重组尿酸酶发酵培养及制备的工艺条件

Study on Fermentation and Preparation of Recombinant Uricase

摘要:

利用含PET-Urate Oxidase表达质粒的重组E.coli JM109(DE3)菌株进行大规模发酵培养,拟建立一种高效、低成本的生产重组尿酸酶的技术工艺路线。取冻存的工程菌接种于含质量分数1%琼脂的种子培养基中进行培养,从划线平板上挑取单菌落接种于摇瓶中继续培养,最后以体积分数5%接种量接种于发酵培养基中,绘制生长曲线;以乳糖作为诱导剂与传统IPTG诱导方法进行发酵培养比较;菌体经高压均质机破碎、质量分数40%~60%硫酸铵分级沉淀,以及Q-Sepharose F.F.层析进行分离纯化。结果显示,乳糖作为诱导剂在300 L的发酵罐中进行培养可获得菌体湿质量2 700 g,目的蛋白质表达量占菌体可溶性蛋白质的30%左右,略高于IPTG诱导的效果;经分离纯化后该酶的纯化倍数可达原来的4.5倍,活性回收率为40%;纯化的重组尿酸酶经SDS-PAGE和HPLC分析,显示单一蛋白质色带和单一洗脱峰。成果为该酶的医学实际应用奠定了理论实验基础。

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.