【作者】 姚婷婷；

【导师】 王正祥；

【作者基本信息】 江南大学 ， 生化与分子生物学， 2006， 硕士

【摘要】 糖化酶,全称葡萄糖淀粉酶(Glucoamylase EC.3.2.1.3),又称淀粉葡萄糖苷酶,功能在于从淀粉、糊精或糖原等碳水化合物的非还原性末端释放β-D-葡萄糖。糖化酶是水解淀粉产生葡萄糖的主要酶类,因此被广泛地应用于食品、医药、发酵等工业,具有很高的商业价值,是我国产量最大的酶制剂产品之一。目前,工业中广泛使用黑曲霉、泡盛曲霉等为生产菌株来提取糖化酶。本文首先利用PCR方法从糖化酶工业生产菌株F0410染色体扩增出包括ITS1、ITS2的部分序列和5.8S rDNA完整序列的保守系列,通过序列测定及比对分析确认该菌种为黑曲霉。根据GenBank上公布的黑曲霉糖化酶基因序列设计引物,通过PCR扩增得到黑曲霉F0410的糖化酶基因,扩增产物为3327 bp,其中糖化酶编码基因为2167 bp,推衍得到的氨基酸序列有640个氨基酸残基。PCR扩增出的glaA基因片段经试剂盒纯化后插入到载体pBC-Hygro的SmaⅠ位点,构建重组质粒pBC-Hygro-glaA。构建了黑曲霉F0410的PEG-原生质体转化系统。用重组质粒pBC-Hygro-glaA进行原生质体转化。经多次转化在潮霉素B抗性平板上共获得123株转化子,从中挑选酶活力提高的6株转化子进行摇瓶发酵和糖化酶基因拷贝数的研究。在染色体整合2-3倍的糖化酶基因对糖化酶的过量合成是适宜的,糖化酶的活力比出发菌株提高了12.7%-23%,当在染色体整合的糖化酶基因拷贝数达到出发菌株的8倍时,糖化酶的活力仅提高了6%。经初筛和复筛,最终获得了酶活力显著提高的转化子GB0506作进一步的研究。初步确定了重组菌GB0506适宜的培养基和发酵条件,最优的摇瓶发酵条件是30 g/L酵母膏,20 g/L棉籽粉,2 g/L硝酸铵和80 g/L工业葡萄糖,培养基pH 5.5,接种量为8%。在优化条件下对重组菌GB0506和出发菌株F0410进行摇瓶发酵,在整个发酵过程中,GB0506的糖化酶活力稳定高于F0410,在144 h发酵终了时,转化子GB0506的糖化酶活力比出发菌株F0410提高了17.5%。更多还原

【Abstract】 Glucoamylase, also known as amyloglucosidase, is an extremely important enzyme for starch hydrolysis processes. It catalyzes the release of D-glucose from the non-reducing ends of starch or related oligo- and poly-saccharide molecules. At present, Aspergillus niger and Aspergillus awamori are commonly used to produce glucoamylase in the industry.Fristly, the conserved sequence including partial ITS1, ITS2 and entire 5.8S rDNA was amplified from the chromosomal DNA of F0410, a glucoamylase industrial producer, and sequenced. The strain was confermed as A. niger by the BLAST analysis of network service. A set of primer was designed according to the public glucoamylase gene in GenBank. A 3.3 kb fragment containing complete glaA was amplified from A. niger F0410 chromosomal DNA by PCR. Sequencing the PCR product showed that the nucleotide sequence of glaA was 2167 bp located in the middle of the amplified 3327 bp DNA fragment. The deduced protein was 640 amino acids. The PCR product was purified and subcloned into the Sma I site of pBC-Hygro, creating pBC-Hygro-glaA.The recombinant plasmid was transformed into A. niger F0410 by PEG-mediated protoplast transformation which was constructed in the research. One hundred and twenty three transformants were obtained on the selective plates containing hygromycin B. Six of the transformants were subjected to characterize extensively the glucoamylase production in shake flask fermentations and glaA copy numbers. The results revealed an optimal integrated copy number of glaA for the maximum yield of glucoamylase. It was found that 2-3 multiples of glaA were optimal for the highest expression of glucoamylase. The transformants carrying more than 8 multiples of glaA were found to have slightly elevated glucoamylase activity. After several times of screening, transformant GB0506 was selected for the further research.Several culture media were tested at different conditions to optimize glucoamylase production with recombinant A. niger GB0506. The optimal shake-flask culture turned out to be 30 g/L yeast extract, 20 g/L cottonseed, 2 g/L ammonium nitrate and 80 g/L glucose at initial pH 5.5 with 8% inoculation. Time course of glucoamylase production with A. niger GB0506 was carried out in shake-flask fermentation at optimal conditions. The glucoamylase yield with the transformant GB0506 was steadily higher than parental strain F0410 during the process. The enzyme secreted by transformant GB0506 was 17.5% higher than parental strain F0410 at the end of fermentation, which was更多还原