Closing the loop for Poly(butylene-adipate-co-terephthalate) recycling: Depolymerization, monomers separation and upcycling

The study demonstrates complete depolymerization of PBAT into its monomers using a thermostable cutinase variant LCC-WCCG, followed by separation and recovery of pure monomers and their recycling/upcycling to achieve a circular plastics system.

was inserted between NcoI and XhoI restriction sites of the expression vector pET28a(+) followed by ligation with T4 DNA ligase and transformation of the ligation mixture into E. coli BL21(DE3).The cells were plated on LB agar plates supplemented with 50 µg mL -1 kanamycin and grown overnight at 37 °C.Plasmids extracted from the transformant colonies were sequenced (GATC Biotech AB, Solna, Sweden), and those with correct sequences were used for protein expression in E. coli BL21(DE3).Glycerol stock of (His 6 -LCC-WCCG-pET28a(+))-E. coli BL21(DE3) was prepared by growing the cells overnight at 37 °C, 200 rpm in LB medium supplemented with 50 µg mL -1 kanamycin, and then mixing with 50% glycerol, prior to distributing in 1 mL aliquots for storage at -80 °C.

Purification of His 6 -LCC-WCCG
Purification of His tagged LCC-WCCG was done by immobilized metal ion chromatography.
The cell pellet obtained above was suspended in the binding buffer (100 mM Tris-HCl, 20 mM imidazole, 0.5 M NaCl, pH 8) at a concentration of 1 gm cells (wet weight) in 10 mL binding buffer and sonicated on ice (5 X, 60 s, cycle 0.5) using UP400S sonicator (Dr.Hielscher GmbH, Stahnsdorf, Teltow, Germany).The cell debris was removed by centrifugation at 14,000 rpm, 30 min, 4 °C (Sorvall LYNX 4000).The clarified cell lysate was filtered through a 0.2 µm filter and loaded on 5 mL HisTrap FF TM Nickel column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) pre-equilibrated with the binding buffer and connected to ÄKTA Start chromatography system controlled by UNICORN software for fast protein liquid chromatography (FLPC, GE Healthcare) at a flow rate of 5 mL min -1 .Unbound proteins were removed by washing with the same buffer, and finally, the bound proteins were eluted with the elution buffer (100 mM Tris-HCl, 0.5 M NaCl, 0.5 M imidazole, pH 8).The purified protein was desalted on a 5 mL HiTrap desalting column (Sephadex G-25 Superfine resin, GE Healthcare) equilibrated with storage buffer (100 mM Tris-HCl, 500 mM NaCl, pH 8), analyzed by SDS-PAGE, and quantified with NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and bicinchoninic acid (BCA) assay.

Analyses HPLC and LC-MS analysis of PBAT monomers and BDO oxidation products
The monomers released from the hydrolysis of PBAT (AA, BDO), and the oxidation reaction products (succinic acid, and 4-hydroxybutanoic acid) were quantified using HPLC (JASCO, Tokyo, Japan) equipped with refractive index detector (ERC, Kawaguchi, Japan), a JASCO intelligent autosampler and a chromatographic oven (Shimadzu, Tokyo, Japan).Separation of AA and BDO was done on an Aminex HPX-87H column (100 x 7.8 mm) connected to a guard column (Biorad, Richmond, CA, USA), maintained at 65 °C and using 0.05 mM H 2 SO 4 as mobile phase at flow rate of 0.6 mL min -1 .SA and 4-HB were separated on Aminex HPX-87H column (300 x 7.8 mm) at 65 °C using 5 mM H 2 SO 4 as mobile phase.TPA was analysed on a C18 column (Kromasil, Sweden) connected to guard column (C18 Kromasil, Sweden) at 40 °C using 20 % acetonitrile with 0.02 % formic acid as mobile phase at 0.6 mL min -1 and JASCO UV detector at 215 nm.Samples were prepared by diluting in DMSO for TPA, AA, and BDO analysis.The detection limits for TPA, AA, and BDO are 0.5 µg ml -1 , 0.05 mg ml -1 , and 0.05 mg ml -1 , respectively.

Scanning Electron Microscopy of PBAT films
PBAT films (6-9 mg, 0.5 × 1.0 cm each), treated with 0.125 µM of the purified LCC-WCCG in 100 mM potassium phosphate buffer pH 8 at 50 °C, 600 rpm for 24 and 48 hours, were examined by scanning electron microscopy.The film incubated under the same conditions in the absence of the enzyme served as control.The films were washed with Milli-Q water and dried by lyophilization.The dried films were critical-point dried (BAL-TEC CPD 030, Bal-Tec AG, Balzers, Liechtenstein), glued onto metal sockets, and sputter-coated with gold/palladium at 1.2 kV, 15 mA, 0.02 mbar (Polaron SC7640 sputter coater, Quorum Technologies Ltd, Kent, UK).The films were examined with a scanning electron microscope (SEM; JEOL JSM-5600LV, Jeol Ltd, Tokyo, Japan) at 7 kV, the areas of interest were photographed and recorded.Control (untreated PBAT) films were treated and examined under the same conditions for comparison.

Fourier-transform infrared spectroscopy (FTIR)
Ten microliter samples from the polymerization reaction between AA and HDMA were placed on the pedestal of a Nicolet™ iS™ 5 FTIR Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), and after the solvent evaporated the percent transmission was collected in the ZeSe window.
Equation S1: Conventional Michaelis-Menten kinetics model Conv V max , refers to the maximum reaction velocity, S 0 is concentration of the substrate (substrate load), Conv K m is Michaelis constant of the conventional kinetic model.
Conv V max , can be calculated from K cat turnover number and E 0 enzyme concentration in the reaction.

Equation S2: Inverse Michaelis-Menten kinetics model
Inv V max is the maximum reaction velocity calculated from inverse kinetic model, E 0 is enzyme concentration used in the reactions, Inv K M is Michaelis constant of the inverse model.
Inv V max can be calculated using Kcat, reactive site density, and E 0 enzyme concentration.

Γ
Equation S3: Reactive site density equation Γ Reactive site density can be calculated from both conventional and inverse kinetic models using equation S3 Conventional Kinetics Inverse kinetics LCC-WCCG

Figure S6
. Activity of LCC-WCCG against PET and PBAT, based on ratio of released TPA (this study) and its specific activity against PBAT (this study) and PET (Tournier et al. 2020). 1 Reactions were done in duplicates using PET or PBAT particles (5 mg mL -1 ) with 0.5 µM of LCC-WCCG in 100 mM Phosphate buffer pH 8. Samples were taken after 24 hours from the reaction.For the specific activity measurements, the reactions were done following the conditions described in table S3.The molar fraction of each dyad was calculated from the 1 H NMR spectrum using the area of the signals corresponding to the TT, AT, TA, and AA units according to Eq. 1-4.The probability factors, the sequence length, and the degree of randomness were calculated using Eq.5-6, Eq. 7-8, and Eq. 9 respectively.
Eq. 1 Eq. 4 Eq. 5 Eq. 6 Eq. 7 Eq. 8 Eq. 9 f XX is the molar fractions of the AA, AT, TA, and TT segments respectively.A X is the area of the integral for the protons (f, j, h, and c) corresponding to the AA, AT, TA, and TT segments respectively.P AT is the probability of encountering a terephthalate unit next to an aliphatic segment.f A is the molar content of adipate units in the polymer.f T is the molar content of terephthalate units in the polymer.L nA = average sequence length of the aliphatic segment.L nT = average sequence length of the aromatic segment.r = Degree of randomness.
Table S2.Sequence distribution of the PBAT samples.

EFigure S1 .
Figure S1.Kinetics measured for LCC-WCCG against PBAT.(A, B) Initial reaction rate curves showing total concentration of the released monomers (millimolar per minute).(A) Conventional kinetics model with variable substrate concentration (0.5-25 mg mL -1 ) and fixed enzyme concentration, and (B) Inverse kinetics model using variable enzyme concentrations (0.01-1.5 µM) and fixed substrate concentration (10 mg mL -1 ).Symbols are experimental data with a standard deviation of triplicates.(C, D) Michaelis-Menten (MM) plots for PBAT hydrolysis by LCC-WCCG from (C) conventional MM kinetics model, the curve shows the reaction rate as a function of PBAT concentration, and (D) inverse MM kinetic model, where the reaction rate is a function of the enzyme concentration.The plots were generated using the data from curves A and B. Error bars represent the standard deviation of reactions performed in triplicates.(E) Kinetic parameters calculated for LCC-WCCG catalysed hydrolysis of the PBAT polymer.The deviation error is generated from fitting triplicate measurements.

Figure S2 .
Figure S2.Overall change in the carbon backbone of the LCC-WCCG structure after 3 ns of MD simulation: (A) Root mean square deviation (RMSD) of backbone of LCC-WCCG structure at different temperatures, (B) Fluctuation of each residue of the LCC-WCCG structure at different temperature points.0.4, 1, and 2.5 ns were selected for running the docking experiment.(C) Superimposed structures

Figure S3 .Figure S4 .
Figure S3.Different oligomers used in the docking studies including smiles and structures.Structures were drawn using ChemDraw 20.1, and 3D structures of the ligands were created using YASARA structure.

Figure S7 .
Figure S7.PBAT (12.5 g L -1 ) depolymerisation using 1.65 M, 2.5 mg LCC-WCCG in 50 mL reaction volume at 70 °C.Reaction was run in a shaking flask with a sealed opening to reduce evaporation.Samples were taken for analysis of the released TPA, pH was adjusted by adding 1 M NaOH while sampling.The polymer weight loss achieved within 7 days was 97.5 %.

Figure S8 .Figure S9 .Figure S10. 1 H
Figure S8.Terephthalic acid separation form PBAT hydrolysate, obtained by treatment of 15 g L -1 PBAT film in 1 Liter volume by LCC-WCCG, (A) gradual decrease in TPA concentration in the solution during stepwise acidification of the solution.(B) HPLC chromatogram of TPA purified from the reaction mixture at pH 2.5.(C) 1 H-NMR spectrum of the purified TPA in DMSO-d 6 .The spectrum indicates nearly 5 % impurities from AA and BDO.

Figure S11. 1 H
Figure S11.1 H NMR spectra in CDCl 3 of the synthesized PBAT using commercial monomers (vPBAT) and recycled monomers (rPBAT), respectively.The 1 H NMR spectrum of a commercially available PBAT (cPBAT) was shown as well.

Figure S13 .
Figure S13.Zoomed-in figure of the 1 H-NMR spectrum of rPBAT in CDCl 3 for sequence distribution calculations.

Figure S14 .
Figure S14.Zoomed-in figure of the 1 H-NMR spectrum of vPBAT in CDCl 3 for sequence distribution calculations.

Figure S20. 1 H
Figure S20. 1 H-NMR analysis in D 2 O for the oxidation of BDO present in nanofiltration (red) and ultrafiltration (blue) permeate fractions using G. oxydans.4-HB signals are assigned, the rest are for AA and BDO, and TPA in case of UF-permeate fraction.

Table S3 .
Comparison of enzymatic degradation of PBAT described in literature and this study.