耐熱β-糖苷酶基因的重组与表达.doc

耐热β-糖苷酶基因的重组与表达 生物技术 200503030160 满光波 指导教师 杨雪鹏 摘 要 非解朊栖热菌HG102耐热β-糖苷酶具有广泛的应用，高效生产该酶制剂显得非常需要。基于上述目的，本课题运用聚合链式反应（PCR），从重组质粒pUC18-tβgly扩增出gly结构基因(1131bp)，经割胶回收、酶切（NdeI和EcoRI）和纯化，与经过相同酶切的高效表达载体pET28(a)连接，并在上游融合6个组氨酸序列；再经转化和鉴定，筛选出阳性重组质粒，命名为pET28-y；把重组质粒转入大肠杆菌BL21细胞，经诱导表达，酶活测定，筛选出表达β-糖苷酶活性的重组子；重组子细胞经优化培养和诱导，离心收集细胞，再经超声破碎、上清加热沉淀和镍柱亲和层析等分离纯化，最后制备出纯度约90%的耐热β-糖苷酶蛋白溶液，酶的发酵活力达150 u/ml。以上结果表明，本课题成功构建了高效表达耐热β-糖苷酶蛋白的重组菌，且重组蛋白易于纯化，这为工业化生产高纯度的糖苷酶打下坚实的基础，也为耐热糖苷酶的应用奠定了坚实的基础。 关键词：基因重组； 融合表达； 酶固定化 ABSTRACT The thermostable Beta-glycosidase of Thermus nonproteolyticus HG102 will be applied in many areas. Therefore, the production of the enzyme with high yield is completely needed. In this research, an approximately 1200 bp DNA encoding the Beta-glycosidase gene designated as y was amplified from recombinant plasmid of pUC18-tβgly by polymer chain reaction (PCR). The DNA was recovered and purified after enzyme digestion (EcoRI and NdeI), and sub-cloned into a bacterial expression vector of pET28(a) which also treated with EcoRI and NdeI resulting in a 6×His-Y fusion gene construction. The SDSelectrophoresis analysis showed that the recombinant protein was over expressed in Escherichia coli BL-21(DE-3). The activity 150 u/ml of the enzyme was achieved. The expressed fusion protein was found almost entirely in the soluble form in cell lysate. The recombinant protein was purified easy by heat precipitation and Ni-NTA colum. These results indicated this method produced a relately high yield of recombinant protein and offered the potential development of the enzyme. Key words: genetic recombination expression enzyme immobilization 一、概述 β-糖苷酶（β-glucosidase，EC3.2.1.21）亦称β-葡糖苷酶，能将各种烷基、芳香基、糖苷基为配质的β-D-葡糖苷进行水解的酶之总称。该种酶广泛存在于各类生物体，水解多种β-糖苷键，具有特殊的生物功能。 耐热酶最适温度从65-125 oC，在应用上有更大优越性，可以提高反应速度、减少杂菌污染、增强对化学试剂的耐受力。 耐热β-糖苷酶工业用途极为广泛，首先是参与纤维素的降解，此过程需要多种内切和外切酶共同参与，耐热糖苷酶水解纤维二糖生成葡萄糖，是纤维降解过程中的限速步骤，高温反应可提高速度，降低成本。第二个用途是通过转糖苷活力，合成各种各样的寡糖、糖苷或糖缀合物，在高温和有机溶剂中效率更高，具有更大优势。 二、研究内容及策略 耐热糖苷酶基因位于重组pHY质粒