Production and Characterization of Manganese Peroxidase (MnP) from Mutant and Native Strains of Coriolus versicolor

Abstract

Economical production of industrial enzymes in higher yields has been an area of intensive research interest for the last many decades. In recent years, scientists have made tremendous progress in enzyme biotechnology that has opened up new opportunities for enzymes in molecular biology as well as industrial applications. Fungi belong to a group of organisms with the ability to produce different types of enzymes. The importance of fungi is not limited to their native products; they are also useful in the development and commercialization of new products through the modern techniques of mutagenesis and molecular biology. Enzyme production in higher yields is important for any biomedical or industrial application of enzymes. White rot fungi are efficient lignin degrading microorganisms that produce high activities of ligninolytic enzymes, including lignin peroxidase, manganese peroxidase and laccase. Manganese peroxidase has got tremendous applications in biomass utilization, bioremediation, pulp and paper industry, food technology, nanobiotechnology and medicine. With increasing new biotechnological and industrial applications of manganese peroxidase there is dire need to search for new and hyperproduccing enzyme sources. The various techniques for developing hyperproducing fungal strains are radiation and chemical mutagenesis, and genetic engineering. Chemical mutagenesis offers the possibility of a wider and more economical technique for developing mutant strains for producing biocatalysts in industry, waste treatment, medicine, and in the development of bioprocess monitoring devices like biosensors. Keeping in view the above mentioned facts, a study was conducted to develop a hyperproducing mutants of Coriolus versicolor IBL-04 through chemical mutagenesis. Two chemical mutagens ethedium bromide (EB) and ethyle methansuphonate (EMS) were used for mutational work and hyperprucing mutants in each case were selected on basis of MnP production in solid state fermentation. The physical and nutritional parameters for the production of MnP by native and selected mutants were optimized through one-factor-at-a-time optimization stratergy. Physico-chemical culture conditions, such as pH, tempature, moisture levels, inoculum size, C:N ratio, mediators and metal ions were optimized and maximum MnP activities by mutant strains EB-60 and EMS-90 were 2796 U mL -1 and 3140 U mL -1 , repectivelty, as compared to 1635 U mL -1 MnP actvity from native strain. An increase of 3.2-fold in enzyme production after mutagenesis and optimization of various parameters was achieved as compared to native strain. The MnPs produced by native and mutant strains were 1.4-5.5 fold purified through ammonium sulphate precipitation, ion exchangechromatography and gel filtration. The purified MnPs from from mutant strains had the molecular masses of around 43 kDa on SDS-PAGE. The enzymes from native , EB-60 mutant and EMS-90 mutant strains of Coriolus versicolor were compared in terms of their pH & temperature optima and kinetic characteristics. The lower K M and higher V max values from selected mutant strains suggested that the MnPs from mutants were more efficient and stable as compared to MnP from the native strain. The results of this work demostrated that the random chemical mutagenesis of Coriolus versicolor IBL-04 significantly enhanced MnP production under optimized laboratory conditions. Higher activities and thermo-stabilities of MnPs from mutant strains suggest the potential of mutant strains for commercial scale MnP production for diverse industrial applications.