1. Biocatalytic Depolymerization of Plastics

2. Engineer for Non-Natural Substrates
Advantages of Biocatalysis
Mild
Selective
Engineer for Non-Natural Substrates

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 Cutin: polyester of hydroxylated Fatty acids Powerful natural
Search for enzymes that can perform
mild and Selective Plastic Biorecycling
Cutin: polyester of hydroxylated Fatty acids
Cutinase
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 Fusarium solani (FsC), Humicola insolens (HiC). Bacterial cutinases: : Cutinases from Leaf and branch compost, Pseudomonas mendocina (PmC),
Cutinase-catalyzed hydrolysis
Chen et al Biochemistry 2013, Biotechnol Adv 31:1754–1767, Ronkvist et al Macromolecules 2009, 42, 6086–6097,

7. Characterization of Cutinase activity
Determination of kinetics constants
Just an introduction to activity measurements
Ronkvist et 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 k2
Catalytic constant K
Substrate affinity
Introduction to enzyme kinetics measurement
K is a function of-
Productive surface adsorption
Polymer chain accessibility
Polymer chain orientation
Scandola et al Macromolecules 1998, 31,

9. Significance of Cutinase Thermostabilization
1. Overcoming chain rigidity
PET (Tg =70oC)
HiC
Enhanced thermostability will enable reactions to be performed at higher temperature, preferably above the glass transition temperature (Tg) where chain segments in amorphous domains have increased mobility.
A slide talking about the significance of thermostabilization , I think thermostabilization makes better sense when you are talking about recycling also the biophysics could be difficult to go over in detail
You can mention that we have spent significant time and learned lot of important information like, global stability, active site stability, kinetic stability, thermodynamic stability , aggregation and also how glycosylation could be used– but its out of focus and the tak will focus on enzyme activity engineering’
Ronkvist et al Macromolecules 2009, 42, 6086–6097

10. Importance of crystallinity crystallinity
Substrates: lcPET (7% crystallinity) vs. boPET (35%)
crystallinity)
Y-axis expanded
lcPET
boPET 7%
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 10

11. lcPET film weight loss PET film Maximum weight loss 5 ± 1%PMC 6 ±4%FSC
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
Ronkvist et al Macromolecules 2009, 42, 6086–6097

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

13. PET recycling - LCC Very thermostable enzyme Tm = 86oC
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
Disulfide bond
Very thermostable enzyme Tm = 86oC
However, very prone to thermal aggregation resulting in low kinetic stability
This slide is an introductory slide for LCC- in previous slide we mentioned it’s a best bacterial cutinase without telling what it is actually
Here you can also mentioned that we are focused on this enzyme for all future activities
Also I higlighes a problem with aggregation – as wanted to introduce LCC-G which wasused in the activity analysis in subsequent slides- so point in LCC_NG is already better – but LCC-G further improves its performance at higher temperature
Sulaiman et al Biochemistry 2014, 53, 1858−1869

14. Introduce glycosylation by expression in Pichia pastoris
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
Active site
Introduce glycosylation by expression in Pichia pastoris
LCC was found to have three glycosylation sites Consensus sequence N-X-S/T where X could be any amino acid except proline
Thus by expression in Pichia we gel LCC_G
N - Native state
U - reversibly unfolded state
I - Irreversibly unfolded state
Agg- Aggregates
Glycosylated Protein stained Purple
Shirke et al. , Biochemistry (2018), 57(7), DOI: /acs.biochem.7b01189

15. Stabilization by glycosylation
1 Assessment of tendency to aggregate
DLS Thermal scan
LCC(NG)
LCC-G
Agg point
70oC
80oC
Glycosylation stabilizes LCC primarily by inhibition of aggregation

16. PET hydrolysis activity
Comparative activity analysis of LCC-G and Nov-HiC
Enzyme (Temp oC) k2
(µmol·cm-2·h-1) K
(µM-1)
LCC-G (70)
1.03
7.64
Nov-HiC (70)
0.51
15.13
LCC-G
e-HiC
You see here I had to use LCC- G to be able to measure the kinetic constants thas why all previous slides to introduce LCC-G
Anyways, this slide is for the comparative PET hydrolysis analysis-
Here you are comparing catalytic constant
And saying LCC has higher k2 value than e-HiC however has lower K ( adsorption constants) likely due to highly charge ( hydrophilic) surface for the hydrophobic polymer PET.
Note - here coparison is with LCC-G I haven’t introduced LCC-G before just didn’t want to go into the details of glycosylation here included a foot note , but tis up to you how to make it clear, it makes sense to use lcc-g as lcc ng doesn’t provide the desired kinetics profile to determine the constants. In the end I have included supplementary slide talking about how lcc-g catalytically performs better than LCC-NG also you have my thesis defense presentation if you have better idea
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 LMWPE Rubber Degrading Enzymes Polystyrene Nylons
Jendrossek, D. & Birke, J. Appl Microbiol Biotechnol (2019) 103:
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
AlkB Cloned Recombinant Cell. J Bioremed Biodegrad 3:145. doi: /
Jendrossek, D. & Birke, J. Appl Microbiol Biotechnol (2019) 103:
S.S. Yang et al Chemosphere Dec;212:
doi: /j.chemosphere Epub 2018 Aug 18.
Nagai et al Appl Microbiol Biotechnol (2014) 98:8751–8761
DOI /s

19. w-hydroxyfatty acids: biobased platform chemicals
Engineered yeast catalyzes w-hydroxylation
Wenchun Xie
Wenhua Hu
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 C O

20. Biobased and Biodegradable medium-density Polyethylene Replacement
Tensile Properties Poly(w-HOC14) Mw
×10-3
PDI
Young’s Modulus (MPa)
elongation at break
(%)
stress at yield (true)A
(MPa) 78
1.5
415
640
50.0
140
1.8
420
730
63.0
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)
126 94
B second heating scan
P(-OH-C18)
123
102

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

22. Thank you!
QUESTIONS?
Award # CBET