- Email: [email protected]
Redox reactions in lignin degradation: interactions between laccase, different peroxidases and cellobiose: quinone oxidoreductase
Journal of Biotechnology, 13 (1990) 189-198 Elsevier
189
BIOTEC 00471
Redox reactions in lignin degradation: interactions between laccase, different peroxidases and cellobiose: quinone oxidoreductase Paul Ander a, Chittra Mishra 2, Roberta L. Farrell 2 and Karl-Erik L. Eriksson 3 1 STFI (Swedish Pulp and Paper Research Institute), Pulp Department, Stockholm, Sweden; 2 Repligen Sandoz Research Corporation, Lexington, Massachusetts, U.S.A.; 3 University of Georgia, Department of Biochemistry, Athens, Georgia, U.S.A. (Received 31 July 1989; accepted 17 October 1989)
Summary Depolymerization of kraft lignin and decarboxylation of vanillic acid was investigated with lignin peroxidase, laccase, horseradish peroxidase (HRP) and manganese-dependent peroxidase (Mn-peroxidase) with and without cellobiose : quinone oxidoreductase (CBQase) and cellobiose. Polymerization of kraft lignin by lignin peroxidase decreased in the presence of CBQase plus cellobiose, whereas vanillic acid decarboxylation by lignin peroxidase, laccase, HRP and Mn-peroxidase was strongly inhibited by CBQase plus cellobiose. Veratryl alcohol oxidation by lignin peroxidase was also inhibited by active CBQase. Kraft lignin; Decarboxylation; Vanillic acid; Veratryl alcohol; CeUobiose : quinone oxidoreductase; Laccase; Lignin peroxidase; Manganese-dependent peroxidase; Horseradish peroxidase
Introduction
Lignin is a complex polymer of phenyl propane units comprising 20-30% of wood and other higher plant tissues. White-rot basidiomycetes degrade lignin more Correspondence to: P. Ander, STFI (Swedish Pulp and Paper Research Institute), Pulp Dept., Box 5604, S-114 86, Stockholm, Sweden. 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
190 rapidly and extensively than other studied microbial groups (Kirk and Farrell, 1987; Leisola and Fiechter, 1985). In 1983, two research groups using the basidiomycete Phanerochaete chrysosporium, independently isolated a lignin peroxidase (ligninase) which is thought to play an important role in lignin degradation (Tien and Kirk, 1983; Glenn et al., 1983). Later a manganese-dependent peroxidase (Mn-peroxidase) was also discovered (Kuwahara et al., 1984). In P. chrysosporium these two iron-porphyrin containing enzymes may work together to degrade lignin but their function in this connection is not well understood. Laccase, which is a copper containing enzyme, also has been implicated in lignin biodegradation although the enzyme in vitro causes both depolymerization and polymerization of lignin (Kawai et al., 1988). Lignin peroxidase catalyzes the oxidation of both phenolic and non-phenolic units by a one electron oxidation mechanism, resulting in the formation of phenoxy radicals and cation radicals. Depending on the substrate these radicals may couple and partly polymerize at least in vitro (Haemmerli et al., 1986; Kirk, 1986). In similarity with laccase, Mn-peroxidase oxidizes phenolic compounds to give phenoxy radicals and quinones, which may undergo coupling and condensation. It has been suggested that extracellular enzymes other than the above-mentioned peroxidases or laccase may be involved in extensive degradation of lignin in vivo. A likely candidate is thought to be cellobiose:quinone oxidoreductase (CBQase) which reduces quinones and possibly phenoxy radicals with simultaneous oxidation of cellobiose to cellobionolactone (Westermark and Eriksson, 1974a, b; Ishihara and Nishida, 1983). The purpose of the present study was to investigate the interaction of CBQase with hgnin peroxidase, laccase, HRP and Mn-peroxidase. Indulin AT, a commercial lignin preparation derived from the digestion of softwoods in kraft pulping, as well as veratryl alcohol and vanillic acid, were used as substrates in these studies. The results indicate that CBQase interacts with the above mentioned oxidases in contrast to the results of Odier et al. (1988).
Materials and Methods
Gel filtration of Indulin AT (Westvaco Corporation, Charleston, SC, U.S.A.), a kraft lignin isolated from the black liquor of a predominant yellow southern pine kraft cook, was done in acetate buffer on a TSK 3000 PW column using Hewlett Packard 1090 High Performance Liquid Chromatography. The polystyrene calibrants used were 200 kDa, 31 kDa and 16 kDa. Reactions were done in a mixture (1 ml total volume) which contained Indulin AT, a crude extract from a white-rot fungus containing laccase (6U), lignin peroxidase (50 U ) + CBQase (20 U) in 50 mM sodium acetate buffer pH 4.0 at 37 ° C. The reaction was terminated after 3 h by the addition of 0.5 ml 6 M NaOH. The reaction mixture was diluted to 3 ml and samples were subjected to gel filtration chromatography. The crude extract of fungal enzymes and CBQase alone were run as controls. For the Indulin AT experiments, pure CBQase was obtained from GOran Pettersson, BMC, Uppsala, Sweden, or was
191 provided by Tom Kelleher (IMS, 20851 Canyon View Drive, Saratoga, CA 95070, U.S.A.). Decarboxylation of 14COOH-vanillic acid to 14CO2 by laccase or the peroxidases was measured as described by Ander and Eriksson (1987). CBQase was isolated from a 10 days shaking cultivation of P. chrysosporium ME-446 on a high nitrogen medium containing 1% Munktell's Cellulose Powder (Ander and Eriksson, 1977) followed by purification on FPLC with a MonoQ column (Pharmacia). The purity was tested with isoelectric focusing and with a SDS-gel (Pharmacia Phast Gel). The CBQase was found to be pure and to give only one protein band with a molecular weight of 60 kDa and with an IP of 4.2. Similar values were earlier obtained by Westermark and Eriksson (1975). Purification and purity tests were done by Bert Pettersson, STFI. The activity of CBQase was determined by measuring reduction of 3,5-di-tert-butyl-o-benzoquinone at 420 nm in the presence of cellobiose (Ander and Eriksson, 1977). Pure laccase (C. versicolor) was a gift of B. Reinhammar, Chalmers Inst. of Technology, Gothenburg, Sweden. Mn-peroxidase was obtained as a crude sample from a 6.5 d standing cultivation of P. chrysosporium ME-446 on a low nitrogen-l% glucose medium (Ander and Eriksson, 1987). The sample probably also contains some lignin peroxidase activity. Comparative measurements of laccase and peroxidase activity using syringaldazine were performed as described (Ander and Eriksson, 1987). The lignin peroxidase used in the decarboxylation experiments and in the tetramethoxybenzene experiments was from a P. chrysosporium BKM cultivation harvested on day 5 (Kirk et al., 1986). Purification of the enzyme was done on a MonoQ anion exchange column (Pharmacia). The isoenzymes used were predominantly H2 (LIP2) with some H1 (LIP3) (Kirk et al., 1986; Farrell et al., 1989). The activity was measured according to Faison and Kirk (1985) in 50 m M Na-tartrate at p H 4 and 3 (see also Fig. 2 and Table 2). Horseradish peroxidase (HRP) type VI was from Sigma (see also Table 2). Oxidation of 1,2,4,5-tetramethoxybenzene (TMB) (Kersten et al., 1985, 1987) by the pure lignin peroxidase was measured with or without the presence of CBQase + cellobiose or as a control with 200 /~g bovine serum albumin (BSA). The assay mixture contained 0.2 mM TMB, 0.4 mM H202, 50 mM Na-tartrate p H 3 or 0.25 M Na-acetate pH 4. Enough lignin peroxidase was added to give an absorbance change at room temperature of 0.2 m1-1 min -1. Reactions were started by adding the peroxide, and were monitored as increased absorbance at 450 nm; the molar extinction coefficient of the cation radical product is 9800 M -1 cm -1 (Kirk et al., 1989). Control reactions were necessary to verify that the rate of cation radical decay is less than the rate of its production. Results
Depolymerization of kraft lignin Indulin AT was incubated with fungal crude extract containing a lignin peroxidase, laccase and Mn-peroxidase with or without the addition of hydrogen
192 |
.lOB
OD 280 nm
I
I
I
I
I
|
A ~1 I B
160
glgnin
A
14B 128 188 08 60 4828"
8-
~
| .
.
.
.
,
,
,L
,.
.
Crude extract+H~O 2 B
.128 188
88 4828" 8
= ,
18
.
,
3~
28
extract÷HzO2 +CBQase+celloblose C Crude
88 68" 48' 28"
8
18
28
68
38
Crude e x t r a c t
alone
48
D 28'
8
.
,
.
.
.
.
,
J8
-
.
.-
.
2B
40"
,
3~
CBQase a l o n e
28.
,.
2b Ttma
38
(mln)
Fig, 1. Treatment of Indulin AT kraft lignin with lignin peroxidase (ligninase) with and without the presence of CBQase + cellobiose followed by gel chromatography on TSK 3000 PW in acetate buffer. A. Lignin alone. B. Lignin plus fungal crude extract containing ligninase (H202 added to give 0.24 raM). C. Lignin plus ligninase, H202, CBQase and cellobiose. D. Fungal crude extract alone. E. CBQase alone.
193 peroxide. End products were analyzed by TSK 3000 PW Gel filtration chromatography. As shown in Fig. 1, untreated Indulin AT was fractionated into 2 major peaks, designated A and B, with a minor peak eluting at about 15 min. The ultraviolet spectra of peaks A and B are essentially the same (data not shown). Indulin AT treated with crude extract plus 0.24 mM H202 giving the effect of lignin peroxidase is shown in Fig. lB. A significant decrease in peak B with an increased broadening of peak A was obtained after 3 h of treatment. No change in the elution profile was observed in the absence of hydrogen peroxide (data not shown). This seems to indicate that some material of peak B was polymerized to form the shoulder on peak A. A significant decrease in both peaks A and B was obtained when lignin peroxidase, H202, CBQase and cellobiose were simultaneously added to the system (Fig. 1C). Also a new minor peak was observed eluting at about 12 min. It may be postulated that the formation of this new peak, which represents a polymer of intermediate size, is due to the decrease in peaks A and B. Since the relative area of the peaks in Fig. 1A-C is 100:99:68, a substantial (32%) loss of 280 nm absorbing material was obtained in the presence of lignin peroxidase/H202 plus CBQ/cellobiose (Fig. 1C). This could be due to strong ring-cleavage reactions in this case. When another method of chromatography was used to analyze products, TSK 4000PW Gel filtration chromatography with dimethylformamide as eluant, the elution profile of Indulin AT was slightly different (data not shown)than from the TSK 3000PW system. The effect of lignin peroxidase plus hydrogen peroxide, with and without CBQase and cellobiose on Indulin AT followed the same trend as the data shown and results discussed.
Decarboxylation of vanillic acid Laccase and different peroxidases are all able to decarboxylate vanillic acid to methoxyquinone (Ander and Eriksson, 1987; Bollag et al., 1982). However, this reaction is strongly inhibited by CBQase as shown in Tables 1 and 2. Laccase-catalyzed decarboxylation decreased from 37.8% to 2.44%, whereas the corresponding figures for Mn-peroxidase was a decrease from 34.3% to 5.54% (Table 1). Incubation with CBQase without cellobiose gave 44.2% and 46.2% decarboxylation, respectively, for the two enzymes. HRP (Table 2) similarly increased decarboxylation of vanillic acid in the presence of CBQase but absence of ceUobiose. The increase in decarboxylation with only CBQase present appears significant and has also been seen during oxidation of 2,2-azino-di(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) with Mn-peroxidase. Oxidation of ABTS by Mn-peroxidase in the presence of CBQase + cellobiose was inhibited 87-93%. To find out if the increase in decarboxylation obtained with CBQase without cellobiose was due to addition of protein in the CBQase sample (200/~g m1-1) we also investigated the effect of 60 #g and 300/tg bovine serum albumin (BSA) on decarboxylation. With laccase, addition of 60 /~g BSA decreased decarboxylation somewhat while with Mn-peroxidase and HRP some stimulation was obtained. This
194 TABLE 1 I N F L U E N C E O F CBQase O N D E C A R B O X Y L A T I O N OF 1 4 C O O H - V A N I L L I C A C I D TO 14CO2 BY L A C C A S E A N D M n - P E R O X I D A S E Addition
% 14CO 2 (% of added 1 4 C O O H - V A ) Laccase
Mn-peroxidase
H 2 0 (0.6 ml) CBQase + cb CBQase without cb
37.8 +1.2 2.44 + 0.21 44.2 + 5.4
34.3 +6.5 5.54 + 0.02 46.2 + 4.0
All assay mixtures contained 3.0 ml 20 m M Na-tartrate pH 4.0, and 0.1 ml cellobiose (cb, 170 mg per 25 ml) a n d / o r 0.5 ml CBQase (743 U m1-1) as indicated in the table. In the laccase assay 0.2 ml (80 /tg) pure laccase was added and the incubation time at 2 8 ° C was 2.5 h with a total volume of 3.8 ml. In the Mn-peroxidase assay 0.5 ml crude peroxidase, 0.3 ml hydrogen peroxide (final conc. 0.03 mM) and a total volume of 4.4 ml was used. Incubation time was 1 h 45 min. In all cases 10/~1 [carboxy-14C]vanillic acid with 25.000 - 30.000 d p m in ethanol was added (13). Vanillic acid decarboxylation by Mn-peroxidase is relatively independent of M n 2 + (Kuwahara et al., 1984; Ander, unpublished results). Duplicate flasks were used.
stimulation was smaller than the one obtained with CBQase without cellobiose. The 300 #g BSA tested with HRP had no effect on vanillic acid decarboxylation. CBQase did not contain any decarboxylating activity with or without the presence of H202 and boiled CBQase did not have any stimulating effect on decarboxylation of the oxidases tested here (data not shown). Decarboxylation of vanillic acid by lignin peroxidase (Table 2) was strongly inhibited by CBQase plus cellobiose both at pH 4 (decrease from 24.7% to 0.46%) and at pH 3 (decrease from 14.8% to 0.58%). However, incubation of CBQase without cellobiose did not increase decarboxylation by lignin peroxidase (Table 2) as was the case for the other enzymes studied.
TABLE 2 I N F L U E N C E O F CBQase ON D E C A R B O X Y L A T I O N LIGNINASE AND HORSERADISH PEROXIDASE Addition
Cellobiose(cb) CBQase + cb CBQase without cb
OF
14COOH-VANILLIC
ACID
BY
% 14CO2 (% of added 14COOH-vanillic acid) Ligninase p H 4.0 0.05 ml
Ligninase p H 3.0 0.05 ml
HRP p H 4.0 0.025 ml
24.7 5-0.7 0.46 + 0.04 15.5 +0.7
14.8 +2.4 0.58 + 0.01 11.6 +0.08
32.8 +3.3 7.27 + 0.50 42.9 -I-4.2
The assay mixtures contained 3.0 ml 20 m M Na-tartrate at p H 4.0 or p H 3.0, 0.1 ml cellobiose (cb, 170 m g per 25 ml) a n d / o r 0.3 ml CBQase (920 U m1-1 at p H 4.0 and 765 U m1-1 at p H 3.0). In the ligninase incubation 0.06 ml ligninase gave 1.19 U m1-1 at p H 4.0 and 2.62 U m1-1 at pH 3.0). H R P (0.025 ml) gave 758 U m1-1 with syringaldazine. Hydrogen peroxide concentration was 0.15 m M in the iigninase incubation and 0.5 m M in the H R P incubation. Incubation time was 1 h 45 rain except for the H R P incubation where it was 1 h. Total volume was 3.67-3.81 ml and temperature 2 8 ° C . In all cases 10/~l [carboxy-14C]vanillic acid with 33,000 d p m was added. Duplicate samples were assayed.
195 A somewhat smaller inhibition of decarboxylation obtained with HRP and CBQase plus cellobiose (from 32.8% to 7.3%) may be due to the very high activity of the HRP used.
Veratryl alcohol oxidation Veratryl alcohol oxidation to veratraldehyde by lignin peroxidase was also measured in the presence of CBQase both at pH 4 and 3 mainly as described by Faison and Kirk (1985). As shown in Fig. 2A and B, CBQase plus cellobiose strongly inhibited lignin peroxidase activity and presumably due to differences in pH optima the effect of CBQase was less at pH 3 than at pH 4. Lignin peroxidase is most active at pH 2.5-3.0 and CBQase at pH 4-5. Control experiments showed that CBQase plus cellobiose could not reduce veratraldehyde back to the alcohol with or without the presence of H202. The presence of only cellobiose had no influence on oxidation of veratryl alcohol by lignin peroxidase.
Oxidation of tetramethoxybenzene To somewhat clarify the possible influence by CBQase on cation radical formation we used 1,2,4,5-tetramethoxybenzene (TMB) which is oxidized by lignin peroxidase/H202 to a yellow relatively stable cation radical which can be detected at 450 nm (Kersten et al., 1985, 1987; Kirk et al., 1989). The data in Table 3 show the appearance of yellow colour at 450 nm indicating that formation or longevity of cation radicals was strongly decreased by the presence of CBQase with cellobiose.
1.000
-OD
31Onto
A
/
Z
/'Q
!
0.800
0.600
0.400
0.200 0.100 0
,2=.,_._.......-..* '
,:o
'
~1o
'
/ ~!o
'
,'.0
'
o
I
11o
Incubation time (rain)
i
2'.0
I
~!o
' ,I0
Fig. 2. Influenceof CBQaseon ligninaseoxidationof veratrylalcoholin 50 mM Na-tartrate. (A) pH 4.0. (B) pH 3.0. e, Ligninase; o, ligninase+CBQase; &, ligninase+CBQase+cellobiose;II, ligninase+ CBQase(2 x ) + cellobiose (2 × ).
196 TABLE 3 OXIDATION OF 1,2,4,5-TETRAMETHOXYBENZENE(TMB) BY LIGNINASE IN THE PRESENCE OF CBQase OR BOVINE SERUM ALBUMIN (BSA) Assay Ligninase+ H202
Ligninase+ H202 + CBQase+ cb Ligninase+ H zO2 + BSA
OD 450 nm pH 3.0 0.659 0.316 0.713
pH 4.0 0.418 0.120 0.418
A stronger effect was obtained at pH 4 probably due to differences in pH-optima for the two enzymes. Another possible explanation for the results in Table 3 is that the 2,5-dimethoxy-p-benzoquinone formed from the cation radical by water addition (Kersten et al., 1987) is reduced by CBQase. However, the results of Kersten et al. (1987) indicate that absorbance at 450 nm is specific for cation radical formation from TMB by lignin peroxidase.
Discussion Earlier it has been reported that CBQase catalyzes specific conversion of cellobiose to form cellobionolactone with the transfer of 2 electrons non-specifically to quinones and CBQase has been suggested to reduce quinones as well as phenoxy radicals (Westermark and Eriksson, 1974a, b). Present studies demonstrate that CBQase with cellobiose has the ability to decrease polymerization of kraft lignin by lignin peroxidase and that vanillic acid decarboxylation by laccase, Mn-peroxidase, H R P or lignin peroxidase is strongly inhibited by CBQase with cellobiose. Formation of phenoxy radicals of vanillic acid is the main and common requisite for evolution of CO 2 from the carboxylic group by the different enzymes (laccase, Mn-peroxidase, HRP, lignin peroxidase) investigated here, but at present it cannot be decided if CBQase really reduces the phenoxy radicals of vanillic acid or if CBQase interacts with the subsequent attack of oxygen or hydroperoxy radicals which are supposed to be the ultimate cause of CO 2 release. The inhibition of lignin peroxidase activity as measured by veratryl alcohol oxidation and by tetramethoxybenzene oxidation further suggests that cation radical formation a n d / o r stability is affected by CBQase plus cellobiose. Odier et al. (1988) recently found that CBQase did not reduce the phenoxy radical generated from acetosyringone by lignin peroxidase and that polymerization of guaiacol and synthetic lignin was not prevented by CBQase. The difference in results for the two studies may partly be due to the lignin substrates or to the fact that in our studies with kraft lignin we used crude enzymes containing other components of white-rot fungal extracellular supernatant. However, in our experiments with vanillic acid, veratryl alcohol and TMB mostly pure enzymes were used. ESR studies, as well as analyses of remaining substrate and products formed during
197
the oxidations in the presence or absence of CBQase plus cellobiose should help to clarify the importance of CBQase in lignin and hgnin model degradation. During fungal degradation of lignin in wood or in cellulose cultures we believe that CBQase is important at least for reduction of quinones (Westermark and Eriksson, 1974a, b; Ander and Eriksson, 1977; Ishihara and Nishida, 1983). In glucose cultures several quinone reducing enzymes are produced to facilitate further degradation (BusweU et al., 1979; Schoemaker et al., 1989). Rapid reduction and metabolism of quinone compounds is probably one way to shift the polymerization/depolymerization equilibrium towards degradation. Our results indicate that CBQase should be one ingredient in an enzyme mixture degrading lignin in vitro.
Acknowledgement The authors wish to acknowledge their great appreciation to Wendy Zimmerman of Repligen Sandoz Research Corporation for conducting the HPLC analysis. For providing CBQase we thank Tom Kelleher, Bert Pettersson and G~Sran Pettersson.
References Ander, P. and Eriksson, K.-E. (1977) Selective degradation of wood components by white-rot fungi. Physiol. Plant. 41,239-248. Ander, P. and Eriksson, K.-E. (1987) Determination of phenoloxidase activity using vanillic acid decarboxylation and syringaldazine oxidation. Biotechnol. Appl. Biochem. 9, 160-169. Ander, P., Eriksson, K.-E. and Yu, H-s. (1983) Vanillic acid metabolism by Sporotrichum pulverulentum: evidence for demethoxylation before ring-cleavage. Arch. Microbiol. 136, 1-6. Bollag, J.-M., Liu, S.-Y. and Minard, R.D. (1982) Enzymatic oligomerization of vanillic acid. Soil Biol. Biochem. 14, 157-163. BusweU, J.A., Hamp, S. and Eriksson, K.-E. (1979) Intracellular quinone reduction in Sporotrichurn puloerulentum by a NAD(P)H:quinone oxidoreductase. Possible role in vanillic acid catabolism. FEBS Lett. 108, 229-232. Faison, B.D. and Kirk, T.K. (1985) Factors involved in the regulation of a ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 49, 299-304 Farrell, R.L., Murtagh, K.-E., Tien, M., Mozuch, M.D. and Kirk, T.K. (1989) Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium. Enzyme Microb. Technol. 11,322-328. Glenn, J.F., Morgan, M.A., Mayfield, M.B., Kuwahara, M. and Gold, M.H. (1983) An extracellular HzO2-requiring enzyme preparation involved in lignin biodegradation by the white-rot basidiomycete Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 114, 1077-1083. Haemmerli, S.D., Leisola, M.S.A. and Fiechter, A. (1986) Polymerisation of lignins by ligninases from Phanerochaete chrysosporium. FEMS Microbiol. Lett. 35, 33-36. Ishihara, T. and Nishida, A. (1983) The role of oxidation and reduction enzymes in lignin biodegradation. In: Higuchi, T., Chang, H-m. and Kirk, T.K. (Eds.). Recent Advances in Lignin Biodegradation Research. Uni Publishers Co Ltd, Tokyo, pp. 134-142. Kawai, S., Umezawa, T. and Higuchi, T. (1988) Degradation mechanisms of phenolic fl-1 substructure model compounds by laccase of Coriolus oersicolor. Arch. Biochem. Biophys. 262, 99-110. Kersten, P.J., Tien, M., Kalyanaraman, B. and Tien, T.K. (1985) The ligninase of Phanerochaete chrysosporium generates cation radicals from methoxybenzenes. J. Biol. Chem. 260, 2609-2612.
198 Kersten, P.J., Kalyanaraman, B., Hammel, K.-E. and Kirk, T.K. (1987) Horseradish peroxidase oxidizes 1,2,4,5-tetramethoxybenzene by a cation radical mechanism. In: Odier, E. (Ed.), Lignin Enzymic and Microbial Degradation. INRA, Paris, pp. 75-78. Kirk, T.K. (1986) The action of ligninase on lignin model compounds and lignin. Proc. TAPPI 1986 Research and Development Conference, Raleigh NC, pp. 73-78. Kirk, T.K., Croan, S., Tien, M., Murtagh, K.E. and Farrell, R.L. (1986) Production of multiple ligninase by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme Microb. Technol. 8, 27-32. Kirk, T.K. and Farrelt, R.L. (1987) Enzymatic "combustion": the microbial degradation of lignin. Annu. Rev. Microbiol. 41,465-505. Kirk, T.K., Tien, M., Kersten, P.J., Hammel, K.-E., Kalyanaraman, B. and Farrell, R.L. (submitted to Methods in Enzymology 1989). Kuwahara, M., Glenn, J.K., Morgan, M.A. and Gold, M.H. (1984) Separation and characterization of two extracellular H202-dependent oxidases from ligninolytic cultures of Phanerochaete chrysosporium. FEBS Lett. 169, 247-250. Leisola, M.S.A. and Fiechter, A. (1985) New trends in lignin biodegradation. Adv. Biotechnol. Processes 5, 59-89. Odier, E., Mozuch, M.D., Kalyanaraman, B. and Kirk, T.K. (1988) Ligninase-mediated phenoxy radical formation and polymerization unaffected by cellobiose:quinone oxidoreductase. Biochimie 70, 847-852. Schoemaker, H.E., Meijer, E.M., Leisola, M.S.A., Haemmerli, S.D., Waldner, R., Sanglard, D. and Schmidt, W.H. (1989) Oxidation and reduction in lignin biodegradation. In: Lewis, N.G. and Paice, M.G., (Eds.). Plant Cell Wall Polymers:Biogenesis and Biodegradation. ACS Symp. Ser. No. 399, Ch. 33, pp. 454-471. Tien, M. and Kirk, T.K. (1983) Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporiurn Burds. Science 221, 661-663. Westermark, U. and Eriksson, K.-E. (1974a) Carbohydrate dependent quinone reduction during lignin degradation. Acta Chem. Scand. B28, 204-208. Westermark, U. and Eriksson, K.-E. (1974b) Cellobiose : quinone oxidoreductase, a new wood-degrading enzyme from white-rot fungi. Acta Chem. Scand. B28, 209-214. Westermark, U. and Eriksson, K.-E. (1975) Purification and properties of cellobiose:quinone oxidoreductase from Sporotrichum puloerulentum. Acta Chem. Scand. B29, 419-424.
189
BIOTEC 00471
Redox reactions in lignin degradation: interactions between laccase, different peroxidases and cellobiose: quinone oxidoreductase Paul Ander a, Chittra Mishra 2, Roberta L. Farrell 2 and Karl-Erik L. Eriksson 3 1 STFI (Swedish Pulp and Paper Research Institute), Pulp Department, Stockholm, Sweden; 2 Repligen Sandoz Research Corporation, Lexington, Massachusetts, U.S.A.; 3 University of Georgia, Department of Biochemistry, Athens, Georgia, U.S.A. (Received 31 July 1989; accepted 17 October 1989)
Summary Depolymerization of kraft lignin and decarboxylation of vanillic acid was investigated with lignin peroxidase, laccase, horseradish peroxidase (HRP) and manganese-dependent peroxidase (Mn-peroxidase) with and without cellobiose : quinone oxidoreductase (CBQase) and cellobiose. Polymerization of kraft lignin by lignin peroxidase decreased in the presence of CBQase plus cellobiose, whereas vanillic acid decarboxylation by lignin peroxidase, laccase, HRP and Mn-peroxidase was strongly inhibited by CBQase plus cellobiose. Veratryl alcohol oxidation by lignin peroxidase was also inhibited by active CBQase. Kraft lignin; Decarboxylation; Vanillic acid; Veratryl alcohol; CeUobiose : quinone oxidoreductase; Laccase; Lignin peroxidase; Manganese-dependent peroxidase; Horseradish peroxidase
Introduction
Lignin is a complex polymer of phenyl propane units comprising 20-30% of wood and other higher plant tissues. White-rot basidiomycetes degrade lignin more Correspondence to: P. Ander, STFI (Swedish Pulp and Paper Research Institute), Pulp Dept., Box 5604, S-114 86, Stockholm, Sweden. 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
190 rapidly and extensively than other studied microbial groups (Kirk and Farrell, 1987; Leisola and Fiechter, 1985). In 1983, two research groups using the basidiomycete Phanerochaete chrysosporium, independently isolated a lignin peroxidase (ligninase) which is thought to play an important role in lignin degradation (Tien and Kirk, 1983; Glenn et al., 1983). Later a manganese-dependent peroxidase (Mn-peroxidase) was also discovered (Kuwahara et al., 1984). In P. chrysosporium these two iron-porphyrin containing enzymes may work together to degrade lignin but their function in this connection is not well understood. Laccase, which is a copper containing enzyme, also has been implicated in lignin biodegradation although the enzyme in vitro causes both depolymerization and polymerization of lignin (Kawai et al., 1988). Lignin peroxidase catalyzes the oxidation of both phenolic and non-phenolic units by a one electron oxidation mechanism, resulting in the formation of phenoxy radicals and cation radicals. Depending on the substrate these radicals may couple and partly polymerize at least in vitro (Haemmerli et al., 1986; Kirk, 1986). In similarity with laccase, Mn-peroxidase oxidizes phenolic compounds to give phenoxy radicals and quinones, which may undergo coupling and condensation. It has been suggested that extracellular enzymes other than the above-mentioned peroxidases or laccase may be involved in extensive degradation of lignin in vivo. A likely candidate is thought to be cellobiose:quinone oxidoreductase (CBQase) which reduces quinones and possibly phenoxy radicals with simultaneous oxidation of cellobiose to cellobionolactone (Westermark and Eriksson, 1974a, b; Ishihara and Nishida, 1983). The purpose of the present study was to investigate the interaction of CBQase with hgnin peroxidase, laccase, HRP and Mn-peroxidase. Indulin AT, a commercial lignin preparation derived from the digestion of softwoods in kraft pulping, as well as veratryl alcohol and vanillic acid, were used as substrates in these studies. The results indicate that CBQase interacts with the above mentioned oxidases in contrast to the results of Odier et al. (1988).
Materials and Methods
Gel filtration of Indulin AT (Westvaco Corporation, Charleston, SC, U.S.A.), a kraft lignin isolated from the black liquor of a predominant yellow southern pine kraft cook, was done in acetate buffer on a TSK 3000 PW column using Hewlett Packard 1090 High Performance Liquid Chromatography. The polystyrene calibrants used were 200 kDa, 31 kDa and 16 kDa. Reactions were done in a mixture (1 ml total volume) which contained Indulin AT, a crude extract from a white-rot fungus containing laccase (6U), lignin peroxidase (50 U ) + CBQase (20 U) in 50 mM sodium acetate buffer pH 4.0 at 37 ° C. The reaction was terminated after 3 h by the addition of 0.5 ml 6 M NaOH. The reaction mixture was diluted to 3 ml and samples were subjected to gel filtration chromatography. The crude extract of fungal enzymes and CBQase alone were run as controls. For the Indulin AT experiments, pure CBQase was obtained from GOran Pettersson, BMC, Uppsala, Sweden, or was
191 provided by Tom Kelleher (IMS, 20851 Canyon View Drive, Saratoga, CA 95070, U.S.A.). Decarboxylation of 14COOH-vanillic acid to 14CO2 by laccase or the peroxidases was measured as described by Ander and Eriksson (1987). CBQase was isolated from a 10 days shaking cultivation of P. chrysosporium ME-446 on a high nitrogen medium containing 1% Munktell's Cellulose Powder (Ander and Eriksson, 1977) followed by purification on FPLC with a MonoQ column (Pharmacia). The purity was tested with isoelectric focusing and with a SDS-gel (Pharmacia Phast Gel). The CBQase was found to be pure and to give only one protein band with a molecular weight of 60 kDa and with an IP of 4.2. Similar values were earlier obtained by Westermark and Eriksson (1975). Purification and purity tests were done by Bert Pettersson, STFI. The activity of CBQase was determined by measuring reduction of 3,5-di-tert-butyl-o-benzoquinone at 420 nm in the presence of cellobiose (Ander and Eriksson, 1977). Pure laccase (C. versicolor) was a gift of B. Reinhammar, Chalmers Inst. of Technology, Gothenburg, Sweden. Mn-peroxidase was obtained as a crude sample from a 6.5 d standing cultivation of P. chrysosporium ME-446 on a low nitrogen-l% glucose medium (Ander and Eriksson, 1987). The sample probably also contains some lignin peroxidase activity. Comparative measurements of laccase and peroxidase activity using syringaldazine were performed as described (Ander and Eriksson, 1987). The lignin peroxidase used in the decarboxylation experiments and in the tetramethoxybenzene experiments was from a P. chrysosporium BKM cultivation harvested on day 5 (Kirk et al., 1986). Purification of the enzyme was done on a MonoQ anion exchange column (Pharmacia). The isoenzymes used were predominantly H2 (LIP2) with some H1 (LIP3) (Kirk et al., 1986; Farrell et al., 1989). The activity was measured according to Faison and Kirk (1985) in 50 m M Na-tartrate at p H 4 and 3 (see also Fig. 2 and Table 2). Horseradish peroxidase (HRP) type VI was from Sigma (see also Table 2). Oxidation of 1,2,4,5-tetramethoxybenzene (TMB) (Kersten et al., 1985, 1987) by the pure lignin peroxidase was measured with or without the presence of CBQase + cellobiose or as a control with 200 /~g bovine serum albumin (BSA). The assay mixture contained 0.2 mM TMB, 0.4 mM H202, 50 mM Na-tartrate p H 3 or 0.25 M Na-acetate pH 4. Enough lignin peroxidase was added to give an absorbance change at room temperature of 0.2 m1-1 min -1. Reactions were started by adding the peroxide, and were monitored as increased absorbance at 450 nm; the molar extinction coefficient of the cation radical product is 9800 M -1 cm -1 (Kirk et al., 1989). Control reactions were necessary to verify that the rate of cation radical decay is less than the rate of its production. Results
Depolymerization of kraft lignin Indulin AT was incubated with fungal crude extract containing a lignin peroxidase, laccase and Mn-peroxidase with or without the addition of hydrogen
192 |
.lOB
OD 280 nm
I
I
I
I
I
|
A ~1 I B
160
glgnin
A
14B 128 188 08 60 4828"
8-
~
| .
.
.
.
,
,
,L
,.
.
Crude extract+H~O 2 B
.128 188
88 4828" 8
= ,
18
.
,
3~
28
extract÷HzO2 +CBQase+celloblose C Crude
88 68" 48' 28"
8
18
28
68
38
Crude e x t r a c t
alone
48
D 28'
8
.
,
.
.
.
.
,
J8
-
.
.-
.
2B
40"
,
3~
CBQase a l o n e
28.
,.
2b Ttma
38
(mln)
Fig, 1. Treatment of Indulin AT kraft lignin with lignin peroxidase (ligninase) with and without the presence of CBQase + cellobiose followed by gel chromatography on TSK 3000 PW in acetate buffer. A. Lignin alone. B. Lignin plus fungal crude extract containing ligninase (H202 added to give 0.24 raM). C. Lignin plus ligninase, H202, CBQase and cellobiose. D. Fungal crude extract alone. E. CBQase alone.
193 peroxide. End products were analyzed by TSK 3000 PW Gel filtration chromatography. As shown in Fig. 1, untreated Indulin AT was fractionated into 2 major peaks, designated A and B, with a minor peak eluting at about 15 min. The ultraviolet spectra of peaks A and B are essentially the same (data not shown). Indulin AT treated with crude extract plus 0.24 mM H202 giving the effect of lignin peroxidase is shown in Fig. lB. A significant decrease in peak B with an increased broadening of peak A was obtained after 3 h of treatment. No change in the elution profile was observed in the absence of hydrogen peroxide (data not shown). This seems to indicate that some material of peak B was polymerized to form the shoulder on peak A. A significant decrease in both peaks A and B was obtained when lignin peroxidase, H202, CBQase and cellobiose were simultaneously added to the system (Fig. 1C). Also a new minor peak was observed eluting at about 12 min. It may be postulated that the formation of this new peak, which represents a polymer of intermediate size, is due to the decrease in peaks A and B. Since the relative area of the peaks in Fig. 1A-C is 100:99:68, a substantial (32%) loss of 280 nm absorbing material was obtained in the presence of lignin peroxidase/H202 plus CBQ/cellobiose (Fig. 1C). This could be due to strong ring-cleavage reactions in this case. When another method of chromatography was used to analyze products, TSK 4000PW Gel filtration chromatography with dimethylformamide as eluant, the elution profile of Indulin AT was slightly different (data not shown)than from the TSK 3000PW system. The effect of lignin peroxidase plus hydrogen peroxide, with and without CBQase and cellobiose on Indulin AT followed the same trend as the data shown and results discussed.
Decarboxylation of vanillic acid Laccase and different peroxidases are all able to decarboxylate vanillic acid to methoxyquinone (Ander and Eriksson, 1987; Bollag et al., 1982). However, this reaction is strongly inhibited by CBQase as shown in Tables 1 and 2. Laccase-catalyzed decarboxylation decreased from 37.8% to 2.44%, whereas the corresponding figures for Mn-peroxidase was a decrease from 34.3% to 5.54% (Table 1). Incubation with CBQase without cellobiose gave 44.2% and 46.2% decarboxylation, respectively, for the two enzymes. HRP (Table 2) similarly increased decarboxylation of vanillic acid in the presence of CBQase but absence of ceUobiose. The increase in decarboxylation with only CBQase present appears significant and has also been seen during oxidation of 2,2-azino-di(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) with Mn-peroxidase. Oxidation of ABTS by Mn-peroxidase in the presence of CBQase + cellobiose was inhibited 87-93%. To find out if the increase in decarboxylation obtained with CBQase without cellobiose was due to addition of protein in the CBQase sample (200/~g m1-1) we also investigated the effect of 60 #g and 300/tg bovine serum albumin (BSA) on decarboxylation. With laccase, addition of 60 /~g BSA decreased decarboxylation somewhat while with Mn-peroxidase and HRP some stimulation was obtained. This
194 TABLE 1 I N F L U E N C E O F CBQase O N D E C A R B O X Y L A T I O N OF 1 4 C O O H - V A N I L L I C A C I D TO 14CO2 BY L A C C A S E A N D M n - P E R O X I D A S E Addition
% 14CO 2 (% of added 1 4 C O O H - V A ) Laccase
Mn-peroxidase
H 2 0 (0.6 ml) CBQase + cb CBQase without cb
37.8 +1.2 2.44 + 0.21 44.2 + 5.4
34.3 +6.5 5.54 + 0.02 46.2 + 4.0
All assay mixtures contained 3.0 ml 20 m M Na-tartrate pH 4.0, and 0.1 ml cellobiose (cb, 170 mg per 25 ml) a n d / o r 0.5 ml CBQase (743 U m1-1) as indicated in the table. In the laccase assay 0.2 ml (80 /tg) pure laccase was added and the incubation time at 2 8 ° C was 2.5 h with a total volume of 3.8 ml. In the Mn-peroxidase assay 0.5 ml crude peroxidase, 0.3 ml hydrogen peroxide (final conc. 0.03 mM) and a total volume of 4.4 ml was used. Incubation time was 1 h 45 min. In all cases 10/~1 [carboxy-14C]vanillic acid with 25.000 - 30.000 d p m in ethanol was added (13). Vanillic acid decarboxylation by Mn-peroxidase is relatively independent of M n 2 + (Kuwahara et al., 1984; Ander, unpublished results). Duplicate flasks were used.
stimulation was smaller than the one obtained with CBQase without cellobiose. The 300 #g BSA tested with HRP had no effect on vanillic acid decarboxylation. CBQase did not contain any decarboxylating activity with or without the presence of H202 and boiled CBQase did not have any stimulating effect on decarboxylation of the oxidases tested here (data not shown). Decarboxylation of vanillic acid by lignin peroxidase (Table 2) was strongly inhibited by CBQase plus cellobiose both at pH 4 (decrease from 24.7% to 0.46%) and at pH 3 (decrease from 14.8% to 0.58%). However, incubation of CBQase without cellobiose did not increase decarboxylation by lignin peroxidase (Table 2) as was the case for the other enzymes studied.
TABLE 2 I N F L U E N C E O F CBQase ON D E C A R B O X Y L A T I O N LIGNINASE AND HORSERADISH PEROXIDASE Addition
Cellobiose(cb) CBQase + cb CBQase without cb
OF
14COOH-VANILLIC
ACID
BY
% 14CO2 (% of added 14COOH-vanillic acid) Ligninase p H 4.0 0.05 ml
Ligninase p H 3.0 0.05 ml
HRP p H 4.0 0.025 ml
24.7 5-0.7 0.46 + 0.04 15.5 +0.7
14.8 +2.4 0.58 + 0.01 11.6 +0.08
32.8 +3.3 7.27 + 0.50 42.9 -I-4.2
The assay mixtures contained 3.0 ml 20 m M Na-tartrate at p H 4.0 or p H 3.0, 0.1 ml cellobiose (cb, 170 m g per 25 ml) a n d / o r 0.3 ml CBQase (920 U m1-1 at p H 4.0 and 765 U m1-1 at p H 3.0). In the ligninase incubation 0.06 ml ligninase gave 1.19 U m1-1 at p H 4.0 and 2.62 U m1-1 at pH 3.0). H R P (0.025 ml) gave 758 U m1-1 with syringaldazine. Hydrogen peroxide concentration was 0.15 m M in the iigninase incubation and 0.5 m M in the H R P incubation. Incubation time was 1 h 45 rain except for the H R P incubation where it was 1 h. Total volume was 3.67-3.81 ml and temperature 2 8 ° C . In all cases 10/~l [carboxy-14C]vanillic acid with 33,000 d p m was added. Duplicate samples were assayed.
195 A somewhat smaller inhibition of decarboxylation obtained with HRP and CBQase plus cellobiose (from 32.8% to 7.3%) may be due to the very high activity of the HRP used.
Veratryl alcohol oxidation Veratryl alcohol oxidation to veratraldehyde by lignin peroxidase was also measured in the presence of CBQase both at pH 4 and 3 mainly as described by Faison and Kirk (1985). As shown in Fig. 2A and B, CBQase plus cellobiose strongly inhibited lignin peroxidase activity and presumably due to differences in pH optima the effect of CBQase was less at pH 3 than at pH 4. Lignin peroxidase is most active at pH 2.5-3.0 and CBQase at pH 4-5. Control experiments showed that CBQase plus cellobiose could not reduce veratraldehyde back to the alcohol with or without the presence of H202. The presence of only cellobiose had no influence on oxidation of veratryl alcohol by lignin peroxidase.
Oxidation of tetramethoxybenzene To somewhat clarify the possible influence by CBQase on cation radical formation we used 1,2,4,5-tetramethoxybenzene (TMB) which is oxidized by lignin peroxidase/H202 to a yellow relatively stable cation radical which can be detected at 450 nm (Kersten et al., 1985, 1987; Kirk et al., 1989). The data in Table 3 show the appearance of yellow colour at 450 nm indicating that formation or longevity of cation radicals was strongly decreased by the presence of CBQase with cellobiose.
1.000
-OD
31Onto
A
/
Z
/'Q
!
0.800
0.600
0.400
0.200 0.100 0
,2=.,_._.......-..* '
,:o
'
~1o
'
/ ~!o
'
,'.0
'
o
I
11o
Incubation time (rain)
i
2'.0
I
~!o
' ,I0
Fig. 2. Influenceof CBQaseon ligninaseoxidationof veratrylalcoholin 50 mM Na-tartrate. (A) pH 4.0. (B) pH 3.0. e, Ligninase; o, ligninase+CBQase; &, ligninase+CBQase+cellobiose;II, ligninase+ CBQase(2 x ) + cellobiose (2 × ).
196 TABLE 3 OXIDATION OF 1,2,4,5-TETRAMETHOXYBENZENE(TMB) BY LIGNINASE IN THE PRESENCE OF CBQase OR BOVINE SERUM ALBUMIN (BSA) Assay Ligninase+ H202
Ligninase+ H202 + CBQase+ cb Ligninase+ H zO2 + BSA
OD 450 nm pH 3.0 0.659 0.316 0.713
pH 4.0 0.418 0.120 0.418
A stronger effect was obtained at pH 4 probably due to differences in pH-optima for the two enzymes. Another possible explanation for the results in Table 3 is that the 2,5-dimethoxy-p-benzoquinone formed from the cation radical by water addition (Kersten et al., 1987) is reduced by CBQase. However, the results of Kersten et al. (1987) indicate that absorbance at 450 nm is specific for cation radical formation from TMB by lignin peroxidase.
Discussion Earlier it has been reported that CBQase catalyzes specific conversion of cellobiose to form cellobionolactone with the transfer of 2 electrons non-specifically to quinones and CBQase has been suggested to reduce quinones as well as phenoxy radicals (Westermark and Eriksson, 1974a, b). Present studies demonstrate that CBQase with cellobiose has the ability to decrease polymerization of kraft lignin by lignin peroxidase and that vanillic acid decarboxylation by laccase, Mn-peroxidase, H R P or lignin peroxidase is strongly inhibited by CBQase with cellobiose. Formation of phenoxy radicals of vanillic acid is the main and common requisite for evolution of CO 2 from the carboxylic group by the different enzymes (laccase, Mn-peroxidase, HRP, lignin peroxidase) investigated here, but at present it cannot be decided if CBQase really reduces the phenoxy radicals of vanillic acid or if CBQase interacts with the subsequent attack of oxygen or hydroperoxy radicals which are supposed to be the ultimate cause of CO 2 release. The inhibition of lignin peroxidase activity as measured by veratryl alcohol oxidation and by tetramethoxybenzene oxidation further suggests that cation radical formation a n d / o r stability is affected by CBQase plus cellobiose. Odier et al. (1988) recently found that CBQase did not reduce the phenoxy radical generated from acetosyringone by lignin peroxidase and that polymerization of guaiacol and synthetic lignin was not prevented by CBQase. The difference in results for the two studies may partly be due to the lignin substrates or to the fact that in our studies with kraft lignin we used crude enzymes containing other components of white-rot fungal extracellular supernatant. However, in our experiments with vanillic acid, veratryl alcohol and TMB mostly pure enzymes were used. ESR studies, as well as analyses of remaining substrate and products formed during
197
the oxidations in the presence or absence of CBQase plus cellobiose should help to clarify the importance of CBQase in lignin and hgnin model degradation. During fungal degradation of lignin in wood or in cellulose cultures we believe that CBQase is important at least for reduction of quinones (Westermark and Eriksson, 1974a, b; Ander and Eriksson, 1977; Ishihara and Nishida, 1983). In glucose cultures several quinone reducing enzymes are produced to facilitate further degradation (BusweU et al., 1979; Schoemaker et al., 1989). Rapid reduction and metabolism of quinone compounds is probably one way to shift the polymerization/depolymerization equilibrium towards degradation. Our results indicate that CBQase should be one ingredient in an enzyme mixture degrading lignin in vitro.
Acknowledgement The authors wish to acknowledge their great appreciation to Wendy Zimmerman of Repligen Sandoz Research Corporation for conducting the HPLC analysis. For providing CBQase we thank Tom Kelleher, Bert Pettersson and G~Sran Pettersson.
References Ander, P. and Eriksson, K.-E. (1977) Selective degradation of wood components by white-rot fungi. Physiol. Plant. 41,239-248. Ander, P. and Eriksson, K.-E. (1987) Determination of phenoloxidase activity using vanillic acid decarboxylation and syringaldazine oxidation. Biotechnol. Appl. Biochem. 9, 160-169. Ander, P., Eriksson, K.-E. and Yu, H-s. (1983) Vanillic acid metabolism by Sporotrichum pulverulentum: evidence for demethoxylation before ring-cleavage. Arch. Microbiol. 136, 1-6. Bollag, J.-M., Liu, S.-Y. and Minard, R.D. (1982) Enzymatic oligomerization of vanillic acid. Soil Biol. Biochem. 14, 157-163. BusweU, J.A., Hamp, S. and Eriksson, K.-E. (1979) Intracellular quinone reduction in Sporotrichurn puloerulentum by a NAD(P)H:quinone oxidoreductase. Possible role in vanillic acid catabolism. FEBS Lett. 108, 229-232. Faison, B.D. and Kirk, T.K. (1985) Factors involved in the regulation of a ligninase activity in Phanerochaete chrysosporium. Appl. Environ. Microbiol. 49, 299-304 Farrell, R.L., Murtagh, K.-E., Tien, M., Mozuch, M.D. and Kirk, T.K. (1989) Physical and enzymatic properties of lignin peroxidase isoenzymes from Phanerochaete chrysosporium. Enzyme Microb. Technol. 11,322-328. Glenn, J.F., Morgan, M.A., Mayfield, M.B., Kuwahara, M. and Gold, M.H. (1983) An extracellular HzO2-requiring enzyme preparation involved in lignin biodegradation by the white-rot basidiomycete Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 114, 1077-1083. Haemmerli, S.D., Leisola, M.S.A. and Fiechter, A. (1986) Polymerisation of lignins by ligninases from Phanerochaete chrysosporium. FEMS Microbiol. Lett. 35, 33-36. Ishihara, T. and Nishida, A. (1983) The role of oxidation and reduction enzymes in lignin biodegradation. In: Higuchi, T., Chang, H-m. and Kirk, T.K. (Eds.). Recent Advances in Lignin Biodegradation Research. Uni Publishers Co Ltd, Tokyo, pp. 134-142. Kawai, S., Umezawa, T. and Higuchi, T. (1988) Degradation mechanisms of phenolic fl-1 substructure model compounds by laccase of Coriolus oersicolor. Arch. Biochem. Biophys. 262, 99-110. Kersten, P.J., Tien, M., Kalyanaraman, B. and Tien, T.K. (1985) The ligninase of Phanerochaete chrysosporium generates cation radicals from methoxybenzenes. J. Biol. Chem. 260, 2609-2612.
198 Kersten, P.J., Kalyanaraman, B., Hammel, K.-E. and Kirk, T.K. (1987) Horseradish peroxidase oxidizes 1,2,4,5-tetramethoxybenzene by a cation radical mechanism. In: Odier, E. (Ed.), Lignin Enzymic and Microbial Degradation. INRA, Paris, pp. 75-78. Kirk, T.K. (1986) The action of ligninase on lignin model compounds and lignin. Proc. TAPPI 1986 Research and Development Conference, Raleigh NC, pp. 73-78. Kirk, T.K., Croan, S., Tien, M., Murtagh, K.E. and Farrell, R.L. (1986) Production of multiple ligninase by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme Microb. Technol. 8, 27-32. Kirk, T.K. and Farrelt, R.L. (1987) Enzymatic "combustion": the microbial degradation of lignin. Annu. Rev. Microbiol. 41,465-505. Kirk, T.K., Tien, M., Kersten, P.J., Hammel, K.-E., Kalyanaraman, B. and Farrell, R.L. (submitted to Methods in Enzymology 1989). Kuwahara, M., Glenn, J.K., Morgan, M.A. and Gold, M.H. (1984) Separation and characterization of two extracellular H202-dependent oxidases from ligninolytic cultures of Phanerochaete chrysosporium. FEBS Lett. 169, 247-250. Leisola, M.S.A. and Fiechter, A. (1985) New trends in lignin biodegradation. Adv. Biotechnol. Processes 5, 59-89. Odier, E., Mozuch, M.D., Kalyanaraman, B. and Kirk, T.K. (1988) Ligninase-mediated phenoxy radical formation and polymerization unaffected by cellobiose:quinone oxidoreductase. Biochimie 70, 847-852. Schoemaker, H.E., Meijer, E.M., Leisola, M.S.A., Haemmerli, S.D., Waldner, R., Sanglard, D. and Schmidt, W.H. (1989) Oxidation and reduction in lignin biodegradation. In: Lewis, N.G. and Paice, M.G., (Eds.). Plant Cell Wall Polymers:Biogenesis and Biodegradation. ACS Symp. Ser. No. 399, Ch. 33, pp. 454-471. Tien, M. and Kirk, T.K. (1983) Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporiurn Burds. Science 221, 661-663. Westermark, U. and Eriksson, K.-E. (1974a) Carbohydrate dependent quinone reduction during lignin degradation. Acta Chem. Scand. B28, 204-208. Westermark, U. and Eriksson, K.-E. (1974b) Cellobiose : quinone oxidoreductase, a new wood-degrading enzyme from white-rot fungi. Acta Chem. Scand. B28, 209-214. Westermark, U. and Eriksson, K.-E. (1975) Purification and properties of cellobiose:quinone oxidoreductase from Sporotrichum puloerulentum. Acta Chem. Scand. B29, 419-424.