Harnessing Colloidal Dispersion for Laccase‐Driven Enzymatic Depolymerization of Polystyrene

Abstract

Polystyrene (PS) is one of the most widely used synthetic polymers, with annual global production of around 20 million tons. However, its robust C─C backbone renders it highly recalcitrant to (bio)chemical depolymerization, and no sustainable re‐/up‐cycling method has yet been developed. Here, we establish a proof‐of‐concept for the efficient depolymerization of PS under mild aqueous conditions, using a laccase–mediator system (LMS) composed of Trametes versicolor laccase, 1‐hydroxybenzotriazole (HBT), and ambient oxygen. To overcome substrate accessibility issues, PS is formulated into colloidally stable nanoparticles, promoting interfacial remote biocatalysis. Under such conditions, up to 99.9% decrease in molar mass is achieved from an initial PS of over 2 million g mol⁻¹, synthesized by ab initio free‐radical emulsion polymerization. This colloidal dispersion strategy is also effective for commercial PS and expanded PS waste processed by post‐dispersion in surfactant‐containing aqueous media. Mechanistic studies suggest that LMS‐mediated depolymerization proceeds via HBT radical diffusion into PS nanoparticles, triggering hydrogen atom transfer (HAT)‐based oxidation and β‐scissions of PS chains. This approach provides an efficient method for PS depolymerization using aqueous conditions, ambient O₂ and a native enzyme without harsh solvents or experimental conditions.

Polystyrene (PS), long considered non‐degradable by biocatalytic pathways, can now be broken down under aqueous conditions using atmospheric air and a laccase–mediator system composed of a commercially available fungal enzyme and 1‐hydroxybenzotriazole as small organic mediator. By formulating PS into stable colloidally nanoparticles, we unlock access for biocatalytic attack, leading to a drastic molar mass reduction. This mild, effective method opens new paths for sustainable plastic depolymerization without harsh chemicals or energy‐intensive processes.

Introduction

Plastics are indispensable in our modern way of living, addressing our needs for construction, health, clothing, packaging, transport, communication, energy, etc.^{[ 1} , ^{2 ]} The global production of synthetic polymers forming the basis of plastics amounts to almost 400 million metric tons per year.^{[ 1} , ^{2 ]} Synthetic polymers, mainly derived from fossil resources, pose however a major ecological concern due to their low recycling rate and accumulation in the environment.^{[ 1 ]} The need to design polymeric materials capable of being degraded after usage, while retaining optimal performance in service, is therefore a key issue. In this context, the recycling of plastic waste into their original monomers, or upcycling into functional derivatives with higher added value, has become a hot topic in the perspective of a circular economy for plastics. Current plastic recycling technologies rely primarily on mechanical recycling, which is an efficient and widely used method, particularly for well‐sorted and contaminant‐free polymers like poly(ethylene terephthalate) (PET).^{[ 3} , ^{4 ]} However, repeated processing can lead to material degradation and a reduction in quality over time.^{[ 3} , ^{4 ]} Chemical recycling of robust polymers, such as polyolefins, can be performed by pyrolysis, gasification, or hydrothermal processes, but such processes are far too energy‐demanding.^{[ 4} , ⁵ , ^{6 ]} More selective and much less energy‐intensive chemical recycling technologies are therefore highly sought for. Recent advances have introduced strategies in order to recycle or upcycle vinyl polymers following a chemical pathway.^{[ 7} , ⁸ , ⁹ , ^{10 ]} For instance, chemical depolymerization of PS via photoconversion has emerged as a promising route to achieve value‐added products.^{[ 11} , ^{12 ]} While these methods offer exciting possibilities, challenges remain in catalyst design, solvent use, cost, and scalability. On the other hand, substantial efforts have been made over the last 20 years to degrade synthetic plastics following a biocatalytic approach.^{[ 13} , ¹⁴ , ^{15 ]} However, evidence of truly effective enzymatic depolymerization has so far been limited to a very small number of synthetic polymers. These concern polymers containing chemical bonds that are relatively easy to hydrolyze, in particular PET,^{[ 16} , ^{17 ]} and also, to a lesser extent, polylactide (PLA)^{[ 18} , ^{19 ]} and some polyamides (Figure 1).^{[ 13 ]} Non‐hydrolyzable polymers constituted of C─C bonds in their main chain, such as polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), or polystyrene (PS), are still recalcitrant to enzymatic deconstruction (Figure 1). Despite of some encouraging findings,^{[ 13} , ²⁰ , ²¹ , ²² , ^{23 ]} Stepnov et al. have recently reported that several of these findings should be taken with great caution because of major experimental pitfalls.^{[ 24 ]} In the end, this is not so surprising, as nature has not yet developed appropriate biocatalytic tools for this purpose, the accumulation of plastics in the environment being quite recent (< 80 years). Here, we tackle the challenge of the enzymatic depolymerization of plastic by focusing on one of the most common polymers, namely, PS. PS reaches a global annual production of ca. 20 million tons.^{[ 2 ]} Due to their remarkable properties,^{[ 25 ]} including lightness, impact resistance, or insulating properties, PS‐based materials are ubiquitous in a broad range of applications, for example, in sound and heat insulation materials for the building industry, or in food packaging. Only a handful of studies have reported the action of microorganisms on PS, such as mealworms’ gut microorganisms,^{[ 22} , ^{26 ]} bacteria,^{[ 22} , ^{27 ]} fungi,^{[ 28} , ²⁹ , ³⁰ , ³¹ , ^{32 ]} and enzymes^{[ 33} , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ^{38 ]} (Table S1), although often with no compelling experimental evidence. Efficient biocatalytic systems for degrading PS are thus still lacking. Given the hydrophobic nature of PS, we hypothesized, based on the known challenges of enzyme access to non‐hydrolyzable polymers,^{[ 33} , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ^{38 ]} that its efficient enzymatic depolymerization could be facilitated by implementing an interfacial mechanism. Another challenge was to select an enzymatic system that would be of sufficiently high redox potential to oxidize the constitutive C─H and C─C bonds of PS. In this context, since filamentous fungi have evolved over millions of years to deconstruct recalcitrant naturally occurring polymers,^{[ 39 ]} we sought to harness their potential as source of powerful biocatalysts to target synthetic PS. Based on these working hypotheses, here we show that a laccase–mediator system (LMS, Figure 2a), namely, the well‐known fungal laccase from Trametes versicolor, used in conjunction with a redox mediator and molecular oxygen, enables to unlock the enzymatic depolymerization of PS, provided that the polymer is formulated into colloidally stable nanoparticles (Figure 1). PS chains were thus arranged into submicron‐sized nanoparticles by a user‐ and environmentally‐friendly PS colloidal dispersion process in aqueous media using a surfactant, in order to promote its interfacing with the LMS. We refer to the proposed system as “interfacial remote biocatalysis”, where laccase indirectly acts through a redox mediator, to distinguish it from interfacial on‐site biocatalysis in which enzymes act directly on the polymer.^{[ 13} , ¹⁶ , ¹⁷ , ¹⁸ , ^{19 ]} We demonstrate that, under these conditions, an initial PS‐based latex synthesized by ab initio free‐radical polymerization (FRP) in emulsion undergoes an unprecedented enzymatic depolymerization. A systematic study was carried out to better rationalize the effect of a number of reaction parameters, including pH, laccase, and redox mediator concentrations, aeration, particle size, or nature of the redox mediator. We further successfully extended this colloidal dispersion strategy to commercial PS samples, including PS waste. All these experimental results finally allowed us to propose an oxidative depolymerization mechanism.

Figure 1.

Overview of enzymatic depolymerization of C─X and C─C polymers. For PS, we compare results reported in previous studies^{[ 33} , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ^{38 ]} with the deconstruction of dispersed PS by the LMS (this work) (see also Table S1). PE: polyethylene; PP: polypropylene; PS: polystyrene; PVC: polyvinyl chloride; HBT: 1‐hydroxybenzotriazole.

Figure 2.

Preliminary experiments evidencing biocatalytic depolymerization of a PS‐based latex by the LMS. a) Simplified reaction mechanism involving the laccase–mediator system (LMS) with 1‐hydroxybenzotriazole (HBT) as mediator and a substrate (here PS). b) PS synthesis by FRP in emulsion. ACPA: 4,4′‐azobis(4‐cyanopentanoic acid); SDS: sodium dodecyl sulfate. c) Schematic representation and transmission electron microscopy (TEM) of the PS latex. d) Enzymatic depolymerization of different PS‐based samples arranged in different forms (latex, powder, film, and biphasic mixture); on the left: pictures of the different reaction systems; in the middle: SEC traces of PS samples after enzymatic treatment; on the right: M _p values of PS after enzymatic treatment; the PS latex was synthesized in 0.5 M sodium phosphate buffer (NaP; pH 6.0) and its initial M _p value was 2 648 000 ± 73 000 g mol⁻¹. PS film and powder were obtained from the PS latex (see Experimental Section). The reaction mixtures containing PS (10 mg mL⁻¹) in sodium phosphate buffer (50 mM, pH 6.0) or in the organic phase for the biphasic systems, and varying additional compounds (see below) were incubated at 40 °C and 200 rpm, under atmospheric air, during 48 h. The varying compounds (indicated in between brackets in the figure), added alone or combined, included: the laccase (2.1 U mL⁻¹), CuSO₄ (10 µM) and HBT (5 mM, 1% v/v residual DMSO). Bars show average values and error bars show standard deviations (n ≥ 3 independent biological replicates); SEC traces represent the average of these replicates. dRI: differential refractive index; M _p: molar mass at peak maximum; Lac: laccase. See also Supporting Information Table S3 for all M _n, M _p, and M _w values and associated reaction conditions.

Results and Discussion

Proof of Concept: Synthesizing PS Latexes Greatly Facilitates Their Enzymatic Depolymerization

In previous studies, PS (Table S1)^{[ 33} , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ^{38 ]} or other recalcitrant vinyl‐based plastics were most often used in the form of powders, films, or membranes, or simply dissolved in an organic solvent, thereby limiting the interaction of the hydrophobic polymer with the enzymatic bio‐catalyst. We thus sought to find a simple way of solving this problem and facilitate the interaction of PS with the LMS. To this end, we dispersed PS chains as nanoparticles in aqueous conditions, i.e., in the form of colloidally stable latex using a surfactant.

Such polymer latexes are widely used in coating applications, like paints or adhesives, due to their unique combination of liquid‐like processability and solid‐like film‐forming properties after drying.^{[ 40 ]} Although FRP of styrene in emulsion and the use of LMS may appear, taken individually, to be trivial systems, we show that the integration of these two methods into a single process has made PS tractable to enzymatic depolymerization. As proof of concept, we first synthesized a PS‐based latex by ab initio FRP in emulsion following a standard procedure.^{[ 41 ]} Briefly, FRP was performed at 70 °C using sodium dodecyl sulphate (SDS) as surfactant and 4,4′‐azobis(4‐cyanopentanoic acid) (ACPA) as initiator, in presence of phosphate buffer at pH 6.0 (Figure 2b,c). The resulting latex proved stable over months, and its analysis by transmission electron microscopy (TEM) indicated the formation of nanoparticles with diameters of around 99 ± 9 nm and a narrow size distribution, while characterization by size exclusion chromatography (SEC) of the PS obtained showed that the PS was of very high molar mass (M _n = 754 000 ± 58 000 g mol⁻¹; M _p = 2 648 000 ± 72 000 g mol⁻¹; Đ = 3.1 ± 0.3; Table S2 entry 1).

The LMS, with the laccase from Trametes versicolor and 1‐hydroxybenzotriazole (HBT, 1) as mediator, was then selected due to its capability of generating mediator radical with high redox potential.^{[ 42} , ^{43 ]} This enzymatic catalytic system was thus expected to oxidize the constitutive C─H and C─C bonds of PS (Figure 2a). Therefore, the PS latex (10 mg mL⁻¹) was incubated as such in the presence of laccase (2.1 U mL⁻¹) and HBT (5 mM) at 40 °C and 200 rpm for 48 h, in presence of O₂ (air atmosphere). Under such reference conditions, we noted a drastic decrease of the M _p value, from 2 648 000 ± 73 000 g mol⁻¹ to 80 000 ± 5000 g mol⁻¹, i.e., a molar mass decrease of 97% (Figure 2d and Table S3). By comparison, using PS samples in the form of powder or film, or a biphasic mixture of an aqueous phase and an organic solvent (ethyl acetate or dichloromethane) to solubilize the PS, led to much more limited depolymerization after enzymatic treatment with the same LMS. Indeed, only a slight shift in the M _p value (final M _p around 2 580 000–2 200 000 g mol⁻¹) was observed (Figure 2d and Table S3). Control experiments then showed that both the laccase and the HBT mediator were necessary, as no effect was observed when using laccase or HBT alone (Figure 2d and Table S3). A similar observation was made after purification of the commercial laccase batch, so that depolymerization could not be ascribed to the presence of an impurity (Figure S1). Moreover, PS chains remained unaffected when CuSO₄ was used in place of the laccase, ruling out a possible catalysis by copper that would be released from the enzyme (Figure 2d and Table S3). These preliminary results underscore that formulation of PS in latex form unlocks its enzymatic depolymerization, resulting in unprecedented molar mass reductions of up to 97%, far exceeding previously reported values (<20%, Table S1).^{[ 33} , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ^{38 ]}

Optimizing the Biocatalytic Depolymerization of PS‐Based Latexes by LMS

To gain deeper insight into the parameters influencing PS depolymerization, a systematic optimization study was conducted and is discussed in detail in the Supporting Information. Notably, we examined the physicochemical features of PS latexes synthesized ab initio by FRP and the conditions of the LMS. We found that the enzymatic treatment consistently yielded a residual molar mass plateau around 80–90 000 g mol⁻¹, independent of the initial molar mass or latex particle size (Table S2 Parts A–C and Figure S2). Importantly, the composition of the polymerization medium ‐particularly phosphate buffer versus water‐ had a marked influence on the reaction outcomes. Thus, direct synthesis of PS latex in concentrated phosphate buffer was found to enhance the enzymatic depolymerization (Figure S3 and Table S2 Part B for more details). In order to pinpoint the rate‐limiting factors of the enzymatic system, we also investigated the role of the main reaction parameters, including reaction time (Figure S4), nature of the mediator (Figure S5), pH (Figure S6), oxygen availability (Figures S7–S9), temperature (Figure S10), as well as concentrations of the PS (Figure S11), laccase (Figure S12), and HBT (Figures S13, S14). For instance, a time‐course monitoring revealed that most of the reaction took place within the first 24 h, while no drastic change occurred between 2 and 10 days of reaction, with a rather small decrease in the M _p value, from 85 000 to 70 000 g mol⁻¹ (Figure S4). Moreover, among a broad panel of 15 mediators, HBT proved the more effective (Supporting Information and Figure S5) with optimal activity observed at pH 5.0–6.0 (see Supporting Information and Figure S6). In addition, increasing the amount of HBT to 30 mM (2% v/v residual DMSO), hereafter referred to as optimized conditions, improved a little further the PS depolymerization (final M _p = 51 000 g mol⁻¹, M _p decrease of 98.1%, see Supporting Information and Figure S14).

Importantly, a steep decrease in molar mass was systematically observed after the first LMS treatment. However, as inactivation of the enzyme and/or HBT may occur at some stage, we envisaged successive additions of laccase and/or HBT. Repeated additions of HBT or laccase alone led to only modest improvements in PS depolymerization (Figures 3a and S15, S16). Reaction ceased after the third HBT addition, consistent with progressive enzyme inactivation. Independent stability assays confirmed a rapid loss of laccase activity over time, particularly in the presence of HBT (Figure S17). In contrast, successive additions of both laccase and HBT enabled molar mass reductions up to 98.9%, corresponding to a decrease of the M _p value down to 28 000 g mol⁻¹ after 7–8 additions (Figure 3). Notably, these conditions led to nanoparticle aggregation, as observed by DLS and TEM analyses (Figure S18 and Table S4), a hallmark of extensive depolymerization.

Figure 3.

LMS‐mediated depolymerization of the PS synthesized by FRP in emulsion upon multiple additions of the laccase and/or HBT. The graphs show: a) M _p values, as determined by SEC, as a function of the number of additions of laccase and/or HBT; b) SEC traces upon treating the PS latex by the LMS, as a function of the number of additions of laccase and HBT. In the initial stage, the reaction mixtures containing the PS (10 mg mL⁻¹) in sodium phosphate buffer (50 mM, pH 6.0), laccase (2.1 U mL⁻¹) and HBT (5 mM; 1% v/v DMSO residual) were incubated at 40 °C and 200 rpm, under atmospheric air, for up to 23 days. For multiple additions of HBT alone or laccase alone, a PS latex with an initial M _p value of 2 194 000 g mol⁻¹ was used; each data point represents an individual experiment. Laccase (25 µL from a 42 U mL⁻¹ stock solution) or HBT (0.5 µL from a 5 M stock solution in DMSO) was added each day during working days, for a total of 8 successive additions in 10 days (multiple additions of HBT only) or of 9 additions in 13 days (multiple additions of laccase only). For multiple additions of laccase and HBT, a PS latex with an initial M _p value of 2 648 000 g mol⁻¹ was used and the experiments were carried out at an initial volume of 5 mL. Laccase (250 µL from a 42 U mL⁻¹ stock solution) and HBT (5 µL from a 5 M stock solution in DMSO) were added once a day, during working days, for a total of 17 additions in 23 days. Samples of 100 µL were taken to monitor the reaction. add: additions. d: days.

Biocatalytic Depolymerization of PS Latexes Prepared by Post‐Dispersion

Having established that PS‐based latexes synthesized under ab initio emulsion conditions can be enzymatically degraded by LMS, we investigated whether commercial PS films or powders, including waste, could be biocatalytically degraded by shaping them into latex form, i.e., by post‐dispersion in aqueous conditions. First, a PS film obtained by drying an ab initio emulsion of FRP‐synthesized latex was redispersed in aqueous conditions, using SDS as surfactant and dichloromethane as organic co‐solvent (Figure 4a). Such formulation, however, resulted in rather high particle size heterogeneity, as evidenced by DLS and TEM analyses (Figure 4b right sight and S19 and Table S5) compared to the initial latex. Yet, when submitting this redispersed PS to the LMS under our reference conditions (Figure 4b and Table S6), a drop of the molar mass by a factor 3 was observed (from M _p = 1 944 000 to 537 000 and to 615 000 g mol⁻¹ for the redispersed PS in phosphate buffer and in water, respectively). These results show that the post‐dispersion process enables a rather efficient biocatalytic depolymerization, although to a lesser extent than with the pristine PS latex (Figure 4b and Table S6). Then, a commercial PS was also shaped into a colloidal dispersion following the same process (Figure S20 for TEM images), which allowed reducing its molar mass by 20% (from M _p = 220 000 to 177 000 g mol⁻¹ for a PS dispersed in phosphate buffer or water; Figure 4c and Table S7). Under the optimized conditions, i.e., using 30 mM HBT and 2% DMSO, the M _p value was further reduced to 170 000 g mol⁻¹ (Figure 4c and Table S7). Successive additions of both laccase and HBT (24 additions over 20.5 days) enabled to reach a M _p value of 140 000 g mol⁻¹, though with a pronounced broadening of the SEC trace toward lower molar masses (molar mass decrease of 71%, 36% and 43% for M _n, M _p, and M _w, respectively; Figure S21). The same colloidal dispersion process was then leveraged to degrade an expanded PS waste (Figure S22 for TEM images), allowing a molar mass reduction of 8% (Figure 4d and Table S8). Several additions of the LMS (laccase + HBT; 25 additions over 40 days) led to a molar mass reduction of 64%, 25% and 40% for M _n, M _p, and M _w, respectively (Figure S23). DLS and TEM analyses of such post‐dispersed PS revealed great heterogeneity in particle size, regardless of whether it was treated with LMS or not (Figures S24, S25, and Tables S9, S10). This particle size distribution of post‐dispersed PS (from 20 to 2,600 nm as measured by TEM) proved much larger than the ones of PS latex synthesized ab initio in emulsion (from 24 to 116 nm), thus entailing a bigger exchange surface area for the latter that may explain its more efficient biocatalytic depolymerization.

Figure 4.

LMS‐mediated depolymerization of redispersed PS, dispersed commercial PS, and dispersed expanded PS waste. a) Redispersion process in aqueous conditions (see Experimental Section for details), in which a PS film is first prepared from the PS latex, before being redispersed. b) SEC traces of redispersed PS in phosphate buffer before and after incubation with the LMS (left side), and TEM images of redispersed PS (right side). The M _p value of the initial PS latex not incubated was 1 944 000 ± 33 000 g mol⁻¹. c) and d) SEC traces of dispersed commercial PS (panel c) and dispersed expanded PS waste (panel d) before and after incubation with the LMS. M _p values of commercial PS and expanded PS waste before incubation were 220 000 ± 2000 g mol⁻¹ and 182 000 ± 1000 g mol⁻¹, respectively. The reaction mixtures contained PS (10 mg mL⁻¹) in sodium phosphate buffer (50 mM, pH 6.0), laccase (2.1 U mL⁻¹), HBT (5 mM, 1% v/v DMSO residual), styrene (0.5 µL, 8.7 mM, 9% compared to PS repeating unit, when specified) or toluene (0.5 µL, when specified), and were incubated at 40 °C and 200 rpm, under atmospheric air, during 48 h. In experiments marked with a star (*) 30 mM of HBT and 2% (v/v) DMSO were used (optimized conditions). Experiments were done at least in triplicate for (b) and (c) and in duplicate for (d). Lac: laccase. Sty: styrene. Tol: toluene. See also Supporting Information Tables S6 to S8 for all M _n, M _p, and M _w values and associated reaction conditions.

This work echoes with studies performed on natural rubber based on polyisoprene in latex form, against which bacteria have evolved rubber oxygenases that act on C═C bonds.^{[ 44 ]} Interestingly, in the latex globules the polyisoprene core is covered by a phospholipid monolayer (and proteins),^{[ 45 ]} which plays a role somehow analogous to SDS used as surfactant in our work, i.e., as interfacial agent. In a recent contribution, Binder et al. reported that the enzymatic depolymerization of oligoisoprenes of synthetic origin could be improved by formulating these oligomers in the form of latexes in absence of surfactant, but instead using either a co‐solvent (n‐hexadecane) or “acoustic emulsification”.^{[ 46 ]} Formulating high molar mass and hydrophobic polymers such as PS into stable and concentrated surfactant‐free latexes is inherently more challenging than for low molar mass oligomers, owing to their reduced solubility and limited colloidal stability. We therefore employed SDS as a surfactant to ensure the colloidal stability of PS latexes. While the use of a surfactant is deemed as deleterious as it could denature enzymes (e.g., SDS is used in polyacrylamide gel electrophoresis to unfold proteins),^{[ 47} , ^{48 ]} here we show that both LMS and SDS are compatible (up to 6 mM SDS were present in our assays). Their combination even provides a synergetic effect to unlock the enzymatic depolymerization of PS.

Beneficial Effect of Adding Styrene on PS Depolymerization

We noticed that PS latex obtained from FRP in emulsion contained around 4% of residual styrene (Table S2). We thus wondered if the presence of styrene could play a role in the biocatalytic depolymerization of PS. By heating the PS latex, the residual styrene decreased from 4.2% to 1.3% first (Evap1 latex; Table S11), and then to 1.0% (Evap2 latex; Table S11). These conditions proved less effective, with final M _p values of 236 000 (Evap1) and 432 000 g mol⁻¹ (Evap2) versus 120 000 g mol⁻¹ for the non‐heated initial latex (Figure S26 and Table S11). Intentionally adding styrene (0.5 µL, 8.7 mM, 9% rel. to PS) to Evap1 and Evap2 latexes restored the initial results (final M _p = 123 000 g mol⁻¹; Figure S27 and Table S11). Yet, adding styrene to the initial latex did not further enhance the reduction of PS molar mass (Figure S26 and Table S11).

Considering that redispersed PS, dispersed commercial and expanded PS are devoid of any residual styrene, we thought to test the effect of the addition of styrene (0.5 µL, 8.7 mM, 9% rel. to PS) and showed that the LMS‐mediated depolymerization was indeed much more effective, reaching respective final M _p values of 74 000, 116 000, and 135 000 g mol⁻¹, versus 537 000, 170 000, and 168 000 g mol⁻¹ without added styrene (Figure 4b–d and Tables S6–S8). Adding various amounts of styrene showed that a concentration ranging from 3.5 to 35 mM (i.e., 3.6 to 36% rel. to PS) was the most beneficial (Figure S27). Addition of toluene did not affect PS depolymerization compared to the laccase + HBT system alone (Figure 4c and Table S7), indicating that toluene does not deactivate the enzyme, also ruling out a solvent effect by styrene, and instead suggesting its direct involvement in the reaction mechanism. We also tested two styrene derivatives, namely, 4‐styrene sulfonic acid sodium salt and α‐methylstyrene, as well as oxidized styrene products such as styrene oxide and benzaldehyde. However, only α‐methylstyrene was found effective (Figure S28 and Table S12). As water‐soluble 4‐styrene sulfonic acid had no impact, we concluded that only hydrophobic styrene derivatives had a favorable effect, likely due to their ability to penetrate the PS nanoparticles. Furthermore, the absence of effect of styrene oxide or benzaldehyde suggests that styrene ‐rather than its potential oxidation products‐ is involved in the reaction mechanism (Figure S28 and Table S12). Given the beneficial effect of styrene addition, we next investigated the impact of multiple additions of laccase, HBT, and styrene (Figures 5 and S29–S31). This successive addition process was explored in an optimization context. This strategy did significantly enhance PS degradation, leading to a reduction of M _p of the emulsion‐synthesized PS to 1,787 g mol⁻¹, corresponding to a remarkable 99.9% decrease for M _p (99.4% and 99.6% molar mass decrease for M _n and M _w, respectively; Figure 5). For dispersed commercial PS and expanded PS waste, depolymerization was also more pronounced, as evidenced by the broadening of the SEC trace toward lower molar masses and the emergence of multiple PS populations, resulting in an M _n decrease of approximately 90% (Figures S30, S31). However, repeated additions of laccase, HBT, and styrene led to progressive latex destabilization, with slow PS precipitation over time. This was accompanied by an increase in particle size, as evidenced by DLS and TEM analyses, and particle coalescence, particularly in emulsion‐synthesized PS (Figure S32 and Table S13). Enhancing latex stability may therefore be a key factor for further improvement and warrants additional investigation. For PS latexes obtained by a post‐dispersion process, a more significant reduction in M _n value compared to M _w was systematically observed by considering multiple addition of laccase, HBT, and ± styrene, thus causing an increase in dispersity. This suggests that LMS activity is less effective at degrading the highest molar mass chains in latexes formulated under post‐dispersion conditions, greatly contrasting with latexes synthesized ab initio, where LMS appears to act more uniformly.

Figure 5.

LMS‐mediated depolymerization of PS synthesized by FRP in emulsion upon multiple additions of laccase, HBT, and styrene. The graphs show: a) SEC traces and b) corresponding M _p, M _n, and M _w values after LMS treatment. Full chromatograms are provided in the Supporting Information Figure S29. The PS latex was synthesized in 0.5 M NaP (pH 6.0) and the initial M _p of the PS was 2 194 000 ± 39 000 g mol⁻¹. Experiment was carried out at a volume of 5 mL. In the initial stage, the reaction mixture containing the PS (10 mg mL⁻¹) in sodium phosphate buffer (50 mM, pH 6.0), laccase (2.1 U mL⁻¹) and HBT (5 mM; 1% v/v DMSO residual) was incubated at 40 °C and 200 rpm, under atmospheric air, for 21 days. Laccase (250 µL from a 42 U mL⁻¹ stock solution), HBT (5 mM, 5 µL from a 5 M stock solution in DMSO) and styrene (5 mM, 5 µL from a 5 M stock solution in DMSO) were added twice a day during working days for a total of 26 additions in 21 days. Samples of 100 µL were taken to monitor the reaction. No styrene was added during the first addition as the latex already contained residual styrene. add: additions. d: days.

Mechanistic Insight Through Pull‐Down Assays and Characterization of Depolymerization Products

To further elucidate the mechanism underlying LMS‐mediated depolymerization of PS, we examined the localization of the reagents in the heterogenous mixture and also analyzed the resulting low molar mass PS‐based species. A pull‐down assay revealed that laccase predominantly remained in solution (> 99%) rather than absorbing onto or being internalized by the PS nanoparticles, regardless of the PS concentration (Figure S33). After separating the PS from the aqueous phase by ultrafiltration, HBT quantification next showed that its concentration decreased with increasing PS levels, but only in the presence of laccase (Figure S34). This result suggests that the oxidized HBT interacts with the PS nanoparticles.

We then performed ¹H NMR analyses of solubilized products from LMS‐treated emulsion‐synthesized PS, redispersed commercial PS and expanded PS, under both reference conditions and repeated LMS (± styrene) conditions (Figures S35–S37). Benzaldehyde and other unidentified compounds were detected, except in commercial and expanded PS under reference conditions. Due to complex NMR spectra, we used GC‐MS and GC‐FID for further analyses (Figures S38, S39), identifying benzaldehyde, acetophenone, benzoic acid, and benzotriazole (a degradation product of HBT) all quantified by GC‐FID but in low yields (<8%; Table S14). Since styrene was present in emulsion‐synthesized PS (∼4%) and in repeated LMS + styrene treatments, a control experiment using styrene alone confirms its breakdown into benzaldehyde, acetophenone, and benzoic acid (Figure S40 and Table S14). This suggests that some degradation products also stem from styrene. However, for commercial and expanded PS, as no styrene was present in the reference conditions and with multi‐additions of laccase + HBT, the oxidized products (benzaldehyde, acetophenone, and benzoic acid) arise from PS depolymerization. We underscore that the low yields are consistent with SEC data, indicating oligomers as the main end‐products. FTIR analysis of the water‐insoluble PS fraction after treatment under reference and repeated LMS (± styrene) conditions revealed new peaks at 1260, 1529, and 1655–1685 cm⁻¹ potentially corresponding to C─O, C═C, and C═O bonds along with O─H stretches in some cases, indicating oxidative cleavage.^{[ 7 ]} These features were observed in emulsion‐synthesized (Figure S41), commercial (Figure S42), and expanded PS (Figure S43). Control FTIR spectra of SDS, laccase, HBT, benzotriazole, benzaldehyde, acetophenone, benzoic acid, and styrene showed distinct signatures from LMS‐degraded PS (Figure S44), ruling out spectral artifacts from residual compounds such as proteins.^{[ 24 ]} To gain further insight into the depolymerization mechanism, we conducted complementary analyses of the oligomeric PS fraction by NMR and MALDI‐TOF MS. The NMR spectra of LMS‐treated PS displayed additional unshielded aromatic (7.3–8.4 ppm) and aliphatic (2.7–3.4 ppm) resonances, consistent with protons in α‐position to carbonyl groups (Figures S45–S47). MALDI‐TOF MS further revealed the presence of multiple oligomeric populations, including chains terminated by one or two carbonyl groups (Figures S48, S49). These data provide strong evidence for oxidative transformations at PS chain ends, in line with a HAT‐type oxidative mechanism.

Additionally, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of PS samples showed a decrease in thermal stability after LMS treatment (Figure S50 and Table S15). The glass transition temperature (T _g) of PS synthesized in emulsion indeed decreased from 104 to 95 °C after enzymatic treatment, while the degradation temperature for 5% mass loss (T _d5%) decreased from 360 to 256 °C. Similarly, T _g and T _d5% respectively decreased from 107 to 97 °C and from 369 to 272 °C for dispersed commercial PS, and from 105 to 99 °C and from 341 to 309 °C for dispersed expanded PS waste. These results are fully consistent with biocatalytic depolymerization causing a drop in molar mass, as revealed by the SEC analyses discussed above.

From a mechanistic perspective, the above results provide several insights. First, they demonstrate that the redox potential of the selected LMS is high enough to oxidize the C─H and C─C bonds in PS. Based on experimental data and kinetic analysis (see Supporting Information for more details), we propose the model shown in Figure 6. Laccase oxidizes HBT in aqueous conditions to form a radical species, which diffuses into PS nanoparticles and initiates PS chain oxidation. We postulate that styrene can participate in electron transfer or radical formation via its conjugated π‐system. Under initial rate conditions, PS depolymerization rate (ca. 10 µM.h⁻¹) is 2–3 orders of magnitude slower than that of HBT oxidation rate (ca. 10³–10⁴ µM h⁻¹), suggesting rate‐limitation by mediator diffusion, or by C─H activation/C─C scission reactions in PS. We postulate that depolymerization proceeds via a HAT mechanism, forming alkyl PS‐derived radicals that react with O₂ to induce β‐scissions of PS chains.^{[ 49 ]} The presence of α‐keto functionalities at the oligomer chain ends support the proposed oxidative mechanism. Ultimately, successive cleavages yield small molecules, such as benzaldehyde, benzoic acid, or acetophenone, which were detected in low amounts by GC, providing direct evidence for the proposed oxidation mechanism and enhancing our understanding of enzymatic PS depolymerization. Benzoic acid would arise from oxidation of the benzylic radical, whereas the primary alkyl radical could abstract a hydrogen atom, forming a PS with a methyl end group, the subsequent oxidation of which would lead to the formation of acetophenone. As noted, however, PS depolymerization remains incomplete, limiting the production of value‐added products. One possibility is that low‐molar mass PS chains confined in hydrophobic nanoparticles are less accessible to LMS, which likely resides at the aqueous‐nanoparticle interface. Additionally, these shorter chains may interact less effectively with the hydrophobic mediator, reducing HAT efficiency. Differences in chain accessibility and the chemical nature of PS chain ends may also affect oxidation susceptibility, potentially making some oligomers more resistant. Further investigation is needed to fully understand these limitations and achieve complete depolymerization to small value‐added products. A seemingly intriguing aspect of this biocatalytic depolymerization process is the conservation of a Gaussian distribution of molar masses over the depolymerization kinetics for PS latexes synthesized ab initio by FRP in emulsion, suggesting random homolytic scissions of inner bonds, rather than chain‐end scissions.^{[ 50} , ^{51 ]} The situation is different, however, for latexes obtained by post‐dispersion, where longer chains appear to be more difficult to cleave than shorter ones. In this case, the distribution of chain size, i.e., dispersity, widens significantly after LMS treatment, with a clearer decrease in M _n value compared to M _w. The reasons for the reduced efficiency of LMS in breaking down the longest chains in post‐dispersed latexes are not entirely clear at this stage. It could be correlated with the heterogeneity of particle size in the case of post‐dispersion latexes, compared with those synthesized ab initio. Studies are underway to elucidate this phenomenon.

Figure 6.

Proposed oxidative HAT‐type depolymerization mechanism of PS by LMS in the presence of dioxygen. “Med^[ox]” refers to the mediator oxidizing the PS chain, and is either HBT^[ox] itself, or a relay mediator such as a styrene radical. On the right‐hand side, the H atom in blue arises from abstraction by the peroxide radical species (O─O^•) of a H atom of another PS chain.

This work provides a robust proof‐of‐concept for the enzymatic depolymerization of vinyl polymers, demonstrating a paradigm shift in their degradation. We show that PS can be enzymatically degraded in aqueous conditions with an unprecedented molar mass reduction of 97% after one single addition of LMS on non‐pre‐oxidized PS, compared to a maximum of 30% in previous studies using a pre‐oxidized PS (Table S1). For the first time we also quantified high‐value products resulting from this enzymatic depolymerization. While these results are encouraging and highlight the potential for sustainable upcycling of vinyl polymers under mild aqueous conditions (40 °C), the process is not directly attractive for industrial recycling, as the main products are low‐molar‐mass PS oligomers of limited market value. Future work is thus needed to increase the yield of added‐value molecules. The beneficial effect of styrene addition would also need to be replaced by more sustainable compounds. Nevertheless, this study establishes an effective depolymerization strategy that forms a crucial starting point for biocatalytic conversion of vinyl polymers. Ongoing research aims to transform these oligomeric intermediates into higher‐value molecules, such as benzoic acid and benzaldehyde, providing a potential sustainable alternative to conventional methods. Drawing inspiration from a recent work carried out using lytic polysaccharide monooxygenases endowed with plastic binding properties,^{[ 52 ]} we think that protein engineering may enable us to design more suitable chimeric enzymes. Indeed, such enzymes might be more likely to penetrate and/or attach to the nanoparticles, where the PS chains are confined, and could thus allow further improvement of this system. We anticipate that the proposed dual and synergetic method, combining a colloidal dispersion process and use of LMS or analogous systems, could reshape the approach to biocatalytic depolymerization of other hydrophobic polymers, such as polyethylene (PE) and polypropylene (PP). Thus, this strategy paves the way toward new solutions for the biocatalytic depolymerization of recalcitrant synthetic polymers.

Conclusion

We demonstrate that combining a colloidal dispersion strategy with an oxidative laccase–mediator system (LMS) enables efficient biocatalytic depolymerization of polystyrene (PS). By formulating PS into colloidally stable submicron‐sized nanoparticles in aqueous media, we achieve up to 99.9% molar mass reduction, far surpassing previous studies, a result that would be unattainable with bulk PS in powder or film form. Importantly, this strategy is effective not only for emulsion‐synthesized PS but also for commercial and expanded PS waste, with molar mass reductions up to 90%. Control experiments confirm the essential roles of both laccase and the mediator. A number of experiments, including product quantifications, pull‐down assays, and time‐resolved molar mass analysis, allow rationalizing the observed trends and proposing an oxidative depolymerization HAT‐type mechanism of PS, involving β‐scissions and formation of small oxidation products. Chain scission occurs randomly within PS backbone, particularly in emulsion‐synthesized latexes. This biocatalytic depolymerization process proceeds at 40 °C in aqueous conditions, without harsh solvents or conditions, and relies on a native and commercially available enzyme. Overall, this work establishes an effective enzyme‐based approach for PS depolymerization via interfacial remote biocatalysis, i.e., an indirect enzymatic process mediated by laccase/HBT, that integrates enzymology, polymer chemistry, and environmental science, representing a significant step forward toward practical vinyl polymer biodegradation. Future efforts will aim at converting the resulting PS oligomers into higher‐value platform chemicals and bioproducts, thereby opening perspectives for sustainable biocatalytic upcycling of vinyl polymers.

Supporting Information

The authors have cited additional references within the Supporting Information.^{[ 33} , ³⁴ , ³⁵ , ³⁶ , ³⁷ , ³⁸ , ⁵³ , ⁵⁴ , ⁵⁵ , ⁵⁶ , ⁵⁷ , ⁵⁸ , ⁵⁹ , ^{60 ]}

Conflict of Interests

The authors declare no conflict of interest.

Supporting information

Supporting Information

Pujol M., Gonsales S. A., Seksek F., Berrin J.‐G., Bissaro B., Taton D., Angew. Chem. Int. Ed.. 2026, 65, e13937, 10.1002/anie.202513937

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.