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An engineered enzyme embedded into PLA to make self-biodegradable plastic

Nature volume 631, pages 884–890 (2024)Cite this article

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Abstract

Plastic production reached 400 million tons in 2022 (ref. 1), with packaging and single-use plastics accounting for a substantial amount of this2. The resulting waste ends up in landfills, incineration or the environment, contributing to environmental pollution3. Shifting to biodegradable and compostable plastics is increasingly being considered as an efficient waste-management alternative4. Although polylactide (PLA) is the most widely used biosourced polymer5, its biodegradation rate under home-compost and soil conditions remains low6,7,8. Here we present a PLA-based plastic in which an optimized enzyme is embedded to ensure rapid biodegradation and compostability at room temperature, using a scalable industrial process. First, an 80-fold activity enhancement was achieved through structure-based rational engineering of a new hyperthermostable PLA hydrolase. Second, the enzyme was uniformly dispersed within the PLA matrix by means of a masterbatch-based melt extrusion process. The liquid enzyme formulation was incorporated in polycaprolactone, a low-melting-temperature polymer, through melt extrusion at 70 °C, forming an ‘enzymated’ polycaprolactone masterbatch. Masterbatch pellets were integrated into PLA by melt extrusion at 160 °C, producing an enzymated PLA film (0.02% w/w enzyme) that fully disintegrated under home-compost conditions within 20–24 weeks, meeting home-composting standards. The mechanical and degradation properties of the enzymated film were compatible with industrial packaging applications, and they remained intact during long-term storage. This innovative material not only opens new avenues for composters and biomethane production but also provides a feasible industrial solution for PLA degradation.

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Fig. 1: PLA-depolymerizing enzyme PAM outperforms proteinase K.
Fig. 2: Structural insight into the PAM PLAase.
Fig. 3: Improvement of PLA depolymerization activity of PAM by site-saturation mutagenesis.
Fig. 4: Comparison of PLA depolymerization performance of ProteinT and aqualysin-I with that of PAM PLAase.
Fig. 5: Improvement in PLA depolymerization activity of the thermostable ProteinT.
Fig. 6: Improved aqueous biodegradation of enzymated PLA material after incorporation of engineered ProteinTFLTIER compared with proteinase K.
Fig. 7: Disintegration of PLA film under home-compost conditions is triggered by the incorporation of ProteinTFLTIER.

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Data availability

Data supporting the findings of this study are available within the article, its Extended Data and Supplementary Information. The atomic coordinates and structure factors of the reported structures have been deposited in the Protein Data Bank under PDB IDs 8C4X and 8C4Z.

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Acknowledgements

We thank the PICT-ICEO facility of the Toulouse Biotechnology Institute, which is part of the Integrated Screening Platform of Toulouse (PICT, IBiSA), for providing access to HPLC and protein purification equipment; the structural biophysics team of the Institute of Pharmacology and Structural Biology (Toulouse, France) and the PICT platform for access to the crystallization facility, as well as the ALBA (Barcelona, Spain) and European Synchrotron Radiation Facility (Grenoble, France) synchrotrons for data collection; and the CRITT Bioindustries from INSA Toulouse for providing access to their equipment and for their help and expertise. We also thank P. Tsvetkov and F. Devred from the Microcalorimetry Pole in the PINT platform of the Institute of NeuroPhysiopathology (Marseille, France) for the nanoDSF analysis and the staff of the PISSARO proteomic platform (University of Rouen, France) for the N-terminal amino acid analysis; B. Chezeau of Bio-Valo laboratory for anaerobic digestion evaluation; J. Jacquin from the biodegradation and microbiology team of the Industrial Technical Centre for Plastics and Composites of Clermont-Ferrand (Clermont-Ferrand, France) for conducting aerobic biodegradation experiments; D. Thizy, A. Beaugeon and V. Legrand from Carbiolice for their help; L.-A. Chabaud, A. Mathé and N. Panel for help in the conception of the cover art. Finally, we thank Toulouse White Biotechnology (UMS INRAE 1337/UMS CNRS 3582) for administrative support. For this work, we were granted access to high-performance computing resources from the regional computing mesocenter CALMIP and the HPC-Regional Center ROMEO. This work was mainly conducted in the cooperative INSA/Carbios laboratory PoPlaB (Polymers, Plastics and Biotechnology) at the Toulouse Biotechnology Institute and was supported by a grant-in-aid for scientific research (THANAPLAST project, OSEO ISI contract number I 1206040W).

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Author notes

  1. These authors contributed equally: M. Guicherd, M. Ben Khaled, M. Guéroult

Authors and Affiliations

  1. Toulouse Biotechnology Institute, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France

    M. Guicherd, M. Ben Khaled, M. Guéroult, J. Nomme, P. Alvarez, E. Kamionka, S. Gavalda, M. Vuillemin, E. Amillastre, D. Labourdette, G. Cioci, I. André, S. Duquesne & A. Marty

  2. Carbios, Clermont-Ferrand, France

    M. Guicherd, M. Guéroult, M. Dalibey, F. Grimaud, S. Gavalda, V. Tournier & A. Marty

  3. Carbiolice, Clermont-Ferrand, France

    M. Noël

  4. Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand

    V. Kitpreechavanich

  5. Center of Innovation and Research in Materials & Polymers, University of Mons, Mons, Belgium

    P. Dubois

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  1. M. Guicherd
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Contributions

I.A., S.D. and A.M. designed and directed the research. V.K. provided the A. keratinilytica T16-1 strain. P.A. and E.A. discovered and characterized the PAM enzyme from A. keratinilytica T16-1. D.L. analysed the RNA short reads and obtained a DNA sequence for the PAM enzyme. M. Guicherd, E.K. and M.V. characterized the performance of PAM and performed engineering, purification and kinetics analysis of PAM enzymes. M.B.K. characterized the performance of ProteinT and performed engineering, purification and kinetics analysis of ProteinT enzymes. M. Guicherd and E.K. performed comparative assessment of PAM and ProteinT enzymes and characterization of the performance of the best mutants. M. Guéroult, S.D. and I.A. performed sequence analysis enabling identification of ProteinT. M. Guéroult and I.A. carried out molecular modelling studies of PAM and ProteinT. J.N., S.G. and G.C. carried out structural and physical characterization of enzymes. M.D. performed PLA powder preparation and characterization, and prepared masterbatches and enzymated PLA films. M.D. and P.D. conducted scanning electron microscopy analyses and performed mechanical tensile studies of films. F.G. performed biodegradation studies of enzymated PLA films. M.N. conducted film disintegration studies under compost conditions, biodegradation studies under industrial compost conditions and anaerobic digestion of PLA films. M. Guicherd, M.B.K., M. Guéroult, V.T., P.D., I.A., S.D. and A.M. wrote the original draft. All authors reviewed and approved the manuscript.

Corresponding authors

Correspondence to I. André or A. Marty.

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Competing interests

M. Guicherd, M. Guéroult, M.D., F.G., S.G., V.T. and A.M. are employees of Carbios. M.N. is an employee of Carbiolice. P.A., E.A., S.D. and A.M. have filed patent WO 2016/062695 (applicants: Carbios, Institut National de la Recherche Agronomique, Institut National des Sciences Appliquées, Centre National de la Recherche Scientifique; patent application extended in various countries or regions, including Europe, United States, China, India and Japan; covering the wild-type PAM described in the manuscript); A.M. has filed patent WO 2016/198652 (applicant: Carbios; additional inventors: E. Guémard and M. Château; patent application extended in various countries or regions, including Europe, United States, China, India and Japan; covering the production of PLA plastic articles comprising a PLA-degrading enzyme and an antic-acid filler, such as calcium carbonate); M. Guicherd, M.B.K., M.V., S.D. and A.M. have file patent WO 2018/109183 (applicant: Carbios; patent application extended in various countries or regions, including Europe, United States, China, India and Japan; covering the optimized ProteinT for degradation of PLA); M.D. has filed patents WO 2019/043145 (applicant: Carbios; additional inventor: E. Guémard; patent application extended in various countries or regions, including Europe, United States, China, India and Japan; covering the use of a liquid composition of enzyme comprising arabic gum for the production of a masterbatch to be introduced in PLA plastic material) and WO 2019/043134 (applicant: Carbiolice; additional inventor: C. Arnault; patent application extended in various countries or regions, including Europe, United States, China, India and Japan; covering a biodegradable PLA plastic material made from a masterbatch and liquid composition of enzyme and arabic gum); M. Guicherd, M. Guéroult, I.A., S.D. and A.M. have filed patent WO 2019/122308 (applicant: Carbios; patent application extended in various countries or regions, including Europe, United States, China, India and Japan; covering the optimized PAM (including PAMFLI); and M.N. has filed patent WO 2021/148666 (applicant: Carbiolice; patent granted in France; covering the use of a masterbatch to improve the mechanical properties of a PLA article comprising such a masterbatch). Confidentiality agreements prevent authors from disclosing newly submitted patents that are not declared. The other authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Isolation of a novel PLAase from Actinomadura keratinilytica T16-1.

a, Experimental workflow to identify the sequence of the PLA depolymerase isolated from A. keratinilytica T16-1 (see Supplementary Fig. 1 for uncropped protein gel). b, Full nucleotide, and amino-acid sequences. The prepeptide and propeptide are underlined by dotted and solid lines, respectively. The first 24 sequenced N-terminal amino-acid residues on mature peptide are shown in italics. The catalytic triad residues are colored in magenta. Disulfide bond-forming cysteines are indicated in green letters and pairs (DS1 and DS2) are circled, numbered, and associated with black lines.

Extended Data Fig. 2 Comparison of enzymes reported.

a, Enzyme identification with UniprotKB and PDB accession number. Sequence identity matrix comparing the amino acid sequence of the mature form of the PAM protein with Proteinase K, Aqualysin-I and ProteinT generated by clustalO. b, Multiple sequence alignment of the corresponding sequence identity matrix. In red are highlighted catalytic residues D, H and S and in green, residues N and T involved in the oxyanion hole formation. In cyan are shown the seven amino acid residues selected for site-saturation mutagenesis in PAM and their equivalent residue in ProteinT. In orange is shown the residue R166 found to be the most favorable in interaction between PAM-PLA4 and its equivalent residue in ProteinT, Y167.

Extended Data Fig. 3 First contact shell of PAM amino-acid residues with PLA4 oligomer.

a, PAM-PLA4 covalent docking model. The 37 first-layer amino acid residues are shown as grey surface. Catalytic residues are highlighted in magenta surface and sticks and saturated positions in light blue surface and sticks. Covalently docked PLA4 fragment model substrate is represented as light yellow sticks. b, List of 37 amino-acid residues of PAM establishing contacts more than 10% of the time with the PLA4 model substrate along Molecular Dynamics simulations. The Shannon information entropy, H(x), calculated from a multiple sequence alignment with PAM homologs using Sequester software developed in-house, is provided. D40 and H71 catalytic residues are highlighted in magenta. Residues selected for site-saturation mutagenesis, based on conservation frequency (0.4 < H(x) value < 0.7), are highlighted in light purple.

Extended Data Fig. 4 Multilevel screening approach followed for PAM variant selection.

Structure-based engineering of PAM PLAase consisted in four levels of screening of PAM mono-variants before recombinational analysis and the evaluation of performances of two triple variants (PAMFLI and PAMFFI). The 1st level of screening evaluates the ability to form a halo on dispersed PLA submicroparticles at pH 9 and 45 °C of 133 PAM mono-variants resulting from the site-saturation mutagenesis of seven selected amino acid positions. The 2nd level of screening, through liquid PLA depolymerization, evaluates PLA depolymerization specific activity of selected variants at pH 9 and 45 °C. The best variants identified entered in the 3rd level of screening that evaluates PLA depolymerization performances at pH 7.5 and 45 °C. Thermostability assessment performed during the 4th level of screening finalizes the selection of the most promising mono-variants of PAM.

Extended Data Fig. 5 Selection of the improved PAM mono-variants from the site-saturation mutagenesis strategy of seven PAM residues (1st level of multilevel screening).

Boxplot distribution of the relative halo diameter sizes of the independent site-saturation mutagenesis of the seven residues of PAM (S101, S103, T105, T106, G133, E159 and I217) as compared to wild type PAM halo. Halo diameter sizes were evaluated after incubation at 45 °C of agarose-immobilized PLA substrate, buffered at pH 9.0 loaded with protein extracts. Individual boxes were drawn using first and third quartiles of the distribution, the median value is shown as an orange line. Boundaries of the whiskers are based on a 1.5 interquartile range value and outliers are shown (open circle). The variant selection threshold was set at 100% of the halo diameter of the PAM reference when the median value of the distribution was lower than 100% but was set at the median value when this latter value was higher than 100% (positions S101 and S103). Detail of the number of selected variants per position is indicated.

Extended Data Fig. 6 Crystal structure of mature ProteinTS224A inactive variant.

a, The monomeric structure is presented as a cartoon representation. The three amino acid residues (D39, H72, S224A) forming the catalytic triad are shown as magenta-colored sticks. Three-dimensional structure of ProteinT with the prodomain and catalytic domain colored in orange and cyan, respectively. Conserved DS1 and DS2 disulfide bonds are labeled and shown as yellow sticks. Calcium ion is colored in pink and magnesium ion in green. b, representation of calcium ion coordination. c, representation of magnesium ion coordination.

Extended Data Fig. 7 Biodegradation assessment of enzymated PLA material.

a, Aqueous biodegradation of enzymated PLA material after a long-term storage of 18 months at room temperature. Depolymerization of PLA + MBs + ProteinTFLTIER film (line) or PLA + MBs + Proteinase K film (dotted line) at pH 9.0 and 45 °C before (light blue) and after (black) a long-term storage of 18 months at room temperature of the films. Each filled symbol represents the mean value ± s.d. (n = 2 independent experiments). b, Aqueous biodegradation of enzymated PLA material after 1 month immersion in yogurt. Depolymerization of PLA + MBs + ProteinTFLTIER film at pH 9.0 and 45 °C, at the time of the film production (blue), after a long-term storage of 18 months at room temperature (black), and after a long-term storage of 18 months at room temperature followed by an additional 1 month immersion in yogurt at 4 °C (red). Each filled symbol represents the mean value ± s.d. (n = 2 independent experiments), except for yogurt at 4 °C (n = 1).

Extended Data Fig. 8 Surface morphology study of PLA films by scanning electron microscopy (SEM).

a, SEM images of PLA + MBs film control after film production (untreated film), after 65 h incubation in buffer at 45 °C, pH 9.0 (aqueous biodegradation assay) and after 1 month immersion in yogurt at 4 °C. b, SEM images of PLA + MBs + Proteinase K film after film production (untreated film) and after 65 h incubation in buffer at 45 °C, pH 9.0 (aqueous biodegradation assay). c, SEM images of PLA + MBs + ProteinTFLTIER film after film production (untreated film), after 65 h incubation in buffer at 45 °C, pH 9.0 (aqueous biodegradation assay) and after 1 month immersion in yogurt at 4 °C. All experiments were performed as independent duplicates and showed the same results as presented.

Extended Data Fig. 9 Biodegradation assessment of PLA films.

a, Aerobic biodegradation of films in industrial compost conditions. Biodegradation of cellulose film (black), PLA + MBs + ProteinTFLTIER film (blue), and PLA + MBs film control (green). The yield of film biodegradation was assessed using the amount of CO2 released following ISO 14855-1:2012 standards. b, Anaerobic biodegradation of PLA films in mesophilic (37 °C) digestion conditions. Anaerobic biodegradation of PLA + MBs + ProteinTFLTIER film (blue) and PLA + MBs film control (black). Each filled symbol represents the mean value ± s.d. (n = 3 independent experiments). The rate of biodegradation was assessed by considering the ratio between the mass of CH4 and CO2 gases produced, and the mass of carbon initially introduced.

Extended Data Table 1 Mechanical properties of PLA films
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Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Tables 1–11, Notes, Methods and references.

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Guicherd, M., Ben Khaled, M., Guéroult, M. et al. An engineered enzyme embedded into PLA to make self-biodegradable plastic. Nature 631, 884–890 (2024). https://doi.org/10.1038/s41586-024-07709-1

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