Biotechnology for the Eucalyptus Biorefinery Ana Gutirrez, Jos C del - - PowerPoint PPT Presentation

Biotechnology for the Eucalyptus Biorefinery

Ana Gutiérrez, José C del Río

IRNAS, CSIC, Seville, Spain

Susana Camarero, Angel T Martínez

CIB, CSIC, Madrid, Spain

7ICEP-May15

Content:

• 1. Introduction
• 2. Eucalypt decaying fungi and their genomes

Lignin vs polysaccharides decay (biopulping) Biodegradation of (pitch forming) wood lipids Why sequence basidiomycete genomes?

• 3. Oxidative enzymes for the eucalypt mill

Ligninolytic peroxidases The laccase-mediator system Towards industrial feasibility Biobleaching + pitch biocontrol

• 4. Recent studies: Enzymes for eucalypt biorefinery

Wood delignification for biofuel production The delignification process as shown by 2D-NMR A new generation of tailor-made enzymes

7ICEP-May15

Biotechnology for the Eucalyptus Biorefinery

KET-based Industrial Leadership

7ICEP-May15

• 1. Introduction (Biotechnology for the Eucalyptus Biorefinery)

BioBased Industries Consortium BBI Public-Private Partnership H2020 (Horizon 2020 RTD programme)

total: € 77 billion

7ICEP-May15

An overview of results obtained at IRNAS (Seville) and CIB (Madrid) during

• ver 15 years working in biotechnology

for eucalypt biorefinery (cellulose and bioethanol production) is presented The aim was to develop biocatalysts for removal of lignin (bottleneck for using wood in biorefineries) and lipophilic extractives (causing pitch troubles in the Cl-free production of cellulose)

Sitosterol Lignin model

7ICEP-May15 Jorge L. Colodette Fernando J. Gomes Teresa Vidal Cristina Valls Blanca Roncero José E. Colom Tiina Liitia Tarja Tamminen Augusto Milanez Javier Romero José L. Francisco Henrik Lund Lisbeth Kalum Kim Borch

These studies were performed in collaboration with cellulose and biotechnology companies, and also included other research/academic partners

Content:

• 1. Introduction
• 2. Eucalypt decaying fungi and their genomes

Lignin vs polysaccharides decay (biopulping) Biodegradation of (pitch forming) wood lipids Why sequence basidiomycete genomes?

• 3. Oxidative enzymes for the eucalypt mill

Ligninolytic peroxidases The laccase-mediator system Towards industrial feasibility Biobleaching + pitch biocontrol

• 4. Recent studies: Enzymes for eucalypt biorefinery

Wood delignification for biofuel production The delignification process as shown by 2D-NMR A new generation of tailor-made enzymes

7ICEP-May15

Biotechnology for the Eucalyptus Biorefinery

These fungi (and their enzymes) are the biocatalysts

• f choice in wood biorefinery

The so-called white-rot fungi are responsible for the natural degradation

• f lignin in forests providing access to

cellulose to other (micro)organisms

Ganoderma australe Selective delignification by Ganoderma australe Ceriporiopsis subvermispora

• 2. Eucalypt decaying fungi and their genomes

7ICEP-May15

5 10 15 20 25 min

Paecilomyces sp.

24 25 26 23 20 21 16 18 14 10 11 13 9 6 7 5 4 3 2 1 22 12 l m j k g b d e h

Ceriporiopsis subvermispora

22 24 25 26 23 20 21 16 18 14 15 10 11 13 9 6 7 5 4 3 2 1 12 a b d e f h i j k l m g

Carbohydrate peaks Carbohydrate peaks

5 10 15 20 25 min

Control wood (Eucalyptus globulus)

22 24 25 26 23 20 21 16 18 19 14 15 10 11 12 13 8 9 6 7 5 4 3 2 1

Carbohydrate peaks

a b c d e f g h i j k l m 5 10 15 20 25 min

OH OMe

G

OMe OH

G

OH OMe

G

OH OMe

G

OH OMe MeO

S

OH OMe MeO

S

OH OMe MeO

S

OH OMe MeO

S

Eucalyptus wood treatment with fungi: Py-GC/MS analysis

Lignin peaks

del Río J.C., A. Gutiérrez, M. J. Martínez, and A. T. Martínez. Py-GC-MS study of Eucalyptus globulus wood treated with different fungi. J.Anal.Appl.Pyrolysis 58/59:441-453, 2001.

7ICEP-May15

4 6 0.5 1.0 1.5 2.0

PAE CSU FTR PRA CVA KEU FOX MHE CPU PCH OPI BAD Control MOL OVA OPI* PPU

7 5

S/G Lignin/Carbohydrate

Basidiomycetes caused the most selective removal of lignin (low lignin/carbohydrate ratio) with simultaneous decrease of its S/G ratio Ceriporiopsis subvermispora (CSU) showed the highest biotechnological potential for biological delignification of Eucalyptus globulus wood Some of the basidiomycetes also removed the recalcitrant lipids respondible for pitch deposits in Eucalyptus TCF pulps and were assayed for simultaneous depitching and delignification 

Basidiomycetes Ascomycetes Deuteromycetes

Eucalyptus wood treatment with fungi: Patterns observed

7ICEP-May15

Pitch-forming lipid classes in Eucalyptus wood and pulps

OH

n-octacosanol

HO

campesterol

O OH O OH HO CH2OH

sitosteryl 3-β-D-glucopyranoside

HO

sitosterol

O O OH O OH HO CH2OH

7-oxositosteryl 3-β-D-glucopyranoside

HO

stigmastanol

O O

sitosteryl linoleate

HO O

7-oxositosterol

CO-O-CH2 CO-O-CH CO-O-CH2

trilinolein

OH O O

stigmasta-3,5-dien-7-one palmitic acid

Gutiérrez, A., J. C. del Río, M. J. Martínez, and A. T. Martínez. 1999b. Fungal degradation of lipophilic extractives in Eucalyptus globulus wood. Appl. Environ.

• Microbiol. 65:1367-1371.

7ICEP-May15

Lipid removal from Eucalyptus globulus chips by Ceriporiopsis subvermispora and other white-rot fungi (and bio-kraft pulping)

Bjerkandera adusta

10 20 30 40 50 60 70 80 7 14 28 49 days

mg/100 g wood

Phlebia radiata

10 20 30 40 50 60 70 80 7 14 28 49 days

mg/100 g wood

Pleurotus pulmonarius

10 20 30 40 50 60 70 80 7 14 28 49 days

mg/100 g wood

Sterols Fatty acids Squalene Steroidal hydrocarbons Steroidal ketones Sterol esters Triglycerides Bjerkandera Ceriporiopsis Phlebia Pleurotus

Up to 60% of wood steroids can be removed in 1-2 week treatments with only 1% loss of chips weight

Weight loss (%) Sterol removal (%) Ceriporiopsis subvermispora

10 20 30 40 50 60 70 80 7 14 28 49 days

mg/100 g wood

Martínez-Íñigo, M. J., A. Gutiérrez, J. C. del Río, M. J. Martínez, and A. T. Martínez. 2000. Time course of fungal removal of lipophilic extractives from Eucalyptus globulus Labill. wood. J. Biotechnol. 84:119-126. Gutiérrez, A., M. J. Martínez, J. C. del Río, J. Romero, J. Canaval, G. Lenon, and A. T. Martínez. 2000. Fungal pretreatment of Eucalyptus wood can strongly decreases the amount of lipophilic extractives during chlorine-free kraft pulping. Environ. Sci. Technol. 34:3705-3709.

7ICEP-May15

Energy consumption during mill-scale refining of Eucalyptus chips from a 50-tonne pile treated with Ceriporiopsis subvermispora (60 d)

See review of Ferraz et al. on "Technological advances and mechanistic basis for fungal biopulping" (Enzyme Microb. Technol. 43: 178, 2008) and subsequent papers

Pretreatment of Eucalyptus grandis chips with Ceriporiopsis subvermispora for bio-TMP and bio-CTMP (bio-kraft, bio-sulfite and bio-organosolv also evaluated) Biopulping enzymatic/chemical mechanisms analyzed

↓18% ↓27%

Enzymes and pH during treatment of Eucalyptus chips (30 d) with Ceriporiopsis subvermispora (5 ppm inoculum with CSL)

Scale-up of Eucalyptus pretreatment (biopulping) with Ceriporiopsis subvermispora (Ferraz & coworkers)

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USDA biopulping trial

Ceriporiopsis subvermispora also yielded good (selectivity) results in pilot-scale treatment of other types of wood However, the known lignin-degrading enzymes (lignin peroxidase and versatile peroxidase ) were never identified in this fungus To solve this paradox, and contribute to delignification applications, the genome

• f Ceriporiopsis subvermispora was

sequenced at JGI (US DOE) and compared with Phanerochaete chrysosporium (the first basidiomycete sequenced as the model lignin-degrading fungus) 

7ICEP-May15

Why sequence Ceriporiopsis subvermispora?

www.jgi.doe.gov

Prof Rafael Vicuña Universidad Católica Santiago de Chile Dr Dan Cullen FPL, USDA Madison, WI, USA

Why sequence Ceriporiopsis subvermispora?

7ICEP-May15

The Ceriporiopsis subvermispora genome

Lignin peroxidase (LiP) from Ceriporiopsis genome Degradation of lignin model dimer by Ceriporiopsis LiP Degradation of synthetic lignin by Ceriporiopsis LiP

7ICEP-May15

The missing enzymes were identified from the genome

Content:

• 1. Introduction
• 2. Eucalypt decaying fungi and their genomes

Lignin vs polysaccharides decay (biopulping) Biodegradation of (pitch forming) wood lipids Why sequence basidiomycete genomes?

• 3. Oxidative enzymes for the eucalypt mill

Ligninolytic peroxidases The laccase-mediator system Towards industrial feasibility Biobleaching + pitch biocontrol

• 4. Recent studies: Enzymes for eucalypt biorefinery

Wood delignification for biofuel production The delignification process as shown by 2D-NMR A new generation of tailor-made enzymes

7ICEP-May15

Biotechnology for the Eucalyptus Biorefinery

FAD H400 H64 H111 H395 H456 C451 H450 H398 H452 H66 H109

T1 T1 T3 T3 T3 T3 T2 T2

H400 H64 H111 H395 H456 C451 H450 H398 H452 H66 H109

T1 T1 T3 T3 T3 T3 T2 T2

Arg343 His322 Heme Asp182 Glu405 Asp183 Glu41 Glu37 His177 Heme Trp172 Arg343 His322 Heme Asp182 Glu405 Arg343 His322 Heme Asp182 Glu405 Asp183 Glu41 Glu37 His177 Heme Trp172 Asp183 Glu41 Glu37 His177 Heme Trp172

heme-peroxidases flavooxidases multicopper laccases

Fungal oxidoreductases include peroxidases,

• xidases and laccases,

classified according to the nature of their cofactors:

• 1 heme molecule
• 1 FAD molecule
• 4 Cu ions
• 3. Oxidative enzymes for

the eucalypt mill

Enzymes have the advantage of being selective and easier to apply Xylanases for bleaching (and lipases) were the first enzymes introduced at the pulp mill The potential of oxidative enzymes in bleaching is higher since they act directly on the lignin

7ICEP-May15

Controversy on lignin-degrading enzymes: Answer from fungal genomes

Hemeperoxidases Class-II Twelve new genomes

Total 31 JGI fungal genomes compared: 344 Class-II and over 5000 oxidoreductases

The comparison of basidiomycete genomes provided evidence on the central role of peroxidases (LiP, VP and MnP families) in ligninolysis since the corresponding genes were present in all the sequenced lignin-degrading (white- rot) species and absent from all the cellulose-degrading (brown-rot) species

7ICEP-May15

FAD H400 H64 H111 H395 H456 C451 H450 H398 H452 H66 H109

T1 T1 T3 T3 T3 T3 T2 T2

H400 H64 H111 H395 H456 C451 H450 H398 H452 H66 H109

T1 T1 T3 T3 T3 T3 T2 T2

Arg343 His322 Heme Asp182 Glu405 Asp183 Glu41 Glu37 His177 Heme Trp172 Arg343 His322 Heme Asp182 Glu405 Arg343 His322 Heme Asp182 Glu405 Asp183 Glu41 Glu37 His177 Heme Trp172 Asp183 Glu41 Glu37 His177 Heme Trp172

heme-peroxidases flavooxidases laccases

Class-II (ligninolytic) peroxidases of the lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) families play a central role in natural degradation of lignin

class-II peroxidases

However, they present serious drawbacks (e.g. inactivation and low expression) that limit their industrial applicability In contrast, laccases (in presence of redox mediators) can be used for delignification due to stability, availability and lack of co-substrates

The heme channel (A) is not involved but both Mn(II) site (B) and lignin site (C) seem to

• xidize POM

POM reoxidation studied using 3 mutated variants: A) E140G/K176G B) E36A/E40A/D175A C) W164S SiW11MnIII Polyoxometalates (POM) are an efficient alternative to chlorine bleaching agents but some of them are difficult to be reoxidized

Three oxidation sites in VP

The filtrate from eucalypt pulp bleaching with SiW11MnIII was up to 98% reoxidized in 6 min when treated with VP and H2O2 50% ClO2 was saved by substituting a D stage in eucalypt bleaching by a VP- reoxidation stage

(a) (b) (a) (b)

POM turnover: a) pink SiW11MnIII; b) yellow SiW11MnII

MnIII

Versatile peroxidase in pulp bleaching (a peroxidase-based application)

Marques, G., J. A. F. Gamelas, F. J. Ruiz-Dueñas, J. C. del Río, D. V. Evtuguin, A. T. Martínez, and

• A. Gutiérrez. 2010. Delignification of eucalypt kraft pulp with manganese-substituted

polyoxometalate assisted by fungal versatile peroxidase. Bioresource Technol. 101:5935-5940.

7ICEP-May15

Cellulose Laccase

The laccase-mediator system (acting on cell-wall lignin)

The mediator radicals oxidize lignin at distance from the enzyme, accessing the narrow pores in plant cell-wall, and increasing its

• xidizing power due to the stability of mediator radicals (their

reduced forms being then oxidized again by the enzyme that uses O2 as final electron acceptor)

7ICEP-May15

 Bourbonnais et al (1990) Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation. FEBS Lett. 267:99-102

O2 DTPA H2SO4 H2O2 NaOH

Digestor Washing filters

O O

Filter Filter Filter Filter Filter

Stock Stock

Q Po P

To drying machine O2 O2

98ºC pH 11 98ºC pH 11 105ºC pH 11 98ºC pH 11

Na2S NaOH Stock Blow tank Wood chips

1) L-O-O-Q-PoP (control: O-O-Q-PoP)

1

2) O-O-L-Q-PoP (control: O-O-a-Q-PoP)

2

3) O-O-LQ-PoP (control: O-O-Q-PoP)

3

Laboratory bleaching reactors (ENCE)

In eucalypt TCF pulp, a laccase-mediator stage modifies residual lignin (NMR) and improves delignification (and bleaching when followed by a peroxide stage)

 discarded

• ther analyses (including 2D-NMR) 

7ICEP-May15

6,8 10,4 5,2 5,7 91,2 84,4 8,6

O-O-L-Q-PoP vs O-O-L/Q-PoP

4 8 12 16

Brown O-O O-O-L/Q O-O-L/Q-PoP Brown O-O O-O-L O-O-L-Q O-O-L-Q-PoP Brown O-O O-O-a O-O-a-Q O-O-a-Q-PoP Brown O-O O-O-Q O-O-Q-PoP 4 8 12 16

35 45 55 65 75 85 95

Brown O-O O-O-L/Q O-O-L/Q-PoP Brown O-O O-O-L O-O-L-Q O-O-L-Q-PoP Brown O-O O-O-a O-O-a-Q O-O-a-Q-PoP Brown O-O O-O-Q O-O-Q-PoP 55,9 87,9 35 45 55 65 75 85 95

Kappa number and brightness analyses of enzymatic and control bleaching sequences

O-O-L-Q-PoP

Best result:

Ibarra et al. 2006. Integrating laccase-mediator treatment into an industrial-type sequence for totally chlorine free bleaching eucalypt kraft pulp. J. Chem. Technol. Biotechnol. 81:1159-1165.

2D-NMR analysis 

7ICEP-May15 ..….. Controls ...….

Eucalypt lignin modification during O-O-L-Q-PoP sequence as shown by 2D-NMR of isolated lignin

O-O-L-E O-O-a-Q-PoP O-O-L-Q-PoP Brown O-O-a O-O-L

Aliphatic signals (lignin linkages)

O-O-L-E O-O-a-Q-PoP O-O-L-Q-PoP Brown O-O-a O-O-L

Aromatic signals (lignin units)

Eucalypt lignin modification during O-O-L-Q-PoP sequence as shown by 2D-NMR of isolated lignin

O-O-L-E O-O-a-Q-PoP O-O-L-Q-PoP Brown O-O-a O-O-L

• Eucalypt residual lignin is rich

in S units (>70%) and β-O-4' bonds (>75% side-chains)

• Enzymatic stage causes
• xidation of S units (>60%)
• Enzyme-altered lignin is

removed by alkali in PoP stage

• Therefore, the kappa number

decreases

• However, peroxide is necessary

for good brightness (>90% ISO)

Eucalypt lignin modification during O-O-L-Q-PoP sequence as shown by 2D-NMR of isolated lignin

Ibarra et al. 2007b. Structural modification of eucalypt pulp lignin in a totally chlorine free bleaching sequence including a laccase- mediator stage. Holzforschung 61:634-646.

7ICEP-May15

Natural vs synthetic laccase mediators

Most laccase mediators are compounds of synthesis, and their use increases the cost of the treatment (enzymes alone are already more expensive than fungal inocula) and introduce toxicity risks Screening of lignin-derived phenols as mediators has shown that these natural (potentially safe and cheap) compounds can be used together with laccase in eucalypt pulp biobleaching Syringaldehyde (SA) and methyl syringate (MS), two phenols forming stable radicals, provide some of the most interesting results in subsequent studies

HAA ABTS VIO HBT PZ TEMPO

• MS

SA

Camarero, S., D. Ibarra, M. J. Martínez, and A. T. Martínez. 2005. Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl. Environ.

• Microbiol. 71:1775-1784.

Camarero, S., D. Ibarra, A. T. Martínez, J. Romero, A. Gutiérrez, and J. C. del Río. 2007. Paper pulp delignification using laccase and natural mediators. Enzyme Microb. Technol. 40:1264-1271.

7ICEP-May15

Towards industrially-feasible delignification by treating eucalypt pulp with Myceliophthora thermophila laccase (MtL) and a phenolic mediator (MS)

Low-cost MtL can be used for eucalypt pulp delignification in combination with methyl syringate (MS) Interesting results were

• btained after lowering the

doses of both MtL and MS, which made enzymatic bleaching with laccase- mediator industrially-feasible

Babot et al. 2011. Towards industrially feasible delignification and pitch removal by treating paper pulp with Myceliophthora thermophila laccase and a phenolic mediator. Bioresource Technol. 102:6717- 6722

7ICEP-May15

Pilot-scale trials showed that an enzymatic stage using low-cost laccase and unexpensive phenolic mediator can be implemented in eucalypt pulp bleaching providing improvements in: 1) consumption of bleaching agents 2) control of pitch lipids Process scale-up at CTP pilot-plant (by Burnett et al)

Scheme of the CTP pilot plant used for enzymatic bleaching

Towards industrially-feasible delignification by treating eucalypt pulp with Myceliophthora thermophila laccase (MtL) and a phenolic mediator (MS)

7ICEP-May15

Enzymatic control of pitch lipids in pulps

HO

sitosterol

HO

campesterol

HO

stigmastanol

O OH O OH HO CH2OH

sitosteryl 3-β-D-glucopyranoside

O O

sitosteryl linoleate

CO-O-CH2 CO-O-CH CO-O-CH2

trilinolein

OH O

palmitic acid

One of the most interesting results

• btained in laccase-mediator treatment of

eucalypt pulps is the removal of pitch- forming lipids, which are not affected by lipases, by the same enzymatic stage removing lignin and improving brightness

Gutiérrez, A., J. C. del Río, D. Ibarra, J. Rencoret, J. Romero, M. Speranza,

• S. Camarero, M. J. Martínez, and A. T. Martínez. 2006a. Enzymatic removal
• f free and conjugated sterols forming pitch deposits in environmentally

sound bleaching of eucalypt paper pulp. Environ. Sci. Technol. 40:3416- 3422. Gutiérrez, A., J. C. del Río, J. Rencoret, D. Ibarra, and A. T. Martínez.

• 2006b. Main lipophilic extractives in different paper pulp types can be

removed using the laccase-mediator system. Appl. Microbiol. Biotechnol. 72:845-851. Gutiérrez, A., J. C. del Río, J. Rencoret, D. Ibarra, A. M. Speranza, S. Camarero, M. J. Martínez, and A. T. Martínez. 2008. Mediator-enzyme system for controlling pitch deposits in pulp and paper production. Patent (USA) 10080210393 / Patent (European) 2008, EP 1 908 876 A1.

7ICEP-May15

2.5 5 7.5 10 12.5 15 17.5 20 min

HO O O OH O O OH HO CH2O H

Lipid removal after O-O-L-Q-PoP and corresponding control

OOaQPoP Control

Blow tank Digestor Washing filters

O O

Filter Filter Stock O2 O2 98ºC pH 11 98ºC pH 11 Stock Filter Filter Filter Stock

Q Po P

To drying machine H2O2 NaOH

O2

DTPA H2SO4

105ºC pH 11 98ºC pH 11

Laboratory reactors (ENCE)

2.5 5 7.5 10 12.5 15 17.5 20 min

HO O O HO O

Blow tank Digestor Washing filters

O O

Filter Filter Stock O2 O2 98ºC pH 11 98ºC pH 11 Stock Filter Filter Filter Stock

Q Po P

To drying machine H2O2 NaOH

O2

DTPA H2SO4

105ºC pH 11 98ºC pH 11

OOLQPoP Laccase-HBT

Laboratory reactors (ENCE)

Pycnoporus cinnabarinus laccase (20 U/g) HBT (1.5%), pH 4, 2h, 50ºC

Lipid removal after O-O-L-Q-PoP and corresponding control

Pitch lipids degradation with laccase and natural mediators

Syringaldehyde (SAD) Acetosyringone (ACS) p-Coumaric acid (PCA) Laccase (20 U/g), pH 4, 12 h, 50 ºC, natural mediators (3%)

Gutiérrez, A., J. Rencoret, D. Ibarra, S. Molina, S. Camarero, J. Romero, J. C. del Río, and A. T. Martínez. 2007. Removal of lipophilic extractives from paper pulp by laccase and lignin-derived phenols as natural mediators. Environ. Sci. Technol. 41:4124-4129.

7ICEP-May15

Content:

• 1. Introduction
• 2. Eucalypt decaying fungi and their genomes

Lignin vs polysaccharides decay (biopulping) Biodegradation of (pitch forming) wood lipids Why sequence basidiomycete genomes?

• 3. Oxidative enzymes for the eucalypt mill

Ligninolytic peroxidases The laccase-mediator system Towards industrial feasibility Biobleaching + pitch biocontrol

• 4. Recent studies: Enzymes for eucalypt biorefinery

Wood delignification for biofuel production The delignification process as shown by 2D-NMR A new generation of tailor-made enzymes

7ICEP-May15

Biotechnology for the Eucalyptus Biorefinery

Enzymatic delignification (biofuel production) recent studies...

7ICEP-May15

Lignin content (%) 4 Cycles LEp

Nearly 50% lignin reduction!

8.6 % KL ControlEp - LEp treatment:

Trametes villosa laccase (and HBT as mediator)

Doses laccase 10 – 50 U/g Doses HBT 2.5 %

Laccase is not only able to attack lignin in pulp but also when applied directly on ground wood, as a biorefinery pretreatment

This resulted in improved hydrolyzability of wood 

7ICEP-May15

4 8 12 12 24 36 48 60

Ethanol / (g/L) Time (h) Time (h)

1 1 2 2 3 3 4 4 5 12 24 36 48 60 72

Glucose/Xylose (g/L) Time (h)

Xyl Glu

Time (h) Glucose/xylose (g/L)

… lignin modifications were "in situ" analyzed by 2D-NMR 

Eucalypt samples pretreated with laccase and mediator were further evaluated for saccharification and fermentation at VTT

Laccase -HBT Laccase -HBT Laccase -HBT

7ICEP-May15

Laccase Control

Interestingly, it is possible to analyze changes in lignin (and polysaccharides) by 2D-NMR without its isolation (whole wood swelled in dimethylsulfoxide-d6)  del Río talk

Chips Knife milling Ball milling Sawdust Powder gel in DMSO-d6

2D-NMR

wood HSQC δC

(ppm)

δH (ppm)

• f interest in enzymatic delignification 

Rencoret et al. 2009. HSQC-NMR analysis of lignin in woody (Eucalyptus globulus and Picea abies) and non-woody (Agave sisalana) ball-milled plant materials at the gel state. Holzforschung 63:691-698

7ICEP-May15

2D-NMR of the treated eucalypt (gel stage) revealed removal of lignin without changes in polysaccharides (blue signals)

CONTROL LMS-10 LMS-50

O O C H 3 H 3 C O O H O H O H 3 C O O C H 3 1 6 5 4 3 2 2 ' 6 ' 5 ' 4 ' 3 ' 1 ' a b g

A

O OCH3 OH 1 6 5 4 3 2 a

G

O OCH3 H3CO OH 1 6 5 4 3 2 a

S

7ICEP-May15

O OCH3 H3CO OH 1 6 5 4 3 2 a

S

O OCH3 H3CO O 1 6 5 4 3 2 a

S’

CONTROL LMS-10 LMS-50

 S' units have been identified (by HMBC NMR) as aromatic acids and ketones

(S' >60% lignin units!)

However, the most noticeable change is the increase of Ca-oxidized S units

Gutiérrez et al. 2012. Demonstration of laccase-mediator removal of lignin from wood and non-wood plant feedstocks. Bioresource Technol. 119:114-122

7ICEP-May15

Recently, wood lignin removal by a commercial laccase (MtL from Novozymes) and a phenolic mediator (MS) is investigated to increase the industrial feasibility

H400 H64 H111 H395 H456 C451 H450 H398 H452 H66 H109

H400 H64 H111 H395 H456 C451 H450 H398 H452 H66 H109

MtL MS The effect of MtL-Ms was being investigated by 2D-NMR directly on the treated (and control) wood and on the lignin (CEL) isolated from the treated (and control) wood 

7ICEP-May15

Reduced lignin content and increased mososaccharide release were obtained

Control MtL-MeS

Wood gels Isolated (CEL) lignins

Control MtL-MeS Rico et al. 2013. Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock. Biotechnol.Biofuels in press

7ICEP-May15

A new generation of tailor-made enzymes

Basic approaches to engineer industrial enzymes:

• Rational design by directed

mutagenesis based on structural, biochemical and sequence information

• Molecular evolution using

random mutagenesis, recombination and high- throughput screening

• Combination in semi-rational

approaches, including site- saturation mutagenesis, etc

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7ICEP-May15

Rational design

The ability to degrade lignin was conferred to a highly-stable peroxidase (Ceriporiopsis MnP6) by introducing a catalytic residue (surface tryptophan present in LiP and VP) Engineering a catalytic site in a stable enzyme is sometimes easier than improving the stability of the enzyme of interest For stabilization, directed evolution is often used 

Directed evolution is based on natural evolution (mutation+recombination+selection) but directs the process by artificial selection and reduces the time scale (from million years to months) by molecular biology and hightrougput methods

7ICEP-May15

Directed evolution

Directed evolution

Directed evolution

7ICEP-May15 the mutated residues are shown (sticks)

In this way, laccases with highly-increased thermal and pH stabilities, and better activities on target substrates (such as redox mediators for industrial delignification) have been obtained

CONCLUSIONS

 Biotechnological tools exist for both eucalypt

"biopulping" and "biobleaching" (the latter including biological control of pitch)

 Some of them have also shown their potential in

biofuel production from eucalypt wood

 Some commercial enzymes are already available

for these applications

 More efficient variants are under development

using enzyme engineering tools

7ICEP-May15

The studies were funded by several EU projects:

www.peroxicats.org www.lignodeco.com.br www.indoxproject.eu

7ICEP-May15

www.biorenew.org

Many thanks for your attention!