Indian Journal of Biotechnology Vol 5, April 2006, pp 184-188 β-Galactosidase from Lactobacillus acidophilus isolated from fermented ragi (Eleusine coracana) S K Akolkar, A D Sajgure and S S Lele* Food and Fermentation Technology Department, Mumbai University Institute of Chemical Technology Matunga, Mumbai 400 019, India Received 2 August 2004; revised 6 May 2005; accepted 5 July 2005 Lactobacillus acidophilus was isolated from fermented millet, Eleusine coracana. It was characterized using several biochemical tests. This strain was found to be homofermentative, slime forming and β-galactosidase producer. Recovery and characterization of β-galactosidase were studied at laboratory scale. Since the enzyme was intracellular various cell lysis methods were studied to achieve maximum enzyme release from cell fragments. Homogenization at a pressure of 2000 psig two passes showed an enzyme release of 2050 U/mL and was found the best method for cell lysis. The cell extract was purified using ultrafiltration and gel permeation chromatography. Specific activity and fold purification of β-galactosidase was found to be 568.61 and 21.2, respectively. Kinetic parameters were determined using ONPG (o-nitrophenyl galactopyranoside) as a substrate. Michaeli’s Menten equation was found to fit the reaction of ONPG using β-galactosidase from L. acidophilus. Vmax and Km were calculated from the Lineweaver Burk plot. Vmax was found to be 4.94 min -1 and Km to be 0.11 mM. Using gel permeation chromatography, the molecular weight was found to be in the range of 450-500 kDa. Keywords: Lactobacillus acidophilus, Eleusine coracana, β-galactosidase IPC Code: Int. Cl. 8 C12N 19/38; C12R1:23 Introduction Ragi (Eleusine coracana Gaertn.) is a millet grown in India as a subsistence crop and is predominantly used by rural population for consumption 1,2 . Fermentation of ragi provides an environment conducive for the growth of lactic acid bacteria (LAB) along with yeast. These microorganisms ferment ragi at different stages. One of the LAB viz. lactobacilli are known to possess health-promoting attributes including anti-carcinogenic 3 . Lactobacilli are indispensable in the manufacture and preservation of many fermented food products and are involved in the protection of young animals against intestinal infections. Lactobacilli are reported to show diverse behaviour and, hence, attracted attention as a potential source of new applications as well as for enzyme production 3,4 . In this work, Lactobacillus was isolated from fermented ragi. The strain was found to be homofermentative, slime forming and produced β- galactosidase or Lactase (β-D-galactohydrolase EC 3.2.1.23) in significant quantities. β-Galactosidase is a hydrolytic enzyme, which hydrolyses the terminal non-reducing β-D-galactoside residues to form β- galactosides to glucose and galactose, which are simple monosaccharides. β-galactosidase is one of the important commercial enzymes having several applications in food and pharmaceutical industry. Lactose free milk for consumption by lactose intolerant persons is produced using this enzyme 5-7 . This enzyme is not commercially manufactured in India. Hence this work was undertaken. The present work focuses on the recovery and characterization of β-galactosidase that was obtained by laboratory scale fermentation of Lactobacillus acidophilus isolated from fermented ragi. Since the enzyme is intracellular, various cell lysis methods were tried to achieve maximum enzyme release from the cells. Molecular weight determination was done by gel permeation chromatography. Michaeli’s Menten equation and Lineweaver-Burk plot were applied to study the kinetic parameters of β- galactosidase. The β-galactosidase obtained in the present study was compared with a commercial sample of β-galactosidase from Kluveromyces fragilis. _______________ *Author for correspondence: Tel: 91-22-24145616, ext-345. Fax: 91-22-2414 5614 E-mail: sslele@udct.org