Study on Fermentation and Preparation of Recombinant Uricase

Abstract:

This manuscript was applied with recombinant plasmid PET-urate oxidase gene expressed from E.coli JM109(DE3) to study the conditions of fermentation and purification of recombinant urate oxidase. The freezing stored engineering bacteria were inoculated to seed culture medium which contained 1% agar for slant cultivation. We used lactose as inducer and fermentation was conducted in 300 L fermenter. Finally,2 700 g wet samples(1 200 U/g) were prepared and the aimed protein expression was about 30% proportions to soluble bacteria. After crushed by high-pressure quality machine,the ammonium sulphate precipitation from 40% to 60% saturation and Q-Sepharose FF ion-exchange chromatography,the purity of the obtained r-Urate Oxidase was up to 97% and 40% of final yield,as well as 30 U/mg of the specific activity. Additionally,the interference enzyme of catalase was also under standard conditions. The purified enzyme can be stored one year at 4 ℃ under sterile procedure and the activity can retain about 85%. The lyophilized powder can also be stored for one year and the activity have not been released. All results of this study could lay the theoretical foundation in medical application.