Exo-β-glucosaminidase from Amycolatopsis orientalis, : Reaction mechanism and Application to industrial sugar production

来自东方无枝酸菌的外切-β-氨基葡萄糖苷酶，：反应机理及其在工业糖生产中的应用

• 批准号: 17580085
• 负责人: FUKAMIZO Tamo
• 金额: $ 2.5万
• 依托单位: Kinki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17580085/
• 关键词: chitosan; glucosaminidase; Reaction Specificity; catalytic residue; real-time monitoring; 質量分析; グルコサミダーゼ; 部位特異的変異; 反応機構; 分泌発現

Exo-β-glucosaminidase gene from Amycolatopsis orientalis was cloned, sequenced, and expressed in Streptomyces lividans TK24. The purified enzyme from the culture supernatant was tested for its reaction specificity. The enzyme was found to hydrolyze the β-1,4-glycosidic linkage of G1cN-G1cNAc in addition to G1cN-G1cN. Site-directed mutagenesis of Asp469 and Glu541 revealed that Asp469 is a proton donor and Glu541 a nuclephile. To more efficiently analyze the emzymatic reaction, we used continuous flow ESI-MS. The time-courses of the enzymatic hydrolysis of oligosaccharide substrates obtained by ESI-MS were found to be consistent with those obtained by HPLC. Furthermore, the amounts of the enzyme and substrate required for such a time-course determination were much lower than those required for determination by HPLC. The continuous flow ESI-MS was found to be efficient for characterizing the enzymatic reaction.

克隆了东方拟无枝酸菌（Amycolatopsis orientalis）外切β-氨基葡萄糖苷酶（Exo-β-glucosaminidase）基因，并在变铅青链霉菌（Streptomyces lividans）TK 24中进行了表达。从培养物上清液中纯化的酶用于其反应特异性的测试。发现该酶除了水解G1 cN-G1 cN外，还水解G1 cN-G1 cNAc的β-1，4-糖苷键。Asp 469和Glu 541的定点突变显示Asp 469是质子供体，Glu 541是亲核试剂。为了更有效地分析酶促反应，我们使用连续流动ESI-MS。ESI-MS获得的寡糖底物的酶促水解的时间过程被发现与HPLC获得的一致。此外，这种时程测定所需的酶和底物的量远低于HPLC测定所需的量。连续流动电喷雾质谱被认为是有效的表征酶促反应。

项目成果

Exo-β-D-glucosaminidase from Amyeolatopsis orientalis : catalytic residues, sugar recognition specificity, kinetics, and synergism.

来自东方拟淀粉的外切-β-D-氨基葡萄糖苷酶：催化残基、糖识别特异性、动力学和协同作用。

• 发表时间: 2006
• 作者: Fukamizo;T. Brzezinski;R.
• 通讯作者: R.

The role of Arg114 at subsites E and F in reactions catalyzed by hen egg-white lysozyme

• DOI: 10.1271/bbb.70694
• 发表时间: 2008-03-01
• 期刊: BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY
• 影响因子: 1.6
• 作者: Kawamura, Shunsuke;Chijiiwa, Yuki;Torikata, Takao
• 通讯作者: Torikata, Takao

Exo-?-D-glucosaminidase from Amycolatopsis orientalis : catalytic residues, sugar recognition specificity, kinetics, and synergism.

来自东方无枝酸菌的外切-β-D-氨基葡萄糖苷酶：催化残基、糖识别特异性、动力学和协同作用。

• 发表时间: 2006
• 期刊: Glycobiology 16
• 作者: Fukamizo;T. et al.
• 通讯作者: T. et al.

ソテツ由来FamilyGH-18キチナーゼの芳香族アミノ酸残基について

关于来自苏铁的FamilyGH-18几丁质酶的芳香族氨基酸残基

• 发表时间: 2007
• 作者: Ohnishi;Fukamizo;深溝 慶
• 通讯作者: 深溝 慶

Structure and Function of Exo-β-Glucosaminidase from Amycolatopsis orientalis

东方无枝酸菌外切-β-氨基葡萄糖苷酶的结构和功能

• 发表时间: 2007
• 期刊: J.Appl.Glycosci. 54
• 作者: Dennhart;N.;Fukamizo;T.;Letzel;T.;深溝 慶;深溝 慶;深溝 慶;深溝 慶;深溝 慶
• 通讯作者: 深溝 慶