D-GLUCOSE DEHYDROGENASE OF GLUCONOBACTER-SUBOXYDANS - SOLUBILIZATION, PURIFICATION AND CHARACTERIZATION
D-GLUCOSE DEHYDROGENASE OF GLUCONOBACTER-SUBOXYDANS - SOLUBILIZATION, PURIFICATION AND CHARACTERIZATION
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DOI：
10.1080/00021369.1981.10864633
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
ADACHI, O
中科院分区：
文献类型：
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作者：
AMEYAMA, M;SHINAGAWA, E;ADACHI, O
从弱氧化葡糖杆菌[氧化葡糖杆菌弱氧化亚种]IFO 12528的细胞膜中将D - 葡萄糖脱氢酶纯化至均一。该酶用Triton X - 100从膜中溶解出来，并用SE - 纤维素和羟基磷灰石柱进行分级分离。在酶纯化的初始步骤中，通过在相当酸性的pH条件下处理酶，去除了与D - 葡萄糖脱氢酶共溶解的其他种类的膜结合脱氢酶。纯化后的酶在分析超速离心和蔗糖密度梯度离心中是均一的。该酶的表观沉降常数为4.2S，通过尿素 - 十二烷基硫酸钠凝胶电泳显示分子量为87,000。对纯化的酶制剂进行的光谱研究表明，一种新的辅基吡咯并喹啉醌参与了该酶的作用。在该酶催化的D - 葡萄糖氧化反应中，吩嗪硫酸甲酯是最佳的电子受体。以铁氰化钾为电子受体时，D - 葡萄糖氧化的最适pH为3.0；以吩嗪硫酸甲酯、2,6 - 二氯酚靛酚和伍斯特蓝为电子受体时，最适pH为6.0。该酶的底物特异性似乎仅限于D - 葡萄糖，除麦芽糖以较低速率被氧化外，其他糖类均不被氧化。从细菌膜典型整合蛋白的角度讨论了该酶的疏水性。
D-Glucose dehydrogenase was purified to homogeneity from the membrane of G. suboxydans [G. oxydans ssp. suboxydans] IFO 12528. The enzyme was solubilized from the membrane with Triton X-100 and fractionated with SE-cellulose and hydroxyapatite columns. Other species of membrane-bound dehydrogenases which were cosolubilized with D-glucose dehydrogenase were eliminated by treating the enzyme at fairly acidic pH in the initial step of enzyme purification. The purified enzyme was homogeneous in analytical ultracentrifugation and sucrose density gradient centrifugation. The enzyme had an apparent sedimentation constant of 4.2 S and showed a MW of 87,000 by urea-sodium dodecyl sulfate gel electrophoresis. The spectral studies with the purified enzyme preparation showed the participation of pyrroloquinoline quinone, a new prosthetic group, in the enzyme. Phenazine methosulfate was the best electron acceptor in D-glucose oxidation with the enzyme. The optimum pH of D-glucose oxidation was at pH 3.0 with potassium ferricyanide, and pH 6.0 with phenazine methosulfate, 2,6-dichlorophenolindophenol and Wurster''s blue as electron acceptor. The substrate specificity of the enzyme seemed to be restricted to D-glucose, and other sugars were not oxidized except for maltose which was oxidized at a low rate. The hydrophobicity of the enzyme was discussed from the standpoint of a typical integral protein of the bacterial membrane.