Characterization of endogenous carbohydrates extracted from recombinant enzyme preparations.

Recombinant proteins were denatured and treated with amyloglucosidase prior to glucose estimation. Heterologous expression were either performed in E. coli BL21(DE3) strains or in glycogen deficient ΔglgCAP cells with the same genetic background. For characterization of endogenous glucans enzyme preparations expressed in BL21(DE3) were denatured and treated with isoamylase. Debranched products were analysed by HPAEC-PAD. For evaluation of data different chain length peaks were grouped. Only DP populations (DP2–7, DP8–13, and DP14–20) were tested for significance among the enzyme preparations.Differences were significant with * P = 0.05, ** P = 0.02 or *** P = 0.01 (rSSI with rBE2 and rBE3). Differences (DP-2-7 and DP8–13) between rBE2 and rBE3 were significant with P = 0.05. Average values and standard deviation of four independent experiments for each preparation are given. a) Ratio between nmol glucose and nmol protein. n.d. – not detectable.在进行葡萄糖定量检测前，需先对重组蛋白（recombinant protein）进行变性处理，并使用淀粉葡萄糖苷酶（amyloglucosidase）进行酶解处理。 异源表达（heterologous expression）可在大肠杆菌BL21(DE3)菌株（E. coli BL21(DE3)）或具有相同遗传背景的糖原缺陷型ΔglgCAP细胞中完成。 为表征内源性葡聚糖（endogenous glucans），对在BL21(DE3)中表达的酶制剂进行变性处理，并使用异淀粉酶（isoamylase）进行脱支处理。脱支产物通过高效阴离子交换色谱-脉冲安培检测法（High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection，简称HPAEC-PAD）进行分析。 为开展数据分析，将不同链长的色谱峰进行分组。仅针对聚合度（Degree of Polymerization，简称DP）区间DP2~7、DP8~13及DP14~20的组分，在各酶制剂之间开展显著性检验。 差异具有统计学意义的标记规则为：*对应P=0.05，**对应P=0.02，***对应P=0.01（该显著性结果适用于rSSI与rBE2、rSSI与rBE3的比较）。rBE2与rBE3之间在DP2~7和DP8~13区间的差异同样具有统计学意义，P=0.05。 文中给出了每种酶制剂四次独立重复实验的平均值与标准偏差。 a) 葡萄糖纳摩尔数与蛋白质纳摩尔数的比值；n.d. 表示未检出（not detectable）。