Crystallization and Characterization of NADP-Dependent D-Glucose Dehydrogenase from Gluconobacter suboxydans
Crystallization and Characterization of NADP-Dependent D-Glucose Dehydrogenase from Gluconobacter suboxydans
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低氧化葡糖杆菌中 NADP 依赖性 D-葡萄糖脱氢酶的结晶和表征
DOI：
10.1271/bbb1961.44.301
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
M. Ameyama
中科院分区：
文献类型：
--
作者：
O. Adachi;K. Matsushita;E. Shinagawa;M. Ameyama
首次从亚氧化氧葡萄糖杆菌IFO 12528胞浆中结晶出nadp依赖性d -葡萄糖脱氢酶（EC 1.1.1.47）。采用DEAE-Sephadex A-50柱层析和blue-dextran Sepharose 4B亲和层析对酶进行了纯化。该酶被纯化约1800倍，总收率为30%。结晶酶制备在圆盘凝胶电泳和超离心分析中均质。该酶对NADP具有高度特异性，对NAD完全无活性。在d-葡萄糖氧化生成d-葡萄糖-δ-内酯的过程中产生的NADPH被存在于生物体同一细胞质组分中的老黄酶再氧化为NADP。在d-葡萄糖脱氢酶、老黄酶和过氧化氢酶存在的情况下，即使反应混合物中存在少量的NADP或NADPH， NADP的循环再生也能顺利进行。
NADP-Dependent D-glucose dehydrogenase (EC 1.1.1.47) was crystallized for the first time from cytosol fraction of Gluconobacter suboxydans IFO 12528. Purification of the enzyme was successfully performed by column chromatography on DEAE-Sephadex A-50 and affinity chromatography by blue-dextran Sepharose 4B. The enzyme was purified about 1,800-fold with an overall yield of 30%. Crystalline enzyme preparation was homogeneous in disc gel electrophoresis and analytical ultracentrifugation. The enzyme was highly specific for NADP and completely inactive with NAD. NADPH yielded in d-glucose oxidation to d-glucono-δ-Iactone was reoxidized to NADP by the old yellow enzyme which existed in the same cytosol fraction of the organism. Cyclic regeneration of NADP occurred smoothly in the presence of d-glucose dehydrogenase, old yellow enzyme and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.