Enzymatic Recycling Research Articles

Fusion of Hydrophobic Anchor Peptides Promotes the Hydrolytic Activity of PETase but not the Extent of PET Depolymerization

Enzymatic recycling of polyethylene terephthalate (PET) has attracted significant attention in recent years. While the fusion of anchor peptides to PET hydrolases is believed to enhance PET hydrolytic activity, a quantitative analysis is yet lacking. Here, we construct four fusion enzymes by fusing anchor peptides (including hydrophobic LCI, LCIM1 and TA2, and hydrophilic EK4) to the C terminus of HotPETase, one of the most active PET hydrolases for high‐crystallinity PET (HC‐PET). Single‐molecule force spectroscopy (SMFS) demonstrates that hydrophobic anchor peptides promote adhesive interactions between the fusion enzymes and the PET surface. This is also validated by the adsorption kinetics and isotherms, and the saturated adsorption capacity remains unaltered compare to HotPETase. At low substrate loadings, the apparent hydrolytic activity of these fusion enzymes is positively related to the hydrophobicity of the anchor peptides. Among them, HotPETase‐LCI stands out as the most effective enzyme for HC‐PET degradation, demonstrating a 1.5‐fold increase in hydrolytic activity. At high substrate loadings, the advantages of fusion with anchor peptides diminish. We conclude that fusion enzymes only facilitate the hydrolytic rates of HC‐PET but have little effect on the final conversion extent.

Manufacturing of the highly active thermophile PETases PHL7 and PHL7mut3 using Escherichia coli

BackgroundThe global plastic waste crisis requires combined recycling strategies. One approach, enzymatic degradation of PET waste into monomers, followed by re-polymerization, offers a circular economy solution. However, challenges remain in producing sufficient amounts of highly active PET-degrading enzymes without costly downstream processes.ResultsUsing the growth-decoupled enGenes eX-press V2 E. coli strain, pH, induction strength and feed rate were varied in a factorial-based optimization approach, to find the best-suited production conditions for the PHL7 enzyme. This led to a 40% increase in activity of the fermentation supernatant. Optimization of the expression construct resulted in a further 4-fold activity gain. Finally, the identified improvements were applied to the production of the more active and temperature stable enzyme variant, PHL7mut3. The unpurified fermentation supernatant of the PHL7mut3 fermentation was able to completely degrade our PET film sample after 16 h of incubation at 70 °C at an enzyme loading of only 0.32 mg enzyme per g of PET.ConclusionsIn this research, we present an optimized process for the extracellular production of thermophile and highly active PETases PHL7 and PHL7mut3, eliminating the need for costly purification steps. These advancements support large-scale enzymatic recycling, contributing to solving the global plastic waste crisis.

Comparative evaluation of the extracellular production of a polyethylene terephthalate degrading cutinase by Corynebacterium glutamicum and leaky Escherichia coli in batch and fed-batch processes

BackgroundWith a growing global population, the generation of plastic waste and the depletion of fossil resources are major concerns that need to be addressed by developing sustainable and efficient plastic recycling methods. Biocatalytic recycling is emerging as a promising ecological alternative to conventional processes, particularly in the recycling of polyethylene terephthalate (PET). However, cost-effective production of the involved biocatalyst is essential for the transition of enzymatic PET recycling to a widely used industrial technology. Extracellular enzyme production using established organisms such as Escherichia coli or Corynebacterium glutamicum offers a promising way to reduce downstream processing costs.ResultsIn this study, we compared extracellular recombinant protein production by classical secretion in C. glutamicum and by membrane leakage in E. coli. A superior extracellular release of the cutinase ICCGDAQI was observed with E. coli in batch and fed-batch processes on a litre-scale. This phenomenon in E. coli, in the absence of a signal peptide, might be associated with membrane-destabilizing catalytic properties of the expressed cutinase. Optimisations regarding induction, expression temperature and duration as well as carbon source significantly enhanced extracellular cutinase activity. In particular, in fed-batch cultivation of E. coli at 30 °C with lactose as carbon source and inducer, a remarkable extracellular activity (137 U mL−1) and cutinase titre (660 mg L−1) were achieved after 48 h. Literature values obtained with other secretory organisms, such as Bacillus subtilis or Komagataella phaffii were clearly outperformed. The extracellular ICCGDAQI produced showed high efficacy in the hydrolysis of PET textile fibres, either chromatographically purified or unpurified as culture supernatant. In less than 18 h, 10 g L−1 substrate was hydrolysed using supernatant containing 3 mg cutinase ICCGDAQI at 70 °C, pH 9 with terephthalic acid yields of up to 97.8%.ConclusionExtracellular production can reduce the cost of recombinant proteins by simplifying downstream processing. In the case of the PET-hydrolysing cutinase ICCGDAQI, it was even possible to avoid chromatographic purification and still achieve efficient PET hydrolysis. With such production approaches and their further optimisation, enzymatic recycling of PET can contribute to a more efficient and environmentally friendly solution to the industrial recycling of plastics in the future.

Exploring enzymatic degradation, reinforcement, recycling, and upcycling of poly(ester)s-poly(urethane) with movable crosslinks

Exploring enzymatic degradation, reinforcement, recycling, and upcycling of poly(ester)s-poly(urethane) with movable crosslinks

Plastic-Degrading Enzymes from Marine Microorganisms and Their Potential Value in Recycling Technologies.

Since the 2005 discovery of the first enzyme capable of depolymerizing polyethylene terephthalate (PET), an aromatic polyester once thought to be enzymatically inert, extensive research has been undertaken to identify and engineer new biocatalysts for plastic degradation. This effort was directed toward developing efficient enzymatic recycling technologies that could overcome the limitations of mechanical and chemical methods. These enzymes are versatile molecules obtained from microorganisms living in various environments, including soil, compost, surface seawater, and extreme habitats such as hot springs, hydrothermal vents, deep-sea regions, and Antarctic seawater. Among various plastics, PET and polylactic acid (PLA) have been the primary focus of enzymatic depolymerization research, greatly enhancing our knowledge of enzymes that degrade these specific polymers. They often display unique catalytic properties that reflect their particular ecological niches. This review explores recent advancements in marine-derived enzymes that can depolymerize synthetic plastic polymers, emphasizing their structural and functional features that influence the efficiency of these catalysts in biorecycling processes. Current status and future perspectives of enzymatic plastic depolymerization are also discussed, with a focus on the underexplored marine enzymatic resources.

Enzymatic depolymerization of polyester: Foaming as a pretreatment to increase specific surface area

AbstractPoly(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 (Tg, 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 Tg, 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 Tg, and high SSA leads to improved monomer conversion. These findings emphasize the significance of amorphization and foaming in enhancing PET enzymatic depolymerization.

Regioselective enzymatic depolymerization of aromatic-aliphatic polyester revealed by computational modelling

Poly(butylene adipate-co-terephthalate) (PBAT) is widely utilized in the production of food packaging and mulch films. Its extensive application has contributed significantly to global solid waste, posing numerous environmental challenges. Recently, enzymatic recycling has emerged as a promising eco-friendly solution for the management of plastic waste. Here, we systematically investigate the depolymerization mechanism of PBAT catalyzed by cutinase TfCutSI with molecular docking, molecular dynamics simulations, and quantum mechanics/molecular mechanics calculations. Although the binding affinities for acid ester and terephthalic acid ester bonds are similar, a regioselective depolymerization mechanism and a “chain-length” effect on regioselectivity were proposed and evidenced. The regioselectivity is highly associated with specific structural parameters, namely Substrate@O4-Met@H7 and Substrate@C1-Ser@O1 distances. Notably, the binding mode of BTa captured by X-ray crystallography does not facilitate subsequent depolymerization. Instead, a previously unanticipated binding mode, predicted through computational analysis, is confirmed to play a crucial role in BTa depolymerization. This finding proves the critical role of computational modelling in refining experimental results. Furthermore, our results revealed that both the hydrogen bond network and enzyme’s intrinsic electric field are instrumental in the formation of the final product. In summary, these novel molecular insights into the PBAT depolymerization mechanism offer a fundamental basis for enzyme engineering to enhance industrial plastic recycling.

Surface-associated residues in subtilisins contribute to poly-L-lactic acid depolymerization via enzyme adsorption.

Poly-L-lactic acid (PLLA) is currently the most abundant bioplastic; however, limited environmental biodegradability and few recycling options diminish its value as a biodegradable commodity. Enzymatic recycling is one strategy for ensuring circularity of PLLA, but this approach requires a thorough understanding of enzymatic mechanisms and protein engineering strategies to enhance activity. In this study, we engineer PLLA depolymerizing subtilisin enzymes originating from Bacillus species to elucidate the molecular mechanisms dictating their PLLA depolymerization activity and to improve their function. The surface-associated amino acids of two closely related subtilisin homologues originating from Bacillus subtilis (BsAprE) and Bacillus pumilus (BpAprE) were compared, as they were previously engineered to have nearly identical active sites, but still varied greatly in PLLA depolymerizing activity. Further analysis identified several surface-associated amino acids in BpAprE that lead to enhanced PLLA depolymerization activity when engineered into BsAprE. In silico protein modelling demonstrated increased enzyme surface hydrophobicity in engineered BsAprE variants and revealed a structural motif favoured for PLLA depolymerization. Experimental evidence suggests that increases in activity are associated with enhanced polymer binding as opposed to substrate specificity. These data highlight enzyme adsorption as a key factor in PLLA depolymerization by subtilisins.

Analysis of Introducing Plastic Waste Enzymatic Recycling for Sustainable Waste Management in Latvia

Economic growth, urbanisation, and consumer consumption habits have contributed to the yearly increase in the municipal solid waste amount. A significant portion of household waste consists of plastic materials and packaging, which currently cannot be fully and endlessly recycled, does not decompose in nature, and degrades into micro- and nanoparticles entering the soil, air, aquatic environments, and organisms. According to an OECD report from 2022, the amount of plastic waste produced globally is expected to triple by 2060, with around half being landfilled and less than one-fifth being recycled. As waste volumes are going to increase, the need to ensure appropriate waste recycling technology is addressed in this study by introducing enzymatic plastic waste recycling in Latvia. Enzymatic recycling has an advantage over mechanical recycling technology because it can depolymerise plastics without degrading the quality of the material. The methodology applied in this study includes data analysis of the waste amount in Latvia, analysis of the existing plastic recycling plant operation, life cycle assessment of existing and proposed methods, and socio-economic impact evaluation. The proposed solution meets the objectives of promoting sustainable plastic waste recycling through the introduction of enzymatic recycling; therefore, it aligns with the European Union’s targets to follow circular economy principles.

Recent progress in enzymatic degradation and recycling of polyurethanes

The widespread production of polymeric materials has precipitated severe environmental challenges, prompting a pressing need for effective solutions. Among these materials, polyurethanes (PUs) stand out significantly due to their versatility in synthetic approaches, manufacturing, properties, and applications. This versatility has led to a high demand for PUs, resulting in significant waste accumulation at the end of their lifecycle. Along with that, the PU’s inherent chemical stability prevents the occurrence of natural hydrolytic processes. Consequently, there is a critical need for sustainable recycling methods, particularly those capable of recovering reusable monomers. Recycling is a leading way to manage waste by reducing plastic pollution and its environmental impact, which can be performed through mechanical, chemical, and biological processes. Biological degradation occurs through the action of enzymes and microbes that can potentially convert the polymers into monomers or other valuable chemicals. This approach presents an opportunity to establish a sustainable and circular economy within the PUs industry while considerably reducing the environmental impact. This review highlights enzymatic degradation as a promising solution for PU recycling by discussing the types of enzymes utilized in the process, potential biochemical pathways for PU degradation, recent achievements, and future remarks for this process.

On the temperature dependence of enzymatic degradation of poly(ethylene terephthalate)

Enzymatic recycling of poly(ethylene terephthalate), PET, has attracted significant attention in recent years. Temperature is a governing factor in the enzymatic degradation of PET, influencing simultaneously the catalytic activity and thermal stability of enzymes, as well as the biodegradability of PET materials from many perspectives. Here we present a detailed examination of the complex and mutual effects of temperature on the degradation of low-crystallinity PET (LC-PET, 7.6%) and high-crystallinity PET (HC-PET, 30%) microparticles using the WCCG variant of the leaf-branch compost cutinase (LCC). The degradation velocity apparently increases exponentially with increasing temperature at temperatures below 65 °C. Arrhenius plots show a sudden reduction in activation energy at temperatures higher than 40 °C, suggesting the onset of the surface glass transition of PET particles. This is more than 20 °C lower than the bulk glass transition temperature, underscoring the interfacial catalytic nature of enzymatic PET degradation. WCCG undergoes substantial conformational changes upon thermal incubation at temperatures ranging from 50 to 70 °C and exhibits enhanced activity, owing to the increased intrinsic catalytic activity and improved adsorption on PET surface. Further increasing the temperature leads to the inactivation of enzymes alongside the rapid recrystallization of amorphous PET, impeding the enzymatic degradation. These findings offer a detailed mechanistic understanding of the temperature dependence of the enzymatic degradation of PET, and may have implications for the engineering of more powerful PET hydrolases and the selection of favorable conditions for industrially-related recycling processes.

Exploring the pH dependence of an improved PETase

Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His. Here, we explore this aspect in the context of LCCICCG, an optimized PETase, derived from the leaf-branch compost cutinase enzyme. We use NMR to identify the dominant tautomeric structure of the six histidines. Five show surprisingly low pKa values below 4.0, whereas the catalytic H242 in the active enzyme displays a pKa value that varies from 4.9 to 4.7 when temperatures increase from 30°C to 50°C. Whereas the hydrolytic activity of the enzyme toward a soluble substrate can be modeled by the corresponding protonation/deprotonation curve, an important discrepancy is found when the substrate is the solid plastic. This opens the way to further mechanistic understanding of the PETase activity and underscores the importance of studying the enzyme at the liquid-solid interface.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion

AbstractSecondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N‐terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug‐binding LmrR and nucleotide‐binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D‐proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1‐benzyl‐1,4‐dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro‐R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR‐based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion.

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N-terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D-proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR-based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts.

Computational design of highly efficient thermostable MHET hydrolases and dual enzyme system for PET recycling

Recently developed enzymes for the depolymerization of polyethylene terephthalate (PET) such as FAST-PETase and LCC-ICCG are inhibited by the intermediate PET product mono(2-hydroxyethyl) terephthalate (MHET). Consequently, the conversion of PET enzymatically into its constituent monomers terephthalic acid (TPA) and ethylene glycol (EG) is inefficient. In this study, a protein scaffold (1TQH) corresponding to a thermophilic carboxylesterase (Est30) was selected from the structural database and redesigned in silico. Among designs, a double variant KL-MHETase (I171K/G130L) with a similar protein melting temperature (67.58 °C) to that of the PET hydrolase FAST-PETase (67.80 °C) exhibited a 67-fold higher activity for MHET hydrolysis than FAST-PETase. A fused dual enzyme system comprising KL-MHETase and FAST-PETase exhibited a 2.6-fold faster PET depolymerization rate than FAST-PETase alone. Synergy increased the yield of TPA by 1.64 fold, and its purity in the released aromatic products reached 99.5%. In large reaction systems with 100 g/L substrate concentrations, the dual enzyme system KL36F achieved over 90% PET depolymerization into monomers, demonstrating its potential applicability in the industrial recycling of PET plastics. Therefore, a dual enzyme system can greatly reduce the reaction and separation cost for sustainable enzymatic PET recycling.

ENZYMES IN THE DEVELOPMENT OF PHYSICO-CHEMICAL METHODS FOR BIOMEDICAL RESEARCH

The paper addresses the possibilities of voltammetry, surface-enhanced Raman spectroscopy (SERS) and mass spectrometry in tandem with enzymatic catalysis. The basic principles of operation of electrochemical biosensors based on oxidases and dehydrogenases are described. It has been shown that biosensors using cyclic enzymatic reactions and substrate recycling have the best sensitivity. The variants of significant improvement of the analytical potential of biosensor analysis due to the use of polymers for effective modification of the electrode surface and non-destructive immobilization of enzymes are illustrated. The data demonstrating how the use of enzyme labels expands the range of bioanalytical applications of SERS are presented. The possibility of highly sensitive measurement of the activity of enzyme labels (peroxidase, alkaline phosphatase, β-galactosidase) using SERS, in fact, opens up a new universal platform for the development of methods for the determination of various antigens. By way of example of the most commonly used proteases, the main trends in the development of the methodology of proteomic studies by mass spectrometry, as well as the role of proteases in the design of mass spectrometric experiments, are considered.

Exploring new galaxies: Perspectives on the discovery of novel PET-degrading enzymes

Polyethylene terephthalate (PET) is a widely used polyester due to its beneficial material properties and low cost. However, PET contributes significantly to the growing problem of plastic waste pollution. Enzymatic PET recycling has emerged as a promising alternative to conventional mechanical and chemical recycling methods. While many PET hydrolases – belonging to the α/β-hydrolase fold superfamily – have been discovered, the wild-type enzymes obtained from natural sources are not optimal for industrial conditions and need to be optimized through rational design or directed evolution to improve their efficiency and stability. This Perspective summarizes case studies of engineered PET hydrolases and proposes a workflow that tightly integrates a variety of in silico and high-throughput approaches for biochemical and structural characterization to accelerate the discovery of PET-degrading enzymes, also with novel structural scaffolds. These biocatalysts could be candidates for developing further innovative plastic recycling techniques.

Enzymatic hydrolysis of single-use bioplastic items by improved recombinant yeast strains

Single-use bioplastic items pose new challenges for a circular plastics economy as they require different processing than petroleum-based plastics items. Microbial and enzymatic recycling approaches could address some of the pitfalls created by the influx of bioplastic waste. In this study, the recombinant expression of a cutinase-like-enzyme (CLE1) was improved in the yeast Saccharomyces cerevisiae to efficiently hydrolyse several commercial single-use bioplastic items constituting blends of poly(lactic acid), poly(1,4-butylene adipate-co-terephthalate), poly(butylene succinate) and mineral fillers. The hydrolysis process was optimised in controlled bioreactor configurations to deliver substantial monomer concentrations and, ultimately, 29 to 78% weight loss. Product inhibition studies and molecular docking provided insights into potential bottlenecks of the enzymatic hydrolysis process, while FT-IR analysis showed the preferential breakdown of specific polymers in blended commercial bioplastic items. This work constitutes a step towards implementing enzymatic hydrolysis as a circular economy approach for the valorisation of end-of-life single-use bioplastic items.

Degradation of polypropylene by fungi Coniochaeta hoffmannii and Pleurostoma richardsiae

The urgent need for better disposal and recycling of plastics has motivated a search for microbes with the ability to degrade synthetic polymers. While microbes capable of metabolizing polyurethane and polyethylene terephthalate have been discovered and even leveraged in enzymatic recycling approaches, microbial degradation of additive-free polypropylene (PP) remains elusive. Here we report the isolation and characterization of two fungal strains with the potential to degrade pure PP. Twenty-seven fungal strains, many isolated from hydrocarbon contaminated sites, were screened for degradation of commercially used textile plastic. Of the candidate strains, two identified as Coniochaeta hoffmannii and Pleurostoma richardsiae were found to colonize the plastic fibers using scanning electron microscopy (SEM). Further experiments probing degradation of pure PP films were performed using C. hoffmannii and P. richardsiae and analyzed using SEM, Raman spectroscopy and Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). The results showed that the selected fungi were active against pure PP, with distinct differences in the bonds targeted and the degree to which each was altered. Whole genome and transcriptome sequencing was conducted for both strains and the abundance of carbohydrate active enzymes, GC content, and codon usage bias were analyzed in predicted proteomes for each. Enzymatic assays were conducted to assess each strain’s ability to degrade naturally occurring compounds as well as synthetic polymers. These investigations revealed potential adaptations to hydrocarbon-rich environments and provide a foundation for further investigation of PP degrading activity in C. hoffmannii and P. richardsiae.

Assessment of Four Engineered PET Degrading Enzymes Considering Large-Scale Industrial Applications.

In recent years, enzymatic recycling of the widely used polyester polyethylene terephthalate (PET) has become a complementary solution to current thermomechanical recycling for colored, opaque, and mixed PET. A large set of promising hydrolases that depolymerize PET have been found and enhanced by worldwide initiatives using various methods of protein engineering. Despite the achievements made in these works, it remains difficult to compare enzymes' performance and their applicability to large-scale reactions due to a lack of homogeneity between the experimental protocols used. Here, we pave the way for a standardized enzymatic PET hydrolysis protocol using reaction conditions relevant for larger scale hydrolysis and apply these parameters to four recently reported PET hydrolases (LCCICCG, FAST-PETase, HotPETase, and PES-H1L92F/Q94Y). We show that FAST-PETase and HotPETase have intrinsic limitations that may not permit their application on larger reaction scales, mainly due to their relatively low depolymerization rates. With 80% PET depolymerization, PES-H1L92F/Q94Y may be a suitable candidate for industrial reaction scales upon further rounds of enzyme evolution. LCCICCG outperforms the other enzymes, converting 98% of PET into the monomeric products terephthalic acid (TPA) and ethylene glycol (EG) in 24 h. In addition, we optimized the reaction conditions of LCCICCG toward economic viability, reducing the required amount of enzyme by a factor of 3 and the temperature of the reaction from 72 to 68 °C. We anticipate our findings to advance enzymatic PET hydrolysis toward a coherent assessment of the enzymes and materialize feasibility at larger reaction scales.