A customized self-assembled synergistic biocatalyst for plastic depolymerization.

The enzymatic degradation of plastic offers a green, sustainable strategy and scalable circular carbon route for solving polyester waste. Among the earlies discovered plastic-degrading enzymes are PET hydrolase (PETase) and MHET hydrolase (MHETase), which act synergistically. To promote the adsorption of enzymes on PET surfaces, increase their robustness, and enable directly depolymerization, we designed hydrophobin HFBI fused-PETase and MHETase. A customized self-assembled synergistic biocatalyst (MC@CaZn-MOF) was further developed to promote the two-step depolymerization process. The tailored catalysts showed better adhesion to the PET surface and desirable durability, retaining over 70% relative activity after incubation at pH 8.0 and 60 °C for 120 h. Importantly, MC@CaZn-MOF could directly decompose untreated AGf-PET to generate 9.5 mM TPA with weight loss over 90%. The successful implementation of a bifunctional customized catalyst makes the large-scale biocatalytic degradation of PET feasible, contributing to polymer upcycling and environmental sustainability.

Advances in plastic mycoremediation: Focus on the isoenzymes of the lignin degradation complex.

This study investigates P. ostreatus and A. bisporus biodegradation capacity of low density polyethylene (LDPE) oxidised to simulate environmental weathering. Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) were used to analyse the degradation of LDPE treated with fungal cultures. Molecular implications of LDPE degradation by P. ostreatus and A. bisporus were evaluated by Reverse transcription followed by quantitative PCR (qRT-PCR) of lac, mnp and lip genes expression. After 90 days of incubation, FT-IR analysis showed, for both fungal treatments, an increasing in the intensity of peaks related to the asymmetric C-C-O stretching (1160 to 1000 cm-1) and the -OH stretching (3700 to 3200 cm-1) due to the formation of alcohols and carboxylic acid, indicating depolymerisation of LDPE. This was confirmed by the SEM analysis, where a widespread alteration of the surface morphology was observed for treated LDPE fragments. Results revealed that the exposure of P. ostreatus to oxidised LDPE treatment led to a significant increase in the expression of the lac6, lac7, lac9, lac10 and mnp2 genes, while A. bisporus showed an over-expression in lac2 and lac12 genes. The obtained results offer new perspectives for a biotechnological use of P. ostreatus and A. bisporus for plastic bioremediation.

Engineered plastic-associated bacteria for biodegradation and bioremediation.

The global plastic waste crisis has triggered the development of novel methods for removal of recalcitrant polymers from the environment. Biotechnological approaches have received particular attention due to their potential for enabling sustainable, low-intensity bioprocesses which could also be interfaced with microbial upcycling pathways to support the emerging circular bioeconomy. However, low biodegradation efficiency of solid plastic materials remains a bottleneck, especially at mesophilic conditions required for one-pot degradation and upcycling. A promising strategy used in nature to address this is localisation of plastic-degrading microbes to the plastic surface via biofilm-mediated surface association. This review highlights progress and opportunities in leveraging these naturally occurring mechanisms of biofilm formation and other cell-surface adhesion biotechnologies to co-localise engineered cells to plastic surfaces. We further discuss examples of combining these approaches with extracellular expression of plastic-degrading enzymes to accelerate plastic degradation. Additionally, we review this topic in the context of nano- and microplastics bioremediation and their removal from wastewater and finally propose future research directions for this nascent field.

Phylum-level studies of bacterial cutinases for unravelling enzymatic specificity toward PET degradation: an in silico approach.

The overwhelming use of PET plastic in various day-to-day activities led to the voluminous increase in PET waste and growing environmental hazards. A plethora of methods have been used that are associated with secondary pollutants. Therefore, microbial degradation of PET provides a sustainable approach due to its versatile metabolic diversity and capacity. The present work highlights the cutinase enzyme's role in PET degradation. This study focuses on the bacterial cutinases homologs screened from 43 reported phylum of bacteria. The reported bacterial cutinases for plastic degradation have been chosen as reference sequences, and 917 sequences have shown homology across the bacterial phyla. The dienelactone hydrolase (DLH) domain was identified for attaining specificity towards PET binding in 196 of 917 sequences. Various computational tools have been used for the physicochemical characterization of 196 sequences. The analysis revealed that most selected sequences are hydrophilic, extracellular, and thermally stable. Based on this analysis, 17 sequences have been further pursued for three-dimensional structure prediction and validation. The molecular docking studies of 17 selected sequences revealed efficient PET binding with the three sequences derived from the phylum Bacteroidota, the lowest binding energy of -5.9 kcal/mol, Armatimonadota, and Nitrososphaerota with -5.8 kcal/mol. The two enzyme sequences retrieved from the phylum Bacteroidota and Armatimonadota are metagenomically derived. Therefore, the present studies concluded that there is a high probability of finding cutinase homologs in various environmental resources that can be further explored for PET degradation.

Discovery and characterization of two novel polyethylene terephthalate hydrolases: One from a bacterium identified in human feces and one from the Streptomyces genus.

Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETaseY239R/F244G/Y250G and ScPETaseA212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation.

Marine degradation and ecotoxicity of conventional, recycled and compostable plastic bags.

Plastic bags are currently a major component of marine litter, causing aesthetical nuisance, and undesirable effects on marine fauna that ingest them or are entangled. Plastic litter also rises concern on the ecotoxicological effects due to the potential toxicity of the chemical additives leached in aquatic environments. Conventional plastic bags are made of polyethylene, either from first use or recycled, but regulations restricting single-use plastics and limiting lightweight carrier bags (<50 µm thickness) have fostered the replacement of thin PE bags by compostable materials advertised as safer for the environment. In this study, we assess the degradation of commercially available plastic bags in marine conditions at two scales: aquariums (60 days) and outdoors flow-through mesocosm (120 days). Strength at break point and other tensile strength parameters were used as ecologically relevant endpoints to track mechanical degradation. Ecotoxicity has been assessed along the incubation period using the sensitive Paracentrotus lividus embryo test. Whereas PE bags did not substantially lose their mechanical properties within the 60 d aquarium exposures, compostable bags showed remarkable weight loss and tensile strength decay, some of them fragmenting in the aquarium after 3-4 weeks. Sediment pore water inoculum promoted a more rapid degradation of compostable bags, while nutrient addition pattern did not affect the degradation rate. Longer-term mesocosms exposures supported these findings, as well as pointed out the influence of the microbial processes on the degradation efficiency of compostable/bioplastic bags. Compostable materials, in contrast toPE, showed moderate toxicity on sea-urchin larvae, partially associated to degradation of these materials, but the environmental implications of these findings remain to be assessed. These methods proved to be useful to classify plastic materials, according to their degradability in marine conditions, in a remarkably shorter time than current standard tests and promote new materials safer for the marine fauna.

A screening method for plastic-degrading fungi.

The growing amount of plastic waste requires new ways of disposal or recycling. Research into the biodegradation of recalcitrant plastic polymers is gathering pace. Despite some progress, these efforts have not yet led to technologically and economically viable applications. In this study, we show that respirometric screening of environmental fungal isolates in combination with scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy can be used to identify new strains with the potential for the degradation of plastic polymers. We screened 146 fungal strains, 71 isolated from car repair shops, an environment rich in long-chain hydrocarbons, and 75 isolated from hypersaline water capable of growing at high concentrations of NaCl. When grown in a minimal medium with no carbon source, some strains produced significantly more CO2 when a pure plastic polymer was added to the medium, some only at high salinity. A selection of these strains was shown by FTIR and Raman spectroscopy to alter the properties of plastic polymers: Cladosporium sp. EXF-13502 on polyamide, Rhodotorula dairenensis EXF-13500 on polypropylene, Rhodotorula sp. EXF-10630 on low-density polyethylene and Wickerhamomyces anomalus EXF-6848 on polyethylene terephthalate. Respirometry in combination with specific spectroscopic methods is an efficient method for screening microorganisms capable of at least partial plastic degradation and can be used to expand the repertoire of potential plastic degraders. This is of particular importance as our results also show that individual strains are only active against certain polymers and under certain conditions. Therefore, efficient biodegradation of plastics is likely to depend on a collection of specialized microorganisms rather than a single universal plastic degrader.

Insights into Plastic Degradation Processes in Marine Environment by X-ray Photoelectron Spectroscopy Study.

The present study employs X-ray photoelectron spectroscopy (XPS) to analyze plastic samples subjected to degradation processes with the aim to gain insight on the relevant chemical processes and disclose fragmentation mechanisms. Two model plastics, namely polystyrene (PS) and polyethylene (PE), are selected and analyzed before and after artificial UV radiation-triggered weathering, under simulated environmental hydrodynamic conditions, in fresh and marine water for different time intervals. The object of the study is to identify and quantify chemical groups possibly evidencing the occurrence of hydrolysis and oxidation reactions, which are the basis of degradation processes in the environment, determining macroplastic fragmentation. Artificially weathered plastic samples are analyzed also by Raman and FT-IR spectroscopy. Changes in surface chemistry with weathering are revealed by XPS, involving the increase in chemical moieties (hydroxyl, carbonyl, and carboxyl functionalities) which can be correlated with the degradation processes responsible for macroplastic fragmentation. On the other hand, the absence of significant modifications upon plastics weathering evidenced by Raman and FT-IR spectroscopy confirms the importance of investigating plastics surface, which represents the very first part of the materials exposed to degradation agents, thus revealing the power of XPS studies for this purpose. The XPS data on experimentally weathered particles are compared with ones obtained on microplastics collected from real marine environment for investigating the occurring degradation processes.

Enzyme_Metal-Organic Framework Composites as Novel Approach for Microplastic Degradation.

Plastic pollution is one of the main worldwide environmental concerns. Our lifestyle involves persistent plastic consumption, aggravating the low efficiency of wastewater treatment plants in its removal. Nano/microplastics are accumulated in living beings, pushing to identify new water remediation strategies to avoid their harmful effects. Enzymes (e. g., Candida rugosa-CrL) are known natural plastic degraders as catalysts in depolymerization reactions. However, their practical use is limited by their stability, recyclability, and economical concerns. Here, enzyme immobilization in metal-organic frameworks (CrL_MOFs) is originally presented as a new plastic degradation approach to achieve a boosted plastic decomposition in aqueous systems while allowing the catalyst cyclability. Bis-(hydroxyethyl)terephthalate (BHET) was selected as model substrate for decontamination experiments for being the main polyethylene terephthalate (PET) degradation product. Once in contaminated water, CrL_MOFs can eliminate BHET (37 %, 24 h), following two complementary mechanisms: enzymatic degradation (CrL action) and byproducts adsorption (MOF effect). As a proof-of-concept, the capacity of a selected CrL_MOF composite to eliminate the BHET degradation products and its reusability are also investigated. The potential of these systems is envisioned in terms of improving enzyme cyclability, reducing costs along with feasible co-adsorption of plastic byproducts and other harmful contaminants, to successfully remove them in a single step.

Improved reference quality genome sequence of the plastic-degrading greater wax moth, Galleria mellonella.

Galleria mellonella is a pest of honeybees in many countries because its larvae feed on beeswax. However, G. mellonella larvae can also eat various plastics, including polyethylene, polystyrene, and polypropylene, and therefore, the species is garnering increasing interest as a tool for plastic biodegradation research. This paper presents an improved genome (99.3% completed lepidoptera_odb10 BUSCO; genome mode) for G. mellonella. This 472 Mb genome is in 221 contigs with an N50 of 6.4 Mb and contains 13,604 protein-coding genes. Genes that code for known and putative polyethylene-degrading enzymes and their similarity to proteins found in other Lepidoptera are highlighted. An analysis of secretory proteins more likely to be involved in the plastic catabolic process has also been carried out.

Biodegradation of polyethylene by the marine fungus Parengyodontium album.

Plastic pollution in the marine realm is a severe environmental problem. Nevertheless, plastic may also serve as a potential carbon and energy source for microbes, yet the contribution of marine microbes, especially marine fungi to plastic degradation is not well constrained. We isolated the fungus Parengyodontium album from floating plastic debris in the North Pacific Subtropical Gyre and measured fungal-mediated mineralization rates (conversion to CO2) of polyethylene (PE) by applying stable isotope probing assays with 13C-PE over 9 days of incubation. When the PE was pretreated with UV light, the biodegradation rate of the initially added PE was 0.044 %/day. Furthermore, we traced the incorporation of PE-derived 13C carbon into P. album biomass using nanoSIMS and fatty acid analysis. Despite the high mineralization rate of the UV-treated 13C-PE, incorporation of PE-derived 13C into fungal cells was minor, and 13C incorporation was not detectable for the non-treated PE. Together, our results reveal the potential of P. album to degrade PE in the marine environment and to mineralize it to CO2. However, the initial photodegradation of PE is crucial for P. album to metabolize the PE-derived carbon.

Interactive impacts of photoaged micro(nano)plastics and co-occurring chemicals in the environment.

In the environment, sunlight or ultraviolet (UV) radiation is considered to be the primary cause of plastic aging, leading to their fragmentation into particles, including micro(nano)plastics (MNPs). Photoaged MNPs possess diverse interactive properties and ecotoxicological implications substantially different from those of pristine plastic particles. This review aims to highlight the mechanisms and implications of UV-induced photoaging of MNPs, with an emphasis on various UV sources and their interactions with co-occurring organic and inorganic chemicals, as well as the associated ecological and health impacts and factors affecting those interactions. Compared to UV-B, UV-A and UV-C were more widely used in laboratory studies for MNP degradation. Photoaged MNPs act as vectors for the transportation of organic pollutants, organic matter, and inorganic chemicals in the environment. Literature showed that photoaged MNPs exhibit a higher sorption capacity for PPCPs, PAHs, PBDEs, pesticides, humic acid, fulvic acid, heavy metals, and metallic nanoparticles than pristine MNPs, potentially causing significant changes in associated ecological and health impacts. Combined exposure to photoaged MNPs and organic and inorganic pollutants significantly altered mortality rate, decreased growth rate, histological alterations, neurological impairments, reproductive toxicity, induced oxidative stress, thyroid disruption, hepatotoxicity, and genotoxicity in vivo, both in aquatic and terrestrial organisms. Limited studies were reported in vitro and found decreased cellular growth and survival, induced oxidative stress, and compromised the permeability and integrity of the cell membrane. In addition, several environmental factors (temperature, organic matter, ionic strength, time, and pH), MNP properties (polymer types, sizes, surface area, shapes, colour, and concentration), and chemical properties (pollutant type, concentration, and physiochemical properties) can influence the photoaging of MNPs and associated impacts. Lastly, the research gaps and prospects of MNP photoaging and associated implications were also summarized. Future research should focus on the photoaging of MNPs under environmentally relevant conditions, exploiting the polydisperse characteristics of environmental plastics, to make this process more realistic for mitigating plastic pollution.

Oceanimonas pelagia sp. nov., a novel biosurfactant-producing and plastic-degrading potential bacterium isolated from marine coastal sediment.

A marine bacterial strain, named NTOU-MSR1T, was isolated from marine sediment of northern coast of Taiwan. This bacterium was Gram-stain-negative, aerobic, and motile, with a single flagellum. Its rod-shaped cells measured approximately 0.5-0.6 µm in width and 1.8-2.0 µm in length. NTOU-MSR1T grew at temperatures ranging from 10 to 45 °C, optimally at 30 °C. The pH range for growth was 7.0-10.0, with optimal growth at pH 7.0-8.0. It tolerated NaCl concentrations up to 12%. The cell membrane predominantly contained fatty acids such C16:1ω7c, C18:1ω7c, and C16:0. The overall genome relatedness indices indicated that strain NTOU-MSR1T had an average nucleotide identity (ANI) of 87.88% and a digital DNA-DNA hybridization (dDDH) value of 35.40% compared to its closest related species, O. marisflavi 102-Na3T. These values fell below the 95% and 70% threshold for species delineation, respectively. These findings suggested that the strain NTOU-MSR1T was a new member of the Oceanimonas genus. Its genomic DNA had a G + C content of 61.0 mol%. Genomic analysis revealed genes associated with the catechol branch of ß- ketoadipate pathway for degrading polycyclic aromatic hydrocarbons, resistance to heavy metal, biosynthesis of polyhydroxybutyrate and the production of glycoside hydrolases (GH19, GH23, and GH103) for chitin and glycan digestion. Additionally, NTOU-MSR1T was capable of synthesizing biosurfactants and potentially degrading plastic. The proposed name for this new species is Oceanimonas pelagia, with the type strain designated as NTOU-MSR1T (= BCRC 81403T = JCM 36023T).

Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes.

Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house E. coli extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.

Cross-linked enzyme aggregates of polyethylene terephthalate hydrolyse (PETase) from Ideonella sakaiensis for the improvement of plastic degradation.

Polyethylene terephthalate (PET) is one of the most produced plastics globally and its accumulation in the environment causes harm to the ecosystem. Polyethylene terephthalate hydrolyse (PETase) is an enzyme that can degrade PET into its monomers. However, free PETase lacks operational stabilities and is not reusable. In this study, development of cross-linked enzyme aggregate (CLEA) of PETase using amylopectin (Amy) as cross-linker was introduced to solve the limitations of free PETase. PETase-Amy-CLEA exhibited activity recovery of 81.9 % at its best immobilization condition. Furthermore, PETase-Amy-CLEA exhibited 1.37-, 2.75-, 2.28- and 1.36-fold higher half-lives than free PETase at 50 °C, 45 °C, 40 °C and 35 °C respectively. Moreover, PETase-Amy-CLEA showed broader pH stability from pH 5 to 10 and could be reused up to 5 cycles. PETase-Amy-CLEA retained >70 % of initial activity after 40 days of storage at 4 °C. In addition, lower Km of PETase-Amy-CLEA indicated better substrate affinity than free enzyme. PETase-Amy-CLEA corroded PET better and products yielded was 66.7 % higher than free PETase after 32 h of treatment. Hence, the enhanced operational stabilities, storage stability, reusability and plastic degradation ability are believed to make PETase-Amy-CLEA a promising biocatalyst in plastic degradation.

Engineering microbiomes to transform plastics.

The design and study of active microbial consortia able to degrade plastics represent an exciting area of research toward the development of bio-based alternatives to efficiently transform plastic waste. This forum article discusses concepts and mechanisms to inform emerging strategies for engineering microbiomes to transform plastics under controlled settings.

Hydrolase and plastic-degrading microbiota explain degradation of polyethylene terephthalate microplastics during high-temperature composting.

This research aims to explore the degradation properties of polyethylene terephthalate (PET) by PET hydrolase (WCCG) in high-temperature composting and its impact on microbial communities. PET degradation, composting parameters and microbial communities were assessed in 220 L sludge composters with PET and WCCG using high-throughput sequencing. Results showed that WCCG addition led to a deceleration of the humification process and a reduction in the relative abundance of thermophilic genera. Potential PET degrading microbiota, e.g. Acinetobacter, Bacillus, were enriched in the plastisphere in the composters where PET reduced by 26 % without WCCG addition. The external introduction of the WCCG enzyme to compost predominantly instigates a chemical reaction with PET, concurently curtailing the proliferation of plastic-degrading bacteria, leading to a 35 % degradation of PET. Both the WCCG enzyme and the microbiota associated with plastic-degradation showed the potential for reducing PET, offering a novel method for mitigating pollution caused by environmental microplastics.

Polyurethane with ß-Selenocarbonyl Structure Enabling the Combination of Plastic Degradation and Waste Upcycling.

Degradable polymers offer a promising solution to mitigate global plastic pollution, but the degraded products often suffer from diminished value. Upcycling is a more sustainable approach to upgrade polymer waste into value-added products. Herein, we report a ß-selenocarbonyl-containing polyurethane (SePU), which can be directly degraded under mild conditions into valuable selenium fertilizers for selenium-rich vegetable cultivation globally, enabling both plastic degradation and waste upcycling. Under oxidation condition, this polymer can be easily and selectively degraded via selenoxide elimination reaction from mixed plastic waste. The degraded product can serve as effective selenium fertilizers to increase selenium content in radish and pak choi. The SePU exhibits excellent mechanical properties. Additionally, we observed the formation of spherulites-like selenium particles within the materials during degradation for the first time. Our research offers a successful application of selenoxide elimination reaction in the field of plastic degradation for the first time, endowing plastics with both degradability and high reusable value. This strategy provides a promising solution to reduce pollution and improve economy and sustainability of plastics.

Exploring Microorganisms from Plastic-Polluted Sites: Unveiling Plastic Degradation and PHA Production Potential.

The exposure of microorganisms to conventional plastics is a relatively recent occurrence, affording limited time for evolutionary adaptation. As part of the EU-funded project BioICEP, this study delves into the plastic degradation potential of microorganisms isolated from sites with prolonged plastic pollution, such as plastic-polluted forests, biopolymer-contaminated soil, oil-contaminated soil, municipal landfill, but also a distinctive soil sample with plastic pieces buried three decades ago. Additionally, samples from Arthropoda species were investigated. In total, 150 strains were isolated and screened for the ability to use plastic-related substrates (Impranil dispersions, polyethylene terephthalate, terephthalic acid, and bis(2-hydroxyethyl) terephthalate). Twenty isolates selected based on their ability to grow on various substrates were identified as Streptomyces, Bacillus, Enterococcus, and Pseudomonas spp. Morphological features were recorded, and the 16S rRNA sequence was employed to construct a phylogenetic tree. Subsequent assessments unveiled that 5 out of the 20 strains displayed the capability to produce polyhydroxyalkanoates, utilizing pre-treated post-consumer PET samples. With Priestia sp. DG69 and Neobacillus sp. DG40 emerging as the most successful producers (4.14% and 3.34% of PHA, respectively), these strains are poised for further utilization in upcycling purposes, laying the foundation for the development of sustainable strategies for plastic waste management.

De novo design of a polycarbonate hydrolase.

Enzymatic degradation of plastics is currently limited to the use of engineered natural enzymes. As of yet, all engineering approaches applied to plastic degrading enzymes retain the natural $\alpha /\beta $-fold. While mutations can be used to increase thermostability, an inherent maximum likely exists for the $\alpha /\beta $-fold. It is thus of interest to introduce catalytic activity toward plastics in a different protein fold to escape the sequence space of plastic degrading enzymes. Here, a method for designing highly thermostable enzymes that can degrade plastics is described. With the help of Rosetta an active site catalysing the hydrolysis of polycarbonate is introduced into a set of thermostable scaffolds. Through computational evaluation, a potential PCase was selected and produced recombinantly in Escherichia coli. Thermal analysis suggests that the design has a melting temperature of >95$^{\circ }$C. Activity toward polycarbonate was confirmed using atomic force spectroscopy (AFM), proving the successful design of a PCase.