Purification and characterization of α-galactosidase from Lactobacillus acidofillus

  • M Farzadi
  • S Khatami
  • M Mousavi
  • N Amirmozafari
Keywords: a-Galactosidase, enzyme purification, Lactobacillus acidofillus, kinetic studies.


α−Galactosidase (α-D-galactoside galactohydrolase [EC]) was obtained from Lactobacillus acidofillus which was grown in modified de Man, Rogosa and Sharpe (MRS) medium, supplemented with raffinose. α-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. Km and Vmax for this enzyme was determined by p-nitrophenyl-α-D-galactoside as substrate, to be about 0.47 mM, and 17.54 μmol/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 α-galactosidase activity was found in the presence of 0.1 mM HgCl.

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


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eISSN: 1684-5315