Cell-assisted synthesis of conducting polymer – polypyrrole – for the improvement of electric charge transfer through fungal cell wall
Graphical abstract
Introduction
Polyphenol oxidases belong to a group of copper-based proteins widely distributed on the phylogenetic scale from bacteria and fungi to higher plants and mammals [1]. The oxidation of aromatic compounds using molecular oxygen is the common feature of this enzyme group. Three types of catalytic activity are distinguished within: (1) o-diphenol:oxygen-oxidoreductase or catechol oxidase (EC 1.10.3.1); (2) p-diphenol: oxygen-oxidoreductase or laccase (EC 1.10.3.2); (3) monophenol monooxygenase or cresolase (EC 1.18.14.1) [2]. Several enzymes display both cresolase and catecholase activity leading to straightforward bioconversion of monophenols or o-diphenols to o-quinones, commonly known as tyrosinases, while p-diphenol oxidation is highly specific for laccases [3].
The structure of all known tyrosinases consists of a type III copper center, where two copper ions are antiferromagnetically coupled and each coordinated by three histidine ligands [4]. Laccases belong to the multi-copper or blue oxidases along with ascorbate oxidase and ceruloplasmin, possessing four copper atoms distributed between three types of spectroscopically different copper centers [5]. Type I or the blue copper center represents the primary redox site where the reducing substrate binds electrons, while at type II/type III copper cluster, molecular oxygen is reduced to water [6]. Thus, four successive electron transfer steps are required for a full catalytic cycle of laccase [7]. The type III copper center is the only common one between laccases and tyrosinases, however they possess an overlapping range of substrates. The main differences in substrate specificity are that only tyrosinase is capable of monophenol hydroxylation [8,9], while only laccase acts upon activated metoxyphenols, such as syringaldazine [10]. Often, these enzymes have been employed for sensitive determination of phenolic compounds in bioelectrochemistry or for bioremediation and biodegradation studies [[11], [12], [13]]. Organic chemistry found polyphenol oxidases effective for developing polymers and medical compounds [[14], [15], [16]]. Further, enzymatic preparation of conducting polymers [17,18], such as polyaniline [[19], [20], [21], [22]] and polypyrrole [[23], [24], [25]] catalyzed by multi-copper oxidases has been achieved. Conducting polymers (CPs) are important materials due to their intrinsic electric properties that have found many applications in the last decades [[26], [27], [28]]. Electrochemical and chemical methods of synthesis are well-established, however, biocatalytic methods are becoming increasingly researched because they meet concerns related to purity and biocompatibility of the final product in respect to biomedical applications, while fulfilling the requirements of ‘green’ chemistry [29,30].
Several other oxidases were involved in the preparation of CPs, mainly peroxidases (e.g. horseradish peroxidase [31], palm tree peroxidase [32], soybean peroxidase [33]) or glucose oxidase [34,35]. The distinction in using phenol oxidases relates to the facility of employing molecular oxygen as oxidant without requirement for additional substrates.
Nonetheless, enzymes are a relatively costly and fragile subclass of biomolecules, subject to inactivation or denaturation due to environmental factors. The potential for biosynthesis of CPs without being restricted to pure enzymes is of increased significance as a wide range of biological components are available such as tissues and whole cells, which have been employed extensively in biosensors and/or biofuel cells [36]. In this respect, enzyme performance can be improved by ensuring a natural environment for catalysis. Although several polymers can be prepared via enzymatic catalysis, the preparation of conducting polymers using microorganism cells as enzyme source is in its preliminary stages [37,38]. In the present work, we propose a ‘green’ pyrrole polymerization/oligomerization method based on the catalytic action of white-rot fungal cultures.
Trametes spp. are remarkable producers of phenoloxidases, highlighted as model green catalysts in literature data on bioremediation [39,40] and bioelectrocatalysis [41,42]. Two Trametes spp. strains: Trametes versicolor (TV) and Trametes pubescens (TP) were selected for this study and cultivated under submerged aerobe conditions. Since polyphenol oxidases can be biosynthesized either intra- or extracellularly, both whole cell cultures and cell-free culture filtrates (supernatants) were used as polymerization bulk solutions. The advantages of the proposed method are multiple: enzyme purification and the susceptibility for enzyme inactivation are subsided whilst a multi-enzyme complex is available to initiate pyrrole oxidation. The use of crude laccase extract from Trametes versicolor (TV) for the synthesis of pharmacological compounds was examined previously in comparison with pure enzyme and better performance was reported when crude extracts were used [43]. This was justified by the production of natural mediators along with polyphenol oxidases within the fungal culture. Since several reports of laccase-catalyzed pyrrole polymerization are available [23,25], it is certainly interesting to use crude enzyme extract as polymerization media. Moreover, whole cell biocatalysis is available for comparison. The entrapment of the copper-oxidases responsible for pyrrole oxidation within the polymeric matrix is highly favorable as extensive amount of research focused on enzyme immobilization within polypyrrole has been reported [44]. Moreover, several electrochemical applications, such as biofuel cells and biosensors based on microbial components would benefit by the association with polypyrrole [45,46].
In this study, the primary observations available upon pyrrole addition in the white-rot fungal cultures will be provided. The enzymatic factors responsible for inducing the bioprocess were assessed. Spectral and electrochemical monitoring of the polymerization/oligomerization process along with microscopic and infrared characterization of the final product are onward described.
Section snippets
Chemicals
All used chemicals were purchased from Sigma-Aldrich or other commercials suppliers and were used without any further purification.
Microorganisms strains and biological conditions
Firstly, three white-rot fungal strains were tested to evaluate their ability for initiating pyrrole polymerization/oligomerization. The strains tested were: Trametes versicolor (TV), Trametes pubescens (TP), Trametes hirsuta (TH) acquired from the Cultures Collection of Faculty of Biology, Alexandru Ioan Cuza University of Iasi, Romania. They are preserved by
Analysis of polymerization media
Several laccases from wood rotting sources such as Trametes spp. have been purified and characterized and the crystal structure of a Trametes versicolor laccase was elucidated [55,56]. However, within the fungal culture, it is difficult to distinguish laccase activity from a background of monophenol oxidase and peroxidase. The strains that proved the ability for pyrrole bioconversion in current research, Trametes versicolor (TV) and Trametes pubescens (TP) displayed high enzyme activity for
Conclusions
Preliminary investigations of the pyrrole polymerization/oligomerization process within cell-containing fungal cultures were provided. White-rot fungal strains belonging to Trametes spp. were cultivated in submerged aerobe conditions and the cell cultures were tested as polymerization bulk solutions for the pyrrole monomer. Two out of three strains proved effective in initiating the pyrrole polymerization and the microorganisms cultures, weather in the presence or absence of cells, provided a
Acknowledgements
AR and AR acknowledges that this research is funded by the European Regional Development Fund according to the supported activity ‘Research Projects Implemented by World-class Researcher Groups’ under Measure No. 01.2.2-LMT-K-718. Support from COST Action M1407 is also acknowledged.
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