Thermal stabilization of embedded proteins

Abstract

Plastic compositions comprise an embedded protein, a salt and optional polymeric protectants configured sufficient to increase thermal stabilization of the protein, such as wherein the polymeric protectants are random heteropolymers, particularly wherein the salt and polymeric protectants provide synergistic effects in thermal stabilization of the protein.

Description

Thermal Stabilization of Embedded Proteins

[0001] Government Support Clause

[0002] This invention was made with government support under grant number W911NF-21-1- 0128 awarded by the Army Research Office, and grant number 2132025 awarded by the National Science Foundation. The government has certain rights in the invention.

[0001] Introduction

[0002] Bioactive plastics e.g., plastics with embedded biotic species such as proteins, are versatile functional materials that leverage nature-optimized designs in human-engineered systems. At present they are mostly pharmaceuticals and smart food packaging with embedded antimicrobial agents. ’ However, bioactive plastics are also excellent for heterogeneous catalysis applications such as water purification, as unlike solution catalysis, solid films can be easily transported and placed in varying environments in situ without facing post-reaction separation issues. The range of synthesizable bioactive plastics was significantly broadened with the development of random heteropolymers (RHPs), amphiphilic copolymers that can stabilize proteins in non-native environments such as organic solvents.3RHPs can make ordinarily fragile proteins compatible with solvent-casted plastic processing by preventing their misfolding and aggregation en route to solid-state encapsulation.4External small molecules aside, the substrate for heterogeneous catalysis could also be the plastic matrix itself. Nanoscopically embedding enzymes into biodegradable plastics is a promising approach to modulating degradation latency, eliminating single-use plastic waste, and mitigating microplastic formation.5-10

[0003] Unfortunately solvent processing using volatile organic compounds is not environmentally sustainable and thus is often legally restricted.11For industrial relevance at scale, proteins must be compatible with traditional melt extrusion and additive manufacturing plastic processing, in which plastic pellets are heated at high temperatures to melt, mixed with additives for a residence time, extruded, then molded or air blown to shape.2,12Proteins must retain folded structure after incubation in high temperature polymer melts for the duration of the residence time to achieve commercially viable bioactive plastics. Residence times are highly dependent on the polymer, extruder type and operating parameters, and are usually minimized because polymers at high temperatures can also thermally or hydrolytically degrade. Poly(lactic acid) (PLA) is a commercially relevant platform for bioactive plastic processing exploration. In 2023, PLA represented 31% of the 2.18 million metric tons of global bioplastics produced annually.13Reported PLA residence times vary from as low as 20 seconds to as high as 8 minutes, with most on average being under 5 minutes.14,15Thus, enzymes that degrade PLAmust retain folded structure after ~5 minutes of residence time in viscoelastic polymer melt above the maximum plastic melt temperature (Tm) i.e., 150-200°C for PLLA.14This temperature is common for most commercial plastics that range in Tmfrom 100-200°C. After extrusion, the enzymes must remain catalytically active at 40-65°C to match compost conditions.16

[0004] Most enzymes denature in aqueous solutions as temperatures approach 60-80°C, and even the most hyperthermophilic proteins are only thermostable up to 117°C.17Above 110°C, molecules such as amino acids and metabolites become highly unstable and hydrophobic interactions significantly weaken. Moreover, these hyperthermophiles evolved in extreme environments, so their optimal operating temperatures are also inconveniently high. The >100°C thermostability gap to Tm,pLLA is unlikely to be bridged solely by enzyme engineering,18’19but rather by the design of protective chaperones. Proteinase K (PK) is the most well demonstrated PLLA degrading enzyme, however, it loses all catalytic activity when heated in solution for 5-10 minutes at 75°C.20Recent works have immobilized PK onto polyacrylamide microparticles to retain 2% activity after 5 minutes at 200°C,19and onto ZIF-8 to retain 61.8% activity after 10 minutes at 160°C. However, no existing result is amenable for practical use, which requires engineering stable PLLA degrading enzymes such as PK for a minimum of 5 minutes of residence time at up to 200°C in PLLA melt.

[0005] RHPs were designed for solution stabilization and have been shown to increase protein thermostability in solution. It was shown that RHP does not actually prevent misfolding but rather chaperones enzyme refolding back to the native structure during the cooling process. However, in solution interactions, hydrophobic effects dominate, it is an open question if that remains true in polymer melts. There are three fundamental ways in which protein stability may differ in polymer melts compared to biological solutions: polarity, water deficiency, and viscosity. Although plastics vary in polarity, in general compared to aqueous solutions, most plastics are hydrophobic. In solutions the hydrophobic effect is dominant. Losing this in polymer melts could cause protein misfolding. However, the large body of research on enzyme stability in organic solutions showed that it might help enzyme rigidification due to the retention of the core hydrophobic shell good for stability, bad for activity (as a certain degree of flexibility is required for function).23,24Hydrophobic plastic matrices at 200°C will be almost if not entirely water deficient. The lack of water will destabilize hydrophobic and hydrogen bonding stabilizing interactions. Finally, considering viscosity, the density of amorphous PLA is 1248 g 1 , an order of magnitude higher than the estimated total concentration of macromolecules in the cytoplasm, that ranges from 50 to 400 g 1”1.26Comparing to enzymes in dilute aqueous solutions, the contrast in molecular crowding is even more stark. High viscosity helps someproteins remain stable and causes others to misfold depending on interactions. Although Nature has evolved proteins to not interact with locally surrounding macromolecules to avoid misfolding, it has done no such fine-tuning for synthetic polymer interactions. Thus, we expect high viscosity to hurt rather than help protein stability. However, this effect may be counterbalanced or exceeded by an increase in enzyme structural stability afforded by immobilization in a viscoelastic state. If dynamics are low, perhaps proteins will not have the flexibility to misfold even in a liquid polymer melt.

[0006] Summary of the Invention

[0007] The invention provides novel methods and compositions comprising thermal stabilization of polymer embedded proteins.

[0008] This invention provides enhancing protein thermal stability and retain protein activities upon heating as high as 250°C. According to the invention, proteins are embedded inside of polymer matrices together with additives, thermally annealed at elevated temperature and cool down before testing protein activities. The additives contain certain fraction of salts and in embodiments protein protectants based on random heteropolymers. In examples, protein activities are assessed via either protein activity solution assay or via degradation of host polymer matrix. Practical applications include functional materials, plastics degradation, protein therapeutics, sensors and other protein-based biotechnology.

[0009] In aspects and embodiments the invention provides:

[0010] 1. A plastic composition comprising a blend of a thermoplastic, a protein and a salt configured to increase thermal stabilization of the protein in the composition.

[0011] 2. A composition herein, further comprising polymeric protectants configured to increase thermal stabilization of the protein.

[0012] 3. A composition herein, further comprising polymeric protectants, wherein the salt and polymeric protectants provide synergistic effects in thermal stabilization of the protein.

[0013] 4. A composition herein, wherein the composition enables preservation of embedded protein activity at high temperatures for downstream / subsequent functional use, such as by biocatalysis of small molecules external to embedded matrix as well as biocatalysis on the matrix itself (plastic degradation).

[0014] 5. A composition of claim 1 , wherein the salt comprises a cation and anion pair that together maintain protein hydration during thermal processing by gradually releasing bound water over the melt processing temperature range, without being deliquescent or acidic, and without interacting with the protein in a manner that induces misfolding or deactivation.

[0015] 6. A composition herein, wherein the composition further comprises co-embedded random heteropolymers (RHP) to:

[0016] increase retained protein activity; and

[0017] reduce salt and / or protein leaching.

[0018] 7. A composition herein, wherein the composition further comprises co-embedded random heteropolymers (RHP), wherein the heteropolymers:

[0019] promote retention of the embedded protein, e.g., in practical applications such as compostable plastics;

[0020] reduce or prevent unwanted porous plastic formation;

[0021] reduce leaching and salt loading;

[0022] improve protein and / or salt dispersion; and

[0023] provide or increase retention of blend properties, such as desired optical and mechanical properties.

[0024] 8. A composition herein, wherein the protein has increased thermal stability at 100- 215°C, including 155°C, 185°C, 200°C and 215°C.

[0025] 9. A composition herein, wherein the protein has thermal stability at 100-215 °C, including 155°C, 185°C, 200°C and 215°C, for a minimum of 5 minutes of residence time at the temperature.

[0026] 10. A composition herein, wherein the salt is present at 0.05%-30wt% or 2-20wt%

[0027] 11. A composition herein, wherein the protein is present at 0.2-25% or 0.5-10wt%, including 2%, 5%, 10% and 20%.

[0028] 12. A composition herein, wherein the protein has increased thermal stability at 100-215°C, including 155°C, 185°C, 200°C and 215°C;

[0029] the protein has thermal stability at 100-215°C, including 155°C, 185°C, 200°C and 215°C, for a minimum of 5 minutes of residence time at the temperature;

[0030] the salt is present at 0.05%-30wt% or 2-20wt%; and

[0031] the protein is present at 0.2-25% or 0.5-10wt%, including 2%, 5%, 10% and 20%.

[0032] 13. A composition herein, wherein the plastic comprises Polyethylene Terephthalate (PET or PETE), High-Density Polyethylene (HDPE), Low-Density Polyethylene (LDPE), Polyvinyl Chloride (PVC or Vinyl), Polypropylene (PP), Polystyrene (PS), polyethylene oxide (PEO) I Polyethylene glycol (PEG), Poly(L-lactic acid) (PLLA), Polylactic acid (PLA), Polycaprolactone (PCL), Polymethylmethacrylate (PMMA) and / or a Polyhydroxy alkanoate (PH A).

[0033] 14. A composition herein, wherein the salt is selected from magnesium, calcium, potassium, sodium, zinc, ammonium, and lanthanide salts, including sulfates, acetates, chlorides, nitrates, etc.

[0034] 15. A composition herein, wherein the salt is selected from MgSO4 , CaiCFhCOOp Na2SO4, ZnSCh, MgCE, Mg(NCh)2, NaCl and KC1, and ammonium nitrate and nitrates of Eu, Nd, and Dy, and preferably MgSO |.

[0035] 17. A composition herein, wherein the protein is an enzyme, such as a protease, lipase, amylase, pullulanase, pectinase, peroxidase, xylase, laccase, transgluaminase, phytase, hydrolase, transferase, redox enzyme, lyase, isomerase, ligase, or xylanase.

[0036] 18. A composition herein, wherein the plastic comprises Polyethylene Terephthalate (PET or PETE), High-Density Polyethylene (HDPE), Low-Density Polyethylene (LDPE), Polyvinyl Chloride (PVC or Vinyl), Polypropylene (PP), Polystyrene (PS), polyethylene oxide (PEO) I Polyethylene glycol (PEG), Poly(L-lactic acid) (PLLA), Polylactic acid (PLA), Polycaprolactone (PCL), Polymethylmethacrylate (PMMA) and / or a Polyhydroxy alkanoate (PH A);

[0037] the salt is selected from Magnesium Sulfate, Calcium Acetate and Potassium Chloride,

[0038] the protein is an enzyme, such as a protease, lipase, amylase, pullulanase, pectinase, peroxidase, xylase, laccase, transgluaminase, phytase, hydrolase, transferase, redox enzyme, lyase, isomerase, ligase, or xylanase.

[0039] 19. A method of making a composition herein, comprising embedding the protein in the thermoplastic, together with the salt, thermally annealing the composition at elevated temperature, and cooling down the composition before detecting activity of the protein, wherein the method may further comprise:

[0040] lyophilizing the protein and the salt together prior to embedding,

[0041] incubating the composition under conditions wherein the protein is demonstrably active, such as an enzyme protein degrading the polymer matrix, and / or

[0042] detecting the activity of the protein.

[0043] 20. A method of using a composition herein, preserving activity of the embedded protein at high temperatures for downstream / subsequent functional use, such as by biocatalysis of small molecules external to embedded matrix as well as biocatalysis on the matrix itself (plastic degradation).

[0044] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0045] Brief Description of the Drawings

[0046] Figs. 1. MgSO i thermostabilizes Proteinase K (PK) in 30kDa Poly(L-Iactic acid) (PLLA30). For all bar plots triplicates were averaged and standard deviations were plotted as error bars. Films were solvent cast using DCM. A. % Retained activity of PK, PK and MgSO4, and control films with no additive after heat treatment for 5 minutes at temperatures ranging from 155-250°C. B. % Mass loss of 8 minute 185°C heat treated films with no additive, 2wt% PK, 10wt% MgSCh, and both after 24 hours in 40°C pH 8.0 tris buffer. C. Activity rate of 2wt% PK co-embedded with varying ratios of MgSCL as-cast (AC) and heat treated for 5 minutes at 155 and 185 °C.

[0047] Figs. 2. Thermostability in polymer melt experimental setup. Solid polymer pellets either go through A. Our experimental assay that is trying to mimic B. industrial melt extrusion process that does not involve organic solvents.

[0048] Fig. 3. MgSO4 thermostabilized Proteinase K (PK). Non- As Cast (AC) normalized data for of Fig. 1, part A.

[0049] Fig 4. 10wt% PK loading in various plastics. Protein thermostability does not improve with increased % loading in polymer films.

[0050] Fig. 5. Bioactive plastic degradation and mechanical performance of PLLA with co-embedded Proteinase K and MgSCL. A. Mass loss after 3 days at 40 °C in 0.01 x Tris pH 8 buffer for PLLA films either melt-extruded or solvent-cast with DCM, then heat-treated for 5 mins, with or without embedded PK-MgSCL Insets: Cross-sectional SEM images showing internal morphology before (0%) and after (25%) degradation. B. Time course of mass loss during PLLA degradation in 0.01 x Tris pH 8 buffer at 40 °C. C. Comparison of degradation in compost at 60 °C between PLLA films embedded with MgSCL, PK, or both. D. DSC graphs of PLLA films showing thermal transitions for PLLA, PK-, MgSCL-, and PK-MgSCL-loaded samples. E. DMA stress-strain curves for extruded PLLA films containing 0%, 0.2%, and 2 wt% PK-MgSCL Insets: photographs of films arranged by increasing % MgSCL, shown under applied weight (pens).

[0051] Fig. 6. SEM images of extruded bioactive PLA films during enzymatic degradation. SEM images of 2 wt% PK + 2 wt% MgSCL in PLLA after 0%, 10%, 25%, and 50% degradation by mass in 40 °C Tris buffer. All images taken at 600 x magnification; scale bars represent 20 pm.

[0052] Fig. 7. Degradation of PLLA films in compost with co-embedded Proteinase K and MgSCL at different temperatures. Mass loss over time for films containing 2.0 wt% PK and 2.0 wt% MgSCL under compost (dot) or buffer and compost (triangle) conditions at 40 °C, 60 °C, and 80 °C.

[0053] Fig 8. 155°C universal deactivation across 24 protein-plastic pairings. The retained activity % of 4 Hydrolases were screened across 6 plastics from 140 to 180 °C.

[0054] Fig. 9. UV-Vis of proteins. Representative spectra of triplicates of each protein shown. A260 / A280 shown in brackets indicating very high purity for AO, BC, BL and PK.

[0055] Fig. 10. Enzyme featurization. A. Comparing surface featurization (top) chemistry, where size corresponds to the ratio of positive : negative surface charges and color corresponds to metal ion presence, (middle) secondary structure, where size corresponds to molecular weight (Mw) and color corresponds to the type of enzyme, (bottom) geometry, where each axis is the % of total dimension for example a perfectly spherical enzyme would be at coordinates [0.33,0.33,0.33], size corresponds to Mw and legend is what kingdom the enzyme’s original organism is from. B. Comparison of binding pocket featurization.

[0056] Fig. 11. 155°C universal deactivation. Non- As cast (AC) normalized version of Fig. 3

[0057] Fig. 12. Generality of MgSCh mediated protein thermal stabilization. For all bar plots, heat treatment was done for 5 minutes, triplicates were averaged, and standard deviations were plotted as error bars. A. % Retained activity of four enzymes (PK, BL, AO, BC) in PLLA85 as-cast (AC) and after 185 °C heat treatment, with or without co-embedded MgSO4. B. % Retained activity of 2 wt% PK with MgSO4 first dissolved in DCM solution of four different polymers (PLLA85, PMMA, PCL, PS) after 185 °C treatment, showing broad polymer compatibility. C. Systematic salt screen for retained PK activity in PLLA85 at AC, 155 °C, 170 °C, and 185 °C, organized by ion kosmotropicity. Gray boxes indicate conditions not tested, while white boxes indicate no activity.

[0058] Fig. 13. Viscosity of polymers used for protein embedding. Viscosity (Pa s) as a function of shear rate (1 / s) for eight polymers measured at 185 °C. PLLA samples of varying molecular weights (30-100 kDa) are shown alongside PMMA, PCL, PS, and LDPE.

[0059] Fig. 14. MgSCh enables thermal stabilization of multiple protein classes in PLA.For all bar plots, heat treatment was performed and lyophilized powders were mixed with polymer by vortexing. Triplicates were averaged with standard deviations shown. A. PK activity after heat treatment at 185 °C for 5 minutes in LDPE, PLLA and PEG with and without MgSCh. B. HRP activity at 185 °C for 10 minutes, in LDPE, PLLA and PEG with and without MgSO4.

[0060] Fig. 15. Molecular insights into MgSO4 protein stabilization in polymer matrices. A. DSC of PK powder and PMMA films with and without MgSO4 and PK. B. CD spectra of PK leached in water from films annealed at 185 °C, with or without MgSO4. C. FTIR spectra of PLLA films with PK and MgSO4 show O-H stretching (left) and H-O-H bending (right) regions.

[0061] Fig. 16. Thermostability of PK in PLLA with MgSCh at 185 °C over time. Retained activity of PK-MgSCh in 85 kDa PLLA following thermal exposure at 185 °C for varying durations. Error bars represent standard deviation of triplicate measurements.

[0062] Fig. 17. Effect of humid environment and additives on thermal stability of embedded PK in PLLA. A. % Retained activity of PK, AO, and BC in PLLA80, after heat treatment in >99% RH air environment. B. % Retained enzymatic activity of PK co-embedded with different additives after 5-minute heat treatments at different temperatures up to 215 °C for MgSO4, PEG8k, CTAB, and AOT.

[0063] Fig. 18. Adding random heteropolymers (RHP) and salts increase retained activity to ~95% post 185C thermal treatment. A. % Retained activity of 2 wt% PK-MgSO4 with various RHPs. B. . Activity rate of 2wt% PK co-embedded with varying ratios of MgSO4 as-cast (AC) and heat treated for 5 minutes at 155 and 185 °C.

[0064] Fig. 19. Thermostability screen with varying salts, plastics, and enzymes. For all bar plots heat treatment was done for 5 minutes, triplicates were averaged, and standard deviations were plotted as error bars. A. Activity rate of 2wt% PK in PLLA80 co-embedded with varying hygroscopic and efflorescent salts. B. Activity rate of 2wt% PK in PLLA80 co-embedded with salts with chloride and nitrate anions.

[0065] Description of Particular Embodiments of the Invention

[0066] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0067] Industrial bioactive plastic processing

[0068] We demonstrated through a series of systematic experiments, fundamental differences between protein thermostability in polymer matrices and melts in comparison to aqueous and organic solutions. We showed that plastic-degrading hydrolases, including proteinase K from Tritirachium album, are thermostable up to 145-155 °C in plastics of varying polarity, encompassing both biodegradable polymers and traditional polyolefins, as well as melts of varying viscosity. Crucially, we exceeded the engineering goal of stabilizing enzymes for PLLA melt processing by co-embedding enzymes with MgSCh. This enabled retention of enzymatic activity even after >8 minutes of residence time in 200 °C PLLA melts. We further validated thisapproach in both solvent-cast and melt-extruded PLLA films. Films containing 0.2-2 wt% proteinase K and 0.2-10 wt% MgSCh catalyzed rapid degradation of their host PLLA matrix, achieving up to 90% mass loss within 3 days at 40 °C in buffer, and -70% in compost at 60 °C in under 8 days. Near-complete degradation (>95%) was observed when compost was hydrated and heated to 80 °C. These effects were consistent across different preparation methods, and SEM images confirmed internal matrix degradation rather than surface erosion. We also demonstrated tunability with 0.2 wt% enzyme-salt loading producing slower but sustained degradation (-95% over 25 days) at 40 °C in buffer, enabling formulation flexibility. Thermal characterization showed that polymer melting and crystallization behavior remain unchanged, while mechanical testing indicated that although strength and stiffness decrease with enzymesalt addition, the films retain structural integrity making them suitable for practical applications. These results show a cheap, simple, and scalable strategy for producing bioactive, compostable plastics with programmable degradation, using enzyme-salt co-embedding to overcome industrial processing barriers. We further showed that the deactivation temperature is universal across the tested hydrolases that span a diverse range of secondary structures, and that the stabilization effects of MgSO4are universal. Control experiments showed that these results are not purely due to local hydration modulation; specific structural change and / or chaperoned refolding are also occurring.

[0069] Thermostable proteins in 200°C polymer melt

[0070] The primary engineering goal of this study is to enhance protein stability during industrial polymer melt extrusion. However, even micro-extruders on a lab scale produce films of at least gram-scale, making melt extrusion inefficient for the repetitive screening and assaying of biological additives due to material wastage, slow processing, and the confounding effects of mixing gradients and mechanical shear. To determine how the temperature, chemistry, and viscosity of stationary polymer melts affect protein thermostability, we developed a proxy thermal processing method. Polymer, enzyme, and any intermolecular chaperones were mixed in organic solvent, solution cast, air dried into films, and then heat treated for a simulated extruder residence time of 5 minutes. After heat treatment, the films were rapidly cooled and assayed for enzymatic activity using a kinetic heterogeneous catalysis colorimetric assay. Enzyme embedded films are soaked in solution of 4-nitrophenyl butyrate (4NPB), an ester bond containing small molecule whose hydrolysis is catalyzed. Product absorbance at 410nm is measured over time, and initial enzyme activity rates are calculated via an initial, linear slope fitting. Activity rates of heat-treated films were normalized by the activity rates of pre-heat treated (as-cast, AC) films of the exact composition, yielding % retained activity (% RA) valuesthat are plotted. Normalization is important as it allows for fair comparison across experiments that used different enzyme batches, enzyme loading %, or even different enzymes, plastics, or small molecules entirely. Varying each of these will result in different extents of activity loss due to misfolding during organic solvent processing, AC catalysis rates, and diffusion pathways to and from solid-state enzymes embedded in hydrophobic films. Thus, normalization removes all the confounding variable for comparison of only thermostability in the polymer matrix / melt.

[0071] Applying this method, we found that 2wt% proteinase K (PK) is outstandingly thermostable in 155°C 30kDa poly(L-lactic acid) (PLLA30), maintaining 59+10% RA at 155 °C. However, PK is fully deactivated by 185°C, and so would not survive melt extrusion in PLLA30. PK stability at 155°C was surprising considering without any optimization, confinement in PLLA30 resulted in thermostability ~80°C higher than Tmin solution. Recently published literature also observed similar results, and may suggest that the molecular crowding effect of densely entangled polymer chains effectively immobilize the enzyme, removing the flexibility required to misfold.19,20Indeed, differential scanning calorimetry (DSC) showed that at 155°C PLLA30 is above Tgbut below the onset Tm. Incredibly, we discovered that the inclusion of just 2wt% MgSO4 enables the retention of 44+11% RA at 185 °C, 15+2% RA at 200 °C, and 6+2% RA at 215 °C, before fully deactivating by 230°C. DSC results further showed that including MgSO in films resulted in a minor 4. 1+4.8% (n=6) decrease in film crystallinity calculated from the total integrated area of cold-crystallization and melting peaks. An exact % crystallinity of films was not calculated as there are enthalpic relaxation peaks at lower temperatures as PLLA30 heats past its Tgthat were not measured. Embedding 10wt% MgSO4 resulted in the PLLA30 melt temperature mode (Tm) only increasing from 173 to 174°C, and the maximum (Tm,max) remained constant at 178°C. Thus, it is safe to conclude that by 185 °C all polymer chains in all films with or without salt are fully melted, and the salt effect improves enzyme stability rather than plastic matrix stability.

[0072] To test if our proxy assay for retained activity could translate to post heat treated plastic degrading activity, we heat treated PLLA30 films for 8 minutes at 185 °C to ensure that all films were fully melted. Films were air cooled then incubated for 24 hours in 40°C pH 8.0 tris buffer solution. Post-incubation films were lyophilized to remove adsorbed water. Films with only 2wt% PK barely decreased in mass by 0.8+0.2%, whereas films with co-embedded 2wt% PK and 10wt% MgSO4decreased in mass by 43.4+10.4% . Macroscopically PK only films gained a brown tint, indicative of misfolded and aggregated proteins, whereas the PK+MgSCE films turned completely opaque white and became brittle, indicative of the formation of internal porosity. However, films embedded with MgSO4lost 3.9+ / -0.2% mass, suggesting that -25% of total salt mass leached out of the film. This was expected as prior literature has intentionallysolution-cast poly(L-lactic acid) with embedded salt and then let the salt leach to create microporous foams.24Given that internal degradation creates nanoporous small molecule transport channels, a higher rate of salt leaching is expected in partially degraded films. Yet, even if we assume 100% of MgSO4 leached out in the PK+MgSO4 films and subtract that from the mass loss, lower bounded PK degradation resulted in 32% PLLA30 mass loss. Additionally, buffer solution pH was measured after removing films with PK+MgSO4 embedded, and it dropped to pH 5. This suggested significant lactic acid production and further confirmed PK was made thermostable after 8 minutes at 185 °C by co-embedding MgSO4 and indicate that degradation rate might have been higher had a larger volume or a continuous flow of buffer solution been used to maintain the optimal pH 8. Notably, this rapid degradation occurred in both solvent-cast and melt-extruded films, confirming that our thermal proxy assay reliably predicts melt-processing outcomes. Moreover, this enzyme-salt strategy supports fine-tuning: even 0.2 wt% additive loadings enable slow but sustained degradation to >95% over 25 days, while higher loadings (2 wt%) achieve nearly 90% degradation within just 3 days. In composting conditions (60 °C with added water), the same 2:2 wt% formulation degraded -70% within 8 days and >95% at 80 °C with mild buffering, confirming industrial and environmental applicability.

[0073] We also demonstrate modulation of mechanical properties of plastic films. For example, depending on the engineering application, foam-like microporous plastics could be desired and produced, however, for plastic packaging where barrier properties are required, this is definitely not the case. We screened the thermostability of PK in PLLA30 with varying MgSO4 loading and found the optimum to be within the 2-20 wt% range but with retained activity at 185 °C as low as 0.05 wt%.

[0074] In this work 2wt% was used to optimize for enzyme activity retention, however in practice the trade-off with film mechanical properties can be considered when picking a desired salt loading. Ultimately, we successfully demonstrated thermostabilization of a functional enzyme in a mass-produced biopolymer using a common salt (MgSO4). These results enable industrially viable, truly compostable and / or bioactive plastics.

[0075] Universal rules governing protein thermostability in polymer melts and matrices

[0076] We next investigated if the remarkable observed protein thermostability in plastic melt that is further enhanced by co-embedding MgSO4is universal or system specific to PK in PLLA30. We systematically screened thermostability across various proteins-polymer pairings. The thermostabilities of 5 hydrolases: PK, AO, BC, BL and HRP were investigated across 6 plastics: PLLA30, PLLA85, PLLA100, PMMA, PCL, PS. The enzymes are relatively pure A260 / A280 -0.6. Turbidity of all enzyme solutions was also low, quantified by absorbance at320nm. Enzyme surfaces features were computationally extracted using the pipeline developed herein. Chemical surfaces of proteins are relatively similar overall, with the largest discrepancy being AO having the most charged surface with -25% charged residues that are more negative. PK, AO, BC, BL are extremely spherical and are <35% beta-sheet, with a more equal distribution (35-45% alpha-helix, 40-60% random coil). Plastics investigated ranged in polarity and in molecular weight (Mn) from ~30-500kDa. The three PLA films and PCL are semicrystalline, while PMMA and PS are amorphous. These features were hypothesized to be predictive of embedded enzyme thermostability and were summarized in Table 1.

[0077] Thermostability screens of PK, AO, BC, and BL across six plastics showed that all 24 combinations had 0% RA after heat treatment by 155-165 °C. Up to 145 °C, AO and PK were generally more thermostable across plastics in comparison to BC and BL. There are 2 notable enzyme-plastic pairing exceptions to this that stand out as more thermostable than the rest: BC- PCL and BL-PMMA. Interestingly, the % RA of PK, BC and BL seems to decrease from 135- 165 °C with a smooth exponential decay, however, AO experiences a sharp step-function drop in % RA between 145 and 155°C. Despite these variations in enzyme thermostability after heat treatment in the 135-145 °C range, the overwhelming result remains that none of the protein nor plastic features are significant when pushing towards Tm,PLA- Importantly, although 165°C happens to be the onset of PLA melting, this temperature marks no significant chain conformation or dynamics transition for any of the other 3 plastics. This screening was also repeated with enzyme loading increased from l-2wt% to 10wt%. The same outcome was observed, demonstrating that thermostability cannot be increased by increasing enzyme loading. These results point to some universal mechanism of deactivation that is independent of surface chemistry interactions and chain density confinement effects.

[0078] Water is the remaining universal factor. We hypothesized that universal misfolding across all protein-plastic pairings was due to evaporation of solid-state water bound to enzyme (hydration shell layer). This temperature would be relatively agnostic to variations in surrounding plastic, enzyme chemistry, and matrix / melt morphology. If this were true, the role of MgSOt could therefore be to modulate the water molecules local to embedded enzymes. MgSCL is a well-known dehydrating agent, can form various highly hydrated states, and thus absorb or release water depending on environmental humidity. We screened various proteinplastic pairings with and without MgSCL to test the universality of its thermostabilizing effect. In short, MgSCL boosted the thermostability of every pairing tested. When plastic is varied from the more polar PLLA30 to the most hydrophobic PS, PK was still thermostabilized by MgSO i, retaining 34 ± 3% RA after 185 °C heat treatment. PK-only control films with no salt under the same conditions were completely deactivated (-0.1% RA). DSC analysis further supported ourhydration hypothesis: when MgSCU was co-embedded with PK in PMMA, a polymer chosen to avoid overlapping transitions, the protein’s melting peak became sharper and shifted from -63.5 °C to -77.5 °C, indicating a more stable folded state. A lower-temperature transition near 60 °C also shifted rightward, consistent with delayed water loss. This behavior was not observed in the absence of MgSCh, and repeated DSC scans under nitrogen showed no recovery of the melting peak, implying refolding does not occur without air or atmospheric moisture. This supports the interpretation that MgSCT stabilizes protein conformation during heating by retaining hydration, rather than through chemical stabilization or polymer matrix changes.

[0079] We further showed that in addition to PK, all of AO, BC, and BL can be thermostabilized in PLLA by MgSO4 up to heat treatment of at least 185 °C, demonstrating universality across hydrolases even with significantly varying secondary structures, species of origin, metal ions, and native substrates. CD spectra confirmed that PK leached from thermally annealed films retained its P-sheet-rich secondary structure only when MgSO4 was present, supporting either preserved folding or chaperoned refolding. FTIR spectra of PLLA films containing PK and MgSO4 showed distinct O-H stretching and H-O-H bending peaks after heating to 185 °C, which disappeared by 230 °C. This closely matched the temperature at which activity is lost, providing strong evidence that protein stabilization is directly linked to retained hydration.

[0080] Additionally, to test whether the remarkable protein thermostability observed in plastic melts generalizes beyond hydrolases, we expanded our study to include horseradish peroxidase (HRP), a heme-containing enzyme with a distinct tertiary structure and catalytic mechanism compared to the serine hydrolases previously tested. By mixing the enzyme, polymer, and MgSCL followed by thermal processing on a hot plate, we embedded HRP in a range of polymers including LDPE, PLLA, and PEO without the use of organic solvent. Enzymatic activity was measured post-treatment. Results show that HRP retains measurable activity after heating to 185 °C only when co-embedded with MgSCh, across all polymers tested. These findings align with those seen for PK and demonstrate that MgSCL’s stabilizing effect is not limited to hydrolases, but extends to functionally and structurally diverse enzymes such as HRP. This supports the generalizability of MgSCh as a thermostabilizing additive for a wide variety of bioactive plastics.

[0081] Salt-enzyme-polymer specific thermostabilizing interactions

[0082] To test the bound hydration water evaporation hypothesis, we did a modified heat treatment procedure where instead of an oven (dry environment) we heat treated in an environment saturated with > 99% relative humidity steam. PK, AO, and BC in PLLA were steam heat treated, with results matching those done in the standard, dry heat treatment setup, asall 3 enzymes fully deactivate by 155 °C. To further probe our hydration hypothesis, we screened PK thermostability in PLLA with several salts known to form highly hydrated states. Although lanthanides are extremely hygroscopic, all 3 tested (nitrates of Eu, Nd, and Dy) deactivated enzymes completely prior to heat treatment, suggesting there was likely a binding interaction that was too strong, causing PK to misfold. Only cobalt acetate (CoAc) successfully managed to retain PK activity after heat treatment at 185 °C. However, it should be noted that the CoAc mixture was extremely insoluble in organic solvent, so it’s likely most of the salt, and a large portion of salt-bound enzyme, did not end up in the plastic films. These results showed that the universal deactivation of all tested hydrolases in all tested plastics at 155-165 °C, as well as the thermostabilization mechanism of MgS04, are not purely water / hydration-driven phenomena.

[0083] To expand on these results, we conducted a systematic salt screen using PK embedded in PLLA85. Salts were selected to span a range of ion kosmotropicity and chaotropicity, organized along the Hofmeister series, and co-embedded with PK at 2 wt%. Only MgSO4 enabled consistent activity retention across all tested temperatures, with PK maintaining high activity even at 185 °C. CaiCHiCOOh also provided stabilization, though with lower retained activity and greater variability. Other salts, including Na2SO4, ZnSO4, MgCL, MgiNOda. NaCl, and ammonium nitrate, failed to improve thermal stability and, in many cases, fully deactivated PK either during solvent casting or upon heating. Notably, MgCL and MgtNCLh, despite sharing the same Mg2+cation as MgSO4, fully deactivated PK as-cast, suggesting that anion identity and salt interaction profile are critical. This rules out a sulfate-only mechanism and highlights that MgSCL's stabilizing effect is not purely hydration-based but also depends on the specific protein-salt interaction environment. The inactivity of PK observed for highly hygroscopic salts like lanthanide nitrates further supports this conclusion. This suggests that while the aformentioned salts destabilize PK, their effect may not be universally deactivating and could potentially stabilize other proteins, as their interactions appear to be governed by specific salt-enzyme compatibility rather than simply hygroscopic behavior. In addition to ionic salts, we screened common surfactants and excipients including PEG8k, CTAB, and AOT to determine whether co-embedding them with PK in PLLA could lead to thermal stabilization. None of these additives improved activity retention at any tested temperature, up to 215 °C. These results collectively show that MgSCL’s stabilizing mechanism is not simply due to hygroscopicity.

[0084] Together, these results show that while hydration plays a central role, the thermostabilization mechanism of MgSCL across a wide range of proteins also involves protein interactions (or lack thereof) that are not replicated by structurally similar salts. This suggeststhat MgSCh’s hygroscopic nature, without being deliquescent or acidic, contributes to maintaining hydration during thermal exposure, releasing bound water gradually over the thermal range relevant to melt processing. These criteria all seem to apply to Ca(CH3COO)2 as well. Of course the foregoing results were for PK embedded in PLLA85, and suitability of a particular salt, for a particular protein, under particular conditions may be determined empirically.

[0085] Random heteropolymers

[0086] Previous works have described the using random heteropolymers as intermolecular chaperones for protein solvent and thermal stability. However, these have primarily been demonstrated for enzymes solution not in embedded solid or viscoelastic states. Moreover, the thermostability has never exceeded 100°C.

[0087] We screened PK (2wt%) thermostability in PLLA with MgSO4 (10wt%) with several RHP compositions (2wt%) and found that a composition 50-20-25-5DMAEMA was able to retain 95% of the as-cast enzymatic activity. 10-60-25-5SPMA yielded a slight increase in thermostability, however all other compositions negligibly changed or reduced the retained activity compared to using MgSO4 alone. All RHP compositions alone without salt did not meaningfully increase PK thermostability in similar conditions.

[0088] Interpretation of Selected Figures

[0089] In Fig. 5, part A, we demonstrate that co-embedding Proteinase K (PK) and MgSOi in 55 kDa PLLA leads to rapid enzymatic degradation, reaching nearly 90% mass loss after just 3 days at 40 °C in Tris Buffer. This effect was consistent across two different approaches: (1) solvent-cast films using DCM followed by oven annealing at 185 °C for 5 minutes, and (2) direct melt extrusion where lyophilized PK, MgSC , and PLLA pellets were blended and processed at 185 °C for 5 minutes. SEM images provide direct evidence that degradation occurs through embedded enzymatic degradation rather than surface-driven degradation shown by the increase in the size and number of pores present in the plastic matrix at higher levels of mass loss.

[0090] Fig. 5, part B shows the time course of mass loss for PLLA films containing varying loadings of PK and MgSCh in O.Olx Tris buffer at 40 °C. When 0.2 wt% of each additive is embedded, degradation is slow but sustained, reaching approximately 95% mass loss over the course of 25 days.

[0091] Fig. 5, part C compares degradation of 2 wt% PK and 2 wt% MgSCh in compost at 60 °C with 60 wt% added water wrt soil. Under these conditions, the formulation with 2 wt% PK and 2 wt% MgSCh achieves rapid degradation, reaching -70% mass loss in approximately 8days. By comparison, the control films only showed small signs of degradation (max 10%). Our data also show by increasing the temperature to 80 °C and adding 0.0 lx Tris buffer to the compost, near complete mass loss is observed (>95%).

[0092] Fig. 5, part D, shows that the thermal transition properties of PLLA remain unchanged, as seen by superimposable DSC traces. Melt temperature (Tm) & crystallization peak (Tc) remain the same, as well as their characteristic shapes. Glass transition (Tg) shows a slight dip for samples containing salts. Overall, this indicates that the embedded additives do not disrupt the polymer’s semi-crystalline structure prior to degradation.

[0093] Finally, the stress-strain plot in Fig. 5, part E shows that increasing the PK-MgSO4 loading in PLLA reduces both the strength and stiffness of the material, but stays within the order of magnitude. The 2 wt% and 0.2 wt% PK-MgSO4 films has lower peak stress, indicating a softer but mechanically weaker polymer. These results indicate that while embedding the enzyme-salt pair makes degradation easier, it reduces the mechanical strength of the material compared to pure PLLA. However, as shown in the insets, the films retain sufficient integrity to remain intact and support weight, demonstrating practical usability despite the reduction in tensile properties. These material properties can be tuned and improved further by engineering optimization (i.e. more consistent blending / composition fine tuning).

[0094] Fig. 12 shows how broad MgSCL’s applications are while being a specific thermal stabilizer for enzymes embedded in polymer matrices. In part A, four different hydrolases / lipases are embedded into PLLA85 with or without 2 wt% MgSOi and subjected to 185 °C for 5 minutes in an oven. Across all enzymes, co-embedding with MgSCL consistently enhances retained activity post-treatment. This broad applicability of MgSCri is likely due to maintenance of hydration shell.

[0095] In Fig. 12, part B, 2 wt% PK-MgSCh is tested in four different plastics (PLLA85, PMMA, PCL, PS), all heat treated at 185 °C. Despite the varying structural properties of these matrices, significant enzyme activity is retained in each case, confirming that MgSCL’s effect is not strongly dependent on the polymer’ s inherent chemistry. This further supports a hydrationbased stabilization mechanism rather than one specified by polymer- specific interactions. This is further supported by our results which show that thermal stabilization is observed across a wide range of viscosities (10-105Pa*s) showing that this effect is not due to confinement in polymer matrices and also helps support the hydration shell theory.

[0096] Fig. 12, part C shows a systematic screen of inorganic salts. We grouped them by their positions in the Hofmeister series. Only MgSO4 and, to a lesser extent, Ca(CH3COO)2 retain PK activity up to 185 °C. Other salts, including common stabilizers such as NaCl, KC1, and ammonium sulfate, result in negligible activity retention. This indicates that MgSO4’shygroscopic nature, without being deliquescent or acidic, contributes to maintaining hydration during thermal exposure, releasing bound water gradually over the thermal range relevant to melt processing. These criteria apply to CaiCfLCOOh as well.

[0097] Fig. 14 expands the scope of proteins MgSCb can thermostabilize at 185°C. In part A, PK retains high enzymatic activity in LDPE, PLLA, and PEO (all three polymers with varying viscosities and chemistry) but only when co-embedded with MgSCL. In part B, a similar trend can be seen with horseradish peroxidase (HRP), a heme-containing enzyme that is structurally distinct from PK. Once again, co-embedding with MgSO4 preserves measurable activity across all polymers except PEO where the difference between with or without MgSO4 isn’t statistically significant.

[0098] Fig. 15 shows that MgSO4 preserves protein structure during high-temperature processing by maintaining hydration giving us a clue on the mechanism by which it thermostabilizes proteins.

[0099] In part A, DSC was used to analyze PK embedded in PMMA, an amorphous polymer chosen to avoid overlapping transitions. When MgSO is co-embedded with PK, the protein’s melting peak becomes sharper and shifts from -63.5 °C to -77.5 °C, indicating a more stable folded state prior to greater temperature increases. Additionally, a low-temperature transition near 60 °C also shifts rightward, consistent with delayed water loss. This behavior is not observed when PK is embedded without MgSCh. This indicates that MgSOt modulates water retention within the polymer. Repeated DSC scans under nitrogen show no recovery of the melting peak in any sample, implying that refolding does not occur without access to air or atmospheric moisture. This supports the interpretation that MgSCL stabilizes protein conformation during heating by retaining hydration, rather than by chemical stabilization or changing the polymer environment.

[0100] Fig. 15, part B further supports this hypothesis using circular dichroism (CD). PK leached into water Polymer films were first heated to 185 °C and soaked in water. The PK that leached out of the polymer showed complete loss of secondary structure in the absence of MgSCkBut, PK lyophilized with MgSOt resembles the native protein spectra that is mainly made up of sheets. This demonstrates that MgSCL either preserves folding or allows proper refolding once rehydrated, or a mixture of both, consistent with our hydration-dependent stabilization mechanism proposal.

[0101] Fig. 15, part C shows FT1R spectra of PLLA films containing PK and MgSCL, focusing on the O-H stretching and H-O-H bending regions. Films heated to 185 °C retain clear hydration peaks, whereas those heated to 230 °C do not. This matches activity data, which shows no activity past 215 °C with MgSC

[0102] Fig. 16 shows that time-dependent heating produces a similar effect where prolonged heating leads to progressive activity loss. This is consistent with gradual dehydration. Putting this information together with our results which indicate a thermal limit for hydration-mediated thermostabilization is at a maximum 1 hour at -215 °C using MgSCh.

[0103] Fig. 17 tests other potential additives in the form of surfactants and hygroscopic agents such as PEG, CTAB, and AOT. None of these options could replicate MgSCh’s effect on enzyme stability / activity. This specificity points to some of MgSOFs unique properties since it is hygroscopic but not deliquescent, it binds to water effectively, and does not alter pH (usually neutral), making it optimal for preserving protein structure during thermal processing. Fig. 17 also shows that simply heating the polymers in a humid environment does not increase enzyme thermostability.

[0104] Methods and materials

[0105] Film Preparation: Proteinase K from Tritirachium album (PK), lipase from Burkholderia cepacia (BC), lipase from Aspergiullus oryzae (AO), and protease from Bacilus licheniformis (BL) were purchased from Sigma- Aldrich and purified following previously established procedures. Concentration of enzyme stock solutions were made ranging between 0.5-1.5 mg / mL of enzyme in Milli-Q (MQ) water and quantified via absorbance at 280 nm. MgSO4, KC1, NH4NO3, NH4C1, MgCl2, Mg(NO3)2, Na2SO4, ZnSO4, cobalt (II) acetate, dysprosium (III) nitrate, neodymium nitrate and europium (III)nNitrate were purchased from Sigma-Aldrich. Salt stock solutions were made in MQ water ranging between 10-100 mg / mL. lyophilized Salt and enzyme solutions were mixed at mass ratios between 0.05: 1 and 100:1, frozen at -80 °C, and lyophilized for a minimum of 8 hours.

[0106] Polycaprolactone (PCL), Polystyrene (PS) and Polymethylmethacrylate (PMMA) were purchased from Sigma Aldrich. Polylactic acid (PLLA) polymers were purchased from PolySciTech. Full plastic details can be found in Table 1. PCL, PLLA and PMMA was dissolved in dichloromethane (DCM) at 4 wt% concentration and stirred for 4 hours to ensure complete dissolution. PS was dissolved in DCM at 6 wt%. The co-lyophilized enzyme and salt complexes were resuspended directly in the polymer solution at 0.1 -2 wt% enzyme loading. Mixtures were vortexed for 5 min before 20 ,u L drops were cast on glass sides to obtain approximately 1 mg films for thermostability assay trials. For larger degradation trials, 20 mg films were cast. All films were air-dried at room temperature. Unless otherwise specified, -85 kDa PLLA was used for thermostability experiments and -30 kDa PLLA was used for degradation experiments.

[0107] Thermostability Assays: Quadruplicates of 1 mg plastic films were used for each temperature trial and were thermally annealed in a Fischer Scientific Isotemp oven for 5-8minutes. A thermocouple was also placed in the oven right above the films to get the most temperature. For 99% RH steam heat treatment, a hotplate was used with 3x 30 mL beakers filled with distilled water and covered with a glass cover to create a saturated humid air environment. Oven to hotplate temperature calibration was done. 4-Nitrophenyl butyrate (4NPB) was used as a small molecule ester substrate to test enzyme activity for all hydrolases. Each film was peeled off glass using a razor blade and placed in individual wells in a 96-well plate filled with 200 L of 0.5 mM 4NPB or 0.7mM N-Suc solution in lx PBS. Absorbance at 410 nm was measured at 30 second intervals for each film for 30 minutes using an infinite M nano plate reader. A regression was performed on the linear region of the enzyme activity plot to determine initial rates.

[0108] PLLA Degradation: ~20 mg films triplicates were thermally annealed in the isotherm oven for 12 minutes, ~6 mg strips were cut and placed in 7 mL glass vials filled with 5 mL of O.Olx Tris pH8.0 buffer. Films were removed from a 40°C water bath after 24 hours, any residual buffer salts were washed with MQ water, lyophilized overnight, and then weighed to determine mass loss.

[0109] Table 1. Key information on the 6 plastics screened. Values in regular font were provided by the supplier, values in italics were taken from literature, values in bold were experimentally measured.

[0110] References(1) Bahrami, A.; Delshadi, R.; Assadpour, E.; Jafari, S. M.; Williams, L. Antimicrobial-Loaded Nanocarriers for Food Packaging Applications. Adv. Colloid Interface Sci. 2020, 278.102140. https: / / doi.Org / 10.1016 / j.cis.2020.102140.(2) Breitenbach, J. Melt Extrusion: From Process to Drug Delivery Technology. Eur. J.Pharm. Biopharm. 2002, 54 (2), 107-117. https: / / doi.org / 10.1016 / S0939-6411(02)00061-9.(3) Panganiban, B.; Qiao, B.; Jiang, T.; DelRe, C.; Obadia, M. M.; Nguyen, T. D.; Smith, A. A. A.; Hall, A.; Sit, I.; Crosby, M. G.; Dennis, P. B.; Drockenmuller, E.; Olvera de la Cruz, M.;Xu, T. Random Heteropolymers Preserve Protein Function in Foreign Environments. Science 2018, 359 (6381), 1239-1243. https: / / doi.org / 10.1126 / science.aao0335.(4) DelRe, C.; Huang, C.; Li, T.; Dennis, P.; Drockenmuller, E.; Xu, T. Reusable Enzymatic Fiber Mats for Neurotoxin Remediation in Water. ACS Appl. Mater. Interfaces 2018, 10 (51), 44216-44220. https: / / doi.org / 10.1021 / acsami.8bl8484.(5) DelRe, C.; Jiang, Y.; Kang, P.; Kwon, J.; Hall, A.; Jayapuma, I.; Ruan, Z.; Ma, L.; Zolkin, K.; Li, T.; Scown, C. D.; Ritchie, R. O.; Russell, T. P.; Xu, T. Near-Complete Depolymerization of Polyesters with Nano-Dispersed Enzymes. Nature 2021, 592 (7855), 558— 563. https: / / doi.org / 10.1038 / s41586-021-03408-3.(6) DelRe, C.; Chang, B.; Jayapuma, I.; Hall, A.; Wang, A.; Zolkin, K.; Xu, T. Synergistic Enzyme Mixtures to Realize Near-Complete Depolymerization in Biodegradable Polymer / Additive Blends. Aclv. Mater. 2021, 33 (49), 2105707. https : / / doi. org / 10.1002 / adma.202105707.(7) Kwon, J.; DelRe, C.; Kang, P.; Hall, A.; Arnold, D.; Jayapuma, I.; Ma, L.; Michalek, M.; Ritchie, R. O.; Xu, T. Conductive Ink with Circular Life Cycle for Printed Electronics. Adv. Mater. 2022, 34 (30), 2202177. https: / / doi.org / 10.1002 / adma.202202177.(8) Kalita, N. K.; Hakkarainen, M. Triggering Degradation of Cellulose Acetate by Embedded Enzymes: Accelerated Enzymatic Degradation and Biodegradation under Simulated Composting Conditions. Biomacromolecules 2023, 24 (7), 3290-3303. https: / / doi.org / 10.1021 / acs.biomac.3c00337.(9) Warner, M. J.; Lassa, J. P.; Narcross, H.; Commisso, A.; Ghosh, K.; Romero, M.; Schwartz, J. M.; Engler, A. C.; Kohl, P. A.; Leguizamon, S. C.; Jones, B. H. Chemical Recycling of Polybutadiene Rubber with Tailored Depolymerization Enabled by Microencapsulated Metathesis Catalysts. ACS Sustain. Chem. Eng. 2023, 11 (39), 14538-14548. https: / / doi.org / 10.1021 / acssuschemeng.3c03907.(10) Maraveas, C.; Kotzabasaki, M. I.; Bartzanas, T. Intelligent Technologies, Enzyme- Embedded and Microbial Degradation of Agricultural Plastics. AgriEngineering 2023, 5 (1), 85- 1 11. https: / / doi.org / 10.3390 / agriengineering5010006.(11) Siemann, U. Solvent Cast Technology - a Versatile Tool for Thin Film Production. In Scattering Methods and the Properties of Polymer Materials’, Springer Berlin Heidelberg: Berlin, Heidelberg, 2005; pp 1-14. https: / / doi.org / 10.1007 / bl07336.(12) Rauwendaal, C. Polymer Extrusion, 5th edition.; Hanser Publications ; Hanser Publication: Munich : Cincinnati, 2014.(13) EUBIO_Admin. Market. European Bioplastics e.V. https: / / www.european- bioplastics.org / market / (accessed 2024-04-29).(14) Velghe, I.; Buffel, B.; Vandeginste, V.; Thielemans, W.; Desplentere, F. Review on the Degradation of Poly(Lactic Acid) during Melt Processing. Polymers 2023, 15 (9), 2047. https: / / doi.org / 10.3390 / polyml5092047.(15) Aldhafeeri, T.; Alotaibi, M.; Barry, C. F. Impact of Melt Processing Conditions on the Degradation of Polylactic Acid. Polymers 2022, 14 (14), 2790. https : / / doi.org / 10.3390 / polym 14142790.(16) Yeh, C. K.; Lin, C.; Shen, H. C.; Cheruiyot, N. K.; Camarillo, M. E.; Wang, C. L. Optimizing Food Waste Composting Parameters and Evaluating Heat Generation. Appl. Sci. 2020, 10 (7), 2284. https: / / doi.org / 10.3390 / appl0072284.(17) Vieille, C.; Zeikus, G. J. Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability. Microbiol. Mol. Biol. Rev. 2001, 65 (1), 1-43. https: / / doi.org / 10.1128 / MMBR.65.Ll-43.2001.(18) Modarres, H. P.; Mofrad, M. R.; Sanati-Nezhad, A. Protein Thermostability Engineering. RSC Adv. 2016, 6 (116), 115252-115270. https: / / doi.org / 10.1039 / C6RA16992A.(19) Guicherd, M., Ben Khaled, M., Gueroult, M. et al. An engineered enzyme embedded into PL.A to make self-biodegradable plastic. Nature 631, 884—890 (2024). https : / / doi.org / 10.1038 / s41586-024-07709- 1(20) Xu, C.; Battig, A.; Schartel, B.; Siegel, R.; Senker, J.; Von Der Forst, I.; Unverzagt, C.; Agarwal, S.; Moglich, A.; Greiner, A. Investigation of the Thermal Stability of Proteinase K for the Melt Processing of Poly( L -Lactide). Biomacromolecules 2022, 23 (11), 4841-4850. https: / / doi.org / 10.1021 / acs.biomac.2c01008.(21) Weng, Y.; Dunn, C. B.; Qiang, Z.; Ren, I. Immobilization of Protease K with ZIF-8 for Enhanced Stability in Polylactic Acid Melt Processing and Catalytic Degradation. ACS Appl. Mater. Interfaces 2023, acsami.3cl l979. https: / / doi.org / 10.1021 / acsami.3cl l979.(22) Tamasi, M. J.; Patel, R. A.; Borca, C. H.; Kosuri, S.; Mugnier, H.; Upadhya, R.; Murthy, N. S.; Webb, M. A.; Gormley, A. J. Machine Learning on a Robotic Platform for the Design of Polymer-Protein Hybrids. Adv. Mater. 2022, 34 (30), 2201809. https: / / doi.org / 10.1002 / adma.202201809.(23) Zaks, A.; Klibanov, A. M. The Effect of Water on Enzyme Action in Organic Media. J. Biol. Chem. 1988, 263 (17), 8017-8021. https: / / doi.org / 10. 1016 / 80021-9258(18)68435-2.(24) Clark, D. S. Characteristics of Nearly Dry Enzymes in Organic Solvents: Implications for Biocatalysis in the Absence of Water. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2004, 359 (1448), 1299-1307. https: / / doi.org / 10.1098 / rstb.2004.1506.(25) Mikos, A. G.; Thorsen, A. J.; Czerwonka, L. A.; Bao, Y.; Langer, R.; Winslow, D. N.; Vacanti, J. P. Preparation and Characterization of Poly(l-Lactic Acid) Foams. Polymer 1994, 35 (5), 1068-1077. https: / / doi.org / 10.1016 / 0032-3861(94)90953-9.(26) Shahid, S.; Hassan, Md. I.; Islam, A.; Ahmad, F. Size-Dependent Studies of Macromolecular Crowding on the Thermodynamic Stability, Structure and Functional Activity of Proteins: In Vitro and in Silico Approaches. Biochim. Biophys. Acta BBA - Gen. Subj. 2017, 1861 (2), 178-197. https: / / doi.Org / 10.1016 / j.bbagen.2016.l l.014.(27) Barias, N. T.; Bahamonde, H. A.; Pimentel, C.; Dommguez-Huidobro, P.; Pina, C. M.; Fernandez, V. Evaluating Leaf Wettability and Salt Hygroscopicity as Drivers for Foliar Absorption. Plants 2023, 72 (12), 2357. https: / / doi.org / 10.3390 / plantsl2122357.(28) Ashraf, H.; Guo, Y.; Wang, N.; Pang, S.; Zhang, Y.-H. Hygroscopicity of Hofmeister Salts and Glycine Aerosols-Salt Specific Interactions. J. Phys. Chem. A 2021, 725 (7), 1589— 1597. https: / / doi.org / 10.1021 / acs.jpca.0cl0710.(29) Choi, M. Y.; Chan, C. K. Investigation of Efflorescence of Inorganic Aerosols Using Fluorescence Spectroscopy. J. Phys. Chem. A 2005, 109 (6), 1042-1048. https : / / doi.org / 10.102 l / jp048103h.(30) Williams, D. F. Enzymic Hydrolysis of Polylactic Acid. Eng. Med. 1981, 70 (1), 5-7. https: / / doi.org / 10. 1243 / EMED_JOUR_1981_010_004_02.(31) Bomscheuer, U., et al. Lipase of Pseudomonas Cepacia for Biotechnological Purposes: Purification, Crystallization and Characterization. Biochim. Biophys. Acta BBA - Gen. Subj. 1994, 7207 (1), 55-60. https: / / doi.org / 10. 1016 / 0304-4165(94)90151-1.

Claims

CLAIMS1 . A plastic composition comprising a blend of a thermoplastic, a protein and a salt configured to increase thermal stabilization of the protein in the composition.

2. A composition of claim 1, further comprising polymeric protectants configured to increase thermal stabilization of the protein.

3. A composition of claim 1, further comprising polymeric protectants, wherein the salt and polymeric protectants provide synergistic effects in thermal stabilization of the protein.

4. A composition of claim 1, wherein the composition enables preservation of embedded protein activity at high temperatures for downstream / subsequent functional use, such as by biocatalysis of small molecules external to embedded matrix as well as biocatalysis on the matrix itself (plastic degradation).

5. A composition of claim 1, wherein the salt comprises a cation and anion pair that together maintain protein hydration during thermal processing by gradually releasing bound water over the melt processing temperature range, without being deliquescent or acidic, and without interacting with the protein in a manner that induces misfolding or deactivation.

6. A composition of claim 1, wherein the composition further comprises co-embedded random heteropolymers (RHP) to: increase retained protein activity; and reduce salt and / or protein leaching.

7. A composition of claim 1, wherein the composition further comprises co-embedded random heteropolymers (RHP), wherein the heteropolymers: promote retention of the embedded protein, e.g., in practical applications such as compostable plastics; reduce or prevent unwanted porous plastic formation; reduce leaching and salt loading; improve protein and / or salt dispersion; and provide or increase retention of blend properties, such as desired optical and mechanical properties.

8. A composition of any of claims 1-7, wherein the protein has increased thermal stability at 100-215°C , including 155°C, 185°C, 200°C and 215°C.

9. A composition of any of claims 1-7, wherein the protein has thermal stability at 100-215°C, including 155°C, 185°C, 200°C and 215°C, for a minimum of 5 minutes of residence time at the temperature.

10. A composition of any of claims 1-7, wherein the salt is present at 0.05%-30wt% or 2-20wt%1 1. A composition of any of claims 1-7, wherein the protein is present at 0.2-25% or 0.5-10wt%, including 2%, 5%, 10% and 20%.

12. A composition of any of claims 1-7, wherein the protein has increased thermal stability at 100-215°C, including 155°C, 185°C, 200°C and 215°C; the protein has thermal stability at 100-215°C, including 155°C, 185°C, 200°C and 215°C, for a minimum of 5 minutes of residence time at the temperature; the salt is present at 0.05%-30wt% or 2-20wt%; and the protein is present at 0.2-25% or 0.5-10wt%, including 2%, 5%, 10% and 20%.

13. A composition of claim 12, wherein the plastic comprises Polyethylene Terephthalate (PET or PETE), High-Density Polyethylene (HDPE), Low-Density Polyethylene (LDPE), Polyvinyl Chloride (PVC or Vinyl), Polypropylene (PP), Polystyrene (PS), polyethylene oxide (PEO) I Polyethylene glycol (PEG), Poly(L-lactic acid) (PLLA), Polylactic acid (PLA), Polycaprolactone (PCL), Polymethylmethacrylate (PMMA) and / or a Polyhydroxyalkanoate (PH A).

14. A composition of claim 12, wherein the salt is selected from magnesium, calcium, potassium, sodium, zinc, ammonium, and lanthanide salts, including sulfates, acetates, chlorides, nitrates, etc.

15. A composition of claim 12, wherein the salt is selected from MgSCL , Ca(CH3COO)2 Na2SC>4, ZnSCL. MgCL, MgiNChh. NaCl and KC1, and ammonium nitrate and nitrates of Eu, Nd, and Dy, and preferably MgSCL.

16. A composition of claim 12, wherein the salt is MgSCL.

17. A composition of claim 12, wherein the protein is an enzyme, such as a protease, lipase, amylase, pullulanase, pectinase, peroxidase, xylase, laccase, transgluaminase, phytase, hydrolase, transferase, redox enzyme, lyase, isomerase, ligase, or xylanase.

18. A composition of claim 12, wherein the plastic comprises Polyethylene Terephthalate (PET or PETE), High-Density Polyethylene (HDPE), Low-Density Polyethylene (LDPE), Polyvinyl Chloride (PVC or Vinyl), Polypropylene (PP), Polystyrene (PS), polyethylene oxide (PEO) / Polyethylene glycol (PEG), Poly(L-lactic acid) (PLLA), Polylactic acid (PLA), Polycaprolactone (PCL), Polymethylmethacrylate (PMMA) and / or a Polyhydroxyalkanoate (PH A); the salt is selected from MgSO4, Ca(CH3COO)2 and KC1; and the protein is an enzyme, such as a protease, lipase, amylase, pullulanase, pectinase, xylase, laccase, transgluaminase, phytase, peroxidase, hydrolase, transferase, redox enzyme, lyase, isomerase, ligase, or xylanase.

19. A method of making a composition of claim 1, comprising embedding the protein in the thermoplastic, together with the salt, thermally annealing the composition at elevated temperature, and cooling down the composition before detecting activity of the protein, wherein the method may further comprise: lyophilizing the protein and the salt together prior to embedding, incubating the composition under conditions wherein the protein is demonstrably active, such as an enzyme protein degrading the polymer matrix, and / or detecting the activity of the protein.

20. A method of using a composition of claim 1, preserving activity of the embedded protein at high temperatures for downstream / subsequent functional use, such as by biocatalysis of small molecules external to embedded matrix as well as biocatalysis on the matrix itself (plastic degradation).

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