Isolation and characterization of L-sorbose dehydrogenase from gluconobacter melanogenus ATCC 15163

Abstract

The L-sorbose dehydrogenase which catalyzes the oxidation of L-sorbose to L-sorbosone was isolated from G. melanogenus ATCC 15163 by fractional sedimentation as a membrane-bound particulate form and its general properties were characterized. It was found that the enzyme consumes oxygen or reduces certain dyes such as PMS, MB, and $K_3Fe(CN)_6$ as electron acceptors for the specific oxidation of L-sorbose to L-sorbosone. The stoichiometry of the enzyme reaction was established; L-sorbose + 1/2 $O_2 → L-sorbosone + $H_2O$. The enzyme activity was assayed colorimetrically by measuring the production of L-sorbosone using MBTH and $FeCl_3$. This colorimetric method was very specific and sensitive. The enzyme was found to be extremely specific toward L-sorbose with a Km value of 7.7 mM and it showed slight binding affinities for D-glucose and D-xylose which have a D-xylo configuration. The maximum activity was obtained at pH 7.5 and 45 ℃. The enzyme activity was completely inhibited by heavy metal ions such as $Ag^{+2}$, $Hg^{+2}$, and $Mo^{+2}$. No requirement of coenzymes and free metal ions was found. It is suggested that the enzyme is linked to the cytochrome system in the respiratory chain. Also, a sequential kinetic mechanism was shown in the binding of two substrate, L-sorbose and oxygen, to the complex in which the anzyme is linked to the respiratory chain.