Purification, Characterization and Immobilization of Lignin Peroxidase Produced by Ganoderma lucidum for Industrial Applications

Abstract

Microorganisms are commonly used in biotechnological and environmental processes through exploitation of their natural catalytic activities. An indigenous white rot fungus Ganoderma lucidum IBL-05 was used for Lignin peroxidase (LiP) production using wheat straw as lignocellulosic substrate in solid state fermentation (SSF). The SSF process for LiP production was improved by optimizing some physical and nutritional parameters at pre-optimized pH 4.5 and temperature 35 o C. By optimization different physical and nutritional factors, LiP production by the fungus was substantially enhanced and the maximum LiP activity (1019.6 IU/mL) was obtained after 48h with Medium III, 60% moisture level, 5mL inoculum size, glucose as and urea as a carbon and nitrogen source in 15:1 C/N ratio, 1mL of 2mM Zn 2+ as metal ion, 1mL of 4mM 4-MMA as LiP mediator. Surfactants like Tween-80, Tween-20 and SDS suppressed LiP synthesis by G. luucidum IBL-05. LiP produced under optimum conditions was purified by 80% of ammonium sulphate precipitation, dialysis, ion exchange chromatography and Sephadex G-200 gel filtration chromatography to 6.4 fold to get 16.8 % yield and 2860.049 U/mg specific activity. The purified LiP was run on SDS-PAGE to determine its molecular organization and molecular mass. The presence of two bands (47 kDa and 28 kDa) on SDS-PAGE confirmed that the enzyme was an oligomeric protein composed of two polypeptide chains. Immobilization of LiP using xerogels of different hydrophobicity enhanced its activity as compared to free LiP. The enzyme entrapped in 1:5 P/T ratio gel was the most active. Immobilized and free. The immobilized LiP showed optimum pH 3 and optimum temperature 55 o C while free LiP showed the same optimum pH 3 but optimum temperature was 40 o C. Immobilized LiP was more thermostable as compared to free LiP. The Km values for free and immobilized LiP were 0.83 and 0.5 mM, respectively and their respective V max values were 545.6 and 639.7 mM/min, respectively. Immobilized LiP was more efficient decolorizer of selected textile dyes and practical textile industry effluents.