本文選題：普魯蘭酶 + 酶學性質(zhì)　； 參考：《齊魯工業(yè)大學》2016年碩士論文

【摘要】：普魯蘭酶(Pululluanase,EC 3.2.1,41)又名I型普魯蘭酶、真普魯蘭酶(True pululluanase)、極限糊精酶(Limit dextrinases)、脫支酶(Debranching enzymes)、支鏈淀粉-6-葡聚糖水解酶(Amylopectin 6-glucanohydrolase),是一種通過內(nèi)切方式水解普魯蘭多糖、支鏈淀粉中的α-1,6-糖苷鍵分別形成以麥芽三糖和線性低聚糖為主的產(chǎn)物的酶。因此在工業(yè)化應用中,普魯蘭酶常與其他淀粉水解酶(如α-淀粉酶、葡萄糖淀粉酶)協(xié)同作用,可以有效地將一些“頑固性”生物質(zhì)降解為可發(fā)酵糖,以用來生產(chǎn)生物燃料和化工原料。本論文以構(gòu)建能夠高效表達普魯蘭酶的基因工程菌為目標,主要研究工作如下:1以Bacillus thuringiensis BtR05基因組DNA為模板,通過常規(guī)PCR技術(shù)獲得其編碼普魯蘭酶的結(jié)構(gòu)基因,將其插入大腸桿菌表達載體pET(K)-Trx,成功構(gòu)建重組表達載體pET(K)-Trx-pul,并在分子伴侶質(zhì)粒pG-KJE8的輔助下,實現(xiàn)了普魯蘭酶基因在大腸桿菌BL21(DE3)中的可溶性表達。2對重組工程菌的進行了發(fā)酵條件優(yōu)化,最佳的發(fā)酵條件為:菌體OD600值達到0.6時進行誘導,誘導溫度為25℃,誘導時間為48 h。經(jīng)過發(fā)酵條件優(yōu)化后酶活提高到32 U/mL左右。3重組普魯蘭酶的酶學性質(zhì)研究表明:其最適作用溫度為45℃,在溫度為35℃-45℃范圍內(nèi)重組酶具有較高的活性,60℃條件下的半衰期為20h;最適pH為6.0,在pH為6.0-7.0范圍內(nèi),重組酶具有較高的活性;重組酶對普魯蘭糖、可溶性淀粉、糊精和支鏈淀粉的水解能力依次降低。4利用自誘導培養(yǎng)基對重組菌進行了自誘導培養(yǎng),經(jīng)25℃培養(yǎng)24 h后,其胞內(nèi)普魯蘭酶酶活達到了27.7 U/mL。對自誘導培養(yǎng)基的成分進行了優(yōu)化,得到的最佳的培養(yǎng)基配方為:Tryptone 2%、Yeast extract 0.25%、甘油1.0%、葡萄糖0.025%、乳糖0.4%。經(jīng)過培養(yǎng)基優(yōu)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.
【學位授予單位】：齊魯工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：Q78;TQ925

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