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Influence of the physical state of lignin on its degradability by the lignin peroxidase of Phanerochaete chrysosporium
Influence of the physical state of lignin on its degradability by the lignin peroxidase of Phanerochaete
chrysosporium Bernard Kurek,* Bernard M o n t i e s t and Etienne Odier* * Laboratoire de Microbiologie and ? Laboratoire de Chimie Biologique, Centre de Biotechnologies Agro-Industrielles, I N R A , Thiverval-Grignon, France
The rate of oxidation of spruce milled wood lignin by Phanerochaete chrysosporium was investigated with precipitated lignin as well as lignin finely dispersed in water (colloidal lignin). 14202 consumption rates in the course of lignin oxidation in the presence of purified lignin peroxidase were much higher with colloidal lignin compared with precipitated lignin. Characterization of reaction products and molecular-size distribution confirms that reaction rate of lignin peroxidase with 14C-lignin is strongly dependent on the physical state of lignin. The lignin peroxidase catalyse mainly repolymerization of colloidal lignin. However, in the presence of veratryl alcohol, both polymerization and depolymerization were observed.
Keywords: Lignin, milled wood; lignin, 14C labelled lignin; colloidallignin; lignin peroxidase; depolymerization; Phanerochaete chrysosporium Introduction Phanerochaete chrysosporium produces several peroxidases involved in lignin biodegradation. ~ The lignin peroxidase catalyses the removal of one electron from aromatic structures with subsequent formation of a cation radical. 2 Cation radicals decompose according to various reactions that depend on the nature of substitutents of the aromatic ring and the presence of molecular oxygen, j The lignin peroxidase oxidizes nonphenolic aromatic structures resistant to oxidation by previously described peroxidases such as horseradish peroxidase. 3,4 For this reason, lignin peroxidase is assumed to be able to attack most aromatic structures in lignin while other peroxidases can oxidize only phenolic moieties. Mechanistic studies with lignin dimeric model compounds establish that lignin peroxidase induces various cleavage reactions including C~-C/3 cleavage 5~/30-4 cleavage, 6,7,9A°and aromatic ring cleavage. 8,t~ The
Abbreviations: LM, milledwoodlignin;LE, enzymaticallyliberated lignin; LCn;colloidalfractionof ligninformedafter n precipitations from DMF solutioninto water; DMF, dimethylformamide Address reprint requests to Dr. Kurek, Laboratoirede Biochimie, Centre de BiotechnologiesAgro-lndustrielles,INRA, 78850Thiverval-Grignon, France Accepted for publication 2 April 1990
©
1990 Butterworth-Heinemann
structure of biodegraded lignin extracted from wood reveals that fungal attack operates through reactions that are catalysed by lignin peroxidase with lignin model compounds. 12-14 Another peroxidase, designated manganese peroxidase, produced by P. chrysosporium oxidizes Mn ++ to Mn +++, which, in turn, can oxidize various structures. 15-~7 Phenolic lignin model compounds can be degraded by manganese peroxidase with subsequent alkyl-phenyl and C~-C/3 cleavage. 18 Wariishi et al. also reported the degradation of nonphenolic model compounds by the Mn peroxidase in the presence of thiols. ~9 However, to the best of our knowledge, there is no report that manganese peroxidase is able to degrade nonphenolic structures or macromolecular lignin in vitro. For this reason, the function of manganese peroxidase in direct lignin degradation is not apparent. Although the mechanism by which lignin model compounds are degraded by lignin peroxidase is now well understood, incubation of lignin with lignin peroxidase under conditions suitable for catalysis does not result in the expected formation of lignin depolymerization products. On the contrary, polymerization of lignin after incubation is apparent. 2°-22 This result can be explained by coupling of phenoxy radicals that are formed by oxidation of lignin peroxidase after cleavage of ether substructures in lignin. 21 Since ether bonds are the major intermonomeric bonds in lignin, Enzyme Microb. Technol., 1990, vol. 12, October
771
Papers attack of etherified units and/3-0-4 cleavage by lignin peroxidase is then expected to result in the formation of a significant proportion of phenols. These phenolics can be further oxidized to phenoxy radicals by lignin peroxidase. Umezawa et al. 23 reported/3-0-4 ether bond cleavage as well as ring opening products through the action of lignin peroxidase on synthetic lignin. The yields of degradation products, however, was not reported. Indeed Kern et al. 22 have shown that lignin peroxidase probably does not induce cleavage reactions in the lignin macromolecule under the conditions of their experiments. However, available data do not provide quantitative information concerning the extent of lignin structure modification during reaction with lignin peroxidase. Because lignin is a three-dimensional structure, degradation is expected to be confined to exposed surfaces, thereby limiting the rate of degradation. In a previous report, 24 we have shown that lignin can be fractionated into precipitated and colloidal lignin by precipitation of a dimethylformamide solution into water. Characterization of the molecular size and chemical structure have shown that colloidal and precipitated lignins do not differ significantly. Colloidal lignins can then be considered as total lignin in a highly dispersed state. Preliminary studies have shown that consumption of H202 by the lignin peroxidase is greater for oxidation of colloidal lignin in water compared with precipitated lignin, suggesting a relationship between lignin reactivity for enzymic oxidation and the dispersion state. In this study, the detailed comparison of oxidation rates by the lignin peroxidase of two different lignin preparation (LM and LE) in the precipitated or colloidal state is investigated. The subsequent modification of the elution profile by size-exclusion chromatography of both precipitated and colloidal lignin labeled in the propane side chain and the aromatic ring was then determined, as well as the formation of volatile and insoluble compounds, indicative, respectively, of C I and C2 compounds and modification in the chemical structure of lignin.
Materials and methods Extraction of spruce lignins: LM (milled wood lignins) and LE lignins were prepared according to Lapierre et al.25 Labeled lignins: 14C-lignins (propane and side chain) were prepared after labeling lignin in 2-year-old spruce (Picea abies) plants with lac-(U)-L-phenylalanine (16.6 MBq mmol -~, CEA, Saclay, France) by infusion of the basal extremity from the cut stems. 26 Fresh xylem was then roughly ground (0.2 to 0.5-mm particle size) and immediately Soxhlet-extracted 48 h with toluene/ethanol (I : 1, v/v) followed by ethanol (24 h) and water (48 h). The labeled cell wall residue obtained was then milled with a Retsch MM-2 Miller (Prolabo, Paris, France) before lignin extraction: 300 mg cell wall residue were milled 72 h in 10-ml agate cups with two 12mm diameter agate balls at 1900 cycles mn -~. The LM
772
Enzyme Microb. Technol., 1990, vol. 12, October
lignin fraction was then extracted with the same procedure as for nonlabeled lignins (see above). Colloidal lignins were prepared by precipitation of lignin in water from a dimethylformamide (DMF) solution (Ref. 24; see also Figure 1): Lignin is allowed to swell in a minimum volume of DMF during one night. More DMF is then added to adjust the lignin concentration to 250 or 125 mg ml -1, respectively, for LM and LE lignins. This solution is added from a glass syringe to distilled water with vigorous agitation to 5 mg/ml lignin final concentration. The suspension is centrifuged at 17,000g during 20 min. The supernatant is then filtered with suction on a nitrocellulose filter 0.45 /zm pore size (Millipore) to eliminate the residual precipitated lignin. The colloidal LC lignin obtained can be stored at 4°C for several months. The LC, lignin is obtained by n repetitions of this procedure according to Figure 1. Lignin peroxidase was purified from static cultures of P. ehrysosporium UK 5 according to Ref. 27. The isozymes with pI 4.6 and 4.8 were used in the experiments. Lignin peroxidase was assayed by the veratryl alcohol assay according to Tien and Kirk. 28 H202 consumption by the lignin peroxidase for oxidation of lignins were determined using a YSI 2510 electrode (Yellow Springs Instrument, Yellow Springs, OH) connected to an Prg-Del amperometric monitor (Taccussel, Paris, France). Incubation of HC-lignin with lignin peroxidase and H202 was as follows. The reaction mixture contained 3 mg precipitated lignin or 0.5 mg colloidal lignin (380 Bq/mg), lignin peroxidase (purified or crude protein mixture) in Na-tartrate buffer 100 mM, pH 3, in a total volume of 3 ml. In some experiments, veratryl alcohol was added as specified. Reactions were started by addition of HzO2. In certain experiments, lignin peroxidase or H202 were added during incubation. Radioactivity in volatile compounds formed upon incubation of JaC-lignin with lignin peroxidase and H202 was determined by liquid scintillation after distillation of the total reaction mixture under 5 mbar (500 Pa) pressure at 40-45°C and collecting the condensation products at -60°C. The nonvolatile reaction products were recovered by solubilization of the residue in DMF after distillation. Molecular-size distribution of lignin and lignin degradation products was analyzed according to Connors et al. 29 using Sephadex LH-60 (Pharmacia, St Quentin en Yvelines, France) in DMF containing LiCI 0. ! M in a 1 × 50-cm column (Pharmacia). Radioactivity in the effluent was assayed after collection of 0.9-ml fractions. Radioactivity was determined by liquid scintillation in Pico-fluor (Packard, Rungis, France) using a Betamatic II counter (Kontron, St Quentin en Yvelines, France).
Results The insoluble PI lignin obtained after precipitation of both LM and LE lignin from a DMF solution (Figure 1) was washed extensively with water in order to elim-
Lignin enzymic degradability according to its physical state: B. Kurek Pmtmatment relate i ~ l k ~ n
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inate residual colloidal lignins. The residual colloidal lignins represent then only 0.2% of initial lignin. H202 consumption in reaction mixtures with lignin peroxidase and washed precipitated lignin from LM or LE was determined. The different colloidal lignins LC, from LM and LE were incubated with lignin peroxidase in presence of hydrogen peroxide (see Figure 1 for nomenclature). Residual I-I~O2 in the reaction mixture was determined with the hydrogen peroxide electrode. Results (Figure 2) show that colloidal lignins from LM or LE give a much faster consumption of H202 with iignin peroxidase than did the corresponding washed precipitated lignins. Consumption of 1-I202 in the presence of colloidal lignins from LM is greater than with colloidal lignins from LE. The different colloidal iignins from LM (LCI, LC2, etc) differ also in reactivity for lignin peroxidase based on H202 consumption rates. LC2 from LM reacts less than do LC1, LC3, and LC4. Figure 2 shows also that differences in reactivity between colloidal lignins and precipitated washed lignins are greater with LM lignins than with LE lignins. The modifications of 14C-lignin after incubation with lignin peroxidase and H202 were examined by determining molecular-size distribution and the formation of volatiles as well as DMF-insoluble labeled compounds. Experiments were run with precipitated or colloidal lignins with or without veratryl alcohol in the reaction mixture. Amounts of lignin peroxidase (crude or purified) were varied as well as was that of H202.
Preliminary experiments were conducted to determine the evolution of lignin peroxidase activity versus time in reaction mixtures. Figure 3 shows that lignin peroxidase activity decreases rapidly in a reaction mixture without veratryl alcohol, and no lignin peroxidase activity could be deteeted after 50 min (Figure 3). In contrast, when veratryl alcohol was added, lignin peroxidase activity was maintained in the reaction mixture during the first 15 min. When addition of H202 LM
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Reoction time (rain) Figure 2 Consumption of H202 in reaction mixtures (3 ml) containing purified lignin peroxidase (9 nkat), precipitated washed lignin (P) from LM and LE (3 mg), colloidal lignin (0.3 rag) LC1, LC2, LC3, and LC4 from LM (left) and LE (right); reaction is in 3 ml tartrate-Na buffer, pH 3 (see Materials and methods)
E n z y m e M i c r o b . T e c h n o l . , 1990, v o l . 12, O c t o b e r
773
Papers Table 1 C o m p o s i t i o n of reaction mixtures containing precipitated LM ~4C-lignin, lignin peroxidase as crude protein mixture, and H2Oz, and radioactivity in volatiles products f o r m e d after incubation a
Run
Veratryl alcohol (/~moles)
Lignin peroxidase as crude protein mixture (nkatb; mg ~)
100
o~.
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--0
75
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•
50
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0.6
0 100 (0.2) then 50 (0.1) after 2 h ~ 50 (0.1) repeated after 2 h~
O
0.7 (0.23) 0.4 (0.13)
c-
0.6 (0.20)
2~
25
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0 0
a All reaction mixtures are in Na-tartrate buffer 100 mM, pH 3, with 0.8 rng (305 Bq) lignin in a final v o l u m e of 3 ml; incubation time is 4 h b Lignin peroxidase activity in the crude protein mixture (nkat) Total protein quantity in parentheses d % of total radioactivity in parentheses e 150 n m o l e s H~O2 also added
10
20
? 30
40
50
Reaction time (min) Figure 3 Evolution of lignin peroxidase activity in reactions mixtures in control experiments m3 and T4 versus time (see Table 2); (0) with veratryl alcohol (1-4); (©) w i t h o u t veratryl alcohol (T3); lignin peroxidase activity is expressed in % of activity at time zero. The a r r o w indicates successive addition of 3 ~mol H~02 every 2 min in order to exhaust veratryl alcohol in reaction mixture
is repeated in the reaction mixture, veratryl alcohol becomes exhausted and lignin peroxidase activity decreases progressively (Figure 3). The first experiment was run with a precipitated 14C-lignin and crude lignin peroxidase (Table 1). Addition of lignin peroxidase and H202 was repeated after 2 h incubation. A similar set of reactions with precipitated '4C-lignin and purified lignin peroxidase was conducted with high enzymic activity in the reaction mixture (Table 2). In a third set of reactions, colloidal J4C-lignin was incubated with lignin peroxidase at the same level as in the first experiment (Table 3). Radioactive volatile compounds were produced from '4C-lignin in all reaction mixtures with lignin peroxidase and H202. Yields of volatiles were always low, particularly in experiments when veratryl alcohol was omitted, whether the lignin substrate was precipitated or colloidal (Tables 1-3). H20, alone caused the formation of volatile compounds in all cases (Tables I-3). Negligible amounts of radioactive volatile com-
pounds were formed when '4C-tignin was incubated with lignin peroxidase in the absence of H202 (Table 3). In several runs, a fraction of ~4C could not be solubilized in DMF after the reaction. That proportion which became insoluble in DMF was 12% with precipitated lignin (Table 2) and 25% with colloidal lignin (Table 3) in the presence of veratryl alcohol. In the absence of veratryl alcohol, the corresponding data are 6.1 and 0.9%, respectively (Tables 2 and 3). Control experiments with H202 alone showed only small amounts of DMF-insoluble compounds after incubation. DMF-insoluble compounds could be solubilized by the addition of NaOH to the DMF (0.05 M final concentration). DMF-soluble reaction products from 14C-lignin were analyzed for molecular-size distribution after incubation with lignin peroxidase in the different experi-
Table 2 C o m p o s i t i o n of reaction mixtures containing precipitated LM 14C-lignin, purified ~ignin peroxidase, and H2Oz, and radioactivity in volatiles and DMF insoluble products f o r m e d after incubation a H202
Run T1 T2 E1 or T3 d E2 or T4d
Veratryl alcohol (~moles)
Lignin peroxidase (nkat)
/~moles
N u m b e r of additions and time elapsed b e t w e e n additions"
0 0 0 60
0 1000 1000 1000
3 0 3 3
1 0 1 10 (2)
Radioactivity (Bq) in volatile reaction products c 3 0.3 1 8
(0.26) (0.03) (0.09) (0.67)
Radioactivity (Bq) in reaction products insoluble in DMF c 21.7 4.5 69 136
(1.9) (0.40) (6.10) (12)
ND: not determined. a All reaction mixtures are in Na-tartrate buffer 100 mM, pH 3, with 3 mg (1140 Bq) lignin in a final v o l u m e of 3 ml; incubation time is 2 h. b Time (min) between t w o additions in parentheses c % of total radioactivity in parentheses d Control e x p e r i m e n t s for lignin peroxidase activity during runs E~ and E2
774 EnzymeMicrob. Technol., 1990,vol. 12, October
Lignin enzymic degradability according to its physical state: B. Kurek
e t al.
Table 3 Composition of reaction mixtures containing 14C-colloidal lignin LC~ from LM, purified lignin peroxidase, and H202, and radioactivity in volatiles and DMF insoluble products formed after incubation a Lignin peroxidase
Run
Veratryl alcohol (#moles) 0 0 0 60
T~ T2 E~ E2
H202
(nkat)
Number of additions and time elapsed between addition@
#moles
Number of additions and time elapsed between addition@
0 60 60 60
0 1 4 d (15) 1
3 0 0.125 d 3
1 0 1 (4) 20 (3)
Radioactivity (Bq) in volatile reaction products c 1.10 0.20 0.16 0.85
Radioactivity (Bq) in reaction products insoluble in DMF c
(0.6) (0.1) (0.08) (0.4)
0 (0) ND 2 (0.9) 40 (24)
ND: not determined. a All reaction mixtures are in Na-tartrate buffer 100 mM, pH 3, with 0.48 mg colloidal iignin in a final volume of 3 ml; incubation time is 2h. b Time (min) between two additions in parentheses c % of total radioactivity in parentheses d After each addition of lignin peroxidase, 0.175 #mole H202 is added; after 4 min, 0.125 #mole H202 is added again
ments. Almost no modification could be observed when precipitated lignin was incubated with lignin peroxidase as the crude enzyme preparation (experiments according to Table 1 and Figure 4). When the same precipitated lignin was incubated with a high amount of lignin peroxidase (Table 1), slight differ-
ences--perhaps not significant--were seen in the elution profile of the reaction products (data not shown). When colloidal lignins were used instead of precipitated lignins, differences in the molecular-size distribution of the DMF-soluble reaction products were drastic (Figure 5). This was particularly evident when veratryl alcohol was present in the reaction mixture. Purified lignin peroxidase in the absence of veratryl
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Figure 4 Distribution of radioactivity by size-exclusion chromatography (Sephadex LH-60 in DMF/LiCI 0.1 M) in labeled LM lignins treated with lignin peroxidase (as a crude protein mixture from ligninolytic cultures of P. chrysosporium) in the conditions detailed in Table I. (O) run E2: with veratryl alcohol; (©) run El: without veratryl alcohol; (A) run T~: control with H202 without lignin peroxidase. Polystyrene standards of 20,000 and 2000 D used for calibration of columns are eluted, respectively, in fractions 12 and 26
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Fraction n u m b e r Figure 5 Distribution of radioactivity by size-exclusion chromatography (Sephadex LH-60 in DMF/LiCI 0.1 M) in labeled colloidal lignin LC1 from LM treated by 60 nkat purified lignin peroxidase in the conditions detailed in Table 3; (Q) run E2: veratryl alcohol; (©) run E1 without veratryl alcohol; (&) run TI: control with H202 without lignin peroxidase: (~) run T2: control with lignin peroxidase and without H202
Enzyme Microb. Technol., 1990, vol. 12, October
775
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Figure 6 Distribution of radioactivity in DMF insoluble lignins products formed after incubation of colloidal lignin with purified lignin peroxidase in the presence of veratryl alcohol in the conditions detailed in Table 3 (run E2; see also Figure 5)
alcohol caused the repolymerization of lignin, while some depolymerization was apparent when veratryl alcohol was added (Figure 5). H202 alone caused minor modifications of the molecular-size distribution of lignin (Figure 5). DMF-insoluble products were analyzed after solubilization by addition of NaOH. Sizeexclusion chromatography showed this fraction consisted of repolymerized lignin products (Figure 6).
Discussion The ability of different lignin preparations to be oxidized by lignin peroxidase varies considerably as judged by the rate of H202 consumption. Colloidal lignins from both preparations, LM and LE, are oxidized much more rapidly than are the corresponding precipitated lignins. We have also reported elsewhere that colloidal lignin partially precipitated by a freeze-thawing cycle is less reactive than is initial colloidal iignin. 24 Altogether, these results show that differences in the physical state of precipitated and colloidal lignins accounts for differences in reactivity. However, differences in the reactivity between various colloidal lignins preparations are also observed. The colloidal lignins from LM are oxidized much more rapidly than are the colloidal lignins from LE. In the same way, the various LC~, LC~, LC3, and LC4 colloidal lignins from LM are, respectively, more and more reactive to the lignin peroxidase oxidation. These differences in reactivity cannot be explained by variations in their chemical composition, which appeared to be practically identical. 24 In return, differences in molecular weight of lignins could account for it. lndeed, gel permeation chromatography studies have shown that the colloidal lignins from the LM fraction are less polymerized than are colloidal lignins from LE. 24 In the same way, LC~ and LC3 from LM are, respectively, slightly enriched in lower molecular weight fractions, compared to LC~. This apparent effect of molecular weight on colloidal lignin reactivity could, however, also be explained 776
Enzyme Microb. Technol., 1990, vol. 12, October
by differences in the physical state of lignin in aqueous media. Indeed, the lower molecular weight lignin would have a greater surface area in colloidal suspension, allowing a greater reactivity. The LC~ colloidal lignin from LM contains about twice as many condensed units as do the LC3 colloidal iignin. 24 However, both colloidal lignins showed comparable reactivity, suggesting that reactivity to lignin peroxidase is not related to the degree of condensation. The sensitivity of the different substructures in the lignin to lignin peroxidase attack deserves, however, further research. Differences in reactivity of highly dispersed and precipitated lignin-to-lignin peroxidase oxidation were confirmed by an analysis of the reaction products. Precipitated lignins are not significantly modified in molecular-size distribution after incubation even with high concentrations of lignin peroxidase. In contrast, the enzyme at 20 nkat/ml strongly affects the elution profile by gel permeation chromatography of colloidal lignins after reaction, leading clearly to polymerization of lignin in the absence of veratryl alcohol. The prominent role of veratryl alcohol in lignin enzymic degradation is confirmed. Our results state that veratryl alcohol protects lignin peroxidase against inactivation caused by H202, 3°,3~ even in the presence of lignin in the reaction mixture, allowing the reaction to proceed longer. In contrast, lignins do not protect the lignin peroxidase. This could indeed explain the greater modification in the elution profile, suggesting partially depolymerization of lignins, when veratryl alcohol was added to the reaction mixture. However, a significant proportion of the radioactivity from the 14Clignin was converted into DMF insoluble products in these experimental conditions and it required separate molecular weight distribution determination. Analysis after dissolution of this sample fraction in a DMFNaOH mixture showed that insoluble lignin consists of repolymerized reaction products. The presence of veratryl alcohol in the reaction mixture could also affect the nature of the lignin degradation endproducts, perhaps according to a cooxidation mechanism. 32,33 This is suggested by the differences in DMF solubility of repolymerized lignin endproducts in the presence or absence of veratryl alcohol in the reaction mixture. However, since veratryl alcohol affects the evolution of lignin peroxidase activity during reaction, no firm conclusion can be drawn; further research is then required to characterize the chemical compositions of repolymerized lignin endproducts in order to confirm the existence of such a mechanism. The results obtained are then consistent with previous reports establishing that lignin peroxidase causes above all repolymerization of lignin. 2°,21 In these studies, however, the formation of such insoluble compounds is not reported and has perhaps gone unsuspected. The formation of volatile compounds was observed in experiments after incubation with lignin peroxidase. In contrast with other studies, the 14C-labeled lignins
Lignin enzymic degradability according to its physical state: B. Kurek et al. used here are labeled in the propane side chain as well as in the aromatic ring. It is likely that the volatile radioactive compounds are derived from C2 fragments by Ca-Cfl or ether bond cleavage by the lignin peroxidase. The formation of volatile 14C-labeled compounds during incubation of lignin with lignin peroxidase and H202 could then indicate that cleavage reactions have taken place. The yield of these products is, however, less than 1% of the total Jac activity of lignins. In addition, control experiments in which lignin was incubated with H202 without lignin peroxidase show higher yields in volatile compounds compared to the experiments with enzyme even in the presence of veratryl alcohol. This indicates that formation of such compounds is not directly related to the extent of modification in the molecular weight distribution of lignins. In conclusion, this study shows that precipitated lignins have a low reactivity to lignin peroxidase oxidation. In contrast, highly dispersed lignins in the colloidal state are easily oxidized and modified. Our results are consistent with previous reports on enzymic hydrolysis of insoluble macromolecules such as starch or cellulose showing that an increase in substrate accessibility for the enzyme results in higher reaction rates 34 Polymerization of lignins seems to be the main reaction catalysed by lignin peroxidase. However, in the presence of veratryl alcohol, some depolymerization of lignin was also apparent. These results deserve further research in order to confirm and to quantify the extent of depolymerization with other analytical techniques than gel permeation chromatography. The evolution of oxidation products from lignin was not controlled in our study as in previous reports. 2°,2~ Further investigation is required to elucidate the mechanism by which coupling reactions could be prevented or diminished to improve the degradation of lignins. Our results strongly suggest that depolymerization could be effective when lignin peroxidase is incubated in media designed for preventing a coupling reaction and allowing good dispersion of lignin. This observation could be of great significance for applications using ligninolytic enzymes for transformation of the lignin polymer.
Acknowledgement This research was co-financed by La Cellulose du Pin (Bordeaux, France).
References Gold, M. H., Wariishi, H. and Valli, K. In: ACS Symposium Series No. 389: Biocatalysis in Agricultural Biotechnology American Chemical Society, Washington, DC, 1989, 127-140
Kersten, P. J., Tien, M., Kalyanaraman, B. and Kirk, T. K. J. Biochem. 1985, 260, 2609-2612 Hammel, K. E., Kalyanaraman, B. and Kirk, T. K. J. Biol. 3 Chem. 1986, 261, 16948-16952 4 Kersten, P. J., Kalyanaraman, B., Hammel, K. E. and Kirk, T. K. In: Lignin Enzymic and Microbial Degradation, Paris, April 23-24 (Les colloqaes de I'I.N.R.A.) I.N.R.A., Paris 5 1987, Vol. 40, 75-80. Tien, M. and Kirk, T. K. Science 1983, 221, 661-663 6 Gold, M. H., Kuwahara, J., Chiu, A. A. and Glenn, J. K. Arch. Biochem. Biophys. 1984, 234, 353-362 7 Habe, T., Shimada, M., Umezawa, T. and Higuchi, T. Agric. Biol. Chem. 1985, 49, 3505-3510 8 Miki, K., Renganathan, V., Mayfield, M. B. and Gold, M. H. FEBS Lett. 1987, 210, 199-203 9 Glenn, J. K., Morgan, M. A., Mayfield, M. B., Kuwahara, M. and Gold, M. H. Biochem. Biophys. Res. Commun. 1983, 114, 1077-1083 10 Kirk, T. K., Tien, M., Kersten, P. J., Mozuch, M. D. and Kalayanaraman, B. Biochem. J. 1986, 236, 279-287 11 Umezawa, T., Shimada, M., Higuchi, T. and Kusai, K. FEBS Lett. 1986, 205, 287-292 12 Chua, M. G., Chen, C. L. and Chang, H. M. Holzforchung 1982, 36, 165-172 Ellwardt, P. C., Haider, K. and Ernst, L. Holzforschung 1981, 13 35, 103-109 14 Tai, D., Terazawa, M., Chen, C. L., Chang, H. M. and Kirk, T. K. In: Recent Advances in Lignin Biodegradation Research Uni, Tokyo, 1983, 44-63. 15 Kuwahara, M., Glenn, J. K., Morgan, M. A. and Gold, M. H. FEBS Lett. 1984, 169, 247-250 16 Paszczynski, A., Huynh, V. B. and Crawford, R. Arch. Biochem. Biophys. 1986, 244, 750-765 17 Glenn, J. K., Akileswaran, L. and Gold, M. H. Arch. Biochem. Biophys. 1986, 251, 688-696 18 Wariishi, H., Valli, K. and Gold, M. H. Biochemistry 1989, 28, 6017-6023 19 Wariishi, H., Valli, K., Renganathan, V. and Gold, M. H. J. Biol. Chem. 1989, 264, 14185-14191 20 Haemmerli, S. D., Leisola, M. S. A. and Fiechter, A. FEMS Microb. Lett. 1986, 35, 33-36 Odier, E., Mozuch, M., Kalyanaraman, B. and Kirk, T. K. 21 Biochimie 1988, 70, 847-852 22 Kern, H. W., Haider, K., Pool, W., de Leeuw, J. W. and Ersnt, L. Holzforschung 1989, 43, 375-384 23 Umezawa, T. and Higuchi, T. FEBS Lett. 1989, 242, 325-329 24 Kurek, B., Monties, B. and Odier, E., Holzforschung, 1990, in press 25 Lapierre, C., Lallemand, J. Y. and Monties, B. Hozforschung 1982, 36, 275-282 26 Crawford, D. L. and Crawford, R. L. Dev. Ind. Microbiol. 1978, 19, 35-49 27 Odier, E., and Defftttre, M. Enzyme Microb. Technol. 1990, 12, 447-452 28 Tien, M. and Kirk, T. K. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 2280-2284 29 Connors, W. J., Sarkanen, S. and McCarthy, J. L. Holzforschung 1980, 34, 80-85 3O Haemmerli, S. D., Leisola, M. S. A., Sanglard, D. and Fiechter, A. J. Biol. Chem. 1986, 261, 6900-6903 31 Tonon, F. and Odier, E. Appl. Environ. Microbiol. 1988, 54, 466-472 32 Harvey, P. J., Gilardi, G. F. and Palmer, J. M. In: Enzyme Systems for Lignocellulose Degradation Elsevier Applied Science, London, 1989, 111-120 33 Harvey, P. J., Schoemaker, H. E. and Palmer, J. M. FEBS Lett. 1986, 195, 242-246 34 Baker, T. I., Quicke, G. V., Bentley, O. G., Johnson, R. R. and Moxon, A. L. J. Anim. Sci. 1959, 18, 655-662
2
Enzyme Microb. Technol., 1990, vol. 12, October
777
chrysosporium Bernard Kurek,* Bernard M o n t i e s t and Etienne Odier* * Laboratoire de Microbiologie and ? Laboratoire de Chimie Biologique, Centre de Biotechnologies Agro-Industrielles, I N R A , Thiverval-Grignon, France
The rate of oxidation of spruce milled wood lignin by Phanerochaete chrysosporium was investigated with precipitated lignin as well as lignin finely dispersed in water (colloidal lignin). 14202 consumption rates in the course of lignin oxidation in the presence of purified lignin peroxidase were much higher with colloidal lignin compared with precipitated lignin. Characterization of reaction products and molecular-size distribution confirms that reaction rate of lignin peroxidase with 14C-lignin is strongly dependent on the physical state of lignin. The lignin peroxidase catalyse mainly repolymerization of colloidal lignin. However, in the presence of veratryl alcohol, both polymerization and depolymerization were observed.
Keywords: Lignin, milled wood; lignin, 14C labelled lignin; colloidallignin; lignin peroxidase; depolymerization; Phanerochaete chrysosporium Introduction Phanerochaete chrysosporium produces several peroxidases involved in lignin biodegradation. ~ The lignin peroxidase catalyses the removal of one electron from aromatic structures with subsequent formation of a cation radical. 2 Cation radicals decompose according to various reactions that depend on the nature of substitutents of the aromatic ring and the presence of molecular oxygen, j The lignin peroxidase oxidizes nonphenolic aromatic structures resistant to oxidation by previously described peroxidases such as horseradish peroxidase. 3,4 For this reason, lignin peroxidase is assumed to be able to attack most aromatic structures in lignin while other peroxidases can oxidize only phenolic moieties. Mechanistic studies with lignin dimeric model compounds establish that lignin peroxidase induces various cleavage reactions including C~-C/3 cleavage 5~/30-4 cleavage, 6,7,9A°and aromatic ring cleavage. 8,t~ The
Abbreviations: LM, milledwoodlignin;LE, enzymaticallyliberated lignin; LCn;colloidalfractionof ligninformedafter n precipitations from DMF solutioninto water; DMF, dimethylformamide Address reprint requests to Dr. Kurek, Laboratoirede Biochimie, Centre de BiotechnologiesAgro-lndustrielles,INRA, 78850Thiverval-Grignon, France Accepted for publication 2 April 1990
©
1990 Butterworth-Heinemann
structure of biodegraded lignin extracted from wood reveals that fungal attack operates through reactions that are catalysed by lignin peroxidase with lignin model compounds. 12-14 Another peroxidase, designated manganese peroxidase, produced by P. chrysosporium oxidizes Mn ++ to Mn +++, which, in turn, can oxidize various structures. 15-~7 Phenolic lignin model compounds can be degraded by manganese peroxidase with subsequent alkyl-phenyl and C~-C/3 cleavage. 18 Wariishi et al. also reported the degradation of nonphenolic model compounds by the Mn peroxidase in the presence of thiols. ~9 However, to the best of our knowledge, there is no report that manganese peroxidase is able to degrade nonphenolic structures or macromolecular lignin in vitro. For this reason, the function of manganese peroxidase in direct lignin degradation is not apparent. Although the mechanism by which lignin model compounds are degraded by lignin peroxidase is now well understood, incubation of lignin with lignin peroxidase under conditions suitable for catalysis does not result in the expected formation of lignin depolymerization products. On the contrary, polymerization of lignin after incubation is apparent. 2°-22 This result can be explained by coupling of phenoxy radicals that are formed by oxidation of lignin peroxidase after cleavage of ether substructures in lignin. 21 Since ether bonds are the major intermonomeric bonds in lignin, Enzyme Microb. Technol., 1990, vol. 12, October
771
Papers attack of etherified units and/3-0-4 cleavage by lignin peroxidase is then expected to result in the formation of a significant proportion of phenols. These phenolics can be further oxidized to phenoxy radicals by lignin peroxidase. Umezawa et al. 23 reported/3-0-4 ether bond cleavage as well as ring opening products through the action of lignin peroxidase on synthetic lignin. The yields of degradation products, however, was not reported. Indeed Kern et al. 22 have shown that lignin peroxidase probably does not induce cleavage reactions in the lignin macromolecule under the conditions of their experiments. However, available data do not provide quantitative information concerning the extent of lignin structure modification during reaction with lignin peroxidase. Because lignin is a three-dimensional structure, degradation is expected to be confined to exposed surfaces, thereby limiting the rate of degradation. In a previous report, 24 we have shown that lignin can be fractionated into precipitated and colloidal lignin by precipitation of a dimethylformamide solution into water. Characterization of the molecular size and chemical structure have shown that colloidal and precipitated lignins do not differ significantly. Colloidal lignins can then be considered as total lignin in a highly dispersed state. Preliminary studies have shown that consumption of H202 by the lignin peroxidase is greater for oxidation of colloidal lignin in water compared with precipitated lignin, suggesting a relationship between lignin reactivity for enzymic oxidation and the dispersion state. In this study, the detailed comparison of oxidation rates by the lignin peroxidase of two different lignin preparation (LM and LE) in the precipitated or colloidal state is investigated. The subsequent modification of the elution profile by size-exclusion chromatography of both precipitated and colloidal lignin labeled in the propane side chain and the aromatic ring was then determined, as well as the formation of volatile and insoluble compounds, indicative, respectively, of C I and C2 compounds and modification in the chemical structure of lignin.
Materials and methods Extraction of spruce lignins: LM (milled wood lignins) and LE lignins were prepared according to Lapierre et al.25 Labeled lignins: 14C-lignins (propane and side chain) were prepared after labeling lignin in 2-year-old spruce (Picea abies) plants with lac-(U)-L-phenylalanine (16.6 MBq mmol -~, CEA, Saclay, France) by infusion of the basal extremity from the cut stems. 26 Fresh xylem was then roughly ground (0.2 to 0.5-mm particle size) and immediately Soxhlet-extracted 48 h with toluene/ethanol (I : 1, v/v) followed by ethanol (24 h) and water (48 h). The labeled cell wall residue obtained was then milled with a Retsch MM-2 Miller (Prolabo, Paris, France) before lignin extraction: 300 mg cell wall residue were milled 72 h in 10-ml agate cups with two 12mm diameter agate balls at 1900 cycles mn -~. The LM
772
Enzyme Microb. Technol., 1990, vol. 12, October
lignin fraction was then extracted with the same procedure as for nonlabeled lignins (see above). Colloidal lignins were prepared by precipitation of lignin in water from a dimethylformamide (DMF) solution (Ref. 24; see also Figure 1): Lignin is allowed to swell in a minimum volume of DMF during one night. More DMF is then added to adjust the lignin concentration to 250 or 125 mg ml -1, respectively, for LM and LE lignins. This solution is added from a glass syringe to distilled water with vigorous agitation to 5 mg/ml lignin final concentration. The suspension is centrifuged at 17,000g during 20 min. The supernatant is then filtered with suction on a nitrocellulose filter 0.45 /zm pore size (Millipore) to eliminate the residual precipitated lignin. The colloidal LC lignin obtained can be stored at 4°C for several months. The LC, lignin is obtained by n repetitions of this procedure according to Figure 1. Lignin peroxidase was purified from static cultures of P. ehrysosporium UK 5 according to Ref. 27. The isozymes with pI 4.6 and 4.8 were used in the experiments. Lignin peroxidase was assayed by the veratryl alcohol assay according to Tien and Kirk. 28 H202 consumption by the lignin peroxidase for oxidation of lignins were determined using a YSI 2510 electrode (Yellow Springs Instrument, Yellow Springs, OH) connected to an Prg-Del amperometric monitor (Taccussel, Paris, France). Incubation of HC-lignin with lignin peroxidase and H202 was as follows. The reaction mixture contained 3 mg precipitated lignin or 0.5 mg colloidal lignin (380 Bq/mg), lignin peroxidase (purified or crude protein mixture) in Na-tartrate buffer 100 mM, pH 3, in a total volume of 3 ml. In some experiments, veratryl alcohol was added as specified. Reactions were started by addition of HzO2. In certain experiments, lignin peroxidase or H202 were added during incubation. Radioactivity in volatile compounds formed upon incubation of JaC-lignin with lignin peroxidase and H202 was determined by liquid scintillation after distillation of the total reaction mixture under 5 mbar (500 Pa) pressure at 40-45°C and collecting the condensation products at -60°C. The nonvolatile reaction products were recovered by solubilization of the residue in DMF after distillation. Molecular-size distribution of lignin and lignin degradation products was analyzed according to Connors et al. 29 using Sephadex LH-60 (Pharmacia, St Quentin en Yvelines, France) in DMF containing LiCI 0. ! M in a 1 × 50-cm column (Pharmacia). Radioactivity in the effluent was assayed after collection of 0.9-ml fractions. Radioactivity was determined by liquid scintillation in Pico-fluor (Packard, Rungis, France) using a Betamatic II counter (Kontron, St Quentin en Yvelines, France).
Results The insoluble PI lignin obtained after precipitation of both LM and LE lignin from a DMF solution (Figure 1) was washed extensively with water in order to elim-
Lignin enzymic degradability according to its physical state: B. Kurek Pmtmatment relate i ~ l k ~ n
Extraction and purlncallon
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Figure 1 Isolation and purification of lignins from cell wall residue and preparation of colloidal lignins used in this study (see also Ref. 24)
inate residual colloidal lignins. The residual colloidal lignins represent then only 0.2% of initial lignin. H202 consumption in reaction mixtures with lignin peroxidase and washed precipitated lignin from LM or LE was determined. The different colloidal lignins LC, from LM and LE were incubated with lignin peroxidase in presence of hydrogen peroxide (see Figure 1 for nomenclature). Residual I-I~O2 in the reaction mixture was determined with the hydrogen peroxide electrode. Results (Figure 2) show that colloidal lignins from LM or LE give a much faster consumption of H202 with iignin peroxidase than did the corresponding washed precipitated lignins. Consumption of 1-I202 in the presence of colloidal lignins from LM is greater than with colloidal lignins from LE. The different colloidal iignins from LM (LCI, LC2, etc) differ also in reactivity for lignin peroxidase based on H202 consumption rates. LC2 from LM reacts less than do LC1, LC3, and LC4. Figure 2 shows also that differences in reactivity between colloidal lignins and precipitated washed lignins are greater with LM lignins than with LE lignins. The modifications of 14C-lignin after incubation with lignin peroxidase and H202 were examined by determining molecular-size distribution and the formation of volatiles as well as DMF-insoluble labeled compounds. Experiments were run with precipitated or colloidal lignins with or without veratryl alcohol in the reaction mixture. Amounts of lignin peroxidase (crude or purified) were varied as well as was that of H202.
Preliminary experiments were conducted to determine the evolution of lignin peroxidase activity versus time in reaction mixtures. Figure 3 shows that lignin peroxidase activity decreases rapidly in a reaction mixture without veratryl alcohol, and no lignin peroxidase activity could be deteeted after 50 min (Figure 3). In contrast, when veratryl alcohol was added, lignin peroxidase activity was maintained in the reaction mixture during the first 15 min. When addition of H202 LM
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Reoction time (rain) Figure 2 Consumption of H202 in reaction mixtures (3 ml) containing purified lignin peroxidase (9 nkat), precipitated washed lignin (P) from LM and LE (3 mg), colloidal lignin (0.3 rag) LC1, LC2, LC3, and LC4 from LM (left) and LE (right); reaction is in 3 ml tartrate-Na buffer, pH 3 (see Materials and methods)
E n z y m e M i c r o b . T e c h n o l . , 1990, v o l . 12, O c t o b e r
773
Papers Table 1 C o m p o s i t i o n of reaction mixtures containing precipitated LM ~4C-lignin, lignin peroxidase as crude protein mixture, and H2Oz, and radioactivity in volatiles products f o r m e d after incubation a
Run
Veratryl alcohol (/~moles)
Lignin peroxidase as crude protein mixture (nkatb; mg ~)
100
o~.
Radioactivity (Bq) in volatile products d
--0
75
o o .7o .__
"R 0 ,.,_ 0
•
50
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T1 E~
---
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0.6
0 100 (0.2) then 50 (0.1) after 2 h ~ 50 (0.1) repeated after 2 h~
O
0.7 (0.23) 0.4 (0.13)
c-
0.6 (0.20)
2~
25
~'O
0 0
a All reaction mixtures are in Na-tartrate buffer 100 mM, pH 3, with 0.8 rng (305 Bq) lignin in a final v o l u m e of 3 ml; incubation time is 4 h b Lignin peroxidase activity in the crude protein mixture (nkat) Total protein quantity in parentheses d % of total radioactivity in parentheses e 150 n m o l e s H~O2 also added
10
20
? 30
40
50
Reaction time (min) Figure 3 Evolution of lignin peroxidase activity in reactions mixtures in control experiments m3 and T4 versus time (see Table 2); (0) with veratryl alcohol (1-4); (©) w i t h o u t veratryl alcohol (T3); lignin peroxidase activity is expressed in % of activity at time zero. The a r r o w indicates successive addition of 3 ~mol H~02 every 2 min in order to exhaust veratryl alcohol in reaction mixture
is repeated in the reaction mixture, veratryl alcohol becomes exhausted and lignin peroxidase activity decreases progressively (Figure 3). The first experiment was run with a precipitated 14C-lignin and crude lignin peroxidase (Table 1). Addition of lignin peroxidase and H202 was repeated after 2 h incubation. A similar set of reactions with precipitated '4C-lignin and purified lignin peroxidase was conducted with high enzymic activity in the reaction mixture (Table 2). In a third set of reactions, colloidal J4C-lignin was incubated with lignin peroxidase at the same level as in the first experiment (Table 3). Radioactive volatile compounds were produced from '4C-lignin in all reaction mixtures with lignin peroxidase and H202. Yields of volatiles were always low, particularly in experiments when veratryl alcohol was omitted, whether the lignin substrate was precipitated or colloidal (Tables 1-3). H20, alone caused the formation of volatile compounds in all cases (Tables I-3). Negligible amounts of radioactive volatile com-
pounds were formed when '4C-tignin was incubated with lignin peroxidase in the absence of H202 (Table 3). In several runs, a fraction of ~4C could not be solubilized in DMF after the reaction. That proportion which became insoluble in DMF was 12% with precipitated lignin (Table 2) and 25% with colloidal lignin (Table 3) in the presence of veratryl alcohol. In the absence of veratryl alcohol, the corresponding data are 6.1 and 0.9%, respectively (Tables 2 and 3). Control experiments with H202 alone showed only small amounts of DMF-insoluble compounds after incubation. DMF-insoluble compounds could be solubilized by the addition of NaOH to the DMF (0.05 M final concentration). DMF-soluble reaction products from 14C-lignin were analyzed for molecular-size distribution after incubation with lignin peroxidase in the different experi-
Table 2 C o m p o s i t i o n of reaction mixtures containing precipitated LM 14C-lignin, purified ~ignin peroxidase, and H2Oz, and radioactivity in volatiles and DMF insoluble products f o r m e d after incubation a H202
Run T1 T2 E1 or T3 d E2 or T4d
Veratryl alcohol (~moles)
Lignin peroxidase (nkat)
/~moles
N u m b e r of additions and time elapsed b e t w e e n additions"
0 0 0 60
0 1000 1000 1000
3 0 3 3
1 0 1 10 (2)
Radioactivity (Bq) in volatile reaction products c 3 0.3 1 8
(0.26) (0.03) (0.09) (0.67)
Radioactivity (Bq) in reaction products insoluble in DMF c 21.7 4.5 69 136
(1.9) (0.40) (6.10) (12)
ND: not determined. a All reaction mixtures are in Na-tartrate buffer 100 mM, pH 3, with 3 mg (1140 Bq) lignin in a final v o l u m e of 3 ml; incubation time is 2 h. b Time (min) between t w o additions in parentheses c % of total radioactivity in parentheses d Control e x p e r i m e n t s for lignin peroxidase activity during runs E~ and E2
774 EnzymeMicrob. Technol., 1990,vol. 12, October
Lignin enzymic degradability according to its physical state: B. Kurek
e t al.
Table 3 Composition of reaction mixtures containing 14C-colloidal lignin LC~ from LM, purified lignin peroxidase, and H202, and radioactivity in volatiles and DMF insoluble products formed after incubation a Lignin peroxidase
Run
Veratryl alcohol (#moles) 0 0 0 60
T~ T2 E~ E2
H202
(nkat)
Number of additions and time elapsed between addition@
#moles
Number of additions and time elapsed between addition@
0 60 60 60
0 1 4 d (15) 1
3 0 0.125 d 3
1 0 1 (4) 20 (3)
Radioactivity (Bq) in volatile reaction products c 1.10 0.20 0.16 0.85
Radioactivity (Bq) in reaction products insoluble in DMF c
(0.6) (0.1) (0.08) (0.4)
0 (0) ND 2 (0.9) 40 (24)
ND: not determined. a All reaction mixtures are in Na-tartrate buffer 100 mM, pH 3, with 0.48 mg colloidal iignin in a final volume of 3 ml; incubation time is 2h. b Time (min) between two additions in parentheses c % of total radioactivity in parentheses d After each addition of lignin peroxidase, 0.175 #mole H202 is added; after 4 min, 0.125 #mole H202 is added again
ments. Almost no modification could be observed when precipitated lignin was incubated with lignin peroxidase as the crude enzyme preparation (experiments according to Table 1 and Figure 4). When the same precipitated lignin was incubated with a high amount of lignin peroxidase (Table 1), slight differ-
ences--perhaps not significant--were seen in the elution profile of the reaction products (data not shown). When colloidal lignins were used instead of precipitated lignins, differences in the molecular-size distribution of the DMF-soluble reaction products were drastic (Figure 5). This was particularly evident when veratryl alcohol was present in the reaction mixture. Purified lignin peroxidase in the absence of veratryl
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Figure 4 Distribution of radioactivity by size-exclusion chromatography (Sephadex LH-60 in DMF/LiCI 0.1 M) in labeled LM lignins treated with lignin peroxidase (as a crude protein mixture from ligninolytic cultures of P. chrysosporium) in the conditions detailed in Table I. (O) run E2: with veratryl alcohol; (©) run El: without veratryl alcohol; (A) run T~: control with H202 without lignin peroxidase. Polystyrene standards of 20,000 and 2000 D used for calibration of columns are eluted, respectively, in fractions 12 and 26
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Fraction n u m b e r Figure 5 Distribution of radioactivity by size-exclusion chromatography (Sephadex LH-60 in DMF/LiCI 0.1 M) in labeled colloidal lignin LC1 from LM treated by 60 nkat purified lignin peroxidase in the conditions detailed in Table 3; (Q) run E2: veratryl alcohol; (©) run E1 without veratryl alcohol; (&) run TI: control with H202 without lignin peroxidase: (~) run T2: control with lignin peroxidase and without H202
Enzyme Microb. Technol., 1990, vol. 12, October
775
Papers
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Figure 6 Distribution of radioactivity in DMF insoluble lignins products formed after incubation of colloidal lignin with purified lignin peroxidase in the presence of veratryl alcohol in the conditions detailed in Table 3 (run E2; see also Figure 5)
alcohol caused the repolymerization of lignin, while some depolymerization was apparent when veratryl alcohol was added (Figure 5). H202 alone caused minor modifications of the molecular-size distribution of lignin (Figure 5). DMF-insoluble products were analyzed after solubilization by addition of NaOH. Sizeexclusion chromatography showed this fraction consisted of repolymerized lignin products (Figure 6).
Discussion The ability of different lignin preparations to be oxidized by lignin peroxidase varies considerably as judged by the rate of H202 consumption. Colloidal lignins from both preparations, LM and LE, are oxidized much more rapidly than are the corresponding precipitated lignins. We have also reported elsewhere that colloidal lignin partially precipitated by a freeze-thawing cycle is less reactive than is initial colloidal iignin. 24 Altogether, these results show that differences in the physical state of precipitated and colloidal lignins accounts for differences in reactivity. However, differences in the reactivity between various colloidal lignins preparations are also observed. The colloidal lignins from LM are oxidized much more rapidly than are the colloidal lignins from LE. In the same way, the various LC~, LC~, LC3, and LC4 colloidal lignins from LM are, respectively, more and more reactive to the lignin peroxidase oxidation. These differences in reactivity cannot be explained by variations in their chemical composition, which appeared to be practically identical. 24 In return, differences in molecular weight of lignins could account for it. lndeed, gel permeation chromatography studies have shown that the colloidal lignins from the LM fraction are less polymerized than are colloidal lignins from LE. 24 In the same way, LC~ and LC3 from LM are, respectively, slightly enriched in lower molecular weight fractions, compared to LC~. This apparent effect of molecular weight on colloidal lignin reactivity could, however, also be explained 776
Enzyme Microb. Technol., 1990, vol. 12, October
by differences in the physical state of lignin in aqueous media. Indeed, the lower molecular weight lignin would have a greater surface area in colloidal suspension, allowing a greater reactivity. The LC~ colloidal lignin from LM contains about twice as many condensed units as do the LC3 colloidal iignin. 24 However, both colloidal lignins showed comparable reactivity, suggesting that reactivity to lignin peroxidase is not related to the degree of condensation. The sensitivity of the different substructures in the lignin to lignin peroxidase attack deserves, however, further research. Differences in reactivity of highly dispersed and precipitated lignin-to-lignin peroxidase oxidation were confirmed by an analysis of the reaction products. Precipitated lignins are not significantly modified in molecular-size distribution after incubation even with high concentrations of lignin peroxidase. In contrast, the enzyme at 20 nkat/ml strongly affects the elution profile by gel permeation chromatography of colloidal lignins after reaction, leading clearly to polymerization of lignin in the absence of veratryl alcohol. The prominent role of veratryl alcohol in lignin enzymic degradation is confirmed. Our results state that veratryl alcohol protects lignin peroxidase against inactivation caused by H202, 3°,3~ even in the presence of lignin in the reaction mixture, allowing the reaction to proceed longer. In contrast, lignins do not protect the lignin peroxidase. This could indeed explain the greater modification in the elution profile, suggesting partially depolymerization of lignins, when veratryl alcohol was added to the reaction mixture. However, a significant proportion of the radioactivity from the 14Clignin was converted into DMF insoluble products in these experimental conditions and it required separate molecular weight distribution determination. Analysis after dissolution of this sample fraction in a DMFNaOH mixture showed that insoluble lignin consists of repolymerized reaction products. The presence of veratryl alcohol in the reaction mixture could also affect the nature of the lignin degradation endproducts, perhaps according to a cooxidation mechanism. 32,33 This is suggested by the differences in DMF solubility of repolymerized lignin endproducts in the presence or absence of veratryl alcohol in the reaction mixture. However, since veratryl alcohol affects the evolution of lignin peroxidase activity during reaction, no firm conclusion can be drawn; further research is then required to characterize the chemical compositions of repolymerized lignin endproducts in order to confirm the existence of such a mechanism. The results obtained are then consistent with previous reports establishing that lignin peroxidase causes above all repolymerization of lignin. 2°,21 In these studies, however, the formation of such insoluble compounds is not reported and has perhaps gone unsuspected. The formation of volatile compounds was observed in experiments after incubation with lignin peroxidase. In contrast with other studies, the 14C-labeled lignins
Lignin enzymic degradability according to its physical state: B. Kurek et al. used here are labeled in the propane side chain as well as in the aromatic ring. It is likely that the volatile radioactive compounds are derived from C2 fragments by Ca-Cfl or ether bond cleavage by the lignin peroxidase. The formation of volatile 14C-labeled compounds during incubation of lignin with lignin peroxidase and H202 could then indicate that cleavage reactions have taken place. The yield of these products is, however, less than 1% of the total Jac activity of lignins. In addition, control experiments in which lignin was incubated with H202 without lignin peroxidase show higher yields in volatile compounds compared to the experiments with enzyme even in the presence of veratryl alcohol. This indicates that formation of such compounds is not directly related to the extent of modification in the molecular weight distribution of lignins. In conclusion, this study shows that precipitated lignins have a low reactivity to lignin peroxidase oxidation. In contrast, highly dispersed lignins in the colloidal state are easily oxidized and modified. Our results are consistent with previous reports on enzymic hydrolysis of insoluble macromolecules such as starch or cellulose showing that an increase in substrate accessibility for the enzyme results in higher reaction rates 34 Polymerization of lignins seems to be the main reaction catalysed by lignin peroxidase. However, in the presence of veratryl alcohol, some depolymerization of lignin was also apparent. These results deserve further research in order to confirm and to quantify the extent of depolymerization with other analytical techniques than gel permeation chromatography. The evolution of oxidation products from lignin was not controlled in our study as in previous reports. 2°,2~ Further investigation is required to elucidate the mechanism by which coupling reactions could be prevented or diminished to improve the degradation of lignins. Our results strongly suggest that depolymerization could be effective when lignin peroxidase is incubated in media designed for preventing a coupling reaction and allowing good dispersion of lignin. This observation could be of great significance for applications using ligninolytic enzymes for transformation of the lignin polymer.
Acknowledgement This research was co-financed by La Cellulose du Pin (Bordeaux, France).
References Gold, M. H., Wariishi, H. and Valli, K. In: ACS Symposium Series No. 389: Biocatalysis in Agricultural Biotechnology American Chemical Society, Washington, DC, 1989, 127-140
Kersten, P. J., Tien, M., Kalyanaraman, B. and Kirk, T. K. J. Biochem. 1985, 260, 2609-2612 Hammel, K. E., Kalyanaraman, B. and Kirk, T. K. J. Biol. 3 Chem. 1986, 261, 16948-16952 4 Kersten, P. J., Kalyanaraman, B., Hammel, K. E. and Kirk, T. K. In: Lignin Enzymic and Microbial Degradation, Paris, April 23-24 (Les colloqaes de I'I.N.R.A.) I.N.R.A., Paris 5 1987, Vol. 40, 75-80. Tien, M. and Kirk, T. K. Science 1983, 221, 661-663 6 Gold, M. H., Kuwahara, J., Chiu, A. A. and Glenn, J. K. Arch. Biochem. Biophys. 1984, 234, 353-362 7 Habe, T., Shimada, M., Umezawa, T. and Higuchi, T. Agric. Biol. Chem. 1985, 49, 3505-3510 8 Miki, K., Renganathan, V., Mayfield, M. B. and Gold, M. H. FEBS Lett. 1987, 210, 199-203 9 Glenn, J. K., Morgan, M. A., Mayfield, M. B., Kuwahara, M. and Gold, M. H. Biochem. Biophys. Res. Commun. 1983, 114, 1077-1083 10 Kirk, T. K., Tien, M., Kersten, P. J., Mozuch, M. D. and Kalayanaraman, B. Biochem. J. 1986, 236, 279-287 11 Umezawa, T., Shimada, M., Higuchi, T. and Kusai, K. FEBS Lett. 1986, 205, 287-292 12 Chua, M. G., Chen, C. L. and Chang, H. M. Holzforchung 1982, 36, 165-172 Ellwardt, P. C., Haider, K. and Ernst, L. Holzforschung 1981, 13 35, 103-109 14 Tai, D., Terazawa, M., Chen, C. L., Chang, H. M. and Kirk, T. K. In: Recent Advances in Lignin Biodegradation Research Uni, Tokyo, 1983, 44-63. 15 Kuwahara, M., Glenn, J. K., Morgan, M. A. and Gold, M. H. FEBS Lett. 1984, 169, 247-250 16 Paszczynski, A., Huynh, V. B. and Crawford, R. Arch. Biochem. Biophys. 1986, 244, 750-765 17 Glenn, J. K., Akileswaran, L. and Gold, M. H. Arch. Biochem. Biophys. 1986, 251, 688-696 18 Wariishi, H., Valli, K. and Gold, M. H. Biochemistry 1989, 28, 6017-6023 19 Wariishi, H., Valli, K., Renganathan, V. and Gold, M. H. J. Biol. Chem. 1989, 264, 14185-14191 20 Haemmerli, S. D., Leisola, M. S. A. and Fiechter, A. FEMS Microb. Lett. 1986, 35, 33-36 Odier, E., Mozuch, M., Kalyanaraman, B. and Kirk, T. K. 21 Biochimie 1988, 70, 847-852 22 Kern, H. W., Haider, K., Pool, W., de Leeuw, J. W. and Ersnt, L. Holzforschung 1989, 43, 375-384 23 Umezawa, T. and Higuchi, T. FEBS Lett. 1989, 242, 325-329 24 Kurek, B., Monties, B. and Odier, E., Holzforschung, 1990, in press 25 Lapierre, C., Lallemand, J. Y. and Monties, B. Hozforschung 1982, 36, 275-282 26 Crawford, D. L. and Crawford, R. L. Dev. Ind. Microbiol. 1978, 19, 35-49 27 Odier, E., and Defftttre, M. Enzyme Microb. Technol. 1990, 12, 447-452 28 Tien, M. and Kirk, T. K. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 2280-2284 29 Connors, W. J., Sarkanen, S. and McCarthy, J. L. Holzforschung 1980, 34, 80-85 3O Haemmerli, S. D., Leisola, M. S. A., Sanglard, D. and Fiechter, A. J. Biol. Chem. 1986, 261, 6900-6903 31 Tonon, F. and Odier, E. Appl. Environ. Microbiol. 1988, 54, 466-472 32 Harvey, P. J., Gilardi, G. F. and Palmer, J. M. In: Enzyme Systems for Lignocellulose Degradation Elsevier Applied Science, London, 1989, 111-120 33 Harvey, P. J., Schoemaker, H. E. and Palmer, J. M. FEBS Lett. 1986, 195, 242-246 34 Baker, T. I., Quicke, G. V., Bentley, O. G., Johnson, R. R. and Moxon, A. L. J. Anim. Sci. 1959, 18, 655-662
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