Bioactive plastic capable of controllable degradation and microplastic elimination

Abstract

By nano-dispersing trace amounts of enzymes and random heteropolymers in plastics, we provide environmentally friendly, fully functional plastics that can eliminate microplastics and achieve controllable degradation.

Description

Technical Field

[0001] Priority: This invention was made with government support under grant number W911NF-13-1-0232 awarded by the Army Research Laboratory, US Department of Defense, and grant number FWP KC3104 awarded by the Office of Basic Energy Sciences, US Department of Energy. The US government retains certain rights in this invention.

Background Art

[0002] Plastics are as useful materials as polymers, but the burden of plastic waste has reached its limit, and it is necessary for all organizations to cooperate and address it promptly. As a new issue, the problem of how to effectively eliminate microplastics has been recognized, but despite years of efforts in plastic recycling, there is no method that can immediately solve this problem. 1 . Landfills 2 and the ocean 3 where plastic waste accumulates is decomposed into microplastics, ingested by various organisms, and carried up the food chain 4,5 causing serious health problems for humans and wildlife. To improve the efficiency of chemical recycling in an economically viable way, there is a need to develop new plastic materials that can be controllably decomposed and quickly eliminate microplastics within the existing manufacturing framework.

[0003] In landfills and aquatic ecosystems, the recycling process of materials is catalyzed by enzymes 6 but the concentration of available enzymes is low, and the surface is eroded under diffusion control through random cleavage of polymer chains, so such a decomposition process from the outer surface takes several years. 7,8 By embedding a catalyst capable of cleaving polymer chains in plastics, the decomposition of plastics can be accelerated. 9,10Also, by physically encapsulating the enzyme, the degradation of plastics can be partially controlled, but microplastics cannot be eliminated. In polymers treated by solution treatment or melt treatment, the enzyme aggregates and loses its important activity 11 When the host material disintegrates, the enzyme elutes 12 When the enzyme is nano-dispersed, the available enzyme increases, the degradation efficiency improves, the elution of the enzyme is suppressed, and even if microplastics are formed, they can be continuously decomposed. Little is known about the behavior of the enzyme encapsulated in a solid phase with a size comparable to that of a single polymer chain, but by regulating the interaction between the enzyme and the host material and the degradation of the polymer, it is possible to control by-products at the molecular level, and as a result, it is considered that a closed-loop resource cycle can be achieved.

[0004] In WO2019 / 143578 previously filed by the present inventors, a random heteropolymer capable of maintaining the function of a protein in a heterogeneous environment is disclosed. Also, US20180142097 relates to a biodegradable polyester that is randomly cleaved. In the controllable degradation process disclosed herein, by utilizing the shape of the active site of the enzyme and interfacial chemistry to manipulate the interaction between the enzyme and the polyester, a processive type of degradation of a single polymer chain is realized.

Summary of the Invention

Problems to be Solved by the Invention

[0005] This specification discloses that by nano-dispersing a trace amount of an enzyme (e.g., lipase) in a plastic (e.g., polycaprolactone (PCL)), it is possible to provide an environmentally friendly and fully functional plastic that can achieve the elimination and controllable degradation of microplastics. By encapsulating the enzyme in nanocapsules, (1) inducing continuous degradation to eliminate 95% of the microplastics; (2) providing a polymer degradation mechanism capable of generating repolymerizable low-molecular-weight by-products by selectively cleaving the ends of the polymer chains rather than randomly cleaving the polymer chains; (3) enabling the melt-processed host matrix to be degraded in a spatially and temporally controllable manner because the degradation of the polymer occurs locally depending on the lamellar thickness regardless of the bulk crystallinity; and (4) making it possible to prepare a conductive ink for 3D printing from which the entire amount of the noble metal filler can be recovered. Therefore, the present invention provides a technically feasible solution that is environmentally friendly and can achieve the elimination of microplastics and the recycling of materials.

Means for Solving the Problems

[0006] In one aspect, the present invention is a bioactive plastic composition comprising an organic polymer, a nano-dispersion of a complex composed of an enzyme that hydrolyzes the organic polymer and a random heteropolymer (RHP), and characterized in that controllable processive-type depolymerization and elimination of microplastics are made possible by hydrolyzing the organic polymer with the enzyme.

[0007] In one embodiment, the complex is uniformly dispersed in the composition, and the size of the complex ranges from 10 nm, 20 nm, or 40 nm to 100 nm, 200 nm, or 500 nm, or the size of the complex present between the lamellae of the crystalline organic polymer ranges from 10 nm, 20 nm, or 40 nm to 100 nm, 200 nm, or 500 nm, and / or the content of the enzyme in the composition ranges from 0.001%, 0.01%, or 0.1% to 0.1%, 1%, or 5%.

[0008] In one embodiment, the RHP contains a plurality of types of monomers selected from methyl methacrylate (MMA), oligo(ethylene glycol) methacrylate (OEGMA), potassium 3-sulfopropyl methacrylate (3-SPMA), and 2-ethylhexyl methacrylate (2-EHMA) in various ratios.

[0009] In one embodiment, the combination of the organic polymer and the enzyme is selected from the combination of polycaprolactone (PCL) and lipase, the combination of polylactic acid (PLA) and proteinase K, and the combination of polyethylene terephthalate (PET) and PET hydrolase.

[0010] In one embodiment, the composition is prepared into a conductive ink for 3D printing capable of recovering most (ranging from 50%, 60%, 70%, 80%, or 90% to 90%, 95%, or 99%) of the noble metal filler.

[0011] In one embodiment, the composition is configured to induce continuous decomposition to eliminate 65%, 90%, 95%, or 99% of the microplastics.

[0012] In one embodiment, the composition is configured to provide a polymer-based degradation mechanism that can generate repolymerizable low-molecular-weight by-products by selectively cleaving the ends of the polymer chains rather than randomly cleaving the polymer chains.

[0013] In one embodiment, the composition is configured such that the degradation of a single polymer chain occurs locally depending on the lamellar thickness, regardless of the bulk crystallinity, so that the processed (melt-processed or solution-processed) host matrix degrades in a spatially and temporally controllable manner.

[0014] In one aspect, the present invention provides a method for controllable degradation, which includes enabling controllable degradation of the polymer and elimination of microplastics by providing the composition disclosed herein under conditions where an enzyme can cleave the main chain of the polymer.

[0015] The present invention includes all combinations of the specific aspects and embodiments described herein, as if they were described in detail. BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

Fig. 1a-1e

[0017]

Fig. 2a-2d

[0018]

Fig. 3a-3f

[0019]

Fig. 4a-4c

[0020]

Fig. 5

[0021]

Fig. 6

[0022]

Fig. 7

[0023]

Fig. 8

[0024]

Fig. 9

[0025]

Fig. 10

[0026]

Fig. 11

[0027]

Fig. 12

[0028]

Fig. 13

[0029]

Fig. 14

[0030]

Fig. 15

[0031]

Fig. 16

[0032]

Fig. 17

[0033]

Fig. 18

[0034]

Fig. 19

Modes for Carrying Out the Invention

[0035] Throughout the following detailed description and the entire specification, unless inappropriate or otherwise stated, "a" and "an" mean one or more, and "or" means "and / or". The examples and embodiments described in this specification are for illustrative purposes only. Therefore, those skilled in the art are suggested that these examples and embodiments can be variously modified or changed, and these modifications and changes are included in the gist and scope of the present application and the scope of the appended claims. All publications, patents, and patent applications (including cited references in these documents) cited in this specification are hereby incorporated by reference in their entirety for all purposes.

[0036] In this specification, it is shown that by using a method using a random heteropolymer (RHP) to nano-disperse an enzyme in plastic, microplastics can be effectively removed without impairing the processability and general properties of the polymer. 13,14By nano-dispersing enzymes in plastics, the available enzymes increase and their stability improves. Therefore, by adding a small amount of enzyme (for example, by adding 0.02 wt% lipase to poly(caprolactone) (PCL)), about 95% of the microplastics in water can be eliminated within 24 hours. Lipase encapsulated in RHP selectively hydrolyzes PCL by cleaving the polymer chain from the end (Figure 1a), generating non-toxic and repolymerizable low-molecular-weight substances. The nano-dispersed RHP-lipase exhibits excellent thermal stability required during melt processing. By controlling the recrystallization of the melted polymer, the degradation can be controlled both temporally and spatially. Also, using such bioactive plastics, a conductive ink for 3D printing of fully functional electronic circuits can be prepared. After continuously operating the electronic circuit 3D printed with this conductive ink, the noble metal filler can be recovered. Furthermore, the controllable degradation of plastics by dispersing a complex composed of RHP and an enzyme can also be applied to another system composed of a plastic and an enzyme. In the disclosure of this specification, it is verified that bioactive plastics are a feasible means for achieving effective plastic recycling and microplastic elimination.

Example

[0037] For the preparation of bioactive plastics with functionality, it is essential to nano-disperse enzymes in plastics. However, when mixing enzymes with the host polymer, there are problems such as the formation of aggregates or the need for a high enzyme concentration of 10 wt% for effective degradation. 9 As the degradation progresses, enzyme aggregates elute 15, microplastics remain. RHP-lipase is well-dispersed in various types of solvents and forms a complex with a size of approximately 285 nm in toluene (Figure 5). However, when only lipase is dispersed in toluene, precipitation occurs. In the PCL film prepared by the solution casting method, it was confirmed by fluorescence microscope images that RHP-lipase was uniformly dispersed (Figure 1b). Transmission electron microscope (TEM) images showed that the nano-dispersed RHP-lipase complex was in the range of approximately 50 nm to approximately 500 nm between the lamellae of crystalline PCL (Figure 1c). Even when the enzyme was incorporated, when its content was 2 wt% or less, only slight changes were observed in the crystallinity (%) and mechanical properties of the bulk of PCL (Figures 6 and 7). In the analysis of the small-angle X-ray scattering (SAXS) profile, no difference was observed in the crystallization of PCL whether lipase was incorporated or not (Figure 8).

[0038] The semi-crystalline PCL containing RHP-lipase (referred to as "PCL-RHP-lipase") is immediately decomposed when immersed in water. Figure 1d shows a series of photographs of a PCL-RHP-lipase film (containing 0.02 wt% lipase) immersed in a buffer at 40 °C as a function of immersion time. As the decomposition progresses, the PCL-RHP-lipase film collapses and microplastic particles are generated. However, when fluorescently labeled lipase was used, it was shown that the lipase was maintained embedded within the microplastic particles of PCL and was well-dispersed within the microplastic particles of PCL (Figure 1e). Control experiments further confirmed that the activity of lipase was maintained within the microplastic particles and that PCL was continuously decomposed. As estimated by gel permeation chromatography (GPC), it was shown that after 24 hours, approximately 95% of the microplastics were decomposed into low-molecular-weight by-products. When the concentration of lipase was titrated, 95% of the microplastics were decomposed when the concentration of lipase was 0.01 wt% or 0.001 wt%.

[0039] If the mechanism by which PCL-RHP-lipase is internally degraded and changes from a bulk state to a nanoscale state can be elucidated in detail, the degradation of PCL can be well controlled, and furthermore, it will be useful for the future design of another type of enzyme-embedded bioactive plastic. PCL-RHP-lipase is degraded from the inside, not by surface erosion. Since the PCL-RHP-lipase film degraded at the same rate regardless of the amount of buffer (1 mL to 1 L), it was found that the degradation was not catalyzed by the enzyme eluted to the outside from the surface, but was consistent with the design that the degradation was catalyzed by the embedded enzyme. Figure 2a shows the SAXS profiles of the as-cast PCL-RHP-lipase film and the PCL-RHP-lipase film with a maximum mass reduction of approximately 25%. As the degradation progressed and the internal degradation proceeded, a nanoporous structure was formed, resulting in an increase in intensity in the low-q region. This result was consistent with the result observed in the cross-sectional image by scanning electron microscopy (SEM) (insert in Figure 2a).

[0040] The degradation of PCL-RHP-lipase showed temperature dependence, which was significantly different from the surface erosion process. PCL-RHP-lipase was rapidly degraded from the inside at 37 °C, but only very slight degradation was observed even after observing for 3 months at 20 °C. On the other hand, when a lipase solution was used, the surface was eroded even at 20 °C, and approximately 50% of PCL could be degraded within several days. Furthermore, since PCL-RHP-lipase can hydrolyze low-molecular-weight esters in solution at 20 °C (Figure 9), it was found that lipase enables the utilization of water to exert its activity. The reason why the degradation of PCL-RHP-lipase was limited at 20 °C is considered to be due to the interaction between the binding of the substrate to the active site of lipase, the mobility / flexibility of the enzyme in the solid matrix, and the density of the PCL chains locally.

[0041] The degradation of PCL-RHP-lipase proceeds by selective cleavage at the polymer chain ends rather than random cleavage of the polymer chains. When GPC analysis was performed, as the degradation progressed, the intensity of the main peak of PCL decreased, but the formation of polymers and oligomers of medium molecular weight was not observed (Figure 2b), indicating that cleavage occurred from the polymer chain ends. 16 When liquid chromatography-mass spectrometry (LCMS) was performed, it was confirmed that the main by-products in the degradation of PCL-RHP-lipase were monomers and small oligomers (oligomers with less than 5 repeating units of caprolactone accounted for the majority) (Figure 2c and Figure 10). On the other hand, when PCL was degraded in a concentrated lipase solution (0.1 mg / mL), it was shown that medium molecular weight by-products were generated due to the degradation of PCL from the outer surface in the GPC chromatogram and LCMS chromatogram, and this by-product was composed of at least 12 repeating units (Figure 10). To investigate the degradation mechanism in more detail, the degradability of a PCL-based triblock copolymer (PS-PCL-PS = 1,500 g / mol - 8,000 g / mol - 1,500 g / mol) capped at both ends with small polystyrene (PS) blocks was evaluated. When immersed in a buffer at 37 °C for 2 days by the same method as described above, only very slight degradation of PS-PCL-PS was observed (Figure 11). The lack of observed degradation of PS-PCL-PS suggested that lipase binds to the polymer chain ends of solid-state PCL. The generation of small water-soluble degradation by-products is very beneficial. Monomers and small oligomers are more efficient in chemical recycling than by-products with large molecular weights. As a proof-of-concept experiment, PCL was repolymerized from the by-products obtained when PCL-RHP-lipase was degraded.

[0042] Based on the fact that selective cleavage from the end of the polymer chain was experimentally observed, the active site of lipase was analyzed to further investigate this. When performing interfacial chemical analysis of the active site of lipase, it was shown that PCL can selectively bind to the active site of lipase mainly through hydrophobic interactions predominantly observed in the binding pocket of lipase (Figure 1a and Figure 12). The catalytic triad of lipase is located at a depth of 1.7 nm from the surface, and the bottom surface near the catalytic triad is narrow, with a width of 4.5 Å 17 . Since the active site of lipase has a deep and narrow structure, bulky substrates are excluded, and it is considered that only the most motile part of PCL (i.e., the end of the polymer chain) can reach the catalytic serine residue located at the fissure-like bottom surface. Considering the three-dimensional structures of solid-state lipase and PCL chains, the selective binding of lipase to the end of the PCL polymer chain is considered favorable.

[0043] By analyzing the changes in the crystalline properties of PCL during the degradation process, the degradation mechanism at the nanoscale was investigated in more detail, and it was suggested that the local lamellar thickness affects the degradation process. Due to the selective degradation of the amorphous phase, the bulk crystallinity increased from 39 ± 1.8% to 47 ± 2.0% in 0 - 1 h (insert in Fig. 2d, black ×). On the other hand, from 1 - 5 h, although it decreased from about 80% to only about 20% compared to the initial film amount, the variation in crystallinity was within the experimental error range, indicating that the crystalline domains were degraded by lipase. Furthermore, the degradation curve was almost linear from 0 - 3 h, but the degradation rate decreased around 3 h. The melting temperature is proportional to the average thickness of the lamellae of the semicrystalline polymer, but the melting temperature increased due to thermal annealing from 0 - 3 h (insert in Fig. 2d, blue □). The degradation rate decreased around 3 h where the peak of the melting temperature was observed, which is thought to be due to an increase in the enthalpic stability of the local lamellae with increased thickness by thermal annealing upon enzyme - mediated degradation. Additionally, past reports have shown that certain enzymes contribute to the reduction of the activation barrier during the decrystallization of a single polymer chain and can degrade the polymer chain processively (i.e., can undergo consecutive hydrolysis reactions without dissociating from the polymer chain). 18,19 Lipase has characteristics common to processive enzymes. Processive enzymes are characterized by interactions via hydrophobic bonds and tunnel - shaped active sites, such that the enzyme slides along a single polymer chain without dissociating from the polymer chain. 18 In semicrystalline polymers, assuming that a single polymer chain spans both crystalline and amorphous domains, the degradation of PCL - RHP - lipase by a mechanism that degrades from the end of the polymer chain depending on the lamellar thickness can be explained as a processive type of degradation of the polymer chain.

[0044] By embedding lipase in plastic, even after 5 hours at a molten state of 80 °C, 40% of the biological activity before the start of the experiment was maintained. Thus, it was found that the thermal stability of lipase was improved and it was compatible with the melting treatment. From the image obtained by superimposing the polarized light microscope image and the fluorescence microscope image (insert in Fig. 3a), it was shown that lipase was incorporated into spherulites rather than being located between spherulites. This was considered to be due to the fact that in the molten state of PCL, the diffusion rate of the enzyme was several orders of magnitude slower than the crystal growth rate. At any recrystallization temperature, the distribution of the enzyme was similar, but changing the treatment conditions had a significant effect on degradation. When the film prepared by the solution casting method was melted for 5 minutes (the decrease in enzyme activity was minimized) and recrystallized at 20 °C, degradation similar to that of the unstretched film was observed. However, when recrystallized at 49 °C, even when the film was immersed in a buffer at 37 °C for several weeks, degradation was minimized (Fig. 3a). This difference in degradability was considered to be due to the difference in the local thermodynamic stability of the crystal domains. In the film crystallized at 49 °C (Tc = 49 °C), although the bulk crystallinity was similar to that of the unstretched film, the melting temperature increased by about 6 °C, indicating that the crystal growth rate was slow and the thickness of the lamellae and amorphous domains increased (Fig. 13). Also, in the film crystallized at 49 °C (Tc = 49 °C), since the thickness of the lamellae increased, the local enthalpy stability increased significantly. In this case, however, when processive degradation of a single PCL chain proceeds, it can be disadvantageous in terms of the energy required for enzyme-catalyzed degradation. On the other hand, in the film crystallized at 20 °C (Tc = 20 °C), it was confirmed that the lamella thickness and degradation rate were similar to those of the unstretched film sample. From the difference in the degradation behavior between the film recrystallized at 20 °C and the film recrystallized at 49 °C, it was found that the lamella thickness was strongly correlated with degradability and was consistent with the processive type of degradation mechanism of a single polymer chain of PCL-RHP-lipase.

[0045] By performing the melting treatment, the degradation of PCL-RHP-lipase can be controlled both temporally and spatially. The degradation rate in a buffer at 37 °C can be controlled by adjusting the crystallization time of PCL-RHP-lipase at 49 °C (Figure 3b). When the PCL-RHP-lipase film was crystallized at 49 °C for 12 hours and the crystallization was stopped at 20 °C, two types of crystal morphologies were shown (Figure 3c). When the film with the two types of crystal morphologies mixed was immersed in a buffer at 37 °C for 24 hours, only the region crystallized at 20 °C decomposed, and the large spherulites grown at 49 °C remained without decomposition while maintaining the original structure (Figure 3d).

[0046] Next, an attempt was made to demonstrate that the degradation of PCL-RHP-lipase can be scaled up using a commercially available lipase mixture (lipase cb ). This commercially available lipase mixture can be used as it is without purification and can be embedded in PCL at the nanoscale. Observed by fluorescence microscope images (Figure 14) and TEM images (Figure 15) and confirmed by dynamic light scattering method (DLS) (Figure 16), the RHP-lipase cb complex formed particles with a size of about 300 nm or less in toluene, but the lipase cb that did not form a complex with RHP did not dissolve in toluene. As the simplest method to control the degradation rate, there is a method of changing the concentration of lipase cb in the unstretched film (Figure 3e). The film thickness also affects the degradation. Up to a thickness of about 1 mm at most, the degradation rate slows down as the film thickness increases and reaches a plateau at a thickness of about 1 mm (Figure 3f). Since water is required for lipase to perform a continuous hydrolysis reaction, it is expected that the degradation of the film depends on its thickness. Therefore, in the degradation of thick materials, the diffusion rate of water into the hydrophobic PCL matrix becomes the rate-limiting factor. The degradation of biomedical plastics is highly important and difficult to control, and 20,21 considering that PCL is a material approved by the FDA for human use 22The system of the present invention that can temporally and spatially control the degradation of thick materials (>1 mm) composed of PCL-RHP-lipase is considered to be extremely useful for biomedical applications.

[0047] PCL-RHP-lipase cb can be used to prepare conductive inks for 3D printing of recyclable flexible electronics. Silver flakes and RHP-lipase cb complexes are mixed in a toluene solution of concentrated (20 wt%) PCL to prepare an ink for 3D printing. The PCL-RHP-lipase cb -silver-containing ink was 3D printed to produce a circuit with high conductivity (Figure 4a), and as expected, the percolation network phenomenon was measured 23,24 (Figure 17). When this printed circuit was incubated in a buffer at 37°C for 4 hours, the PCL matrix was degraded by lipase, so the percolation network of silver flakes was destroyed and the current flowing through the printed circuit became zero (Figure 18). Also, PCL-RHP-lipase cb circuits were stored at room temperature for 7 months and a voltage of 5 V was applied for 1 month, and degradation in a buffer at 37°C was observed, indicating that the embedded enzyme has long-term stability and resistance to electrically induced denaturation and inactivation. After decomposing the PCL-RHP-lipase cb -silver circuit, the silver flakes can be easily recovered with high purity (Figure 4b) and reused without loss of conductivity (Figure 4c). In particular, considering the increasing importance of 3D printing technology for functional materials, inks containing complexes of RHP and enzymes are attractive for 3D printing of high-performance plastics with efficiently recyclable expensive fillers and other applications utilizing the catalytic action of enzymes 25-27 .

[0048] The concept of using enzyme nanodispersions for plastic degradation is applicable to other combinations of plastics and enzymes. Proteinase K degrades poly(lactic acid) (PLA), 28 whereas lipase cannot. This is thought to be because proteinase K has a more open hydrophilic binding pocket than lipase, and this binding pocket can form a hydrogen bond network with the repeating units of the ester groups of PLA (Figure 19). When the RHP - proteinase K complex is dispersed in PLA and immersed in a buffer at 37 °C for 10 days, more than 50% degradation of PLA is observed. Since PLA - RHP - proteinase K could be degraded, it was demonstrated that the method of the present invention of embedding enzymes in plastics can also be applied to important plastics that are beginning to be used as alternatives to non - degradable polyolefins used in daily necessities such as packaging. 29 .

[0049] By embedding fillers with catalytic activity in plastics, controllable degradation as needed and desirable recyclability can be achieved. The behavior of enzymes embedded at the nanoscale in a solid matrix can be significantly different in terms of substrate binding, by - products, mechanism of action, etc. As a route to regulate the degradation rate and degradation pathway of plastics, it is effective to utilize the spatial position of catalytically active particles within the plastic and the crystalline properties of the plastic. Considering recent advances in synthetic biology and genomic information, there are great advantages for the development of new degradable plastics and environmentally friendly materials. On the other hand, if plastics embedded with enzymes can be rationally designed, it is possible to provide a technically immediately feasible method that can control the entire polymer life cycle and eliminate microplastics.

[0050] Method Materials: The Amano PS lipase derived from Burkholderia cepacia and the proteinase K derived from Tritirachium album were purchased from Sigma-Aldrich. In the studies using the purified enzymes, the enzymes were purified using the methods reported in the past. 30 In the studies using commercially available enzyme mixtures, the purchased ones were used as they were. PCL (80,000 g / mol, PDI < 2) and PLA (85,000 - 160,000 g / mol) were purchased from Sigma-Aldrich, and PS-PCL-PS was purchased from Polymer Source. All of these were used without purification. The random heteropolymer (about 70,000 g / mol) was synthesized according to past reports. 13 For the display of the crystal structure of lipase, the Protein Data Bank entry ID: 3lip was used, and for the display of the crystal structure of proteinase K, the Protein Data Bank entry ID: 1ic6 was used. In the substrate binding analysis, the lipase with entry ID: 1ys1 and the proteinase K with entry ID: 3prk were used. The 3D printing of the ink consisting of PCL-RHP-lipase-silver was performed by 3D printing a toluene solution of PCL (20 wt%) containing RHP-lipase and silver flakes at room temperature. The conductivity was measured with a self-made DC measuring instrument.

[0051] Decomposition: The RHP-lipase complex was mixed with an aqueous solution, freeze-dried overnight, and directly resuspended in a toluene solution of 4 wt% PCL (or the RHP-proteinase K complex was directly suspended in a dichloromethane solution of 4 wt% PLA). The resulting suspension was applied to a microscope slide and dried, and the dried film was peeled off the slide and immersed in sodium phosphate buffer (25 mM). The film was taken out at a predetermined time point, rinsed, and dried under reduced pressure. For the film composed of PCL-RHP-lipase, the remaining amount was measured using a balance up to a maximum of 5 hours. In the estimation of the residual concentration of microplastics in the unstretched film after 24 hours, since the particle size of the microplastics was small and the remaining amount was also small, it was impossible to measure the weight with a balance. Therefore, the peak at 11 - 15 minutes measured by gel permeation chromatography (GPC) was manually integrated, and its area was divided by the peak area of a commercially available PCL sample (it was estimated that about 95% of the microplastics were excluded). As a proof-of-concept experiment, after the decomposed PCL by-products were recovered from the enzyme and buffer salts by liquid-liquid extraction and filtration, the method 31 reported in the past was used for repolymerization. In the experiment using PLA-RHP-proteinase K, the degradation was measured by weighing the remaining amount with a balance at the times shown in the graph.

[0052] Characteristic evaluation: Dynamic light scattering measurements were performed at a scattering angle of 90° using a Brookhaven BI-200SM light scattering system. Differential scanning calorimetry (DSC) was measured at a scanning rate of 2 °C / min while raising the temperature from 25 °C to 70 °C. The crystallinity (%) was determined by dividing the melting enthalpy of the sample by 151.7 J / g (the melting enthalpy of 100% crystalline PCL). 32 In the uniaxial tensile test, a test piece was prepared by casting a PCL solution into a self-made standard dumbbell-shaped Teflon mold. TEM images were taken at an acceleration voltage of 120 kV using a JEOL 1200 microscope. The amorphous domains of RHP-lipase and PCL were stained using a 5 wt% ruthenium tetroxide solution.

[0053] In the test by small-angle X-ray scattering method (SAXS), the sample was cast in a Teflon beaker to prepare a film with a thickness of about 300 μm. The obtained film sample was decomposed and dried under reduced pressure for at least 16 hours, and then the SAXS method was performed using beamline 7.3.3 of the Advanced Light Source (ALS) synchrotron radiation facility at Lawrence Berkeley National Laboratory. X-rays with a wavelength of 1.24 Å were irradiated for 2 seconds.

[0054] In the low-molecular assay (performed to estimate thermal stability), the film was immersed in a buffer solution of 0.5 mM 4-nitrophenyl butyrate. Hydrolysis was quantified by monitoring the activity using ultraviolet-visible spectroscopy for 20 minutes.

[0055] Fluorescence microscopy observation was performed using a U-MWBS3 mirror unit that transmits an excitation wavelength of 460 - 490 nm. According to the manufacturer's procedure, commercially available NHS-fluorescein (5 / 6-carboxyfluorescein succinimidyl ester) was used to label lipase. A centrifugal ultrafiltration filter with a molecular weight cut-off of 10,000 g / mol was used to centrifuge the labeled lipase solution to remove excess dye from the labeled lipase.

[0056] Measurements by gel permeation chromatography (GPC) were performed using a THF solution with a combined concentration of residual film and by-products of 2 mg / mL. 2 μL of this solution was injected into an Agilent PolyPore column (7.5×300 mm). Measurements by liquid chromatography-mass spectrometry (LCMS) were performed by resuspending the supernatant of the decomposition product in a mixed solvent of acetonitrile and water (67 vol%:33 vol%) and flowing it through an Agilent InfinityLab EC-C18 column (2.7 μm). The mass spectrum showed the combination of the main peaks observed in the liquid chromatogram. For GPC measurement and LCMS measurement, the decomposition products were lyophilized overnight and then resuspended in an appropriate solvent.

[0057] References 1 Law, K. L. & Thompson, R. C. Microplastics in the seas. Science 345, 144-145, doi:10.1126 / science.1254065 (2014). 2 Geyer, R., Jambeck, J. R. & Law, K. L. Production, use, and fate of all plastics ever made. Sci Adv 3, e1700782, doi:10.1126 / sciadv.1700782 (2017). 3 Jambeck, J. R. et al. Plastic waste inputs from land into the ocean. Science 347, 768-771, doi:10.1126 / science.1260352 (2015). 4 Catarino, A. I., Macchia, V., Sanderson, W. G., Thompson, R. C. & Henry, T. B. Low levels of microplastics (MP) in wild mussels indicate that MP ingestion by humans is minimal compared to exposure via household fibres fallout during a meal. Environ Pollut 237, 675-684, doi:10.1016 / j.envpol.2018.02.069 (2018). 5 Setala, O., Fleming-Lehtinen, V. & Lehtiniemi, M. Ingestion and transfer of microplastics in the planktonic food web. Environ Pollut 185, 77-83, doi:10.1016 / j.envpol.2013.10.013 (2014). 6 Wei, R. & Zimmermann, W. Microbial enzymes for the recycling of recalcitrant petroleum-based plastics: how far are we? Microb Biotechnol 10, 1308-1322, doi:10.1111 / 1751-7915.12710 (2017). 7 Wei, R. et al. Turbidimetric analysis of the enzymatic hydrolysis of polyethylene terephthalate nanoparticles. Journal of Molecular Catalysis B: Enzymatic 103, 72-78, doi:10.1016 / j.molcatb.2013.08.010 (2014). 8 Mueller, R. J. Biological degradation of synthetic polyesters - Enzymes as potential catalysts for polyester recycling. Process Biochem 41, 2124-2128, doi:10.1016 / j.procbio.2006.05.018 (2006). 9 Ganesh, M., Dave, R. N., L’Amoreaux, W. & Gross, R. A. Embedded Enzymatic Biomaterial Degradation. Macromolecules 42, 6836-6839, doi:10.1021 / ma901481h (2009). 10 Zan, L. et al. Solid-phase photocatalytic degradation of polystyrene with modified nano-TiO2 catalyst. Polymer 47, 8155-8162, doi:10.1016 / j.polymer.2006.09.023 (2006). 11 Klibanov, A. M. Why are enzymes less active in organic solvents than in water? Trends Biotechnol 15, 97-101, doi:10.1016 / S0167-7799(97)01013-5 (1997). 12 Klibanov, A. M. Improving enzymes by using them in organic solvents. Nature 409, 241-246, doi:10.1038 / 35051719 (2001). 13 Panganiban, B. et al. Random heteropolymers preserve protein function in foreign environments. Science 359, 1239-1243, doi:10.1126 / science.aao0335 (2018). 14 DelRe, C. et al. Reusable Enzymatic Fiber Mats for Neurotoxin Remediation in Water. Acs Appl Mater Inter 10, 44216-44220, doi:10.1021 / acsami.8b18484 (2018). 15 Khan, I., Nagarjuna, R., Dutta, J. R. & Ganesan, R. Enzyme-Embedded Degradation of Poly(epsilon-caprolactone) using Lipase-Derived from Probiotic Lactobacillus plantarum. Acs Omega 4, 2844-2852, doi:10.1021 / acsomega.8b02642 (2019). 16 Kobayashi, S., Uyama, H. & Takamoto, T. Lipase-catalyzed degradation of polyesters in organic solvents, a new methodology of polymer recycling using enzyme as catalyst. Biomacromolecules 1, 3-5, doi:10.1021 / bm990007c (2000). 17 Pleiss, J., Fischer, M. & Schmid, R. D. Anatomy of lipase binding sites: the scissile fatty acid binding site. Chem Phys Lipids 93, 67-80, doi:10.1016 / S0009-3084(98)00030-9 (1998). 18 Horn, S. J. et al. Costs and benefits of processivity in enzymatic degradation of recalcitrant polysaccharides. P Natl Acad Sci USA 103, 18089-18094, doi:10.1073 / pnas.0608909103 (2006). 19 Beckham, G. T. et al. Molecular-Level Origins of Biomass Recalcitrance: Decrystallization Free Energies for Four Common Cellulose Polymorphs. J Phys Chem B 115, 4118-4127, doi:10.1021 / jp1106394 (2011). 20 Washington, M. A. et al. The impact of monomer sequence and stereochemistry on the swelling and erosion of biodegradable poly(lactic-co-glycolic acid) matrices. Biomaterials 117, 66-76, doi:10.1016 / j.biomaterials.2016.11.037 (2017). 21 Li, J., Stayshich, R. M. & Meyer, T. Y. Exploiting Sequence To Control the Hydrolysis Behavior of Biodegradable PLGA Copolymers. Journal of the American Chemical Society 133, 6910-6913, doi:10.1021 / ja200895s (2011). 22 Woodruff, M. A. & Hutmacher, D. W. The return of a forgotten polymer-Polycaprolactone in the 21st century. Prog Polym Sci 35, 1217-1256, doi:10.1016 / j.progpolymsci.2010.04.002 (2010). 23 Valentine, A. D. et al. Hybrid 3D Printing of Soft Electronics. Adv Mater 29, doi:10.1002 / adma.201703817 (2017). 24 Stauffer, D. & Aharony, A. Introduction to Percolation Theory: Second Edition. (CRC Press, 1994). 25 Muth, J. T. et al. Embedded 3D Printing of Strain Sensors within Highly Stretchable Elastomers. Adv Mater 26, 6307-6312, doi:10.1002 / adma.201400334 (2014). 26 Zarek, M. et al. 3D Printing of Shape Memory Polymers for Flexible Electronic Devices. Adv Mater 28, 4449-4454, doi:10.1002 / adma.201503132 (2016). 27 Lind, J. U. et al. Instrumented cardiac microphysiological devices via multimaterial three-dimensional printing. Nat Mater 16, 303-308, doi:10.1038 / Nmat4782 (2017). 28 Li, S. M. & McCarthy, S. Influence of crystallinity and stereochemistry on the enzymatic degradation of poly(lactide)s. Macromolecules 32, 4454-4456, doi:10.1021 / ma990117b (1999). 29 Farah, S., Anderson, D. G. & Langer, R. Physical and mechanical properties of PLA, and their functions in widespread applications - A comprehensive review. Adv Drug Deliver Rev 107, 367-392, doi:10.1016 / j.addr.2016.06.012 (2016). 30 Bornscheuer, U. et al. Lipase of Pseudomonas-Cepacia for Biotechnological Purposes - Purification, Crystallization and Characterization. Bba-Gen Subjects 1201, 55-60, doi:10.1016 / 0304-4165(94)90151-1 (1994). 31 Schindler, A., Hibionada, Y. M. & Pitt, C. G. Aliphatic Polyesters.3. Molecular-Weight and Molecular-Weight Distribution in Alcohol-Initiated Polymerizations of Epsilon-Caprolactone. J Polym Sci Pol Chem 20, 319-326, doi:10.1002 / pol.1982.170200206 (1982). 32 Wurm, A. et al. Crystallization and Homogeneous Nucleation Kinetics of Poly(epsilon-caprolactone) (PCL) with Different Molar Masses. Macromolecules 45, 3816-3828, doi:10.1021 / ma300363b (2012).

Claims

1. A bioactive plastic composition comprising: an organic polymer; a nano-dispersion of a complex composed of an enzyme that hydrolyzes the organic polymer and a random heteropolymer; wherein hydrolysis of the organic polymer by the enzyme enables depolymerization and elimination of microplastics; the complex is uniformly dispersed in the composition; the size of the complex ranges from 10 nm to 500 nm; the size of the complex is the hydrodynamic diameter; the content of the enzyme in the composition ranges from 0.001 wt% to 5 wt%; and the random heteropolymer contains a plurality of types of monomers selected from methyl methacrylate (MMA), oligo(ethylene glycol) methacrylate (OEGMA), 3-sulfopropyl potassium methacrylate (3-SPMA), and 2-ethylhexyl methacrylate (2-EHMA) in various proportions.

2. The composition according to claim 1, wherein the size of the complex ranges from 20 nm to 500 nm.

3. The composition according to claim 1, wherein the size of the complex ranges from 40 nm to 500 nm.

4. The composition according to claim 1, wherein the size of the complex ranges from 10 nm to 200 nm.

5. The composition according to claim 1, wherein the size of the complex ranges from 20 nm to 200 nm.

6. The composition according to claim 1, wherein the size of the complex ranges from 40 nm to 200 nm.

7. The composition according to claim 1, wherein the size of the complex ranges from 10 nm to 100 nm.

8. The composition according to claim 1, wherein the size of the complex ranges from 20 nm to 100 nm.

9. The composition according to claim 1, wherein the size of the complex ranges from 40 nm to 100 nm.

10. The composition according to claim 1, wherein the content of the enzyme in the composition ranges from 0.01 wt% to 5 wt%.

11. The composition according to claim 1, wherein the content of the enzyme in the composition ranges from 0.1 wt% to 5 wt%.

12. The composition according to claim 1, wherein the content of the enzyme in the composition ranges from 0.001 wt% to 1 wt%.

13. The composition according to claim 1, wherein the content of the enzyme in the composition ranges from 0.01% by weight to 1% by weight.

14. The composition according to claim 1, wherein the content of the enzyme in the composition ranges from 0.1% by weight to 1% by weight.

15. The composition according to claim 1, wherein the content of the enzyme in the composition ranges from 0.001% by weight to 0.1% by weight.

16. The composition according to claim 1, wherein the content of the enzyme in the composition ranges from 0.01% by weight to 0.1% by weight.

17. The composition according to claim 1, wherein the content of the enzyme in the composition is 0.1% by weight.

18.

19. The composition according to claim 1, wherein the size of the complex present between the lamellae of the crystalline organic polymer ranges from 10 nm to 500 nm.

20. The composition according to claim 1, wherein the size of the complex present between the lamellae of the crystalline organic polymer ranges from 20 nm to 500 nm.

21. The composition according to claim 1, wherein the size of the complex present between the lamellae of the crystalline organic polymer ranges from 10 nm to 200 nm.

22. The composition according to claim 1, wherein the size of the complex present between the lamellae of the crystalline organic polymer ranges from 20 nm to 200 nm.

23. The composition according to claim 1, wherein the size of the complex present between the lamellae of the crystalline organic polymer ranges from 40 nm to 200 nm.

24. The composition according to claim 1, wherein the size of the complex present between the lamellae of the crystalline organic polymer ranges from 10 nm to 100 nm.

25. The composition according to claim 1, wherein the size of the complex present between the lamellae of the crystalline organic polymer ranges from 20 nm to 100 nm.

26. The composition according to claim 1, wherein the size of the complex present between the lamellae of the crystalline organic polymer ranges from 40 nm to 100 nm.

27.

28. The composition according to any one of claims 1 to 26, wherein the combination of the organic polymer and the enzyme is selected from the combination of polycaprolactone (PCL) and lipase, the combination of polylactic acid (PLA) and proteinase K, and the combination of polyethylene terephthalate (PET) and PET hydrolase.

28. The composition according to any one of claims 1 to 26, which generates low molecular weight by-products that can be repolymerized by decomposition.

29. Use of the composition according to any one of claims 1 to 28 in the preparation of a conductive ink for 3D printing.

Images