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

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。

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.