Laccases (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductases) are multi-copper proteins that use molecular oxygen to oxidize various phenolic and non-phenolic aromatic compounds by a radical-catalyzed reaction mechanism. Laccases can also recognize as glycoproteins since carbohydrates take part in the chemical stabilization of enzymatic molecules 12. Fungi produce laccases for degradation of polymeric phenolic compounds which are the main source of carbon. Molecule of laccase is too large for direct contact with the phenolics present in the inner part of such biopolymers as lignin or the lignocellulose complex, so the process of degradation runs very slowly and needs to cooperate with specific co-catalizators. They can enhance laccase activity by using low molecular compounds which accelerate the catalytic properties of enzyme by improving their affinity of specific radical forms to phenolic biopolymers 3. Fungal laccases, as well known biocatalysts, are readily used in many branches of industrial technology. Among them the depolymerization 4 and biobleaching of lignin 5, decolorization of artificial dyes in the textile industry 16 or of natural pigments in food technology 7 could be mentioned. Laccases can also oxidize xenobiotic compounds 8 and remove of lipophilic compounds from paper pulp 9. Another important activity of laccase is demethylation of non-phenolic lignins 1011. At the beginning of this process, non-phenolic lignin particles, rich in methoxylic compounds as veratrate or anisate, are degraded by laccase to phenolic compounds mainly via demethylation and hydroxylation and these processes are accelerated in combination with redox co-catalysts known as mediators 1213. Many popular mediators of laccase are recognized also among the natural products of lignin and humus degradation and syringaldehyde, acetosyringone, vanillic, p-coumaric and ferulic acids can be mentioned 1415. These natural mediators represent an alternative to synthetic mediators which are more efficient but unfortunately more toxic for environment and more expensive in exploitation.

Commonly used synthetic laccase mediators belong to phenolics. Most of them contain N-hydroxy-groups as 1-hydroxybenzotriazole (HBT), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and N-hydroxy-acetanilide (NHA) or contain pirimidine origin such as violuric acid (VA), which can disturb the organization of nucleic acids 1617. Also transition metal complexes, such as K₄Mo(CN)₈ or K₄W(CN)₈), were suggested as a new class of laccase mediators for pulp bleaching 1819. For industrial purposes, both synthetic and metal-complex mediators must be used in large quantities, which is not very safe for the environment and is very costly. To diminish these difficulties, various methods have been tested and among them, electrochemical methods 2021 and production of laccase recombinants could be mentioned. In our earlier papers, the opportunity of using the low doses of laccase effectors during fungal cultivation was tested 22. The possibility to enhance laccase efficiency by the incubation with high dilutions of synthetic mediators is now presented. These experiments are based on our earlier results with changes of plant peroxidase activity in the presence of very low doses of selected phenolics 2324. The oscillating character of the mentioned changes allowed us to indicate the dilutions of the tested aromatic substances which reveal a maximum and a minimum effect on enzymatic activity. These changes had been tested using spectrophotometrical and luminometrical methods 25. In the present study the technique of electrophoresis (PAGE) of native extracellular laccase from cultures of T. versicolor and C. unicolor was used to compare of isozymic patterns of laccase growing with or without low doses of mediators, ABTS and HBT.

The presented results of activation or inhibition of extracellular laccase in laboratory cultures under the influence of diluted synthetic mediators ABTS and HBT are in accordance with the action of the law of hormesis. This law describes the opposing biological effect of diluted effectors and concerns regulation of the speed of biological response to a given stimulus based on the phenomenon of feedback and allosteric properties of complex protein systems 37. During gradual dilution, the intensity of a biological effect oscillates in a sinusoidal manner in accordance with a characteristic hormetic curve, which changes dynamically in time. The universality of the oscillatory influence of high dilutions on processes taking place in human and animal organisms has been pointed out in a growing number of studies, for instance those cataloged in the years 2001-2008 by Calabrese 3738.

In the experiments on fungal laccase, studies of the influence of the individual mediator dilutions in the order in which they had been prepared made it possible to distinguish those which activated laccase from those which inhibited it, and the use of two fungal species additionally showed that each of them had an individual pattern of the sine curve. This is an important observation, from which it follows that the results obtained in the present study cannot be put directly into practice without constructing appropriate hormetic curves in the conditions of a given laboratory. Also, there are yet other species of industrial fungi that need to be studied in which selective use of highly diluted mediators may strengthen the ability to produce laccase. The studies presented here show that among the molar dilutions, a clearly activating effect was exerted by those of the order of picomoles, corresponding to the 100^-6 mol/l concentration of the mediator. Use of picogram, instead of gram amounts of a mediator in industry may lead to a significant reduction of industrial-scale use of mediators without lowering the effects of their action, which is promising not only on account of the general reduction of production costs but also because of a decreased pollution of the environment. It can also be mentioned that diluted mediators may act on fungal cultures without additional sterilization procedures if high percent ethanol is used in preparing the dilutions. These dependencies allow applying experimentally selected dilutions of fungal laccase mediators for intensification of oxidation processes during transformation of laccase substrates such as phenols and methoxyphenols.

The process of incubation of high-molecular-weight lignin polymers with laccase applied in the paper industry is long-lasting and its supplementation with diluted effectors may have a practical use in shortening the contact of wood with a fungus. In accordance with the results shown in Fig. 6, an activating concentration of ABTS which corresponds to dilution n = 6 is within the range of 100^-6 mol/l and is thus a picogram quantity. Even repeated supplementation with such a strongly-diluted effector will remain safe for the environment and will considerably decrease the costs of applying these useful laccase-activating compounds.

It seems that this remains true for both native laccase from in vitro cultures and for the pure enzyme, after its isolation and purification. PAGE data indicate that the effect of the diluted effectors reaches as far as the molecular level of cells, increasing the processes of isozyme production in the fungal cell. A well-visible strengthening of the bands of laccase activity, corresponding to the process of intensification of laccase induction by the most active dilutions, dependent on the type of mediator and kind of fungal strain, was observed during our study. We also noted a simultaneous augmentation of the pool of laccase secreted extracellularly from cells to the culture medium.

Although the double action of biologically active substances, dependent on the degree of dilution, has been confirmed in numerous experiments, this phenomenon has not received a complete explanation within the accepted scientific canons. Currently, more and more studies point to the possibility of explaining the biological effect of low doses by means of physical and chemical instruments 3940414243. Probably, in the future it will be possible to use those instruments to explain the phenomenon, described in this work, of reaction continued for dilutions greater than the Avogadro number. This phenomenon was already signaled earlier in the 1980s by French researchers 44 for human and animal organisms and has now also been demonstrated for plant material 45. Also similar results with fungal material or pure enzymes were presented earlier by us, and in all those experiments the conformity and repeatability of the results were very high. All these data seem to constitute a prelude to recognizing hormesis, so far considered in molecular categories, to be an initial stage of the dose-response dilution process which smoothly passes into the range of action of homeopathic dilutions greater than the Avogadro number. Although the phenomenon has not yet been explained by contemporary science (cf. 46), the growing number of common observations enforces the need to acknowledge the facts and the possibility of their practical implementation just as unfamiliarity with the scientific basis of fermentation did not prevent the Ancients from consuming large amounts of wine. The physico-chemical character of the discussed phenomenon seems important for the biology and biotechnology of fungal laccase because of its practical consequences in reducing the toxic effect of industrially applied laccase mediators on the environment.

During cultivation of two fungal strains, Trametes versicolor and Cerrena unicolor, in the presence of high diluted laccase mediators (ABTS, HBT), changes in the production and activity of extracellular laccase in the culture medium were observed. The profile of the activity had the character of a sine curve with distinct maxima and minima. These changes were also noticed in the enzymatic patterns after specific visualization of laccase activity on PAGE gel, and the correlation between maximum activities on the sine curve and the abundance of laccase-active protein bands was very distinct. The selected dilutions of the mediators caused similar changes in the demethylation abilities of pure laccase isolated from T. versicolor. All these results strongly bear out the new features of diluted substances against selected enzymes tested in in vitro and in vivo conditions. The biphasic role of high diluted low molecular effectors on the laccase system of fungal cells is best understood as an instance of hormesis, in which every substance after dilution can act in vivo either as an inhibitor or as an activator of a biological process dependent on the rate of dilution. As for laccase applications, the possibility of radically diminishing the amount of its mediators in industrial processes with simultaneous augmentation of its production during fungal cultivation is very real. For this reason, the mentioned results are very interesting both from the economic point of view and from the perspective of environmental protection against chemical pollution.

The presented study should be treated mainly as a report on the newly discovered ability of selected dilutions of commonly used mediators to change the activity of laccase. Practical application of these results in industrial processes will require from factories adaptation of this knowledge to their own experimental conditions.

The authors declare that they have no competing interests.

EM conceived of the study, and participated in its design and coordination. JKR carried out the PAGE assay and coordinate experiments. AJW participated in the sequence alignment. All authors read and approved the final manuscript.