Isolation and Characterization of the Cyanide-Resistant and Azide-Resistant Catalase of Lactobacillus plantarum

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Abstract
Johnston , M. A. (Cornell University, Ithaca, N.Y.), and E. A. Delwiche . Isolation and characterization of the cyanide-resistant and azide-resistant catalase of Lactobacillus plantarum . J. Bacteriol. 90: 352–356. 1965.— Lactobacillus plantarum T-1403-5 has been shown to possess a very active cyanide- and azide-resistant catalase. By means of fractional ammonium sulfate precipitation, removal of nucleic acids with protamine sulfate, adsorption on calcium phosphate gel, and p H gradient chromatography on diethylaminoethyl cellulose, the catalase “activity” was purified approximately 14-fold. The purified enzyme preparation was insensitive to the heme poisons cyanide and azide, the metal chelating agents ethylenediaminetetraacetate and o -phenanthroline, and the sulfhydryl binding agent p -chloromercuribenzoate. The purified enzyme moved at a uniform rate in the electrophoretic field (isoelectric point, p H 4.7). The ultraviolet-light absorption spectrum was negative for heme-iron components, and fluorescence measurements yielded negative results with regard to flavin components. Acriflavin and Atabrine had no effect on enzyme activity. The nonheme catalase displayed a much broader p H range of activity than the heme-iron catalase of a control culture of Escherichia coli and the azide-sensitive catalase developed by L. plantarum NZ48 when grown in the presence of preformed hematin. The nonheme catalase was more resistant to heat inactivation. No retention of the enzyme on a chromatographic column could be obtained with Sephadex 200, nor could the enzyme be separated from crystalline beef-liver catalase by the gel filtration technique. Sedimentation was obtained in a centrifugal field of 144,000 × g for 12 hr.