Crystallization and Properties of 5-Keto-d-gluconate Reductase from Gluconobacter suboxydans
Crystallization and Properties of 5-Keto-d-gluconate Reductase from Gluconobacter suboxydans
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低氧化葡糖杆菌 5-酮-d-葡萄糖酸还原酶的结晶和性质
DOI：
10.1271/bbb1961.43.75
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
M. Ameyama
中科院分区：
文献类型：
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作者：
O. Adachi;E. Shinagawa;K. Matsushita;M. Ameyama
首次从低黄葡糖酸菌IFO 12528的无细胞提取物中纯化并结晶了5-酮-d-葡糖酸还原酶（EC 1.1.1.69）。经DEAE-Sephadex A-50、blue-dextran Sepharose 4 B柱层析和DEAE-Sephadex A-50 pH梯度层析，获得了较好的纯化效果。该酶纯化了约1200倍，总收率为40%。通过添加d-葡萄糖酸盐或5-酮基-d-葡萄糖酸盐，酶对加热和储存更加稳定。2-酮基-d-葡糖酸盐对稳定酶没有作用，并且证实该酶是与由葡糖酸盐或2-酮基-d-葡糖酸盐而不是5-酮基-d-葡糖酸盐稳定的2-酮基-d-葡糖酸盐还原酶不同的实体。还用结晶酶证实，5-酮基-d-葡糖酸还原酶被认为具有与NADP的再生结合将细胞内5-酮基-d-葡糖酸还原为d-葡糖酸的功能。
5-Keto-d-gluconate reductase (EC 1.1.1.69) was purified and crystallized for the first time from cell-free extract of Gluconobacter suboxydans IFO 12528. Purification of the enzyme was successfully performed by column chromatography on DEAE-Sephadex A–50, blue-dextran Sepharose 4B, followed by pH gradient chromatography on DEAE-Sephadex A–50. The enzyme was purified about 1200-fold with an overall yield of 40%. The enzyme was much stabilized against heating and storage by adding either d-gluconate or 5-keto-d-gluconate. 2-Keto-d-gluconate had no effect to stabilize the enzyme and the enzyme was confirmed to be a different entity from 2-keto-d-gluconate reductase stabilized by gluconate or 2-keto-d-gluconate but not 5-keto-d-gluconate. It was also confirmed with crystalline enzyme that 5-keto-d-gluconate reductase is considered to have a function to reduce intracellular 5-keto-d-gluconate to d-gluconate in combination with regeneration of NADP.