【作者】 冯涛；

【导师】 廖飞；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2016， 硕士

【摘要】 采用易错PCR(error-prone PCR,epPCR)技术对天然苛求芽孢杆菌胞内尿酸酶三段氨基酸区域(A1-V150;V150-D212;Q160-L322)对应编码基因进行体外随机突变,在不影响PCR产物生成量的前提下,通过增加反应体系中MnCl2浓度至1.5 mM,提高突变率至4.4%;通过epPCR反应后快速加入2.0 mM EDTA-Na2溶液消除高浓度Mn2+对连接效率的影响;采用同源重组克隆方法实现表达载体突变体库的构建。突变体单克隆接种于48孔细胞培养板进行细胞培养及诱导表达,诱导后细胞悬液以1:9体积比加至含1.0 M Tris-HCl(pH 9.0)缓冲液的96孔细胞培养板(含0.1%吐温-20,1.0 mM 4-氨基苯甲脒二磷酸盐缓冲液),室温下于微孔板快速振荡器中裂解7.5-10.5h;使用150μM底物在酶标仪上于298 nm检测尿酸酶反应过程;分析酶动力学过程确定裂解液中尿酸酶活性Vm/Km。动力学过程分析法确定尿酸酶活性Vm/Km对初始底物浓度不敏感,且定量上限为经典初速度法的3倍。基于所述高通量筛选系统,选取活性之比分别为1.8倍(天然尿酸酶VS L322D-SN-6His)和3.3倍(天然尿酸酶VS E2R/L322D-SN-6His)的两对尿酸酶突变体作为测试菌株,应用受试者工作曲线(Receiver Operation Curve,ROC)比较用Vm/Km识别活性提高一倍以上阳性突变体的可靠性。当活性之比为3.3时,对应碱裂解方法曲线下面积(Area Under Curve,AUC)为0.957;与经典超声裂解法相当(AUC=0.978)。将尤登指数最大时对应Vm/Km作为判断尿酸酶突变体活性提高1倍以上的参考阈值,相当于以模板尿酸酶活性均值加上3.6倍标准偏差。用所建立定向进化系统,对近400个单克隆高通量筛选,获得一株活性提高的阳性尿酸酶协同突变体L171I/Y182F/Y187F/A193S。此突变体活性较天然尿酸酶提高了约60%,但其生理条件下热稳定性较差,仅为天然酶的1/70。综上所述,所建立的定向进化系统可以用来进行苛求芽孢杆菌尿酸酶的体外定向分子改造筛选高活性突变体。更多还原

【Abstract】 For directed evolution of Bacillus fastidiosus uricase, Mutants were from three libraries(A1-V150; V150-D212; Q160-L322) generated by error-prone PCR. The mutation rate was raised to 4.4% with final 1.5 mM MnCl2. Final 2.0 mM EDTA-Na2 was used to alleviate the effects of Mn2+ on ligation efficiency.After induced expression in 48-well microplates, transformed Escherichia coli cells were lyzed by 1.0 M Tris-HCl buffer at pH 9.0 plus 0.1% tween-20 and 1.0 mM 4-aminobenzamidine in 96-well microplates at 25 °C for 7.5~10.5 h; uricase reaction was monitored with 0.15 mM uric acid in 96-well plates by absorbance at 298 nm, to estimate Vm/Km by kinetic analysis of reaction curve. Uricase Vm/Km was resistant to initial uric acid levels with an upper limit three fold over that of initial rates.To recognize the one of higher activity in a uricase pair whose ratio of specific activities was 1.8 or 3.3, the area-under-the-curve by receiver-operator-curve analysis was comparable to that with cell lysates prepared by sonication treatment. A cutoff for the maximum Youden index was developed to recognize positive mutants of one-fold higher activity.Indeed, one mutant bearing four mutations and higher activity was discovered from the library. Hence, the proposed system was practical for HTS of uricase mutants.更多还原