Purification Of A New Manganese Peroxidase Of The White-rot Fungus Schizophyllum Sp. F17, And Decolorization Of Azo Dyes By The Enzyme

White-rot fungus Schizophyllum sp. F17 was isolated from a decayed wood chip pile in the vicinity of Hefei, China. The fungus was very effective to decolorize a wide range of structurally diverse synthetic dyes including azo, anthraquinone, heterocyclic, and triphenylmethane dyes. The aim of this work was to isolate and immobilize manganese peroxidase(MnP), produced by Schizophyllum sp. F17 as the major ligninolytic enzyme on pinewood solid-state cultures, to detect the potential of MnP to decolorize different azo dye structures, which could help to elucidate the biochemical mechanisms of dye decolorization by fungi.(1) The crude enzyme was concentrated by ultrafiltration, then fractionation of manganese peroxidase was performed by Sephadex G-75 gel filtration chromatography followed by DEAE-Cellulose anion exchange chromatography. This purification attained 3.8 % activity yield with a purification factor of 9.2. According to data on sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE), the molecular weight of the enzyme was 48.7 kDa. The isoelectric point of the purified MnP is 3.60. The enzyme is a glycoprotein with 18% carbohydrate content which was estimated by the phenol sulfuric acid method using dextroglucose as the standard. The N-terminal amino acid sequence of MnP was VTCPDGVNTATNAAA. The optimum pH and temperature of purified MnP for oxidation of DMP were 6.8 and 35℃, respectively. This enzyme was stable in the pH range 4.0-7.0, at 25℃for 1 h incubation period. The K_m values of MnP for Mn²⁺, H₂O₂, guaiacol, ABTS and DMP were 35.2, 6.7, 13.1, 47.8 and 122.9μmol/L at pH 4.5, respectively. The effect of different chemicals on MnP activity was tested. Among the heavy metal ions tested, only Ag⁺ caused a strong inhibition. No activity loss was observed with MnP for these incubation periods with Cu²⁺, Ba²⁺, Zn²⁺, Pb²⁺, and Co²⁺. Our examination indicated that Fe³⁺ and Fe²⁺ at a concentration of 0.1 mM did not significantly affect MnP activity, but at 1.0 mM concentration displayed inhibitory properties towards our MnP. Among other metal ions tested, MnP was also resistant to presence of up to 1.0 mM Na⁺, but it was inhibited in the presence of 10 mM Na⁺. Mg²⁺, Al³⁺ and K⁺ did not cause any alteration in the activity. The enzyme showed a high percentage of activity in reaction mixtures containing of 10 mM Mn²⁺ and Ca²⁺. MnP activity was almost completely inhibited by dithiothreitol(DTT) and potassium ferro cyanide at the indicated concentrations. Partial inhibition was observed using SDS, NaN₃, EDTA, and thiourea. The azo dyes were efficiently decolorized using the MnP purified. The initial degradation rates were 7.74, 1.61 and 1.38 mg L^-1 min^-1 for Congo Red, Orange G and Orangeâ…£, respectively.(2) With sodium alginate, gelatin, or chitosan as a carrier, and glutaraldehyde as crosslinking agent, MnP was co-immobilized by embed-crosslinked method and adsorb-crosslinked method. The immobilization conditions and partial properties of the three immobilized enzymes were investigated. The optimum conditions of the 3 immobilizations of MnP were: sodium alginate embed-crosslinked immobilization(the sodium alginate concentration was 1%, the glutaraldehyde concentration was 3%, and the CaCl₂ concentration was 0.1 mol/L); gelatin adsorb-crosslinked immobilization(the ratio of the gelatin particles and the enzyme solution was 1 g/l mL, the glutaraldehyde concentration was 0.2%, and the cross-linking time was 10 min); Chitosan adsorb-crosslinked immobilization 3% chitosan was used to form the beads, the glutaraldehyde concentration was 0.2%, the ratio of the chitosan beads and the enzyme solution was 1.4 g/l mL, and the cross-linking time was 70 min). The activity recovery rate of the 3 immobilized enzymes reached 70%, 67%, and 64%, respectively. Compared with the free enzyme, the optimum pH values and temperatures of the three immobilized MnPs carried by alginate, gelatin, and chitosan were respectively shifted from 7.0 to 5.0, 5.0, 3.0 and from 35℃to 75℃, 55℃, 75℃.The thermostabilities of the three immobilized MnPs was much better than native enzyme. The chitosan-immobilized enzyme was stable in the wide range of pH 2.2 to pH 11. The enzyme activities of the three immobilized MnPs decreased less than 5% even after repeated use for 6-9 times. The ability of decolourizing azo dyes in static and shaky situation by gelatin-immobilized MnP approached to the free enzyme, and there was no loss of enzyme activity during 2 repeated batch reaction.