Abstract
This chapter discusses the determination of mannitol (cytochrome) dehydrogenase. D-Mannitol: cytochrome oxidoreductase is extracted from Acetobacter suboxydans as a particulate preparation that contains several other enzymatic activities. Mannitol dehydrogenase can be assayed by measuring the rate of oxygen uptake at the optimum pH (5.0). It can be assayed anaerobically by linking the dehydrogenation of mannitol to phenol blue and measuring the rate of reduction of the dye (decrease in absorbance at 655 mμ) at pH 5.4. Acetobacter suboxydans (American Type Culture Collection, No. 621) is grown at 30° for 3 days in a medium containing 0.5% of Difco yeast extract, 0.025 M potassium phosphate buffer, pH 5.5, and 2.5% of glycerol (or mannitol or sorbitol). The cultures (100 ml of medium in 500-ml flasks) are aerated by continuous shaking. The cells are harvested by centrifuging and washed three times with water. The enzyme oxidizes a wide range of acyclic polyols containing the D- erythro configuration (Bertrand–Hudson rule).
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