Purification and properties of α,α-trehalase from the mucosa of rat small intestine

Abstract

α,α-Trehalase (EC 3.2.1.28, α,α-trehalose glucohydrolase) was solubilized from the microvillous membrane of the intestinal mucosa of rats with Triton X-100 and butanol. It was purified 6350-fold by gel filtration on Sephadex G-150 and chromatography on DE-52 and hydroxyapatite. The purified enzyme, with a specific activity of about 127 units per mg of protein, showed almost a single band of protein and activity on polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 96 000 on Sephadex G-150 and 90 000 and sodium dodecyl sulfate-polyacrylamide gel electrophoressi. Its pH optimum was 5.5–5.7 and its K m value for trehalose was 5.4 mM. Its activity was inhibited 30 and 100% by 1 mM p-chloromercuribenzoate and 0.1 mM HgCl 2, respectively and 30% by 1 mM MgCl 2. Moreover, its activity was inhibited completely by 10 mM tris(hydroyxmethyl)aminomethane and about 60% by 10 mM sucrose and cellobiose. The enzyme showed a high specificity for trehalose.