【作者】 陶雪莹；

【导师】 喻子牛； 李泳春；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 硕士

【摘要】 糖苷酶是作用于各种糖苷或寡糖并催化糖苷键水解的水解酶类总称。糖苷酶第13家族中的α-淀粉酶有重要的商业价值,可广泛应用于淀粉加工处理、酿造业、酒精生产、纺织业、去污剂以及其它工业。其中琼脂酶分成α-琼脂酶和β-琼脂酶,后者可产生新琼寡糖;新琼寡糖在食品、化妆品、医疗卫生等方面有重要作用。本论文分别从假交替单胞菌(Pseudoalteromonas)中克隆到了淀粉酶基因和琼脂酶基因,并对它们进行了表达及特性鉴定。从海洋微生物假交替单胞菌菌株MY-1中克隆得到一个α-淀粉酶基因(amyA),并将该基因在大肠杆菌中进行表达。基因amyA包括一个2,007bp的开放阅读框,所编码蛋白质由668个氨基酸组成,分子量为74kDa;等电点为5.15。amyA基因所编码蛋白质AmyA的氨基酸全序列与其它淀粉酶序列比对结果显示,该蛋白与来自于嗜冷假交替单胞菌Pseudoalteromonas haloplanktis的前蛋白原具有86%的同源性。蛋白序列包含了一个α-淀粉酶催化区域和Amy C区域。将淀粉酶AmyA纯化到电泳纯,并对纯化后蛋白进行生化特性鉴定。结果显示:AmyA在pH 7.0和40℃条件下酶活性最高。此外,对AmyA进行了底物及相应产物分析,该淀粉酶可将可溶性淀粉及各种麦芽寡糖水解生成分子量更小的不同寡糖,同时麦芽糖是该淀粉酶水解可溶性淀粉及各种麦芽寡糖的共同产物。此外,实验结果证明还证明,钙离子在淀粉酶AmyA的酶催化反应中起到重要作用。本实验还从另一假交替单胞菌菌株JT-6中克隆到一个胞外β-琼脂酶基因(agaA6),并将该基因在大肠杆菌中进行了表达。基因agaA6包含一个1,338bp的开放性阅读框,所编码蛋白由445个氨基酸残基组成,分子量为50kDa.该基因所编码蛋白AgaA6的氨基酸全序列与来自于詹森菌属(Janthinobacterium sp.)菌株SY12的琼脂酶基因agaA所编码蛋白有99%的同源性。该蛋白AgaA6在结构上由三部分组成:信号肽序列、糖苷水解酶家族16的β-琼脂酶催化域、糖底物结合域。融合蛋白rAgaA6在大肠杆菌中得到超量表达并纯化至电泳纯。纯化后的融合酶最适温度为40℃,最适pH为9.0。该酶为内切型β-琼脂酶,可将琼脂糖水解成不同的低聚糖。更多还原

【Abstract】 Glycosidases catalyze the hydrolysis of the glycosidic linkage,α-Amylases are classified as family 13 of the glycosidases;they are among the most important commolercial enzymes,have wide applications in starch processing,brewing,alcohol production, textile,detergent and other industries.Agarases,catalyzing the hydrolysis of agar,are characterized asα-agarases,β-agarases;β-agarases produce neoagarooligosaccharides which have potential applications for the food,cosmetic,pharmaceutical and medical industries.In this study,two glycosidase genes were cloned from Pseudoalteromonas species;they were expressed and characterized.A gene（amyA） encoding an extracellularα-amylase from marine bacterium, Pseudoalteromonas sp.MY-1,was cloned and expressed in Escherichia coli.It comprised an open reading frame of 2,007 base pairs and encodes a protein of 668 amino acids with a predicted molecular weight of 74 daltons and a pI of 5.15.The entire amino acid sequence of amyA gene showed 86%similarity to theα-amylase preproprotein from Pseudoalteromonas haloplanktis.It consists of a signal peptide,α-amylase catalytic domain and an Amy_C domain.The recombinant amylase was purified to homogeneity and biochemically characterized.The enzyme revealed maximum activity at pH 7.0 and 40℃.The enzyme hydrolyzed soluble starch and some maltooligosaccharides to several oligosaccharides,and maltose was the commolon product from different substrates.The results also suggest that Ca²⁺ plays important roles inα-amylase.A gene（agaA6） encoding an extracellular agarase from Pseudoalteromonas sp.JT-6,was cloned,sequenced and expressed in Escherichia coli.It comprised an open reading frame of 1,338 base pairs and encodes a protein of 445 amino acids with a predicted molecular weight of 50 daltons.The entire amino acid sequence of this agarase gene showed 99% identity with the agaA gene from Janthinobacterium sp.SY12.It consists of a signal peptide,a glycoside hydrolase family 16β-agarase domain and a carbohydrate-binding domain.The recombinant agarase was overexpressed and purified to homogeneity. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant enzyme were around 40℃and 9.0.The enzyme was an endo-typeβ-agarase and hydrolyzed agarose to several oligosaccharides.更多还原