1. The Enzyme platform (WP3)
Vincent Eijsink
Anikó Várnai, Jane Agger
& Group members

2. W2G – From MSW to oligomeric & monomeric feedstocks
200 kg
750 L
DERIVATISATION & SCREENING
50 L

3. MSW components targeted in W2G
Converting plant-derived biomass (cellulose, hemicellulose, lignin) to oligomeric products has been a key target in Waste2Go. To achieve this, enzyme technology was indispensable.

4. MSW components targeted in W2G

5. MSW components targeted in W2G
Cellulose: simple (?) homopolymer, simple enzyme cocktails for conversion (?), homogenous products (glucose and glucose oligomers), recalcitrant and insoluble

6. MSW components targeted in W2G
Hemicelluloses and pectin: complex, mixed heteropolymers, heterogeneous products that are soluble and sometimes charged, complex enzyme machineries, options for fine-tuning

7. MSW components (not) targeted in W2G
Lignin: a polyaromate that is not particularly amenable to controlled (chemical) modification

8. Enzyme challenges
Recalcitrance of cellulose

9. Enzyme challenges Recalcitrance of cellulose
Product inhibition of cellulases and degradability of intermediate products

10. Enzyme challenges Recalcitrance of cellulose
Product inhibition for cellulases
Complexity of hemicellulose processing
Ox4

11. Enzyme challenges, a recent revolution: oxidative processes contribute to polysaccharide conversion
G. Vaaje-Kolstad et al., Science 330: (2010)

12. Enzyme challenges, a recent revolution: oxidative processes contribute to polysaccharide conversion

13. Enzyme challenges, a recent revolution: oxidative processes contribute to polysaccharide conversion
Proc Natl Acad Sci USA, Volume 111, pages (2014).

14. The W2G substrates (cellulose) and their pretreatment
Thermo-mechanical: Rotoclavation at GWB Materials:
Alternative pretreatment: Steam explosion (SE) at NMBU Materials:
7 / 14 min
170 / 190 / 210 °C
Dry, mixed recycling
(DMR)
Confidential paper waste
(CWP)
Shredded
office paper
(SOP)
Cut cardboard
(CCB)
Shredded
Newspaper
(SNP)

15. The enzymatic targets for cellulose processing
1. Conversion to soluble oligomeric products Biobased surfactants & other
glyco-conjugates
2. Conversion to monomers Benchmarking & fermentation-based downstream products
3. Conversion to oxidized cello-oligomers Novel products
Needs:
For 1: Endo-acting cellulases that produce longer cellodextrins and that show little product inhibition (!)
For 2: Commercial cellulase cocktails
For 3: LPMOs & Cellobiose DeHydrogenase (CDH) & any (?) cellulase

16. The enzyme platform Enzyme selection
Enzyme production and purification
Functional testing & benchmarking
Engineering for improved properties
Upscaling (WP4)
The Waste2Go joint CPI – NMBU enzyme production pipeline

17. The enzyme platform – Waste2Go strains
Activity
Enzyme
Host
Scale so far
Endoglucanase
Ta Cel5A from Thermoascus aurantiacus
P. pastoris - GAP
750 L
Af Cel12A from Aspergillus fumigatus
Ma Cel45A from Melanocarpus albomyces
LPMO
Nc LPMO9C (2916) from Neurospora crassa
P. pastoris - AOX
Nc LPMO9F (3328) from Neurospora crassa
Sc LPMO9C from Streptocmyces coelicolor
Escherichia coli
1 L
CDH
Mt CDH from Myriococcum thermophilum
Deacetylase
An CDA9 from Aspergillus niger
Cj CE2B from Cellvibrio japonicus

18. Optimizing the input materialpH pH
DMR, PAPER, TIMBER, HOUSEHOLD, FILTER PAPER
Best conditions selected
Substrate: 5% DM; pH 5.0; 50 °C; 4 h; Cellic CTec2: 5 mg/g dw

19. Functional testing – Oligomeric products
Maximum yields low: < 1% of total dw
Substrate: 5% DM; pH 5.0; 50 °C; 4 h; EG: 5 mg/g dw

20. Functional testing – Cellobionic acid-type products
Final pH:
Substrate: 7.5% DM TM-DMR (batch 2015-Jul); pH 5.0, 58 °C, 72 h
Interesting results, but low yields

21. Functional testing – Complete saccharification
Good conversion process

22. Functional testing enzymatic processes – Conclusions
The pretreated W2G raw materials are very degradable Scale up (WP5) and progress to WP7
Producing soluble oligomeric cellodextrins in large amounts seems like a too ambitious goal Focus on the insoluble material using the innovative solubilization and separation technologies of WP6 (separate talk)
&
Experiments with other cellulose substrates & cellulose acetate
Start working on hemicelluloses; show proof-of-concept

23. Other cellulosic substrates.
NB! 10-25% conversion yield
Substrate: 1% DM; pH 5.0; 50 °C; 24 h; EG, Shearzyme: 1 mg/g dw PASC: 0.1% DM

24. Cellulose acetate. Acetylation DP Different product profile:
DP\Ac 1 2 3 4 5 6 7 8 9 10 11 DP
TaCel5A
AfCel12A
MaCel45A
Different product profile:
TaCel5A: shorter products, high DS → less inhibited by acetylation
MaCel45A: longer products, low DS → more inhibited by acetylation

25. Hemicellulose concepts - I
Woody MSW (hardwood)
Insoluble (cellulose)
Pretreatment (Steam explosion) and separation
Monomers (glucose)
Mixture of neutral and acidic xylo-oligosaccharides (DP≤5 with acetylations)
Enzymatic treatment (GH10, CE6)
Enzymatic treatment (cellulase preparation)
Neutral xylo-oligosacchardes
Acidic xylo-oligosacchardes
Separation
Further processing
Soluble (hemicellulose, xylan)

26. Hemicellulose concepts - I
More enzymes needed !

27. Hemicellulose concepts – II – Step 1
Plant-derived food MSW (vegetables)
Soluble (hemicellulose, xyloglucan)
Pretreatment (extraction) and separation
Mixture of neutral xyloglucan-oligosaccharides (DP7-10)
Enzymatic treatment AfCel12A
(optional: β-galactosidase)
Further processing
Commercial xyloglucan
Can be done with W2G enzymes

28. Hemicellulose concepts – II – Step 2
Oligosaccharide (ex. XG7)
Reductively aminated oligosaccharide (XG7-NH2)
Chemical reduction and amination
Cyanoborohydride, sat NH4HCO3
Biosurfactant (oligosaccharide-lipophilic coupled compound, XG7-NH-C18)
Chemical coupling of aliphatic acid to oligosaccharide
Purification

29. Making a green, biobased surfactant – proof of concept
Water soluble biobased surfactant

30. Spin-off: completely novel glycoconjugates via C4 oxidation
LPMO
Reductant; O2
Cellulose
Cello-oligomers EG
Glc
1 (Reducing end)
4 (Non-reducing end) n 1 4
Oxidised cello-oligomers
Glc4ox
Glc1A
EGs + LPMOs
Cello-oligomers and oxidised cello-oligomers

31. The Enzyme platform – Conclusions
Good selection of enzymes in great quantities. Good saccharification of WP2 materials. The project was too ambitious as to producing cellulose oligomers directly, but innovative WP6 technologies, and alternative substrate/processing strategies are opening up possibilities. Great potential for hemicellulose Hemicellulose-based and cellulose-based glyco-conjugates have been or are being produced Future work: cellulose-(di)acetate, oxidized cellulose-derived products, hemicellulose, novel glyco-conjugates, e.g. from acidic xylo- oligosaccharides.