Fig. 1. The temperature effects on the hydrolysis of LC-PET by WCCG. (a) The dependence of LC-PET hydrolytic velocity of WCCG on temperature. The reaction velocity was measured by monitoring the rate of release of soluble products in the first hour. (b) The corresponding Arrhenius plot. The activation energies, Ea1,LC and Ea2,LC, were calculated based on the slopes over the temperature ranges of 30 to 40 °C, and 50 to 70 °C, respectively. Error bars represent the standard deviations of three measurements.

Fig. 2. Thermal activation of WCCG after incubation at various temperatures. (a) Relative activity of the thermally-treated WCCG toward LC-PET. WCCG was pre-incubated at temperatures ranging from 50 to 85 °C for 1 to 6 h. The hydrolytic activity was assayed at 60 °C based on the concentrations of soluble products released in the first hour. (b) The activity-temperature dependence of the non-treated and thermally-treated WCCG. (c) Conventional Michaelis-Menten plots of LC-PET hydrolysis catalyzed by the non-treated and the thermally-treated WCCG, and the soluble WCCG*. The hydrolysis was carried out with 18 nmol L-1 enzyme at 60 °C in 1 mL phosphate buffer (50 mmol L-1, pH 8.0). Error bars represent the standard deviations of three measurements.

Fig. 3. Conformational changes of WCCG after thermal treatment. Far-UV CD spectra of WCCG* (a) and the changes in the secondary structure contents (b) of the WCCG* after 4-h incubation at 50, 55, 60, 65, 70, 75, 80 and 85 °C. Representative structures of WCCG after heat treatment, showcasing β-sheet extension (c) and α-helix uncoiling (d). The thermally-treated WCCG and non-treated WCCG were depicted in red and blue, respectively. (e) Fluorescence spectra of ANS-WCCG* complex after 4-h incubation at 50, 55, 60, 65, 70, 75, 80 and 85 °C. (f) Representative histograms of detachment force distribution of non-treated WCCG and WCCG* interacting with PET surfaces. A Gaussian fit is added to show the mean adhesion force.

Fig. 4. The temperature effects on the hydrolysis of HC-PET by WCCG. (a) The Arrhenius plot for the enzymatic hydrolysis of HC-PET. The inset shows the dependence of reaction velocity on temperature. The reaction velocity was measured by monitoring the concentration of soluble products released in the first hour. (b) Conventional Michaelis-Menten kinetics of HC-PET hydrolysis catalyzed by the non-treated WCCG, thermally-treated WCCG, and WCCG*. The hydrolysis was carried out with 18 nmol L?1 of enzyme at 60 °C in 1 mL phosphate buffer (50 mmol L?1, pH 8.0). Error bars represent the standard deviations of three measurements.

Fig. 5. The time-course changes in the degradation velocity of LC-PET (a) and HC-PET (b) by WCCG for reactions at 60, 65, and 70 °C. The insets show the corresponding changes in the crystallinity level of LC-PET and HC-PET after incubation in the same reaction buffer for 7 h. Error bars represent the standard deviations of three measurements.