Application of microbial β-glucan phosphorylases

构建了热纤梭菌纤维二糖磷酸化酶（CBR 2）和纤维糊精磷酸化酶（CDR 2）基因的表达载体。E.转化的大肠杆菌表现出相应的酶活性。通过对培养温度、培养时间、宿主菌等条件的优化，测定了E.大肠杆菌转化子的最低抑菌浓度分别为130和930 mg/l，分别是C.热纤这些酶经电泳纯化至均一。重组酶的酶学性质和理化性质与天然酶无明显差异。用马铃薯α-葡聚糖磷酸化酶水解淀粉制备葡萄糖-1-磷酸。以1-磷酸葡萄糖为葡萄糖供体，葡萄糖为受体，利用CBF和CDF的逆反应，以总收率1.73%的淀粉为原料，制备了纤维低聚糖混合物。此外，纤维低聚糖的酶促合成标记在其还原端的发色团进行了检查。结果表明，CBB 2和CDB 3不仅可用于纤维寡糖的酶促合成，还可用于纤维寡糖类似物的酶促合成，为纤维素酶的研究奠定了基础。

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.