【摘要】：β-葡萄糖苷酶(β-glucosidase;EC.3.2.1.21)全稱β-D-葡萄糖苷水解酶,屬于纖維素酶的一種,被認(rèn)為是纖維素酶水解的限速酶。本論文以黑曲霉3.213為實驗對象,對其產(chǎn)酶曲線、蛋白質(zhì)標(biāo)準(zhǔn)曲線、葡萄糖標(biāo)準(zhǔn)曲線進(jìn)行了測定;bgl基因的擴(kuò)增和序列分析;同源重組敲除原bgl基因;構(gòu)建異源耐熱β-葡萄糖苷酶表達(dá)載體pPHT-bgl;表達(dá)質(zhì)粒轉(zhuǎn)化黑曲霉3.213和對轉(zhuǎn)化的黑曲霉3.213進(jìn)行產(chǎn)酶曲線的測定。具體結(jié)論如下:1.通過對其產(chǎn)酶標(biāo)準(zhǔn)曲線的繪制測得黑曲霉3.213最佳產(chǎn)酶時間為168 h,這點(diǎn)具有最高酶活1.704 U/mL。2.采用真菌基因組提取試劑盒法和CTAB法提取黑曲霉3.213的基因組DNA,以其為模板,設(shè)計引物,進(jìn)行PCR擴(kuò)增得到大小為2 083 bp的目的基因片段。將序列進(jìn)行blastn比對,同源性達(dá)到100%,表明成功將黑曲霉3.213的bgl基因擴(kuò)增。而且對其進(jìn)行序列分析和多重序列比對。3.運(yùn)用三段式PCR方法與同源重組對黑曲霉3.213原bgl基因進(jìn)行了敲除,導(dǎo)入潮霉素抗性基因(PHT),通過潮霉素選擇性篩選和PCR檢測確定將原基因敲除,消除了原基因?qū)δ康幕虻母蓴_。4.將TtrpC進(jìn)行PCR,質(zhì)粒pUC19與其產(chǎn)物連接方式為EcoRⅠ、HindⅢ雙酶切,得到中間質(zhì)粒;將中間質(zhì)粒與PglaA雙酶切連接;然后將其與PHT通過雙酶切進(jìn)行連接,得到的產(chǎn)物通過雙酶切與bgl基因進(jìn)行連接,最后構(gòu)建出黑曲霉3.213的高效表達(dá)載體pPHT-bgl.5.用混合酶解液懸浮適量菌絲,振蕩酶解3 h。過濾收集原生質(zhì)體,通過一系列方法進(jìn)行外源DNA的轉(zhuǎn)化,將表達(dá)質(zhì)粒通過原生質(zhì)體轉(zhuǎn)化黑曲霉3.213。對其進(jìn)行PCR檢測,目的條帶大小約為2 300 bp,可初步認(rèn)為異源耐熱β-葡萄糖苷酶在黑曲霉3.213中進(jìn)行表達(dá)。6.比較原菌株與轉(zhuǎn)化后菌株的產(chǎn)酶能力的變化,可通過數(shù)據(jù)處理得出其產(chǎn)酶最高點(diǎn)比原菌株增加了33.5%,初步完成了異源耐熱β-葡萄糖苷酶在黑曲霉3.213中的高效表達(dá)。
[Abstract]:尾 -glucosidase (EC.3.2.1.21) is a kind of cellulase, which is regarded as the rate-limiting enzyme of cellulase hydrolysis. In this paper, Aspergillus Niger 3.213 was used as the experimental object. The enzyme production curve, protein standard curve and glucose standard curve were used to determine the amplification and sequence analysis of BGL gene, homologous recombination knockout of the original bgl gene. The pPHT-bgl; expression vector of Heterothermic 尾 -glucosidase was constructed to transform Aspergillus Niger 3.213 and the transformed Aspergillus Niger 3.213 to determine the curve of enzyme production. The concrete conclusion is as follows: 1. The optimum enzyme production time of Aspergillus Niger 3.213 was 168h, which had the highest enzyme activity of 1.704 U / ml. 2. The genomic DNA, of Aspergillus Niger 3.213 was extracted by fungal genomic extraction kit method and CTAB method. Primers were designed and amplified by PCR to obtain the target gene fragment of 2083 bp. The sequence was compared with blastn, and the homology was 100%, which indicated that the bgl gene of Aspergillus Niger 3.213 was amplified successfully. And the sequence analysis and multiple sequence alignment. 3. The primordial bgl gene of Aspergillus Niger 3.213 was knocked out by three-segment PCR method and homologous recombination. The hygromycin selective screening and PCR detection were used to determine the knockout of the proto gene by introducing hygromycin resistant gene (PHT), which eliminated the interference of proto gene to the target gene. The PCR, plasmid pUC19 was ligated with its product by double digestion of EcoR 鈪，

本文編號：2241343