Abstract

a-Galactosidase (a-D-galactoside galactohydrolase [EC 3.2.1.22]) was obtained from Lactobacillus acidofillus which was grown in modified de Man, Rogosa and Sharpe (MRS) medium, supplemented with raffinose. a-Galactosidase was released from the cells by ultrasonic treatment, then precipitated by ammonium-sulfate and further purified with Sephadex G-200 and DEAE cellulose chromatography with a 18.5-fold increase in specific activity and 28% recovery. K_mand V_max for this enzyme was determined by p-nitrophenyl-a-D-galactoside as substrate, to be about 0.47 mM, and 17.54 mmol/min per mg of protein, respectively. Maximum enzymatic activity occurred at pH 5.5 and temperature at 45°C. The enzymatic activity was retained at least for 30 min, at temperatures of 25 - 55°C, but there was inactive temperature at about 60°C. Galactose was able to decrease the enzyme activity by a factor of 63%. Among the sugars tested, fructose, glucose, sucrose, lactose and mannose reduced the enzyme activity only slightly (less than 10% of the control). A strong inhibition of a-galactosidase activity was found in the presence of 0.1 mM HgCl.

Key words: a-Galactosidase, enzyme purification, Lactobacillus acidofillus, kinetic studies.

Abbreviation

MRS, de Man, Rogosa and Sharpe medium; SDS, sodium dodecylsulphate.