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Enzyme selection, optimization, and production toward biodegradation of post–consumer poly(ethylene terephthalate) at scale
Poly(ethylene terephthalate) (PET) is one of the world's most widely used polyester plastics. Due to its chemical stability, PET is extremely difficult to hydrolyze in a natural environment. Recent discoveries in new polyester hydrolases and breakthroughs in enzyme engineering strategies have inspired enormous research on biorecycling of PET. This study summarizes our research efforts toward large-scale, efficient, and economical biodegradation of post-consumer waste PET, including PET hydrolase selection and optimization, high-yield enzyme production, and high-capacity enzymatic degradation of post-consumer waste PET. First, genes encoding PETase and MHETase from Ideonella sakaiensis and the ICCG variant of leaf-branch compost cutinase (LCC ICCG ) were codon-optimized and expressed in Escherichia coli BL21(DE3) for high-yield production. To further lower the enzyme production cost, a pelB leader sequence was fused to LCC ICCG so that the enzyme can be secreted into the medium to facilitate recovery. To help bind the enzyme on the hydrophobic surface of PET, a substrate-binding module in a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM) was fused to the C-terminus of LCC ICCG . The resulting four different LCC ICCG variants (LCC, PelB-LCC, LCC-PBM, and PelB-LCC-PBM), together with PETase and MHETase, were compared for PET degradation efficiency. A fed-batch fermentation process was developed to produce the target enzymes up to 1.2 g L –1 . Finally, the best enzyme, PelB-LCC, was selected and used for the efficient degradation of 200 g L –1 recycled PET in a well-controlled, stirred-tank reactor. Furthermore, the results will help develop an economical and scalable biorecycling process toward a circular PET economy.
Evaluation of enzymatic depolymerization of PET, PTT, and PBT polyesters
Millions of tons of waste polyester plastics, including polyethylene terephthalate (PET), polytrimethylene terephthalate (PTT), and polybutylene terephthalate (PBT), end up in the environment as soil and water contaminants. Recent advances in enzymatic polyester degradation have motivated researchers toward biorecycling of plastic wastes. Leaf-branch compost cutinase (LCC) enzymes have been proven to be effective in the biodegradation of PET. This study focuses on enzymatic depolymerization of waste PET, PTT, and PBT materials by using an ICCG variant of LCC (LCC ICCG ) produced from Escherichia coli BL21(DE3). The degradation efficiency of the polyesters was determined by the monomer terephthalic acid (TPA) released from the depolymerization reaction. It was found that the most efficient depolymerization was achieved for PET, followed by PTT and PBT. A kinetic model based on Langmuir adsorption and the Michaelis-Menten equation was developed to describe the enzymatic depolymerization of PET, PTT, and PBT with various enzyme and substrate loadings. The model simulation results revealed that the LCCICCG enzyme loading should be linearly increased as the work capacity of the polyester substrate increases. A specific enzyme loading of 0.91 mg/g PET is suggested to achieve 90% depolymerization of PET within three days. Furthermore, the experimental data and model simulation results can be used to help further engineer the enzyme and process to achieve a complete biodegradation of polyester wastes at large scale.
Melt Processing Pretreatment Effects on Enzymatic Depolymerization of Poly(ethylene terephthalate)
Poly(ethylene terephthalate) (PET) is a common thermoplastic material, used in a wide variety of applications (i.e., bottles, fabrics, packaging, electronics, and automotive components). Increasing demand for PET has precipitated a need for improved recycling technology, especially for single-use PET waste. Recently, enzymatic depolymerization has shown promise as an environmentally responsible alternative for PET chemical recycling that yields economically useful products (e.g., terephthalic acid, adipic acid, and ethylene glycol). However, the depolymerization system still suffers from low rates on crystalline PET substrates, and effects of realistic waste streams are not known. In our work, PET waste is pretreated using an ultra-high-speed twin-screw extruder system. PET substrates were modified by various processing pretreatments to allow enzymes better access to depolymerize substrate materials. The effect of varying throughput and mechanical shear on structural properties of the PET waste was analyzed using molecular weight and thermal characterizations. These pretreated samples exhibit modifications in molecular weight, glass transition temperature, crystallinity, and specific surface area. The unpurified leaf-branch compost cutinase enzyme produced from the fed-batch fermentation of Escherichia coli BL21(DE3) was used in enzymatic depolymerization, where a faster reaction was observed as crystallinity was decreased and the specific surface area was increased. The rate of terephthalic acid production was also significantly higher for samples processed at lower mechanical shear with higher throughputs. As a result, this work demonstrates the potential for tailoring pretreatments in pursuit of faster and more energy efficient PET recycling using enzymes, with facile adaptation to the industrial scale for the circular economy.
High-Capacity Enzymatic Degradation of Postconsumer Poly(ethylene terephthalate): Modeling and Experimental Investigations
Enzymatic degradation is considered as one of the key steps for biorecycling of polyethylene terephthalate (PET), a widely used plastic. Recently, we have developed an optimized leaf-branch compost cutinase PelB-LCC ICCG and demonstrated ∼80% degradation of 200 g L −1 recycled PET (RPET) within two days in bioreactors. In this study, continued research efforts were made to achieve a complete degradation of up to 300 g L −1 RPET in bioreactors. First, low-capacity reaction conditions with ≤5 g L −1 PET were examined to identify the key variables for PET degradation with PelB-LCC ICCG , including temperature, enzyme loading, and product inhibition. To better understand and further optimize the enzymatic degradation process, a mechanism-based model was established to describe the kinetics of PET degradation and formation of the main product terephthalic acid (TPA) and byproducts MHET and BHET in high-capacity bioreactors. Model simulation suggested that a minimal enzyme loading of ∼1.4 mg PelB-LCC ICCG g −1 RPET is required to achieve a nearly complete degradation of RPET within 48 h, which was used to guide more high-capacity experiments with 100−300 g L −1 RPET in fully controlled 1 L bioreactors. A higher temperature (≥65 °C) was found not only to enable rapid degradation in the beginning but also to induce a gradual increase in RPET’s crystallinity and significantly slow down the degradation after 48 h. A high loading of RPET solids and the accumulation of the produced insoluble TPA pose a big challenge on mixing and mass transfer in the stirred bioreactor, which can be addressed by increasing the stirring speed. The results pave the way toward biorecycling of PET at a large scale.
Eight Up-Coming Biotech Tools to Combat Climate Crisis
Biotechnology has a high potential to substantially contribute to a low-carbon society. Several green processes are already well established, utilizing the unique capacity of living cells or their instruments. Beyond that, the authors believe that there are new biotechnological procedures in the pipeline which have the momentum to add to this ongoing change in our economy. Eight promising biotechnology tools were selected by the authors as potentially impactful game changers: (i) the Wood–Ljungdahl pathway, (ii) carbonic anhydrase, (iii) cutinase, (iv) methanogens, (v) electro-microbiology, (vi) hydrogenase, (vii) cellulosome and, (viii) nitrogenase. Some of them are fairly new and are explored predominantly in science labs. Others have been around for decades, however, with new scientific groundwork that may rigorously expand their roles. In the current paper, the authors summarize the latest state of research on these eight selected tools and the status of their practical implementation. We bring forward our arguments on why we consider these processes real game changers.
Engineering Enzymes for Environmental Sustainability
The development and implementation of more efficient and sustainable technologies is key to delivering our net–zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO2) and have been embedded into new efficient metabolic CO2 fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, PET. Finally, biocatalytic approaches for accessing petroleum–based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Finally, through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of more environmentally sustainable approaches, in order to protect our planet.
Enzymatic depolymerization of polyester: Foaming as a pretreatment to increase specific surface area
Abstract Poly(ethylene terephthalate) (PET) is widely used for its high strength‐to‐weight ratio, gas barrier properties, and chemical resistance. The growing PET use highlights the demand for a better recycling system. Enzymatic recycling, alongside mechanical and chemical methods, is eco‐friendly and yields properties similar to virgin PET. Substrate properties ( T g , crystallinity, and specific surface area [SSA]) and enzyme stability significantly impact conversion efficiency. Higher SSA and lower crystallinity tend to yield improved depolymerization when employing leaf compost‐cutinase (LCC‐ICCG) enzymes. This study explored melt extrusion and foaming as pretreatment techniques to modify PET structural properties, using a low‐cost chemical foaming agent (CFA). The monomer conversion rate and efficiency during depolymerization were measured and related to the processing, extrudate micro‐ and meso‐structure, and polyester type. Pretreated PET substrates showed reduced T g , crystallinity, density, and enhanced SSA, resulting in a 90% mass loss for foamed RPET and VPET substrates within 2 days. In contrast, PET with ~30% of cyclohexanedimethanol comonomer exhibited a nearly 50% lower depolymerization rate, with zero BHET production. It indicates that the combination of low crystallinity, low T g , and high SSA leads to improved monomer conversion. These findings emphasize the significance of amorphization and foaming in enhancing PET enzymatic depolymerization.
In‐Situ Product Removal for the Enzymatic Depolymerization of Poly(ethylene terephthalate) via a Membrane Reactor
Poly(ethylene terephthalate) (PET) is a common single-use plastic and a major contributor to plastic waste. PET upcycling through enzymatic depolymerization has drawn significant interests, but lack of robust enzymes in acidic environments remains a challenge. This study investigates in-situ product removal (ISPR) of protons and monomers from enzymatic PET depolymerization via a membrane reactor, focusing on the ICCG variant of leaf branch compost cutinase. More than two-fold improvements in overall PET depolymerization and terephthalic acid yields were achieved employing ISPR for an initial PET loading of 10 mgPET ml buffer −1 . The benefit of ISPR was reduced for a lower initial loading of 1 mgPET ml buffer −1 due to decreased need for pH stabilization of the enzyme-containing solutions. A back-of-envelop analysis suggests that at a modest dilution ratio, ISPR could help achieve savings on caustic base solutions used for pH control in a bioreactor. Our study provides valuable insights for future ISPR developments for enzymatic PET depolymerization, addressing the pressing need for more sustainable solutions towards plastic recycling and environmental conservation.
Effects of Copolymer Structure on Enzyme-Catalyzed Polyester Recycling
Polyesters are an omnipresent material used for a variety of applications (e.g., bottles, packaging, textile, windshield), roughly comprising 8% of plastics produced worldwide. Enzymatic recycling is an emerging solution to deal with the increasingly diverse polyesters that are not suitable for mechanical recycling. However, enzyme activity and efficiency are still the limiting factors impeding enzymatic recycling for different plastic waste forms. The effects of thermal and structural properties (e.g., glass transition temperature, crystallinity, specific surface area), which are determined by chemical composition and preprocessing, directly influence enzyme recycling efficiency. This work investigates two extrusion methods (single screw and twin-screw extrusion) to pretreat a range of copolyesters (RPET, PETG, Ecozen, Tritan and PBT) and modify their properties (i.e., glass transition temperature (T g ), crystallinity (%), and molecular weight (M n )). A PET-specific enzyme, leaf-branch compost cutinase (LCC ICCG ), produced from a fed-batch fermentation of Escherichia coli BL21(DE3), was used for the enzymatic depolymerization of different polyesters. Several copolyesters showed improved depolymerization after pretreatment, as measured by rate and amount of monomers produced. Furthermore, those that did not depolymerize were found to have exceptionally high glass transition temperature or percent crystallinity, highlighting the importance of these physical parameters on conversion efficiency.
Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis
Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1–2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.
A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds
Not Available
The reaction mechanism of the Ideonella sakaiensis PETase enzyme
Abstract Polyethylene terephthalate (PET), the most abundantly produced polyester plastic, can be depolymerized by the Ideonella sakaiensis PETase enzyme. Based on multiple PETase crystal structures, the reaction has been proposed to proceed via a two-step serine hydrolase mechanism mediated by a serine-histidine-aspartate catalytic triad. To elucidate the multi-step PETase catalytic mechanism, we use transition path sampling and likelihood maximization to identify optimal reaction coordinates for the PETase enzyme. We predict that deacylation is likely rate-limiting, and the reaction coordinates for both steps include elements describing nucleophilic attack, ester bond cleavage, and the “moving-histidine” mechanism. We find that the flexibility of Trp185 promotes the reaction, providing an explanation for decreased activity observed in mutations that restrict Trp185 motion. Overall, this study uses unbiased computational approaches to reveal the detailed reaction mechanism necessary for further engineering of an important class of enzymes for plastics bioconversion.