【作者】 张亮；

【导师】 花尔并；

【作者基本信息】 天津科技大学 ， 生物工程（专业学位）， 2015， 硕士

【摘要】 淀粉和纤维素是自然界中最廉价、最丰富的可再生资源,糖化酶和内切葡聚糖酶是降解它们最关键的酶类。为了利用丰富的淀粉和纤维素资源,就必须获得性质优良的糖化酶和内切葡聚糖酶。真菌来源的糖化酶和内切葡聚糖酶是目前淀粉酶和纤维素酶研究的主要部分。过氧化氢酶是一种重要的工业酶制剂,它能催化分解过氧化氢生成水和氧气,在食品、医药,纺织等行业具有广泛的应用潜力。因此,开发出真菌来源的新型过氧化氢酶具有重要的研究价值。本实验室(蛋白质表达及分子生物学实验室)前期对粉红粘帚霉菌全基因组进行了测序,获得了粉红粘帚霉全基因组的序列信息,进而为新型酶基因的研究奠定了基础。本研究通过采用氨基酸序列比对方式,并结合相关序列的功能注释信息,从粉红粘帚霉全基因组中挖掘到了5个相关酶的基因(糖化酶基因g3440,内切葡聚糖酶基因45家族g8456和g1028,内切葡聚糖酶基因5家族g204,过氧化氢酶基因g2092)。为了实现分泌型表达,对各基因编码氨基酸的序列进行了信号肽检测,证实了5个基因的氨基酸序列均含有信号肽。本实验采用基因工程技术手段,构建了5个基因的pGm重组质粒,并将其分别转化黑曲霉G1进行重组表达。获得了三个基因的成功表达,其分别是：糖化酶基因g3440,内切葡聚糖酶基因g1028,过氧化氢酶基因g2092。测定了三种酶粗酶液的活性及相关酶的性质,结果表明：糖化酶活性达292.85 U/mL,最适温度和pH分别为50℃和5.0,该酶的耐热性较差,pH稳定性较好；内切葡聚糖酶活性达218.17 U/mL,最适温度和pH分别为55℃和6.0；过氧化氢酶活性达640.15U/mL,最适温度和pH分别60℃和6.0。更多还原

【Abstract】 Starch and Cellulose are the most abundant and cheap biomass resources. Glucoamylase is a key hydrolase in starch degradation, and endoglucanase is also important for cellulose hydrolysis. In order to take advantage of abundant starch and cellulose, we need to get new glucoamylases and endoglucanases with good properties. Glucoamylases and endoglucanases from fungi are the main part of the current research. Catalase is an important industrial enzyme which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This enzyme has great potential for a wide range of applications, such as food, textile and pharmaceutical industries. Therefore, the development of new catalases from fungi has important research value.Previously, our laboratory (Protein Expression and Molecular Biology Laboratory) sequenced the whole genome of Gliocladium roseum, and obtained all sequence information, which laid the foundation for the study of enzymes by genome mining. In this study, five relevant enzyme genes (glucoamylase gene g3440; family 45 endoglucanase gene g8456 and g1028; family 5 endoglucanase gene g204 and catalase gene g2092) were obtained by using the amino acid sequence alignment methods and functional annotation information. In order to realize the secretory expression, the signal peptides of each gene were identified and were confirmed. In this study, for the purpose of achieving recombinant expression, we constructed five pGm recombinant plasmids by using genetic engineering techniques and transformed them into Aspergillus niger host strain G1. Finally, we achieved the successful expression of three genes, and these three genes are:glucoamylase gene g3440, endoglucanase gene g1028, and catalase gene g2092. Meanwhile, we measured the activity and enzymatic properties of crude enzyme preparations. Results demonstrated that:glucoamylase activity was up to 292.85 U/mL, the optimum temperature and pH were 50℃ and 5.0, respectively. Although it has poor thermal resistance, it has a good pH stability; the endoglucanase activity reached 218.17 U/mL, the optimum temperature and pH were 55℃ and 6.0, respectively; the catalase activity reached 640.15 U/mL, the optimum temperature and pH were 60℃ and 6.0, respectively.更多还原