权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis of Thermostability and Function of Olygomeric α-Glucosidases

寡聚α-葡萄糖苷酶的热稳定性和功能分析

基本信息

批准号：
11660095
负责人：
SUZUKI Yuzuru
金额：
$ 2.3万
依托单位：
Kyoto Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The gene that coded for the subunit of an molecular weight (Mr) 540,000 tightly-subunit-associated homohexameric α-glucosidase II (α-D-glucoside glucohydrolase, EC 3.2.1.20) with both exo-α-1, 4-glucosidase and oligo-1, 6-glucosidase (dextrin 6-α-D-glucanohydrolase, EC 3.2.1.10) activities produced by Bacillus thermoamyloliquefaciens growing at 30 to 66℃ was expressed in Escherichia coli HB101. The resulting homohexameric enzyme had a half-life of 10 min at 80℃. Its purification and characterization showed that the enzyme was identical with the native one except for the latter deleting 7 N-terminal residues found in the former. The primary sequence of the subunit with 787 residues and an Mr of 91,070 deduced from the gene was 32% and 24-34% identical to the corresponding sequences of the subunit (770 residues ; Mr 88,620) of an Mr 530,000 subunit-dissociable/reassociable α-glucosidase III specific for maltosaccharides from the same microorganism and of 15 α-glucosidases in the glycosyl hydrolase family 31 from 14 eukaryotic origins and the archaeon Sulfolobus solfataricus 98/2, respectively.From the sequence analysis by the neural network method of Rost and Sander[Rost, B.and Sander, C., Proteins : Struct. Funct. Genet.. 19, 55-72 (1994)], we inferred that α-glucosidase II like α-glucosidase III might make each subunit of 3 secondary structural regions, i.e., one N-terminal β region, one central α/β or α+β region with two catalytic residues Asp407 and Asp484 (equivalent to Asp383 and Asp451 in α-glucosidase III), and one C-terminal β region, and that this architecture of a β(α/β)β or β(α+β)β sandwich motif might hold for other α-glucosidases in the family 31.

在大肠杆菌HB 101中表达了编码分子量（Mr）为540，000的紧密亚基缔合的同型六聚体α-葡萄糖苷酶II（α-D-葡萄糖苷葡萄糖水解酶，EC 3.2.1.20）的基因，该基因由在30 - 66℃下生长的热解淀粉芽孢杆菌产生，具有外切-α-1，4-葡萄糖苷酶和寡聚-1，6-葡萄糖苷酶（糊精6-α-D-葡萄糖苷水解酶，EC 3.2.1.10）活性。所得同六聚体酶在80℃下的半衰期为10 min。纯化和鉴定结果表明，该酶除N端7个残基缺失外，其余与天然酶完全相同。该亚基的一级序列为787个氨基酸残基，分子量为91，070，与相应的亚基序列的同源性分别为32%和24- 34（770个残基;先生88，620）的先生530，000个亚单位的可解离/可再结合的α-葡萄糖苷酶III对来自相同微生物的麦芽糖醇具有特异性，根据通过Rost和Sander的神经网络方法的序列分析[Rost，B.和Sander，C.，蛋白质：结构、功能Genet.. 19，55-72（1994）]，我们推断α-葡糖苷酶II像α-葡糖苷酶III一样可能使每个亚基具有3个二级结构区，即，一个N-末端β区，一个具有两个催化残基Asp 407和Asp 484（相当于α-葡糖苷酶III中的Asp 383和Asp 451）的中心α/β或α+β区，以及一个C-末端β区，并且这种β（α/β）β或β（α+β）β夹心基序的结构可能适用于家族31中的其他α-葡糖苷酶。