Study On Manganese Peroxidase Of Phanerochaete Chrysosporium

Extracellular manganese peroxidase which is secret by white-rot Basidiomycetes is a major component of the extracellular lignin-degrading systems. Manganese peroxidase from a model strain of the white-rot Basidiomycetes-Phanerochaete Chrysosporium is a H2O2-requiring heme glycoprotein of Mr=4.5 ×104-4.7×104Dalton.The main purpose of this dissertation lies in establishing the optimum culture components and the optimum fermentation conditions that Phanerochaete Chrysosporium produce manganese peroxidase,and lies in that clone of manganese peroxidase was acquired by gene engineering means.the groundwork that manganese peroxidase albumen hetero-source expression in fitting fungal expression system was established. The mainly researching contents of the dissertation include two aspects as blow:1 .The most agreeable conditions that Phanerochaete Chrysosporium produce manganese peroxidase was established.Under static culture condition, the optimum culture components by optimizing culture which produce manganese peroxidase showed as blow(L-1):Glucose 10g, ammonium tartrate 2mmol, Tween 80 1g, acetate buffer (pH4.0) lOmmol, VB1 1mg , Mn2+ 9.9×10~6 g. The culture temperature was 34℃.Under above conditions,the strain produced MnP activity up to 1200 U/L during 5 days stationary culture ,which increased almost 17 times than that of initially fermentation culture.The most agreeable culture condition which produce manganese peroxidase was optimized farther. The volume of liquid culture is 30mL(250 mL Erlenmeyer flasks),the pH of the fementable limiting nitrogen culture is 4.5,the inoculation volume is 6.7×105 entries spores per milliliter,the culture temperature was 37 ℃ .Under above conditions,the strain produced MnP activity up to 1452U/L during 5 days stationary culture, which increased almost 20% than that of initially fermentation condition.2. Molecular clone of cDNA encoding manganese peroxidase (MnP2). total RNA of 5.776 was withdrawed from the fungi silk which is educated in the most agreeable conditions and RT-PCR was proceeded regarding it as template, cDNA encoding specialized fragment whose size is about 1.3kb was amplified successfully just as anticipation.Amplified produut was extracellularly recombined with pUC19 vector after purification,and was transformed into E. coli JM109,then,the MnP2 gene cloning was obtained after screening and enzyme-cut identify.The analysis through SDS-PAGE showed that recombination plasmidpUC19-mnp2/JM109 have a more albumen fragment than that of volume plasmid pUC19/JM109 and its size is about 4.5 X 104Dalton,the same as that of manganese peroxidase(MnP2) monomer come from Phanerochaete Chrysosporium which proved clone is success.