Studies On The Properties Of Manganese Peroxidase Of Schizophyllum Sp. F17, And Optimization Of Process Variables For The Enzyme

White-rot fungus manganese peroxidase (MnP) is one of the extracellular glycolsylated heme proteins produced by fugus which has remarkable potential for degrading and decolorizing azo dyes. This study was carried out by manganese peroxidase(MnP) which was obtained by a certain white-rot fungus Schizophyllum sp. F17. The Schizophyllum sp. F17 medium was solid-state fermentation selecting rice hull. The enzyme was purified and the properties of this MnP was also learned in this work. Response surface methodology (RSM) here was conducted in order to optimize this catalytic process of the MnP. The work began to explore the clone of this mnp gene.(1) Studies on the properties of manganese peroxidase.The MnP in this study was isolated and purified from Schizophyllum sp.F17. The crude enzyme was concentrated by ultraliltration, then fractionation of MnP was performed Sephadex G-75 gel filtration chromatography followed by DEAE-cellulose anion exchange chromatography. The protein concentration of this purification was 23μg/ml. The optimum pH of purified MnP was pH 4.5 as the Mn2+ was the substrate. The half-life of the MnP in the presence of 0.1 mM H2O2 was 4-5 min. The Km values of MnP for Mn2+ and H2O2 were 13.1μM,5.2μM which means this enzyme has much better effect in the oxidation-reduction reaction.(2) Studies on process variables for the enzyme.There are three main factors that decide the MnP activity:concentrations of Mn2+, H2O2 and amounts of enzyme itself. By the one-factor-at-a-time analysis this study shows:As the Mn2+ concentration reached 1.2mM, the MnP had the maximum activity then slowly decreased over the concentration of 1.2mM. MnP showed its strong activity (more than 80% of the maximum) at a board concentration of H2O2 ranging from 0.1 to 0.2mM. The maximum was observed when the H2O2 was 0.1 mM, but decreased sharply at the concentration over 0.25mM. The optimum MnP dosage for the enzyme activity was 0.4ml, The activity increased with the increase in amount of purified enzyme used up to 0.4ml, however amounts above 0.4 ml decreased the MnP enzyme activity.The MnP catalytic cycle process is a complicated system. The three main factors that decide the enzyme activity of MnP are concentrations of H2O2, Mn2+and enzyme itself. It is important to analyze the factors as a whole and understand the combination interactions among these three factors. Based on the the one-factor-at-a-time analysis results, A two-level-three-full factorial Central Composite Design (CCD) was designed to assesses the influence of the three main factors on the MnP activity. The result shows that the concentration of H2O2 and the interaction between the H2O2 and MnP played the most important roles for the enzyme activity. Response surface methodology (RSM) was conducted by this design shows that in different concentrations of H2O2, the MnP had larger activity when the concentration of Mn2+ was higher; in different amounts of MnP, the MnP activity became larger when the model had more Mn2+. However, when the concentration of Mn2+ was low, the change of enzyme activity was significant if the amounts of enzyme increased; MnP activity could reach its maximum if the concentration of H2O2 should be near 0.15mM and amounts of enzyme should be 0.55ml.(3) Azo dye decolorization by MnP.Based on the conclusion of the RSM, in the 3ml reaction system,when the pH was 4.5, the concentration of Mn2+and H2O2 were 1.6mmol/L,0.15mmol/L, amounts of enzyme was 0.42ml, the the concentration of dye azo was 1mM, the decolorization for Orange G reached nearly 35% after 60 min of treatment as well as for the Congo red.(4) The gene clone of MnP. Seek MnP proteins sequence by NCBI, then compare different MnP protein consensus sequence in GeneBank database by the method of the BLAST. Choose the consensus sequences as follows:PFDSTP and GGGADGS. The corresponding degenerated primes sequence were 5' GGNGGNGGNGCNGAYGGNTC 3'and 5'NGGNGARTCRAANGG 3'. PCR was carried out with the degenerated primes of MnP and template of the genome of Schizophyllum sp. F17. The reaction system was:DNA template 1μl,10×PCR buffer 1.5μl,25mmol/L MgCl2 2.5μl, 10mmol/L dNTPs 2μl, upstream primeμl, downstream prime 1μl, Taq 0.5μl. The reaction condition was 94℃4min and the 94℃30s,55℃30s,72℃60s with 30 cycles, at last 72℃10min. A small DNA consensus sequence with 573bp was obtained.