本文選題：黑曲霉　切入點：α-葡萄糖苷酶　出處：《深圳大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：α-葡萄糖苷酶屬于外切糖苷酶,能夠催化底物的非還原末端的裂解,以釋放α-D-葡萄糖,同時被認為在水解淀粉產(chǎn)生不可發(fā)酵糖的過程中起重要作用,此外α-葡萄糖苷酶能將游離的葡萄糖殘基轉(zhuǎn)移到另一糖類底物上,從而生產(chǎn)低聚異糖類。來自α-葡萄糖苷酶的轉(zhuǎn)苷用于產(chǎn)生葡萄糖基化合物或低聚異麥芽糖。黑曲霉α-葡萄糖苷酶的生產(chǎn)通常伴隨著糖化酶,糖化酶的存在使α-葡萄糖苷酶可以利用的淀粉減少,從而抑制了α-葡萄糖苷酶的水解和轉(zhuǎn)化底物的能力。糖化酶是具有外切酶活性的酶,主要催化淀粉,糖原和寡糖中的α-1,4-糖苷鍵水解,它也以較低的速率水解α-1,6-糖苷鍵,糖化酶在工業(yè)生產(chǎn)中也經(jīng)常受到α-葡萄糖苷酶的轉(zhuǎn)苷反應(yīng)的影響,導(dǎo)致葡萄糖產(chǎn)量的顯著降低。由于可以水解相同的底物,因此α-葡萄糖苷酶和糖化酶可能競爭性地彼此抑制。在本研究中,通過靶向基因缺失敲除黑曲霉的糖化酶基因,構(gòu)建具有較高α-葡萄糖苷酶活性的黑曲霉突變菌株,并研究糖化酶對α-葡萄糖苷酶活性的影響。本文采用同源重組的方法得到黑曲霉糖化酶基因缺失突變體,首先在黑曲霉基因組的上下游分別擴增約1,000 bp左右的片段,同時從質(zhì)粒pBC-Nours.R載體中擴增得到諾爾絲菌素抗性基因,通過Overlap PCR構(gòu)建基因敲除表達盒GA1-NR和NR-GA2,并通過原生質(zhì)體轉(zhuǎn)化獲得具有抗性基因的轉(zhuǎn)化子,在含有125μg/mL諾爾絲菌素的培養(yǎng)基上篩選轉(zhuǎn)化子,并通過PCR進一步證明篩選的陽性轉(zhuǎn)化子。對得到的黑曲霉糖化酶基因缺失突變菌株ΔGA進行表型分析,生長情況測定,α-葡萄糖苷酶和糖化酶酶活測定,α-葡萄糖苷酶基因在黑曲霉突變菌株中的相對表達量的測定以及α-葡萄糖苷酶轉(zhuǎn)苷作用的分析。根據(jù)對突變菌株ΔGA的分析,由于糖化酶基因的缺失,培養(yǎng)基上的ΔGA突變體的生長速度在一定程度上減緩。但在生長發(fā)育如菌落表型,形態(tài)和色素沉著中沒有明顯的缺陷。此外,對α-葡萄糖苷酶酶活的測定可知,相比于出發(fā)菌株,突變菌株ΔGA的α-葡萄糖苷酶活性升高了63.08%,而突變菌株損失部分糖化酶活性,糖化酶酶活降低了21.14%,說明糖化酶基因的敲除有利于α-葡萄糖苷酶活力的增強。并且通過RT-qPCR證實了在ΔGA中α-葡萄糖苷酶基因表達水平提高至出發(fā)菌株的2.53倍,說明糖化酶基因的敲除有利于α-葡萄糖苷酶基因表達量的增加。高效液相色譜法檢測突變菌株ΔGA與出發(fā)菌株的轉(zhuǎn)苷產(chǎn)物,其中ΔGA轉(zhuǎn)苷生產(chǎn)異麥芽糖的濃度為出發(fā)菌株的2.00倍,潘糖的濃度為出發(fā)菌株的1.59倍,說明糖化酶基因的敲除有利于α-葡萄糖苷酶轉(zhuǎn)苷產(chǎn)物濃度的提升。綜上所述,本研究實現(xiàn)了黑曲霉糖化酶基因的敲除,獲得了黑曲霉突變菌株ΔGA。進一步證明糖化酶對α-葡萄糖苷酶的活性具有負面的影響作用,并獲得了具有更高α-葡糖苷酶活性的黑曲霉HE01突變株ΔGA,為工業(yè)上提供具有可應(yīng)用價值的α-葡萄糖苷酶生產(chǎn)菌株。
[Abstract]:Alpha glucosidase belongs to exoglycosidases can catalyze the cleavage of the non reducing end, with the release of alpha -D- glucose, and are thought to have not played an important role in the process of fermenting sugar in hydrolysis of starch, the alpha glucosidase residue free glucose can be transferred to other sugars the substrate, thereby producing Isomalto sugars. Alpha glucosidase from the glucoside to produce glucose based compounds or Isomaltooligosaccharide. Aspergillus niger glucoamylase production is usually accompanied by the existence of glucoamylase, alpha glucosidase can use starch reduced, thereby inhibiting the ability hydrolysis and substrate conversion of alpha glucosidase. Glucoamylase has exonuclease activity of the enzyme, which catalyses the hydrolysis of starch, glycogen and -1,4- alpha glycosidic bond of oligosaccharide, it is to lower the rate of hydrolysis of -1,6- alpha glycosidic bond, saccharifying enzyme in Effect of industrial production is also transferred by reaction of alpha glucosidase, resulting in significantly lower glucose production. Due to the hydrolysis of the same as alpha glucosidase and saccharifying enzyme could competitively inhibit each other. In this study, the lack of glucoamylase gene knock in Aspergillus niger to the target gene, construct high alpha glycosidase activity of grape Aspergillus niger mutant strains, and study the effect of enzyme on a-glucosidase activity. The Aspergillus niger glucoamylase gene deletion mutant by homologous recombination method, first in the downstream of Aspergillus niger genome were amplified approximately about 1000 BP at the same time amplified fragment, Noel silk bacterium receive resistant gene from plasmid pBC-Nours.R, gene knockout expression box of GA1-NR and NR-GA2 by Overlap PCR, and through protoplast transformation with resistance gene Because of the transformants, on the culture medium screening of transformants containing 125 mu g/mL nourseothricin, and through PCR further proved the positive transformants. Screening of Aspergillus niger glucoamylase gene deletion mutant obtained Delta GA phenotype analysis, determination of growth, activity determination of alpha glucosidase and saccharifying enzyme the enzyme alpha glucosidase gene in Aspergillus niger mutant strains in the relative expression amount and determination of alpha glucosidase in turn effect analysis. According to the analysis of the mutant strain GA, due to the lack of Glucoamylase Gene, GA mutant culture based on the growth slowed to a certain extent but in the growth and development such as colony morphology and pigment phenotype, no obvious defects in pigmentation. In addition, the determination of alpha glucosidase activity shows that, compared to the original strain, the enzyme activity of the mutant strain GA alpha grape increased by 63.08%, while the mutation Glucoamylase activity strains loss part, glucoamylase activity decreased by 21.14%, indicating the Glucoamylase Gene knockout is enhanced alpha glucosidase activity. And was confirmed by RT-qPCR in Delta GA of alpha glucosidase gene expression level increased to 2.53 times that of the original strain, the Glucoamylase Gene Knockout for alpha glucosidase gene expression increased. HPLC analysis of mutant strain GA and delta glucoside conversion products, including the concentration of GA in production of isomaltose is 2 times that of the original strain, the sugar concentration of Pan was 1.59 times of the strains, indicating Glucoamylase Gene Knockout for alpha glucosidase in turn product concentration increase. In conclusion, this study realizes the Glucoamylase Gene knockout of Aspergillus niger, Aspergillus niger mutant strain was obtained GA. further proved the glucoamylase activity of alpha glucosidase It has a negative effect and has obtained a HE01 strain GA of Aspergillus niger, which has higher activity of alpha glucosidase. It provides a valuable strain producing glucosidase for the industry.

【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78;Q55

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