黑曲霉中糖化酶基因的敲除及对α-葡萄糖苷酶活力的影响研究

发布时间：2018-03-04 06:40

本文选题：黑曲霉　切入点：α-葡萄糖苷酶　出处：《深圳大学》2017年硕士论文　论文类型：学位论文

【摘要】：α-葡萄糖苷酶属于外切糖苷酶,能够催化底物的非还原末端的裂解,以释放α-D-葡萄糖,同时被认为在水解淀粉产生不可发酵糖的过程中起重要作用,此外α-葡萄糖苷酶能将游离的葡萄糖残基转移到另一糖类底物上,从而生产低聚异糖类。来自α-葡萄糖苷酶的转苷用于产生葡萄糖基化合物或低聚异麦芽糖。黑曲霉α-葡萄糖苷酶的生产通常伴随着糖化酶,糖化酶的存在使α-葡萄糖苷酶可以利用的淀粉减少,从而抑制了α-葡萄糖苷酶的水解和转化底物的能力。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-糖苷键水解,它也以较低的速率水解α-1,6-糖苷键,糖化酶在工业生产中也经常受到α-葡萄糖苷酶的转苷反应的影响,导致葡萄糖产量的显着降低。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竞争性地彼此抑制。在本研究中,通过靶向基因缺失敲除黑曲霉的糖化酶基因,构建具有较高α-葡萄糖苷酶活性的黑曲霉突变菌株,并研究糖化酶对α-葡萄糖苷酶活性的影响。本文采用同源重组的方法得到黑曲霉糖化酶基因缺失突变体,首先在黑曲霉基因组的上下游分别扩增约1,000 bp左右的片段,同时从质粒pBC-Nours.R载体中扩增得到诺尔丝菌素抗性基因,通过Overlap PCR构建基因敲除表达盒GA1-NR和NR-GA2,并通过原生质体转化获得具有抗性基因的转化子,在含有125μg/mL诺尔丝菌素的培养基上筛选转化子,并通过PCR进一步证明筛选的阳性转化子。对得到的黑曲霉糖化酶基因缺失突变菌株ΔGA进行表型分析,生长情况测定,α-葡萄糖苷酶和糖化酶酶活测定,α-葡萄糖苷酶基因在黑曲霉突变菌株中的相对表达量的测定以及α-葡萄糖苷酶转苷作用的分析。根据对突变菌株ΔGA的分析,由于糖化酶基因的缺失,培养基上的ΔGA突变体的生长速度在一定程度上减缓。但在生长发育如菌落表型,形态和色素沉着中没有明显的缺陷。此外,对α-葡萄糖苷酶酶活的测定可知,相比于出发菌株,突变菌株ΔGA的α-葡萄糖苷酶活性升高了63.08%,而突变菌株损失部分糖化酶活性,糖化酶酶活降低了21.14%,说明糖化酶基因的敲除有利于α-葡萄糖苷酶活力的增强。并且通过RT-qPCR证实了在ΔGA中α-葡萄糖苷酶基因表达水平提高至出发菌株的2.53倍,说明糖化酶基因的敲除有利于α-葡萄糖苷酶基因表达量的增加。高效液相色谱法检测突变菌株ΔGA与出发菌株的转苷产物,其中ΔGA转苷生产异麦芽糖的浓度为出发菌株的2.00倍,潘糖的浓度为出发菌株的1.59倍,说明糖化酶基因的敲除有利于α-葡萄糖苷酶转苷产物浓度的提升。综上所述,本研究实现了黑曲霉糖化酶基因的敲除,获得了黑曲霉突变菌株ΔGA。进一步证明糖化酶对α-葡萄糖苷酶的活性具有负面的影响作用,并获得了具有更高α-葡糖苷酶活性的黑曲霉HE01突变株ΔGA,为工业上提供具有可应用价值的α-葡萄糖苷酶生产菌株。
[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.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q55

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