Recent advances in studies of the mechanisms of microbial degradation of lignins

Recent advances in studies of the mechanisms of microbial degradation of lignins RONALD L. CRAWFORD Gray Freshwater Biological Institute, Department o f Microbiology, University o f Minnesota, P.O. Box 100, Navarre, Minnesota 55392, USA

and DON L. CRAWFORD Department o f Bacteriology and Biochemistry, Idaho Agricultural Experiment Station, University o f Idaho, Moscow, Idaho 83843, USA

Summary. Major advances in our understanding o f the biochemical and enzymological mechanisms o f lignin biodegradation have been made in the past three years. Research has principally involved two ligninolytic microorganisms, the white rot fungus Phanerochaete chrysosporium and the actinomycete Streptomyces viridosporus. Research has been centred on attempts to identify the microbial catalysts that mediate lignin decay in these two microbes. Emphasis has been on studies concerned with isolating specific lignin catabolic enzymes and/or reduced forms o f oxygen involved in attacking the lignin polymer. The possibility that lignin degradation might be non-enzymatic and mediated by extracellular reduced oxygen species such as hydrogen peroxide (H202), superoxide (02 "-), hydroxyl radical (" OH) or singlet oxygen (102) has been investigated with both microorganisms. Using methods which have not always been unequivocal, the question o f involvement o f reduced oxygen species in lignin degradation by P. chrysosporium has been examined exhaustively. Evidence for the involvement o f H202 is conclusive. However, there is little evidence to support the involvement o f other extracellular reduced oxygen species, including "OH,\direetly in the process o f lignin degradation. Scavenger studies have been inconclusive because o f questions o f their specificity. I f activated oxygen species are involved, the activated oxygen is probably held within the active site o f a n enzyme molecule. With S. viridosporus, scavenger studies also strongly indicate that extracellular reduced oxygen species are not involved in lignin degradation since scavengers generally do not significantly affect the ligninolytic system. The involvement o f specific enzymes in lignin degradation by both P. chrysosporium and S. viridosporus has now been confirmed. With P. chrysosporium, ligninolytic enzymes recently discovered include extracellular non-specific peroxidases and oxygenases. They show numerous activities including dehydrogenative, peroxidatic, oxygenative and C a - C O cleavages o f lignin side chains. A t least one P. chrysosporium enzyme, a unique H202-requiring oxygenase, has been purified to homogeneity. Evidence has been presented to show that S. viridosporus also produces a ligninolytic enzyme complex involved in demethylation o f lignin's aromatic rings and in

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the oxidation o f lignin side chains and cleavage o f {J-ether linkages within the polymer. The combined activites o f these enzymes generate water-soluble polymeric modified lignin fragments, which are then slowly degraded further by S. viridosporus. The (J-ether cleaving enzyme complex is probably membrane associated, but it is not extracellular. These first isolations o f ligninolytic enzymes have changed the course o f basic research on lignin biodegradation. N e w research priorities are already emerging and include e n z y m e purifications, kinetic studies, enzyme reaction mechanism studies and screenings Jbr more enzymes. In addition, genetic studies are being carried out with both P. chrysosporium and S. viridosporus. Genetic manipulations include not only classical mutagenesis techniques, but also recombinant DNA techniques such as protoplast fusion. This latter technique has already been used to generate overproducers o f the ligninolytic enzyme complex in S. viridosporus and it has been successfully used to recombine mutant strains o f P. chrysosporium. Keywords: Lignin; biodegradation; Streptomyces viridosporus; Phanerochaete chrysosporium ; enzymes; hydroxyl radical; activated oxygen

Introduction Efforts to arrive at an understanding of the molecular mechanisms of lignin biodegradation have been under way for more than 80 years. Progress in some areas (e.g. the chemical changes that occur within the lignin macromolecule during microbial decay) has been slow, but steady. Progress in other areas, however, has been minimal and very sporadic. A particularly good example o f the latter case is the failure over decades by workers in the field to identify and characterize the microbial catalysts that mediate lignin decay. As late as 1981 it was stated that: 'One area o f lignin biodegradation research that has seen little . . . progress concerns the . . . mechanisms of lignin decay. Most of our evidence concerning lignin-transforming enzymes is indirect . . . we have yet to . . . isolate and characterize any protein that has been shown unequivocally to be a component of 0141 --0229/84/100434--09 $03.00 © 1984 Butterworth & Co. (Publishers) Ltd

Microbial degradation of lignins: R. L. Crawford and D. L. Crawford

a microbial lignolytic.., system', t How things have changed during the last three years! There have been two prominent theories as to how microorganisms might degrade the complex, aromatic, macromolecular substance lignin. These hypotheses centre on either the mediation of lignin decay by classical microbial enzymes,1 or by 'active oxygen' species2 such as hydroxyl radicals (-OH), superoxide anion (O2-'), singlet oxygen (102)and hydrogen peroxide

(H2Ch). Up until about 1980 it was assumed by most workers in the field that lignin decay is mediated by microbial enzymes. This assumption is logical. Biodegradation as a phenomenon of nature is almost always an enzymatic event. It is true that lignin is rather resistant to microbial attack because the lignin macromolecule is complex and composed of large numbers of methoxylated benzene derivatives. There is, however, a large literature describing oxidative microbial enzymes that degrade methoxylated benzene rings, albeit only low molecular weight, watersoluble molecules.3 Also, what has been learned concerning the chemical changes brought about within the lignin polymer during its decay by aerobic microorganisms j,a-6 is consistent with the operation of oxidative enzymes such as hydroxylases7 demethylases,s and dioxygenases3 similar to those microbes use to degrade simple benzenoid molecules. Yet, into the 1980s no such lignin-specific enzymes were being discovered despite much searching among the lignin-degrading fungi and other lignolytic microorganisms. A number of enzyme activities, particularly against model compounds, were described and were suggested as playing important roles in lignin decay, but evidence was always unconvincing.9 Most investigators attributed these failures to inadequacies in methodology, particularly to the lack of suitable enzyme assay procedures. Persistence in the search thus seemed to be our only alternative. By about 1980 some investigators were considering alternatives to the 'lignase' hypothesis. Although it had been discussed informally among various scientists prior to his paper, Hall 2 was the first to suggest in print that lignin decay might be mediated by a non-enzymic agent he dubbed 'activated oxygen'. This term describes reduced oxygen species such as hydrogen peroxide (HuO2), superoxide (O2-'), hydroxyl radical (.OH), and singlet oxygen (102). Of these species, hydroxyl radical is the most reactive and thus the most promising candidate for being a lignin oxidant. Hall's suggestion, based on little evidence, created a bandwagon effect as researchers in numerous laboratories set out to prove (or disprove) the 'activated oxygen hypothesis' of lignin decay. The idea of the involvement of a reactive oxygen species in lignin biodegradation has certain attractions. First, lignin is a high molecular weight, insoluble polymer that cannot readily enter microbial cells. Lignin's biodegradation must almost certainly involve the activities of extracellular catalysts, and a reduced oxygen species could fit this requirement, assuming it were produced and excreted by a ligninolytic microorganism. Also, lignin is very complex, both chemically and stereochemically. A highly non-specific ligninolytic system is thus required. Again, something like hydroxyl radical fits the bill. Many enzymes involved in the catabolism of low molecular weight aromatic molecules require cofactors such as NAD(P)H. This is fine for cytoplasmic enzymes, which can use cofactors recirculated within the cell. However, it seems unreasonable (from an

energy conservation standpoint) that a microbe would excrete such cofactors into its growth medium in order to decay lignin. Again, excretion of a reduced oxygen species as a ligninolytic catalyst obviates the need for extracellular cofactors. Analyses of lignin residues following microbial attack indicate that lignin degradation by white-rot fungi and actinomycetes is largely oxidative.4,1° Attack on lignin by reduced oxygen species would be oxidative. A number of wood decay fungi (brown-rotters and white-rotters) are known to produce substantial amounts of one reduced oxygen species, hydrogen peroxide. 11-12 This H202 could be a source of even more powerful oxidants such as hydroxyl radical. Radicals have, in fact, been detected in biological systems such as macrophages and polymorphonuclear leucocytes.13 Overall, the reduced oxygen theory is quite attractive. Perhaps the most attractive aspect of this theory (as of 1980) was that it would explain why we had not discovered ligninolytic enzymes - they were perhaps figments of our imagination. We will now discuss the evidence accumulated during the past three years concerning lignin decay mechanisms, particularly as they relate to enzymes and/or reduced forms of oxygen. This research involves principally two microbes, the white-rot fungus Phanerochaete chrysosporium and the actinomycete Streptomyces viridosporus. Finally, we will attempt to put this work into an overall perspective, reaching some conclusions and speculations regarding the questions, 'How do microorganisms degrade lignin?' and 'What are the implications of recent discoveries to further research?' Lignin s t r u c t u r e and i m p o r t a n c e The structure of the phenylpropanoid lignin polymer, and its importance to the biopheric carbon/oxygen cycles, have been discussed in recent reviews 1's-6 and will not be discussed in detail here. Adler 14 and Nimz 15 have published detailed schematic representations of softwood (spruce) and hardwood (birch) lignins. It is sufficient to reiterate a few key points. First, the quantitative importance of lignin as a renewable aromatic polymer is difficult to overstate. Lignocellulosic materials comprise as much as 95% of the earth's land-produced biomass.6 About a quarter of that amount is lignin, a recalcitrant polymer that is not only resistant to microbial degradation but also acts as a physical barrier to inhibit biodegradation of the cellulosic components of lignocellulose,t In addition, lignin degrading abilities are restricted to a few microorganisms, including certain fungi and a few bacteria and actinomycetes. 1,4,6 Lignin is a heterogeneous phenylpropanoid polymer containing a diverse range of stable carbon/carbon bonds and aryl ether linkages, s While lignins from most hardwoods and softwoods contain a predictable complement ofphenylpropane units derived from coniferyl, sinapyl and coumaryl alcohol monomers, grass lignins also have these, but are also complexed with significant quantities of esterified phenylpropanoid acids. 1,16 As a result, the name 'lignin' really defines a structurally variable water-insoluble polymer that is particularly difficult to study as a substrate for microorganisms. However, based upon its universal phenolic subunit structure, lignin from almost any vascular plant may be potentially converted into similar phenolic intermediates or modified polymers. As Higuchi has stated: 'Demethylation, hydroxylation, side-chain shortening and ring cleavage of lignin could satisfactorily alter polymeric lignin for chemical modifications. 's

Enzyme Microb. Technol., 1984, vol. 6, October

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Review

Two important lignin-degrading microorganisms Phanerochaete chrysosporium The white-rot fungus Phanerochaete chrysosporium has become the organism of choice for many workers studying lignin biodegradation. 1'4'5 As recently pointed out by Kirk, n by 1978 P. chrysosporium (=Sporotrichum pulverulentum) had been studied and selected as 'a suitable experimental organism for detailed study'. Most of the recent papers concerned with the biochemistry and enzymology of lignin degradation by white-rot fungi have involved this particular organismJ ,18 P. chrysosporium is a sporeforming, thermotolerant fungus which grows rapidly at 37°C and completely mineralizes lignin to carbon dioxide and water. Cultural parameters such as 02 concentration, the presence or absence of utilizable growth substrate other than lignin, medium pH, the presence or absence of agitation, buffering agents used and medium nitrogen content all markedly affect lignin degradation by white-rot fungi, and all of these parameters have been optimized for

P. chrysosporium. ] Streptomyces viridosporus Actinomycetes have long been thought to have some role in lignin biodegradation. In 1971, Kirk 9 reviewed the early literature and concluded that some bacteria probably attacked lignin, but the chemistry of that attack was unknown. As late as 1978, reviewers still questioned the importance of bacteria in the degradation of lignin. 19 However, by 1980 workers utilizing new 14C-radioisotopic lignin biodegradation assays had confirmed that certain bacteria decomposed lignin to C02 and water-soluble catabolites.4 In 1978, Streptomyces species were conclusively confirmed as lignin decomposers, using [14C] lignin degradation assays. 2° Since then, several Streptornyces species have been shown to degrade lignin to C02 and water-soluble compounds.21-26 The most studied actinomycete is Streptomyees viridosporus T7A, an isolate obtained from Idaho soil by D. L. Sinden.27 Research with this strain has been extensive and includes studies of the chemistry of softwood lignins after partial degradation by this actinomycete,l° isolation and characterization of low-molecular phenolic intermediates of lignin degradation,28 comparison of decomposition rates of softwood, hardwood and grass lignoceltuloses,29 and the characterization of a quantitatively major modified water-soluble polymeric lignin generated during growth on lignocellulose by S. viridosporus.3° The strain has also been genetically manipulated, using protoplast fusion techniques, to generate recombinant strains which overproduce the polymeric intermediate.3] Several relevant enzymes have been observed in this strain, including an aromatic aldehyde oxidase that may be involved in low molecular weight lignin fragment catabolism in S. viridosporus,32 and recently an enzyme complex shown to cleave /3-ether linkages in lignin substructure model compounds.33

Involvement of reduced oxygen species in the biodegradation of lignin Phanerochaete chrysosporium The ligninolytic system of Phanerochaete chrysosporium is known to be one component of this organism's secondary metabolic phase. 34 That is, lignin degradative systems are active only after primary growth ceases. The most convenient way to turn on the ligninolytic system in

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Phanerochaete chrysosporium

is to induce secondary growth by nitrogen starvation, using a low-nitrogen growth medium.3s Using this trick to turn the ligninolytic system off and on (assayed by determining the abilities of whole cultures to convert [14C]lignin to 14CO2), it has been possible to correlate other physiological parameters with ligninolytic activity. Several investigators have attempted to correlate the production of reduced oxygen species with lignin-degrading ability in Phanerochaete chrysosporium Burds. Nakatsubo et al.36 suggested in an early active oxygen paper that singlet oxygen (102) plays an integral role in lignin biodegradation. Their evidence included the following observations: (a) a non-biological riboflavin/light/O2 photochemical IOz-generating system and ligninolytic cultures of Phanerochaete chrysosporium both cleaved a lignin substructure model compound, giving the same oxidation products (Figure 1); (b) a singlet oxygen scavenger (anthracene 9,10-bisethanesulphonate) inhibited degradation of the model by ligninolytic cultures and the photochemical system; (c) anthracene 9,10-bisethanesulphonate prevented lignin oxidation, but not glucose oxidation by whole cells; and (d) ultraviolet fluorescence and absorbance of the scavenger were reduced in cultures, as would be expected following reaction of the anthracene derivative with 10 2. Unfortunately, these aqueous active oxygen systems are quite complex, and reactive species other than 102 might interact with scavengers like anthracene 9,10-bisethanesulphonate. Further experimentation 37 indicated that singlet oxygen was probably not involved in the fungal degradation of lignin model compounds. When photochemical oxidations were performed in Dz O rather than H20, there was no rate enhancement. Enhancement of reaction rates of singlet oxygen reactions in D20 as compared to H20 are considered diagnostic of 102 involvement. 38 Thus, something other than 102 appears to be the catalyst mediating oxidation of the model compound. Also, another 102-generating system (H202/sodium hypochlorite) examined during the second study gave different oxidation products from fungal cultures when incubated with the model compound used in the first study. Taken together, the two studies indicate that free 102 probably does not play a role in lignin degradation by Phanerochaete chryso-

sporium. Hydroxyl radicals are much more reactive than other forms of reduced oxygen, thus several investigators have 3,30

OH HO~~r~oc~ OR I~10

?

~~~H

3

OR

CHO OCH3 .~ ~OCH 3 -t- °~ "OH --,~- OCH5 "OR OR OR 2t2o

4

Figure 1 Cleavage of a lignin model compound by Phanerochaete chrysosporium and a photochemical system. 1,2, 3, 4: R = CH2CH3; la, 2a, 3a: R = I"CH3. (Nakatsubo, etaL 198136)

Microbial degradation of lignins: R. L. Crawford and D. L. Crawford

examined the possible role of .OH as a ligninolytic catalyst. Since H202 is a potential source of .OH, Forney eta/. 39 examined the hypothesis that H202-derived "OH might play a role in lignin degradation by Phanerochaete chrysosporium. When the fungus was grown in low-nitrogen medium, an increase in H202 production by cell extracts was observed. This increase in specific activity of a peroxidegenerating system coincided with the appearance of ligninolytic activity ([14C]lignin~14C02). Assays using the conversion of 2-keto-4-thiomethylbutyric acid (KTBA) to ethylene4° suggested that -OH might be produced by ligninolytic cultures. Cell extracts appeared to produce •OH from H202, based upon this assay. The radical species being formed in cultures hydroxylated p-hydroxybenzoic acid forming protocatechuic acid,41 and the radical trap 5,5-dimethyl-l-pyrroline-N-oxide42 was converted to its nitroxide radical by ligninolytic cells. Both of these observations may be indicative of reactions catalysed by •OH. The reactions were inhibited by various •OH-scavenging agents (e.g. mannitol and benzoate), as was fungal lignin degradation. When azide was added to reaction mixtures to suppress endogenous catalase activity, the various "OH type reactions were stimulated. In other studies,n3-44 these authors provided even more indirect evidence that production of both H202 and -OH correlated with lignin degradation abilities in Phanerochaete chrysosporium. It was suggested that glucose oxidase might be the main source of H202, and that peroxide production was localized in unique periplasmic 'microbodies'. Kutsuki and Gold 4s in several well controlled studies also examined the possible involvement of "OH in lignin degradation by Phanerochaete chrysosporium. They confirmed that the fungus produces ethylene from 2-keto-4thiomethylbutyric acid (KTBA), and also from methional.46 Both observations are indirect evidence for fungal production of .OH. Ethylene was produced under conditions when the fungus was competent to degrade [14C]lignin to 14C02. As in the work of Reddy, a variety of known -OH scavengers (4-O-methylisoeugenol, thiourea, salicylate and mannitol) significantly inhibited both fungal ligninolytic activity and ethylene production from KTBA and methional. A mutant strain of Phanerochaete chrysosporium that cannot degrade lignin47 was unable to produce ethylene from either KTBA or methional. A wild-type revertant of the mutant regained the ability to produce ethylene. Exogenously added catalase inhibited ethylene production and lignin degradation by the fungus. Methional inhibited lignin degradation by the fungus (by competing for -OH?) but not glucose degradation. Finally, lignin inhibited production of ethylene from methional. B e s e t al. 48 also suggested that activated oxygen is involved in lignin degradation. Using Phanerochaete chrysosporium and specifically [14C]lignin labelled poplar lignocellulose, these authors confirmed much of the work discussed above. Fungal lignin degradation, using natural lignoceUulose as substrate, was inhibited up to 80% by •OH scavengers such as mannitol, thiourea and benzoate. Scavengers of 102 also caused some inhibition of lignin degradation, but so did paraquat (which is thought to enhance production of 02-" from 02 in vivo). These authors found that Phanerochaete chrysosporium produces some ethylene without the addition of ethylene precursors, but that ethylene evolution is dramatically stimulated by addition of methional. Mannitol or thiourea inhibited production of ethylene from methional as well as lignin degradation. Since benzoate is decarboxylated by "OH,49

the authors examined benzoate decarboxylation by the Phanerochaete. The fungus decarboxylated benzoate, and the decarboxylation was inhibited by "OH scavengers and lignocellulose. There was a good correlation between benzoate decarboxylation activity and ligninolytic activity. Gold et al. so recently isolated a novel mutant of Phanerochaete chrysosporium that is incapable of evolving 14C02 from [14C]lignins. It is, however, capable of generating ethylene from KTBA. The authors concluded from this observation that the generation of ethylene from KTBA is not a reliable assay for lignin-degrading abilities of woodrotting fungi. It should be noted, however, that there are probably many steps between [14C]lignin and 14C02. The fungal mutant might contain a genetic lesion at a late step of lignin catabolism. Hydroxyl radicals, if produced, are probably used during early phases of decay as an initial oxidant. Thus, a degradation negative mutant might still be a producer of "OH. Frick and Crawford sl examined the demethylation of the lignin model polymer polyguaiacols2 by ligninolytic Phanerochaete chrysosporium and various active oxygengenerating systems. The methoxyl group (14C-labelled)of polyguaiacol was cleaved by both hydroxyl radical produced with Fenton's reagent and by photosensitized oxidation (Rose Bengal sensitized and unsensitized photooxidations). However, a xanthine oxidase/xanthine system, which is reported to produce "OH,46's3 did not demethylate polyguaiacol. If hydroxyl radical is generated in ligninolytic cultures ofPhanerochaete chrysosporium, it may be formed by a Fenton type reaction using enzymatically produced hydrogen peroxide. The availability of Fe 2÷ in aerobic cultures, however, would be very low and addition of Fe z÷ should stimulate the reaction. At concentrations below 1 mM, Fe 2÷ had very little effect on fungal demethylating activity in ligninolytic cultures. At higher concentrations Fe 2* inhibited 14C02 formation from 14C-[OCHa]polyguaiacol. Rates of methoxyl release from polyguaiacol by ligninolytic Phanerochaete chrysosporium were compared with rates of ethylene production from methional. Concentrated culture filtrates of the Phanerochaete retained their activity to produce ethylene from methional. More of this activity was retained than demethylating activity; thus, the activities are mediated by different catalysts. Finally, horseradish peroxidase in the presence of hydrogen peroxide catalysed the formation of ethylene from methional quite well. The authors concluded that the use of ethylene production from methional or KTBA as an indicator of •OH in ligninolytic cultures is highly suspect. It is noteworthy that Bors et aL 64 recommended eight years ago that release of ethylene from methional should not be used to indicate -OH since the mechanism of the reaction was not understood. Shimda and Higuchi ss used various radical scavengers and cytochrome P-450 inhibitors to examine inhibition of lignin biodegradation in Phanerochaete chrysosporium. Various extracellular and membrane-bound enzyme fractions obtained from ligninolytic cultures were shown to produce ethylene from KTBA in the presence of H202. Thus, it is clear that numerous enzymes produced by Phanerochaete chrysosporium will mimic the activity of •OH against molecules like KTBA, methional, and possibly ,benzoate. Kirk and Tien s6 extended their previous studies of active oxygen species involvement in lignin degradation by Phanerochaete chrysosporium. They concluded that the fungus uses an oxidizing species that is similar to that Enzyme Microb. Technol., 1984, vol. 6, October

437

Review generated by Fenton's reagent, but that it is not free -OH. Finally, Faison and Kirk s7 reexamined the relationship between lignin degradation and production of reduce:. oxygen species by Phanerochaete chrysosporium. They again confirmed that the kinetics of H202 production coincided with the appearance of the ligninolytic system. Hydrogen peroxide production was markedly enhanced by growing the fungus under 100% 02, mimicking what is seen with ligninolytic activity. Catalase inhibited lignin degradation, as did superoxide dismutase. The production of "OH (assayed by measuring decarboxylation of benzoate) did not, in these author's hands, correlate with ligninolytic activity. However, lignin degradation was inhibited, as seen many times in previous work, by compounds known to react with "OH. The authors concluded that H202 and possibly 02-" are involved in lignin degradation, but that their role is probably indirect. Clearly, the question of involvement of reduced oxygen species in lignin degradation by Phanerochaete chrysosporium has been examined exhaustively. Methods used have not always been unequivocal. However, the evidence is sufficient to make fairly reliable conclusions, at least concerning certain points. The evidence for involvement of H202 in lignin degradation by Phanerochaete chrysosporium is conclusive. It is formed in concert with the ligninolytic system, and when it is destroyed by catalase, lignin degradation is strongly inhibited. Many laboratories, particularly Reddy's group, have confirmed this relationship. There is, however, little evidence for involvement of other reduced oxygen species, including -OH, directly in the processes oflignin degradation. Scavenger studies are inconclusive because of questions of their specificity. The presence (or absence) of enzymes, particularly peroxidases, produced by Phanerochaete chrysosporium is sufficient to account for most observations reported for model compounds, KTBA, methional, and for ligninolytic-negative mutant strains of the fungus. In fact, we have now completed the circle, coming back to enzymes as being the probable ligninolytic catalysts. These enzymes will undoubtedly have characteristics that mimic activated oxygen species, but the activated oxygen will be held within the active site of a protein molecule. The first group of these ligninolytic enzymes from Phanerochaete chrysosporium have been discovered and they are the topic of a following section.

Streptomyces viridosporus Evidence species are

also indicates that activated oxygen not involved in lignin degradation by S. viridosporus. Crawford et al. 33 carried out a series of experiments to determine the effects of active oxygen scavengers on lignin degradation by S. viridosporus and found no inhibitory effects under conditions which strongly inhibited lignin degradation by P. chrysosporium. The singlet oxygen scavenger anthracene 9,10-bisethanesulphonic acid (AES) reacts with singlet oxygen, forming an endoperoxide, and has been used to show that lignin degradation by P. chrysosporium is inhibited until the AES has been completely converted to the endoperoxide.36 As shown in Table 1 and Figure 2, AES had no effect on lignocellulose degradation by S. viridosporus, as measured by lignocellulose weight loss, lignin loss, carbohydrate loss or degradation of [14C]lignin to 14C02. As lignin was degraded there was no apparent conversion of AES to the endoperoxide (Table 1), as judged by no loss of the 438

Enzyme Microb. Technol., 1984, vol. 6, October

I0

/ / 8

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/°: l I

× N 10

io i ii

//,p -

0

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Time (days) Figure 2 Effects of anthracene 9,10-bisethanesulphonic acid (AES) on degradatiot~ of [~4C]lignin lignocellulose to 14C02 by S. viridosporus and P, chrysosporium, o, S. viridosporus plus AES; e, S. viridosporus minus AES; D, p. chrysosporium plus AES; l , p. chrysosporium minus AES. Data taken from Crawford, et al. 33

characteristic fluorescence of AES.36 In contrast, lignin degradation by P. chrysosporiurn was inhibited. Inhibition was significant as shown by Klason lignin assay (Table 1) and by transitory reduction i n 14C02 evolution from [14C]lignin. P. chrysosporium also converted AES to the endoperoxide (Table 1). These results lead to the conclusion 33 that there is no extracellular release of singlet oxygen during lignin degradation by S. viridosporus. Similarly, "OH scavengers such as benzoate, thiourea and o-hydroxybenzoate, which markedly inhibited lignin degradation by P. chrysosporium,4s,s7 do not appear to inhibit lignin degradation by Streptomyces. As shown in Table 2, when these scavengers were added to ligninolytic cultures of three different Streptomyces, including S. viridosporus, they had different effects on the cultures. A [14C]lignin to 14C02 assay showed effects varying from slight inhibition to significant enhancement of lignin degradation. For S. viridosporus, benzoate and thiourea enhanced 1 4 C 0 2 e v o l u t i o n from lignin while o-hydroxybenzoate slightly inhibited degradation. In contrast, all three scavengers markedly inhibited lignin degradation by P. chrysosporium, which was used as a positive control. In these experiments, all four cultures were decomposing lignin at a maximum rate immediately prior to addition of scavengers (at 214h incubation). Directly after addition of specific scavengers, control cultures (=no scavenger added) continued to mineralize lignin at a similar, but slowly decreasing rate. The enhancing or inhibitory effects of scavengers discussed above were, therefore, calculated

Microbial degradation of lignins: R. L. Crawford and D. L. Crawford Table 1

Effects of anthracene 9,10-bisethanesulphonic acid (AES) on corn stover lignocellulose degradation by Streptomyces viridosporus and Phanerochaete chrysosporiuma, b Conversion Organism

endoperoxide

Lignocellulose weight loss (%)

No AES added S. viridosporus P. chrysosporium

N.a. N.a.

30,8 ± 1.8 51.2 ± 1.8

10.6 ± 3.4 c 41.1 -+ 2.3 d

39.2 +- 2.9 56.7 ± 1.7

AES present S. viridosporus P. chrysosporium Sterile control

No Yes No

30.6 ± 1.0 52.1 ± 1.7 0.0 ± 0.1

8.0 ± 0.5 c 35.5 ± 3.1 d 0.0 -+ 0.1

41.6 + 2.2 55.3 + 5.1 3.4 + 01

of AES to

Lignin loss

Carbohydrate loss (%)

a Cultures were grown for 16 days at 37°C on 500 mg lignocellulose in 250 ml flasks using the dampened lignocellulose culture system. Values are averages of two replicates + standard deviation b Taken from Crawford et el. 25 c Not significantly different dsignificantly different N.a., not applicable

using the average ]4CO2 evolution rates for controls between 184 and 280hours incubation as the 100% value. The 53-80% inhibition with P. chrysosporium contrasted markedly to the lack of inhibition with S. viridosporus and suggested a different mechanism of lignin degradation for the actinomycete as compared to the fungus. The compound 2-keto-4-thiomethylbutyric acid (KTBA) reacts with -OH to produce ethylene and has been used to show that -OH may be present in ligninolytic cultures of P. chrysosporium. 4s Crawford et el. 33 confirmed that ligninolytic P. chrysosporium cells generated ethylene from KTBA. However, lignin-degrading Streptomyces did not. The data on active oxygen scavenger effects on Streptomyces lead to two principal conclusions. First, it is clear that Streptomyces do not generate significant extracellular activated oxygen species as a component of their ligninolyric system. Second, their mechanisms for enzymatic attack on lignin may differ from that of P. chrysosporium (see below).

Involvement of enzymes in the biodegradation of lignin Phanerochaete chrysosporium Many complex reactions occur during decay of lignin by white-rot fungi (for example, see ref. 58). Reactions known to occur include, (a) cleavage of

/3-O-4-1inkages, (b) cleavage of C~-C~ side-chain bonds, (c) oxidative fission of aromatic rings, (d) demethylations, and (e) numerous other mostly oxidative transformations. Until 1983 there had been no enzymes described in Phanerochaete chrysosporium (or any other microbe) that catalysed any of these reactions. In 1983, several laboratories reported breakthroughs in the enzymology area. Kirk and Tiens6 reported their observations of C~-C~ cleavage of sidechains of non-phenolic /3-1 lignin model compounds by an extracellular enzyme produced by ligninolytic cultures of Phanerochaete chrysosporium. The purified enzyme requires H202, has a molecular weight of 42000, and shows no stereoselectivity. It shows both peroxidase and oxygenase activities, is sensitive to azide (probably contains a metal complex), and has maximum activity at pH 3.0. The enzyme is active not only against /3,1 models, but also/3-O-4 models (Figure 3), spruce lignin and birch lignin. It oxidizes benzylic hydroxyl moieties forming benzylic carbonyls, a reaction seen commonly during microbial decay of lignin no matter what the microorganism involved. 1 When lignins are oxidized by the enzyme, single-ring products are released following cleavage of Ca-C# bonds in free lignin end groups, and the lignins are partially depolymerized. Much of the above information recently has been published in a more rigorous form.s9 CH2OH CH2OH

Table 2

Effects of hydroxyl radical scavengers on the rates of degradation of [14C]lignin lignocellulose to 14CO2 by Streptomyces strains and Phanerochaete chrysosporiuma, c

~s02 /

HO"~C D

Per cent change in IaCO 2 evolution rate after addition of scavengerb Organism

BEN

TH I

OH B

P. chrysosporium S, viridosporus S. badius S. setonii

--70 + 33 +44 -9

--53 + 27 +3 -12

--80 --4 - 13 -13

a]4co2 evolution rate prior to addition was determined between hours 184 and 214 of incubation, The rate after addition was determined between hours 214 and 280. The per cent change in rate was calculated using the rates for unamended control cultures between 184 and 280 hours as the 100% values. All values are averages of three replicates bBEN, benzoate (0.1% w/v final concentration); THI, thiourea (0.08% w/v final concentration); OHB, o-hydroxybenzoate (0.14% w/v final concentration) c Data taken from Crawford etal. 2s

o.

(OC'13)

(H3CO)~oR (OCRH:)lk,i

P

lsOH

(H3CO} ~O~R (OCH3)

-, R ~ OCH3

"X,~131I OCH3

1

c.o

OH

~1-

(OCH3)

~k ~ . ~ D

+.o 3/]]I

13[

/

R

OCH 3 I][

R= %~" OCH 3 OC2Hs

Figure 3 Degradation of lignin model compounds by an enzyme from Phanerochaete chrysosporium (Kirk and Tien, 1983 s6)

Enzyme Microb. Technol., 1984, vol. 6, October

439

Review

Gold et aL so independently described an enzyme produced by ligninolytic cultures ofPhanerochaete chrysosporium that appears to be similar to or the same as the enzyme described by Tien and Kirk. The enzyme of Gold et al. requires H202 and shows a high degree of nonspecificity. It attacks model compounds (Figure 4), functions as an oxygenase, decolorizes certain polymeric dyes and liberates ethylene from KTBA. A ligninolytic-negative mutant of Phanerochaete chrysosporium does not produce the enzyme. Another report concerning this enzyme has appeared recently.6° This report confirms that the enzyme degrades polymeric lignin. Frick and Crawford sl reported their discovery of a relatively stable enzyme in culture filtrates of ligninolytic Phanerochaete chrysosporium. The enzyme demethylates lignin model polymers such as polyguaiacol.52 The methyl moiety of the substrate appears to be released as methanol or formaldehyde, not carbon dioxide or carbon monoxide. The demethylase requires H202 and shows other similarities to known peroxidases. The authors suspect the enzyme to be important during fungal demethylation of lignin. It probably is a different enzyme from that of Kirk et al. and Gold et al. It is being purified for in-depth studies of its properties. In summary, Phanerochaete chrysosporium's ligninolytic catalysts appear increasingly to be true enzymes, not free active oxygen species. At least some oftheseligninolytic enzymes are non-specific peroxidases, and may be very few in number. They show numerous types of activities, including (a) dehydrogenative, (b) peroxidatic, (c) oxygenative and (d) Ca-Ct3 cleavages of lignin sidechains. Activated oxygen, is of course, involved during catalysis, but the oxygen appears to be held in the active site of extracellular proteins.

S t r e p t o m y c e s viridosporus Increasingly strong evidence suggests that S. viridosporus produces an enzyme complex involved in the cleavage of B-ether linkages in lignin, by a mechanism distinct from that discussed above for P. chrysosporium. The multienzyme complex of S. viridosporus was originally discovered using a lignin substructure model, veratrylglycerol B-guaiacyl ether (VE), as a substrate incubated with S. viridosporus cells growing on lignocellulose. 33 The model was quickly degraded, and guaiacol was released as a B-ether cleavage product. At the time of that first report, the reaction sequence leading to guaiacol production was unknown. However, Thede and Crawford 61 CH3 CH == CH

1802, extracellular enzyme, H202

generating system

~OCH 3 OCH2CH3 I CH2OH ~C @ ~ ) C H 3 HCOH @OCH 2CH3 OCH2CH3

CH3 I~lC -18OH I~C OH '-'t~ ' ~ OCH3 OCH2CH3 R

lsO2 , extracellular

CHO

CHO

~OCH3 OCH2CH3 Ill H2C-OH HC lSOH

geneen~Ytimp' aH2s t% m ~'OCH2CH3 4" @ OCH2CH3 OCH3

4 Reactions catalysed by an extracellular enzyme of ligninolytic Phanerochaete chrysosporium (Gold et al. 1983 so) Figure

440

Enzyme Microb. Technol., 1984, vol. 6, October

H HO-CH

.co OH-CH OMe OMe OMe

OH ~OMe

H HO-CH I

. .co O-:C OR

x OMe OMe

R=Me,H

OH X=COOH,CHO

Figure 5 Pathway of veratrylglycerol f3-guaiacyl ether metabolism and/3-ether cleavage by ligninolytic Streptomyces viridosporus cells

have now elucidated the reaction sequence almost completely, and it is shown in Figure 5. Intermediates of VE metabolism were identified by a combination of techniques utilizing thin-layer chromatography, high performance liquid chromatography, and ultraviolet spectroscopy. Each intermediate was confirmed by comparison with authentic standard. Non-ligninolytic S. viridosporus cells are unable to metabolize VE at a significant rate when the ether is supplied as a carbon source in the absence of lignin.33 When cells are growing on lignocellulose the compound is rapidly metabolized. We have found low levels of the VE catabolism enzymes in uninduced cells, but ligningrown cells have much higher activities (ref. 33 and unpublished results). VE is initially demethylated to form the guaiacylglycerol/3-guaiacyl ether. Then, the a-hydroxyl group of the propane side-chain is oxidized to the acarbonyl. This reaction has also been shown to be catalysed by the P. chrysosporium enzyme of Tien and Kirk. s6 This side-chain oxidation prepares the B-ether linkage for cleavage, which then proceeds quite rapidly, releasing guaiacol as a cleavage product. We have not yet isolated the phenylpropane cleavage product, which is quite transitory. However, this intermediate is degraded through vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillic acid (4-hydroxy-3-methoxybenzoic acid). Considerable evidence suggests that this VE-metabolizing enzyme complex also plays a direct role in lignin catabolism by S. viridosporus. First, the enzyme induction pattern indicates lignin to be an inducer of the system. APPLs will also induce the enzymes. Second, the chemistry of lignin degradation by S. viridosporus 1°,3° and the production of the water-soluble polymeric APPL intermediate 3° are readily explained by the action of this B-ether cleaving enzyme system. Partially degraded lignin and water-soluble APPLs are enriched in a-carbonyl groups and in free phenolic hydroxyl groups. Thus, APPLs are polymeric fragments, probably generated primarily as a result of the B-ether cleavage reactions, 3° although further oxidation of the APPLs occurs after their initial release from the polymer (unpublished data). We are now in the process of examining the activity of the enzyme system on lignin to confirm its direct involvement in lignin catabolism. Recently, we have shown that certain mutant strains of S. viridosporus which are enhanced in APPL-producing ability 31 also produce higher levels of the enzyme complex (unpublished data). Also, Streptomyces setonii,6z,63 which partially mineralizes lignin while not producing significant amounts of APPL, does not produce the B-ether cleaving enzymes when growing on lignin (unpublished data). These findings strongly

Microbial degradation of lignins: R. L. Crawford and D. L. Crawford

support the hypothesized role of the enzyme system in lignin catabolism and in APPL production. Ether cleavage activity is not stimulated by H202, but we have not yet shown whether HzO2 is required for activity. Evidence is sufficient to hypothesize the pathway and mechanism for attack on lignin by S. viridosporus. The mechanism is probably strictly enzymatic and involves a lignin-induced enzyme system which probably does not require H202 as does the Phanerochaete enzyme. Also, unlike the fungal enzyme, the Streptomyces pathway does not catalyse C~-C~ cleavage. Instead, enzymatic attack first involves Ca oxidation of lignin side-chains which generates ot-carbonyls. This makes the /3-ether linkages more susceptible to cleavage, either hydrolytically or by a monooxygenase reaction mechanism. Hydrolytic cleavage seems more likely, although we have not yet confirmed that mechanism. It is possible that the final cleavage reaction is non-enzymatic; however, data obtained using active and inactivzted whole cells incubated with the c~-carbonyl intermediate of VE metabolism strongly suggest that the ether cleavage reaction is enzyme catalysed (unpublished data). Prior to the/3-ether cleavage reaction, demethylation of ring structures occurs. As discussed above (see Figure 5), we have identified an enzyme activity which demethylates VE at the pars position prior to ether cleavage. Such demethylations would also help to prepare the /3-carbon of propane side for the ether cleavage reaction. Chemical evidence 1°'3° indicates that other oxidations of lignin are carried out by S. viridosporus. These include further side-chain oxidations which generate aromatic carboxylic acid groups in residual lignins and APPLs. Esterified aromatic acids are also removed from the APPI_s over a period of time (unpublished data). The net result of ~-ether cleavage and these other concomitant oxidations is the release of the polymeric, water-soluble lignin fragments we have named Acid Precipitable Polymeric Lignin (APPL). 3° APPL is quantitatively the most important lignin degradation intermediate produced by S. viridosporus and it is initially released in amounts approximating the total lignin removed from the lignoceUulose substrate. 33 APPL yields also correlate with the biodegradability of the specific lignocellulose.33 The ultimate fate of APPLs produced by S. viridosporus remains to be determined. Low molecular weight phenolic catabolites are also generated as S. viridosporus degrades lignin, but they are produced in minor amounts, 2s probably from peripheral monomeric units or esterified aromatic acids. They are probably not mechanistically vital to the lignin catabolism pathway. The APPL-generating /3-ether cleaving enzyme system is probably lightly bound to the cytoplasmic membrane of this actinomycete.33 Although it is cell-associated in cellfree extracts, sonication and repeated cell lysis by use of a pressure cell frees the activity into the soluble cytoplasmic fraction of the extracts. At least an association with the cell surface would appear vital to the function of this enzyme system since it must act on an insoluble polymer. F u t u r e research priorities Future research priorities with both P. chrysosporium and S. viridosporus will probably emphasize more enzyme characterizations and genetic manipulations of the two organisms. Kinetic studies with purified enzymes, characterization of new enzymes and overproduction of selected

enzymes are research objectives already under study in several laboratories. Genetic manipulation of both organisms, utilizing recombinant DNA techniques as well as classical mutagenesis, are also active research areas. Gold et al. 47'64 have isolated pleotrophic mutants of P. chrysosporium lacking phenol oxidase and unable to evolve 14CO2 from [14C]lignins and [14C]lignin substructure model compounds. They utilized classical mutagenesis techniques which became useful after the development of a medium that induced colonial growth in this fungus.6s Glenn and Gold 66 have since utilized this mutant in studies with several polymeric dyes which are not attacked by the mutant, but are decolorized by ligninolytic cells of the wild-type strain. The mutant does not produce the H:O2requiring lignin catabolic enzyme,s° These dyes, which are apparently mistaken for lignin by P. chrysosporium, may be useful as a tool in biochemical and genetic studies of lignin degradation of P. chrysosporium, so and possibly other lignin-degrading microbes which attack lignin by a similar mechanism. Recently, Gold et al. 67 successfully formed viable protoplasts of P. chrysosporium and were able to carry out intraspecies protoplast fusion, regenerating stable recombinant organisms. Protoplast fusion will be a powerful recombinant DNA technique for breeding new hybrid lignin-degrading strains. New enzyme studies continue as well. Keller and Reddy 6a have isolated enzymatic activity that they conclude is glucose oxidase involved in H202 production by ligninolytic P. chrysosporium cells. Tien and Kirk 69 have reported the purification and additional mechanistic information on the lignin-degrading H2Oz-requiring oxygenase they were the first to isolate s6,s9 from P. chrysosporium. Current research with ligninolytic Streptomyces is emphasizing biochemical and genetic studies. Pettey and Crawford 31 have utilized interspecies and intraspecies protoplast fusion to generate stable, enhanced lignindegrading recombinants derived from self-crosses of S. viridosporus T7A and interspecies crosses between S. viridosporus T7A and S. setonii 75Vi2. The enhanced lignin-degrading activity was thought to result from a gene amplification phenomenon. Selected recombinants generated between 155 and 264% more APPL from corn stover lignocellulose than was produced by the wild-type S. viridosporus strain grown under similar conditions. Crawford et al. 7° have now shown that stable APPLoverproducing strains ofS. viridosporus can also be produced by ultraviolet irradiation mutagenesis. In this latest work, they also found that APPL-overproducing recombinants and mutants had in common an enhanced level of activity of the fl-ether cleaving enzyme system discussed previously. In new chemical studies of lignin degradation by ligninolytic Streptomyces, Borgmeyer and Crawford 71 have found that S. badius 252, a known lignin-degrading actinomycete,21 produces an APPL which is chemically distinct from that produced by S. viridosporus T7A. Whereas the latter is a structurally modified water-soluble lignin, the former is apparently a polyphenol formed by repolymerization of low molecular weight lignin degradation intermediates. S. badius produces an extracellular laccase which may be involved in this repolymerization. S. viridosporus produces no detectable laccase. S. badius produces its unique APPL only in liquid shake cultures, but only after establishment of growth in semisolid medium. These findings show that different Streptomyces can be expected to

Enzyme Microb. Technol., 1984, vol. 6, October

441

Review exhibit different patterns o f lignin catabolism that will require considerable additional study for a c o m p l e t e elucidation.

37 38 39

References 1 2 3 4

5 6 7 8 9 I0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

27 28 29 30 31 32 33

34 35 36

442

Crawford, R. L. Lignin Biodegradation and Transformation John Wiley and Sons, Inc., New York, 1981 Hall, P.L. EnzymeMicrob. Technol. 1980,3,170-176 Dagley, S. Surv. Prog. Chem. 1977, 8, 121-170 Crawford, D. L. and Crawford, R. L. Enzyme Microb. Technol. 1980, 2, 11-22 Higuchi, T.Experientia 1982, 38, 159-166 Janshekar, H. and Fiechter, A. Adv. Biochem. Eng. Biotechnol. 1983, 27,119-178 Harelund, W. A., Crawford, R. L., Chapman, P. J. and Dagley, S.J. Bacteriol. 1975, 121,275-285 Ribbons, D. W. FEBSLett. 1970, 8, 101-104 Kirk, T. K. Annu. Rev. Phytopathol. 1971, 9, 185-210 Crawford, D. L., Barder, M. J., Crawford, R. L. and Pometto 1II, A. L. Arch. Microbiol. 1982, 131,140-145 Koenigs, J. W. Wood Fiber 1974, 6, 66-72 Highley, T. L. Mater. Orgo 1982, 17,205-214 Sagone, A. L., Decker, M. A., Wells, R. M. and Democko, C. A. Biochim. Biophys. Acta 1980, 628, 90-97 Adler, E. Wood Sci. Technol. 1977, 11,169-218 Nimz, H. Angew. Chem. 1974, 86,336-344 Broda, P. and Paterson, A. Nature 1983, 306,737-738 Kirk, T. K. in Recent Advances in Lignin Biodegradation Research (Higuchi, T., Kirk, T. K. and Chang, H. M., eds), Uni Pub1. Co. Ltd, Tokyo, 1983, pp. 1-11 Higuchi, H., Chang, H. M. and Kirk, T. K., eds Recent Advances in Lignin Biodegradation Research, Uni Pub1. Co. Ltd, Tokyo, 1983 Ander, P. and Eriksson, K. E. Prog. Ind. Microbiol. 1978, 14, 1-58 Crawford, D. L. Appl. Environ. Microbiol. 1978, 35, 10411045 Crawford, D. L. and Sutherland, J. B. Dev. Ind. Microbiol. 1979, 20, 143-151 Phelan, M. B., Crawford, D. L. and Pometto Ili, A. L. Can. J. Microbiol. 1979, 25, 1270-1276 Sutherland, J. B., Blanchette, R. A., Crawford, D. L. and Pometto III, A. L. Curr. MicrobioL 1979, 23,123-126 Crawford, D. L. and Sutherland, J. B. in Lignin Biodegradation: Chemistry and Applications Vol H (Higuchi, T. and Chang, H. M., eds), CRC Press, Boca Raton, Florida, 1980, pp. 95-101 Crawford, R. L., Crawford, D. L. and Dizikes, G. J. Arch. Microbiol. 1981, 129, 204-209 MacDonald, M°, Raeder, U., Liwicki, R., Haylock, R., McCarthy, A., Paterson, A., Birch, O., Ramsey, L. and Broda, P. in Proe. Biotechnol. in the Pulp and Paper Industry Symp. London, England, PIRA, Randalls Rd, Leatherhead, Surrey, England, 1983, pp. 174-177 Sinden, D. L.M.S. Thesis University of Idaho, 1979 Crawford,D. L.BiotechnoL Bioeng. Syrup. 1981, 11,275-291 Antai, S. P. and Crawford, D. L. Appl. Environ. Microbiol. 1981, 42, 378-380 Crawford, D. L., Pometto III, A. L. and Crawford, R. L. Appl. Environ. Microbiol. 1983, 4 5 , 8 9 8 - 9 0 4 Pettey, T. M. and Crawford, D. L. Appl. Environ. Microbiol. 1984, 4 7 , 4 3 9 - 4 4 0 Crawford, D. L., Sutherland, J. B., Pometto 111, A. L. and Miller, J. L A r c h . Microbiol. 1982, 131,351-355 Crawford, D. L., Pometto III, A. L. and Deobald, L. A. in Recent Advances in Lignin Biodegradation Research (Higuchi, T., Kirk, T. K. and Chang, H. M., eds), Uni Publ. Co. Ltd, Tokyo, 1983, pp. 78-95 Keyser, P., Kirk, T. K. and Zeikus, J. G. J. Bacteriol. 1978, 73,294-306 Kirk, T. K., Schultz, E., Connors, W. J., Lorenz, L. F. and Zeikus, J. G. Arch. Microbiol. 1978, 117,277-285 Nakatsubo, F., Reid, I. D. and Kirk, T. K. Biochem. Biophys. Res. Commun. 1981, 102,484-491

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Kirk, T. K., Nakatsubo, F. and Reid, I. D. Biochem. Biophys. Res. Commun. 1983, 111,200-204 Kearns, D. R. in Singlet Oxygen Academic Press, New York, 1979, pp. 115-137 Forney, L. J., Reddy, C. A., Tien, M. and Aust, S. D. J. Biol. Chem. 1982, 257, 11455-11462 Cohen, G. and Cedarbaum, A. I. Arch. Biochem. Biophys. 1980, 199, 438-447 Walling, C. Acc. Chem. Res. 1975, 8, 125-131 Finkelstein, E., Rosen, G. M. and Rauekman, E. J. Arch. Biochem. Biophys. 1980, 200, 1-16 Forney, L. J., Reddy, C. A. and Pankratz, H. S. Appl. Environ. Microbiol. 1982, 44,732-736 Reddy, C. A., Forney, L. J. and Kelly, R. L. in Recent Advances in Lignin Biodegradation Research Uni Pub1. Co. Ltd, Tokyo, 1983, pp. 153-163 Kutsubi, H. and Gold, M. H. Bioehem. Biophys. Res. Commun. 1982, 109, 320-327 Beauchamp, C. and Fridovich, I. J. Biol. Chem. 1970, 245, 4641-4646 Gold, M. H., Mayfield, M. B., Cheng, T. M., Krisnangkura, K., Shimada, M., Enoki, A. and Glenn, J. K. Arch. Microbiol. 1982, 132, 115-122 Bes, B., Ranjeva, R. and Boudet, A. M. Biochimie 1983, 65, 283-289 Winston, G. W. and Cedarbaum, A. I. Biochemistry 1982, 12, 4265-4270 Gold, M. H., Glenn, J. K., Mayfield, M. B., Morgan, M. A. and Kutsubi, H. in Recent Advances in Lignin Biodegradation Research Uni Publ. Co. Ltd, Tokyo, 1983, pp. 219-232 Frick, T. D. and Crawford, R. L. in Recent Advances in Lignin Biodegradation Research Uni Publ. Co. Ltd, Tokyo, 1983, pp. 143-152 Crawford, R. L., Robinson, L. E. and Foster, R. D. Appl. Environ. Microbiol. 1981, 41, 1112-1116 Fridovich, h and Handler, P. J. Biol. Chem. 1958, 233, 15811585 Bors, W., Lengfelder, E., Saran, M., Fuchs, C. and Michel, C. Biochem. Biophys. Res. Commun. 1976, 70, 81-87 Shimada, M. and Higuchi, T. in Recent Advances in Lignin Biodegradation Research Uni Publ. Co. Ltd, Tokyo, 1983, pp. 195-208 Kirk, T. K. and Tien, M. in Recent Advances' in Lignin Biodegradation Research Uni Publ. Co. Ltd, Tokyo, 1983, pp. 233-245 Faison, B. and Kirk, T. K. Appl. Environ. Microbiol. 1983, 46, 1140-1145 Tai, D., Terasaura, M., Chen, C. L., Chang, H. M. and Kirk, T. K. in Recent Advances in Lignin Biodegradation Research Uni Publ. Co. Ltd, Tokyo, 1983, pp. 44-63 Tien, M. and Kirk, T. K. Science 1983, 221,661-663 Glenn, J. K., Morgan, M. A., Mayfield, M. B., Kuwahara, M. and Gold, M. H. Biochem. Biophys. Res. Commun. 1983, 114, 1077-1083 Thede, B. and Crawford, D. L. Abstr. 84th Annu. Meeting Am. Soe. Microbiol. 1984, p. 196 Pometto III, A. L., Sutheriand, J. B. and Crawford, D. L. Can. J. Microbiol. 1981, 27,859-863 Sutherland, J. B., Crawford, D. L. and Pometto III, A. L. Can. J. Microbiol. 1983, 29, 1253-1257 Gold, M. H., Cheng, T. M. and Mayfield, M. B. Appl. Environ. Mierobiol. 1982, 44,996-1000 Gold, M. H. and Cheng, T. M. Appl. Environ. Microbiol. 1978, 35, 1223-1225 Glenn, J. K. and Gold, M. H. Appl. Environ. Mierobiol. 1983, 45, 1741-1747 Gold, M. H., Cheng, T. M. and Alic, M. AppL Environ. Microbiol. 1983, 46,260-263 Kelly, R. L. and Reddy, C. A. Abstr. 84th Annu. MeetingAm. Soc. Microbiol. 1984, p. 172 Tien, M. and Kirk, T. K. Abstr. 84th Annu. MeetingAm. Soc. Microbiol. 1984, p. 173 Crawford, D. L., Pettey, T. M. and Deobald, L. A. Bioteehnol. Bioeng. Syrup. 1984, in press Borgmeyer, J. R. and Crawford, D. L. Abstr. 84th Annu. Meeting Am. Soc. Microbiol. 1984, p. 173