HPLC/RI与HPLC/ESI-MS方法研究细菌D-97酶合成海藻糖的过程

Study on the Production of Trehalose by Bacterium D-97 Endocellular Enzymes Using HPLC/RI and HPLC/ESI-MS

通过高效液相色谱 /示差折光检测系统 (HPLC/RI)分析可获得细菌D 97利用糊精或淀粉水解物合成海藻糖的基本生物学信息 ,包括微生物培养碳源对细菌D 97胞内海藻糖合成酶系的影响以及该酶系利用不同种类或不同分子链长度的麦芽寡糖合成海藻糖的能力及作用过程。采用HPLC与RI及电喷雾电离质谱 (ESI MS)联用并结合其他生物学手段对由细菌D 97获得的纯酶组分 (酶A)作用产物进行定量和定性分析 ,从而基本明确了D 97胞内酶合成海藻糖的过程。

The mechanism in which trehalose is produced from dextrin or starch hydrolyzate by endocellular enzymes of bacterium D 97 can be elucidated by high performance liquid chromatography (HPLC) with differential refraction detection (RI) basically, including the effect of the different carbon sources on the endocellular trehalose producing enzymes in bacterium D 97 and the possibility or ability of the endocellular enzymes to produce trehalose using maltooligosaccharides of different chain lengths. After purification of endocellular enzymes of bacterium D 97, two enzymes (called Enzyme A and Enzyme B) related to trehalose synthesis were found. The unknown oligosaccharides produced by Enzyme A were analyzed with the HPLC/RI and HPLC/ESI MS(electrospray ionization mass spectrometry). The results showed the relative molecular masses of the unknown oligosaccharides were the same as those of the enzymatic reaction substances (maltotriose, maltotetraose and maltopentaose) respectively. In combining with other results of biological experiments, these unknown oligosaccharides had been identified basically. There was no reduction power in these unknown oligosaccharides and only one trehalose residue exited in the molecular chain of these unknown oligosaccharides.