Structural Modifications of
Lignosulphonates
Dimitri Areskogh
Doctoral Thesis Royal Institute of Technology School of Chemical Science and Engineering Department of Fibre & Polymer Technology Division of Wood Chemistry and Pulp Technology Stockholm 2011
AKADEMISK AVHANDLING Som med tillstånd av Kungliga Tekniska Högskolan framläggs till offentlig granskning för avläggande av teknologie doktorsexamen, fredagen den 13 maj 2011 kl. 10.00 i sal D3, Lindstedsvägen 3, KTH, Stockholm. Avhandlingen försvaras på engelska. Fakultetsopponent: Prof. Arthur J Ragauskas Georgia Institute of Technology, Atlanta, Georgia, USA © Dimitri Areskogh Stockholm 2011 TRITA‐CHE Report 2011:26 ISSN 1654‐1081 ISBN 978‐91‐7415‐923‐3
“Reading is the basics for all learning.” —Presidential candidate George W. Bush, Reston, Virginia, March 28, 2000
Abstract
Lignosulphonates are by‐products from the sulphite pulping process for the manufacture of specialty dissolving pulps and paper. During the liberation of the cellulose, the lignin is fractionated and solubilised through covalent addition of sulphonic acid groups at various positions in the structure. The formed sulphonated lignin, lignosulphonate is then further isolated and refined. The amphiphilic nature of lignosulphonates has enabled them to be used as additives to various suspensions to improve their dispersion and stability. The by far largest utilisation of lignosulphonates is as dispersants in concrete. Here, lignosulphonates act by dispersing cement particles to prevent flocculation, un‐even particle distribution and reduced strength development. The dispersion is achieved through steric and electrostatic repulsion of the cement particles by the lignosulphonate polymer. This behaviour is intimately linked with the overall size and amount of charged groups in the dispersing polymer. Traditional modifications of lignosulphonates have been limited to removal of sugars, filtration and fractionation. These modifications are not sufficient for utilisation of lignosulphonates in high‐strength concrete. Here synthetic dispersants and superplasticisers are used which are considerably more efficient even at low dosages. To compete with these, additional modifications of lignosulphonates are likely to be necessary. The molecular weight and functional group composition have been identified and described as the most interesting parameters that can be modified. Currently, no suitable method exists to increase the molecular weight of lignosulphonates. Oxidation by the natural radical initiating enzyme laccase is an interesting tool to achieve such modifications. In this thesis several aspects of the mechanism through which this enzyme reacts with lignin and lignosulphonate structures have been elucidated through model compound studies. Further studies showed that laccase alone was a highly efficient tool for increasing the molecular weight of commercial lignosulphonates at low dosages and in short incubation times. Immobilisation of the laccase to a solid support to enable re‐utilisation was also investigated. Modification of functional group composition of lignosulphonates was achieved through ozonolysis and the Fenton’s reagent, a mixture of hydrogen peroxide and iron(II)acetate. Introduction of charged carboxylic groups was achieved through opening of the benzyl rings of lignosulphonates. It was found that a two‐stage process consisting of laccase oxidation followed by ozonolysis was an efficient technique to create a polymer enriched with carboxylic acid groups with a sufficient molecular size. Oxidation by the Fenton’s reagent was shown to yield similar modifications as the combined laccase/ozonolysis treatment albeit with less pronounced results but with a large level of control through variation of a number of reaction parameters. The Fenton’s reagent can therefore be an interesting alternative to the aforementioned two‐stage treatment. These modifications are interesting for large‐scale applications not only because of their simplicity in terms of reaction parameters but also because of the ubiquity of the used enzyme and the chemicals in the pulp and paper industry.
Sammanfattning
Lignosulfonater är bi‐produkter från sulfitprocessen som används för framställning av ren dissolvingmassa för vidare produktion av regenererad cellulosa. Under denna process löses ligninet upp i kokvätskan genom introduktion av sulfonsyragrupper, vilket medför att ligninet blir vattenlösligt och kan därför separeras från massan. Den amfifatiska strukturen hos lignosulfonaterna som innehåller både hydrofila och hydrofoba grupper har gett lignosulfonaterna unika egenskaper. Lignosulfonater används idag som dispergeringsmedel för olika typer av suspensioner för att förbättra deras dispersion och stabilitet. Det överlägset största användningsområdet av lignosulfonater är som dispergeringsmedel för betongtillverkning. Här används lignosulfonaterna för att dispergera cementpartiklarna för att ge betongen bra flyt och undvika partikelaggregation. Denna dispersion sker främst genom sterisk och elektrostatisk repulsion av de laddade cementpartiklarna. Dessa två fenomen är intimt förknippade med storleken och laddningen hos den dispergerande polymeren. De traditionella modifieringarna av lignosulfonater har varit begränsade till eliminering av socker, filtrering och fraktionering. I högstyrke‐betong ställs andra krav på dispersion vilket medför att man använder syntetiska dispergeringsmedel med väldigt specifika egenskaper vilka är avsevärt effektivare. För att konkurrera med dessa, är ytterligare modifieringar av lignosulfonater nödvändiga. Molekylvikten och andelen laddade grupper har därför identifierats som de två mest lämpliga egenskaperna för modifiering av lignosulfonater. För tillfället finns det inga metoder för att öka molekylvikten hos lignosulfonater. En intressant metod är polymerisering genom enzymatisk oxidering med hjälp av det radikal‐initierande enzymet lackas. I denna avhandling har flera aspekter av reaktionsmekanismen hos detta enzym undersökts och kartlagts via studier med modellkomponenter. Oxidation av tekniska lignosulfonater visade att detta enzym är mycket kapabelt till att öka molekylvikten markant även vid låga doseringar. Tekniker för att tillåta återanvändning av enzymet genom immobilisering till en support har undersökts. Modifiering av mängden laddade grupper hos lignosulfonater skedde genom ozonering och reaktion med Fentons reagens, en blandning av väteperoxid och järn(II)acetat. Laddade karboxylgrupper visade sig bildas genom reaktioner där bensylgrupper i lignosulfonaterna bröts upp. En kombination av oxidation med lackas följt av ozonering visade sig vara en mycket intressant tvåstegsmodifiering vilket gav upphov till en högmolekylär polymer berikad med laddade karboxylgrupper. Oxidering med Fentons reagens visade sig ge liknande resultat som en kombinerad lackas och ozonbehandling med märkbart lägre effektivitet men dock med en större grad av kontroll. Detta reagens skulle kunna vara ett intressant alternativ till den ovan nämnda tvåstegsmodifieringen. Dessa modifieringar är intressanta för storskalig användning då dessa är bör vara lätta att implementera men också eftersom både enzymet och kemikalierna är väl bekanta för pappers‐ och massaindustrin.
List of Publications
This thesis is based on the following papers: I. Oxidative polymerisation of models for phenolic lignin end‐groups by laccase Areskogh, D.; Li, J.; Nousiainen, P.; Gellerstedt, G;, Sipilä, J. and Henriksson, G. Holzforschung, 2009, 64, 21–34. II. Sulfonation of phenolic end groups in lignin directs laccase‐initiated reactions towards cross‐linking. Areskogh, D,; Li, J.; Nousiainen, P.; Gellerstedt, G.; Sipilä, J. and Henriksson, G. Industrial Biotechnology 2010, 6, 50‐59. III. Investigation of the Molecular Weight Increase of Commercial Lignosulfonates by Laccase Catalysis. Areskogh, D.; Li, J.; Gellerstedt, G. and Henriksson, G. Biomacromolecules, 2010, 11, 904–910. IV. Structural modification of commercial lignosulphonates through laccase catalysis and ozonolysis. Areskogh, D.; Li, J.; Gellerstedt, G. and Henriksson, G. Industrial Crops and Products 2010, 32, 458‐466. V. Immobilisation of laccase for polymerisation of commercial lignosulphonates. Areskogh, D. and Henriksson, G. Process Biochemistry 2011, 46, 1071‐1075 VI. Fenton’s reaction: a simple and versatile method to structurally modify commercial lignosulphonates. Areskogh, D. and Henriksson, G. Nordic Pulp & Paper Research Journal, 2011, 26, 90‐98Author’s Contribution
Paper I‐VI: Principal author. Formulated research strategies with Prof. Gunnar Henriksson.
Abbreviations
In alphabetical order: C3A Calcium aluminate, (CaO)3Al2O3. C2S Dicalcium silicate, (CaO)2SiO2 C3S Tricalcium silicate, (CaO)3SiO2C‐S‐H Calcium silicate tetrahydrate (CaO)3(SiO2)2∙4∙H2O
ECF Elemental Free Chlorine
FT‐IR Fourier Transform Infrared Spectroscopy
GC/MS Gas Chromatography/Mass Spectrometry
Gypsum Calcium sulphate dihydrate, CaSO2⋅2∙(H2O)
HSQC‐NMR Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance LCC Lignin‐carbohydrate complex LMS Laccase‐mediatory system MALDI‐TOF MS Matrix‐Assisted Laser Desorption/Ionisation Time‐of‐Flight Mass Spectrometry Me‐ Methyl group, CH3‐
MeO‐ Methoxy group, CH3O‐
MtL Myceliophthora thermophila laccase SEC Size Exclusion Chromatography TCF Totally Chlorine Free TvL Trametes villosa laccase
Table of Contents
I Introduction ... 1 I.1 Aim ... 2 II Background ... 3 II.1 Structure and Chemistry of Lignin ... 3 II.2 Lignosulphonates ... 4 II.2.1 Production and Structural Properties ... 4 II.2.2 Utilisation ... 6 II.2.3 Concrete Admixtures ... 6 II.2.4 Specialty Markets ... 10 II.2.5 Modified lignosulphonates ... 10 II.3 Laccase ... 11 II.3.1 A Blue Multi‐Copper Oxidase ... 11 II.4 Structure and Catalytic Properties ... 11 II.5 Occurrence and Role in Nature ... 13 II.6 The laccase‐mediator system ... 14 II.7 Applications ... 15 II.7.1 Laccase in the pulp and paper industry ... 16 II.7.2 Laccase in the textile industry ... 16 II.7.3 Laccase in alternative applications ... 17 III Experimental ... 18 III.1 Materials ... 18 III.1.1 Enzymes ... 18 III.1.2 Lignosulphonates ... 18 IV Results and Discussion ... 19 IV.1 Model compound studies of the laccase reaction mechanism (Paper I & II) ... 19 IV.1.1 The 1‐position ... 20IV.1.2 The Cα position ... 23
IV.1.3 Conclusions from the model compound studies ... 25
Laccase oxidation of lignosulphonates (Paper III) ... 26
IV.1.5 The influence of the enzyme ... 29 IV.2 Structural modifications of lignosulphonates through laccase oxidation and ozonolysis (Paper IV) ... 31 IV.2.1 Laccase oxidation of DP401 and DP795 ... 31 IV.2.2 Ozonolysis of DP401 and DP795 after laccase oxidation ... 32 IV.3 Immobilisation of TvL (Paper V) ... 35 IV.3.1 Oxidation of DP851 by immobilised TvL ... 36 IV.3.2 Lignosulphonate adsorption ... 38 IV.3.3 Deactivation of the immobilised TvL ... 39 IV.4 Lignosulphonate structural modification by the Fenton’s reagent (Paper VI) ... 40 IV.4.1 Fenton’s reagent at acidic pH ... 41 IV.4.2 Which cation does what? ... 43 IV.4.3 Fenton’s reagent at alkaline pH ... 43 IV.4.4 The effect of lignosulphonate concentration ... 44 IV.4.5 The effect of hydrogen peroxide concentration ... 45 V Conclusions and Further Perspectives ... 47 VI Acknowledgements ... 49 VII References ... 50 VIII Errata‐list ... 55
1
I
Introduction
The industrial utilisation of cellulose for papermaking from lignocellulosic plants has in many ways transformed the role of lignin from a necessity in nature to a reject in the industry. Not only is lignin one of the most abundant biopolymers on Earth, second only to cellulose and possibly chitin 1, it is also a bafflingly complex plant constituent of wood. While the other major wood constituent, cellulose occupies a prominent place as a valued industrial product, lignin has been reduced to an obscure role as a low‐quality fuel or chemical. The reborn realisation in the pulp and paper industry that wood can be used for a variety of products besides paper and fuel has led to the adoption of the biorefinery concept, where the biomass feedstock is processed into a spectrum of products such as fuel, chemicals, materials and energy through sustainable processes.T
his concept is nothing new to the petroleum industry which has been producing fuels, power and chemical products from petroleum for the last two centuries 2. Ironically, the idea of maximising the utilisation of the wood feedstock was exploited already in 1898 by Simonsen in his efforts to convert sawdust to ethanol 3. Over the decades, this knowledge was however forgotten, but the concept of a biorefinery experienced a rebirth some 30 years ago and it is emerging today as a future model of operation for the pulp and paper industry to expand its business into new fields. The National Renewable Energy Laboratory (NREL), a facility of the U.S. Department of Energy for research and development in renewable energy and energy efficiency, states that “to maximize the value generated from a heterogeneous feedstock, refineries make use of all component fractions, producing a variety of co‐products in the process … and also has tremendous potential to benefit society” 4. The role of lignin in a biorefinery is yet to be defined, but it is clear that is essential to exploit its value. Although the use of lignin as a replacement or complement to fossil fuels makes sense in a traditional paper mill, the concept of a biorefinery is likely to change this. An excellent example of this is the water‐soluble lignosulphonates, a by‐product from the sulphite pulping process. Their unique properties have led to utilisation of lignosulphonates in a variety of fields as additives or binders and as a precursor for further chemical processing. Due to the limited quality and performance, lignosulphonates have faced significant competition from synthetic plasticisers as concrete additives. Traditional modifications of lignosulphonates to improve their performance have been limited to filtration and fractionation to obtain lignosulphonates in more specific molecular weight ranges. Although sufficient to give moderate improvements in performance, the improvements are not enough to compete with their synthetic equivalents as concrete dispersing additives. Other modifications are likely to be necessary. No methods suitable for industrial integration to increase the average molecular weight of lignosulphonates exist to date. To achieve such an increase, it might be interesting to utilise biotechnical tools such as enzymes. One particular group appear promising, the natural radical‐2 initiating laccases. The recent rapid advancements in heterologous expression and production have solved the problem of the low availability of the enzyme for industrial applications. What remains to be solved is how laccases can be applied on lignosulphonates and what benefits are to be gained.
I.1 Aim
The goal of this work described in this thesis is to investigate the various modifications of lignosulphonates achieved by laccase and conventional bleaching chemicals. Several aspects of laccase oxidation of lignosulphonates are explored through studies with model compounds and experiments with lignosulphonates. The main targets for modification are the average molecular weight and functional group composition. The proposed modifications of technical lignosulphonates are potentially important for improving their performance as additives to concrete and also for expanding their industrial utilisation.3
II Background
II.1 Structure and Chemistry of Lignin
Lignin is next to cellulose the most abundant biopolymer on Earth, manifesting its presence on all corners of the planet Earth, on land as well as sea 5. Fortification of the cell wall by lignin is considered to be the key innovation in the evolution of terrestrial plants from their aquatic ancestors nearly half a billion years ago. The heterogeneous and highly complex aromatic polymer loosely termed “lignin” encrusts cellulose microfibrils and other components in the cell wall preventing collapse and lending biomechanical support to the cell wall and allowing plants to overcome gravity and rise above the ground. The widespread colonisation of terrestrial eco‐ regions by plants with lignified cell walls is excellent evidence of the success of such a design. No exact primary chemical and structure of lignin currently exists. In a broader sense, lignin can be described as an aromatic polymer of methoxylated phenylpropanoid units linked to each other through carbon‐carbon and and ether bonds. Several models of lignins have been proposed over the years 6‐9. The fact that only models and no actual structural determination of lignin in situ are available indicates not only the complexity of lignin but also the lack of methods to extract lignin in its’ native form. With current techniques, only fragments can be extracted and this means that the current knowledge of lignin can be considered at best fragmental. Attempts at obtaining the full picture of the structure of lignin have so far yielded only a determination of spruce secondary wall with Raman spectroscopy 8. The lignin polymer is optically inactive 10 although it contains several chiral centres and is generally considered to be branched to facilitate cross‐linking to other cell wall components to form lignin‐carbohydrate complexes (LCCs)11,12. The LCCs serve as anchoring points where the carbohydrates and lignin are covalently bonded to each other and are important for the unique physical properties that wood exhibits as a natural composite material. The lignin polymer is synthesized by the polymerisation mainly of three different cinnamyl alcohols; p‐coumaryl, coniferyl and sinapyl alcohol. These lignin monomers, monolignols, are produced within the wood cell and exported to the cell wall where they are polymerised. The monolignols are the product of the phenylpropanoid pathway starting from the amino acid phenylalanine. The end product of the monolignol polymerisation is the lignin polymer (Scheme 1). The hydroxylation and methylation reactions in the phenylpropanoid pathway ultimately determine which monolignols is formed because the three lignols differ only in the number of methoxyl groups.4 OH N H2 O phenylalanine O H OH p-coumaryl alcohol O H OH OMe coniferyl alcohol sinapyl alcohol O H OH OMe MeO O O H MeO OH OH O O OMe OH O H OH O O H O Lignin O H O CH3 OH O MeO OH O O OH O OMe MeO O MeO OH O MeO O H OH Lignin OH OMe O H O H OMe OH Scheme 1: Phenylpropanoid pathway for lignin biosynthesis in vascular plants.
II.2 Lignosulphonates
II.2.1 Production and Structural Properties
Lignosulphonates are by‐products of the sulphite pulping process for the manufacture of specialty dissolving pulps and paper. The wood chips are digested with acidic calcium bisulphite for 6‐10 hours at 100‐130 °C in a batch‐wise cooking process. During this process, the native lignin is broken down through the degradation of the randomly distributed ether bonds throughout the structure 13. The fragments are solubilised in the cooking liquor through covalent addition of sulphonic acid groups at various positions in the lignin structure (Figure 1). The sulphonic acid groups are stabilised by the presence of calcium ions. After the competition of the cooking stage, the insoluble cellulose is separated from the solubilised lignin by filtration. Further processing of the cooking liquor involves precipitation of the sulphonated lignin through the addition of excess calcium hydroxide (Howard process), the evaporation of water and residual sulphite (in the form of sulphur dioxide) and dilution with fresh water followed by ultrafiltration to remove low‐molecular‐weight fractions and sugar monomers. The purified calcium lignosulphonate is pH‐adjusted to a specific pH and evaporated to a dry matter content suitable for spray drying and packaging (Scheme 2). Scheme 2: Simplified flow chart of lignosulphonate production from spent cooking liquor from the sulphite process.Spent cooking liquor
Ca(OH)2 pH adjustment Precipitation (Howard process) Ultrafiltration pH adjustment Dilution Evaporation Evaporation Spray drying
5 The co‐existence of the hydrophilic sulphite groups and hydrophobic aromatic structures provide lignosulphonates with unique amphiphilic properties. The overall structure of a lignosulphonate is not known. Several models have been proposed over the years suggesting that lignosulphonate behaves as a coiled or expanded polyelectrolyte at either high or low concentration 14. These polyelectrolytes are likely to associate in solution and the high‐molecular weight fractions are more branched than the low‐molecular weight ones. O H MeO S Ca+ O S O Ca2+ O O MeO O H OH OMe O H S O O -O OMe O -O Ca 2+ LS LS O Ca2+ OH OH S O -O O O H MeO O H OMe S O O O -O MeO LS Ca2+ S O Ca2+ Ca2+ O O O O MeO OH OH OMe O H S O O -O OMe OH OMe O H OH OMe S O O O -S O O -O -O Ca2+ LS OH O LS OH O LS OH Figure 1: Tentative calcium lignosulphonate structure. The sulphonic acid groups are stabilised by calcium ions. Residual lignosulphonate chains are denoted as LS. This model has been expanded to describe lignosulphonates as micelle‐type microgels 15 with a non‐charged core consisting of cross‐linked aromatic chains with all the charged groups relocated to or near the surface to facilitate interactions with the aqueous surroundings (Figure 2A) . These particles are likely to exist in solution as irregularly shaped in a wide range of sizes 15. This model has been further elaborated and supported with high‐resolution microscopic imagery 16‐19. An expansion of this model was proposed following the discovery of a monolayer formation of various lignins when spread on a liquid surface 20 and the sandwich‐like arrangement of lignins in the secondary cell wall of wood cells 21. The earlier model of a spherical micelle was revised to a flat disc‐like molecule to better conform to these findings 22. No efforts to relate this new structure to the well‐known behaviour of lignosulphonates in solution have been made to date. Additional studies of lignosulphonates under electric fields displayed a conformational change from a compact sphere to a non‐free unwinding coil 23. A recent investigation suggests that lignosulphonates are randomly branched cross‐linked polyelectrolytes 24 (Figure 2B). According to this suggestion, lignosulphonates consist of a long continuous chain acting as a backbone with short side chains. The side chains are possibly further branched and may be reconnected to the backbone forming closed loops 24. The breaking of ether bonds that occurs during the cooking stage due to acidic hydrolysis and the subsequent
6
introduction of sulphonate groups to the structure is assumed to occur at positions that form the short side chains.
Figure 2: Proposed lignosulphonate structural models as globular micellar-type microgels 15 (A), randomly branched polyelectrolytes 24 (B) and ellipsoidal flat particles 25 (C).
It is therefore assumed that the longer backbone is more hydrophobic while side-chains are hydrophilic due to the presence of covalently linked sulphonate groups. This model was based on scaling analysis that showed how the intrinsic viscosity changed with molecular weight, something which is not valid for the microgel model where the gel increases in size but more or less keeps its overall shape with no change in intrinsic viscosity. A randomly branched
polyelectrolyte will change from a spherical to an elongated shape as the molecular weight is increases with subsequent changes in intrinsic viscosity. Recent small angle X-ray scattering and rheological studies of lignosulphonates in aqueous solutions have shown that low-molecular-weight lignosulphonates form ellipsoidal compact and flat particles 25 (Figure 2C).
II.2.2 Utilisation
The annual global production of lignosulphonates amounted to 1.8 million metric tonnes in 2005 26, but this accounts for less than 2% of all the lignin produced in the pulp and paper industry. The by far largest utilisation of lignosulphonates worldwide which accounts for as much as 90% of the worldwide production is as concrete admixtures 27 and for energy production during the pulping process 28.
II.2.3 Concrete Admixtures
Chemical admixtures have been used in concrete formation throughout the history of
construction. As early as several hundred years B.C., Roman masons added blood and eggs to cement and water pastes to improve their mixing 29. The modern cement formula, Portland cement, was formulated during the mid-1800’s.
7 Despite the use of cement since the industrial revolution in the early 1800’s, the mechanism of the cement setting when mixed with water is still only partially understood. This process involves a series of complicated hydration and crystallisation reactions where the cement particles are interlocked and form a strong water‐insoluble binder. The actual benefits of admixtures in concrete in modern times were unintentionally discovered in the United States after the Second World War in two separate events. One event tells of a leaking bearing that released heavy oil into a grinding mill where concrete was produced. This resulted in the discovery of air‐entraining compounds 29. The second event tells of an employee at the Department of Transportation who had the idea of colouring the concrete in the black central lane of a three‐lane‐highway so that the drivers would notice when they were switching lanes. The Department of Transportation contractor chose, after a recommendation by a sub‐contractor, to use a lignosulphonate‐based dispersant to improve dispersion of the Carbon Black, the colouring agent used to obtain the distinct black colour. After several years it was observed that the state of the central lane on this particular three‐way highway was significantly better than that of the two outer lanes 30. Microscopic studies of the concrete revealed evenly distributed air‐ bubbles throughout the concrete which provided the concrete with greatly improved durability against freezing and thawing. Further studies revealed that lignosulphonates also reduced the tendency of the cement particles to flocculate without any further addition of water.
The most important components of Portland cement are calcium silicates C2S and C3S and calcium aluminates C3A. They comprise over 80% of the total content of Portland cement and are essential for the strength development in the formed concrete. Both silicates react with water during the hydration process to form calcium hydroxide and calcium silicate hydrate gel, C‐S‐H. These three components play an instrumental role during the initial steps of Portland cement hydration further setting and strength development. This process can be broken down to five steps 31: 1. Initial mixing (0‐10 min). The cement particles enter into solution and hydration reactions with C2S, C3S, C3A and water start, as a result of which C‐S‐H is formed: If the rapid C3A hydration reactions are allowed to proceed unhindered, setting occurs too quickly and the formed concrete is not able to develop strength. Therefore, gypsum is added to the Portland cement composition to slow down the C3A hydration. 2. Dormant period (10 min – 3 h). The hydration reactions are significantly slowed down. Flocculation of unhydrated silicates occurs at this stage. 3. Initial setting (3h – 9h). The hydration reactions are rapidly started again as silicates start to precipitate.
C2S: 2 (CaO)2(SiO2) + 5 H2O → (CaO)3(SiO2)2 · 4 H2O + Ca(OH)2 C3S: 2 (CaO)3(SiO2) + 7 H2O → (CaO)3(SiO2)2 · 4 H2O + 3 Ca(OH)2
8 4. Hardening. (9h – 15h) Most of the hydrate gel formation occurs in this highly exothermic stage which in turn accelerates further hydration reactions. 5. Slowdown (15h – 2 days). The hydration proceeds more slowly due to the extensive covering of the cement particles by the hydrate gel which hinders water from penetratating the calcium hydrate silica gel to reach unhydrated parts of the particles. When the water can no longer reach unhydrated areas or when it is consumed, the hydration stops. Water plays a crucial role in concrete preparation; it provides the cement mixture with certain rheological properties and takes part in hydration. Cement particles with their surface charges are highly prone to flocculation when in contact with a polar solvent such as water. Flocculation is detrimental not only for the uniformity of the cement mixture but also for the hydration process which begins as soon as the mixture comes into contact with water. The formation of flocculation aggregates entraps certain amounts of water which become unavailable to lubricate and ensure a flowability of the mixture. To achieve a certain workability of the concrete mixture, more water must be added than is necessary to achieve full hydration of the cement particles 31. When the excess water that does not participate in the hydration evaporates, porous cavities are formed within the paste and these lead to weakening of the mechanical properties and durability of the concrete. The addition of plasticisers and water‐reducing agents is therefore crucial. Approximately 50% of the lignosulphonates produced worldwide are used as concrete admixtures. Here lignosulphonates serve several purposes; they achieve workability of the concrete mixture through dispersion of concrete particles, they reduce amount of water necessary to achieve a certain workability of the mixture to improve the strength of the set concrete and they accelerate or retard the setting and they entrain air in the concrete. Lignosulphonates actively participate in the hydration of the cement minerals in Portland cement by irreversible adsorption to and incorporation into the calcium silicate hydrate gels 32,33. The adsorption retards the C2S, C3S and C3A hydration 34. The initial setting phase can thus be significantly prolonged. Retardation of the C3S and C3S hydration is very pronounced even at low lignosulphonate concentrations. C3A hydration is highly important as this process is rapid and it significantly affects the early hydration and setting of the cement. In the final stage of hydration, lignosulphonates are incorporated into the structure of the calcium silicate hydrate gel and are thus removed from the solution 34. As a consequence of the lignosulphonate adsorption to the cement particle surfaces, steric and electrostatic repulsion between the individual particles occurs. Steric repulsion prevents particle flocculation by forcing the particles apart. The electrostatic repulsion is being achieved through presence of charged groups in the lignosulphonate structure (Figure 3). This mode of action of dispersants was first described by Uchikawa et al. 35 and has been generally accepted. Using lignosulphonates, concrete with a compressive strength of 40‐50 MPa can be produced 36. With the rapidly increasing demands of high‐strength concrete with a compressive strength of 100 MPa and above (during the construction of The Petronas Towers in Kuala Lumpur 120 MPa
9
silica fume concrete was used 37), lignosulphonates are being displaced by expensive synthetic dispersants and water-reducing agents which achieve significantly greater water reduction and compression strength while maintaining low levels of air entrainment and high workability of the concrete.
The entrainment of air and excessive retardation are two problems limiting the increase of dosage lignosulphonate in a cement mixture. Current-generation lignosulphonates differ significantly in their purity and sugar content to minimise the unwanted reactions in concrete and to allow for further water reduction 38.
Figure 3: Cement particle flocculation and entrapment of water within the floc. Steric and electrostatic repulsion of the particles is achieved through rearrangement of the charged additive in relation to the cement
particle surface charges. The individual cement particles are pushed apart and the integrity of the floc is compromised resulting in liberation of the entrapped water.
The synthetic equivalents, the so-called superplasticisers, are based mainly on two groups of non-renewable petrochemicals. Some examples of the most utilised superplasticisers are
polymelamine formaldehyde sulphite (PMS), β-naphthalene sulphonic acid formaldehyde (BNS) and methacrylic acid–methacrylate ω-methoxypolyethylenglycol 39 (Figure 4).
Figure
formaldehyde and (3) methacrylic acid–methacrylate ω-methoxypolyethylenglycol superplasticisers. The ratio of monomeric composition of (3) is indicated by a, b and c.
The high efficiency of superplasticisers is attributed to the large amount of charged groups, up to 0.4 per available position, 31 and their tailored molecular weight, which increases the repulsive forces by ensuring that there is a constant surplus of charged groups present on the
superplasticiser molecule, despite the number of active sites on the cement particle 40-42. Synthetic superplasticisers allow a water reduction of up to 40% and are far superior to lignosulphonates, which allow only a 15% reduction 43. The difference in efficacy between superplasticisers and
Entrapped water HO3S C H3 H 1 2 3 n NH N NH O N N C H3 CH3 N H SO3H n COOH C H3 O O O SO3H CH3 CH3 CH3 CH3 C H3 c a b n
10 lignosulphonates, new standards and a constant production of new‐generation superplasticisers has resulted in a decrease in the use of lignosulphonates 44. The ability of lignosulphonates to function as plasticisers, dispersing agents or surfactants is intimately linked with their molecular weight of the lignosulphonate and the presence of charged groups within the macromolecule. An increase in molecular weight and purity of the lignosulphonate enhances the plasticising effect as well as reducing the viscosity of the concrete 45. High molecular weight lignosulphonates are highly efficient in this sense, even at very low dosages. These lignosulphonates could be a potential plasticising admixture for self‐compacting concrete 45, a form of concrete that does not require vibration for placing and compaction, but is able to flow under its own weight, completely filling formworks and achieving full compaction 46.
II.2.4 Specialty Markets
The use of lignosulphonates as a precursor for the production of chemicals has had only limited success. The conversion of lignosulphonates to vanillin is presently still the most successful process of this kind, despite the fact that it was discovered as early as 1874 47. This process was a prerequisite for introducing vanillin as a bulk ingredient in the food industry but, in comparison with the amount of lignosulphonates produced by the pulping industry and considering the global demand for vanilla, the production of vanillin from lignosulphonates accounts for a far too small market to be able to absorb the vast amount of produced lignosulphonates 48. The current synthetic production of vanillin from guaiacol is more than sufficient to saturate the existing markets. The dispersing and binding properties of lignosulphonates have been explored in various fields such as animal food pellet formation, gypsum board production, pigment dispersion, complexing agents, sludge containers, scale‐inhibitors in wastewater treatment, emulsion stabilisers in oil drilling muds and expanders in lead acid batteries 27.II.2.5 Modified lignosulphonates
The modification of lignosulphonates has traditionally been limited to the purification, ultrafiltration and sugar removal performed during the production. The structural similarities to conventional lignins extracted during Kraft pulping clearly suggest that the traditional bleaching chemicals used in a modern pulp mill can be employed to achieve various modifications in terms of molecular size and functional group composition. The toolbox of bleaching chemicals and the understanding of their mechanisms on lignin structures is extensive and relatively well understood, and this permits specific modifications should they be employed on lignosulphonates. Significantly fewer enzymatic tools are available for lignin modification due to the nature of the lignin itself. The most promising is the oxidoreductive radical‐initiating enzyme laccase.11
II.3 Laccase
II.3.1 A Blue Multi‐Copper Oxidase
The laccase belongs to the group of oxidoreductases, the first class of enzymes in the enzyme classification system (E.C. 1). Strictly defined, oxidoreductases are capable of performing electron transfer from a donor to an acceptor. Further classification of oxidoreductases divides this class into sub‐groups based on the donor and the acceptor. Laccase is found in the group of oxidoreductases active on diphenols and similar substrates (E.C. 1.10), utilising oxygen as electron acceptor (E.C. 1.10.3). The members of the oxidoreductases share one unique feature; they all belong to the group of multi‐copper oxidases with several centres where copper atoms are stabilised 49. Copper is one of the most widespread transition metals in nature. The rich abundance in nature is intimately linked to its oxidation/reduction potential and the key role which oxidation/reduction reactions play in nature. Due to the high toxicity, even at a very low concentration, elaborate stabilisation of copper is required for it to reside in living organisms. It is the presence of copper and the interplay between the Cu(I) and Cu(II) oxidation states that allows the organisms in which it resides to perform complicated oxidation and reduction reactions. Multi‐copper oxidases contain two mono‐nuclear centres (type‐1 and ‐2, containing one copper ion) and one di‐nuclear centre (type‐3, containing two copper ions), each having unique spectroscopic features. The type‐1 centre shows high absorption in the visible region (giving the enzyme a distinct blue colour when in solution), type‐2 centre has undetectable absorption and the type‐3 centre with its pair of copper ions coordinated anti‐ferromagnetically shows strong absorption in the near‐ultraviolet region 50. Multi‐copper oxidases are found in all three life domains, prokaryotes, eukaryotes and archae. In plants and fungi, they are involved in lignin formation and degradation and in yeast and mammals with iron metabolism 51.II.4 Structure and Catalytic Properties
The majority of fungal laccases are monomeric, dimeric or tetrameric glycosylated protein complexes. Glycosylation is believed to play an important role during its secretion, susceptibility towards degradation and thermal stability 52. Besides glycosylation, laccases contain covalently linked carbohydrate units (ranging from 10‐45% of the total molecular mass) which are assumed to contribute to the conformational stability by protecting them from proteolysis and deactivation by radicals 53,54. The overall molecular weight of laccases varies from 50 to 100 kDa. Several three‐dimensional crystal structures of different fungal laccases have been determined 55‐ 60. They all show a striking structural homology but with some minor differences in loop organisation and in the appearance of the substrate‐binding pocket. The overall three‐12
dimensional structure of a laccase consists of three consecutively connected cupredoxin-like domains twisted in a tight globule (Figure 5A). The copper ion binding site T1 is located in the third domain and the T2/T3 site is located between the first and third domains (Figure 5B). This site contains amino acid ligands from both domains 61. Structural sequence comparison of more than 100 laccases identified four conservative regions which are specific to all laccases containing a cysteine and ten histidine residues 62 (Figure 5C). Together they form a compartment in which copper ions are located.
Multiple isoforms of the same laccase are known to be secreted by several white-rot fungi 63,64. These can differ significantly in stability, optimal pH and temperature and substrate affinity. They are encoded by gene families and can be either constitutively expressed or induced by external factors 65,66.
Laccases have been shown to have remarkably low substrate specificity and large differences in range of oxidised substrates can be found in different laccases. These enzymes are all capable of oxidising of wide array of organic and inorganic substrates, including polyphenols, various substituted phenols and aromatic amines.
Figure 5: (A) Ribbon diagram of Trametes hirsuta laccase determined by X-ray chrystallography at 1.8 Å resolution 61 with the three-domain organisation and copper ions clearly distinguishable. (B) The active site
consisting of four copper ion sites (T1 through T4) is located between the domains. (C) The four copper ions are stabilised almost excusively by adjacent histidine residues.
The active site of laccases consists of three copper ion sites designated type-1, -2 and 3 (T1 through T3). The type 2 and 3 sites forms a type 2/3 (T2/T3) cluster. The four copper ions located in these sites are consequently designated Cu T1 through Cu T4. A two-site ping-pong bi-bi reaction mechanism for laccase has been proposed 67. The reaction begins with the oxidation of substrate by the Cu T1 which accepts one electron from the substrate and transfers it to the T2/T3 cluster where dioxygen binds. Upon binding to the T2/T3 cluster, dioxygen accepts the electrons from T1 and is reduced to two molecules of water. This reduction requires four electrons which mean that four one-electron transfers have to be made from the T1 site (Figure 6). The
extraordinary ability of laccase to reduce oxygen to water without the formation of radicals or anions is unusual in biological systems.
C) B) A) T4 Cu T3 Cu T2 Cu T1 Cu N HN His 395 N N H N NH N NH N N H N NH N N H N NH N N H N N H O H T3 Cu SH T1 Cu T2 Cu T4 Cu His 458 His 454 Cys 453 His 452 His 400 His 111 His 398 His 64 His 66 His 109 H
13 Figure 6: Schematic representation of the laccase reaction mechanism. In order to complete the full reduction of one molecule of dioxygen to water, four substrate molecules are oxidised. The key to laccase activity is the redox potential of the T1, T2/T3 copper sites. The potential of T1 site has been determined for a large number of laccases to be within 400‐800 mV 68. The potentials for T2 and T3 sites are less investigated and are so far known only for plant laccase from Rhus vernicifera (390 mV and 460 mV for T2 and T3 respectively) and fungal laccase from Trametes hirsuta (400 mV for T2) 69,70. Based on the potential of the T1 site, laccases are divided into low‐, medium‐ and high‐redox potential enzymes.
II.5 Occurrence and Role in Nature
The first known isolation of laccase was performed by Yoshida in 1883 from the sap of the lacquer tree Rhus vernicifera 71. He was able to isolate a thermolabile compound with what he referred to as diastatic properties which consumed oxygen when it was mixed with urushiol. He drew the conclusion that this diastase‐like compound was responsible for the lacquer drying process that occurs naturally. More than a decade later, the name “laccase” was coined by Bertrand who isolated and purified this compound from the sap of the lacquer tree 72 and also for the first time demonstrated their presence in fungi 73 . He also introduced the concept of metalloenzymes by erroneously claiming that this newly discovered laccase contained manganese, a conclusion he drew from the large abundance of manganese in the sap itself. It was however later shown that laccase in fact contained copper 74,75. Laccases are widely distributed among terrestrial life forms such as plants and fungi. While their role in fungi has been extensively studied and is still a topic under discussion, laccases in plants have been less studied. It is well known that in lacquer trees grown in Eastern Asia laccase is involved in various defence mechanisms as a response to physical injury to the bark of the tree. A white sap (lacquer) is excreted which is oxidised when in contact with oxygen with the subsequent polymerisation of the phenols in the sap so that a highly resistant protective structure is formed. Laccase has been isolated not only from trees such as sycamore and loblolly pine 76 but also from a variety of vegetables and fruits 77 and green shoots of tea 78. 4 x T3 Cu(II) H2O T1 Cu(II) T2 Cu(II) T4 Cu (II) T3 Cu(I) T1 Cu(I) T2 Cu(I) T4 Cu (I) OH OH Lignin OMe OH O Lignin OMe 4 x O214 In the fungi kingdom, laccases are present in all seven phylums. The enzyme has been attributed a role in a variety of cellular processes including delignification, sporulation, plant pathogenesis, pigment production and fruit‐body ripening 79. The most widely studied laccases originate from the white‐rot basidiomycetes which are highly efficient lignin degraders. It is due to their abundant presence in the white‐rot fungi that laccases traditionally have been regarded as an important participant in the lignin‐degradation process. Numerous studies of wood decay have identified other enzymes that participate in the delignification process. Among these are lignin peroxidases and manganese peroxidases, responsible for oxidation of both phenolic and non‐ penolic units, glucose oxidase and glyoxal oxidases for H2O2 production and cellobiose‐quinone oxidoreductase for quinine reduction and hydroxyl radical generation 80. It has been demonstrated that there is no unique mechanism for lignin degradation and that the setup of the required enzymatic tools differs greatly between various microorganisms. Laccases have also been associated with lignin biosynthesis participating as an oxidant of monolignols, a role traditionally reserved solely for peroxidases81‐83. This theory was first proposed by Freudenberg et al. 83 who saw that laccase was able to oxidise lignin monomers to form dimers of similar structure to those produced through the chemical degradation of lignins. Their theory was abandoned when no successful detection of laccases in plant tissues was achieved. Later investigations were however able to reignite this discussion when it was demonstrated that laccase alone could polymerise monolignins in the complete absence of peroxidases 84. It was also suggested that laccases were involved in the very early stage of lignin biosynthesis and that peroxidases were involved at later stages 84. The investigation of lignin biosynthesis (extensively reviewed by Lewis et al. 85) has provided indirect evidence of the involvement of a variety of peroxidases, laccases and other oxidases, suggesting that such a complex reaction as lignin biosynthesis cannot simply involve one specific group of oxidases. This question remains open and is still being widely discussed. Although many enzymes are able to oxidise monolignols in vitro, no unequivocal proof of the involvement of any specific group of oxidases in lignin biosynthesis in vivo has been presented through loss‐of‐fuction experiments in transgenic trees 86. Laccases active on lignin have been reported to be mostly extracellular, although the occurrence of intracellular laccases in white‐rot fungi has been demonstrated 87,88. Froehler and Ericsson also proposed a role for the intracellular as a precursor to the extracellular laccase with little or no difference between them other than their location in the cell 87.
II.6 The laccase‐mediator system
The heterogeneity and complexity of lignin together with the low redox potential of laccases allow them, in contrast to other lignolytic enzymes, to oxidise only those phenolic units in lignin which have an ionisation potential within the range of the T1 site 89,90. To explain how laccase is still able to be active on such a complex polymer, the laccase mediator theory was developed. Earlier it was believed that laccase was involved only in lignin degradation by oxidising phenolic end groups in lignin to phenoxy radicals with subsequent linkage breakage. In later experiments,15 the presence of low‐molecular‐weight co‐substrates, the degradation of non‐phenolic structures was observed 63. The term mediator was soon coined to describe the role of these co‐substrates. Their significantly higher oxidation potential (>900 mV) was shown to be essential for expanding the activity of laccase on non‐phenolic substrates. Since the first experiment where ABTS, diammonium salt of 2,2ʹ‐azinebis(3‐ethylbenzothiazoline‐6‐sulfonic acid) was demonstrated to enhance the enzymatic ability of laccase 63, the number of compounds that can be converted by laccase has increased significantly. Over the years, a large number of synthetic and naturally occurring mediators have been proposed and successfully tried with laccase. Consequently, the role of laccase in nature has also been expanded or revised 63,91. A laccase mediator participates in a cyclic reaction with laccase (Figure 7) where a high‐potential mediator intermediate is formed through laccase oxidation. This intermediate participates in non‐ enzymatic reactions with other substrates not accessible to or oxidisable by laccase alone. Upon oxidising the substrate, the mediator returns to its reduced state, thus closing the cycle 92. This cycle should, ideally, be repeated a number of times before the mediator is degraded. It should be noted that the chemistry by which laccase reacts with the mediator is significantly different to the mediator‐substrate chemistry. The misconception that the redox potential of the laccase increases when a mediator is used is thus not valid 93. Figure 7: The laccase mediator cycle as first proposed by Bourbonnais 63. In‐depth studies of a number of mediators such as ABTS 94,95 various N‐O‐ and N‐OH‐containing mediators such as HBT (1‐hydroxybenzotriazole), violuric acid, N‐hydroxyacetanilide and various polyoxymetals (such as [SiW11V1O40]5–)92,96‐98 show that no compounds currently fit the criteria of an ideal mediator. All the proposed compounds either exhibit low stability during the enzymatic activation or show no or very low ability to regenerate and thus participate in a series of cycles. Despite the recent findings that a few compounds are actually able to participate in a substantial amount of cycles 99,100 it appears that a true laccase redox mediator is yet to be discovered.
II.7 Applications
The potential of laccase as an industrial biocatalyst appears to be significant. Laccase is one of the few oxidoreductive enzymes that do not require expensive co‐factors other than dioxygen and recent developments in heterologous expression have enabled the large‐scale production of the enzyme. Consequently, the enzyme has found its way into a number of industrial processes including paper processing, the prevention of wine decolouration, bioremediation and textile dye oxidation. Extensive research into the laccase‐mediator system has resulted in a massive effort to find technical and industrial applications for such systems. H2O O2 Laccaseox Laccase Mediator ox Mediator Substrateox Substrate16
II.7.1 Laccase in the pulp and paper industry
Lignocellulose is a natural substrate for laccase and, provided that mediators are used, laccases have the potential for breaking non‐phenolic units in lignin without disrupting the integrity of the cellulose which is closely linked to lignin in native wood 11. This is particularly interesting in the pulp and paper industry. The enormous global production volumes of this industry means that even a minor improvement achieved by a LMS process step would have huge implications. The current industrial preparation of pulp and paper, the Kraft and sulphite processes relies on the separation and degradation of lignin from cellulose through cooking and bleaching with conventional chemicals. While the recovery and reuse of the chemicals in the cooking stage is practised, the negative environmental impact of the bleaching stage is well documented. Although recent developments have yielded significantly less detrimental processes with the abolition of elemental chlorine in the early 1990’s and the development of the ECF and TCF bleaching processes there appears still to be a need to develop new methods based on laccase. Extensive studies have been performed to develop and evaluate LMS for Kraft pulp bleaching 101‐ 103. The results appear promising and they have already found practical application in the Lignozym© process 104. The use of LMS with flax pulps has also been explored with successful results 105, displaying the versatility of these systems. There is however one major obstacle for the successful implementation of LMS in a modern paper mill; the mediator. There is, to date, no readily available and cheap mediator that performs several oxidation cycles. In addition, large quantities of mediator are required to achieve a bleaching performance comparable to that of conventional chemical processes. For instance, the Lignozym© process 104 requires a mediator amount of up to 2% of the dry weight of the pulp. In a normal Kraft mill producing 1 000 ‐ 3 000 tonnes of pulp per day, a daily mediator consumption of 60 tonnes is required. Considering the significant cost of the enzyme and the mediator as well as the minimal environmental footprint of a modern pulp mill, the incentive to replace conventional bleaching chemicals with a LMS process step is low. Despite more than two decades of research and development of a LMS bleaching stage there has as yet been no commercial adaptation of this process on an industrial scale. The ability of laccase to form reactive radical species on lignin end groups can also be utilised for fibre modification. It has been successfully demonstrated that laccase facilitates adhesion of fibres during the manufacturing of wood composite materials such as fibreboards 106,107. Functionalisation of lignocellulotic fibres by grafting various phenolic acid derivatives onto Kraft pulp fibres is an another possibility use for laccase 108,109. Although promising, these are areas of laccase utilisation that have not yet reached industrial levels.II.7.2 Laccase in the textile industry
The textile industry consumes large volumes of water and chemicals for textile processing. Each year, more than 700 000 tonnes of dyestuff are produced 110 which are resistant to a variety of chemicals making them difficult to decolourise and detoxify. Current ways of treating dye waste17 water are ineffective and expensive 111 making them an ideal target for processes based on laccase oxidation which have been demonstrated to be very capable of degrading dyes of various structures 112,113. Utilisation of laccase in the textile industry is an expanding field and covers not only effluent treatment but also textile bleaching and dye synthesis 114. In 1996, Novozymes (Bægsverd, Denmark) launched the first laccase preparation to be used in the fabric industry under the name DeniLite®. This preparation utilises a redox mediator (phenothiazine‐ propionate). The product was followed by the laccase‐mediator formulation under the trade name Zylite® (Zytex Pvt. Ltd., Mumbai, India). Both formulations are used for the removal of indigo from denim jeans clothing.
II.7.3 Laccase in alternative applications
In addition to the previously discussed industrial applications, laccase is of interest in a variety of fields and applications. In the food industry, laccase can be used for drink clarification and as biosensor for the monitoring of phenol formation 115. In the baking industry, laccase is interesting for its ability to cross‐link biopolymers in various doughs 116. Other highly interesting applications of laccases are in nanobiotechnology for electroimmunoassay sensors 117, organic synthesis 118 , biofuel production 117 and keratinous fibre dying 119.18
III Experimental
Detailed description of the materials, methods and analytical apparatus is given in the related papers.
III.1 Materials
III.1.1 Enzymes
Two laccases provided by Novozymes (Bagsværd, Denmark) were used in all the experiments. The laccases, denoted as NS51002 and NS51003, originated from Trametes villosa (TvL) and
Myceliophthora thermophila (MtL) respectively. The two enzymes had different temperature and
pH optima as well as redox potentials E0 (50°C, pH 5, E0 780 mV for TvL, 40°C, pH 7.5, E0 480 mV
for MtL.
III.1.2 Lignosulphonates
Four lignosulphonate salts supplied by Borregaard LignoTech (Sarpsborg, Norway) were used in all the experiments. The salts were characterised by the supplier by standard methods (Table 1). The lignosulphonates were used without further purification.
Table 1: Characteristics of the lignosulfonate salts used in laccase oxidation experiments described in Papers III-VI.
Salt Raw Material Process Counter-ion Mw(Da) Phenolic content (%) Organic sulphur (%)
DP398 Softwood Filtered Ca2+ 28 400 1.9 5.7
DP399 Softwood Ion exchanged Ultrafiltered Na+ 46 500 2.1 6.2
DP400 Softwood Desulphonated Ultrafiltered
Oxidised Na
+ 9 000 1.9 3
DP401 Hardwood Heat treated Filtered Ca2+ 5 900 1.4 4.7
The lignosulphonate DP398 was provided from different batches and was thus also denoted DP795 and DP851.
19
IV Results and Discussion
IV.1 Model compound studies of the laccase reaction mechanism (Paper I &
II)
The reaction mechanism of laccase has been extensively studied over the years both in lignin degradation 104,120‐124 and in biosynthesis 83‐85. Model compound studies of laccases have demonstrated the efficacy of the laccase‐mediator systems with an array of mediators, both natural and synthetic. These experiments were however aimed to understand and expand the degradative aspects of laccase activity, and the potential of laccase for polymerisation has attracted significantly less attention. Experiments with mediators such as ABTS 125, polyoxalates 126, ferulic acid 127, HBT 128 and also in the absence of mediators 129‐132 have clearly demonstrated that laccase is a highly potent tool for the polymerisation of phenol containing compounds. To elucidate the polymerisation mechanisms of laccase in the present work, a number of lignin end‐group compounds were subjected to oxidation by two laccases, TvL and MtL (Figure 8). The oxidation experiments were conducted under the optimal conditions for the respective enzymes in Tris‐buffer under constant oxygen saturation. The products were analysed with a variety of mass spectrometric and spectroscopic tools. OMe OH OH MeO OMe OH OH MeO OMe OH OH Me MeO OMe OH Me 1 Vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) 2 Syringyl alcohol (4-hydroxy-3,5-dimethoxybenzyl alcohol) 3 -methylsyringyl alcohol (4-(1-hydroxyethyl)-2,6-dimethoxy phenol) 4 4-methyl syringol (4-methyl-2,6-dimethoxy phenol) OMe OMe MeO OH OMe OH O MeO OMe OMe Me 5 Vanillin (4-hydroxy-3-methoxy benzaldehyde) 6 3,4,5-trimethoxybenzyl alcohol 7 1,2,3-trimethoxy-5-methyl benzene 1 2 3 4 5 6 OMe OH SO3H 8Vanillyl sulfonic acid (4-hydroxy-3-methoxyphenyl-methanesulfonic acid) OMe OH SO3H Me OMe OH SO3H
MeO MeO OMe
OH
SO3H
Me
9 Syringyl sulfonic acid (4-hydroxy-3,5-dimethoxyphenyl
- methanesulfonic acid)
10
-Methylvanillyl sulfonic acid (1-(4-hydroxy-3-methoxyphenyl)
ethanesulfonic acid)
11
-Methylsyringyl sulfonic acid (1-(4-hydroxy-3,5-dimethoxyphenyl) ethanesulfonic acid) 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 Figure 8: Model compounds oxidised by MtL and TvL in Papers I and II. The nomenclature is as follows; the aromatic carbons in the benzyl ring are numbered clockwise from 1 to 6 starting with the carbon to which the side‐chain is attached. The side‐chain carbons are denoted with greek letters α, β and γ. To distinguish atoms on a secondary monolignol, an index ‘ (prime) is added.
20 The end groups in lignin occupy several interesting positions which to varying degrees affect the coupling pattern when they are subjected to oxidation reactions by laccases. The traditional understanding of the polymerisation mechanism of laccase can be summarised in three steps: oxidation of a phenolic substrate through one‐electron abstraction, generation of a resonance‐ stabilised phenoxy radical, and thereafter a spontaneous, non‐enzyme catalysed coupling and rearrangement. The phenoxy radical formed in the first step is characterised by a relatively long life‐time and stability due to the delocalisation of the unpaired electron to various positions along the benzylic ring and side‐groups. As a consequence, the phenoxy radical can participate in a variety of reactions depending on the position of the unpaired electron. The generally low redox potential of laccases limits their action to phenolic substrates. This was evident as neither of the two laccases TvL and MtL was able to alone oxidise the non‐phenolic trimethoxy benzyl alcohol 6. Although the laccase‐mediator system (LMS) has been demonstrated to be a highly efficient tool for oxidation of non‐phenolic lignin units 63,91, the chemistry of those reactions is not within the scope of this thesis and has not been investigated. It should however be noted that the mechanisms of the LMS are significantly different from those of laccase alone. Whereas laccase oxidation relies on the abstraction of phenolic hydrogen, the LMS is able to react with more inert hydrogens such as Hα and Hβ.
