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

克隆了东方Amycolatopsis orientalis的外显子β-glucosaminidase基因，测序并在lividans链霉菌TK24中表达。从培养上清液中纯化的酶检测其反应特异性。除了G1cN-G1cN外，该酶还水解G1cN-G1cNAc的β-1,4-糖苷键。Asp469和Glu541的定点突变表明，Asp469是质子供体，而Glu541是亲核试剂。为了更有效地分析酶促反应，我们使用了连续流ESI-MS。ESI-MS测定的低聚糖底物酶解的时间过程与HPLC测定的结果一致。此外，这种时间过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ESI-MS是表征酶促反应的有效方法。

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.