α-葡萄糖苷酶Tcur-1739和AmyS的克隆表达、纯化及性质研究

发布时间：2018-03-01 06:32

  本文关键词： α-葡萄糖苷酶 嗜热 表达 纯化 表征　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的:α-葡萄糖苷酶(EC.3.2.1.20,α-Glucosidase),属于淀粉水解酶类,能特异性水解低聚糖如麦芽糖、麦芽三糖等。同时,它还具有转糖苷作用,是工业中生产异麦芽糖、异麦芽三糖、异麦芽四糖等低聚异麦芽糖的关键酶。α-葡萄糖苷酶在啤酒酿造过程中生成的异麦芽糖、潘糖和异麦芽三糖等低聚异麦芽糖可以改善啤酒的口感,是工业应用中很重要的一类酶。与常温α-葡萄糖苷酶相比,耐热型的酶具有热稳定性好、活力高、应用范围广以及纯化成本低等优点,更适于工业化应用。因此本论文拟对来自于嗜热菌Thermomonospora curvata Henssen 1957和Bacillus licheniformis Chester 1901的α-葡萄糖苷酶Tcur-1739和Amy S进行克隆、表达和纯化,对酶学特性进行了表征,为α-葡萄糖苷酶在工业中的应用提供了新选择。实验方法:用PCR完成α-葡萄糖苷酶Tcur-1739和Amy S基因的扩增,构建表达载体p ET-21a-tcur-1739和p ET-28a-amy S,并用E.coli.BL21(DE3)表达出重组蛋白,经阴离子交换柱Q及Ni~(2+)螯合柱纯化,随后进行性质表征,并测定了酶的动力学常数Km和kcat值。结果:得到的蛋白序列和NCBI序列吻合,纯度大于93%,Tcur-1739和Amy S的最适p H均为7.0,Tcur-1739和Amy S的最适温度分别为50℃和80℃,Tcur-1739对底物对硝基苯酚α-D-葡萄糖吡喃苷(p NP-α-Glu)的Km值为0.23m M,kcat值为2.94×106 s-1。Amy S对底物p NP-α-Glu的Km值为1.34m M,kcat值为1.88×103s-1。结论:本实验成功地构建并得到了两个高活性的α-葡萄糖苷酶,并纯化得到了电泳纯蛋白,进而进行了性质表征,其中Tcur-1739对底物p NP-α-Glu具有很高的活性,Amy S则表现出了很好的热稳定性及良好的催化特性。
[Abstract]:Background & objective: 伪 -glucosidase EC.3.2.1.20, 伪 -Glucosidase, which belongs to starch hydrolase, can specifically hydrolyze oligosaccharides such as maltose, maltotriose and so on. The key enzyme of isomaltooligosaccharide, such as isomaltose, 伪 -glucosidase, isomaltose, isomaltooligosaccharide and isomaltotriose produced during beer brewing, can improve the taste of beer. Compared with normal temperature 伪-glucosidase, thermostable enzyme has the advantages of good thermal stability, high activity, wide application range and low purification cost. Therefore, the 伪 -glucosidase Tcur-1739 and Amy S from the thermophilic bacteria Thermomonospora curvata Henssen 1957 and Bacillus licheniformis Chester 1901 were cloned, expressed, purified and characterized. This study provides a new choice for the application of 伪 -glucosidase in industry. Methods: the Tcur-1739 and Amy S genes of 伪 -glucosidase were amplified by PCR, the expression vector p ET-21a-tcur-1739 and p ET-28a-amy S were constructed, and the recombinant protein was expressed by E.coli.BL21DE3. After purification by anion exchange column Q and Ni~(2, the properties of the enzyme were characterized, and the kinetic constants km and kcat of the enzyme were determined. Results: the obtained protein sequence coincided with the NCBI sequence. The optimum pH values of Tcur-1739 and Amy S for purity greater than 93C and Amy S are 7.0 Tcur-1739 and 80 鈩，

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