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