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Chemoenzymatical grafting of acrylamide onto lignin

Journal of Biotechnology79(2000)173–183

Carsten Mai*,Oleg Milstein,Aloys Hu¨ttermann

Institut fu¨r Forstbotanik der Uni6ersita¨t Go¨ttingen,Bu¨sgenweg2,D-37077Go¨ttingen,F.R.G,Germany

Received16August1999;received in revised form15February2000;accepted25February2000

Abstract

Laccase(E.C.1.10.3.2.)from white-rot basidiomycete Trametes6ersicolor and dioxane peroxides were essential in the copolymerization of acrylamide and a derivative with lignin in a dioxane-H2O(7:3)mixture.Both a solubility test and an elemental analysis of the eluted and separated fractions provided evidence of grafting.Gel permeation chromatography on Sephadex G-100showed that the side chain and the lignin backbone migrate as one unit through the column.Copolymers obtained were characterized by UV-VIS-spectroscopy as well as FT-IR,and13C-CPMAS-NMR spectroscopy.Freeze-dried copolymers of lignin and acrylamide appeared as homogeneous?bril-like particu-late.The mechanism of the enzymatical grafting is discussed.?2000Elsevier Science B.V.All rights reserved. Keywords:Trametes6ersicolor;Laccase;Lignin;Acrylamide;Copolymerization

https://www.doczj.com/doc/8e10113186.html,/locate/jbiotec

1.Introduction

Almost50×106tons of the lignin produced annually as a by-product of the pulp and paper industry are burned;only small amounts are used as additives in several industrial processes.A large volume of expensive petrochemicals,of which there is not a long term supply,could be substi-tuted in the production of engineering plastics if an economical way existed to transform lignin into a marketable product.The grafting of lignin with synthetic polymers has the potential of mak-ing the production of a new class of engineering plastics possible(Glasser,1989)and at the same time enhancing the biodegradability of the result-ing polymer(Milstein et al.,1992,Chen et al., 1996).Current approaches to grafting synthetic polymers onto lignin involve radical polymeriza-tion initiated by free radical reactions(chemical or radiation)of an appropriate monomer,particu-larly acrylates as well as other vinyl monomers, on any of the available lignins(Koshijima and Muraki,1968;Phillips et al.,1972;Meister et al., 1984a,b,1991).

Although lignin is a recalcitrant aromatic bio-polymer,it could be degraded in the environment by white rot fungi,particularly by the concerted action of their extracellular phenoloxidases and subsequent radical process initiated by https://www.doczj.com/doc/8e10113186.html,c-case(E.C.1.10.3.2.)is one of the enzymes that causes a one-electron oxidation of the phenolic sites of lignin with a concomitant reduction of

*Corresponding author.Tel.:+49-551-393484;fax:+49-

551-392705.

E-mail address:cmai@gwdg.de(C.Mai)

C.Mai et al./Journal of Biotechnology79(2000)173–183 174

molecular oxygen to H2O.In contrast to its as-sumed role in vivo,the obvious net result of the lignin transformation by laccase in vitro was an increase of the molecular weight(Hu¨ttermann, 1978;Kirk and Farrell,1987;Milstein et al.,1993, 1994)caused by the coupling of generated phe-noxy radicals.It has been reported that laccase not only polymerized lignin in aqueous dioxane but was also able to copolymerize lignin of a different origin with low molecular weight com-pounds(Milstein et al.,1994).It has been recently shown that laccase in the presence of organic peroxides of different types elicited free radical copolymerization of acrylamide and lignin(Mai et al.,1999).

In this paper we report on the grafting of acrylamide onto the lignin matrix catalyzed by fungal laccase in combination with dioxane perox-ide in aqueous organic solvent mixtures as well as on the veri?cation of the copolymerization.It was assumed that the enzymatical transformation of lignin by a radical generating system could provide an alternative biotechnological method for the conversion of lignin into a marketable product.

2.Materials and methods

2.1.Source of lignin

Organosolv lignin(OL)obtained by deligni?ca-tion of beech and spruce pulp with methanol–wa-ter at195°C or methanol–water–caustic soda at 170°C was obtained from Organocell,Gesellschaft fu¨r Zellstoff-und Umwelttechnik mbH,Munich, FRG.

2.2.Enzyme

Extracellular laccase was isolated and puri?ed from the culture?ltrates of the white rot fungus Trametes6ersicolor ATCC11235according to Fa?hraeus and Reinhammar(1967).The?nal puri?ed fraction of laccase was entrapped in polydextran beads of Sepharose CL-6B(Pharma-cia,Sweden)(Milstein et al.,1989).Obtained dry enzymatic preparation contained80mg of protein·g?1of powder and enzymatic activity9.9 Kat·g?1of preparation measured in potassium-ci-trate buffer,0.1M,pH4.5with syringaldazine as the substrate.

2.3.Synthesis of N-(-4-methoxyphenyl)-

2-propenamide

An UV-active chromophore was synthesized ac-cording to Yokota et al.(1964)and Meister et al. (1991).p-Methoxyaniline(13.6g)and triethyl-amine(11.4g)were solved in60ml of acetonitrile and the solution was cooled to0°C.Ten milliliters of acryloyl chloride was added dropwise.During this step the reaction temperature was kept below 1°C.The reaction mixture was then stirred for1h at0°C.Triethylamine hydrochloride was removed by?ltration and the acrylamide derivative was precipitated in water.After recrystallization from ethanol–water and freeze drying12.1g(62%)of N-(4-methoxyphenyl)-2-propenamide(mp:95°C) were obtained.The structure of the product was con?rmed by1H-NMR.

2.4.Acrylamide copolymerization with lignin Organosolv lignin(0.8g)and acrylamide(6.4g) were dissolved in40ml of a dioxane–water(70%) mixture.An aliquot(20ml)of this solution was incubated at25°C on a gyrator shaker with a7 cm rotation radius operating at20rpm for48h with the amount of immobilized laccase prepara-tion as shown in Section

3.A thermo-inactivated enzyme preparation served as control.About10 ml of distilled water was added afterwards and the resulting mixture was dialyzed(Spectrapore6, Serva,Sweden,3500Da molecular cut off)with distilled water for48h.The content of the dialysis bag was concentrated on a rotary evaporator to one third of its initial volume.The remaining mixture was lyophilized to recover the lignin-acry-lamide copolymer.

Dioxane peroxides(2-hydroperoxy-1,4-dioxacy-clohexane)(DP)were formed by autoxidation induced by exposure of the solvent in a pyrex ?ask to daylight for20days.Peroxide containing dioxane was blended with freshly distilled dioxane to obtain the necessary level of DP.The concen-

C.Mai et al./Journal of Biotechnology79(2000)173–183175 tration of peroxides was determined iodometri-

cally.To compare enzymatically and free radical

copolymerization processes,graft polymerization

of acrylamide onto lignin was performed follow-

ing the procedure described by Meister et al.

(1984a,b).

2.5.Production of chromophore containing

terpolymerizate

In8ml of aqueous dioxane(70%)with a perox-

ide content of0.18g·1?1,160mg of lignin were

dissolved and0.7g of acrylamide and0.3g of

N-(4-methoxyphenyl)-2-propenamide were added.

The reaction was initiated by adding18mg of

immobilized laccase preparation to the reaction

mixture.The same procedure as described for

lignin acrylamide polymerization was then

followed.

The copolymer of acrylamide and N-(4-

methoxyphenyl)-2-propenamide,a reference com-

pound,was prepared using Fenton’s reagent as

the initiator.Acrylamide(390mg),108mg of

N-(4-methoxyphenyl)-2-propenamide,2mg of

FeC12·4H2O and10mg ascorbic acid were dis-

solved in8ml Dimethylsulfoxide.The polymer-

ization was started with42m l of a30%hydrogen

peroxide solution.The reaction mixture was

stored for24h in a water bath at50°C.After48

h of dialysis with distilled water the product was

recovered and freeze dried.

2.6.Estimation of acrylamide/lignin content in

copolymerizate

The acrylamide proportion in the copolymer-

izate was calculated from the total nitrogen con-

tent of the sample,

Ap,wt%=Np,wt% Na,wt%

×100

where Ap=acrylamide content in the copolymer-izate;Np=nitrogen content in the copolymer-izate;Na=nitrogen content in the initial acrylamide monomer.

In the lignin tested no nitrogen content was detectable.The OL proportion in the copolymer (OLp)was calculated from the total carbon(C) content in a copolymer:OLp,wt%=

(Cp wt%-2.57×Np wt%)

67.7

×100

where Cp=C content in the copolymerizate;

2.57×Np=C wt%of acrylamide in the recov-ered product;67.7=C wt%of the lignin tested.

2.7.Elemental analysis

The elemental composition of lignin,the poly-mer product and the GPC eluted fraction of the products were determined with an elemental ana-lyzer(EA1108;Carlo Erba Instruments).

2.8.Molecular mass distribution

The pattern of molecular mass distribution of lignin and its graftpolymers were analyzed by gel permeation chromatography(GPC).Lyophilized preparation of the polymer was dissolved in0.02 M NaOH at a concentration of0.85mg·l?1.An aliquot of1ml was run through a Sephadex G-100column(2.3×25cm bed size)with0.02M NaOH as the eluent.The void and total volumes of the packed bed of the column were19.0and 94.0ml,respectively.The eluent product was monitored at280nm(Uvicord,LKB).Fractions of5ml were collected to determine the nitrogen content.

2.9.Spectroscopy

Solid state,high resolution13C-NMR cross po-larization/magic angle spinning(CPMAS)was recorded on a Bruker MSL(25.1MHz)spectro-meter with chemical shifts relative to tetramethyl-silane.Infrared spectra were recorded on a Bio–Rad Digi Lab FTS7FT-IR spectrometer as KBr pellets.The applied polymer sample amounted to 1mg·250mg KBr?1.

2.10.Light and electron micrographs

Freeze-dried preparations of the copolymeriza-tes were examined either with the binoocular mi-croscope Zeiss model SV8or the scanning electron microscope(SEM)Philips model515. SEM observation was done as the copolymer preparations were mounted on SEM stubs and

C.Mai et al./Journal of Biotechnology79(2000)173–183 176

sputter coated with gold to a thickness of about 10nm.

2.11.Chemicals

All chemicals used in this work were obtained from Merck(Darmstadt,FRG)and were of an analytical degree of purity.3.Results and discussion

The data in Table1compare the graft copoly-merization ef?ciency of acrylamide onto a lignin backbone in the free radical reaction according to Meister et al.(1984a,b)with the reaction caused by the addition of laccase in aqueous dioxane (70%)and DP both in the presence of and with-out CaCl2.The acrylamide content and the yield of the chemically synthesized copolymer were78.9 and97.7%,respectively in comparison to58.7and 32.7%for the corresponding values of the enzy-matically generated copolymer(Table1).The presence of DP in the reaction initiated by laccase was necessary in order to produce a signi?cant amount of the copolymerizate.In the reaction mixture with an undetectable amount of peroxides (with freshly distilled dioxane)the yield of the product recovered was only slightly higher than the amount of lignin initially applied.A recent comparative study has shown that DP among different aliphatic or cyclic organic peroxides was the most ef?cient co-factor of laccase initiated copolymerization(Mai et al.,1999).It was also reported that both the yield of produced copoly-mer and the portion of incorporated acrylamide in the product was correlated with the concentra-tion of peroxides(Mai et al.,1999).The highest yield was obtained at a DP concentration of170 mg·l?1in the reaction mixture(Mai et al.,1999).

A thermoinactivated enzyme was not able to generate a copolymer product(Fig.1A).

The addition of CaCl2to the reaction mixture with laccase and170mg·l?1of DP caused a further increase in yield(59.3%)and an increase in incorporated acrylamide(75.6%)when com-pared to the highest yield of a pure enzymatic reaction(Table1).The yield of the chemically synthesized product was97.7%with a78.9%acryl-amide incorporation rate(Table1).The results show that the composition of the catalytic milieu and additives(CaCl2)cause a signi?cant impact on the yield as well as on the incorporation rate of acrylamide in the polymer produced.Calcium chloride has been reported to be an ef?cient pro-moter of Kraft lignin graft polymerization with acrylamide in a free radical polymerization pro-cess(Meister and Patil,1985).It was reported

Fig.1.Pattern of molecular weight distribution of OL trans-formed by laccase(1A-2)and lignin–acrylamide copolymer-izates(1B-1,2,3);OL(1A-1)incubated with a thermoinactivated laccase.Pattern of molecular weight distri-bution of lignin–acrylamide copolymerizates produced by lac-case in the presence of170mg·l?1peroxides of dioxane(1 B-1)and170mg·l?1peroxides of dioxane supplemented with CaCl2(1B-2)in the reaction media.Pattern of molecular weight distribution of the copolymerizate obtained in free radical polymerization reaction(1B-3).Arrows indicate void-(V o)and total(V t)volumes of the applied GPC column.

C.Mai et al./Journal of Biotechnology79(2000)173–183 178

Table2

Solubility of the product recovered from the mixture of lignin and acrylamide incubated in aqueous dioxane(70%)with laccase and dioxane peroxides

Precursors and Solubility at Solubility

at pH2

pH9

copolymer

Lignin S a

Acrylamide

Lignin–acrylamide b PS

Lignin–acrylamide c

Lignin–acrylamide d S

a S,soluble;I,insoluble;PS,partly soluble.

b DP concentration in the reaction media90mg l?1.

c DP concentration in the reaction media170mg l?1.

d Copolymer generated in fre

e radical polymerization reac-tion according to Meister et al.(1984a,b).bonds.Still,the concentration o

f phenoxy radicals as a consequence of lignin oxidation by laccase measured with EPR is reported to be higher than either in the untreated lignin or in the lignin oxidized chemically(Meister et al.,1984a,b;Felby et al.,1997;Barsber

g and Thygesen,1999).Pre-sumably,the abundance of radicals generated by laccase on the lignin backbone provides suf?cient grafting potentials despite the competitive lignin crosslinking process.

3.1.Veri?cation of copolymerization

A comparison of the solubility of lignin,poly-acrylamide and their copolymers in an alkaline or acidic aqueous mixture has provided strong evi-dence that the resulted copolymer is not simply a blend of the initial products(Table2).

The polyacrylamide homopolymer is soluble in water,aqueous base and acid;whereas the lignin tested could only be dissolved in an aqueous base solution.A blend comprised of the lignin tested and acrylamide/polyacrylamide is readily soluble in0.02M NaOH.It was predicted that acidi?ca-tion of this solution would cause precipitation of lignin,leaving polyacrylamide homopolymer in the supernatant;however,the reaction products recovered from the mixture of lignin and acryl-amide incubated in aqueous dioxane(70%)with laccase and dioxane peroxides(170mg·l?1)did not precipitate in either a neutral or an acidic solution(Table2).The same pattern of solubility was shown in the graft polymer generated in the free radical polymerization reaction according to Meister et al.(1984a,b)(Table2).Interestingly. the copolymers generated enzymatically in the presence of a reduced amount of DP(below100 mg·l?1)were only partially soluble in neutral or acidic aqueous mixtures(Table2).It has been shown that laccase and dioxane peroxides at a concentration of170mg·l?1generated the maxi-mal yield and the maximal rate of acrylamide incorporation onto the lignin backbone(Mai et al.,1999).Lower concentrations of DP reduced the grafting rate(less than50%of acrylamide was grafted)and the yield of the enzymatically gener-ated copolymer(Mai et al.,1999).Obviously,the grafting of acrylamide onto the lignin molecule

that CaCl2can form an intermediate complex with peroxide species and can accelerate its de-composition either to alkoxy or peroxide radicals (Meister et al.,1984a,b).

The addition of CaCl2causes a higher rate of chain initiation resulting in a higher total yield; however,this presumably occurs at the expense of a higher rate of acrylamide homopolymer synthesis.

Although phenoxy radicals formed by laccase are able to reduce peroxide species to alkoxy radicals which initiate the polymerization of acryl-amide,the initiation system of CaCl2and Ce4+ previously reported is more effective than the chemo-enzymatic system applied in the present study in the initiation of a‘living’polyacrylamide chain(Meister et al.,1984a,b).The chain combi-nation with lignin radicals produces the grafted copolymer.However,because of the high rate of chain initiation there are other statistical possibili-ties for the termination of a‘living’polyacry-lamide chain(disproportionation,combination of acrylamide radicals,hydrogen abstraction)which cause homopolymer formation.The application of laccase should reduce the of homopolymer production due to a higher rate of phenoxy radi-cal generation.

Enzymatic crosslinking(i.e.homopolymeriza-tion)of lignin is a competitive reaction to grafting since enzymatically generated phenoxy radicals are consumed by the formation of intra-lignin

C.Mai et al./Journal of Biotechnology79(2000)173–183179

did,to a certain degree,turn it into a water soluble product.

The GPC data of the tested OL and the reac-tion product showed that the copolymerizate was not simply a blend of lignin and polyacrylamide (Fig.1A and B).Incubation of OL or the mixture of lignin and acrylamide in aqueous dioxane with DP and laccase caused a sharp increase of mole-cular mass of both the lignin tested and the copolymer product recovered(Fig.1A and B). The elution pattern of the copolymers was moni-tored at280nm;this was the absorption maxi-mum of phenolic sites in lignin moiety of the analyzed product.The area of each chro-matogram corresponds to the lignin content in the product since the acrylic side chain of the copoly-mers eluted from the column could not be de-tected by UV detector at280nm.

Although the same quantity of copolymer and of OL was used in chromatography,the UV absorbance of the high molecular weight(HMW) portion of eluted copolymers was lower than that of the corresponding fraction of eluted lignin(Fig. 1A and B).The lower UV absorbance of the copolymer displayed a direct relationship to the decreased lignin content in the graft resulting from the incorporation of acrylamide onto the lignin matrix(Table1).An extensive decrease at 280nm in the HMW of the copolymer generated enzymatically in the presence of CaCl2was ex-pected since CaCl2enhanced the rate of acryl-amide grafting onto the lignin backbone and con-currently diminished the speci?c lignin content in the product(Table1,Fig.1B-2).The permeate of the chemically synthesized products showed the lowest UV absorbance(Fig.1B-3)in comparison to the absorbance of permeates of enzymatical products;this corresponded to the lower lignin content in these products(Table1).

The detection of nitrogen in the permeate re-vealed incorporated acrylamide.About90%of the nitrogen found in the laccase generated co-polymerizate was detected in an HMW portion of the product,independent of the rate and ef?ciency of the polymerization process.Rechromatography of the separated and dialyzed HMW fraction of the eluted copolymer changed neither the pattern of molecular mass distribution nor the N/C ratio of the fractions(data not shown).

To photometrically detect side chains contain-ing acrylamide in the presence of lignin,a terpoly-mer that contained the acrylamide derivate N-(4-methoxyphenyl)-2-propenamide,an UV-ac-tive chromophore,was synthesized.Thus,the side chains of the eluted polymer could also be moni-tored at280nm simultaneously with the lignin backbone of the copolymerizate(Fig.2).Higher UV-absorbance(at280nm)of the HMW portion of the terpolymer permeate in comparison to the same fraction of lignin–acrylamide copoly-mer was due to the absorption of the N-(4-methoxyphenyl)-2-propenamide chromophore which occurred in addition to that of lignin.

A differential spectrum of acrylamide–lignin copolymer and lignin–acrylamide-N-(4-meth-oxyphenyl)-2-propenamide terpolymer revealed the presence of the chromophore in the eluted https://www.doczj.com/doc/8e10113186.html,puted differential spectra obtained by substracting the absorbance spectra of5ml fractions of HMW permeate of acrylamide–lignin copolymer(Fig.2-2)from the absorbance spectra of the same fractions of lignin–acrylamide-N-(4-

Fig.2.Pattern of molecular weight distribution of lignin–acryl-amide-N-(4-methoxyphenyl)-2-propeneamide and lignin–acry-lamide copolymerizates generated by laccase(2–1,2–2);In-sert:Comparison of the UV absorbance differential of the HMW fraction(19–34ml)of lignin–acrylamide-N-(4-methoxyphenyl)-2-propeneamide copolymer(2–3)and the UV absorbance spectra of the chromophore(2–4).Arrows indi-cate void-(V0)and total-(V t)volumes of the applied GPC column..

C.Mai et al./Journal of Biotechnology79(2000)173–183

180

Fig.3.FT-IR and13C-NMR-CP-MAS spectra of lignin–acrylamide copolymerizates.3A FT-IR spectra of lignin–acrylamide copolymerizates recovered from the mixture of lignin and acrylamide incubated in aqueous dioxane(70%)with laccase,dioxane peroxide and CaCl2;(3A-1)DP concentration170mg·l?1,(3A-2)170mg·l?1DP and CaCl2;3B13C-NMR spectra of lignin–acrylamide copolymerizates recovered from the mixture of lignin and acrylamide incubated in aqueous dioxane(70%)with laccase and either with DP(170mg·l?1)or with DP(170mg·l?1)and CaCl2(3B-1and3B-2,respectively).Copolymerizate generated in free radical reaction(3B-3).Insert:Tabulated integration data of the speci?c regions of the NMR spectra.

methoxyphenyl)-2-propenamide terpolymer(Fig. 2-1)displayed an absorbance pattern with a max-imum at252nm(Fig.2-3),which was similar to the spectra of pure N-(4-methoxyphenyl)-2-prope-namide(Fig.2–Fig.4.The peak of eluted lignin–acrylamide–N-(4-methoxyphenyl)-2-propenamide terpolymer(Fig.2-1)appeared to be eluted at a larger volume(Kav=0.08)than the peak of the lignin–acrylamide copolymer(Fig.2-2)(Kav= 0.03).

3.2.Spectroscopic characterization of the products

Fig.3A shows the FT-IR-spectra of the lignin/ acrylamide copolymer generated by laccase in aqueous dioxane(70%)with dioxane peroxides both with and without the addition of CaCl2to the reaction media.The spectra show a series of characteristic bands which can be assigned to speci?c structures of both components making up the copolymer.Both of the copolymers analyzed show a strong peak at1512cm?1caused by aromatic skeletal vibrations of the lignin moiety of the product.Absorption bands caused by the polyacrylamide portion of the polymers are to be found at3343and3197cm?1(doublet of CONH2hydrogen stretch vibration)and1668and 1610cm?1(doublet of CONH2carbonyl stretch vibration).The increase of these signals with the copolymer synthetized in the reaction mixture that was supplemented with CaCl2are in agree-ment with the increasing rate of acrylamide incor-poration shown in this product(Table1).

The13C-CPMAS NMR spectra of copolymers generated either in a free radical reaction or enzy-matically with or without the addition of CaCl2 are shown in Fig.3B.The spectra of enzymati-cally generated copolymers showed a similar pat-tern to that of the graft product obtained in a free radical reaction.The most intensive signals ap-peared at40ppm from the aliphatic C in the polyacrylamide backbone and at170ppm for the carbonylic C of the amide group of the copoly-mers;the same signals appeared regardless which method of graft synthesis was used.The integra-tion data of the areas of the NMR spectra(Fig. 3B insert)in the regions of210–160,150–100and 90–0ppm caused by the speci?c resonance of carboxylic,aromatic and aliphatic carbon,respec-tively,were in agreement with the lignin–acryl-amide ratio in the polymer products recovered (Table1).

C.Mai et al./Journal of Biotechnology79(2000)173–183181

3.3.Light and SEM microscopy obser6ations Freeze-dried copolymerizate preparations ap-peared as a?uffy,?ne beige rather brittle material with shining surfaces.Both light and SEM micro-graphs have demonstrated an abundance of?bril-like particulate in the copolymerizate preparations (Fig.4A–F).Fibrils appeared either straight(Fig.

Fig.4.Light and SEM micrographs.Lignin–acrylamide copolymerizates(4A,B);lignin–acrylamide-N-(4-methoxyphenyl)-2-prope-namide copolymer(4C,D);free radical reaction polymerizates(4E,F).Bars1mm(A,C,E)and0.1mm(B,D,F).

C.Mai et al./Journal of Biotechnology79(2000)173–183 182

4B)or with multiple branches(Fig.4B).The ?brils were:5m m in diameter and over200m m long.The?bril-like morphology of the enzymati-cally synthesized polymerizate was predominant; whereas in the preparations formed by a free radical polymerization,lamellate aggregates were in abundance.Similar morphological structures were not observed in the original lignin prepara-tion nor in the lignin transformed by laccase when both preparations were lyophilized in the same way as were the copolymers.These?ndings are in agreement with previously reported elec-tron microscopic observations of OL from spruce and poplar which revealed globular lignin associ-ations(Fengel et al.,1981).SEM observation of the preparations of laccase initiated copolymer-izates at large magni?cation did not reveal addi-tional substructures.A prerequisite for the formation of a?bril-or crystal-like structures is the regularity of the polymer chain.This is espe-cially important with regard to the composition as well as to the regularity from a steric point of view(Vollmert,1973).Thus,it appears that acry-lamide grafted onto the lignin macromolecule provides the resulting copolymer a certain degree of regularity with respect to the composition of the macromolecule and causes the copolymerized product to take on a crystal-like form.

4.Conclusion

The results presented have shown that laccase of Trametes6ersicolor(E.C.1.10.3.2)in the pres-ence of dioxane peroxides initiates the grafting of acrylamide onto organosolv lignin dissolved in an organic solvent to an extent comparable with that of the free radical reaction.

The FTIR and NMR-spectroscopic analysis of the enzymatically synthesized product as well as its GPC and UV-spectroscopic characteristics re-vealed some similarities with the product gener-ated by free radical polymerization and suggest a covalent bonding between acrylamide and lignin moieties.

We assume that the reactivity of phenoxy radi-cals,generated by laccase catalysis,is not suf?-cient to start side chain polymerization;alkoxy radicals generated by reduction of peroxides are supposed to initiate homopolymerization of acryl-amide.A covalent bonding could then be formed by a‘quenching’reaction of the radical end of the growing,‘living’polyacrylamide homopoly-mer with a phenoxy radical of the lignin back-bone.Alkoxyradicals were probably formed by reduction of peroxides mediated by enzymatically generated phenoxy radicals of lignin or by some reactive oxygen species such as a superoxide radi-cal,whose presence in the reaction media con-taining lignin and laccase has been reported (Milstein et al.,1994).

Our data provide the?rst characterization of products generated by lignin biocatalytic transformation in the presence of conventional plastic monomers.The description represents a biotechnological alternative for lignin utilization and a means of generating new engineering plas-tics.

Water soluble lignin graftpolymers and poly-acrylamide cross-linking gels could provide an alternative to conventional acrylic homopolymer products such as thickeners,drilling muds or soil conditioners.Moreover,copolymerization of syn-thetic side chains onto naturally occurring back-bones could be considered as a way of producing materials which are more biodegradable than the commercial acrylic homopolymers which are cur-rently available.Further research is necessary to elucidate the reaction mechanism and the biodegradability of the copolymerizate. Acknowledgements

The authors wish to thank Dr H.-D.Lu¨de-mann and Dr R.Fru¨nd for the measurement and interpretation of the CPMAS NMR spectra,H.J. Feine for the assistance by the FT-IR spectra recording and W.Tambour for excellent graphics work.

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