High-Level Expression And Purification And Partial Characterization Of Coprinus Cinereus Peroxidase In Pichia Pastoris

Coprinus cinereus peroxidase(CiP) is a type II peroxidase secreted by Basidiomycetes, which have a broad application prospects in industrial wastewater treatment and enzyme immunoassay reactions.CiP was successfully expressed by Pichia pastoris in this experiment, and by integrating chaperones and 5 L fermenter condition optimization to further improve the expression of the target protein, finally, a preliminary separation and purification and some enzymatic properties were researched to the target protein,the main conclusions are as follows:1.CiP was successfully expressed by Pichia pastoris. After cultivation by shake flask, the protein analysised by SDS-PAGE, and the target band identified as CiP by mass spectrometry.Enzyme activity reached 867 U/mL and protein concentration was 2.1 g/L after 5 L cultivation by 5 L fermentor. The endoplasmic reticulum chaperone oxidoreductase 1 (Erol) and disulfide isomerase (PDI) were separately and simultaneously integrated into the X33/pPIC9k-CIP, a separate integration of Erol will reduce the expression of CiP, while the integration of PDI alone and Erol and PDI together will increase the expression of CiP, the enzyme activity increased 2.43,2.62 times higher than the blank after shake flasks cultivation,while the enzyme activity increased 1.94,3.90 times higher and specifity activity increased 1.21,1.46 times higher after cultivation by 5 L fermentor,respectively. Finally the protein concentration reached 4.4 g/L and enzyme activity reached 3379 U/mL by 5 L fermentor when Erol and PDI were intergrated the X33/pPIC9k-CIP together.2.The fermentation process were optimized in 5 L fermentor by the strain X33/pPIC9k-CIP which co-expression of the chaperones Erol and PDI. The experimental results showed that when mentanol and sorbitol mixed to 20:1(w/w), pH 6.0, temperature at 28℃ during the induction, the enzyme activity reached 3963 U/mL, compared to 1200 U/mL have been reported, which is the highest.3.The precedure of the separation and purification of CiP were establishment, the broth centrifugated at 8000 r/min by 5 min, then desalted by 10 kDa molecular weight dialysis bag, Sephadex G25 desalting column to further desalting, finally, DEAE Sepharose Fast Flow column ion-ion exchange chromatography, the enzymatic activity was 50% recoveryed thoughout the purification process. After analyzed by SDS-PAGE,meeting the requirement of study of pharmacokinetics. In the aspect of CiP characterization, the potimum pH was 4.5, and the optimum reaction temperature was 25℃.