Purification and characterization of a Bacillus sp. SAM1606 thermostable α-glucosidase with transglucosylation activity
Purification and characterization of a Bacillus sp. SAM1606 thermostable α-glucosidase with transglucosylation activity
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具有转葡萄糖基活性的芽孢杆菌 SAM1606 耐热 α-葡萄糖苷酶的纯化和表征
DOI：
10.1007/bf00221229
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发表时间：
1994
影响因子：
5
通讯作者：
Y. Shibano
中科院分区：
文献类型：
--
作者：
M. Nakao;T. Nakayama;M. Harada;A. Kakudo;H. Ikemoto;S. Kobayashi;Y. Shibano
我们从转化嗜热芽孢杆菌α-葡萄糖苷酶基因的大肠杆菌中分离纯化了一种新的α-葡萄糖苷酶。SAM1606。该酶以启动子蛋白的单聚体和多聚体形式存在，相对分子质量为64,000，等电点为4.6。我们分离了这种酶的一种单体形式，并对其进行了表征。在已知的α-葡萄糖苷酶中，该酶在广泛的底物专一性和高热稳定性方面是独一无二的。该酶能有效地降解黑糖、麦芽糖、异麦芽糖、蔗糖和海藻糖等多种α-d-吡喃葡萄糖苷。在55°C和pH 6.0时，蔗糖的分子活度(Ko)和米氏常数(Km)分别为54.6 S−1和5.3 mM。最适pH为5.5，最适温度为75℃。该酶具有较高的葡萄糖转移活性：在60℃下与1.8M蔗糖反应70h，得到含茶籽糖的低聚糖，最高得率为35%(w/w)。该酶具有很高的热稳定性(在pH 7.2下稳定到65℃，10分钟)，允许在高温下进行葡萄糖转移反应，这将有利于蔗糖连续生产低聚糖。
We purified a novel α-glucosidase to homogeneity from anEscherichia colirecombinant transformed with the α-glucosidase gene from thermophilicBacillussp. SAM1606. The enzyme existed as mono- and multimeric forms of a promoter protein with a relative molecular weight of 64,000 and isoelectric point of 4.6. We isolated a monomeric form of the enzyme and characterized it. The enzyme was unique among the known α-glucosidases in both broad substrate specificity and high thermostability. The enzyme hydrolysed a variety ofO-α-d-glucopyranosides such as nigerose, maltose, isomaltose, sucrose, and trehalose efficiently. The molecular activity (kO) and the Michaelis constant (Km) values at 55°C and pH 6.0 for sucrose were 54.6 s−1and 5.3 mm, respectively. The optimum pH and temperature for hydrolysis were pH 5.5 and 75°C, respectively. The enzyme exhibited a high transglucosylation activity: it reacted with 1.8msucrose at 60°C for 70 h to yield oligosaccharides containing theanderose in a maximum yield of 35% (w/w). High thermostability of the enzyme (stable up to 65°C at pH 7.2 for 10 min) permits the transglucosylation reaction at high temperatures, which would be beneficial for continuous production of oligosaccharides from sucrose.