Induction and Characterization of β-Galactosidase in an Extreme Thermophile

Abstract

A thermostable β-galactosidase (EC 3.2.1.23; β- d galactoside 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 α-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 p H for activity were determined to be 80 C and p H 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic β-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K m of 2.0 × 10 −3 m for the chromogenic substrate o -nitrophenyl β- d -galactopyranoside (ONPG) and a K 1 of 7.5 × 10 −3 m 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 × 10 5 daltons was estimated by elution of the enzyme from Sephadex 4B.