摘要: 本文利用酶工程的操作方法，对一株表达重组内切葡聚糖酶的菌株EG/BL21(DE3)进行了原核诱导表达，并对重组内切葡聚糖酶进行了纯化和活性分析。论文对重组菌株EG/BL21(DE3)内切葡聚糖酶的表达和纯化方法进行探索，为了提高内切葡聚糖酶基因在大肠杆菌中的表达量，通过实验研究了重组菌的诱导条件。优化的重组菌的培养和表达条件是：在37℃，培养到OD值是0.5时加入0.1mM的IPTG开始诱导，诱导培养4h重组蛋白表达量达最大。对诱导过的菌体进行破碎，使用含有His-标签的Ni柱浓缩纯化了重组酶。利用打孔实验和刚果红染色验证重组酶具有较高的内切葡聚糖酶活性，使用DNS法测定了纯化后的酶液活性达到2245U/mL。
关键词: 内切葡聚糖酶；重组酶；诱导表达；纯化
Abstract: In the paper, induced prokaryotic expression of a recombinated endo-glucanase strain of EG/BL21(DE3) was studied with enzyme engineering techniques, and purification and activity analysis was conducted to the recombinated endo-glucanase. Expression and purification method of the recombinated endo-glucanase strain of EG/BL21(DE3) was conducted here. In order to improve the quantity expression of endo-glucanase gene in E.coli, the induction conditions of the recombinated bacteria was studied through the experiment. The optimized culture and expression conditions of the recombinated bacteria are as follows : culture in 37 ℃; it is the time to add 0.1mM IPTG when the cultivated OD value is 0.5; the expression quantity is the largest when recombinated protein have been inducted 4 h. The induced bacteria need to be broken and then the recombinase was purified and concentrated with the Ni column which has His - tags. Punching experiments and Congo red staining shows, the recombinated enzyme has higher endo-glucanase activity, and the use of DNS method for the determination of purified enzyme activity shows the activity reached 2245 u/mL.
Keywords: Endo-glucanase Recombinant enzyme Induced expression Purification
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