Purification and Properties of Putrescine Oxidase of Micrococcus rubens

A procedure for obtaining a highly purified preparation of the putrescine oxidase of Micrococcus rubens has been developed. The method involved fractionation with ammonium sulfate and separation on successive columns of DEAE-cellulose, DEAE-sephadex and hydroxylapatite. The enzyme was purified about 200-fold from the cell extract and the final preparation showed a symmetric peak in the ultracentrifugal analysis. The purified enzyme was yellow in color and showed absorption maxima at about 380 and 460 mμ. The yellow color was lost by the substrate as well as by sodium dithionite, and was subsequently restored by oxygenation. The purified enzyme contained 12.2 mμ moles of flavin adenine dinucleotide (FAD) per mg of protein. The enzyme was inhibited by p-chloromercuric benzoate.

The putrescine oxidase could oxidatively degrade spermidine into 1,3-diaminopropane and γ-aminobutyraldehyde stoichiometrically, and no ammonia was liberated.