In our previous study, we created PETase mutant S245I, which was shown to exhibits a higher enzymatic activity than that of WT PETase using 4-nitrophenyl dodecanoate as a substrate. In this study, HPLC analysis of digested PET film revealed the presence of intermediate product of PET depolymerization, MHET, suggesting that PET cannot be completely depolymerized by S245I PETase alone. Therefore, we added a second enzyme, MHETase to develop a dual-enzyme system to enhance complete PET degradation. The dual enzyme system is developed in the form of chimeric protein linking S245I PETase and MHETase with a linker or enzyme cocktail containing S245I PETase and MHETase mixed in a single reaction.
Our results demonstrated that PET degradation achieved by enzyme cocktails of S245I PETase and MHETase synergized the degrading efficiency compared with that achieved by single enzyme, S245I PETase alone.
Additionally, chimeric protein of S245I PETase-MHETase was successfully expressed and purified. Preliminary studies suggest that they have PET depolymerization activity. Therefore, our results clearly shows that enzyme cocktail consisting of engineered mutant, S245I PETase, which outperforms WT PETase, further synergizes PET depolymerization with the addition of MHETase. It provides insights into the potential use of the dual enzyme system in industrial PET biodegradation. The detail of the findings are also outlined in the section Experiments and Results.
To study the effect of enzyme cocktail consisting of two enzymes or the S245I PETase-MHETase chimeric protein on PET degradation, commercial PET films were immersed in enzyme solutions containing S245I PETase and MHETase in a 1:1 or 1:2 ratio or 8μL and 16μL chimeric proteins (Figure 1). We analyzed the eluent after the PET film digestion by HPLC and digested PET films were observed using SEM.
Figure 1. Experimental procedure of PET film digestion.
Maximum intensity: 7756.7 cps at 4.63 min
Figure 2a. HPLC profile of products released from PET film digestion using 4 μg S245I PETase and 4 μg MHETase
Maximum intensity: 8166.7 cps at 4.62 min
Figure 2b. HPLC profile of products released from PET film digestion using 4 μg S245I PETase and 8 μg MHETase
Maximum intensity: 4906.7 cps at 4.63 min
Figure 2c. HPLC profile of products released from PET film digestion using 4 μg S245I PETase only
Maximum intensity: 2193.3 cps at 4.63 min
Figure 3. HPLC profile of products released from PET film digestion without using enzyme (negative control)
HPLC data demonstrated that the enzyme cocktail with S245I PETase and MHETase synergizes PET degradation. A mixture of S245I PETase and MHETase in a 1:2 ratio shows similar degradation activ ity with that in a 1:1 ratio but both exhibited better degradation activity than the S245I PETase alone as more TPA were determined in the cocktails (Figure 2a – c). Comparing the extent of PET degradation by S245I PETase alone, addition of MHETase synergizes depolymerization process by increase constituent monomer, TPA, up to 1.7 folds.
Figure 4a. PET film after incubation with 4μg S245I PETase and 4μg MHETase
Figure 4b. PET film after incubation with 4μg S245I PETase and 8μg MHETase
Figure 4c. PET film after incubation with 4μg S245I PETase only
Figure 4d. PET film after incubation with 4μg buffer only
Consistent with HPLC data, the pitting of PET film surface resulting from the digestion of 1:1 and 1:2 ratio of S245I PETase and MHETase is much more significant than from the digestion of S245I PETase only (Figure 4a – c)
Our HPLC and SEM results demonstrated that cocktail mixtures of 1:1 or 1:2 ratio of S245I PETase and MHETase synergize PET depolymerization process compared with S245I PETase alone.
On the other hand, chimeric protein S245I PETase-MHETase was successfully expressed and purified. Preliminary data of HPLC and SEM suggested it exhibits PET depolymerization activity (Please see Experiments and Results). However, other unwanted proteins were also co-purified, making comparison of hydrolytic activity of S245I PETase and MHETase cocktail and chimera difficult. We speculate that chimeric protein may have a better hydrolytic activity as the fusion protein would bring PETase degraded products in close proximity with MHETase. We will optimize our protein extraction protocol and review the design of linker in order to obtain a single chimeric protein for comparison.
Chemical recycling of plastic wastes into monomers is an emerging field of innovative technology. It represents an ultimate closed-loop system to create a circular plastic economy. Our dual enzyme system using engineered PETase mutant, S245I PETase, and MHETase synergizes PET depolymerization, adding value and contribution to the plastic recycling ecosystem. To bring this to life, we have to scale up the production process and address the degradation potential and productivity of the enzyme system. Factors such as time needed for whole bottle degradation, enzyme concentration required per gram of PET, optimal ratio of two enzymes and the cost of enzymes and reactions need to be taken into consideration. Furthermore, it is also important to demonstrate the use of resulting purified TPA and EG monomers to synthesize PET, and ultimately into bottles to complete the recycling loop.
