Fermentation, Purification And Characterization Of Laccase Produced By Marine-derived Pestalotiopsis Sp. J63

Laccase, containing coppers, is a kind of ligninolytic enzyme. It has a wide rage of substrates, so it can be applied to various kinds of industries. A fungus which has the ability to produce abundant laccases was isolated from the East China Sea. It was identified as Pestalotiopsis sp. and designated as J63. The submerged fermentation characters of Pestalotiopsis sp. J63were studied. One kind of laccase isozymes (Laccase1) was isolated and its enzyme properties were studied.Pestalotiopsis sp. J63could take good use of agricultural residues to produce laccases. Adding3g/L maltose,20g/L rice straw and0.09mM phenol to the culture could lead the laccase activity to5791.7U/L. Two laccase isozymes (Laccase1and Laccase2) were detected by Native-PAGE. Phenol, guaiacol and veratryl alcohol could enhance the laccase activity dramatically by both reinforcing the production of Laccase1and inducing the secretion of Laccase2. Salts could enhance laccase activity, especially that H3BO3, SrCCl2·6H2O, NaF and KBr could increase laccase activity for more than200%. The optimal laccase producing temperature was26℃. When the temperature decreased to18℃,655.6U/L laccase activity remained.An efficient purification procedure was established to separate and purify laccases from the culture broth of Pestalotiopsis sp. J63. Flocculation and ultrafiltration could remove the suspended substances and condense the culture. The appropriate conditions for DEAE Sepharose Fast Flow anion exchange chromatography were pH6.0NaH2PO4-Na2HPO4buffer,0.5ml/min flow rate and four-steps elution. The active fractions from DEAE-Sepharose Fast Flow column were collected to feed the Phenyl Sepharose Fast Flow column to perform the hydrophobic interaction chromatography. The final purification factor and recovery rate were14.54and15.27%. One single band (Laccase1) was observed by the SDS-PAGE and Native-PAGE. The molecular weight of Laccase1was52.4kDa.The optimal Laccase1catalytic temperature is60℃and Laccase1was also stable at60℃.46.5%laccase activity remained after incubating at60℃for180min. The optimal pH for Laccase1was3.0and it was stable from pH3.5to6.0. After36h incubation at such pH buffer, more than50%laccase activity remained. The effect of12kinds of salts on laccase production was detected. All of them could enhance laccase activity except FeSO4·7H20. ZnSO4·7H2O, KBr, KAl(SO4)2·12H2O and MnCl2·4H2O could increase laccase activity for more than10%. The Km and Vmax value of Laccase1toward substrate ABTS were104.5μM and5.34μM/min under the condition of pH4.5and20℃.