Molecular Characterization of Microbial Oxidoreductases and their Application in Polymer Biodegradation

Abstract

The detoxification of xenobiotic organic compounds by various microorganism through oxidative coupling is facilitated with oxidoreductases. With the help of energy yielding biochemical reactions these microbes extract energy for their metabolic pathway. They promote the transfer of electrons from a reduced organic substrate to another chemical compound. Enzymatic bioremediation of toxic organic pollutant is the very effective strategy in complex environmental conditions. Oxidoreductases enzyme have a significant potential for the bioremediation of the xenobiotic compounds. Various electron donor complex polymeric substrates containing Phenol and aromatic amines oxidized by peroxidase in the presence of H2O2 while O2 in the case of dioxygenase. Fungi are important among microorganisms for production of extracellular peroxidases. The present study aimed for production and characterization of peroxidases (laccases and lignin peroxidases) and dioxygenase for polymer biodegradation. Fungal strains named as Nk-1, Nk-2, Nk-3 and Nk-4 were selected and processed for different optimization studies. NK- 1 was identified 18s rRNA sequencing as Phanerocheate chrysosporium was further selected for enzyme production. Maximum enzyme production was observed at temperature at 30°C, pH 5.5. Statistical analysis indicates the significance of model and components based on F value and P value <0.05. Purification of enzymes was done by column chromatography. The molecular weight estimation was carried out by Sodium dodecyl sulphate SDS. Fourier transform infrared (FTIR) spectroscopy of enzyme treated substrate revealed the structural changes as compared to control (without enzyme treatment). The decrease in weight (0.08g) was observed in enzymes treated film then control (0.1g). FTIR spectroscopy of enzyme treated plastic film revealed the structural changes as compared to control (without enzyme treatment) i.e. a new peak appeared at 3367 cm -1 (Alkenyl C-H stretch). The peak at wavelength 1633 cm-1 (C=C stretching vibration of aromatic ring) in treated sample was sharp and larger as compared to the control. A visible change in surface of PVC films in term of surface erosion was observed by Scanning Electron Microscopy (SEM) after enzyme treatment. Peroxidase enzymes have wide range of applications from biodegradation of recalcitrant compounds to paper and pulp, cosmetic, food and textile industry. Enhanced production from food waste as cheap substrate was studied. Biosynthesis of lignin peroxidase, manganese peroxidase and laccase enzymes from food waste as a substrate is possible and their production can be enhanced by optimizing different factors. The native way of synthesizing dioxygenase, laccase and lignin peroxidase are not enough to the current industrial demands mostly because of their small amount of yield and the high cost of purification or synthesis. Heterologous expression may allow the production of laccase and lignin peroxidase in higher amount and with desired characteristics. In the present research study, the heterologous expression of fungal lignin peroxidase and laccase were performed in the cells of DHα5 strain of E. coli bacteria. With the help of shake flask experiment, the enzyme assay for enzymes was performed and a variation in the enzyme activity was observed during incubation time. Molecular weight of lignin peroxidase and laccase were found to be 46 kDa and 66 kDa respectively, as determined by the SDS-PAGE analysis. The enhanced production of fungal lignin peroxidase and laccase can be achieved through heterologous expression in bacterial host so that the biodegradation of recalcitrant plastic waste can be speed up.