Thompson J, Gentry-Weeks C R, Nguyen N Y, Folk J E, Robrish S A
Laboratory of Micobial Ecology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA.
J Bacteriol. 1995 May;177(9):2505-12. doi: 10.1128/jb.177.9.2505-2512.1995.
6-Phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase (6-phospho-alpha-glucosidase) has been purified from Fusobacterium mortiferum ATCC 25557. p-Nitrophenyl-alpha-D-glucopyranoside 6-phosphate (pNP alpha Glc6P) served as the chromogenic substrate for detection and assay of enzyme activity. The O2-sensitive, metal-dependent phospho-alpha-glucosidase was stabilized during purification by inclusion of dithiothreitol and Mn2+ ion in chromatography buffers. Various 6-phosphoryl-O-alpha-linked glucosides, including maltose 6-phosphate, pNP alpha Glc6P, trehalose 6-phosphate, and sucrose 6-phosphate, were hydrolyzed by the enzyme to yield D-glucose 6-phosphate and aglycone moieties in a 1:1 molar ratio. 6-Phospho-alpha-glucosidase (M(r) of approximately 49,000; pI of approximately 4.9) is activated by Fe2+, Mn2+, Co2+, and Ni2+, and the maximum rate of pNP alpha Glc6P hydrolysis occurs at 40 degrees C within the pH range 7.0 to 7.5. The sequence of the first 32 amino acids of 6-phospho-alpha-glucosidase exhibits 67% identity (90% similarity) to that deduced for the N terminus of a putative phospho-beta-glucosidase (designated ORF f212) encoded by glvG in Escherichia coli. Western blots involving highly specific polyclonal antibody against 6-phospho-alpha-glucosidase and spectrophotometric analyses with pNP alpha Glc6P revealed only low levels of the enzyme in glucose-, mannose-, or fructose-grown cells of F. mortiferum. Synthesis of 6-phospho-alpha-glucosidase increased dramatically during growth of the organism on alpha-glucosides, such as maltose, alpha-methylglucoside, trehalose, turanose, and palatinose.
6-磷酸-O-α-D-吡喃葡萄糖基：6-磷酸葡糖水解酶（6-磷酸-α-葡糖苷酶）已从死亡梭杆菌ATCC 25557中纯化出来。对硝基苯基-α-D-吡喃葡萄糖苷6-磷酸（pNPαGlc6P）用作检测和测定酶活性的显色底物。在纯化过程中，通过在色谱缓冲液中加入二硫苏糖醇和Mn2+离子，使对O2敏感、依赖金属的磷酸-α-葡糖苷酶得以稳定。各种6-磷酸-O-α-连接的葡糖苷，包括麦芽糖6-磷酸、pNPαGlc6P、海藻糖6-磷酸和蔗糖6-磷酸，被该酶水解，以1:1的摩尔比产生6-磷酸-D-葡萄糖和糖苷配基部分。6-磷酸-α-葡糖苷酶（分子量约为49,000；pI约为4.9）被Fe2+、Mn2+、Co2+和Ni2+激活，pNPαGlc6P水解的最大速率在40℃、pH值7.0至7.5范围内出现。6-磷酸-α-葡糖苷酶前32个氨基酸的序列与大肠杆菌中由glvG编码的假定磷酸-β-葡糖苷酶（命名为ORF f212）N端推导的序列具有67%的同一性（90%的相似性）。涉及针对6-磷酸-α-葡糖苷酶的高度特异性多克隆抗体的蛋白质免疫印迹以及用pNPαGlc6P进行的分光光度分析表明，在以葡萄糖、甘露糖或果糖生长的死亡梭杆菌细胞中，该酶的水平很低。在该生物体以α-葡糖苷（如麦芽糖、α-甲基葡糖苷、海藻糖、松二糖和帕拉金糖）为碳源生长期间，6-磷酸-α-葡糖苷酶的合成显著增加。
