Laccase BioBleaching Review

Matyas Kosa Georgia Institute of Technology School of Chemistry and Biochemistry 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

2 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: 647-653 Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352 Bourbonnais, R. P. (1992) Applied Microbiology and Biotechnology 36: 823-827 3 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 4 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 above

Hammel, E. K. (2008) Current Opininon in Plant Biology 11: 349-355 Hofrichter, M. (2002) Enzyme and Microbial Technology 30: 454-466

Smith, A. T. (2009) Proceedings of the National Academy of Sciences 106: 16084-16089* 5 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 NOT tyrosine • Electrode potential not enough to oxidize nonphenolic lignin -> mediators – Low potential: <470 mV – Medium pot.: 470 mV – 730 mV – High Pot.: >730 mV Laccase oxidizing veratryl alcohol? Baldrian, P. (2006) FEMS Microbiology Reviews 30: 215-242 Martinez, A. T. (2005) International Microbiology 8: 195-204 Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412 Through ABTS! Bourbonnais, R. P. (1990) FEBS Letters 267: 99-102 6 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 7 LACCASE STRUCTURE

3 domains all with -barrel topology.

Garavaglia, S. C. (2004) Journal of Molecular Biology 342: 1519-1531 Lyashenko, A. V. (2006) Acta Crystallographica Section F: Structural Biology and Crystallization Communications F62: 954-957 8 FOLDS, ACTIVE-SITE POSITION Melanocarpus albomyces Laccase SURFACE OF LACCASE

Active-site hydrophilic

hydrophobic

binding pocket with 2,6-dimethoxyphenol

binding pocket

Fold accommodate and enables connection between the binding site Active-site and the active site. Kallio, J. (2009) Journal of Molecular Biology 392: 895-909 9 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

10 BLEACHING CONSIDERATIONS I.

Kraft-pulp (washed) Lignin: residual (native and Changes in lignin Kraft) (& carbohydrate) Lignin model compounds structures + Laccase Bleached pulp Laccase Mediator System (LMS)

LACCASE BIOBLEACHING

11 BLEACHING CONSIDERATIONS II.

• 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

12 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 water Baldrian, P. (2006) FEMS Microbiology Reviews 30: 215-242 Quintanar, L. (2007) Accounts of Chemical Research 40: 445-452 Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412 13 ACTIVE SITE STRUCTURE 1. His 1. ENTRY or substrate oxidation site w/ T1Cu, Cys X is an axial ligand 2. 2. His-Cys-His bridge that connects T1Cu to the trinuclear cluster ~13 Å e- 3. EXIT or O2 reduction OH site, T3Cu’s connected by –OH bridge ~4.3 Å

HOH 3. Baldrian, P. (2006) FEMS Microbiology Reviews 30: 215-242 Quintanar, L. (2007) Accounts of Chemical Research 40: 445-452 Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412 14 SUBSTRATE OXIDATION SITE Phe Planar-triagonal geometry: because of phenylalanine as ~0.7-0.8 V axial ligand. When Met is absent Cys-Cu bond shows increased covalency (and ligand field strength). Met Met-Cu -> long bond; Cys- Cu -> short bond => 4 coor- dinate (tetrahedral) T1 site. ~0.4-0.6 V 3-coordinate T1 sites show substantially higher reduction potentials than 4- . Substrate (S) + Cu++ => S + Cu+ coordinate ones!

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??? Quintanar, L. (2007) Accounts of Chemical Research 40: 445-452 Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412 15 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 T1Cu Quintanar, L. (2007) Accounts of Chemical Research 40: 445-452 Morozova, O. V. (2007) Biochemistry (Moscow) 72: 1396-1412 16 MECHANISM

• Solvent exposed N: of His and another Glu or Asp Active-site Binding-site residue H-bond to the Asp or Glu substrate • The cleft otherwise is hydrophobic •H+ is withdrawn by the acid •e- is withdrawn by T1Cu through His and forwarded to the 2,6-DMP trinuclear center

Garzillo, A. M. (2001) Journal of Protein Chemistry 20: 191-201 Garavaglia, S. C. (2004) Journal of Molecular Biology 342: 1519-1531 Kallio, J. (2009) Journal of Molecular Biology 392: 895-909 17 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: 1519-1531 Baldrian, P. (2006) FEMS Microbiology Reviews 30: 215-242 Shleev, S. (2008) Angewandte Chemie International Edition 47: 7270-7274 18 WHOLE ACTIVE SITE

Research Groups

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

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

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

19 PREDICTED “MECHANISM” 120: oxidized resting state 1: fully reduced enzyme 0 2: peroxide intermediate 3: native intermediate 1 4: native intermediate 2

4 3 Not fully understood, states 0-2 are observed + in 4 all Cu is 2+

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

Shleev, S. (2006) Biochimie 88: 1275-1285 Rulisek, L. (2005) Inorganic Chemistry 44: 5612-5628 20 EFFECTS OF pH Non-phenolic substrate e.g. ABTS • 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 Phenolic substrate e.g. 2,6-DMP phenoxy compounds -> higher activity. Then as pH increases more the above effect kicks in: Bell shaped pH profile

21 ACTIVE-SITE SUMMARY T and pH optimum!

Reaction with 2,6-DMP

fungi 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!

Entry site: T1Cu: H-C-H: Exit site: Trinuclear cluster, O2 substrate is catalytic e- are molecule binds in and through oxidized activity transported the peroxide and native states while T1 site depends from T1 to gets reduced to 2 water is reduced on multiple trinuclear cl. molecules with 4 e- transported (x4) factors but from substrates

not E0 22 LACCASE SIZE PROBLEMS

Archibald, F. S. (1997) Journal of Biotechnology 53: 215-236 23 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…

24 BLEACHING CONSIDERATIONS III SUBSTRATE OXIDATION: • Lignin model compounds • Mediators • Native lignin in pulp • Kraft lignin • Residual lignin • In bleaching: mediator(s) and residual lignin after Kraft cycle

Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390 25 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 26 NATURAL MEDIATORS

acetosyringone syringaldehyde acetovanillone

534 542 E0 [mV] vs. SCE* • Secreted extracellularly by fungi • Present in situ as common secondary plant metabolites • Released in large amounts during the microbial degradation of lignocellulose

Gonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629 *Standard Calomel Electrode 27 SYNTHETIC MEDIATORS

2,2’-azino-bis(3-ethyl-benzothiazoline 1-hydroxybenzotriazole Violuric acid -6-sulphonic acid) ABTS HBT VLA

E0 [mV] vs. SCE* 441 663

• These compounds are target substrates of laccases • They can mediate lignin or veratryl alcohol (VA) oxidation only after being oxidised by laccase or an electrode

Gonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629 *Standard Calomel Electrode 28 ELECTROCHEMISTRY • Cyclic voltammetry: with Current [A] 0.2 mM ABTS in pH=4 ABTS2+

+. buffer the potential of the Ea ABTS ABTS

electrochemical cell is Ea i continuously increased by a 20 mV/s until 1000 mV is ic 2+ ic ABTS reached vs AG/AgCl Ec ABTS+. electrode ABTS • Then E is decreased with same rate Potential [mV]

• Current is monitored Ea = anodic-oxidation potential

E (413 mV) -> large & Ec = cathodic-reduction potential

ic/ia ~ 1 shows stabile (Ec + Ea)/2=E0 vs St.El. intermediates! Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390 29 ABTS-VA

ia(ABTS+VA) ia ~2x ia(ABTS)

ia(ABTS)

(E0=1175 mV) Electrode surface

* 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. Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390 Andrieux C. P. (1986) Journal of Electroanalytical Chemistry 205: 43-58 30 HBT-VA

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

Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390 Gonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629 31 MEDIATOR CATALYTIC EFFICIENCY (CE)

•ia(ABTS+VA)= ik anodic peak current (catalytic current) of the compound i (ABTS+VA) a acting as catalyst in the presence of substrate •i(ABTS)= i is the ia(ABTS) a c diffusion controlled peak current of the catalyst • k will be proportional to

ik/ic and it describes CE

Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390 Gonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629 32 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!

Gonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629 Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390 33 MEDIATOR (ABTS)-LACCASE

• Laccase efficiently oxidizes ABTS mainly to ABTS+.

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

Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390 34 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

Gonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629 Bourbonnais, R. (1998) Biochimica et Biophysica Acta 1379: 381-390 35 “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

Gonzalez Arzola, K. (2009) Electrochimica Acta 54: 2621-2629 36 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 @ 18 different positions) were used in H2 O 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: 1917-1921 Kawai, S. (1985) Agricultural Biological Chemistry 49(8): 2325-2330 Kawai, S. (1988) FEBS Letters 236: 309-311 Kawai, S. (1988)Archives of Biochemistry and Biophysics 262: 99-110 37 OXIDATION MECHANISM 1. Formation of radicals Aryl cation radical Benzylic radical I. 1,3-dihydroxy-2-(2,6- dimethoxyphenoxy)-1-(4-ethoxy- 3-methoxyphenyl) propane

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

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

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

a b c II. III. 38 OXIDATION MECHANISM 2. C-C cleavage

NON-PHENOLIC IN ALL CASES!

IV.

d

IV. 2,6-DMP “generating” a mediator V.

V. 4-ethoxy-3-methoxybenzoic acid

Kawai, S. (2002) Enzyme and Microbial Technology 30: 482-489 39 OXIDATION MECHANISM 3. -ether cleavage

VI. b c

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

Kawai, S. (2002) Enzyme and Microbial Technology 30: 482-489 40 OXIDATION MECHANISM 4. aromatic ring (phenolic)

4,6-di(tert-butyl) guaiacol muconic acid muconolactone methyl ester (MAME) Can be found in lot of literature on lignin degradation with laccase. NO AROMATICS-> NO QUINONES (CHROMOPHORES)-> NO COLOR

Kawai, S. (1988) FEBS Letters 236: 309-311 41 OXIDATION MECHANISM 5. aromatic ring (non-phenolic)

VII. a

b VIII. VII. 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane-2,3-cyclic carbonate VIII. 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane-1,2-cyclic carbonate NO AROMATICS-> NO QUINONES (CHROMOPHORES)-> NO COLOR Kawai, S. (2002) Enzyme and Microbial Technology 30: 482-489 Umezawa, T. (1987) FEBS Letters 218: 255-260 42 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., O2 pressure (pO2) and number of consecutive stages

43 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 44 BOURBONNAIS-PAICE-ABTS t T Laccase ABTS pH [3-6] [0.5, 1, 2, 4 h] [22-80°C] [1-25 U/g] [0.1-2.5%]

~ 2% decrease from optimum @ optimum around the higher the optimum @ 60 LA 17->15 5 5 better: 2.5  ~ 2% decrease from optimum @ optimum around the higher the optimum @ 50 LA-E 14->12 5 1 better: 2.5

only slight increase LA ~0.5% b With pO2 the experience is it doesn’t LA-E ~2% increase really effect  above 100 kPa so there’s no reason to go higher. LA 1.7->10.5 continuous d% All seems really good except that LA-E 16->27 continuous ABTS is pretty expensive. How about other mediators?

Bourbonnais, R. (1996) TAPPI Journal 79: 199-204 45 OTHER SYNTHETIC MEDIATORS

N-acetyl-N- • Delignification with laccase- phenylhydr HBT LMS can remove 20- oxylamine (NHA) 30% more lignin than ABTS LMS • LA-E with NHA, HBT and HBT VLA removed 19, 20 and 37 % of lignin from the starting pulp respectively • VLA reacts with C5- VLA condensed phenolic units as well!

Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352 46 NATURAL MEDIATORS

acetosyringone • LA-E with PCA actually AS increased , while SA and AS decreased it with

syringaldehyde ~1.5 % however they SA both underperformed HBT (~4%)

• Brightness increased p-coumaric acid ~15%, but still under PCA HBT (25%)

Camarero, S. (2007) Enzyme and Microbial Technology 40: 1264-1271 47 E-STAGE EFFECT

The 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). NO QUINONES (CHROMOPHORES)-> NO COLOR

Moldes, D. (2008) Bioresource Technology 99: 8565-8570 Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352 48 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-MS

Sealey, J. (1998) Enzyme and Microbial Technology 23: 422-426 Chakar, F. S. (2000) Holzforschung 54: 647-653 Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352 49 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:

C-3,4 of C-3,4 of G and -O-aryl -COOH substituted G demethylated G MeO- C units units

~13% growth, see Slight ~10% Change ~20% decrease ~37% decrease BAR @ E stage Increase decrease

Even better if an O2 (O) stage is included after pulping! Sealey, J. (1998) Enzyme and Microbial Technology 23: 422-426 Chakar, F. S. (2000) Holzforschung 54: 647-653 Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352 50 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 51 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 BS Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352 52 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) mediators

Camarero, S. (2007) Enzyme and Microbial Technology 40: 1264-1271 Sealey, J. (1998) Enzyme and Microbial Technology 23: 422-426 Moldes, D. (2008) Bioresource Technology 99: 8565-8570 Chakar, F. S. (2000) Holzforschung 54: 647-653 Bourbonnais, R. (1996) TAPPI Journal 79: 199-204 Chakar, F. S. (2004) Canadian Journal of Chemistry 82: 344-352 53