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In 2019, our team created a successful PETase mutant, S245I which exhibits higher enzymatic activity than that of WT PETase using 4-nitrophenyl dodecanoate as a substrate. To explore further insights of the performance of our engineered PETase mutant, this year, we perform PET film digestion to compare the PET hydrolytic activities of both WT PETase and S245I PETase. After incubation of equal concentration (9 ug) of purified proteins with PET film at 30oC for 96h, we performed High Performance Liquid Chromatography (HPLC) analysis of PET digestion eluents and Scanning Electron Microscope (SEM) of digested PET film.
Figure 1a. PET film after incubation with WT PETase
Figure 1b. PET film after incubation with the PETase mutant, S245I
Figure 1c. Buffer-only control of PET film
The pitting resulting from the digestion of PETase mutant, S245I is much more significant than that from the digestion of WT PETase. Consistent with enzyme assay results shown in 2019, S245I PETase exhibits a higher depolymerization activity.
Products released from PET films after digestion with WT PETase and S245I PETase were detected and quantified by HPLC.
Figure 2a. HPLC spectrum of TPA standard.
Figure 2b. HPLC spectrum of MHET standard.
Figure 2c. HPLC spectrum of products released from the PET film digested with WT PETase.
Figure 2d. HPLC spectrum of products released from the PET film digested with S245I PETase.
Table 1. Quantification of products released from PET film digestions
HPLC profiles demonstrated that the detection peaks representing TPA monomer and MHET intermediate product formed during PET film digestion have retention times at 4.64 minutes (Figure 2a) and 5.17 minutes (Figure 2b) respectively.
A dual-enzyme system of PETase and MHETase is developed in the form of chimeras or enzyme cocktails. For the chimeric constructs covalently linking PETase and MHETase, C-terminus of both WT PETase and S245I PETase were linked to the N-terminus of MHETase using a 12 amino acid serine-glycine linker, forming WT PETase-MHETase and S245I PETase-MHETase chimeric constructs. The chimeric constructs were then cloned into the expression vector pET-21b digested with NdeI and XhoI. pET-21b has a C-terminal His-Tag for subsequent protein purification. Besides, gene encoding MHETase from Ideonella sakaiensis was also cloned into NdeI and XhoI digested pET-21b for subsequent protein purification and used in enzyme cocktail experiments.
Colony PCRWe transformed the three recombinant plasmids, MHETase-pET-21b, WT PETase-MHETase-pET21b and S245I PETase-MHETase-pET 21b in E. coli C41 (DE3) and TOP10 competent cells. We performed colony PCR to confirm the presence of insert DNA in plasmids after transformation.
Figure 3. Colony PCR screening of constructs transformed in C41(DE3) competent cells.
Figure 4. Colony PCR screening of constructs transformed in TOP10 competent cells.
Figure 3 and Figure 4 showed that PCR products with the correct size of 498 bp were amplified in all constructs confirming the presence of MHETase, WT PETase-MHETase and S245I PETase-MHETase in the expression vector pET-21b.
Proteins of WT PETase, MHETase and S245I PETase were expressed in E. coli C41(DE3) and induced by the addition of 0.5mM final concentration of IPTG in a 100mL LC medium shake at room temperature for 16 – 20h. Proteins were extracted and purified. The size of WT PETase and S245I PETase is 30kDa and that of MHETase is 65kDa.
Figure 5. SDS PAGE of purified MHETase, WT PETase and S245I PETase proteins.
We performed Bradford protein assay to determine the concentration of our purified proteins. The resulting concentrations of WT PETase, S245I PETase, and MHETase are at 1.35 mg/mL, 1.61 mg/mL, and 0.77 mg/mL respectively.
Figure 6. BSA standard curve. The line of best fit is drawn, with the R2 value being 0.994.
To analyze the degradation rate of PET by enzyme cocktails, commercial PET film was used as the substrate for enzyme assay. The PET film was prepared in a square form with a length of 6 mm. It was then soaked in 300μL of pH 9.0 glycine-NaOH buffer with 4μg or 8μg of enzymes.
In the enzyme cocktails, 4μg WT PETase or S245I PETase were mixed with 4μg and 8 μg MHETase. A solution with only 4μg WT PETase and a solution with only S245I PETase was used as a control to compare the PET degradation rate of single-enzyme systems and dual-enzymes systems.
Table 2. Combinations of WT PETase, S245I PETase and METase in different enzyme cocktails.
The reaction mixture was incubated at 30oC for 96h. The reaction mixture was terminated by dilution of the aqueous solution with 160mM phosphate buffer (pH 2.5) containing 10% (v/v) DMSO followed by heating at 85oC for 15 min, after which the PET film was removed from the reaction mixture. Then the samples were centrifuged at 13,200 rpm for 10 min. 20μL of the supernatant was analyzed by HPLC. The film was washed with 1% SDS and 20% ethanol in distilled water.
Maximum intensity: 36000cps at 4.62 min
Figure 7. HPLC profile of 25μM TPA standard
Maximum intensity: 1736.7 cps at 4.64 min
Figure 8a. HPLC profile of products released from PET film digestion using 4 μg WT PETase and 4 μg MHETase
Maximum intensity: 3986.7 cps at 4.62 min
Figure 8b. HPLC profile of products released from PET film digestion using 4 μg WT PETase and 8 μg MHETase
Maximum intensity: 16000 cps at 4.63 min
Figure 8c. HPLC profile of products released from PET film digestion using 4 μg WT PETase only
Maximum intensity: 7756.7 cps at 4.63 min
Figure 9a. 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 9b. 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 9c. 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 10. HPLC profile of products released from PET film digestion without using enzyme (negative control)
Figure 11. A standard curve of TPA determined by HPLC.
Figure 12. Concentration of TPA released from PET film digestion using different enzyme cocktails. S1: 4μg WT PETase and 4μg MHETase; S2: 4μg WT PETase and 8μg MHETase: S3: 4μg WT PETase only S4: 4μg S245I PETase and 4μg MHETase; S5: 4μg S245I PETase and 8μg MHETase: S4: 4μg S245I only S7: buffer only
HPLC profiles demonstrated that the detection peak representing TPA monomer has a retention time at 4.62 minutes (Figure 7).
HPLC profiles of products released from PET film digestions using WT PETase and MHETase demonstrated that a mixture of WT PETase and MHETase in a 1:2 ratio exhibited better depolymerization performance than that in a 1:1 ratio as more monomers were released (Figure 8a and Figure 8b). However, the extent of depolymerization achieved by WT PETase alone was the highest (Figure 8c) suggesting that the addition of MHETase inhibited or slowed down the PET degradation. We cannot exclude the possibility that the fusion of MHETase to WT PETase impaired activity of WT PETase and subsequent PET depolymerization. Thus, the experimental result needs to be further investigated.
On the other hand, 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 activity with that in a 1:1 ratio but both exhibited better degradation activity than the mixture with S245I PETase alone as more TPA were determined in the cocktails (Figure 9a – c).
A standard curve of TPA was obtained by HPLC analysis of 9 standard solutions (0.098μM, 0.195μM, 0.397μM, 0.78μM, 1.56μM, 3.125μM, 6.25μM, 12.5μM and 25μM) using serial dilution (Figure 11). PET film digestion eluent has a background intensity of 2193 cps as determined by HPLC (Figure 10). TPA concentration released from PET film digestion using different enzyme cocktails demonstrated that the mixture with S245I PETase only released 2.02 ng/mL TPA, while the mixture with 1:1 and 1:2 ration of S245I PETase and MHETase released 3.49 ng/mL and 3.44 ng/mL (Figure 12). 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. The findings revealed that our engineered mutant, S245I PETase which outperforms WT PETase, further synergize PET depolymerization in the presence of MHETase.
Figure 13a. PET film after incubation with 4μg WT PETase and 4μg MHETase
Figure 13b. PET film after incubation with 4μg PETase, WT PETase and 8μg MHETase
Figure 13c. PET film after incubation with 4μg WT PETase only
Figure 14a. PET film after incubation with 4μg S245I PETase and 4μg MHETase
Figure 14b. PET film after incubation with 4μg S245I PETase and 8μg MHETase
Figure 14c. PET film after incubation with 4μg S245I PETase only
Figure 15. PET film after incubation with buffer only
Consistent with the result from HPLC, the pitting of PET film surface resulting from the digestion of WT PETase only is much more significant than from the digestion of WT PETase and MHETase cocktails (Figure 13a – c). Similarly, 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 14a – c). Consistent with enzyme assay results shown in 2019, S245I PETase exhibits a higher depolymerization activity. No pitting of PET film surface was observed in mixture with buffer only (Figure 15). 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.
