Production And Immobilization And Application Of Peroxidases Of Phanerochaete Chrysosporium

Lignin peroxidase(Lip)and manganese peroxidase(Mnp)are the main peroxidases of the white-rot fungus Phanerochaete chrysosporium. In this thesis,the peroxidase's prodution conditions by P.ch in N-limiting was studied.After simple purification,the two peroxidases were immobilized on polylurethane foams,then,the immobilized peroxidases was applied in asymmetric synthesis.Lip and Mnp have the similar optimal producing conditions by P.ch in N-limiting culture.The optimal temperature was respectively 37℃and 34℃during fungus growth and enzymes producing period;the optimal seed amount was 4×10⁷/flask;the optimal rotating speed was respectively 140rpm and 100rpm during fungus growth and enzymes producing period; the optimal concentration of Mn²⁺was 150ppm;the optimal volume was 100mL/500mL flask,after 48h,the optimal volume decanted was 50%; the optimal frequency of flushing pure O₂ was 4min/day.Under optimal conditions,the maximum Lip and Mnp activity was respectively 350U/L and 137U/L.Peroxidases were immobilized On polylurethane foams.Compared with the free enzyme,the immobilized enzyme were better in the thermal, pH and operational stability.The activity of the immobilized Lip was 1.45U/g(dry foam)and the Mnp 0.49U/g(dry foam).The recovery of enzyme activity were 34.79%and 27.78%respectively.The thermo-stabilities of the immobilized peroxidases were better than of the free enzyme,the loss of the Lip and Mnp were both under 20%at 50℃.The immobilized peroxidases were stable respectively in the range of pH 2.0～5.0 and pH3.0～6.5.Morever,the immobilized peroxidases had good operational stability,which could be repeatedly used for 6 times and the loss activity of them were under 3%.Asymmetric oxidation of thioanisole to sylfoxide catalyzed by immobilized peroxidases was studies.The configuration of product was S; the best organic co-solvent was acetonitrile;the optimal reaction temperature was 25℃;the optimal concentration of thioanisole was 0.8mmol;the optimal rotating speed was 140rpm;the optimal feeding amount of H₂O₂ was 0.4mL 0.12%.Under the optimal conditions,the conversion rate of the substrate was 34%,the yield of the sulfoxide was 30%and ee was 37%.