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Induction and Characterization of ß-Galactosidase in an Extreme Thermophile

^a Department of Botany and Microbiology, Montana State University, Bozeman, Montana 59715

ABSTRACT

A thermostable ß-galactosidase (EC 3.2.1.23; ß-Dgalactoside galactohydrolase) was found to be inducible in an extreme thermophile resembling Thermus aquaticus. Enzyme induction was achieved by the addition of lactose, galactose, or the -galactoside, melibiose, to growing cultures. The addition of glucose to induced cultures had a repressive effect on further enzyme synthesis. The enzyme was purified 78-fold, and the optimum temperature and pH for activity were determined to be 80 C and pH 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic ß-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K_m of 2.0 x 10^–3M for the chromogenic substrate o-nitrophenyl ß-D-galactopyranoside (ONPG) and a K₁ of 7.5 x 10^–3M for lactose. The Arrhenius energy of activation (for the hydrolysis of ONPG) was calculated to be 13.7 kcal/mole. A molecular weight of 5.7 x 10⁵ daltons was estimated by elution of the enzyme from Sephadex 4B.

¹ Present address: Department of Microbiology and Biochemistry, Idaho State University, Pocatello, Idaho 83201.

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