1材料与方法-BioPublisher.doc

C-端引入二硫键对黑曲霉木聚糖酶XynZF-2热稳定性的影响 （新乡医学院 生命科学技术学院，合成生物学改造工程与应用实验室，河南 新乡 453000） 摘要：同源比对黑曲霉XZ-3S木聚糖酶基因xynZF-2氨基酸序列，模拟构建木聚糖酶三维结构，确定能够提高酶热稳定性的最佳突变位点。在C-端引入二硫键，突变xynZF-2 205位点的色氨酸和52位点的丙氨酸为半胱氨酸，获取突变基因T205C-A52C，表达于大肠杆菌BL21（DE3）。酶学性质比较发现，突变酶XynZF-T205C-A52C的最适温度为50℃，比原酶XynZF-2提高了10℃50℃ 保温5 min，突变酶相对酶活性为55.36%，原酶相对酶活性为32.62%原酶与突变酶最适pH均为50， 但相同pH下突变酶的相对酶活性较原酶高 5. 0～9.0 3. 0～9.C-端引入二硫键黑曲霉木聚糖酶XynZF-2热稳定性 关键词：木聚糖酶；热稳定性；定点突变；二硫键 Effect of introduction of disulfide?bonds in C-terminal structure on the thermalstability of xylanase XynZF-2 from Aspergillus niger LI Jingyi ，REN Bingjie ， CAI Liutengzi ，ZHANG Mishuai ，ZHOU Chenyan* (Synthetic Biology Remaking Engineering and Application Laboratory, School of Life Science and Technology，Xinxiang Medical University，Xinxiang 453003，China) Abstrct：To locate the optical mutated site of xynZF-2 from Aspergillus niger XZ-3S and acquire higher thermalstability mutated xylanase, the active sites was predicted and the 3D structure of xylanase XynZF-2 was constructed by the software of bioinformatics. The sites were substituted by site-directed mutagenesis (T205C and A52C). Hence, the xynZF-T205C-A52C corresponding gene was cloned in the pET-28a vector and expressed in BL21（DE3）cells. Subsequently, it showed ?difference by comparing the enzymatic properties of mutated and original xylanase. The optimum temperature of the variant XynZF-T205C-A52C was increased from 40℃ to 50℃，and the variant XynZF-T205C-A52C retained about % activity (the XynZF-2 retained 32.62% activity) after treatment at 50℃ for 5 minutes. The optimum pH of mutated xynlase was same to XynZF-2 (pH=5). Both mutated and original xylanase had a pH of 5.0, compared with the reletiave enzyme activity of original xylanase, the mutated xylanase was raised; pH stability interval of mutated xylanase was changed from 5.0~9.0 to 3.0~9.0. The result implied that mutanting Thr205 and Glu52 into Cyr205 and Cys52 respectively and the introduction of disulfide?bonds in the C-terminal structur