Optimization Of Rhizoctonia Sp. SYBC-M3 Fermentation For Manganese Peroxidase Production And The Enzyme Characterization

Manganese peroxidase (MnP) is one of the key enzymes on lignin degradation. MnP can oxidize a variety of aromatic substrates. MnP have been researched and applicated in the pulp biobleaching and other aspects. However, the synthesis of MnP now seems to be limited to basidiomycete, and it still has been in the laboratory stage. So, to screen MnP high-producing strains and study on their Properties, it is meaningful for increasing strain resources and the potential application.A fungus were screened in producing MnP from nature. Through morphological and molecular identification, it was classified as Rhizoctonia, and it was named as Rhizoctonia sp. SYBC-M3.The effect of different carbons, nitrogens, surfactants and Mn²⁺ on the MnP production of Rhizoctonia sp. SYBC-M3 was investigated. A single-factor method and orthogonal experiment has been used to optimize the medium of Rhizoctonia sp. SYBC-M3. The optimized mediu was: soybean meal 20 g/L, peptone 15 g/L, Tween-20 0.5 g/L, Mn²⁺ 1.5 mmol/L, initial pH 4.5. The MnP activity reached a higher degree, up to 2759 U/L. The crude enzyme had a optimal temperature of 55℃, and a optimum pH of 5.0. Its temperature and pH stability were pretty good. It still had a high activity by maintaining in pH 9.0 for 24 h.MnP was purified by ammonium sulfate precipitation, DEAE-32 chromatography and Sephadex G-100 gel filtration. MnP was purified 12.6 folds. Its recovery rate was 38.64%. the optimal pH of the MnP in 50 mmol/Lmalonate buffer was 4.5. It showed high MnP activity from pH 4.0 to 7.0. The optimal catalytic temperature of MnP was 55℃; MnP showed stable activity in the following 50℃. It quickly deactived at 60℃. Zn²⁺, Ca²⁺ and Mg²⁺ promoted the reactive rate of the enzyme, but Fe³⁺, Cu²⁺ and Co³⁺ restrained the reaction. Km value and Vmax of the two substrates, H₂O₂ and Mn²⁺, was determined: Km value of H₂O₂ was 25.3μmol/L; Vmax of H₂O₂ was 537 U/mg protein; Km value of Mn²⁺ was 53.9μmol/L; Vmax of Mn²⁺ was 748 U/mg protein. It showed MnP activity when ABTS, DMP, veratryl alcohol and guaiacol were used as substrates.