HPLC在研究微杆菌D-97胞内酶合成海藻糖机制中的应用

微杆菌D 97胞内酶对麦芽糖的水解微弱 ,并且合成海藻糖的酶反应对磷酸盐无依赖性。采用氨基键合柱HPLC对酶作用的初始底物麦芽寡糖混合物 (Ⅰ )、酶作用 2 4h后未糖化的底物 (Ⅱ )及其糖化水解后的底物 (Ⅲ )分别进行检测对照 ,底物Ⅰ麦芽五糖占34 1 5 % ,底物Ⅱ海藻糖、三糖和五糖分别占 1 7 68%、37 63 %和 3 0 8% ,底物Ⅲ的 2种主要产物葡萄糖和海藻糖各占 77 57%、2 0 2 0 %。通过上述 3种底物二糖至六糖浓度变化的比较 ,发现 DP 值 >4的麦芽寡糖为微杆菌D - 97胞内酶合成海藻糖的较适底物 ,并推断麦芽五糖生成海藻糖的基本方式为 :麦芽五糖→麦芽三糖 +海藻糖。以麦芽五糖为底物的酶反应研究进一步证实了上述作用方式。对于筛选得到的野生菌酶 ,HPLC对照检测从 1个角度为其酶合成海藻糖基本机制研究提供 1种方法。

The Application of HPLC in Investigating Trehalose Synthetic Mechanism by Endocellular Enzymes of Microbacterium D-97

Endocellular enzymes of bacterium D 97 show a weak hydrolytic activity towards maltose. Enzymatic synthetic reaction of trehalose using maltooligosaccharides obtained from partial hydrolysis of corn starch shows that it is a phosphate independent process HPLC analysis of maltooligosaccharides as initial substrate (Ⅰ), 24?h endocellular enzymes treated substrates, unsaccharified(Ⅱ)and saccharified(Ⅲ)reveal 34 15% maltopentaose in substrateⅠ,17 68%, 37 63% and 3 08% trehalose, triose and pentaose respectively in substrateⅡand 77 57%, 20 20% glucose and trehalose respectively in substrateⅢ It is therefore concluded, by comparisons of the concentration changes in maltooligosaccharides of DP2～6, that those of DP>4 are the suitable substrates and that the basic mode of producing trehalose from maltopentaose is as follows: maltopentose→maltotriose + trehalose The results of enzymatic reaction using maltopentaose also demonstrate the basic mechanism above