activity. Experiments were duplicated. The mean values are plotted and the standard errors are reported.

*2.3. Thermo-Stability of the Crude Glucoamylase Extract at Optimal Reaction Temperatures*

Since high GA activity was observed at 55, 60 and 65 °C, thermo-stability of the crude GA extract at these temperatures was further investigated in order to determine the optimal digestion temperature for the subsequent food waste hydrolysis experiment. The residual enzyme activity after heated at 55, 60 and 65 °C for over 90 min is shown in Figure 3. The rate constant (kd, min<sup>í</sup><sup>1</sup> ) for the first-order thermal deactivation was determined from the slope of the deactivation time course as shown in Figure 3 using Equation (1) [18], where Et is the residual GA activity after heat treatment for time t. E0 is the initial enzyme activity before heat treatment. The half-life of thermal deactivation (t1/2) was determined according to Equation (2) [19]. The thermal deactivation of the crude GA extract exhibited a linear relationship showing that it followed first-order kinetics as reported [20]. The thermal deactivation rate constant kd (min<sup>í</sup><sup>1</sup> ) of the crude GA extract at 55 °C was found approximately 10 times slower than the rates at 60 and 65 °C, suggesting the crude GA is more thermo-stable at 55 °C. The kd of the crude GA extract at 55 °C was 2.20 × 10<sup>í</sup><sup>3</sup> in comparison with 2.13 × 10<sup>í</sup><sup>2</sup> and 2.17 × 10<sup>í</sup><sup>2</sup> at 60 and 65 °C, respectively. The half-life (t1/2) of the enzyme extract at 55 °C was 315 min in comparison with 32.5 and 31.9 min for 60 and 65 °C, respectively (Table 2). Since the enzymatic activity of the crude GA extract at 55 °C was close to the activity at 60 and 65 °C, but more stable, it was adopted as the optimal digestion temperature for the subsequent food hydrolysis experiment.

$$\ln\left(\mathbf{E}\_{\mathrm{t}}\mathbf{E}\_{\mathrm{0}}\right) = -\mathbf{k}\_{\mathrm{d}}\mathbf{t} \tag{1}$$

$$\mathbf{t}\_{1/2} = \ln(\mathcal{Z}) / \mathbf{k}\_{\mathrm{d}} \tag{2}$$

**Figure 3.** Thermal deactivation of crude GA extract at (¡) 55 °C, () 60 °C and (Ÿ) 65 °C over 90 min at pH 5.5. Experiments were duplicated. The mean values are plotted and the standard errors were reported.

**Table 2.** Deactivation constant (kd) and half-lives (t1/2) of the crude GA extract at 55, 60 and 65 °C at pH 5.5.


*2.4. Application of Crude Glucoamylase Extract on Mixed Food Waste Hydrolysis for Glucose Production*

In the reality, mixed food waste is rich in salt [21] that may inhibit the enzymatic hydrolysis. To verify if the crude GA extract produced was applicable to food waste digestion for glucose production, it was used to hydrolyse the food waste which was collected from a local restaurant, under its optimal digestion conditions (at pH 5.5 and 55 °C). Increasing concentration of enzyme was added to the food waste and the time required for hydrolysis to produce glucose was determined (Figure 4). Significant difference in glucose concentration was only observed for the first hour but not after. In all cases, the food waste hydrolysis by the enzyme extracts was completed in 1 h. At the end of the hydrolysis, approximately 12 g/L glucose was produced and that was corresponding to approximately 53 g glucose produced from 100 g of mixed food waste (d.b.), while no production of glucose occurred in a control without crude GA extract. The amount of glucose produced from 100 g of mixed food waste hydrolysis was consistent with our previous study using a different hydrolysis approach [5].

**Figure 4.** Hydrolysis of mixed food waste for glucose production in the presence of crude GA extract with (z) no enzyme, (¡) 7.1 U/mL, () 14.2 U/mL and (Ÿ) 28.4 U/mL at pH 5.5 and 55 °C for 3 h. Experiments were duplicated. The mean values are plotted and the standard errors are reported.

The two commonly used approaches for cereal-based waste and food waste hydrolysis to produce glucose rich solution, include simultaneous fungus culturing and hydrolysis with the enzymes actively secreted [5,8,16,22,23] or direct addition of enzyme solution to digest the substrate [24]. In the first case, food waste hydrolysis is usually completed after 24 h [5,8,16,22,23]. In this study, the latter approach was adopted. Food waste hydrolysis by the crude GA extract was completed in 1 h under optimal conditions found. Similar experiment has been reported by *Yan et al.* using commercial GA for food waste hydrolysis. In their studies, food waste hydrolysis was completed in 2.5 h when GA to substrate ratio reached 80–140 U/g food waste [24]. When the GA to substrate ratio in the solution is reduced to 7.4 U/g substrate, 24 h was needed for complete substrate hydrolysis [16]. The higher efficiency for food waste hydrolysis (in 1 h) in this study was likely due to the higher initial GA to substrate ratio.
