The Enzyme platform (WP3)

Vincent EijsinkAnikó Várnai, Jane Agger

& Group members

200 kg

750 L

50 L

DERIVATISATION &

SCREENING

W2G – From MSW to oligomeric & monomeric feedstocks

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.

MSW components targeted in W2G

MSW components targeted in W2G

Cellulose: simple (?) homopolymer, simple enzyme cocktails for conversion (?), homogenous products (glucose and glucose oligomers), recalcitrant and insoluble

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

MSW components (not) targeted in W2G

Lignin: a polyaromate that is not particularly amenable to controlled (chemical) modification

Enzyme challenges

Recalcitrance of cellulose

Enzyme challenges

Recalcitrance of celluloseProduct inhibition of cellulases and degradability of intermediate products

Enzyme challenges

Recalcitrance of celluloseProduct inhibition for cellulasesComplexity of hemicellulose processing

Ox4

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

G. Vaaje-Kolstad et al., Science 330:219-222 (2010)

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

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

Proc Natl Acad Sci USA, Volume 111, pages 6287-6292 (2014).

The W2G substrates (cellulose) and their pretreatment

Thermo-mechanical:

Rotoclavation at GWB

Materials:

Alternative pretreatment:

Steam explosion (SE)

at NMBU

Materials:

Dry, mixedrecycling

(DMR)

Confidentialpaper waste

(CWP)

Shreddedoffice paper

(SOP)

Cutcardboard

(CCB)

ShreddedNewspaper

(SNP)

7 / 14 min170 / 190 / 210 °C

The enzymatic targets for cellulose processing1. 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 cocktailsFor 3: LPMOs & Cellobiose DeHydrogenase (CDH) & any (?) cellulase

The enzyme platform

1. Enzyme selection2. Enzyme production and purification3. Functional testing & benchmarking4. Engineering for improved properties5. Upscaling (WP4)

The Waste2Go joint CPI – NMBU enzyme production pipeline

The enzyme platform – Waste2Go strainsActivity Enzyme Host Scale so

far

Endoglucanase

Ta Cel5A from Thermoascus aurantiacus

P. pastoris - GAP

750 L

Af Cel12A from Aspergillus fumigatus

P. pastoris - GAP

750 L

Ma Cel45A from Melanocarpus albomyces

P. pastoris - GAP

750 L

LPMO Nc LPMO9C (2916) from Neurospora crassa

P. pastoris - AOX

750 L

Nc LPMO9F (3328) from Neurospora crassa

P. pastoris - AOX

750 L

Sc LPMO9C from Streptocmyces coelicolor

Escherichia coli

1 L

CDH Mt CDH from Myriococcum thermophilum

P. pastoris - GAP

750 L

Deacetylase

An CDA9 from Aspergillus niger

Escherichia coli

1 L

Cj CE2B from Cellvibrio japonicus

Escherichia coli

Optimizing the input material

Substrate: 5% DM; pH 5.0; 50 °C; 4 h; Cellic CTec2: 5 mg/g dw

W2G (1.1.)1

W2G (1.1.)2

W2G (1.)2.1

W2G (1.)2.2

W2G (1.)2.3

W2G (1.)2.4

W2G 2.2.2

W2G 2.3.2

W2G 3.3.1-

A

W2G 3.3.1-

B

W2G 3.8.1

W2G 3.9.1

W2G 4.2.1

W2G 5.1.1

FP05

101520253035

Conv

ersio

n(%

of d

w) pH

6.4-6.9

pH 5.2-5.5

DMR, PAPER, TIMBER, HOUSEHOLD, FILTER PAPER

Best conditions selected

Functional testing – Oligomeric products

Substrate: 5% DM; pH 5.0; 50 °C; 4 h; EG: 5 mg/g dw

W2G (1.1.)1

W2G (1.1.)2

W2G (1.)2.1

W2G (1.)2.2

W2G (1.)2.3

W2G (1.)2.4

W2G 2.2.2

W2G 2.3.2

W2G 3.3.1-A

W2G 3.3.1-B

W2G 3.8.1

0.00.20.40.60.81.01.2

CellopentaoseCellotetraoseCellotrioseCellobioseGlucoseCo

nver

sion

(% o

f dw

)

TaCel5A

W2G (1.1.)1

W2G (1.1.)2

W2G (1.)2.1

W2G (1.)2.2

W2G (1.)2.3

W2G (1.)2.4

W2G 2.2.2

W2G 2.3.2

W2G 3.3.1-A

W2G 3.3.1-B

W2G 3.8.1

0.00.20.40.60.81.01.2

CellopentaoseCellotetraoseCellotrioseCellobioseGlucoseCo

nver

sion

(% o

f dw

)

AfCel12A

Maximum yields low: < 1% of total dw

Functional testing – Cellobionic acid-type products

Final pH: 4.8-5.2

no enzyme 1-2916 1-CDH

5-2916 1-CDH

1-2916 5-CDH

5-2916 5-CDH

5-EG 5-EG 1-2916 1-

CDH

5-EG 5-2916 1-

CDH

5-EG 1-2916 5-

CDH

5-EG 5-2916 5-

CDH

0.000.050.100.150.200.250.300.350.40

Glc2 Glc2-1A Glc3 Glc3-1A Glc4 Glc4-1A

Conc

entr

ation

(g/l

anh

ydro

-Glc

)

Substrate: 7.5% DM TM-DMR (batch 2015-Jul); pH 5.0, 58 °C, 72 h

Interesting results, but low yields

Functional testing – Complete saccharification

Good conversion process

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

Other cellulosic substrates.

W2G-3.3.1-A (TM-SOP)

SE-SNP 210/14

SE-CCB 210/14

SE-SOP 210/7

SE-birch (W2G)

BALI spruce

BALI bagasse

Cellulose fibrous

Filter paper

Avicel

PASC Wh-1

PASC Avi

SigmaCell (cotton)

0

10

20

TaCel5A Cellotetraose

Cellotriose

Cellobiose

Glucose

Conv

ersio

n(%

of d

w)

W2G-3.3.1-A (TM-SOP)

SE-SNP 210/14

SE-CCB 210/14

SE-SOP 210/7

SE-birch (W2G)

BALI spruce

BALI bagasse

Cellulose fibrous

Filter paper

Avicel

PASC Wh-1

PASC Avi

SigmaCell (cotton)

0

10

20

AfCel12A Cellotetraose

Cellotriose

Cellobiose

Glucose

Conv

ersio

n(%

of d

w)

W2G-3.3.1-A (TM-SOP)

SE-SNP 210/14

SE-CCB 210/14

SE-SOP 210/7

SE-birch (W2G)

BALI spruce

BALI bagasse

Cellulose fibrous

Filter paper

Avicel

PASC Wh-1

PASC Avi

SigmaCell (cotton)

0

10

20

MaCel45A Cellotetraose

Cellotriose

Cellobiose

Glucose

Conv

ersio

n(%

of d

w)

Substrate: 1% DM; pH 5.0; 50 °C; 24 h; EG, Shearzyme: 1 mg/g dw PASC: 0.1% DM

NB! 10-25% conversion yield

Cellulose acetate.

DP\Ac 0 1 2 3 4 5 6 7 8 9123456789

1011

TaCel5AAfCel12AMaCel45A

DP

Acetylation

• Different product profile:• TaCel5A: shorter products, high DS → less inhibited by

acetylation• MaCel45A: longer products, low DS → more inhibited by

acetylation

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-oligosacchar

des

Separation

Further processing

Further processing

Soluble (hemicellulose,

xylan)

Hemicellulose concepts - I

More enzymes needed !

Hemicellulose concepts – II – Step 1

Plant-derived food MSW

(vegetables)

Soluble (hemicellulose,

xyloglucan)

Pretreatment (extraction) and

separation

Mixture of neutral

xyloglucan-oligosaccharide

s (DP7-10)

Enzymatic treatment AfCel12A

(optional: β-galactosidase)

Further processing

Commercial xyloglucan

Can be done with W2G enzymes

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

Making a green, biobased surfactant – proof of concept

Water soluble biobased surfactant

Spin-off: completely novel glycoconjugatesvia C4 oxidation

LPMOReductant; O2

Cellulose

Cello-oligomers

EG

Glc GlcGlc Glc Glc Glc1 (Reducing end)4 (Non-reducing end)

n

Glc Glc Glc 14

n

Oxidised cello-oligomers

Glc4ox

Glc Glc 14

nGlc Glc

Glc1A

14

n

EGs + LPMOs

Glc Glc Glc 14

n

Glc4ox

GlcGlc1A

14

nGlc Glc

Glc1A

14

n

Cello-oligomers and oxidised cello-oligomers

Glc4ox

Glc Glc 14

n

Reductant; O2

The Enzyme platform – ConclusionsGood 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.