The Optimization Production, Purification And Dye Decolorization Of Manganese Peroxidase From Wood-degrading Fungus Lenzites Gibbosa

Manganese peroxidase (MnP; EC1.11.1.13) a H2O2dependent heme sugar protease is a common and efficient peroxidase for degradating lignin widely exists in the white rot fungus. Recently, the research at home and abroad on MnP mostly concentrates on the basic research and molecular biology, and the research on purification of MnP is confined to a few kinds of white rot fungus such as Phanerochaete chrysosporiu, Trametes versicolor, Ceriporiopsis subvermispora etc., so using high yield strains to explore new purification method of MnP is practical and significant to improve its application and production. Lenzites gibbosa a MnP high yield strain was used as the material in our research which aims to improve the production and purity of MnP and to provide a strong technical support for its large-scale producing and basic application in the future.In the research, L. gibbosa was cultured statically at30℃in medium LNAS contaning Mn2+and poplar sawdust,which was set as the initial culture condition, then two times orthogonal and several single-factor tests were conducted to optimize medium compositions and culture condition of L. gibbosa for producing MnP. Results indicated that the most optimal culture condition within the fixed factors and levels was fructose5g/L, Mn2+200μmol/L tartaric acid ammonium15m mol/L, Tween-800.25mL/L, MgSO4·7H2O0.35g/L, mineral solution lOmL/L, vitamin solution1mL/L and pH4.5in the improved LNAS medium; culture temperature27℃; and110mL culture solution volume at150r/min shaking condition, and the produced manganese peroxidase activity could reach to314.52U/L, which was8.4times higher than that produced at initializing culture condition. MnP yield produced by L. gibbosa could be enhanced in a great deal in the optimizing culture condition.The decolorization effect of culture fluid of L. gibbosa upon4different kinds of dyes alizarin red, neutral red, Congo red and crystal violet were studied respectively. The result showed that decolorization efficiency of L. gibbosa on Alizarin Red (anthraquinone) was100%in1hour which mean that the decolorization on Alizarin Red was thorough and rapidly; decolorization efficency for neutral red and Congo red were22.17%and19%in1hour,33.63%and25.09%in36hours respectively, which indicate the degradation on these two dyes were poor; decolorization efficiency for crystal violet was only0.018%in36hours mean that L. gibbosa has no effect almost on violet. The results above show that L. gibbosa has a strong ability decolorizing anthraquinone compounds.Salt fractionation method, ion exchange column chromatography, ultrafiltration. gel filtration chromatography, poly water absorption method, freeze-drying method and some other methods were used for purification of MnP. Some of these methods were optimized to make it suitable for specific purification of MnP. A single MnP protein band was detected in the SDS-PAGE electrophoresis which indicates that the MnP has been purified basically.The enzymatic properties of MnP, including the determination of molecular weight, the optimal reaction temperature, thermal stability, the MnP preservation, the optimal reaction pH, pH stability, determination of kinetic constants and amino acid sequencing etc. were studied respectively. The results showed that the apparent molecular weight of MnP is45.39kDa;the optimum reaction temperature is about35℃,and in temperature4℃-40℃the enzyme has good catalytic stability; the optimum reaction PH is3.5,and in PH2-3the enzyme activity is most stable when it can be kept for more than10hours; the Km of MnP at21℃with2,6-DMP as substrate is6.124mmol; two conserved amino acid sequences of MnP were found in the samples by use of protein sequencing analysis and the sequences are LTFHDAIGISPK and TVPEPFDTVDSILAR. The retrieval results from the NR database confirmed that the purified enzyme is MnP from T. gibbosa indeed and it is possible that LiP may be secreted by T. gibbosa too.The decolorization ability of the purified MnP enzyme solution on four dyes were studied. In this experiment the degradation system refered to the method of determination of enzyme activity, and2control groups were set:one is the replacement of enzyme solution with buffer solution and the other is the replacement of H2O2with buffer solution. All of these reactions were set in the cuvettes, and the dye concentration and decolorization rate were calculated finally:the dye decolorization efficiency of purified MnP upon4kinds of dyes were96.29%for azo dye Congo red,100%for heterocyclic dye neutral red,51.82%for three phenyl methane crystal violet and77.51%for anthraquinone alizarin red respectively, and the conclusion was that purified enzyme had a better decolorization efficiency with the assistance of H2O2, and the decolorizing time was within2hours.