麦芽四糖淀粉酶基因优化表达及酶学性质分析  

Optimization of Expression and the Character Ization of a G4-amylase Enzyme

作　　者：赵云[1] 朱蓓霖[1] 汪正华[1] 周杰[1] 吴自荣[1] 黄静[1] 

机构地区：[1]华东师范大学生命科学学院,上海200241

出　　处：《中国生物工程杂志》2013年第5期100-106,共7页China Biotechnology

摘　　要：麦芽四糖淀粉酶是一种新型外切淀粉酶,从淀粉的非还原末端特异地顺序切割第4个α-1,4糖苷键,产物为麦芽四糖,广泛应用于食品、医疗保健等领域。对来自嗜糖假单胞菌(Pseudomonas saccharophila)的麦芽四糖淀粉酶基因序列进行优化,优化前后基因序列同源性达75%。将优化合成的成熟肽基因克隆至原核表达载体pET32a(+)上,转化大肠杆菌BL21(DE3),经IPTG诱导,重组蛋白主要以包涵体形式存在。包涵体经变性、复性、多步纯化,获得有活性的麦芽四糖淀粉酶。将麦芽四糖淀粉酶与不同来源的淀粉水解反应,结果表明,该酶能与7种不同来源的淀粉反应产生单一的麦芽四糖。经SDS-PAGE电泳,DNS法和硅胶板薄层色谱分析法(TLC)进行酶学性质分析,结果表明麦芽四糖淀粉酶的分子量约为57kDa,纯化后的酶液最适反应温度为45℃,最适反应pH为8.0。研究结果为麦芽四糖淀粉酶的研究和开发提供依据和参考。G4-amylase catalyses hydrolysis of 1,4-α-D-glucosidic linkages in amylaceous polysaccharides to remove successive mahotetraose residues from the non-reducing chain ends. As a new type of exo-amylase, it is widely used in food, medical care and other fields. The enzyme＇ s gene sequence from Pseudomonas stutzeri was optimized and it showed 75% homology with the wild gene sequence. The optimized gene was synthesized and then cloned into expression vector pET32a （ ＋ ）. The recombinant protein was shown to be expressed in Escherichia coli BL21 （DE3） as inclusion bodies. By protein refolding and purification, gel electrophoresis homogeneous and active protein which had a molecular weight of 57kDa （ by SDS-PAGE）. Hydrolysis product analysis of seven kinds sources of starch showed that only mahotetraose were presented. By DNS determination. The optimum temperature is 45 ℃, the optimal pH is 8.0. This research provided a new idea for heterologous expression of this particular food-enzyme using genetic engineering.

关 键 词：麦芽四糖淀粉酶 嗜糖假单胞菌 基因优化 酶学性质 

分 类 号：Q75[生物学—分子生物学]