本文選題：黑曲霉　切入點(diǎn)：糖化酶　出處：《深圳大學(xué)》2016年碩士論文

【摘要】：糖化酶是一種具有外切酶活性的胞外酶,能從淀粉的非還原性末端依次水解α-1,4葡萄糖苷鍵,在制造葡萄糖、氨基酸、抗生素等發(fā)酵工業(yè)上有著廣泛的應(yīng)用。利用黑曲霉生產(chǎn)的糖化酶具備產(chǎn)量高,活性好,安全性高的特點(diǎn),多數(shù)黑曲霉的糖化酶能水解80%以上的淀粉。本研究選用生產(chǎn)糖化酶的黑曲霉作為出發(fā)菌株,但黑曲霉生產(chǎn)糖化酶的同時(shí)伴隨著α-葡萄糖苷酶的產(chǎn)生。α-葡萄糖苷酶也水解糖類底物非還原末端的α-1,4葡萄糖苷鍵,釋放出葡萄糖或?qū)⒂坞x的葡萄糖殘基轉(zhuǎn)移到另一糖類底物形成α-1,6葡萄糖苷鍵,從而得到非發(fā)酵性的低聚異麥芽糖或糖脂、糖肽等。α-葡萄糖苷酶能和糖化酶競(jìng)爭(zhēng)底物,導(dǎo)致糖化酶可利用的淀粉底物量變少,且α-葡萄糖苷酶反應(yīng)生成物的增多影響了糖化酶作用底物的能力,從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,探討生產(chǎn)高純度糖化酶的黑曲霉基因改造方法,使α-葡萄糖苷酶不再成為黑曲霉產(chǎn)糖化酶的伴隨產(chǎn)物,從而提高糖化酶純度并降低工業(yè)純化成本,為以后設(shè)計(jì)新的效率更好的高產(chǎn)糖化酶的研究方法提供技術(shù)支持。本文采用Split-Marker技術(shù)敲除黑曲霉HE01中的α-葡萄糖苷酶基因。首先從菌株中擴(kuò)增得到α-葡萄糖苷酶基因上游約1 kb左右的片段α-glu E1(995 bp)和下游約1 kb左右的片段α-glu E2(1 013bp),從質(zhì)粒p BC-Nours.R載體中擴(kuò)增得到諾爾絲菌素抗性基因Nourseothrcin Resistant(NR,1 776bp)。通過Overlap PCR分別構(gòu)建α-glu E1-NR(2 771 bp)和NR-α-glu E2(2 789 bp)基因敲除表達(dá)盒,采用原生質(zhì)體轉(zhuǎn)化技術(shù)將敲除表達(dá)盒轉(zhuǎn)入黑曲霉HE01中。根據(jù)同源重組的原理,出發(fā)株(ST)的α-葡萄糖苷酶基因會(huì)被選擇性標(biāo)記基因替換,原生質(zhì)體轉(zhuǎn)化后利用125μg m L-1諾爾斯菌素抗性平板篩選轉(zhuǎn)化子。通過PCR技術(shù)分別擴(kuò)增諾爾絲菌素抗性基因上游和下游兩側(cè)的序列來篩選陽性轉(zhuǎn)化子。隨后對(duì)獲得的α-葡萄糖苷酶基因缺失的黑曲霉突變株(Δα-glu)進(jìn)行表型分析。表型分析主要包括菌株生長(zhǎng)情況測(cè)定,糖化酶酶活以及α-葡萄糖苷酶酶活測(cè)定和糖化酶基因在突變株中相對(duì)表達(dá)情況分析。本文從原生質(zhì)體轉(zhuǎn)化中得到15株黑曲霉轉(zhuǎn)化子,驗(yàn)證篩選到10株陽性轉(zhuǎn)化子,轉(zhuǎn)化率約為66.67%。在察氏固體培養(yǎng)基中,菌絲形態(tài)測(cè)量結(jié)果表明,突變株菌絲的橫向延伸長(zhǎng)度大于出發(fā)株;在可溶性淀粉固體培養(yǎng)基中,菌絲生長(zhǎng)情況表明,出發(fā)株與突變株降解可溶性淀粉的能力沒有明顯差異;在察式和可溶性淀粉液體培養(yǎng)基中,出發(fā)株和突變株的生物量測(cè)定結(jié)果表明,出發(fā)株的菌絲生長(zhǎng)速度大于突變株。對(duì)出發(fā)株和陽性轉(zhuǎn)化子進(jìn)行搖瓶發(fā)酵,測(cè)得出發(fā)株的糖化酶酶活達(dá)到最高時(shí)為6 143.42 U m L-1,α-葡萄糖苷酶基因敲除株(突變株,Δα-glu)的最高糖化酶酶活為7 437.73 U m L-1,糖化酶活力約提高了21.06%;出發(fā)株的α-葡萄糖苷酶最高酶活為2 068.59 U m L-1,突變株為573.54 U m L-1,α-葡萄糖苷酶酶活力下降了72.31%。說明α-葡萄糖苷酶基因的敲除有利于糖化酶活力的增強(qiáng)。此外,熒光定量PCR結(jié)果顯示,突變株的糖化酶基因相對(duì)出發(fā)株表達(dá)量上調(diào),說明α-葡萄糖苷酶基因的敲除有利于糖化酶基因表達(dá)量的增加。綜上所述,本文實(shí)現(xiàn)了α-葡萄糖苷酶基因的敲除,獲得了高產(chǎn)糖化酶的黑曲霉HE01突變株Δα-glu。省去了黑曲霉生產(chǎn)糖化酶工藝中去除α-葡萄糖苷酶的流程,節(jié)約了糖化酶純化的成本,且為工業(yè)提供了純度更高且活性更好的糖化酶生產(chǎn)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.

【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q936

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉宇;譚成玉;趙瑩;吳迪;馬馳宇;胡建恩;孔亮;劉遠(yuǎn);李偉;;新疆鹽生植物對(duì)α-葡萄糖苷酶抑制活性的研究[J];天然產(chǎn)物研究與開發(fā);2010年02期

2 楊震;胡先望;陳朋;梁寧;嚴(yán)曉娟;;α-葡萄糖苷酶應(yīng)用開發(fā)現(xiàn)狀[J];甘肅科學(xué)學(xué)報(bào);2011年02期

3 喬文濤;血清α-葡萄糖苷酶和N-乙酰-β-D-氨基葡萄糖苷酶同工酶的測(cè)定[J];國(guó)外醫(yī)學(xué).臨床生物化學(xué)與檢驗(yàn)學(xué)分冊(cè);1993年04期

4 戴玲;趙幟平;沈業(yè)壽;李劍峰;李明珠;;丹皮多糖對(duì)α-葡萄糖苷酶作用的影響[J];生物學(xué)雜志;2006年02期

5 康文藝;張麗;;五種苦苣苔科植物α-葡萄糖苷酶抑制活性研究[J];天然產(chǎn)物研究與開發(fā);2010年01期

6 胡先望;楊震;陳朋;梁寧;嚴(yán)曉娟;;α-葡萄糖苷酶的研究進(jìn)展[J];甘肅科學(xué)學(xué)報(bào);2011年01期

7 易醒;桂菲菲;尹紅梅;盧艷梅;肖小年;;澤瀉對(duì)鼠源α-葡萄糖苷酶的抑制作用[J];天然產(chǎn)物研究與開發(fā);2013年12期

8 王青云;林親錄;劉永樂;王發(fā)祥;;幾種α-葡萄糖苷酶結(jié)構(gòu)與活性位點(diǎn)的預(yù)測(cè)和比較[J];計(jì)算機(jī)與應(yīng)用化學(xué);2010年05期

9 司曉晶;霍世欣;施雅;呂敬慈;;中藥提取物對(duì)酵母和鼠腸α-葡萄糖苷酶的抑制作用[J];上海大學(xué)學(xué)報(bào)(自然科學(xué)版);2009年04期

10 常星;康文藝;;內(nèi)蒙古產(chǎn)白刺、小果白刺和霸王α-葡萄糖苷酶抑制活性[J];天然產(chǎn)物研究與開發(fā);2012年02期

相關(guān)會(huì)議論文 前10條

1 劉艷菊;;α-葡萄糖苷酶提取方法的研究[A];第十屆全國(guó)中藥和天然藥物學(xué)術(shù)研討會(huì)論文集[C];2009年

2 翟虹;蔡超云;楊櫻芝;吳美欣;汪波;;吲哚�；苌锏暮铣杉捌鋵�(duì)α-葡萄糖苷酶的抑制活性[A];中國(guó)化學(xué)會(huì)第29屆學(xué)術(shù)年會(huì)摘要集——第22分會(huì)：化學(xué)生物學(xué)[C];2014年

3 汪林發(fā);晏菊芳;宋小禮;上官瑞燕;楊大成;;含萘丁美酮結(jié)構(gòu)單元的β-氨基酮衍生物的合成及其α-葡萄糖苷酶抑制活性的初步研究[A];有機(jī)合成創(chuàng)新—產(chǎn)業(yè)化的新動(dòng)力——中國(guó)化學(xué)會(huì)全國(guó)第三屆有機(jī)合成化學(xué)與過程學(xué)術(shù)討論會(huì)論文摘要集[C];2010年

4 陸璐;高裙裙;錢云霞;馬珂珂;王麗慧;張耀東;;芒果苷對(duì)α-葡萄糖苷酶抑制作用[A];第十三屆全國(guó)光化學(xué)學(xué)術(shù)討論會(huì)論文集[C];2013年

5 蔡超云;饒麗;肖志尊;林成功;何嘉韻;汪波;;查爾酮衍生物的合成與α-葡萄糖苷酶的抑制活性[A];中國(guó)化學(xué)會(huì)第29屆學(xué)術(shù)年會(huì)摘要集——第22分會(huì)：化學(xué)生物學(xué)[C];2014年

6 張海鳳;董亞琳;張琰;;沒食子酸的α-葡萄糖苷酶抑制作用及降糖機(jī)制研究[A];2010年中國(guó)藥學(xué)大會(huì)暨第十屆中國(guó)藥師周論文集[C];2010年

7 吳慧平;張U，

本文編號(hào)：1658556