Fructose Dehydrogenase Membrane Bound

D-Fructose ^ 5-keto-D-fructose

D-Fructose oxidation to 5-keto-D-fructose (5KF) is catalyzed by membrane-bound fructose dehydrogenase (FDH, EC 1.1.99.11), which contains a covalently bound FAD as the primary coenzyme. FDH was purified from the membrane fraction of G. industrius IFO 3260. FDH was solubilized from the membrane with 1% Triton X-100 and further purified to a homogeneous state [86]. The purified FDH had a sedimentation coefficient of 5.8S and the total molecular mass was estimated to be 140 kDa by gel filtration. The enzyme was dissociated into three different subunits, 67 kDa, 50 kDa, and 19 kDa, during gel electrophoresis. The largest subunit was positive to enzyme activity staining in a mixture containing PMS, nitroblue tetrazolium, and D-fructose.

When an unstained gel was irradiated with a fluorescent light, intense fluorescence was observed only with the largest subunit, indicating the presence of a covalently bound FAD as the coenzyme. The second subunit (50 kDa) was characterized as the subunit carrying cytochrome c. Regarding the smallest subunit, as with ADH III, GADH, KGDH, and other membrane-bound dehydrogenases, the actual function and properties of the subunit were unknown. However, if the smallest subunit is deleted, the enzyme activity of the individual enzymes is also halted and no accumulation of the corresponding oxidation product outside the cells is observed. Thus, the smallest subunit is essential to enzyme activity as well as to the oxidative fermentation.

It should be noted that only D-fructose was oxidized by FDH during the study of substrate specificity with various substrates. When D-fructose was oxidized in the presence of the same concentration of substrate analogs such as D-glucose, D-mannose, D-fructose-6-phosphate, D-fructose-1,6-diphosphate, 5-keto-D-fructose, D-glucose-6-phosphate, D-glucose-1-phosphate, D-gluconate, 2-keto-D-

gluconate, and 5-keto-D-gluconate, the reaction rate of D-fructose oxidation was not affected at all. These properties of FDH suggest that FDH could be beneficial as the enzyme for D-fructose microdetermination. As mentioned below, an apparent Michaelis constant determined at pH 4.5 was found to be 10 mmol L-1. The optimum pH for D-fructose oxidation was found at pH 4.0-4.5. The reaction product of D-fructose oxidation was identified to be 5-keto-D-fructose, which has no negative effect on D-fructose oxidation.

As has already mentioned above, the most characteristic point of oxidative fermentation is that D-fructose oxidation by FDH continues until the D-fructose added initially in the reaction mixture is oxidized completely to the reaction product; there is no reaction equilibrium, unlike NAD(P)-dependent dehydrogenases. With respect to the enzymatic method for D-fructose determination, a coupling enzymatic method involving hexokinase (EC 2.7.11), phospho-D-glucose isomerase (D-glucose-6-phosphate ketol isomerase, EC 5.3.1.9), and D-glucose-6-phosphate dehydrogenase (EC 1.1.1.49) is the sole reliable method so far reported

[87]. However, this assay method depends largely on the purity of the enzymes used and therefore is rather expensive. Difficulty in preparing the required enzymes without any contaminants could make this assay system complicated and potentially troublesome. A trace of D-glucose present in the samples for D-fructose assay invites inevitable confusion, because hexokinases react widely with many aldoses. The preparation of highly purified D-fructokinase that reacts with only D-fructose is almost impossible. Hence, the use of FDH is highly recommended instead of the method above, because FDH reacts only with D-fructose so can be used for the rate assay as well as for the end point measurement.

In this chapter, it is emphasized that PQQ- and FAD-dependent dehydroge-nases obtained from the membrane fraction of acetic acid bacteria and other aerobic bacteria are useful and convenient enzymes for biosensors monitoring ethanol, acetaldehyde, D-glucose, D-gluconate, 2-keto-D-gluconate, and so on. In the same way, FDH can be the enzyme for D-fructose measurement for clinical purposes

[88]. FDH could be beneficial for agriculture, for example in fruit production to check the best timing for ripening, and in fermentation industries for process control in brewing. FDH is now available on the market.

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