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Near-complete depolymerization of polyesters with nano-dispersed enzymes

Nature volume 592, pages 558–563 (2021)Cite this article

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Abstract

Successfully interfacing enzymes and biomachinery with polymers affords on-demand modification and/or programmable degradation during the manufacture, utilization and disposal of plastics, but requires controlled biocatalysis in solid matrices with macromolecular substrates1,2,3,4,5,6,7. Embedding enzyme microparticles speeds up polyester degradation, but compromises host properties and unintentionally accelerates the formation of microplastics with partial polymer degradation6,8,9. Here we show that by nanoscopically dispersing enzymes with deep active sites, semi-crystalline polyesters can be degraded primarily via chain-end-mediated processive depolymerization with programmable latency and material integrity, akin to polyadenylation-induced messenger RNA decay10. It is also feasible to achieve processivity with enzymes that have surface-exposed active sites by engineering enzyme–protectant–polymer complexes. Poly(caprolactone) and poly(lactic acid) containing less than 2 weight per cent enzymes are depolymerized in days, with up to 98 per cent polymer-to-small-molecule conversion in standard soil composts and household tap water, completely eliminating current needs to separate and landfill their products in compost facilities. Furthermore, oxidases embedded in polyolefins retain their activities. However, hydrocarbon polymers do not closely associate with enzymes, as their polyester counterparts do, and the reactive radicals that are generated cannot chemically modify the macromolecular host. This study provides molecular guidance towards enzyme–polymer pairing and the selection of enzyme protectants to modulate substrate selectivity and optimize biocatalytic pathways. The results also highlight the need for in-depth research in solid-state enzymology, especially in multi-step enzymatic cascades, to tackle chemically dormant substrates without creating secondary environmental contamination and/or biosafety concerns.

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Fig. 1: Biocatalysis with embedded enzyme for polymer degradation.
Fig. 2: Characterization and degradation of PCL-RHP-BC-lipase.
Fig. 3: Embedded BC–lipase depolymerizes polyesters via chain end-mediated processive degradation.
Fig. 4: Enzyme protectants (RHPs) associate with the embedded enzyme to retain activity during melt processing and thermal treatment to program degradation.

Data availability

All data are available in the main text or the Supplementary Information. Additional requests can be made to the corresponding author.

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Acknowledgements

This work was primarily supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, Materials Sciences and Engineering Division (DOE-BES-MSE) under contract DE-AC02-05-CH11231, through the Organic−Inorganic Nanocomposites KC3104 programme (enzyme–plastic composite design and degradation mechanism; C.D., L.M., A.H., I.J., R.O.R., T.X.). Y.J. and T.P.R. acknowledge support from DOE-BES-MSE under the same contract number, through the Adaptive Interfacial Assemblies Towards Structuring Liquids KCTR16 programme (tensiometry studies on RHP/enzyme/polymer complexation). C.D.S. acknowledges Laboratory Directed Research and Development funding from Berkeley Lab (integration with organic waste infrastructure). The US Department of Defense, Army Research Office supported Z.R. (lipase stabilization) under contract W911NF-13-1-0232. C.D. was partially supported by a National Defense Science and Engineering Graduate (NDSEG) Fellowship (Bioremediation). T.X. also acknowledges support from a Bakar Fellowship at University of California Berkeley (compost tests). Scattering studies at the Advanced Light Source were supported by the US Department of Energy, Office of Science, Office of Basic Energy Science under contract DE-AC02-05CH11231.

Author information

Authors and Affiliations

  1. Department of Materials Science and Engineering, University of California, Berkeley, Berkeley, CA, USA

    Christopher DelRe, Yufeng Jiang, Aaron Hall, Ivan Jayapurna, Zhiyuan Ruan, Le Ma, Kyle Zolkin, Tim Li, Robert O. Ritchie & Ting Xu

  2. Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Christopher DelRe, Yufeng Jiang, Junpyo Kwon, Le Ma, Robert O. Ritchie, Thomas P. Russell & Ting Xu

  3. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    Philjun Kang & Ting Xu

  4. Department of Mechanical Engineering, University of California, Berkeley, Berkeley, CA, USA

    Junpyo Kwon & Robert O. Ritchie

  5. Energy Analysis & Environmental Impacts Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Corinne D. Scown

  6. Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA, USA

    Thomas P. Russell

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  1. Christopher DelRe
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Contributions

T.X. conceived the idea and guided the project. C.D. and T.X. analysed degradation rate and by-product experiments, material characterization experiments, and determined the different enzyme mechanisms. C.D., Y.J., T.P.R. and T.X. analysed interfacial-tension data. C.D., P.K. and T.X. analysed the diblock degradation experiments. J.K. and R.O.R. analysed mechanical properties. P.K., Z.R. and A.H. synthesized and characterized the RHPs. I.J. analysed RHPs and enzyme surfaces and repeated the analysis of the degradation results. L.M. performed electron microscopy experiments. A.H. carried out repolymerization experiments. K.Z. and T.L. assisted with degradation-rate quantification and enzyme active-site analysis. C.D.S. provided insights on integration with organic-waste infrastructure.

Corresponding author

Correspondence to Ting Xu.

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Competing interests

T.X., C.D. and J.K. have filed a PCT patent application. A.H. is the founder and CEO of Intropic Materials.

Additional information

Peer review information Nature thanks Jayati Ray Dutta, Mattheos Koffas and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Characterization of enzyme-embedded PCL.

a, DLS results for RHP and purified BC–lipase in toluene (the solvent used to cast PCL) with an average hydrodynamic diameter of 285 nm ± 35 nm (n = 5) (the error indicates standard deviation). b, DSC results for PCL and PCL–RHP–BC-lipase as-cast films. c, SAXS curves of PCL and PCL–RHP–BC–lipase as-cast films.

Extended Data Fig. 2 PCL–RHP–BC–lipase by-product analysis.

Liquid chromatogram of the degradation by-products for degradation by confined and dissolved (surface erosion) BC–lipase.

Extended Data Fig. 3 Degradation by confined CA–lipase with shallow active site.

a, GPC curve of the degradation of PCL–RHP–CA–lipase, showing a shift and broadening of the main peak, indicative of random chain scission. b, Zoomed-in version of a illustrating the peak shift and broadening.

Extended Data Fig. 4 Enzyme environment dictates biocatalytic reaction kinetics.

a, PCL degradation by BC–lipase dissolved in solution (surface), nanoscopically embedded in PCL with RHP, and embedded with Tween 80, a small-molecule surfactant, as microparticles. b, Hydrolysis of 4-nitrophenyl butyrate, a small-molecule ester, by BC–lipase in solution or confined in PCL (error bars represent one standard deviation; n ≥ 3).

Extended Data Fig. 5 Model interfacial-tension experiment to explain intermolecular interactions among enzyme, protectant and matrix.

a–c, When all three components are initially mixed in toluene (a, left) and then a water interface is introduced (a, right), RHP–lipase complexes immediately interact with PCL at the interface, as shown by the fluorescence microscopy image taken ~20 s after shaking the vial to produce an emulsion (b) and the long delay time in interfacial-tension reduction that is seen only for PCL–RHP–lipase (c).

Extended Data Fig. 6 Characterization of semi-crystalline properties of melt-processed PCL–RHP–BC–lipase.

a, DSC curves of PCL–RHP–BC–lipase with different recrystallization conditions (the film with recrystallization temperature Tc = 49 °C has a crystallinity of 41% ± 1.2% compared to 39% ± 1.8% for the as-cast film). The increase in melting temperature from ~58 °C to ~64 °C indicates a substantial thickening in crystalline lamellae for Tc = 49 °C films, which was confirmed by SAXS. b, SAXS profiles of as-cast and Tc = 49 °C films of PCL–RHP–BC–lipase. The increase of long periods (shift to lower q), combined with the negligible difference in the bulk per cent crystallinity according to DSC data, confirms a thickening in crystalline lamellae after crystallizing at Tc = 49 °C.

Extended Data Fig. 7 Confirming enzyme does not denature at high temperatures.

Small-molecule ester hydrolysis by embedded BC–lipase as a function of temperature (red), overlaid with the PCL–RHP–BC–lipase degradation rate. The small-molecule activity remained high at 60 °C but was not quantified because the film shrivelled owing to melting, and thus was much thicker than films at lower temperatures, making quantification incomparable to all other temperatures.

Extended Data Fig. 8 Quantifying segmental hydrophobicity of different RHPs.

a, Hydropathy plots for RHPs with 60:10 MMA:EHMA composition. b, Hydropathy plots for RHPs with 50:20 MMA:EHMA composition. c, Hydropathy plots for RHPs with 20:50 MMA:EHMA composition. d, Average segmental HLB value for each RHP composition. Error bars indicate standard deviation, n ≥ 3.

Extended Data Fig. 9 Characterizing embedded enzymes for more commercially relevant plastics.

a, Crystal structure of proteinase K with the same colour-coding scheme as that used for lipases in the main text (Fig. 3). b, GPC curve of PLA–RHP–proteinase K (‘ProK”) as cast and after depolymerizing in buffer; c, Interfacial-tensiometry experiment results for a DCM–water interface with PLA, RHP and proteinase K in the DCM phase. d, Photograph of ABTS small-molecule assay in malonate buffer after ~10 min, demonstrating that laccase embedded in polystyrene (PS) retains the ability to oxidize a small molecule. Similar results were found for manganese peroxidase and for both enzymes embedded in polyethylene. e, Interfacial-tensiometry experiment results for a toluene–water interface with PS, RHP and either laccase or manganese peroxidase (‘MnP’) in the toluene phase.

Extended Data Table 1 Summary of all polymer–enzyme blends employed in the study
Full size table

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DelRe, C., Jiang, Y., Kang, P. et al. Near-complete depolymerization of polyesters with nano-dispersed enzymes. Nature 592, 558–563 (2021). https://doi.org/10.1038/s41586-021-03408-3

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