Keto Dgluconate Dehydrogenase Membrane Bound

2-Keto-D-gluconate ^ 2,5-diketo-D-gluconate

The enzyme catalyzing 2-ketogluconate oxidation, yielding 2,5-diketo-D-gluconate, has been characterized as a flavohemoprotein with three different subunits [99]. From the membrane fraction of Gluconobacter melanogenus IFO 3293, 2-keto-D-gluconate dehydrogenase (KGDH) is solubilized and purified with high yield to a homogeneous state examined by the criteria of electrophoresis and analytical ultracentrifugation. The purified KGDH is homogeneous in analytical ultracentrifugation, with a sedimentation coefficient of 5.9 S, and also in native-PAGE, with a single protein band. After solubilization of the enzyme performed with 2% Na-cholate and 0.2 mol L-1 KCl, two-step column chromatography gave a purified enzyme. The molecular mass was measured to be 133 kDa and SDS-PAGE shows the presence of three different subunits of 61 kDa (flavoprotein), 47 kDa (cytochrome c), and 25 kDa. The flavoprotein contained a covalently bound FAD [93].

Purified KGDH has a characteristic deep rose-red color due to the cytochome component. The typical reduced cytochrome c type absorption spectrum has maxima at 554, 523, and 417 nm. The cytochrome component of the enzyme is reduced on addition of either 2-keto-D-gluconate or sodium dithionite. A

successful purification of KGDH came from the finding of fundamental differences in hydrophobicity between D-gluconate dehydrogenase (GADH) (see Section 1.4.2) and KGDH. KGDH was not solubilized in the absence of a chaotropic agent, KCl. With Brij 35 and Tween 80, even in the presence of KCl, KGDH was scarcely solubilized, but GADH was solubilized with a final recovery of 100%. Thus, the membrane fraction of G. melanogenus IFO 3293 was treated first with 2% Brij 35 and 0.2 mol L-1 KCl to remove GADH, and after centrifugation the precipitate containing KGDH was collected. The resulting membrane precipitates were suspended in 0.01 mol L-1 Tris-HCl, pH 8.0, and treated with Triton X-100, Na-cholate, or Na-deoxycholate for 3 h at 5 °C. KGDH was solubilized in the presence of 0.3 mol L-1 KCl together with 2% Triton X-100. Alternatively, solubilization of KGDH was carried out overnight with 2% Na-cholate and 0.2 mol L-1 KCl, which was favorable to the following purification procedure involving ammonium sulfate fractionation.

The enzyme activity of KGDH is most active at pH 4.0, and 2-keto-D-gluconate is the only substrate oxidized by the enzyme. Similar compounds such as 5-keto-D-gluconate, 2-keto-D-galactonate, and 2-keto-D-gulonate are inert. KGDH catalyzes 2-keto-D-gluconate oxidation to 2,5-diketo-D-gluconate, which is an important step of bioconversion in a novel pathway to L-ascorbate developed by Sonoyama et al. [100]. D-Glucose was first converted to Ca-2,5-diketo-D-gluconate by a mutant strain of Erwinia sp. with 94.5% yield after 26 h cultivation. Then, a mutant strain of Coprynebacterium sp. reduced 2,5-diketo-D-gluconate stereospe-cifically with 2,5-diketo-D-gluconate reductase to Ca-2-keto-L-gulonate with a yield of 84.5%. The occurrence of KGDH is known in strains of Erwinia, Pantoea, and Pseudomonas, as well as Gluconobacter. Ameyama and Kondo noted the importance of 2,5-diketo-D-gluconate as the precursor of D-lyxuronic acid [101].

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Heal Yourself With Qi Gong

Qigong also spelled Ch'i Kung is a potent system of healing and energy medicine from China. It's the art and science of utilizing breathing methods, gentle movement, and meditation to clean, fortify, and circulate the life energy qi.

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