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Laccase BioBleachingReview

Matyas KosaGeorgia Institute of TechnologySchool of Chemistry and Biochemistry

2

OUTLINE• Introduction• Occurrence in microbes, structure of laccase &

active site, enzyme activity• Active site – substrate oxidation• Mediator resources• CHEMISTRY; oxidative bleaching reactions

between Laccase Mediator System (LMS) &– Lignin– Model compounds

• Process parameters while laccase bleaching• Residual lignins

3

INTRODUCTION

• Laccase BioBleaching could be an environment friendly alternative to conventional methods

• No oxidative degradation on carbohydrates, more pulp more paper

• Laccase “size-problems”, unable to diffuse into pulp fibers

• Mediators (ABTS)Chakar, F. S. (2000) Holzforschung 54: 647-653Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352Bourbonnais, R. P. (1992) Applied Microbiology and Biotechnology 36: 823-827

4

NATURAL OCCURRENCE• Trees: polymerization• Fungi: degradation or “rot”• Ascomycetes: soft rot, stain fungi• Basidiomycetes: white rot, brown rot• Selective for hemicelluloses/lignin in middle

lamella and secondary cell wall using natural mediators

• Enzymes of delignification:– Laccase– Lignin peroxidase, Manganese peroxidase,

versatile peroxidase– (aryl-alcohol oxidase/dehydrogenase, quinone

reductase) Martinez, A. T. (2005) International Microbiology 8: 195-204

5

PEROXIDASES• Oxidants must be:

– Strong enough to attack nonphenolic lignin structures– Small enough to penetrate lignin– Extracellular systems to produce H2O2 (required for enzyme

oxidation)

• Lignin Peroxidase (LiP): degrades nonphenolic units up to 90%, uses veratryl alcohol as “mediator”, “real ligninase” => high E0 (>1.4 V*)

• Mn Peroxidase (MnP): generates Mn3+ as a diffusible oxidizer (chelated by organic acids), that in turn generates peroxide radicals (and others: phenoxi etc)

• Versatile Peroxidase (VP): uses both aboveHammel, E. K. (2008) Current Opininon in Plant Biology 11: 349-355Hofrichter, M. (2002) Enzyme and Microbial Technology 30: 454-466

Smith, A. T. (2009) Proceedings of the National Academy of Sciences 106: 16084-16089*

6

LACCASE• Laccase = benzenediol: oxygen oxidoreductase (or p-

diphenol: dioxygen oxidoreductase) EC 1.10.3.2.

• It catalyzes the reduction of O2 to H2O while oxidizes (typically) a p-dihydroxy phenol or e.g. polyphenols and methoxy substituted phenols like lignin, but NOTtyrosine

• Electrode potential not enough to oxidize nonphenolic lignin -> mediators– Low potential: <470 mV– Medium pot.: 470 mV – 730 mV– High Pot.: >730 mV

Baldrian, P. (2006) FEMS Microbiology Reviews 30: 215-242Martinez, A. T. (2005) International Microbiology 8: 195-204Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412

Bourbonnais, R. P. (1990) FEBS Letters 267: 99-102

Laccase oxidizing veratryl alcohol?

Through ABTS!

7

ENZYME PROPERTIES, ACTIVITY• Only catalyze thermodynamically favorable

reactions towards an equilibrium between substrates and products @ given conditions (T, pH, starting conc. etc.)

• Enzyme activity => 1 U (unit): the amount of enzyme that catalyzes the conversion of 1 mol substrate /min (SI: 1 katal = 1 mol s-1)

• Kinetic parameters: kcat turnover number, KM Michaelis equilibrium const, kcat/KM; (Usually the larger kcat the better as well as for kcat/KM, however KM‘s value would need more discussion. Here the smaller the better…)

Fersht, A. (1999). Structure and mechanism in protein science. New York, W. H. Freeman and Company

8

LACCASE STRUCTURE

Garavaglia, S. C. (2004) Journal of Molecular Biology 342: 1519-1531Lyashenko, A. V. (2006) Acta Crystallographica Section F: Structural Biology and Crystallization CommunicationsF62: 954-957

3 domains all with -barrel topology.

9

FOLDS, ACTIVE-SITE POSITIONSURFACE OF LACCASE

binding pocket with 2,6-dimethoxyphenol

hydrophilic

hydrophobic

Active-site

Fold accommodate and enables connection between the binding site and the active site.

Active-site

binding pocket

Kallio, J. (2009) Journal of Molecular Biology 392: 895-909

Melanocarpus albomyces Laccase

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SO FAR…

• Selective delignification by white rot fungi• Ligninolytic enzymes: laccase, peroxidase• Laccase:

– 3 domains provide accommodation for the binding/active sites, efficiency is important kcat

– Relatively low E0 (three categories) but large size, hence MEDIATORS are needed

• Can be utilized to bleach Kraft-pulp

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BLEACHING CONSIDERATIONS I.

Kraft-pulp (washed)

Lignin: residual (native and Kraft)

Lignin model compounds

LaccaseLaccase Mediator System (LMS)

Changes in lignin (& carbohydrate)structures

+

LACCASE BIOBLEACHING

Bleached pulp

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• Laccase active site mechanism(s)• Mediator types• Laccase efficiency [E0 (?), kcat, KM…]• Parameters affecting efficiency and substrate

specificity• Laccase-Mediator-Lignin “oxidation-line”

chemistry, step-by-step, direct-indirect• Laccase production, mediator resources• Environment for bleaching and its efficacy

BLEACHING CONSIDERATIONS II.

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ACTIVE-SITE• Laccases are in the Multi Copper Oxidase

(MCO) family• All MCO’s contain four Cu ions in their active

sites:– 1 type 1 (T1) Cu, optic absorption @ 600 nm, causes

“blue” color in the enzyme solution, EPR active, substrate oxidation site

– 1 type 2 (T2) Cu, EPR active– 2 type 3 (T3) Cu, ions coupled through –OH bridge ->

diamagnetic, no EPR acivity, UV 330 nm detection• T2+(2)T3= trinuclear site of O2 reduction

to waterBaldrian, P. (2006) FEMS Microbiology Reviews 30: 215-242Quintanar, L. (2007) Accounts of Chemical Research 40: 445-452Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412

14

ACTIVE SITE STRUCTURE1. ENTRY or substrate

oxidation site w/ T1Cu, X is an axial ligand

2. His-Cys-His bridge that connects T1Cu to the trinuclear cluster ~13 Å

3. EXIT or O2 reduction site, T3Cu’s connected by –OH bridge ~4.3 Å

His

Cys

OH

HOH

e-

1.

2.

3. Baldrian, P. (2006) FEMS Microbiology Reviews 30: 215-242Quintanar, L. (2007) Accounts of Chemical Research 40: 445-452Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412

15

SUBSTRATE OXIDATION SITEPhe

Met

Planar-triagonal geometry: because of phenylalanine as axial ligand. When Met is absent Cys-Cu bond shows increased covalency (and ligand field strength).

Met-Cu -> long bond; Cys-Cu -> short bond => 4 coor-dinate (tetrahedral) T1 site.

3-coordinate T1 sites show substantially higher reduction potentials than 4-coordinate ones!

~0.7-0.8 V

~0.4-0.6 V

Substrate (S) + Cu++ => S. + Cu+

Quintanar, L. (2007) Accounts of Chemical Research 40: 445-452Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412

Site directed mutagenesis showed that the axial ligand of the T1 copper ion has no significant effect on redox potential of the T1 site of laccases! Then what does???

16

E0 & REACTIVITY• Factors, such as solvent accessibility, dipole orientation

and H-bonding will contribute to the tuning of E0!• Does E0 affect reactivity? NO. It specifies the type of

substrates that a given enzyme can oxidize (E0 has to be lower). Then what determines reactivity?

• Parameters associated with reactivity, efficiency (kcat):– Side-chains present in the binding site that enhance:

laccase-substrate complex formation, orientation of this complex for appropriate electron transfer (ET) towards T1

– More solvent exposed T1 site, easier access by substrate

– Changes in His-ligand distances to T1CuQuintanar, L. (2007) Accounts of Chemical Research 40: 445-452Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412

17

MECHANISM• Solvent exposed N: of His

and another Glu or Asp residue H-bond to the substrate

• The cleft otherwise is hydrophobic

• H+ is withdrawn by the acid

• e- is withdrawn by T1Cu through His and forwarded to the trinuclear center

Garzillo, A. M. (2001) Journal of Protein Chemistry 20: 191-201Garavaglia, S. C. (2004) Journal of Molecular Biology 342: 1519-1531Kallio, J. (2009) Journal of Molecular Biology 392: 895-909

2,6-DMP

Asp or GluActive-site Binding-site

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BRIDGE• Bridge is formed by a His-

Cys-His bridge• According to modeling

pathways after knowing the crystal structure:– e- goes through Cys-S,

Cys-C=O, H-bond, His-N’s then to the trinuclear cluster

• Electrons are used to reduce O2 to H2O

Garavaglia, S. C. (2004) Journal of Molecular Biology 342: 1519-1531Baldrian, P. (2006) FEMS Microbiology Reviews 30: 215-242Shleev, S. (2008) Angewandte Chemie International Edition 47: 7270-7274

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WHOLE ACTIVE SITE

O2 reductive cleavage, structure of radical containing active site intermediates. Discovered by EPR, Quantum and molecular mechanical studies.

Shin, W. (1996) Journal of the American Chemical Society 118: 3202-3215Palmer, A. E. (2001) Journal of the American Chemical Society 123: 6591-6599Solomon, E. I. (2001) Angewandte Chemie International Edition 40: 4570-4590Lee, S.-K. (2002) Journal of the American Chemical Society 124: 6180-6193Solomon, E. I. (2004) Chemical Reviews 104: 419-458Rulisek, L. (2005) Inorganic Chemistry 44: 5612-5628Quintanar, L. (2007) Accounts of Chemical Research 40: 445-452

Research Groups

Shleev, S. (2006) Biochimie 88: 1275-1285Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412Shleev, S. (2008) Angewandte Chemie International Edition 47: 7270-7274

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PREDICTED “MECHANISM”0: oxidized resting state1: fully reduced enzyme2: peroxide intermediate3: native intermediate 14: native intermediate 2

Shleev, S. (2006) Biochimie 88: 1275-1285Rulisek, L. (2005) Inorganic Chemistry 44: 5612-5628

0

1 2

34 Not fully understood, states 0-2 are observed + in 4 all Cu is 2+

1-4: 02 + 4e- + 4H+ = 2H2O

21

EFFECTS OF pH• Non-phenolic substrates

loose only electron, however as pH increases HO- will bind to the trinuclear cluster decreasing activity: linear dependence on pH

• Phenolics release H+, as the pH increases more phenoxy compounds -> higher activity. Then as pH increases more the above effect kicks in:Bell shapedpH profile

Non-phenolic substrate e.g. ABTS

Phenolic substrate e.g. 2,6-DMP

22

ACTIVE-SITE SUMMARY

Entry site: substrate is oxidized while T1 site is reduced (x4)

T1Cu: catalytic activity depends on multiple factors but not E0

H-C-H:e- are transported from T1 to trinuclear cl.

Exit site: Trinuclear cluster, O2molecule binds in and through the peroxide and native states gets reduced to 2 water molecules with 4 e- transported from substrates

Reaction with 2,6-DMPfungi E0 [mV] kcat [1/s]

T.t. 790 109

T.p. 742 24000

P.o. 740 120100

R.l. 730 7400

Other substrate -> different activities!

T and pH optimum!

23

LACCASE SIZE PROBLEMS

Archibald, F. S. (1997) Journal of Biotechnology 53: 215-236

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ACTIVITY PROPERTIES• Activity really depends on the following factors:

– Side-chains present in the binding site that enhance: laccase-substrate complex formation, orientation of this complex for appropriate electron transfer (ET) towards T1

– More solvent exposed T1 site, easier access by substrate– Changes in His- distances to T1Cu

• Laccase cannot reach lignin in cell-walls

Can it be that laccase evolve(-d) to oxidize “small” molecules (mediators) by increasing its active site E0 and specifically changes its binding/active site structures for enhanced electron transfer?! “Host-range” mutation, where range is not lignin but the most abundant relatively high E0 mediator…

25

BLEACHING CONSIDERATIONS III

• Lignin model compounds• Mediators• Native lignin in pulp• Kraft lignin• Residual lignin• In bleaching: mediator(s) and

residual lignin after Kraft cycle

SUBSTRATE OXIDATION:

Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390

26

LACCASE OR LMS• Using lignin from totally different sources

with different kind of mediators and laccase preparations, and combining these in basically every possible way, the results show:

• IF ONLY LACCASE IS USED THE LIGNIN WILL POLYMERIZE

• IF LACCASE MEDIATOR SYSTEM (LMS) IS USED THEN THE LIGNIN WILL DEPOLYMERIZE

Shleev, S. (2006) Enzyme and Microbial Technology 39: 841-847

27

NATURAL MEDIATORS

• Secreted extracellularly by fungi• Present in situ as common secondary plant

metabolites• Released in large amounts during the

microbial degradation of lignocellulose

acetosyringone syringaldehyde acetovanillone

E0 [mV] vs. SCE*

*Standard Calomel Electrode

534 542

Gonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629

28

SYNTHETIC MEDIATORS

• These compounds are target substrates of laccases

• They can mediate lignin or veratryl alcohol (VA) oxidation only after being oxidised bylaccase or an electrode

E0 [mV] vs. SCE*

*Standard Calomel ElectrodeGonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629

2,2’-azino-bis(3-ethyl-benzothiazoline-6-sulphonic acid) ABTS

1-hydroxybenzotriazoleHBT

Violuric acidVLA

663441

29

ELECTROCHEMISTRY• Cyclic voltammetry: with

0.2 mM ABTS in pH=4 buffer the potential of the electrochemical cell is continuously increased by 20 mV/s until 1000 mV is reached vs AG/AgCl electrode

• Then E is decreased with same rate

• Current is monitoredE (413 mV) -> large & ic/ia ~ 1 shows stabile intermediates!

ABTS ABTS+.

ABTS2+

ABTS+.ABTS

ABTS2+ic

ia

Ea = anodic-oxidation potential

Ec

Ea

Current [A]

Potential [mV]

(Ec + Ea)/2=E0 vs St.El.

Ea

Ec = cathodic-reduction potential

ic


30

ABTS-VA

ia ~2x ia(ABTS)

ia(ABTS)

ia(ABTS+VA)

* Homogeneous redox catalysis (HRC): 1. electrochemical generation of a chemically stable molecular oxidant-> 2. diffuse into solution able to oxidize the substrate in place of the electrode. It is usually possible to carry out oxidation with a smaller overpotential than required directly

Reaction is driven by the irreversible two electron oxidation of VA to V-aldehyde. Regenerates cation radical.

Ele

ctro

de s

urfa

ce

(E0=1175 mV)

Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390Andrieux C. P. (1986) Journal of Electroanalytical Chemistry 205: 43-58

31

HBT-VA

hydrogen atom transfer

ia/ic large: way more than 1, because HBT is instabile or because of a different reduction -> like HAT. There are evidence for both reasoning.

Gonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390

32

MEDIATOR CATALYTIC EFFICIENCY (CE)• ia(ABTS+VA)= ik anodic

peak current (catalytic current) of the compound acting as catalyst in the presence of substrate

• ia(ABTS)= ic is the diffusion controlled peak current of the catalyst

• k will be proportional to ik/ic and it describes CE

ia(ABTS)

ia(ABTS+VA)


33

MEDIATOR vs. ENHANCER• Most compounds described as

laccase-mediators are not strictly redox mediators, since their oxidized intermediates are electrochemically unstable

• Consequently, only a small number of redox cycles occur during their catalytic oxidation

• These compounds have to be continually replenished in the media hence the term ‘laccase enhancer’ is more precise


HBT: enhancer, continuous presence of potential is required!


34

MEDIATOR (ABTS)-LACCASE

• Laccase efficiently oxidizes ABTS mainly to ABTS+.

• There is oxidation of VA @ laccase E (585 mV), however it is really slow


35

CE ON VA AND KL• Both groups

measured the catalytic efficiency (CE) of the given mediators and enhancers on both the model compound VA and on Klason lignin (KL) as well


36

“BEST” MEDIATORS• Electrodes were used and higher potentials than

laccase could produce!• HOWEVER: The redox potential of the

mediators seems to play a negligible role in the catalytic efficiency of lignin oxidation; their effectiveness is likely to depend on the chemical reactivity of the radical formed after their initial step of oxidation.

• Oxidation of the different components of lignin activate cascade reactions between phenolic and non-phenolic compounds!=>further oxidation


37

A DIFFERENT APPROACH• Between 1984 and 1988 Higuchi and his group

conducted oxidation-degradation experiments on lignin model compounds with fungal extracellular media

• They used mass-spectrometry to analyze reaction products

• Different models (synthesized with 18O @ different positions) were used in H2

18O and H2O to figure out cleavage mechanisms

• Later they began to use mediators as well, but stayed with the same analytical logic/methods

Umezawa, T. (1984) Agricultural Biological Chemistry 48: 1917-1921Kawai, S. (1985) Agricultural Biological Chemistry 49(8): 2325-2330Kawai, S. (1988)Archives of Biochemistry and Biophysics 262: 99-110

Kawai, S. (1988) FEBS Letters 236: 309-311

38

OXIDATION MECHANISM 1. Formation of radicals

I. 1,3-dihydroxy-2-(2,6-dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl) propane

I.

II. 2-(2,6-dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)-3-hydroxypropanonepropane

II.

III. 1-(4-ethoxy-3-methoxyphenyl)-3-hydroxypropanonepropane

III.a b c

Aryl cation radicalBenzylic radical

Kawai, S. (2002) Enzyme and Microbial Technology 30: 482-489

39

OXIDATION MECHANISM 2. C-C cleavage

d

IV. 2,6-DMP

IV.

V. 4-ethoxy-3-methoxybenzoic acid

V.


“generating” a mediator

NON-PHENOLIC IN ALL CASES!

40

OXIDATION MECHANISM 3. -ether cleavage

b c

VI. 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane

VI.


41

OXIDATION MECHANISM 4. aromatic ring (phenolic)

Kawai, S. (1988) FEBS Letters 236: 309-311

4,6-di(tert-butyl)guaiacol

muconic acid methyl ester (MAME)

muconolactone

Can be found in lot of literature on lignin degradation with laccase.

NO AROMATICS-> NO QUINONES (CHROMOPHORES)-> NO COLOR

42

OXIDATION MECHANISM 5. aromatic ring (non-phenolic)

a

b

VII. 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane-2,3-cyclic carbonateVIII. 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane-1,2-cyclic carbonate

VII.

VIII.

Kawai, S. (2002) Enzyme and Microbial Technology 30: 482-489 Umezawa, T. (1987) FEBS Letters 218: 255-260

NO AROMATICS-> NO QUINONES (CHROMOPHORES)-> NO COLOR

43

LMS BLEACHING OPERATION• Laccase Mediator System (LMS) bleaching

stage is assigned: LA• Alkali extraction stage, usually NaOH: E• Bleaching efficiency is measured with: KAPPA-

number (), brightness (b), delignification % (d%) pulp & paper physical properties (e.g. tear index)

• Influencing parameters: T, pH, time (t), pulp source, laccase conc., mediator conc., O2pressure (pO2) and number of consecutive stages

44

BOURBONNAIS-PAICE-ABTS

• Observations of Bourbonnais and Paice, with the following conditions: 10% washed Kraft-pulp consistency, 2h, pH 5, 300 kPa (~3 Atm) O2, 1% ABTS and 5U enzyme per g pulp

• Physical properties -> minimal change, except slight 2% decrease in tear index

starting ~17% (SW) decreases 25% after LA-E and by 55% if stages are repeated!Sulfite-pulp it is 50% after only LA-E!

Bourbonnais, R. (1996) TAPPI Journal 79: 199-204

45

BOURBONNAIS-PAICE-ABTS

Bourbonnais, R. (1996) TAPPI Journal 79: 199-204

t[0.5, 1, 2, 4 h] pH [3-6] T

[22-80°C]Laccase

[1-25 U/g]ABTS

[0.1-2.5%]

LA ~ 2% decrease from

17->15optimum @ 5 optimum @ 60 optimum around

5the higher the better: 2.5

LA-E ~ 2% decrease from 14->12

optimum @ 5 optimum @ 50 optimum around

1the higher the better: 2.5

bLA only slight increase

~0.5%

LA-E ~2% increase

d%LA 1.7->10.5 continuous

LA-E 16->27 continuous

With pO2 the experience is it doesn’t really effect above 100 kPa so there’s no reason to go higher.

All seems really good except that ABTS is pretty expensive. How about other mediators?

46

OTHER SYNTHETIC MEDIATORS

• Delignification with laccase-HBT LMS can remove 20-30% more lignin than ABTS LMS

• LA-E with NHA, HBT and VLA removed 19, 20 and 37 % of lignin from the starting pulp respectively

• VLA reacts with C5-condensed phenolicunits as well!

N-acetyl-N-phenylhydroxylamine (NHA)

HBT

VLA

Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352

47

NATURAL MEDIATORS• LA-E with PCA actually

increased , while SA and AS decreased it with ~1.5 % however they both underperformed HBT (~4%)

• Brightness increased ~15%, but still under HBT (25%)

acetosyringone AS

syringaldehydeSA

p-coumaric acidPCA

Camarero, S. (2007) Enzyme and Microbial Technology 40: 1264-1271

48

E-STAGE EFFECTThe alkaline (E) stage isn’t just an extraction stage, it enhances brightness by reacting with quinones as well.BAR = benzylic acid rearrangement.

Moldes et al tested what happens if the E stage is replaced by a peroxide (P) stage. The result: decrease is less than half compared to E, but the brightness increased by 16% (12% in E).

Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352Moldes, D. (2008) Bioresource Technology 99: 8565-8570

NO QUINONES (CHROMOPHORES)-> NO COLOR

49

RESIDUAL LIGNIN

• Isolation procedure: 4.15 w/V% solids (pulp) in solution of 9:1 p-dioxane:water, 2 h of boiling then double filtration, pH neutral with NaHCO3 -> evaporation under reduced pressure to ~10% of solution. Water addition and 1N HCl to pH 2.5, then filtration and wash.

• Analysis by NMR or Pyrolysis GC-MSSealey, J. (1998) Enzyme and Microbial Technology 23: 422-426Chakar, F. S. (2000) Holzforschung 54: 647-653Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352

50

RESIDUAL STRUCTURES-1. before LMS: between 70-100 (10-14%) after LMS: down to around 40-50 (6-7%)

• LA-E with ABTS then 13C-NMR, Sealey et al:

-COOHC-3,4 ofsubstituted G

units

C-3,4 of G anddemethylated G

units

-O-aryl C MeO-

Change ~13% growth, see BAR @ E stage ~20% decrease ~37% decrease Slight

Increase~10%

decrease

Even better if an O2 (O) stage is included after pulping!Sealey, J. (1998) Enzyme and Microbial Technology 23: 422-426Chakar, F. S. (2000) Holzforschung 54: 647-653Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352

51

RESIDUAL STRUCTURES-2.• Chakar et al (2000), 31P-NMR:

– If E, P, O stages are used w/ HBT brightness is really enhanced (~6-7%)

– ~50% increase in –COOH vs brownstock (BS), ~7.5% increase vs only E-stage applied

– Phenolic –OH in C5-noncondensed: 2.5 times the decrease in E-P-O vs E an overall 42%

– Phenolic –OH in C5-condensed: 4.5 times the decrease in E-P-O vs E an overall 37%

– Aliphatic –OH slight increase

Chakar, F. S. (2000) Holzforschung 54: 647-653

52

RESIDUAL STRUCTURES-3.• Chakar et al (2004), 31P-NMR:

– LA-E with HBT and VLA– -COOH: 40% (HBT) 70% (VLA) increase vs

BS– Noncondensed C5: HBT 11%, VLA 41%

decrease vs BS– Condensed C5: HBT 2.5%, VLA 15%

decrease vs BS– Aliphatic: HBT 6%, VLA 12% decrease vs BS-O-aryl: HBT 1-2%, VLA 10% increase vs BS

– Methoxyl: HBT 7%, VLA 6% decrease vs BSChakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352

53

SUMMARY ON RESIDUAL LIGNIN• All results will strongly depend on what stages are

included either before (O) or after (E, P, O) the LA stage as well as the type of mediator used! Repeating stages will increase efficiency as well!

• -COOH content will considerably increase, see E-stage chemistry

• The use of LA-E-P-O or VLA as mediator will significantly increase the removal of C5 condensed units

• Significant decrease in noncondensed C5 units, less significant decrease in methoxyl and aliphatic-OH groups

• Slight increase in -O-aryl structures• Around 30% increase in S/G ratio with both natural

(SA) and synthetic (HBT) mediatorsCamarero, S. (2007) Enzyme and Microbial Technology 40: 1264-1271Moldes, D. (2008) Bioresource Technology 99: 8565-8570Bourbonnais, R. (1996) TAPPI Journal 79: 199-204

Sealey, J. (1998) Enzyme and Microbial Technology 23: 422-426Chakar, F. S. (2000) Holzforschung 54: 647-653Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352

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