Characterization of Invertase Purified from Prevotella oralis Ig4a

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Intracellular and extracellular invertase-like enzymes were purified from Prevotella oralis (formerly Bacteroides oralis) Ig4a by combination of several ion-exchange chromatography and gel filtration steps. The intracellular enzyme was purified as an electrophoretically homogeneous protein, and the extracellular enzyme was partially purified. The molecular weights of intra- and extracellular enzymes were estimated to be 79,000 and 53,000, respectively, by SDS-polyacrylamide gel electrophoresis. Both enzymes specifically cleaved sucrose and raffinose, but not melezitose and other polysaccharides tested, implying that the enzymes are invertases (EC 3.2.1.26; β-D-fructofranoside fructohydrolase). The intra- and the extracellular invertases had optimal pHs of approximately 6.5 and 5.5, optimal temperature of 35°C and 55°C, and Km values of 14.2 mM and 66.7 mM, respectively. These enzymes were inhibited strongly by Hg²⁺, and weakly inhibited by Zn²⁺ and Fe³⁺. The antiserum against the purified intracellular invertase revealed by Ouchterlony analysis that the two invertases were antigenically related