National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
Appl Microbiol Biotechnol. 2012 Feb;93(4):1513-22. doi: 10.1007/s00253-011-3515-9. Epub 2011 Aug 2.
A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of D-glucose and β-D-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k(cat) = 67 s(-1) and K(m) = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using D-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on D-xylose, 1,5-anhydro-D-glucitol, D-galactose, and methyl-α-D-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with D-xylose and methyl-α-D-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-D-glucosyl-D-glucose:phosphate β-D-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.
一株新型植酸梭菌磷酸化酶的特性研究。该酶属于糖苷水解酶家族 65（GH65）（Cphy1874）。在无机磷酸盐存在的情况下，重组 Cphy1874 蛋白对棉子糖表现出磷酸解活性，导致 D-葡萄糖和β-D-葡萄糖 1-磷酸（β-G1P）的释放，同时导致端基构型的反转。该酶对棉子糖的磷酸解活性的动力学参数为 k(cat) = 67 s(-1)和 K(m) = 1.7 mM。该酶除了对昆布二糖表现出较弱的磷酸解活性外，不能磷酸解典型 GH65 酶的底物，如海藻糖、麦芽糖和海藻糖 6-磷酸。它在以 D-葡萄糖为受体和β-G1P 为供体的反转反应中表现出最高的反转磷酸解活性，并且在反应的早期，产物主要是棉子糖。该酶在 D-木糖、1,5-脱水-D-葡萄糖醇、D-半乳糖和甲基-α-D-葡萄糖苷的反转磷酸解活性依次降低。所有主要产物均为α-1,3-葡糖苷二糖，尽管与 D-木糖和甲基-α-D-葡萄糖苷的反应会产生大量的α-1,2-糖苷作为副产物。我们提议 3-α-D-葡糖基-D-葡萄糖：磷酸β-D-葡萄糖基转移酶作为该 Cphy1874 蛋白的系统名，并用棉子糖磷酸化酶作为简称。
