Application of microbial β-glucan phosphorylases

构建了热胞梭菌纤维素二糖磷酸化酶（CBPase）和纤维素糊精磷酸化酶（CDPase）基因的表达载体。经这些质粒转化的大肠杆菌表现出相应的酶活性。在优化培养温度、培养时间和宿主菌株等条件后，大肠杆菌转化菌的CBPase和CDPase产量分别为130和930 mg/l，分别是热细胞杆菌的30倍和440倍。这些酶经电泳纯化均质。重组酶的酶学性质和理化性质与天然酶无显著差异。研究了酶法将淀粉转化为纤维素低聚糖的方法。以马铃薯α-葡聚糖磷酸化酶为原料，以淀粉为原料进行磷解反应制备葡萄糖-1-磷酸。以葡萄糖-1-磷酸为葡萄糖供体，葡萄糖为受体，通过CBPase和CDPase的逆反应，以淀粉为原料，得到了总收率为1.73%的纤维低聚糖混合物。此外，酶促合成纤维素低聚糖在其还原性末端被发色团标记。DPase以葡萄糖-1-磷酸为供体，对硝基-β- d -葡萄糖苷为受体合成对硝基苯基纤维素低聚糖，产率为59%。本研究表明，CBPase和CDPase不仅可以用于纤维素低聚糖的酶促合成，还可以用于纤维素低聚糖类似物的酶促合成，这对纤维素酶的研究具有重要意义。

The expression vectors for cellobiose phosphorylase (CBPase) and cellodextrin phosphorylase (CDPase) genes from Clostridium thermocellum were constructed. E. coli transformed by these plasmid exhibited the corresponding enzyme activity. After optimizing the conditions such as cultivation temperature or time and a host strain, amounts of CBPase and CDPase produced by E. coli transformants were 130 and 930 mg/l, respectivery, which corresponding 30- and 440- fold higher than those by C. thermocellum. These enzymes were purified to homogeneity on electrophoresis. There was no defference on enzymatic and physicochemical properties between the recombinant and native enzymes.The method of enzymatic conversion of starch to cellooligosaccharides were examined. Glucose-1-phosphate was prepared from starch by phsphorolysis reaction with potato α-glucan phosphorylase. Using reverse reaction of CBPase and CDPase with glucose-1-phosphate as a glucose donor and glucose as an acceptor, cellooligosaccharides mixture was produced with the total yield from starting material (starch) of 1.73%. Also, enzymatic synthesis of cellooligosaccharide labeled at its reducing end by chromophore was examined. DPase was able to synthesize p-nitrophenyl-cellooligosaccharides using glucose-1-phosphate as a glucose donor and p-nitrophyl-β-D-glucoside as an acceptor with the yield of 59%.Here, we showed that CBPase and CDPase can be used for enzymatic synthesis of not only cellooligosaccharides but also cellooligosaccharide analogue which could be useful for the studies on cellulases.