# Enzymatic depolymerization of polylactide into repolymerizable oligomers

Alaric Catard, Isabelle Chevalot, Sandrine Hoppe

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Alaric Catard, Isabelle Chevalot, Sandrine Hoppe. Enzymatic depolymerization of polylactide into repolymerizable oligomers. 16th International Symposium on Biocatalysis and Biotransformations (BIOTRANS 2023), Jun 2023, Oléron, France. hal-04664902

HAL Id: hal-04664902

https://hal.science/hal-04664902v1

Submitted on 31 Jul 2024

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# Enzymatic depolymerization of polylactide into repolymerizable oligomers

A. Catard, I. Chevalot and S. Hoppe

Université de Lorraine, CNRS, LRGP, F-54000 Nancy, France

<figure><img src="image_1.png" /></figure>

UNIVERSITÉ
DE LORRAINE

## Context & Scientific Issues

<figure><img src="image_2.png" /></figure>

* Bio-plastic made from lactic acid.

* Lactic acid is produced by fermentation of biorenewable resources.

A possible chemical
recycling pathway?

* Hardly degrade in the environment.

* There are no recycling industries.

* Mechanical recycling has a lot of drawbacks (e.g. limited cycles, loss of properties...).

<figure><img src="image_3.png" /></figure>

* This pathway requires 4 steps to come back to polylactide.

* The production of lactide from lactic acid accounts for 30% of virgin PLA production cost [1].

* Crystalline regions of polymers are hardly accessible for enzymes when the polymer is not dissolved in a solvent [2].

* Solvents used in chemical recycling are often harmful.

## A two steps process:

<figure><img src="image_4.png" /></figure>

Cyclo-

<figure><img src="image_5.png" /></figure>

Poly(L-lactide)
(PLA)

Cyclic
oligomers

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Explore the ability of the immobilised lipase B from Candida antarctica (Novozyme 435®) to catalyze the depolymerization of PLLA into cyclic oligomers by shifting thermodynamic equilibrium towards depolymerization by varying parameters (temperature, [PLLA]...)

<figure><img src="image_8.png" /></figure>

Comparison of sustainable solvents to solubilize PLLA.

<figure><img src="image_9.png" /></figure>

Determine Novozyme 435® stability after incubation in the solvents at high temperatures (110 °C < T < 140 °C).

<figure><img src="image_10.png" /></figure>

Evaluate PLLA's degradation after Novozyme 435® treatment at high temperatures in the sustainable solvents.

## Objectives

## I - Solubility of PLLA in green solvents:

<figure><img src="image_11.png" /></figure>

## II - Stability of N435® in green solvents:

<figure><img src="image_12.png" /></figure>

## III - Enzymatic depolymerization of PLLA in green solvents:

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

Size Exclusion
Chromatography
(SEC) Analysis

<figure><img src="image_18.png" /></figure>

The authors acknowledge the Club Biocatalyse en Synthèse Organique (CBSO) for the grant that enabled this participation at the Biotrans 2023 congress.

## Methodology

## Results

## I - Solubility of PLLA in green solvents:

<table><thead><tr><th></th><th>Diethyl carbonate (DEC)</th><th>Limonene (LIM)</th><th>Anisole (ANI)</th><th>Cyrene® (CYR)</th><th>γ-valerolactone (γ-VL)</th></tr></thead><tbody><tr><td>Solubility (in g.L<sup>-1</sup>)</td><td>60</td><td>0</td><td>&gt; 120</td><td>55</td><td>&gt; 120</td></tr></tbody></table>

* Inability of limonene to solubilize PLLA at 100 °C.

* Diethylcarbonate (DEC), anisole (ANI), Cyrene® (CYR) and γ-valerolactone (γ-VL) enable good dissolution of PLLA and are promising candidates for enzymatic depolymerization of the latter.

## II - Stability of N435® in green solvents:

<figure><img src="image_19.png" /></figure>

Relative activity of N435 after incubation in DEC during 48 hours at different temperatures.

<figure><img src="image_20.png" /></figure>

Relative activity of N435 after incubation in ANI and DEC different times at 120 °C.

* There is no residual activity of N435 after incubation at 120 °C in Cyrene® and γ-valerolactone.

* Incubation in DEC leads to a decrease of N435 activity until total inactivity after 24h at 120°C

* Incubation of N435 in ANI leads to the highest residual activity even after 48h at 120 °C.

## III - Enzymatic depolymerization of PLLA in green solvents:

<figure><img src="image_21.png" /></figure>

SEC curves of different conditions of N435 catalyzed depolymerization of PLLA in DEC.

<figure><img src="image_22.png" /></figure>

SEC curves of different conditions of N435 catalyzed depolymerization of PLLA in ANI.

<table><thead><tr><th>Zone Sample</th><th>1</th><th>2</th></tr></thead><tbody><tr><td>A)</td><td>M<sub>w</sub> = 1.234x10<sup>5</sup> Da</td><td>M<sub>n</sub> = 9.909x10<sup>4</sup> Da</td><td>*</td><td>*</td></tr><tr><td>B)</td><td>M<sub>w</sub> = 1.0x10<sup>5</sup> Da</td><td>M<sub>n</sub> = 7.963x10<sup>4</sup> Da</td><td>M<sub>w</sub> = 7.177x10<sup>5</sup> Da</td><td>M<sub>n</sub> = 4.693x10<sup>5</sup> Da</td></tr><tr><td>C)</td><td>M<sub>w</sub> = 6.879x10<sup>4</sup> Da</td><td>M<sub>n</sub> = 5.388x10<sup>4</sup> Da</td><td>*</td><td>*</td></tr><tr><td>D)</td><td>**</td><td>**</td><td>*</td><td>*</td></tr></tbody></table>

<table><thead><tr><th>Zone Sample</th><th>1</th><th>2</th></tr></thead><tbody><tr><td>A)</td><td>M<sub>w</sub> = 9.784x10<sup>4</sup> Da</td><td>M<sub>n</sub> = 7.080x10<sup>4</sup> Da</td><td>**</td><td>**</td></tr><tr><td>B)</td><td>M<sub>w</sub> = 2.1x10<sup>5</sup> Da</td><td>M<sub>n</sub> = 1.615x10<sup>5</sup> Da</td><td>M<sub>w</sub> = 4.528x10<sup>5</sup> Da</td><td>M<sub>n</sub> = 3.825x10<sup>5</sup> Da</td></tr><tr><td>C)</td><td>M<sub>w</sub> = 2.672x10<sup>5</sup> Da</td><td>M<sub>n</sub> = 2.325x10<sup>5</sup> Da</td><td>*</td><td>*</td></tr></tbody></table>

* Conversion of PLLA into small oligomers is favored using higher times and amounts of N435.

* In ANI, the presence of N435 enabled to depolymerize PLLA into short oligomers.

* It is supposed that the depolymerization occurs in the end-chain of the polymer by conversion of high molecular weight chains into short chains without having intermediary molecular weight chains.

## Conclusion & Perspectives

This work explored the possibility to set up the depolymerization of polylactide catalyzed by the immobilised lipase B of Candida antarctica in green solvents temperatures higher than 120 °C. Firstly, in order to avoid harmful or oil-based solvents to solubilize PLLA, different sustainable or renewable solvents have been tested for their ability to solubilize PLLA. Among solvents tested, diethylcarbonate, anisole, Cyrene® and γ-valerolactone showed good ability to solubilize semi-crystalline PLA reaching solubilities higher than 120 g.L⁻¹, Novozyme435® only retained residual hydrolytic activity into diethylcarbonate and anisole at 120 °C. N435 maintained relatively good activity after long time incubation in anisole at 120 °C.

Finally, DEC and ANI have been selected to carry out the enzymatic depolymerization of polylactide. Higher conversion of PLLA into short oligomers have been done in DEC and ANI showing the ability of N435 to catalyze the depolymerization in these solvents and using these temperatures. Further analysis (e.g. ¹H NMR) will be done in order to characterize the oligomers obtained (linear vs cyclic) and new experiments will be set up in order to determine the best conditions to get the highest conversion of polylactide into cyclic oligomers, which can be repolymerizable by Ring Opening Polymerization (ROP).

<footnote>* Previously dried during 24 hours in desiccator.</footnote>

<footnote>** Previously dried during at least 24 hours with 3Å molecular sieve.</footnote>

<footnote>* Insufficient LS signal ** Insufficient LS and RI signals</footnote>

<footnote>* Insufficient LS signal ** Insufficient LS and RI signals</footnote>

<footnote>[1] Van Wouwe P, Dusselier M, Vanleeuw E, Sels B. Lactide Synthesis and Chirality Control for Polylactic acid Production. ChemSusChem. 10 mai 2016;9(9):907-21</footnote>

<footnote>[2] Kawai F, Kawabata T, Oda M. Current State and Perspectives Related to the Polyethylene Terephthalate Hydrolases Available for Biorecycling. ACS Sustainable Chem Eng. 22 juin 2020;8(24):8894-908.</footnote>