【作者】 李昆；

【导师】 王正祥；

【作者基本信息】 江南大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 糖化酶是目前最重要的工业酶制剂之一,广泛应用于将淀粉糖化产物作为原料的发酵工业。Aspergillus niger CICIM F0410是国内生产糖化酶的代表菌株之一。本文克隆了A. niger F0410糖化酶基因的启动子,对其核苷酸序列进行了测定,在大肠杆菌中鉴定了该启动子的功能,并进一步探讨了其在糖化酶工业生产菌株遗传改良中的应用。本文首先通过PCR扩增到A. niger F0410糖化酶基因启动子PglaA并测定其核苷酸序列,通过分析核苷酸序列并与GenBank上登录号为X00712的黑曲霉糖化酶基因启动子序列进行比对,结果表明,两启动子核苷酸序列同源性为99.45%,有5个位置的碱基变化,包括1个位点缺失和4个位点替换。将克隆得到的糖化酶基因启动子PglaA替换质粒pRS303K上KmR基因启动子,构建成糖化酶基因启动子功能检测质粒pRS-PglaA-KmR,将pRS-PglaA-KmR转入E. coli JM109中,得到重组菌E. coli(pRS-PglaA-KmR)。通过对重组菌的氨基糖苷磷酸转移酶基因活性检测,表明PglaA在E. coli中具有驱动KmR基因表达的活性。采用不同诱导物对该启动子诱导发现,葡萄糖、蔗糖、乳糖、麦芽糖和玉米淀粉,可以不同程度增强PglaA的强度。用糖化酶基因启动子PglaA替换质粒pRS303H上HygR基因启动子,构建成重组质粒pRS-PglaA-HygR,通过PEG介导的原生质体转化方法转化糖化酶工业生产菌株A. niger F0410,通过潮霉素B作为筛选标记获得转化子,并通过PCR对转化子进行了进一步确认,结果表明成功将重组质粒pRS-PglaA-HygR整合到工业菌株A. niger F0410染色体中。通过摇瓶发酵实验和潮霉素B抗性稳定性实验,获得一株与出发菌株产糖化酶活力无明显改变、抗性稳定的转化子GAH14,对其进行亚硝基胍诱变,筛选潮霉素B抗性提高的转化子进行摇瓶发酵实验,并测定其糖化酶酶活。经初筛和复筛,最终获得了糖化酶活力比出发菌株提高了8.8%的转化子GAN12。更多还原

【Abstract】 Glucoamylase is one of the most important industrial enzymes. It has been widely applied in fermentation industry that need glycosylated starch as a raw material. Aspergillus niger F0410 is an industrial glucoamylase producing strain. This research focused on cloning, sequence analyzing and functional identification of glaA promoter from A. niger F0410. The application of this promoter in improving the glucoamylase yield of A. niger F0410 was further carried out and discussed.Fristly, the PglaA fragment was amplified from the chromosomal DNA of A. niger F0410 by polymerase chain reaction. The DNA sequence was further sequenced. Sequence alignment with the PglaA sequence of another A. niger (GenBank accession number X00712) showed that the homologous of two promoter nucleotide sequences reached 99.45 percent, five sites of PglaA sequence has been changed, including one-site loss and four sites replacement.The PglaA was subsequently cloned into the up-stream of the KmR gene encoding region of the plasmid pRS303K to replace the promoter of KmR, yielded the hybrid plasmid pRS-PglaA-KmR. The recombinant plasmid was transformed into Escherichia coli JM109 cells, resulted in the recombinant strain E. coli(pRS-PglaA-KmR). That the activity of KmR was detectable in LB plate containing kanamycin showed that PglaA has function in E. coli. Glucose, sucrose, lactose, maltose or corn starch could enhance the strength of PglaA with defferent degrees.For strain improvement, the recombinant plasmid pRS-PglaA-HygR was constructed and genetic transformed into A. niger F0410 using a PEG-mediated method. With a selectable marker gene encoding hygromycin resistance, a cluster of transformants with pRS-PglaA-HygR randomly integrated into the genome of A. niger F0410 were gained and confirmed by diagnosis PCR.A transformant with superior character, named GAH14, is selected. After NTG mutagenizing, the mutant strains with improved hygromycin resistance are picked out for further fermentation study. After screening for several times, we obtain a strain named GAN12, and the activity of its glucoamylase 8.8% greater than the activity of initial strain.更多还原