Detection and characterization of sorbitol dehydrogenase from apple callus tissue

Sorbitol dehydrogenase (L-iditol:NAD+ oxidoreductase, EC 1.1.1.14) has been detected and characterized from apple (Malus domestica cv. Granny Smith) mesocarp tissue cultures. The enzyme oxidized sorbitol, xylitol, L-arabitol, ribitol, and L-threitol in the presence of NAD. NADP could not replace NAD. Mannitol was slightly oxidized (8% of sorbitol). Other polyols that did not serve as substrate were galactitol, myo-inositol, D-arabitol, erythritol, and glycerol. The dehydrogenase oxidized NADH in the presence of D-fructose or L-sorbose. No detectable activity was observed with D-tagatose. NADPH could partially substitute for NADH. Maximum rate of NAD reduction in the presence of sorbitol occurred in tris(hydroxymethyl)aminomethane-HCl buffer (pH 9), or in 2-amino-2-methyl-1,3-propanediol buffer (pH 9.5). Maximum rates of NADH oxidation in the presence of fructose were observed between pH 5.7 and 7.0 with phosphate buffer. Reaction rates increased with increasing temperature up to 60 C. The Km for sorbitol and xylitol oxidation were 86 millimolar and 37 millimolar, respectively. The Km for fructose reduction was 1.5 molar. Sorbitol oxidation was completely inhibited by heavy metal ions, iodoacetate, p-chloromercuribenzoate, and cysteine. ZnSO4 (0.25 millimolar) reversed the cysteine inhibition. It is suggested that apple sorbitol dehydrogenase contains sulfhydryl groups and requires a metal ion for full activity.