Purification and Properties of NAD⁺-dependent Sorbitol Dehydrogenase from Bacillus fructosus

Abstract

  Sorbitol dehydrogenase (EC 1.1.1.14), which catalyzes the NAD⁺-linked interconversion of D-sorbitol and D-fructose, was purified and crystallized from cell-free extracts of Bacillus fructosus grown on D-sorbitol as a sole carbon source. The crystalline enzyme was homogeneous on disc electrophoresis and ultracentrifugation. The molecular weight was 102,000 by the sedimentation equilibrium method. The enzyme acted specifically on D-sorbitol, and showed an optimum pH at 9.0. The K_m values for D-sorbitol and NAD⁺ were 1.1×10^-2 M and 2.2×10^-4 M, respectively. The enzyme activity was inhibited by p-chloromercuribenzoate, Ag⁺, Hg²⁺, and Cu²⁺.