1. Confirmation of PET hydrolytic activities of WT PETase and engineered mutant, S245I PETase studied in iGEM 2019

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 amount (9 μg) of purified proteins with PET film at 30°C for 96h, we performed High Performance Liquid Chromatography (HPLC) analysis of PET digestion eluents and Scanning Electron Microscope (SEM) of digested PET film.

(A) SEM images of degraded PET films

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.

(B) HPLC profile of the products released from the PET films

Products released from PET films after digestion with WT PETase and S245I PETase were detected and quantified by HPLC.

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.

HPLC profiles of products released from PET film digestions using a single enzyme, WT PETase or S245I PETase revealed incomplete PET hydrolysis as considerable amounts of intermediate product, MHET, which showed a peak at 5.07 minutes were detected (Figure 2c and Figure 2d). Thus, we hypothesized that the presence of a second enzyme, MHETase, which hydrolyzes MHET into TPA, in the PET depolymerization system synergizes the degradation rate of PET into its constituting monomers. Therefore, to completely depolymerize PET, we aimed to develop dual-enzyme systems consisting of PETase and MHETase in this project.

2. Confirmation of the presence of insert DNA in plasmid constructs

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 PCR
We 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 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.

3. PET hydrolytic activities of PETase and MHETase cocktails

(A) Protein expression and purification of WT PETase, S245I PETase and MHETase

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.

(B) Bradford protein assay

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.

(C) PET film digestion

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.

The reaction mixture was incubated at 30°C 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 85°C 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.

(D) HPLC of eluents of digested PET film with different enzyme cocktails

(i) TPA Standard

Maximum intensity: 36000cps at 4.62 min

(ii) Products released from the PET film incubated with WT PETase and MHETase cocktails

Maximum intensity: 1736.7 cps at 4.64 min

Maximum intensity: 3986.7 cps at 4.62 min

Maximum intensity: 16000 cps at 4.63 min

(iii) Products released from the PET film incubated with S245I PETase and MHETase cocktails

Maximum intensity: 7756.7 cps at 4.63 min

Maximum intensity: 8166.7 cps at 4.62 min

Maximum intensity: 4906.7 cps at 4.63 min

(iv) Products released from the PET film incubated with buffer only

Maximum intensity: 2193.3 cps at 4.63 min

(v) Quantification of TPA released from PET film digestion with different enzyme cocktails

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 ratio 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 increasing constituent monomer, TPA, up to 1.7 folds. The findings revealed that our engineered mutant, S245I PETase, which outperforms WT PETase, further synergizes PET depolymerization in the presence of MHETase.

(D) SEM of digested PET film incubated with different enzyme cocktails

(i)PET film incubated with WT PETase and MHETase cocktails

(ii) PET film incubated with S245I PETase and MHETase cocktails

(iii) PET film incubated 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.

4. PET hydrolytic activities of PETase-MHETase chimeras

(A) Protein expression and purification of WT PETase-MHETase and S245I PETase-MHETase

Proteins of PETase-MHETase chimeras were expressed in E. coli C41(DE3). Using the same protein extraction method as that of PETase and MHETase as described in section 3(A) in which chimeric protein was induced by the addition of 0.5mM final concentration of IPTG in a 100mL LC medium shake at room temperature for 16 – 20h, no WT PETase-MHETase chimeric protein was expressed. The size of WT PETase-MHETase is 95kDa.

To optimize the condition for protein expression, we changed the induction condition in which the 400mL culture medium was induced with 0.5mM IPTG and shook at 16°C for 24 - 30h.

Protein extraction protocol was also revised. After induction, 400 mL bacterial culture was divided into two 200 mL solutions and harvested the cells by centrifugation at 5,000 rpm at 4°C for 15 minutes. The cell pellets were resuspended by adding 20 mL of lysis buffer. We further sonicated the suspension 5 times for 15 cycles; each cycle consists of 10s with sonication followed by 10s without sonication. The sonication power is 6-8 W. After centrifuging at 13,000 rpm for 20 min at 4°C, 20mL supernatants were collected. 2 mL Ni-NTA resin was washed with lysis buffer for 5 times with short spins at 3,000 rpm and kept on ice. Then, 10 mL of the supernatant was mixed with 1 mL Ni-NTA resin and then shook on ice for 1 hour at 50 rpm. After rinsing the Nickel column 3 - 4 times with lysis buffer, the mixture was loaded to a column. The column was then washed 3 times with wash buffer and 3 times with 2 mL elution buffer. For SDS-PAGE, we mixed 15 µL of purified proteins and 5 µL 4x loading dye and loaded into the wells. The size of PETase-MHETase chimera is 95kDa, with PETase and MHETase are 30kDa and 65 kDa respectively. If our proteins are successfully expressed and purified, they should give a thick band in the area corresponding to their mass in the SDS-PAGE.

The results of SDS-PAGE demonstrated the presence of purified WT PETase, S245I PETase, MHETase WT PETase-MHETase, and S245I PETase-MHETase. It also showed that PETase-MHETase chimeras are successfully expressed and purified despite the presence of multiple bands. The protein induction and extraction protocol will be optimized in the future to obtain a single band of chimeric proteins.

(B) PET film digestion

To analyze the degradation activity of engineered chimeras, 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 8µL and 16µL of enzymes, MHETase, WT PETase-MHETase and S245I PETase-MHETase. A mixture with buffer only was used as a negative control. We incubated the mixtures for 96h at 30°C.

(C) HPLC of eluents of digested PET film with different chimeras

We analyzed the eluent after the PET film digestion, and showed that the solution contained the constituent monomer of PET, TPA

(i) TPA Standard

Maximum intensity: 2600 cps at 4.64 min

(ii) Products released from the PET film incubated with MHETase only

Maximum intensity: 1050 cps at 4.71 min

Maximum intensity: 1050 cps at 4.66 min

(iii) Products released from the PET film incubated with S245I PETase-MHETase

Maximum intensity: 1160 cps at 4.66 min

Maximum intensity: 1080 cps at 4.67 min

(iv) Products released from the PET film incubated with WT PETase-MHETase

Maximum intensity: 1340 cps at 4.67 min

Maximum intensity: 990 cps at 4.65 min

(v) Products released from the PET film incubated with buffer only

Maximum intensity: 680 cps at 4.69 min

(vi) Quantification of TPA released from PET film digestion with different chimeras

Equal volume of extracted chimera proteins was used to digest PET film in order to investigate their PET depolymerization activity. HPLC data showing that trace amounts of TPA were detected in eluents of PET film digestion with WT PETase-MHETase and S245I PETase-MHETase suggesting that chimeras exhibit PET depolymerization activity (Table 1). However, their degradation rate could not be compared as the concentration of chimeric proteins added were different due to the presence of impurities as indicated by multiple bands observed in SDS-PAGE.

(D) SEM of digested PET film with different chimeras

(i) PET film incubated with MHETase only

(ii) PET film incubated with S245I PETase-MHETase chimera

(iii) PET film incubated with WT PETase-MHETase chimera

(iii) PET film incubated with buffer only

Consistent with the result from HPLC in which chimeric proteins exhibited PET depolymerization activity and released trace amount of TPA, the pitting of PET film surfaces resulting from the digestion of S245I PETase-MHETase and WT PETase-MHETase was observed (Figure 24 and Figure 25). No pitting of PET film surface was observed when PET film was incubated with buffer only solution (Figure 26). However, pitting of PET film could also be observed in the PET film digested with MHETase only (Figure 23). This result was not consistent with HPLC data which showed negligible amounts of TPA. The effect of digestion of PET film using MHETase will be further investigated.

5. SDS-PAGE and Western blot analysis

Since a few bands were obtained in purified chimeric proteins, WT PETase-MHETase and S245I PETase-MHETase, SDS-PAGE and western blot were performed to confirm the identity of purified proteins. The purified proteins, WT PETase, S245I PETase and MHETase were also included in this experiment. All the genes encoding PETase, MHETase and their chimeras were cloned into the expression vector pET-21b which has a C-terminal His-Tag. Therefore, His-Tag antibody was used for the verification of expressed proteins.

Our SDS-PAGE results showed that all purified proteins were expressed (Fig. 27). Clear bands with correct sizes were observed in the western blot of all purified proteins (Figure 28). In addition to the band of 96kDa, which matched the size of chimeric proteins, an extra band with the size matching to that of MHETase was observed in the chimeric proteins. This suggests that some chimeric proteins were cleaved during the process of protein purification. Since MHETase were linked to the C-terminal of WT PETase and S245I PETase in the WT PETase-MHETase and S245I PETase-MHETase chimeric constructs respectively, MHETase was attached to His-Tag which was located near the C-terminal of expression vector pET 21b.Thus cleaved MHETase was detected in both chimeric proteins using His-Tag antibody.

These results demonstrated that our chimeric proteins, WT PETase-MHETase and S245I PETase-MHETase were successfully expressed and purified. They exhibited PET depolymerization activity as seen by trace amounts of TPA detected in HPLC analysis and the degraded PET film surfaces observed under SEM.

To further characterize the chimeric proteins, protein induction and extraction protocols need to be further optimized in order to obtain single purified proteins for subsequent PET digestion experiments. Besides, the length and amino acid sequence of protein linker also affect chimeric protein production. An effective protein linker provides suitable space between two proteins which will decrease their intrusion, improve folding and ultimately improve protein production and activity. In our project, a 12 amino acid serine-glycine linker was used to covalently link C terminus of WT PETase or S245I PETase and N terminus of MHETase to form chimeric proteins. To improve production efficiency and reduce cleavage between two proteins, linkers of different amino acid lengths and combination need to be tested in order to find out the proximity of the two enzymes which would provide the highest PET hydrolytic activity.

Taken together, our data suggest that single enzyme of PETase mutant, S245I exhibits a higher depolymerization activity than that of WT PETase. The detection of considerable amounts of intermediate products, MHET after PET film digestion extend our idea to the development of dual enzyme system for complete PET depolymerization. The PET degradation achieved by enzyme cocktails of WT PETase and MHETase was inhibited while that of S245I PETase and MHETase was synergized compared with that achieved by single enzyme, WT PETase or S245I PETase alone. Therefore, it clearly shows that our engineered mutant, S245I PETase which outperforms WT PETase, further synergize PET depolymerization in the presence of MHETase. Additionally, chimeric proteins of PETase and MHETase were successfully expressed and purified. Preliminary studies suggest that they have PET depolymerization activity.

These findings suggest that enzymatic synergism in dual enzyme PET degrading system is of importance to industrial use. Improved hydrolysis rate compared to single enzyme reduce the use of enzymes and hydrolysis time and thus the cost of PET depolymerization. More importantly, rapid and complete PET degradation leads to ways of closing the circle from production to waste.