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Biocatalytic Depolymerization of Plastics

Biocatalytic Depolymerization of Plastics. Advantages of Biocatalysis. Bio-chemo Recycling Approaches. Challenge: conventional plastics were not designed to be enzymatically degraded. STRATEGIES: i) Use of cutting edge chemical catalysts for plastic pretreatments to:

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Biocatalytic Depolymerization of Plastics

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  1. BiocatalyticDepolymerization of Plastics

  2. Advantages of Biocatalysis

  3. Bio-chemo Recycling Approaches Challenge: conventional plastics were not designed to be enzymatically degraded STRATEGIES: i) Use of cutting edge chemical catalysts for plastic pretreatments to: • decrease polymer molecular weight • Introduce functional handles • Combine microbes and/or enzymes that will work in concert to enhance the efficiency of plastic decomposition. iii) Use other processes that will enhance enzyme access to the plastic substrate. • Physical disintegration to high surface area particles • decrease crystallinity • Increase accessibility (low contents of solvents such as ionic liquids, THF for polystyrene) iv) Protein engineering to enhance the activity of plastic degrading enzymes. • Achieve process efficiency for commercial implementation.

  4. Microbial enzyme production Selection of microorganisms Isolates on plastics

  5. Cutinase Search for enzymes that can perform mild and Selective Plastic Biorecycling Cutin: polyester of hydroxylated Fatty acids Powerful natural hydrolase In nature, catalyzes hydrolysis of cutin, the shiny outer layer of leafs What role can these enzymes play in hydrolysis of synthetic polyesters?? 5

  6. Cutinase: Powerful Hydrolases on Commercial Plastics PET Cellulose acetate PVac • Fungal cutinases: Cutinases from Fusariumsolani(FsC), Humicolainsolens(HiC). Bacterial cutinases: : Cutinasesfrom Leaf and branch compost, Pseudomonas mendocina (PmC), Cutinase-catalyzed hydrolysis Chen et al Biochemistry 2013, BiotechnolAdv 31:1754–1767, Ronkvistet al Macromolecules 2009, 42, 6086–6097,

  7. Characterization of Cutinase activity Determination of kinetics constants Ronkvistet al Macromolecules 2009, 42, 6086–6097

  8. Characterization of Cutinase activity Mechanistic understanding of the reaction in terms of kinetic constants Heterogeneous enzyme kinetics - Adsorption followed by catalysis Constant Substrate concentration – cm2/ml Eq. 1 K is a function of- • Productive surface adsorption • Polymer chain accessibility • Polymer chain orientation Scandola et al Macromolecules 1998, 31, 3846 3851

  9. Significance of Cutinase Thermostabilization 1. Overcoming chain rigidity Enhanced thermostabilitywill enable reactions to be performed at higher temperature, preferably above the glass transition temperature (Tg) where chain segments in amorphous domains have increased mobility. HiC PET (Tg=70oC) Ronkvistet al Macromolecules 2009, 42, 6086–6097

  10. Importance of crystallinity crystallinity Substrates: lcPET (7% crystallinity) vs. boPET (35%) crystallinity) Y-axis expanded 7% lcPET boPET lcPET boPET 35% 30 x decrease At pH 8.0, determined by pH-stat, using 10 mM NaOH as titrant, with 13 cm2/mL of either lcPET (7% crystallinity) or boPET (35 % crystallinity) and 0.13 mg/mL of either HiC, PmC or FsC at 70, 50 and 40 ˚C, respectively. 10 Ronkvist et al Macromolecules 2009, 42, 6086–6097

  11. lcPET film weight loss 250 mm thick 1.5 x 1.5 cm2 0.056 cm2/mg (4.5cm2/80mg) PET film Maximum weight loss 10 mm enzyme concentration • 95 ± 5% HIC • 5 ± 1%PMC • 6 ±4%FSC Other more efficient Enzymes for PET hydrolysis?? Hydrolysis products: Terephaphalic acid & ethylene glycol 2.25 cm2/mL 10% crystalline PET in 10% glycerol, with 0.2 mg/mL of either HiC at 75 oC in 1 M Tris pH 8.5, PmC at 50 oC in 0.1 M Tris pH 8.5 , or FsC at 50 oC in 0.1 M Tris pH 7.5 Ronkvistet al Macromolecules 2009, 42, 6086–6097

  12. Scanning electron micrographsHiC-PET incubations for 2-days Controls HiC Increasing magnification

  13. PET recycling - LCC • LCC- Leaf and Branch Compost Cutinase • Identified using metagenomic approach • Crystal structure available PDB code- 4EBO • Bacterial origin based on homology with bacterial cutinase. Active site • Very thermostable enzyme Tm = 86oC • However, very prone to thermal aggregation resulting in low kinetic stability Disulfide bond Sulaiman et al Biochemistry 2014, 53, 1858−1869

  14. Stabilization by glycosylation Leaf and branch compost cutinase (LCC) (bacterial origin) Glycosylation at consensus sequence N-X-S/T where X could be any AA except proline 3 glycosylation sites Introduce glycosylation by expression in Pichia pastoris Active site N - Native state U - reversibly unfolded state I - Irreversibly unfolded state Agg- Aggregates Glycosylated Protein stained Purple Shirke et al. , Biochemistry (2018), 57(7), 1190-1200. DOI:10.1021/acs.biochem.7b01189

  15. Stabilization by glycosylation 1 Assessment of tendency to aggregate DLS Thermal scan Glycosylation stabilizes LCC primarily by inhibition of aggregation

  16. PET hydrolysis activity Comparative activity analysis of LCC-G and Nov-HiC LCC-G e-HiC • PET film weight loss was determined • at 70 °C 1 µM enzyme, pH 8. PET hydrolysis assay - 2 cm2/ml lc-PET (7%crystalline), Enzyme conc. (0.02µM- 2µM in 0.5mM Tris solution, the required pH was maintained using 10-50mM NaOH by pH stat at respective temperature and pH 8

  17. Practical Route to PET Biorecycling to Monomers?? Next steps: Further improvement in activity and stability (thermodynamic and kinetic

  18. Conclusions Jendrossek, D. & Birke, J. Appl Microbiol Biotechnol (2019) 103: 125. https://doi.org/10.1007/s00253-018-9453-z Its time to launch programs where we integrate chemical, enzyme catalysts and pretreatment technologies to create practical solutions to plastic waste recycling. • LMWPE • Rubber Degrading Enzymes • Polystyrene • Nylons Yoon MG, Jeon HJ, Kim MN (2012) Biodegradation of Polyethylene by a Soil Bacterium and AlkBCloned Recombinant Cell. J BioremedBiodegrad 3:145. doi:10.4172/2155-6199.1000145 Jendrossek, D. & Birke, J. ApplMicrobiolBiotechnol (2019) 103: 125. https://doi.org/10.1007/s00253-018-9453-z S.S. Yang et al Chemosphere. 2018 Dec;212:262-271. doi: 10.1016/j.chemosphere.2018.08.078. Epub 2018 Aug 18. Nagai et al Appl Microbiol Biotechnol (2014) 98:8751–8761 DOI 10.1007/s00253-014-5885-2.

  19. O C O Engineered yeast catalyzes w-hydroxylation WenchunXie Wenhua Hu w-hydroxyfatty acids: biobasedplatform chemicals Monomers Chemical intermediates Biomass derived Fatty acid feedstock Modify chain length and unsaturation esters ethers amines alkenes esters amides more Derivatives to access multiple functionalities w-hyroxytetradecanoic acid (w-HOC14) Journal of the American Chemical Society 2010, 132, 15451–15455

  20. Biobased and Biodegradable medium-density Polyethylene Replacement Tensile Properties Poly(w-HOC14) A calculated after correction for the cross-sectional area. DSC melting transition HB (J/g) Tm (oC)B Mw P(-OH-CX) P(-OH-C14) 140 000 126 94 B second heating scan P(-OH-C18) 170 000 123 102

  21. Compostability test on P(w-HOC14) film Compost test ISO14855 120 110 100 90 80 70 Cellulose Biodegradation % 60 50 40 30 20 10 0 0 10 5 15 20 25 30 35 40 45 50 55 Time (days)

  22. Thank you! QUESTIONS? Award # CBET-1067415

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