Biocatalytic enzyme processes for CO2
conversion and lignin modification
Anne S. Meyer [email protected]
Paris, 2016 Conversion of Biomass:Green Chemistry and Innovative Processes
L’Institut Francais du Danemark
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
New challenges: Global challenges
Biocatalytic conversion of CO2
Enzymatic upgrading of lignin
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Carbon cycle within the ”Methanol Economy”
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Biocatalytic conversion of CO2 to methanol
New Biotechnol 2015 32, 319-327
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
CO2 as a substrate to produce methanol
New Biotechnol 2015 32, 319-327
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
It works! But methanol conc. is low
New Biotechnol 2015 32, 319-327
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
The bottleneck
New Biotechnol 2015 32, 319-327
More efficient enzymes are needed
We currently work on the co-factor regeneration system
Concept CO2 to Methanol works!
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Vision: CO2 conversion at exhaust site
The biocatalytic CO2 technologies are genuinely disruptive
Unique green technology that have potential for export globally
Huge financial potential for European companies
Very interested in collaboration in this area
DTU Chemical Engineering Technical University of Denmark
Enzymatic lignin modification
Biorefinery vision
• Useful molecules from biomass
• Useful upgrading of lignin
• What do the enzymes do?
• How do the enzymes work?
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
White rot fungi grow on lignocellulose
Ganoderma lucidum Phanerochaete chrysosporium Trametes versicolor
“The G. lucidum genome encodes one of the richest sets of wood degradation
enzymes among all sequenced basidiomycetes” (..and the set includes laccases)
Genome sequence of the model medicinal mushroom Ganodermalucidum. Nature Comm. 3:913 | DOI: 10.1038/ncomms1923
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Enzymatic modification of lignin by laccases?
Laccase EC 1.10.3.2, AA1
4 benzenediol + O2 ⇄ 4 benzosemiquinone + 2 H2O
or: 4 monophenols + O2 ⇄ 4 quinones + 2 H2O
Trametes versicolor laccase
Fungal laccases: 520-550 aa
60-70 kDa, pI 4
Role of laccases in nature ?
Lignification, delignification
Fungal virulence?
Plant laccases: ”Stress management”
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Laccase •Broad electron donor substrate specificity
•Oxygen in, water out, hence 4 e- transfer
O2 2 H2O
4 e-
Trametes versicolor laccase
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Fungal laccases high redox potential
Crit Rev Biotechnol. 2016, 36(1), 70-86
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Laccase catalytic mechanism (amazing!)
Crit Rev Biotechnol. 2016, 36(1), 70-86
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Enzymatic modification of lignin by laccases?
Crit Rev Biotechnol. 2016, 36(1), 70-86
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Various theories built on MODEL COMPOUNDS
Crit
Rev B
iote
chnol. 2
016, 36(1
), 7
0-8
6
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Lignin
BioPr Biosyst Eng 2015; 38: 2285-2313
Genuine lignin is different:
Phenolic–OH’s have been ”used” for bonding
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Can laccases catalyze bond cleavage in lignin? Biotechnol Adv, 2015, 33:13-24
Activation of lignin structure
Electron withdrawal from subunit in lignin
Radical formation
Increase in reactivity
Bond cleavage
Coupling
Attachment of
phenylpropanoids
and/or mediators
Modification
Change in functional group:
also:
acetylation and demethylation
Depolymerization
Consecutive cleavage of
linkages within the lignin
polymer
Decrease in average
molecular weight
Release of lignin
substructures
Polymerization
Continuing attachment
Increase in average
molecular weight
Grafting
Single attachment
Changes in properties of
lignin e.g. solubility
(a) (b)
Cleaves lignin model compounds
Laccase cannot act on ligninwithout a mediator
With presence of a mediatordifferent reactions with ligninmay occur : New upgrading routes!
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Laccase-oxidation with mediators
Laccase oxidation
O2 Laccase(red)
H2O Laccase(ox)Phenol-Lignin
Phenol-Lignin(ox)
H2O
O2 Laccase(red)
Laccase(ox)Mediator(red)
Mediator(ox) Non Phenol-Lignin
Non Phenol-Lignin(ox)
Laccase-mediator system oxidation
(1-hydroxybenzotriazole)
HBT
1-hydroxybenzotriazole
HPI
N-hydroxyphthalimide
TEMPO
2,2,6,6-tetramethyl-
Piperidine1-yloxy
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
RT: 1.02 - 34.94
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
5
10
15
20
25
30
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45
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55
60
65
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100
Relati
ve Ab
undanc
e
24.771.22
15.84
18.48
27.697.90
16.09 22.4532.909.67 12.27 17.20
24.3619.41 25.266.8214.39 20.07 27.30
33.074.19 25.891.78 28.04 33.758.50 21.44 28.206.231.87 7.34 31.1822.85 30.0111.70 28.9314.659.49 14.233.70 10.843.43 5.56
NL:1.95E7
TIC MS 150504-21
HBT
RT: 0.90 - 34.95
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
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85
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100
Relati
ve Ab
undanc
e
24.77
15.84
18.49
27.69
16.09 32.88
7.90 22.44
17.20
9.6712.28
25.2619.41
14.39 19.55 33.066.83 26.8628.046.234.20
8.51 33.741.79 20.9618.30 22.84 28.19 31.181.88 14.657.54 11.71 30.719.50 11.093.96 14.232.25
5.56
NL:1.83E7
TIC MS 150504-16
RT: 0.94 - 34.95
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
5
10
15
20
25
30
35
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45
50
55
60
65
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75
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95
100
Relati
ve Ab
unda
nce
24.78
15.84
18.48
27.69
16.097.8932.89
24.3522.4417.1912.27
9.66 19.40 25.266.81 27.3014.38 20.07
33.0725.894.18 28.04
8.50 33.7521.431.7928.2022.616.221.87 31.1830.0111.71 14.657.34 9.50 13.663.94 10.843.70 5.56
NL:2.72E7
TIC MS 150504-19
HBT
”Grafting” of mediators! Tvl
Ongoing work
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
RT: 0.94 - 35.07
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
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75
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85
90
95
100
Relat
ive A
bund
ance
24.77
21.81
15.84
18.49
16.097.90
17.20 22.459.68 12.28 19.4214.39 25.27 27.71
32.9026.8619.554.21 6.83 33.0725.89
28.058.52 21.44 34.226.241.81 22.851.90 32.0431.2130.6814.6511.709.507.55 10.86 14.213.722.27 5.58
NL:1.92E7
TIC MS 150504-13
RT: 0.98 - 34.99
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive A
bund
ance
24.78
21.80
15.84
18.48
16.097.90
22.44 27.6917.20
32.8912.279.6719.41 25.27 27.30
14.3920.07 26.876.83 33.074.21
28.046.23 8.51 33.7521.431.79 1.88 22.85 31.1911.70 30.0014.64 29.117.54 9.513.72 10.86 13.665.582.25
NL:2.77E7
TIC MS 150504-08
RT: 1.02 - 34.99
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Relat
ive A
bund
ance
24.78
15.84
18.48
27.6816.09 32.88
22.457.9117.2012.289.67
19.4125.27
14.39 20.076.83 27.3026.87 33.066.24 28.048.514.21 33.7420.971.79 22.841.88 31.1728.19
23.6011.727.54 14.65 30.009.513.97 10.84 13.663.73 5.58
NL:2.36E7
TIC MS 150504-06
HPI
HPI
”Grafting” of mediators! Tvl
Ongoing work
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
HPI grafts more than HBT
0%
5%
10%
15%
20%
25%
30%
35%
40%
HBT HPI HBOt NHPI
LWSR BOL
med
iato
r ar
ea/L
ign
in (
S+G
are
a)
Blank TvL PoL
Ongoing work
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Fungal growth
Cultivation media Ganoderma
lucidum
Polyporus
brumalis
Polyporus
ciliatus
Trametes
versicolor
LigninMM + ± ± ±
MEA ++ - - -
SCB MM ++ + + +
MEA +++ + + +
Laccase discovery for lignin modification
44 white rot fungi, 4 could grow on lignin
Identification of a laccase fom Ganoderma lucidum CBS229.93 for enhancing cellulase catalyzed lignocellulose degradation EMT, 2013, 53: 378-385
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
0
0.5
1
1.5
2
2.5
3
3.5
SCB SCB+AV SCB SCB+AV SCB SCB+AV SCB SCB+AV
G. lucidum P. ciliatus P. brumalis T. versicolor
U/m
L
MEA
MM
Did these fungi produce laccase?
Identification of a laccase fom Ganoderma lucidum CBS229.93 for enhancing cellulase catalyzed lignocellulose degradation EMT, 2013, 53: 378-385
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
1h 3h 5h 24h
Glu
cose
conc.
(m
M)
Treatment time (h)
CellicTMCtec1_0
CellicTMCtec1_LacGl1
CellicTMCtec1_Tv
Identification of a laccase fom Ganoderma lucidum CBS229.93 for enhancing cellulase catalyzed lignocellulose degradation EMT, 2013, 53: 378-385
Boosting of cellulases? SC-Bagasse
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
LacGL1 on barley straw and wheat straw
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 3 5 24 30
Glu
cose
co
nce
ntr
atio
n [
g/L]
Time [h]
CellicCtech2
T.versicolor
LacGL1
LacJooSS
A. bisporus
P.ostratus
Cellulase 2
0
0.5
1
1.5
2
2.5
1 3 5 24 30G
luco
se c
on
cen
trat
ion
[g/
L]Time [h]
CellicCtech2
T.versicolor
LacGL1
LacJooSS
A. bisporus
P.ostratus
Cellulase 2Cellulase 2
Wheat strawBarley straw
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
Enzyme engineering
Structure, functionality, and tuning up of laccases for lignocellulose and other industrial applications. Crit. Rev. Biotechnol. DOI:10.3109/07388551
Center for BioProcess Engineering DTU Chemical Engineering Technical University of Denmark
• Laccases with mediators can modify lignin, but cannot without mediators
• A novel laccase from Ganoderma lucidum improves cellulase can catalyzeimproved glucose release from biomass
• The G. lucidum laccase investigated/engineered to understand mechanism
• Interested in collaboration to understand function of fungal laccases in nature
Enzymatic lignin modification
Acknowledgments
J. D. Mikkelsen
Jianquan Luo
Manuel Pinelo
Fauziah Marpani
Anna K. Sitarz
Peter Højrup (SDU)
Dayanand Kalyani
Mateusz Lezyk
Michael K. Nielsen
Line Munk
Chr. Nyffenegger
Sine Larsen (KU)
Mirjam Kabel (WUR)
Arjen Punt (WUR)
Danish Advanced Technology Foundation
Danish Strategic Research Council
Globalization Funds DTU
THANKS !
L’Institut Francais du Danemark