本文關(guān)鍵詞： α-葡萄糖苷酶 嗜熱 表達(dá) 純化 表征　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的:α-葡萄糖苷酶(EC.3.2.1.20,α-Glucosidase),屬于淀粉水解酶類,能特異性水解低聚糖如麥芽糖、麥芽三糖等。同時(shí),它還具有轉(zhuǎn)糖苷作用,是工業(yè)中生產(chǎn)異麥芽糖、異麥芽三糖、異麥芽四糖等低聚異麥芽糖的關(guān)鍵酶。α-葡萄糖苷酶在啤酒釀造過(guò)程中生成的異麥芽糖、潘糖和異麥芽三糖等低聚異麥芽糖可以改善啤酒的口感,是工業(yè)應(yīng)用中很重要的一類酶。與常溫α-葡萄糖苷酶相比,耐熱型的酶具有熱穩(wěn)定性好、活力高、應(yīng)用范圍廣以及純化成本低等優(yōu)點(diǎn),更適于工業(yè)化應(yīng)用。因此本論文擬對(duì)來(lái)自于嗜熱菌Thermomonospora curvata Henssen 1957和Bacillus licheniformis Chester 1901的α-葡萄糖苷酶Tcur-1739和Amy S進(jìn)行克隆、表達(dá)和純化,對(duì)酶學(xué)特性進(jìn)行了表征,為α-葡萄糖苷酶在工業(yè)中的應(yīng)用提供了新選擇。實(shí)驗(yàn)方法:用PCR完成α-葡萄糖苷酶Tcur-1739和Amy S基因的擴(kuò)增,構(gòu)建表達(dá)載體p ET-21a-tcur-1739和p ET-28a-amy S,并用E.coli.BL21(DE3)表達(dá)出重組蛋白,經(jīng)陰離子交換柱Q及Ni~(2+)螯合柱純化,隨后進(jìn)行性質(zhì)表征,并測(cè)定了酶的動(dòng)力學(xué)常數(shù)Km和kcat值。結(jié)果:得到的蛋白序列和NCBI序列吻合,純度大于93%,Tcur-1739和Amy S的最適p H均為7.0,Tcur-1739和Amy S的最適溫度分別為50℃和80℃,Tcur-1739對(duì)底物對(duì)硝基苯酚α-D-葡萄糖吡喃苷(p NP-α-Glu)的Km值為0.23m M,kcat值為2.94×106 s-1。Amy S對(duì)底物p NP-α-Glu的Km值為1.34m M,kcat值為1.88×103s-1。結(jié)論:本實(shí)驗(yàn)成功地構(gòu)建并得到了兩個(gè)高活性的α-葡萄糖苷酶,并純化得到了電泳純蛋白,進(jìn)而進(jìn)行了性質(zhì)表征,其中Tcur-1739對(duì)底物p NP-α-Glu具有很高的活性,Amy S則表現(xiàn)出了很好的熱穩(wěn)定性及良好的催化特性。
[Abstract]:Background & objective: 偽 -glucosidase EC.3.2.1.20, 偽 -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, 偽 -glucosidase, isomaltose, isomaltooligosaccharide and isomaltotriose produced during beer brewing, can improve the taste of beer. Compared with normal temperature 偽-glucosidase, thermostable enzyme has the advantages of good thermal stability, high activity, wide application range and low purification cost. Therefore, the 偽 -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 偽 -glucosidase in industry. Methods: the Tcur-1739 and Amy S genes of 偽 -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，

本文編號(hào)：1550834