作　　者： ; ; ; ;

机构地区： 华南理工大学生物科学与工程学院

出　　处： 《生物化学与生物物理进展》 2010年第2期200-207,共8页

摘　　要： 为了获得高效的脂肪酶毕赤酵母表面展示系统,利用来自酿酒酵母絮凝素蛋白Flo1的N端874个氨基酸残基(FS)和C端的1101个氨基酸残基(FL)作为锚定蛋白分别构建了2套载体系统.带有前肽的米黑根毛霉脂肪酶(ProRML)克隆到构建的2套展示载体中,使米黑根毛霉脂肪酶(RML)分别以N端锚定或C端锚定的方式实现在毕赤酵母细胞表面的展示.利用RMLC端的Flag标签,通过流式细胞术和激光扫描共聚焦显微镜检测2套系统中RML在酵母表面的展示情况.研究发现,N端锚定于酵母表面的展示酶FSR以pNPC为底物时,水解活力达到了105.3U/g,大约为C端锚定的展示酶FLR活力的2倍.同时FSR比FLR具有更宽的温度、pH作用范围和更好的热稳定性.与游离酶和固定化酶相比,展示酶FSR也表现出更为优良的热稳定性.结果提示,基于Flo1N端锚定的展示系统更适合展示活性中心近C端的脂肪酶,推动了展示酶的进一步研究和开发. To obtain a high efficiency Pichia pastoris cell surface display system,two new systems based on two different anchor proteins derived from Saccharomyces cerevisiae Flo1 protein（Flo1p） were constructed respectively.The N-terminal anchor system could make the foreign lipase displayed on the P.pastoris cell surface with its C terminus free by fusion with an anchor protein containing N-terminal flocculation functional domain of Flo1p（874 residues,FS）,which was able to adhere to the cell surface via noncovalent interaction with the mannan chain of the cell wall.Conversely,the foreign lipase can kept its N terminus free in another C-terminal anchor system,which utilizes a GPI-attachment signal domain located at C-terminal region of Flo1p（1 101 residues,FL） as anchor protein.Using these systems above,recombinant R.miehei lipase with a pro region（ProRML）,which had its active site near the C-terminus,was displayed on the P.pastoris cell surface,and two surface-displayed RML,named as FSR and FLR,were obtained.Cell-surface display of the RML via Fs or FL anchor system was confirmed by flow cytometer and laser scanning confocal microscope.A strong fluorescence was clearly observed in recombinant yeast cells harboring pKFSR（pKFS-RML）,but no fluorescence was detected in the yeast cells harboring pKFLR（pKFL-RML）.The hydrolytic activity of FSR reached 105.3 U/g·[dry cell weight] with p-Nitrophenyl caprylate（pNPC） as the substrate,which is 2 times as high as that of FLR.In addition,the cell-surface display systems based on FS or FL endowed the displayed RML with different enzymatic properties.The surface-displayed RML with its C-terminus free（FSR） showed a better catalytic performance at temperature,pH and thermostability than the surface-displayed RML with its N-terminus free（FLR） did.The results suggest that the surface display of RML based on FS anchor system is more promising and more effective,especially for N-terminal immobilization of target enzyme whose catalytic site is near the C te

关 键 词： 酵母表面展示 毕赤酵母 米黑根毛霉脂肪酶 端锚定系统 端锚定系统

领　　域： [生物学] [生物学]