苏云金芽孢杆菌BTR05普鲁兰酶基因的克隆与表达

发布时间：2018-06-13 04:27

本文选题：普鲁兰酶 + 酶学性质　；参考：《齐鲁工业大学》2016年硕士论文

【摘要】：普鲁兰酶(Pululluanase,EC 3.2.1,41)又名I型普鲁兰酶、真普鲁兰酶(True pululluanase)、极限糊精酶(Limit dextrinases)、脱支酶(Debranching enzymes)、支链淀粉-6-葡聚糖水解酶(Amylopectin 6-glucanohydrolase),是一种通过内切方式水解普鲁兰多糖、支链淀粉中的α-1,6-糖苷键分别形成以麦芽三糖和线性低聚糖为主的产物的酶。因此在工业化应用中,普鲁兰酶常与其他淀粉水解酶(如α-淀粉酶、葡萄糖淀粉酶)协同作用,可以有效地将一些“顽固性”生物质降解为可发酵糖,以用来生产生物燃料和化工原料。本论文以构建能够高效表达普鲁兰酶的基因工程菌为目标,主要研究工作如下:1以Bacillus thuringiensis BtR05基因组DNA为模板,通过常规PCR技术获得其编码普鲁兰酶的结构基因,将其插入大肠杆菌表达载体pET(K)-Trx,成功构建重组表达载体pET(K)-Trx-pul,并在分子伴侣质粒pG-KJE8的辅助下,实现了普鲁兰酶基因在大肠杆菌BL21(DE3)中的可溶性表达。2对重组工程菌的进行了发酵条件优化,最佳的发酵条件为:菌体OD600值达到0.6时进行诱导,诱导温度为25℃,诱导时间为48 h。经过发酵条件优化后酶活提高到32 U/mL左右。3重组普鲁兰酶的酶学性质研究表明:其最适作用温度为45℃,在温度为35℃-45℃范围内重组酶具有较高的活性,60℃条件下的半衰期为20h;最适pH为6.0,在pH为6.0-7.0范围内,重组酶具有较高的活性;重组酶对普鲁兰糖、可溶性淀粉、糊精和支链淀粉的水解能力依次降低。4利用自诱导培养基对重组菌进行了自诱导培养,经25℃培养24 h后,其胞内普鲁兰酶酶活达到了27.7 U/mL。对自诱导培养基的成分进行了优化,得到的最佳的培养基配方为:Tryptone 2%、Yeast extract 0.25%、甘油1.0%、葡萄糖0.025%、乳糖0.4%。经过培养基优化后酶活提高到50.7 U/m L左右。
[Abstract]:Pululluanase (EC 3.2.1,41) is also known as I prulan, True pululluanase, Limit dextrinases, debranching enzyme (Debranching enzymes), and amylopectin -6- glucan hydrolase (Amylopectin). It is an alpha hydrolysis in amylopectin and amylopectin. 6- glycosides form enzymes that are mainly products of malt three sugar and linear oligosaccharides. Therefore, in the industrial application, pullulinase often cooperates with other starch hydrolases, such as alpha amylase, glucoamylase, and can effectively degrade some "stubborn" biomass into fermentable sugar to produce biofuels and chemicals. The main research work is as follows: 1 using the Bacillus thuringiensis BtR05 genome DNA as a template, the structural gene encoding prulan enzyme was obtained by conventional PCR technology and inserted into the Escherichia coli expression vector pET (K) -Trx, and the recombinant form was successfully constructed. With the aid of pET (K) -Trx-pul, and with the aid of molecular chaperone plasmid pG-KJE8, the soluble expression.2 of the prulic gene in Escherichia coli BL21 (DE3) was realized to optimize the fermentation conditions for the recombinant strain. The optimum fermentation condition was that the strain was induced at 0.6 when the strain of the fungus reached 0.6, the induction temperature was 25, and the induction time was 48 h.. The enzymatic properties of the recombinant pullulan after the optimization of fermentation conditions to about 32 U/mL.3 showed that the optimum reaction temperature was 45 C, the recombinant enzyme had high activity at the temperature of 35 C -45 C, the half-life of the enzyme at 60 C was 20h, the optimum pH was 6, and the recombinant enzyme had higher activity in the pH 6.0-7.0 range. The hydrolysis ability of recombinant enzyme to pullulan, soluble starch, dextrin and amylopectin decreased in turn by.4 using self inducible medium for self induction culture. After 24 h culture at 25 C, the enzyme activity of pullulan reached 27.7 U/mL. for the optimization of the self induced medium, and the best medium formula was obtained. As follows: Tryptone 2%, Yeast extract 0.25%, glycerol 1%, glucose 0.025%, and lactose 0.4%. increased by 50.7 U/m L after optimization of medium.
【学位授予单位】：齐鲁工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;TQ925

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