Mannitol oxidase: partial purification and characterization of the membrane-bound enzyme from the snail Helix aspersa

Abstract

Mannitol oxidase, a membrane-bound oxidase has been purified 250-fold from snail digestive gland tissue. The activity is solubilized by a number of ionic, non-ionic, and zwitterionic detergents. Purification of the solubilized enzyme was by polyethylene glycol fractionation and column chromatography using anionic exchange resins, hydroxylapatite, and gel filtration. The enzyme is stabilized by glycerol and remains active for at least one week at -20 degrees. Hydrogen peroxide is the oxygen reduction product and a mannose/hydrogen peroxide stoichiometry of 0.86 was found. D-Arabinitol and D-mannitol were the most active substrates of those tested. Results with these and other substrates suggest that the configuration around carbons-2 and -4 is critical for binding and reactivity. The apparent Km for D-mannitol is 6 mM and for oxygen, 40 microM. The pH optimum for the enzyme is between 8 and 8.5 and the isoelectric point is 5.4-5.6.