©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Veratryl Alcohol Oxidase from Pleurotus ostreatus Participates in Lignin Biodegradation and Prevents Polymerization of Laccase-oxidized Substrates (*)

(Received for publication, November 1, 1994; and in revised form, December 14, 1994)

Liberato Marzullo Raffaele Cannio Paola Giardina Maria Teresa Santini (1) Giovanni Sannia (§)

From the Dipartimento di Chimica Organica e Biologica, Università degli Studi di Napoli ``Federico II,'' Via Mezzocannone, 16, 80134 Napoli, Italy and Laboratorio di Ultrastrutture, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Oxidative enzymes (laccases and peroxidases) isolated from the culture media of different fungi are involved in the basic mechanism of ligninolysis via radical intermediates. However, experiments aimed at reproducing natural biodegradation in vitro have been unsuccessful so far since the single biocatalysts alone are not able to solubilize lignins because of the simultaneous recondensation of these intermediates. FAD oxidases can prevent this side reaction in lignin depolymerization by reducing quinonoids and radical compounds. This study investigates the possible role of a laccase and a FAD-dependent aryl alcohol oxidase (veratryl alcohol oxidase, VAO) excreted by the basidiomycete Pleurotus ostreatus. In fact, we found that VAO is able to reduce synthetic quinones, laccase-generated quinonoids, and phenoxy radicals with concomitant oxidation of veratryl alcohol to veratryl aldehyde. This cooperative action of laccase and VAO also prevented the polymerization of phenolic compounds and reduced the molecular weight of soluble lignosulfonates to a significant extent.


INTRODUCTION

The biological mechanism of lignin degradation has been extensively studied because of the heterogeneous polymeric structure of phenylpropane subunits recalcitrant to normal hydrolytic treatments and because of the complexity of the enzymatic systems involved. It has been proposed that two classes of extracellular enzymes, phenol oxidases (laccases) and lignin peroxidases, isolated from the culture media of different ligninolytic fungi, participate in the mechanism of ligninolysis through their ability to catalyze the cleavage of carbon-carbon and/or carbon-oxygen bonds in lignin model compounds in vitro. Lignin peroxidases catalyze the oxidation of various aromatic substrates to produce aryl cation radicals, whereas laccases oxidize phenolic substrates to phenoxy radicals. These radicals spontaneously rearrange themselves, leading to the fission of carbon-carbon or carbon-oxygen bonds of the alkyl side chains or to the cleavage of aromatic rings. Nevertheless, experiments aimed at reproducing natural biodegradation in vitro showed that these two biocatalysts are able to depolymerize high molecular weight lignins but that low molecular weight products accumulated simultaneously recondense(1, 2) . This side effect could be avoided in vivo by mere cell uptake or reducing processes that switch off the reactivity of the small molecules formed. It has been suggested that the latter of the functions mentioned could be carried out by some FAD-dependent oxidases able to reduce quinonoids or radical compounds. In fact, the synergistic effect of laccase and glucose oxidase was proposed by Green (3) in Polyporus versicolor and confirmed by Sklarz and Leonowicz (4) using lignosulfonates as the substrates; a similar effect was also present in Rigidoporus lignosus(5) . Another FAD oxidase, cellobiose:quinone oxidoreductase from Phanerochaete chrisosporium, is thought to be involved in preventing the repolymerization in lignin degradation, but its role is still debated (6, 7) .

Among the FAD-dependent oxidases, aryl alcohol oxidases have also been detected and isolated from different ligninolytic fungi(8, 9, 10, 11, 12, 13) . These enzymes catalyze the oxidation of aryl alpha- and alpha-beta-unsaturated -alcohols to the corresponding aldehydes with concomitant reduction of O(2) to H(2)O(2). In all of the species examined, these enzymes have been identified in the culture broth in the later stage of growth when secondary metabolic pathways are presumably switched on by starvation. The metabolic role proposed for these enzymes has been that of providing H(2)O(2) to be used by peroxidases, which have been assumed to be principally responsible for lignin degradation. In Pleurotus ostreatus, at least three different phenol oxidases and an extracellular aryl alcohol oxidase, namely a veratryl alcohol oxidase (VAO), (^1)have been characterized(8, 14) . The only glucose oxidase purified and characterized was found intracellularly(15) , and no cellobiose:quinone oxidoreductase nor any lignin peroxidase activity have so far been detected. To study the role of VAO in prevention of polymerization of phenolic compounds and in lignin degradation, its activity was assayed on laccase-treated soluble lignins, and its specificity on both synthetic and laccase-generated quinonoid species was examined.


MATERIALS AND METHODS

Enzymes and Assays

Homogenous laccase and VAO were isolated from the culture broth of P. ostreatus as previously described(8, 14) . Laccase was assayed in 50 mM sodium phosphate, pH 6.0, using 2 mM 2,2`-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid as substrate, following the increase in absorbance at 420 nm (^M = 36,000). VAO was assayed in 50 mM sodium phosphate, pH 6.0, using 4 mM veratryl alcohol as substrate following the increase in absorbance at 310 nm (^M = 9,300). Enzyme activity was expressed in international units. In the experiments described below, pure enzymes were used as judged by SDS-polyacrylamide gel electrophoresis analysis.

Inhibition of Guaiacol Polymerization by VAO

Inhibition of laccase-catalyzed polymerization of guaiacol was determined in 1 ml of 50 mM sodium phosphate, pH 6.0, containing 10 mM guaiacol, 12 mM veratryl alcohol, and 24 milliunits of laccase. A total of 72 milliunits of VAO or the equivalent amount in protein of bovine serum albumin (BSA) was added after 15 min. Product formation was followed spectrophotometrically at 465 nm.

Inhibition of Ferulic Acid Polymerization by VAO

Inhibition of laccase-catalyzed polymerization of ferulic acid was determined in 1 ml of 50 mM sodium phosphate, pH 6.0, containing 10 mM ferulic acid, 12 mM veratryl alcohol, and 75 milliunits of laccase. 600 milliunits of VAO were added after 5 min. Aliquots of 150 µl were withdrawn after 15, 45, and 75 min of incubation, blocking the reaction by the addition of thioglycolic acid to 0.5 mM and flash freezing the solution in an acetone/dry ice mixture. Product formation was analyzed by gel filtration chromatography in 100 mM glycine-NaOH, pH 9.0, 200 mM NaCl on a Bio-Gel P2 (Bio-Rad) (1 times 46 cm) connected to an fast protein liquid chromatography system (Pharmacia Biotech Inc.) (flow rate, 10.8 ml/h).

Assay of the Quinone Reduction and Test of the Effect of Oxygen and Veratryl Alcohol on VAO Activity

The 2,6-dichlorophenol-indophenol (DCIP)-reducing activity of VAO was assayed at 25 °C under vacuum in 50 mM sodium phosphate, pH 6.0. The activity was measured following the decrease in absorbance at 575 nm in a solution of 4 mM veratryl alcohol and 0.085 M DCIP.

The effect of oxygen and veratryl alcohol on the oxidoreductive cycle was carried out in 50 mM sodium phosphate, pH 6.0, containing 0.085 M DCIP, 0.64 units of laccase and 1 unit of VAO with and without 4 mM veratryl alcohol. Assay mixtures were purged from oxygen by vacuum and flushing of oxygen-free N(2). DCIP absorbance at 575 nm was followed under vacuum for 30 min. Samples were re-exposed to air, and their absorbance was followed for a further 15 min.

Monitoring of Quinonoid Species Formation/Reduction from Lignosulfonate in Laccase/VAO Reaction Mixtures

A 1% solution of Ultrazine sodium (gift from Borregaard Lignotech, Sarpsborg, Norway) in distilled water was partially purified from low molecular weight species by ultrafiltration on Amicon using a PM10 membrane (Diaflo cut-off 10,000, Amicon Corp., Danvers, MA). The assay mixture contained 8 mM veratryl alcohol, 10 optical density units (A) of ultrafiltered Ultrazine, and 2.9 units of laccase in 50 mM sodium phosphate, pH 6.0.

The detection of the changes in the concentration of the quinones was performed spectrophotometrically at 390 nm (wavelength corresponding to the maximal average absorbance of the quinonoids formed as determined by spectrophotometric analysis) for 30 min before and 30 min after the addition of 2.4 units of VAO or the equivalent protein amount of BSA in the control reaction.

Polymerization/Depolymerization Assay on High Molecular Weight Lignosulfonates

Ultrazine sodium was fractionated on a Bio-Gel P-100 (Bio-Rad) gel filtration chromatography in 50 mM NH(4)HCO(3), pH 8.5. The fractions corresponding to the exclusion volume and to the first included peak were collected in two distinct pools (A and B, respectively), and absorption was determined at 280 nm. The reaction mixtures (1 ml) contained 0.5 optical density units/ml fractionated Ultrazine, 0.6 units of laccase, and 4 mM veratryl alcohol in 50 mM sodium phosphate, pH 6.0. After 15 min of incubation at 25 °C, 0.4, 2.8, and 4.0 units of VAO were added, and aliquots (150 µl) were withdrawn after 15, 30, 60, and 120 min and 16 h. The reactions were then stopped by acidification to pH 1 with 37% HCl. Samples were analyzed using gel filtration chromatography in 50 mM sodium phosphate, pH 8.0, on a TSK-GEL G2000 SW column (30 cm times 7.8 mm, inner diameter, Toso Haas, Stuttgart, Germany) connected to a gold high performance liquid chromatography system (Beckman Instruments) (flow rate, 1 ml min).

Electron Paramagnetic Resonance Spectroscopy

The phenoxy-radical acetosyringone was prepared in 50 mM sodium phosphate buffer, pH 6.0, containing 1.2 mM acetosyringone, 4 mM veratryl alcohol, and 13 milliunits/ml laccase. After 1 min of incubation at 25 °C, 0.1, 0.2, 0.35, and 0.5 units/ml of VAO were added to the reaction mixture and quickly flushed with N(2). In the control experiment, either distilled water or the equivalent protein concentrations of bovine serum albumin were added instead of VAO.

EPR measurements were conducted by aspirating the samples directly in gas-permeable Teflon tubing (Zeus Industrial Products, Raritan, NJ), which was then inserted in an open-ended quartz capillary tube. Spectra were recorded at 25 ± 0.1 °C in a Varian E4 X-Band spectrometer operated at 9.08 GHz, a modulation frequency of 100 KHz, a modulation amplitude of 0.5 G, microwave power of 10 mW, a time constant of 1 s, and a scan time of 4 min. The values were expressed as the average of the results of five different experiments performed under N(2) gas flow.


RESULTS

VAO-catalyzed Quinone Reduction

The laccase-catalyzed oxidation of guaiacol produces a brown-colored and scarcely soluble polymer. The different course of the reaction in the presence and in absence of VAO is shown in Fig. 1. The 465-nm absorbance decreases after the addition of VAO, and the precipitate formation is blocked. The experiment demonstrates that VAO, in the presence of veratryl alcohol, reduces the intermediates of this reaction, preventing the phenoxy radical coupling.


Figure 1: Inhibition of polyguaiacol formation by VAO. Laccase oxidation of guaiacol was followed in the absence (plota) and in the presence (plotb) of VAO. The dashedline indicates the time of VAO (b) or BSA (a) addition.



The specificity of VAO toward quinonoid species was tested using DCIP as cosubstrate in the oxidation of veratryl alcohol under conditions of oxygen depletion. DCIP was converted into N-(4-hydroxyphenyl)-3,5 dichloro-4 hydroxyaniline as revealed by the decrease in absorption at 575 nm (Fig. 2A). This dihydroform produced by VAO under vacuum was reoxidized to DCIP by laccase after air reintroduction as shown by the rapid increase in absorbance at 575 nm in Fig. 2A. Moreover, DCIP is also a substrate of laccase and can in fact be oxidized to phenoxy radical when incubated in the presence of oxygen (Fig. 2B), as shown by the hypsochromic shift in its absorption maximum to 510 nm. When the complete reducing system VAO/veratryl alcohol was present, the rate of oxidation by laccase of DCIP was notably decreased.


Figure 2: Influence on laccase oxidation and VAO reduction of DCIP by oxygen and veratryl alcohol. DCIP was incubated with laccase and VAO in the presence (A) and in the absence (B) of veratryl alcohol. In the absence of both the cosubstrates, neither laccase nor VAO could catalyze any reaction on DCIP. Laccase-catalyzed oxidation of DCIP was observed after readmission of air, as shown by the hypsochromic shift in its absorption maximum from 575 to 510 nm. The presence of veratryl alcohol (A) in DCIP-enzyme mixtures under vacuum brought about complete reduction of DCIP. The readmission of air resulted in a quick reappearance of DCIP absorption spectrum.



Inhibition of Ferulic Acid Polymerization by VAO

Gel filtration experiments showed that ferulic acid is completely polymerized by laccase after 80 min and that no influence of veratryl alcohol could be detected in the conditions used. The addition of VAO to the reaction mixture after 5 min was able to inhibit the formation of high molecular weight products. In fact, chromatographic analysis of the reaction products after 80 min of laccase/VAO incubation showed a reduction in the area of the ferulic acid peak of only about 15%.

Formation/Reduction of Quinonoid Species from Lignosulfonates by Laccase/VAO System

The quinone-reducing activity of VAO was tested on quinones produced by the reaction of laccase on lignosulfonates. Quinone formation rates from lignosulfonates were found to be laccase concentration dependent (data not shown). In the assay condition used, VAO proved efficient in reducing the heterogeneous quinones produced; in fact, a slow decrease in the absorbance at 390 nm was observed when VAO was added after 30 min of laccase incubation (Fig. 3).


Figure 3: VAO reduction of quinones produced from lignosulfonates by laccase. Laccase reaction was followed monitoring the absorbance at 390 nm before and after addition of VAO (dashedline) or BSA used as a blank control.



Polymerization/Depolymerization Assays on High Molecular Weight Lignosulfonates

Incubation of two distinct fraction pools of high molecular weight lignosulfonates (pool A and B, see ``Materials and Methods'') with laccase and VAO resulted in depolymerization as shown by gel filtration analysis. Similar results were obtained using both pools, but the most extensive depolymerization was observed when pool B was incubated with a 1:7 mixture of laccase/VAO. Moreover, in all cases, the formation of products in single enzyme (laccase)- or coupled system (laccase/VAO)-catalyzed reactions was dependent on the concentrations of both enzymes (data not shown) and on the incubation time. The decrease observed in high molecular weight species and the increase of the depolymerization products could not be quantitatively related by directly measuring the peak areas, presumably because of the heterogeneity of the extinction coefficients of the species examined. It is worth noting that the overall lower area of the peaks in the chromatogram of the 120-min reaction with laccase in the absence of VAO corresponded to the onset of polymer precipitation observed. On the other hand, the coupled laccase/VAO reaction allowed the almost complete fission of the substrate with no precipitate formation. Fig. 4shows the gradual disappearance of the substrate and the complex pattern of products.


Figure 4: Analysis of laccase/VAO-treated high molecular weight lignosulfonates. High molecular weight lignosulfonates incubated with laccase (a) and with VAO after a 15-min laccase incubation (b) were analyzed by gel filtration high performance liquid chromatography. Chromatographic profiles are reported for laccase and laccase/VAO reaction products at total incubation times as follows: A, 30 min; B, 45 min; and C, 120 min.



Reduction of Laccase-generated Radicals by VAO

To verify whether VAO catalyzes the reduction of phenoxy radicals, the laccase-generated acetosyringone phenoxy radical was used as a VAO cosubstrate in EPR experiments. This radical was chosen because of its considerably high stability(16) . In fact, an EPR signal could still be recorded after 12 min from its laccase-catalyzed formation under the experimental conditions described. The presence of VAO increased the rate of disappearance of the radical signal to an extent directly correlated to the enzyme concentration (Table 1). The rate of disappearance of the radical was due to VAO activity and not to a mere quenching effect mediated by an unspecific protein, as indicated by the fact that the presence of bovine serum albumin instead of VAO did not affect the half-life of the radical.




DISCUSSION

Laccase oxidative reactions on monomeric and polymeric phenol compounds proceed first through the formation of very unstable radical intermediates, which can undergo rapid association and hence polymerization. To reproduce lignin depolymerization in vitro using only one oxidative enzyme is therefore a limited approach. The systems so far assessed have shown that lignin depolymerization requires the action of more than one enzyme and, in some cases, the contribution of mediator molecules(17) . The quenching of the reactive intermediates produced in the reactions catalyzed by both laccases and ligninases, i.e. the prevention of their recondensation, is indeed a necessary step in directing the overall process toward efficient depolymerization. The present work was aimed at verifying whether VAO from P. ostreatus was able, like other FAD-dependent oxidoreductases, to reduce quinonoids or radical compounds and to ascertain its involvement in lignin biodegradation per se or coupled with the oxidative activity of laccase excreted by the same fungus P. ostreatus.

The spectrophotometric assays performed clearly revealed that VAO efficiently reduces both synthetic DCIP and guaiacyl quinonoids generated by laccase. In all experiments, the efficiency of the coupled enzyme system was found to be dependent on the partial pressure of oxygen, since the laccase activity is blocked by O(2) depletion, and the presence of O(2) interferes with the quinonoid reducing activity of VAO.

Phenoxy radicals produced by laccase can be reduced by the action of VAO/veratryl alcohol as demonstrated using laccase-generated acetosyringone radical as a model system in the EPR experiments described. These experiments clearly indicate that the overall oxidoreductive process can be summarized in a simple scheme. An illustrative example is drawn in Fig. 5, where the schematic flow diagram shows that laccase-oxidized lignol-like molecules, such as DCIP, can be channeled by the VAO/veratryl alcohol system through a cycle that prevents the polymerization route.


Figure 5: Schematic diagram of laccase-VAO cycle and its implications in the prevention of lignol substrate polymerization. DCIP was chosen as a model of monophenol/quinonoid substrate of both enzymes.



The efficiency of the VAO/veratryl alcohol system in the prevention of polymerization was analyzed comparing the retention profiles of gel filtration chromatography of the species formed in the laccase-catalyzed reaction using ferulic acid as substrate. The reducing activity of VAO was able to completely reverse the ferulic acid oxidation by laccase in the early stage of the reaction and consequently to prevent the precipitation of multimerization products. This effect was probably due not only to limited association but also to an increased solubility of the oligomers blocked in reduced phenolic forms by VAO.

The radical/quinonoid reduction and the consequent ``freezing'' of species of low degree of polymerization are responsible also for the depolymerization of lignosulfonates when the VAO/veratryl alcohol system acts in cooperation with laccase. The results of chromatographic analysis of the reaction products indicate that VAO plays an important role in in vitro biodegradation of lignin models but only when strictly associated with an oxidative enzyme, such as laccase, able to promote the process of breakdown on the complex polymeric matrix. A similar and until now unclear role can hence be proposed also for the in vivo function of this enzyme. In fact, VAO shows a broad range of substrate specificity (8) toward alcohols that can be, like the radical and/or quinonoid cosubstrates, excised from lignin or endogenously synthesized by the fungus itself. Veratryl alcohol is just a representative substrate that has been shown to be released in culture media by another basidiomycete, P. chrisosporium, as a product of a synthetic pathway starting from glucose(18, 19) . Nevertheless, this molecule seems to play a different and not unique role in these kinds of ligninase-expressing basidiomycetes since it serves as a radical mediator in the ligninase-catalyzed oxidation (17) or as a mere cosubstrate for the consumption of poisoning H(2)O(2), preventing the enzyme inactivation (20, 21, 22) . Veratryl alcohol can therefore function as a ``shuttle'' for the electron transfer between the buried peroxidase heme and both the inaccessible polymeric substrates (23) and non-substrate synthetic molecules(24) . A recent analysis has revealed the presence of p-anysil alcohol, a compound similar to veratryl alcohol for VAO substrate specificity(8) , as one of the most representative aromatic alcohols in the culture broth of Pleurotus spp., including P. ostreatus(25) . Moreover, the recycling of the aryl aldehydes to aryl alcohols could be ensured by the NADPH-dependent aryl aldehyde dehydrogenases recently found intracellularly in a different Pleurotus sp., namely Pleurotus eryngii(26) , although, at the moment, no experimental evidence has been reported on the existence of transport systems of VAO-oxidized substrates. It is worth noting that genetic and enzyme analyses revealed no gene/protein of the ligninase family in P. ostreatus(8, 27, 28) , indicating the possibility of lignin-degradative pathways in which ligninases are not involved. The aryl alcohols would then be provided by the fungus to VAO to explicate an ``ancillary'' support for the early degrading action of laccases or analogous oxidative enzymes.


FOOTNOTES

*
This work was supported in part by grants from Ministero dell'Università e della Ricerca Scientifica and Consiglio Nazionale delle Ricerche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to the memory of our much missed friend and colleague, Gianpaolo Nitti.

§
To whom correspondence should be addressed. Tel.: 39-81-7041241; Fax: 39-81-7041202.

(^1)
The abbreviations used are: VAO, veratryl alcohol oxidase; BSA, bovine serum albumin; DCIP, 2,6-dichlorophenol-indophenol; EPR, electron paramagnetic resonance.


ACKNOWLEDGEMENTS

We acknowledge Prof. Pietro L. Indovina (Università degli Studi di Napoli ``Federico II'') for the useful comments on the EPR experiments and Prof. Gennaro Marino (Università degli Studi di Napoli ``Federico II'') for the helpful discussions. We also thank Giovanna Morelli and Maria Laura Sorge for technical assistance and Patricia Reynolds for manuscript revision.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.