Biocatalytic enzyme processes for CO conversion and lignin

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


Carbon cycle within the ”Methanol Economy”


Biocatalytic conversion of CO2 to methanol

New Biotechnol 2015 32, 319-327


CO2 as a substrate to produce methanol



It works! But methanol conc. is low



The bottleneck


More efficient enzymes are needed

We currently work on the co-factor regeneration system

Concept CO2 to Methanol works!


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?


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


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”


Laccase •Broad electron donor substrate specificity

•Oxygen in, water out, hence 4 e- transfer

O2 2 H2O

4 e-

Trametes versicolor laccase


Fungal laccases high redox potential

Crit Rev Biotechnol. 2016, 36(1), 70-86


Laccase catalytic mechanism (amazing!)



Enzymatic modification of lignin by laccases?



Various theories built on MODEL COMPOUNDS

Crit

Rev B

iote

chnol. 2

016, 36(1

), 7

0-8

6


Lignin

BioPr Biosyst Eng 2015; 38: 2285-2313

Genuine lignin is different:

Phenolic–OH’s have been ”used” for bonding


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!


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


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

35

40

45

50

55

60

65

70

75

80

85

90

95

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

80

85

90

95

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

40

45

50

55

60

65

70

75

80

85

90

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


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

70

75

80

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


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


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


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?



-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


Boosting of cellulases? SC-Bagasse


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


Enzyme engineering

Structure, functionality, and tuning up of laccases for lignocellulose and other industrial applications. Crit. Rev. Biotechnol. DOI:10.3109/07388551


• 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

[email protected]

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

Documents

Biocatalytic enzyme processes for CO conversion and lignin