Preparation of High-Mannose type Oligosaccharides with mannosidases.

本研究的目的是开发N-类高甘露糖型糖链骨架寡糖的制备方法。首先，我们分离了一种产生α-1，2-甘露糖苷酶的土壤细菌。将酶纯化至均一并表征。该酶的分子量为380 Kda，由190 Kda蛋白的两个同源亚基组成。该酶特异性水解α-1，2-甘露糖苷连接的寡糖，并释放D-甘露糖。该酶不水解pNP-或甲基-α-甘露糖苷。利用该甘露糖苷酶以D-甘露糖为底物，通过酶的反向作用制备α-1，2-甘露二糖和α-1，2-甘露三糖。为了得到α-1，3-甘露寡糖，我们还发现了一种新的α-1，3-甘露聚糖特异性酶。从土壤样品中分离到一株革兰氏阴性细菌，菌株9，该生物体分泌α-1，3-甘露聚糖酶以产生α-1，3-甘露寡糖。酶被部分纯化。反应的最终产物是α-1，3-甘露二糖。上述结果表明，α-1，2-和α-1，3-甘露寡糖可以使用两种细菌酶进行酶促制备。

The aim of this study is to develop the preparation method of the skeleton oligosaccharides of N-liked high-mannose type suger chains. First, we isolated a soil bacterium that produced alpha-1,2-mannosidase extracellulary. The enzyme was purified to homogeneity and characterized. The enzyme had a molecular weight of 380 Kda consisting of two homologous subunits of 190 Kda protein. The enzyme hydrolyzed specifically alpha-1,2-mannosidic linked oligosaccharides and D-mannose was liberated. The enzyme did not hydrolyze pNP- or methyl-alpha-mannoside. Using this mannosidase, the alpha-1,2-mannobiose and alpha-1,2-mannotriose were prepared by the reverce action of the enzyme with D-mannose as substrate. To obtainalpha-1,3-linked manno-oligosaccharides, we also found a new enzyme specific for alpha-1.3-mannan. A gram-negative bacterium, strain No. 9 was isolated from the soil samples and the organism secreted alpha-1,3-mannanase to produce alpha-1,3-manno-oligosaccharides. The enzyme was partially purified. The final product of the reaction was alpha-1,3-mannobiose. Above results indicate that alpha-1,2- and alpha-1,3-manno-oligosaccharides can be prepared enzymatically using the two bacterial enzymes.