Kinetic studies of extracellular α galactosidase from Citrobacter freundii

Abstract

Extracellular a-galactosidase producing bacteria were isolated from soil. Bacteria that showed a-galactosidase activity were identified as Escherichia coli, Klebsiella pneumonrae and Citrobacter freundii by morphological and biochemical tests. Citrobacter freundii showed the highest enzyme production of 19 milliunitsl ml after 36 h r s of cultivation i n p H 8 phosphate buffer containing peptone. a-Galactosidase from Citrobacter freundii was purified by ammonium sulphatefractionation and DEAE ion exchange chromatography. One a-galactosidase activity peak was observed indicating the presence of a single enzyme form. A 164 fold purification was obtained with a yield of 8%. Polyacrylamide gel electrophoresis of the enzyme showed 2 protein bands. The kinetic properties of the enzyme were studied using p-nitrophenyl a-D-galactopyranoside. The Michaelis constant and maximum reaction velocity obtained were 2.85 x M and 14/mmol/min/mg of protein respectively. Studies on the effect of pH on enzyme activity showed a broad pH optimum from 6.0 to 8.0 with maximum activity a t pH 7.5 a t 2g°C. The enzyme was stable between pH 5.5 to 8.0. The optimum enzyme activity was observed a t 40% a t pH 7.5. The enzyme was stable upto 40 OC. The enzyme preparation did not contain any invertase activity.