High-efficiency depolymerization/degradation of polyethylene terephthalate plastic by a whole-cell biocatalyst

Abstract

Polyethylene terephthalate (PET) is the most abundantly produced plastic due to its excellent performance, but is also the primary source of poorly degradable plastic pollution. The development of environment-friendly PET biodegradation is attracting increasing interest. The leaf-branch compost cutinase mutant ICCG (F243I/D238C/S283C/Y127G) exhibits the best hydrolytic activity and thermostability of all known PET hydrolases. However, its superior PET degradation is highly dependent on its preparation as a purified enzyme, which critically reduces its industrial utility. Herein, we report the use of rational design and combinatorial mutagenesis to develop a novel ICCG mutant RITK (D53R/R143I/D193T/E208K) that demonstrated excellent whole-cell biocatalytic activity. Whole cells expressing RITK showed an 8.33-fold increase in biocatalytic activity compared to those expressing ICCG. Thermostability was also improved. After reacting at 85 °C for 3 h, purified RITK exhibited a 12.75-fold increase in depolymerization compared to ICCG. These results will greatly enhance the industrial utility of PET hydrolytic enzymes and further the progress of PET recycling.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-023-03557-4.

Introduction

Due to the mass production and use of plastics and the lack of appropriate recycling technology, plastic pollution has become topic of general interest. Since the 1950s, plastics have been produced and applied on a massive scale. By 2015, approximately 5 billion tons of plastics had been discarded into the environment (Geyer et al. 2017). By 2018, annual global plastic production had increased more than 20-fold, reaching 359 million tons, and will reach an estimated 34 billion tons by 2050 (Geyer et al. 2017; PlasticEurope 2019). Polyethylene terephthalate (PET) is widely used in the packaging, automotive, construction, and electrical industries because of its simple synthesis, low cost, stability, and durability (Urbanek et al. 2021). In 2016 the global PET output reached 50 billion tons (Bornscheuer 2016), and the global PET market size was valued at USD 34.15 billion in 2019 (Grand View Research Inc. 2020).

While plastic is widely used and brings many benefits, it also causes severe ecological damage due to its difficult natural depolymerization, and even threatens the marine ecosystem and rainforest reserves. Over 800 animal species are jeopardized, and about 90% of seabirds are estimated to have ingested plastic (Sussarellu et al. 2016; Wilcox et al. 2015). In addition, plastic pollution may endanger human health. Microplastics have been found in human lungs and stool samples (Schwabl et al. 2019). An in vitro study showed that microplastics can destroy membrane integrity, induce inflammation and cytotoxicity, and dysregulate gene expression (Gangadoo et al. 2020). To prevent pollution caused by man-made products, the circular economy will soon become a global concept, and plastic recycling will become a major focus (European Commission 2020). The incineration of plastic waste usually releases toxins, such as furan and dioxin, which cause secondary pollution (Li et al. 2001). Mechanical recycling requires an initial sorting process, and cannot process temperature-sensitive plastics. Chemical recovery requires the use of strong acids or bases and expensive catalysts, often producing additional environmental pollutants. Furthermore, product mixtures generated during chemical recovery are difficult to separate (Garcia and Robertson 2017). Environment-friendly PET biodegradation represents a potential new strategy (Tournier et al. 2020).

Because the glass transition temperature of PET is very high (70 °C), the PET chain shows greater fluidity, and the ester bond is more easily broken by enzymatic degradation. Therefore, the discovery and development of heat-resistant enzymes has received increasing interest (Kawai 2021). Cui et al. (2021) used the GRAPE strategy to increase the thermostability of a PETase from Ideonella sakaiensis 201-F6, and obtained a DuraPETase mutant with a Tm of 77 °C. Chen et al. (2020) constructed an IsPETase-displaying yeast whole-cell biocatalysis towards highly crystallized PET that was dramatically increased (about 36-fold) compared with that of purified IsPETase. Then et al. (2015) strengthened the disulfide bond of TfCut2 from a thermophilic actinomycetes (Thermobifida fusca KW3) to confer PET degradation activity at 70 °C. Wei et al. demonstrated that the single variant TfCut2 (G62A) and the double variant TfCut2 (G62A/I213S) exhibited the highest activities in hydrolyzing PET films. Both variants reduced the weight of PET films by more than 42% after 50 h of hydrolysis, corresponding to a 2.7-fold increase compared to TfCut2 (Wei et al. 2016). Son et al. (2019) designed a IsPETase triple mutant (S121E/D186H/R280A), with a Tm of 57.62 °C, 8.81 °C higher than that of IsPETase. In 2011, Sulaiman et al. used a metagenomic approach to isolate a new cutinase LCC from leaf-branch compost that exhibited high activity at 70 °C (Sulaiman et al. 2012, 2014; Wei et al. 2019). Tournier et al. (2020) obtained ICCG with greatly improved thermostability through directed evolution of LCC, increasing its Tm value by 9.3 °C.

For industrial applications, easy availability of enzymes is an important factor to promote production. Although ICCG has excellent thermostability, its PET depolymerization activity is low when used in whole-cell biocatalysis. High activity in complex production conditions is prerequisite for the industrial application of enzymes. Shirke et al. (2018) found that the large surface charge of LCC promoted aggregation in the natural state, and that LCC aggregation reduced enzyme stability. Consequently, many researchers are studying the direct use of whole cells as an alternative to purified enzymes to degrade PET. Moog et al. (2019) modified Phaeodactylum tricornutum through genetic engineering to secrete a single-point mutant of a PETase into the extracellular medium. This measure successfully achieved the simultaneous expression and secretion of a PETase mutant and facilitated PET degradation. This discovery provided a new solution for PET degradation and recovery. Xue et al. (2021) fused a chitin-binding domain from Chitinolyticbacter meiyuanensis SYBC-H1 to the C-terminus of the LCC ICCG variant, improving degradation performance by up to 19.6% compared with its precursor enzyme without the binding module. Yan et al. (2021) subsequently developed a whole-cell degradation technology that degrades PET while producing LCC by Clostridium thermopellum. Compared with standard enzyme purification, the cost of LCC whole-cell technology is lower, but degradation efficiency is inadequate. After 14 days of reaction, only 31 mg of amorphous PET can be degraded. Therefore, the design of an enzyme that can react efficiently at high temperature without prior protein purification would be of great industrial value.

Materials and methods

Materials

Terephthalic acid (TPA, purity 99.0%) and mono-(2-hydroxyethyl) terephthalate (MHET, purity 98.0%) were purchased from Macklin (Shanghai, China). Goodfellow PET (Gf-PET) film with a thickness of 250 µm and a crystallinity percentage of 7.7% was purchased from Goodfellow Ltd (Bad Nauheim, Germany, product number ES301445) (Tournier et al. 2020). Gf-PET film was prepared in circular samples which served as substrate for enzymatic degradation, with each sample having a diameter of 10 mm and a weight of 22.8 ± 0.5 mg. No further treatment was made on the Gf-PET samples.

Library construction and screening

Aiming to increase ICCG thermostability to enable rapid PET depolymerization by whole-cell biocatalysis at high temperatures, we applied three algorithms, FoldX (force field-based energy function) (Guerois et al. 2002), Rosetta_ddg (force field-based energy function) (Kellogg et al. 2011), and Consensus Analysis (phylogeny-based method) (Wheeler et al. 2014) to improve protein stability. After energy calculation using FoldX and Rosetta, mutations with high frequencies in the Consensus Analysis were selected to construct the mutagenesis library (Table S2).

The LCC ICCG variant (Tournier et al. 2020) was commercially synthesized with codon optimization for expression in Escherichia coli cells (Tsingke, Hangzhou, China). The nucleotide sequence corresponding to the signal peptide was removed from the synthetic DNA. The synthesized gene was cloned into pET-24b at NdeI and XhoI digestion sites and transformed in E. coli JM109 (DE3). The mutagenesis library was rationally constructed through energy calculations and consensus analysis.

The Ala165 residue in ICCG (PDB ID: 6THT) (Tournier et al. 2020) was mutated to Ser165 by Chimera (Pettersen et al. 2004). Energy calculations with FoldX (Guerois et al. 2002) and Rosetta_ddg (Kellogg et al. 2011) were performed. All positions of the protein sequence were mutated in silico to all proteinogenic amino acids. The relative folding free energy changes (∆∆G^Fold) predicted by FoldX and Rosetta_ddg algorithms were calculated using Eq. 1 as follows:

$$\mathrm{\Delta }\mathrm{\Delta }{G}^{\mathrm{Fold}}=\mathrm{\Delta }{G}_{\mathrm{mutation}}^{\mathrm{Fold}}-\mathrm{\Delta }{G}_{\mathrm{ICCG}}^{\mathrm{Fold}}.$$ 1

In Eq. 1, the ΔG^Fold represents the free energy difference between folded and unfolded structures. For FoldX, standard settings were used, and each calculation was repeated five times to obtain better averaging. We used the settings described by Kellogg et al. (2011) for Rosetta_ddg (options -ddg::local_opt_only true -ddg::opt_radius 8.0 -ddg::weight_file soft_rep_design ddg::iterations 50 -ddg::min_cst false -ddg::mean true -ddg::min false -ddg::sc_min_only false -ddg::ramp_repulsive false).

Searching for ICCG-like proteins was performed using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) server using the position-specific iterated BLAST method (Altschul et al. 1997). Multiple alignment was performed by Muscle (Edgar 2004). The frequencies of each letter in the target position were calculated via the Skylign online tool (http://skylign.org) (Wheeler et al. 2014).

Single point mutations were constructed with the ICCG plasmid as a template (Tsingke, Hangzhou, China). ICCG variants were generated by amplifying the full-length plasmid by polymerase chain reaction (PCR) using 2 × Phanta Max Master Mix (Vazyme, Nanjing, China) with designed primers. The primer sequences for mutagenesis are listed in Table S1. PCR products were incubated with DpnI (Takara, Japan), T4 polynucleotide kinase (Takara, Japan), and T4 DNA ligase (Takara, Japan) to digest the original DNA template and to form circular plasmids, which were then transformed into E. coli JM109 (DE3). All ICCG variants were confirmed by sequencing.

The plasmids were individually transformed into E. coli JM109 (DE3) cells. From an overnight microplate culture in Luria–Bertani medium at 37 °C, a 1/1000 dilution in auto-induction medium was performed in 96-deep well plates for a 24 h expression cultivation at 21 °C. E. coli cells were harvested by centrifugation (12,000×g, 1 min) and resuspended in 1 mL potassium phosphate buffer (100 mM, pH 8.0).

One milliliter resuspensions of enzyme (ICCG or variants) were incubated with a Gf-PET sample and 1 mL potassium phosphate buffer (100 mM, pH 8.0). After reacting for 24 h at 72 °C, 20 μL reaction solution was mixed with 80 μL DMSO, passed through a 0.22 μm filter, and centrifuged at 12,000×g for 10 min before application to a high-performance liquid chromatography system (Shimadzu LC16) equipped with an Diamonsil C18 column (4.6 × 250 mm, 5 μm). The C18 column was eluted with a mobile phase of 40% methanol and 60% phosphate buffer (50 mM KH₂PO₄, pH 3.8) at 1 mL/min. The effluent was monitored at a wavelength of 240 nm. Quantities of PET hydrolytic products MHET and TPA were calculated based on standards with a known concentration. Enzymatic activity was calculated by the sum of released MHET and TPA.

Enzymatic assay for Gf-PET degradation

Plasmids were individually transformed into E. coli JM109 (DE3) cells grown in Luria–Bertani medium at 37 °C with agitation of 220 rpm. When OD₆₀₀ reached ∼ 0.8, the expression of recombinant proteins was induced by 1 mM isopropyl β-d-thiogalactopyranoside at 16 °C for 24 h. The cells were harvested by centrifugation at 6000×g for 10 min, and resuspended in lysis buffer (25 mM TrisHCl, 150 mM NaCl, 20 mM imidazole, pH 7.5). Cells underwent ultrasonic disruption and lysate clarification by centrifugation (10,000×g, 30 min, 4 °C). The soluble fraction was purified using the His-tagged Protein Purification Kit (CWBIO, Beijing, China) according to the manufacturer’s description. Purified proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The protein concentration was measured using the Bradford method with bovine serum albumin as the standard (Bradford 1976).

Five hundred microliters of 120 μg/mL of enzyme (ICCG or variants) in 100 mM potassium phosphate (pH 8.0) was incubated with a Gf-PET sample and 1.5 mL potassium phosphate buffer (100 mM, pH 8.0) at indicated temperatures with agitation at 200 rpm for an indicated period of time. After reacting, 20 μL reaction solution was mixed with 80 μL DMSO, passed through a 0.22 μm filter, and centrifuged at 12,000×g for 10 min before undergoing high-performance liquid chromatography (HPLC).

Reaction condition optimization

The effects of temperature and pH on enzymatic activity were calculated by the sum of released MHET and TPA. All samples were analyzed in triplicate in each independent experiment, and mean values and standard deviations were calculated.

For the thermal stability assay, 500 μL of 120 μg/mL of enzyme (ICCG or RITK) in 100 mM potassium phosphate (pH 8.0) was incubated with a Gf-PET sample and 1.5 mL potassium phosphate buffer (100 mM, pH 8.0) at different temperatures (72, 75, 80, 85 °C) for 24 h. After reacting, 20 μL reaction solution was mixed with 80 μL DMSO, passed through a 0.22 μm filter, and centrifuged at 12,000×g for 10 min before undergoing HPLC.

For enzyme concentration assays, 500 μL of 20 μg/mL, 60 μg/mL, 180 μg/mL, 300 μg/mL, or 420 μg/mL of enzyme (ICCG or RITK) in 100 mM potassium phosphate (pH 8.0) was incubated with a Gf-PET sample and 1.5 mL potassium phosphate buffer (100 mM, pH 8.0) at 72 °C for an indicated period of time. After reacting, 20 μL reaction solution was mixed with 80 μL DMSO, passed through a 0.22 μm filter, and centrifuged at 12,000×g for 10 min before undergoing HPLC.

For the cell concentration assays, 1 mL of 10 mg/mL, 20 mg/mL, 40 mg/mL, 80 mg/mL, or 160 mg/mL of cell suspension (ICCG or RITK) in 100 mM potassium phosphate (pH 8.0) was incubated with a Gf-PET sample and 1.5 mL potassium phosphate buffer (100 mM, pH 8.0) at 72 °C for 24 h. After reacting, 20 μL reaction solution was mixed with 80 μL DMSO, passed through a 0.22 μm filter, and centrifuged at 12,000×g for 10 min before undergoing HPLC.

For the optimum pH assay, 500 μL of 120 μg/mL of enzyme in 100 mM potassium phosphate (pH 8.0) was incubated with a Gf-PET sample and 1.5 mL of either tris–HCl buffer (100 mM, pH 8.0, 8.5, 9.0) or potassium phosphate buffer (100 mM, pH 6.0, 7.0, 8.0) at 72 °C for an indicated period of time. After reacting, 20 μL reaction solution was mixed with 80 μL DMSO, passed through a 0.22 μm filter, and centrifuged at 12,000×g for 10 min before undergoing HPLC.

PET depolymerization assay using 1 g sample-PET as substrate

ICCG (whole-cell, 2200 mg) and RITK (whole-cell, 2200 mg) were prepared in 110 mL of 100 mM potassium phosphate buffer at pH 8.0, corresponding to the concentration of 20 mg/mL. The purified ICCG (enzyme, 4.95 mg) and RITK (enzyme, 4.95 mg) were prepared in 110 mL of 100 mM potassium phosphate buffer at pH 8.0, corresponding to the concentration of 45 μg/mL. The whole-cell and pure enzymes were combined with a 1 g sample-PET in a 500 mL conical flask at 72 °C for 27 h. Temperature was regulated by water bath immersion and maintained by constant agitation at 200 rpm. After reacting, 200 μL reaction solution was mixed with 800 μL DMSO, passed through a 0.22 μm filter, and centrifuged at 12,000×g for 10 min before undergoing HPLC.

Results

Screening of higher stability variants

Whole-cell screening assays revealed that the depolymerization activities of whole cells expressing D53R, D53T, R143I, D193T, and E208K mutants were improved significantly over those expressing ICCG. Furthermore, D53R was 7.53-fold more active than ICCG.

The screened dominant single mutants were then mutated into double mutants. Whole cells expressing either D53R/D193T or D53T/E208K were 8.62- and 8.52-fold more active than those expressing ICCG, respectively. Consequently, we constructed triple and quadruple mutants of ICCG, and found that whole cells expressing either of two triple (D53R/D193T/E208K[RTK], D53T/R143I/E208K[TIK]) or two quadruple mutants (D53R/R143I/D193T/E208K [RITK] and D53T/R143I/D193T/E208K [TITK]) demonstrated at least ninefold increases in depolymerization activity (Fig. 1).

Fig. 1.

A Gf-PET sample was used as substrate at 72 °C, with a potassium phosphate reaction buffer (100 mM, pH 8.0). After 24 h of reaction, the total released hydrolysates were measured three times. The relative activities of whole cells expressing single and iterative mutants were compared with that of whole-cell ICCG. Assays were performed in triplicate. Mean values ± standard deviations are shown

The total products of highly active triple and quadruple mutants were very similar after 24 h of reaction. Therefore, we purified the four enzymes and assayed their 3-h product yields. The depolymerization rate of RITK was highest, 1.54-fold higher than ICCG (Fig. 2).

Fig. 2.

A Gf-PET sample was used as substrate at 72 °C with a potassium phosphate reaction buffer (100 mM, pH 8.0). The products of triple and quadruple mutants (30 μg/mL) were compared with those of ICCG. Assays were performed in triplicate. Mean values ± standard deviations are shown

Optimization of reaction conditions

To further validate industrial applicability, we explored the effects of whole-cell concentrations, enzyme concentrations, reaction temperatures, and reaction pH levels on the performance of RITK. When whole-cell ICCG catalyzed the reaction for 3 h, no product was detected. When the reaction time was extended to 24 h, both RITK and ICCG whole cells displayed their highest depolymerization efficiencies at a cell concentration of 20 mg/mL. The depolymerization activity of RITK whole cells was 9.41-fold higher than that of ICCG whole cells (Fig. 3).

Fig. 3.

Depolymerization of PET by whole cells expressing ICCG and RITK with different cell concentrations. Reactions were conducted at 72 °C in potassium phosphate reaction buffer (100 mM, pH 8.0). The total product was determined 24 h after hydrolysis at indicated cell concentrations. Assays were performed in triplicate. Mean values ± standard deviations are shown

When studying the depolymerization activity of purified ICCG and RITK, we found that after 24 h of reaction at concentrations greater than 5 μg/mL, the products of ICCG and RITK did not increase with higher enzyme concentrations. After 3 h of reaction, the depolymerization efficiency of ICCG and RITK at an enzyme concentration of 45 μg/mL reached maximum. However, further increases of enzyme concentration decreased the depolymerization activity of ICCG, but did not change that of RITK (Fig. 4).

Fig. 4.

Depolymerization of PET by ICCG and RITK at different enzyme concentrations. Reactions were conducted at 72 °C, in potassium phosphate reaction buffer (100 mM, pH 8.0). Total product was determined 3 h and 24 h after hydrolysis at indicated cell concentrations. Assays were performed in triplicate. Mean values ± standard deviations are shown

The thermostability of RITK was higher than that of ICCG. At a reaction temperature of 80 °C, the depolymerization capacity of RITK was about twofold higher than that of ICCG whether the reaction time was 3 h or 24 h. When reacting at 85 °C for 3 h, the total depolymerization product of RITK was 12.75-fold higher than that of ICCG (Fig. 5). RITK had good pH stability. When pH was in the range of 8.0–9.0, depolymerization activity did not fluctuate greatly (Fig. 6).

Fig. 5.

ICCG- and RITK-catalyzed PET depolymerization at different temperatures. Reactions were conducted in potassium phosphate buffer (100 mM, pH 8.0) with a reaction enzyme concentration of 30 μg/mL. Total product was determined 3 h and 24 h after hydrolysis at indicated temperatures. Assays were performed in triplicate. Mean values ± standard deviations are shown

Fig. 6.

RITK-catalyzed PET depolymerization at different pH levels. Reactions were conducted in potassium phosphate buffer (100 mM, pH 8.0) and with a reaction enzyme concentration of 30 μg/mL. Total product was determined 3 h and 24 h after hydrolysis at indicated pH levels. Assays were performed in triplicate. Mean values ± standard deviations are shown

Time-resolved PET depolymerization assays

We further evaluated the degradation activity of ICCG and RITK whole-cell biocatalysts. The depolymerization curves of ICCG and RITK pure enzymes were used as controls to explore the depolymerization activity of whole-cell catalysts compared with pure enzymes at indicated time points. The starting time of ICCG whole cells was significantly delayed, while that of RITK whole cells remained unchanged. Even within 9 h of reaction, the depolymerization efficiency of RITK (whole-cell, 20 mg/mL), ICCG (enzyme, 45 μg/mL), and RITK (enzyme, 45 μg/mL) was similar. After 24 h of reaction, the depolymerization products of RITK whole cells (41.32 mM) were 84.31% of those of ICCG pure enzyme degradation (49.01 mM), while the depolymerization products of ICCG whole cells were only 8.92% of that of ICCG (Fig. 7).

Fig. 7.

30-h time-courses of sample-PET (22.8 ± 0.5 mg) reactions, showing total released MHET and TPA, in reactions with ICCG (whole-cell, 20 mg/mL), RITK (whole-cell, 20 mg/mL), ICCG (enzyme, 45 μg/mL), or RITK (enzyme, 45 μg /mL). Reactions were conducted in potassium phosphate buffer (100 mM, pH 8.0) in duplicate. Error bars represent the standard deviations of the replicate measurements

Depolymerization of PET using ICCG and RITK

We further assayed the depolymerization activity of RITK in a small-scale reaction. The depolymerization curves of ICCG and RITK pure enzymes were used as controls to explore the depolymerization activity of whole-cell biocatalysts compared with pure enzymes at indicated time points. The degradation rates of RITK and ICCG pure enzymes decreased, and the degradation rate of ICCG before 18 h was lower than that of RITK. RITK depolymerized more than 80% of the plastic after 18 h of reaction, and more than 90% after 24 h. RITK whole-cell generation of depolymerization products was lower than those of pure enzymes, but was 7.39-fold higher than that of ICCG whole cells at 24 h (Fig. 8). RITK whole-cell biocatalysis degraded 66.15% of PET after 27 h.

Fig. 8.

27-h time-courses of 1 g sample-PET reactions, showing total released MHET and TPA, in reactions with ICCG (whole-cell, 20 mg/mL), RITK (whole-cell, 20 mg/mL), ICCG (enzyme, 45 μg/mL), or RITK (enzyme, 45 μg/mL). Reactions were conducted in potassium phosphate buffer (100 mM, pH 8.0) in duplicate. Error bars represent the standard deviations of the replicate measurements

Discussion

Plastic recycling is essential to control pollution. Biodegradation is considered the most promising method due to its environmental benefits. However, the biocatalytic degradation of PET plastic waste is still problematic. For example, the reaction rate of enzymatic degradation of low crystallinity PET is much higher than that of high crystallinity PET. However, excessive temperature rapidly transforms PET crystallinity from low to high levels. Consequently, the Tm value of purified enzyme is too high. The use of purified enzymes for PET degradation or the addition of PET waste treatment steps will obviously increase the costs of industrial application, which is economically untenable. Our experimental group is committed to using whole-cell biocatalysis for PET degradation. Most researchers believe that high temperature can improve the efficiency of enzymatic degradation of PET. Furthermore, Son et al. (2019) designed a variant with a Tm value that was increased by 8.81 °C and with PET degradation activity that was enhanced 14-fold at 40 °C compared with IsPETase. In our laboratory, the RITK mutant exhibited higher thermostability than ICCG. The amount of PET depolymerization products generated by the whole-cell RITK was up to 84.31% of that generated by the ICCG pure enzyme. Moreover, in a small assay reaction, RITK whole-cell biocatalysis was 7.39-fold higher than that of ICCG whole cells. These findings suggest that RITK whole-cell biocatalysis may offer an economically feasible alternative to pure enzymes for plastic recycling.

Compared with the pure enzyme reaction reported by Tournier et al. (2020), the whole-cell degradation method adopted by our laboratory is simpler and exhibits a very good reaction rate. Furthermore, Moog et al. (2019) engineered the photosynthetic microalgae Phaeodactylum tricornutum and showed that amorphous copolymer polyethylene terephthalate was slightly degraded after 7 days of reaction at 30 °C. Only the degradation products of TPA and MHET could be detected by scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC). Also, the living whole-cell degradation method developed by Yan et al. (2021) degraded only about 31 mg of plastic film after 14 days of reaction. Our technique is faster, as more than 20 mg of a piece of Gf-PET film (22.8 ± 0.5 mg) can be degraded per day by the whole-cell RITK (20 mg/mL) in a 2 mL reaction system. Compared with the improvement of thermostability reported by Zeng et al. (2022) and Son et al. (2019), our methods not only increased thermostability, but also enhanced protein stability in complex environments. We believe that the low activity of ICCG whole cells may be related to the protein aggregation noted by Shirke et al. (2018). In their study, the adhesion time of ICCG whole cells in the depolymerization of sample PET was prolonged and extended with the increase of the system. Compared to the ICCG variant, four positive charges were added through the RITK mutation. According to the recent report that fusing a zwitterionic polypeptide consisting of alternating-charged glutamic acid (E) and lysine (K) residues to the C-terminus of PETase leaded to the improved catalytic performance (Chen et al. 2021), the catalytic activity improvement of the whole-cell RITK may be related to the charge conversion.

In summary, our research team used rational design to generate an RITK mutant with significantly improved thermostability. Whole cells that express RITK can rapidly depolymerize PET. Our results show that whole-cell generated PET depolymerases are highly active without purification, which will promote their development and use in the plastic recycling industry.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

PET

Polyethylene terephthalate

TPA

Terephthalic acid

MHET

Mono-(2-hydroxyethyl) terephthalate

Gf-PET

Goodfellow PET

BLAST

Basic Local Alignment Search Tool

NCBI

National Center for Biotechnology Information

HPLC

High-performance liquid chromatography

Author contributions

YF: visualization, writing—original draft, and writing—review and editing. ZW: supervision, conceptualization, and writing—review and editing. ZC: conceptualization and software. KC: methodology and data curation. JH: formal analysis.

Funding

The authors declare that no funds, grants, or other supports were received during the preparation of this manuscript.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent to publish

Not applicable.