【作者】 陈静；

【导师】 陈惠；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 本实验室之前已筛选分离得到一株米曲霉gif-10,并且从中克隆得到了p-葡萄糖苷酶的基因组序列,为了让p-葡萄糖苷酶的酶活力以及酶学性质得到提高,以及为以后纤维素酶共表达提供基础平台,本研究要构建p-葡萄糖苷酶的酵母工程菌。通过实验得到如下结果：1、通过TRIZOL法提取了米曲霉gif-10的总RNA,并以它为模板,扩增得到了p-葡萄糖苷酶的cDNA序列,该cDNA序列长度为2586 bp,其中GC含量为54.4%,AT含量为45.6%,编码861个氨基酸,其中前20个氨基酸编码信号肽。通过序列比对分析发现,β-葡萄糖苷酶基因bgl与Aspergillus oryzae RIB40的β-葡萄糖苷酶基因序列的同源性为99%。2、引入酶切位点Sac Ⅱ和Xba Ⅰ,将带有酶切位点的目的基因片段与质粒pPICZaA连接,转化,并进行序列测定。序列测定正确后电转入酵母X33,并对酵母转化子进行筛选、鉴定,挑选出6个酵母转化子,酶活力分别为5.95、6.10、5.77、6.88、6.22、5.99。选取4号酵母工程菌进行后续实验,并命名为pPICZaA-bgl。3、将得到酵母工程菌进行培养诱导,最佳诱导时间为96h,连续10次连续培养诱导酵母工程菌,其生长密度以及p-葡萄糖苷酶酶活性方面基本保持稳定,有很好的遗传稳定性。4、通过硫酸铵沉淀、镍柱纯化,SDS-PAGE检测有大小约100 KD的单一蛋白条带,而β-葡萄糖苷酶蛋白大小理论值为94 KD,因为其具有14个潜在糖基化位点,所以大小正确,得到了纯化的β-葡萄糖苷酶蛋白。纯化后的p-葡萄糖苷酶酶活为17.19U/mL,蛋白含量0.286 mg/mL；比活为6Q.10 U/mg。5、对p-葡萄糖苷酶酶学性质分析得知,它的最适温度为50℃,在20～50℃的环境中,酶活力稳定性好；最适pH为5.0,在环境pH为4.0～6.0时,其酶活力能够维持较高水平。通过底物特异性分析,β-葡萄糖苷酶对于水杨苷和纤维二糖的底物特异性强；金属离子对其酶活影响的实验结果显示,激活作用最明显的为MnCl2,抑制作用最明显的为CuCl2。更多还原

【Abstract】 There have an Aspergillus oryzae gif-10 in our laboratory. the genomic sequence of the β-glucosidase was cloned from it. In order to improve the β-glucosidase enzyme activity and enzymatic properties and base cellulase coexpressing the basic platform, this study will build a yeast engineered bacteria of β-glucosidase. The results of the experimental are as follows:1、We extracted the total RNA of Aspergillus oryzae gif-10 by TRIZOL method,and use it as a template, we get the cDNA sequence of the β-glucosidase. the total length of the cDNA sequence is 2586bp, the GC content of the gene sequence is 54.4%, encode 861 amino acids. Compared with the GenBank database analysis found that the β-glucosidase gene bgl with the β-glucosidase gene in Aspergillus oryzae RIB40 sequence homology is 99%.2、Adding the restriction sites Sac Ⅱ and Xba Ⅰ.Put the target gene fragment with restriction sites and the plasmid pPICZaA ligation, transformation, and sequence. Then electricity transferred into the yeast X33, and the yeast transformants screened, identified, selected six yeast transformants.The enzyme activity of them were 5.95,6.10,5.77,6.88,6.22,5.99. Select the No.4 Yeast Strain for the following experiments, and was named pPICZaA-bgl.3、Put the yeast engineered bacteria culture, the best time of induction is 96h,10 consecutive continuous culture induced the yeast engineered bacteria,the growth density and beta-glucosidase enzyme activity remained stable, it have a good genetic stability.4、We have a single protein of about 100KD by precipitation with ammonium sulfate, purified by nickel column and by SDS-PAGE detection, the size of the protein theoretical value is 94KD, because it have glycosylation sites, so the size is the correct one.Purified β-glucosidase activity is 17.19U/mL, the protein content is 0.286mg/mL; enzyme activity is 60.10U/mg.5、The enzymatic properties of β-glucosidase analysis showed that the optimum temperature is 50℃,in the Environment of 20℃ to 50℃,it has a good stability of the enzyme activity; the optimum pH is pH 5.0, in environmental of pH 4.0-6.0, its enzyme activity can hold the high level.β-glucosidase substrate specificity for salicin and cellobiose is strong; Different metal ions shows different effects on β-glucosidase activity., the activating effect is most obvious is MnCl2, inhibition of the most obvious is CuCl2.更多还原