Proximate analysis of lignocellulosic biomass and its utilization for production, purification and characterization of ligninolytic enzymes by Aspergillus flavus

Ligninolytic enzymes are employed for the production of second-generation biofuel to minimize fuel crisis. Additionally, they play a crucial role in global carbon cycle and a variety of applications in food, agriculture, paper and textile industries. On a large scale production of ligninolytic enzymes, microorganisms can be cultured on lignocellulosic wastes. In the present study, proximate analysis including acid detergent lignin (ADL), acid detergent cellulose (ADC), acid detergent fiber (ADF) and acid insoluble ash (AIA) were performed for Platanus orientalis (chinar), Bauhinia variegata (orchid tree), Pinus roxburghii (chir pine), wheat straw and wheat husk. Platanus orientalis was selected as substrate because of higher lignin contents for the production of ligninolytic enzymes by Aspergillus flavus. Solid State Fermentation was used and Response Surface Methodology was employed for optimizing various parameters and enzymes production. Maximum production was achieved at temperature 32 degrees C, fermentation period 120 hours, pH 4.5, inoculums size 3.5 mL, substrate mesh size 80 mm, substrate size 7 g. Maximum purification of laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) was achieved with 50%, 60% and 40% ammonium sulfate respectively and it was enhanced by gel filtration chromatography. Characterization of enzymes shows that Laccase has 35 degrees C optimum temperature, 4.5 pH, 0.289 mM Km and 227.27 mu M/ml Vmax. Manganese peroxidase has 30 degrees C optimum temperature, 5.5 pH, 0.538 mM Km and 203.08 mu M/ml Vmax. Lignin peroxidase has 30 degrees C optimum temperature, 3 pH, 2 mM Km and 2000 uM/ml Vmax. Protein concentrations found in crude extracts and partially purified enzymes are respectively: laccase 1.78 and 0.71 mg/ml, MnP 1.59 and 0.68 mg/ml. LiP, 1.70 and 0.69 mg/ml.