The Research On Optimization Of Fermentation Conditions For Manganese Peroxidase Production By Fomes Lignosus And Its Enzymological Character

In this dissertation, we focuse on the lignin degredation enzymes produced by Fomes lignosus. First of all, we optimized the culture conditions and used the fermentor cultivation to expend production.Then use salting chromatography and other technologies to purify Manganese Peroxidase (MnP) and do further study on it.The effect of different conditions on manganese peroxidase production by Fomes lignosus are studied in this paper.The culture conditions are optimized.The medium compositions are optimized with orthogonal experimental design.The optimal culture conditions are pH5, temperature 34℃, with in 150mL culture medium of 250mL shake flask, glucose:Peptone(c/c)20:1,Mn2+0.3mmol/L, ABTS 1.0mmol/L, and rotation rate 160r/min. The manganese peroxidase activity reaches 1042.76U/L. Comparing with single factor way to optimize culture,the activity of MnP has improved 18.4%.use the fermenting jar to add supplemental material feeing the Fomes lignosus. parameters such as the oxygen dissolving in the jar, rotation rate and the temperature all can be better controlled.The bacterium body density Improved obviously, the activity of MnP has reached 1042.76 U/L in swaying bottle training process, and the activity that in the jar fermenting trains can reach 1222.34 U/L, has improved 17.2%. dry weight of Fomes lignosus in Sway bottle training process maintains basically in 1.7 g/L, dry weight of Fomes lignosus in fermenting jar can reach 7.074 g/L, have a wide margin rise.Manganese peroxidase from F.lignosus has been purified by some steps:over concentration,10% PEG deposition, chromatography and SDS-PAGE. The two column chromatograph were DEAE-cellulose DE52 and Sephadex TM G-75. Finally two MnP isoenzymes (MnPI and MnPⅡ) were purified from the liquid cultivation of F.lignosus, with the yield was 2.4% and 3.6%. Both isoenzymes had approximate molecular weight as determined by SDS-PAGE,46.26kDa for MnPI and 44.41kDa for MnPⅡ. The characteristics of MnPI and MnPII were similarity. They have the same optium temperature 35℃and optimum pH 4.5. The activity of MnPⅠand MnPⅡwere stable from 20℃to 40℃, but they differ in stability of pH(MnPⅠwas below 5.0 and MnPⅡwas below 5.5). DMP was the substrate, the Km values of MnPⅠand MnPⅡto DMP were 40.5μmol/l and 33.9μmol/l respectively.