CHARACTERIZATION OF LIGNINOLYTIC ENZYMES PRODUCED BY SCHYZOPHYLLUM COMMUNE IN SOLID STATE CULTURES FOR INDUSTRIAL APPLICATIONS

Abstract

The potential of an indigenous white rot fungus Shyzophylum commune IBL-06 for the production of ligninolytic enzymes in solid state fermentation of banana stalk was investigated. The production process was further improved by optimizing some physical parameters (incubation time, moisture level, pH, temperature, inoculums size) and nutritional factors (carbon and nitrogen sources, carbon: nitrogen ratio, mediators and metal ions). By optimization of different parameters the maximum activities of enzyme synthesized by S. Commune IBL-06 were 3745 IU/mL of MnP, 2700 IU/mL of LiP and 345 IU/mL of Laccase after 3 days incubation at рH 4.5 and 35°C temperature with inoculum size, 3mL; moisture content, 60%; C: N ratio, 20:1(glucose and ammonium nitrate as carbon and nitrogen supplements), 1mM MnSO4 as mediator, 1mL and 1.25mM MgSO4 .7H2Oa, 1mL. The enzymes produced under optimum conditions were purified by (NH4SO4)2 precipitation, dialysis and Sephadex G-100 gel filtration chromatography. The purified enzymes were run on SDS-PAGE and characterized through kinetic studies. The purified MnP was a monomeric protein with mass of 40 kDa. The optimum pH and temperature for MnP were 5 and 40°C with 0.29 mM KM and 450mM/min Vmax using MnSO4 as substrate. The enzyme was activated by 1mM CuSO4 but was inhibited by CaCl2, EDTA, TEMED, β- Marcaptoethanol, AgNO3 and Pb(NO3)2. The molecular weight of purified LiP was 43KDa and it displayed a single band on SDS-PAGE. LiP showed optimum pH 5.0, optimum temperature, 35°C; KM, 0.5 mM and Vmax, 400 mM/min using varatryl alcohol as substrate. The enzyme was inhibited by CuSO4, MnSO4, CaCl2, EDTA, TEMED, β-Marcaptoethanol, AgNO3, Pb(NO3)2. Molecular mass of Laccase was 63 kDa and it had optimum рH 6.0, optimum temperature 40°C, KM value 0.25mM and Vmax 80mM/min using ABTS as substrate. The laccase activity was enhanced by 2mM CuSO4, and was inhibited by MnSO4, CaCl2, EDTA, TEMED, β-Marcaptoethanol, AgNO3, Pb(NO3)2. Crude ligninase extract decoulorized Novasol direct blue dye to 80%, followed by Novasol direct yellow dye to 60%, Novasol direct red to 38% and Novasol direct black to 37%. The effluent from Magna textile industry was maximally decolorized to 87% in 24 hours, followed by effluents from Crescent, Arzoo and Chenab textile industries. S. Commune IBL-06 produced high activities of MnP and LiP having higher catalytic activities as compared to most of the previously reported enzymes.