黑曲霉中α-葡萄糖苷酶基因的敲除及对糖化酶活力的影响研究

发布时间：2018-03-24 13:43

本文选题：黑曲霉　切入点：糖化酶　出处：《深圳大学》2016年硕士论文

【摘要】：糖化酶是一种具有外切酶活性的胞外酶,能从淀粉的非还原性末端依次水解α-1,4葡萄糖苷键,在制造葡萄糖、氨基酸、抗生素等发酵工业上有着广泛的应用。利用黑曲霉生产的糖化酶具备产量高,活性好,安全性高的特点,多数黑曲霉的糖化酶能水解80%以上的淀粉。本研究选用生产糖化酶的黑曲霉作为出发菌株,但黑曲霉生产糖化酶的同时伴随着α-葡萄糖苷酶的产生。α-葡萄糖苷酶也水解糖类底物非还原末端的α-1,4葡萄糖苷键,释放出葡萄糖或将游离的葡萄糖残基转移到另一糖类底物形成α-1,6葡萄糖苷键,从而得到非发酵性的低聚异麦芽糖或糖脂、糖肽等。α-葡萄糖苷酶能和糖化酶竞争底物,导致糖化酶可利用的淀粉底物量变少,且α-葡萄糖苷酶反应生成物的增多影响了糖化酶作用底物的能力,从而抑制了糖化酶水解淀粉的效率。因此,本文敲除黑曲霉的α-葡萄糖苷酶基因,探讨生产高纯度糖化酶的黑曲霉基因改造方法,使α-葡萄糖苷酶不再成为黑曲霉产糖化酶的伴随产物,从而提高糖化酶纯度并降低工业纯化成本,为以后设计新的效率更好的高产糖化酶的研究方法提供技术支持。本文采用Split-Marker技术敲除黑曲霉HE01中的α-葡萄糖苷酶基因。首先从菌株中扩增得到α-葡萄糖苷酶基因上游约1 kb左右的片段α-glu E1(995 bp)和下游约1 kb左右的片段α-glu E2(1 013bp),从质粒p BC-Nours.R载体中扩增得到诺尔丝菌素抗性基因Nourseothrcin Resistant(NR,1 776bp)。通过Overlap PCR分别构建α-glu E1-NR(2 771 bp)和NR-α-glu E2(2 789 bp)基因敲除表达盒,采用原生质体转化技术将敲除表达盒转入黑曲霉HE01中。根据同源重组的原理,出发株(ST)的α-葡萄糖苷酶基因会被选择性标记基因替换,原生质体转化后利用125μg m L-1诺尔斯菌素抗性平板筛选转化子。通过PCR技术分别扩增诺尔丝菌素抗性基因上游和下游两侧的序列来筛选阳性转化子。随后对获得的α-葡萄糖苷酶基因缺失的黑曲霉突变株(Δα-glu)进行表型分析。表型分析主要包括菌株生长情况测定,糖化酶酶活以及α-葡萄糖苷酶酶活测定和糖化酶基因在突变株中相对表达情况分析。本文从原生质体转化中得到15株黑曲霉转化子,验证筛选到10株阳性转化子,转化率约为66.67%。在察氏固体培养基中,菌丝形态测量结果表明,突变株菌丝的横向延伸长度大于出发株;在可溶性淀粉固体培养基中,菌丝生长情况表明,出发株与突变株降解可溶性淀粉的能力没有明显差异;在察式和可溶性淀粉液体培养基中,出发株和突变株的生物量测定结果表明,出发株的菌丝生长速度大于突变株。对出发株和阳性转化子进行摇瓶发酵,测得出发株的糖化酶酶活达到最高时为6 143.42 U m L-1,α-葡萄糖苷酶基因敲除株(突变株,Δα-glu)的最高糖化酶酶活为7 437.73 U m L-1,糖化酶活力约提高了21.06%;出发株的α-葡萄糖苷酶最高酶活为2 068.59 U m L-1,突变株为573.54 U m L-1,α-葡萄糖苷酶酶活力下降了72.31%。说明α-葡萄糖苷酶基因的敲除有利于糖化酶活力的增强。此外,荧光定量PCR结果显示,突变株的糖化酶基因相对出发株表达量上调,说明α-葡萄糖苷酶基因的敲除有利于糖化酶基因表达量的增加。综上所述,本文实现了α-葡萄糖苷酶基因的敲除,获得了高产糖化酶的黑曲霉HE01突变株Δα-glu。省去了黑曲霉生产糖化酶工艺中去除α-葡萄糖苷酶的流程,节约了糖化酶纯化的成本,且为工业提供了纯度更高且活性更好的糖化酶生产菌株。
[Abstract]:Glucoamylase is a kind of exonuclease activity of extracellular enzymes from the non reducing end of starch hydrolysis in -1,4 alpha glucoside bond, amino acid in the manufacture of glucose, such as antibiotics, fermentation industry has been widely used. The use of Aspergillus niger glucoamylase production with high yield, good activity, high security features the majority of Aspergillus Niger, saccharifying enzyme can hydrolysis of starch 80%. This study selected glucoamylase production by Aspergillus niger as the starting strain, but the Aspergillus niger glucoamylase production with alpha glucosidase. Alpha glucosidase is water solution of sugar substrate nonreducing terminal alpha -1,4 glucoside bond and the release of glucose residues or glucose free transfer to another substrate to form a -1,6 sugar glucoside bond, resulting in non fermented Isomaltooligosaccharide or glycolipid, glycopeptides. Alpha glucosidase and saccharifying enzyme Competition leads to substrate, saccharifying enzyme available starch increased and the quantity of material foundation, alpha glucosidase reaction affects the ability of saccharifying enzyme substrates, thereby inhibiting efficiency of glucoamylase hydrolysis of starch. Therefore, the knockout of Aspergillus niger alpha glucosidase gene, gene of Aspergillus niger the transformation method of producing high purity of glucoamylase, the alpha glucosidase will no longer be accompanied with glucoamylase produced by Aspergillus niger glucoamylase, so as to improve the purity and reduce industrial purification cost, provide technical support for the research methods of high yield glucoamylase after the better efficiency of the new design. This paper uses the Split-Marker technique to knock out the black Niger HE01 in alpha glucosidase gene. Firstly amplified alpha glucosidase gene upstream about 1 KB fragment from E1 alpha -glu strain (995 BP) and downstream about 1 KB fragment of alpha -glu E2 (1 013bp), Get Nourseothrcin amplification Noel silk bacterium resistance gene Resistant from plasmid P BC-Nours.R vector (NR, 1 776bp). Alpha -glu E1-NR were constructed by Overlap PCR (2771 BP) and NR- E2 (2789 BP) alpha -glu gene knockout expression cassette by protoplast transformation technology to knockdown the expression cassette into Aspergillus niger HE01. According to the principle of homologous recombination, strain (ST) of the alpha glucosidase gene will be selective marker gene replacement, protoplast transformation after using 125 g m L-1 Knowles avermectin resistant plate transformants. Nourseothricin resistance gene sequences both upstream and downstream of the screening positive transformants respectively. Amplified by PCR technology. Then the obtained alpha glucosidase gene deletion mutant strain of Aspergillus niger (delta alpha -glu) phenotype analysis. Phenotypic analysis mainly includes the determination of the growth of strains, saccharification enzyme activity and alpha glucose Neuraminidase enzyme activity assay and Glucoamylase Gene mutation analysis in relative expression lines. In this paper, 15 strains of Aspergillus niger transformants from protoplast transformation, 10 strains were selected to verify the positive transformants, the conversion rate is about 66.67%. in Czapek medium, the mycelium morphology measurement results show that the transverse extension length the mutant strain is greater than in the mycelium; soluble starch solid medium, showed that the growth of mycelium, there was no significant difference between mutant strains and the ability of degradation of soluble starch and soluble starch; in Chahar type liquid medium of strain and mutant strains of biomass showed that the mycelium growth rate of strain more than the starting strains and mutants. Positive transformants in shake flask fermentation, glucoamylase activity reached the highest measured strain was 6143.42 at U m L-1, alpha glucosidase gene knockout mutant strains (-gl, alpha Delta U) the highest enzyme activity was 7437.73 U m L-1, glucoamylase activity increased by about 21.06%; the strain of alpha glucosidase activity was highest 2068.59 U m L-1 573.54 U m, mutant L-1, alpha glucosidase enzyme activity decreased by 72.31%. alpha glucosidase gene the knockout can strengthen the activity of glucoamylase. Moreover, fluorescence quantitative PCR results showed that the mutant strain of Glucoamylase Gene Relative strains up-regulated, indicating the alpha glucosidase gene knockout for Glucoamylase Gene expression increased. In summary, this paper implements the alpha glucosidase gene knockout, obtained high yield of glucoamylase from Aspergillus niger HE01 mutant alpha Delta -glu. eliminates the need for removal of alpha glucosidase from Aspergillus niger glucoamylase production process in the process, saving the cost of purification of glucoamylase, and provides the industry with higher purity and activity of saccharification better Enzyme producing strain.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q936

【相似文献】