(Received for publication, November 1, 1994; and in revised form, December 14, 1994)
From the
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.
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 - and
-
-unsaturated
-alcohols to the corresponding aldehydes
with concomitant reduction of O
to
H
O
. 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
O
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), (
)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.
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. 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.
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.
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 gas flow.
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.
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.
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.
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 depletion, and the presence of O
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
HO
, 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.
Dedicated to the memory of our much missed friend and colleague, Gianpaolo Nitti.