Characterization of Catalytic Phenotype of \beta-Galactosidase From LacI Mutant, E. Coli CSH-36, as a Tool For The Management of Lactose Intolerance

Authors: AYŞEGÜL AYYILDIZ

Abstract: Objectives: Lactose intolerance is a common public health problem with 5-100% prevalance among different populations of the world. Use of \beta-galactosidase to reduce the lactose content in food or consumption of special products of milk or exogeneous lactase enzyme are among the approaches taken to manage lactose intolerance. Recombinant \beta -galactosidase as well as \beta -galactosidase derived from different sources have variances in their bioefficiency. Thermodynamic properties of \beta -galactosidase determines the industrial efficiency. In this \beta -galactosidase from E. coli CSH-36, a constitutive expressor of enzyme was investigated for enzyme biofunctionality. Methods: The enzyme was purified by cell lysis, ammonium sulphate precipitation and anion exchange chromatography. Purity was assesed by SDS-PAGE and Western Blotting and the native molecular weight was determined by rate zonal sedimentation. Results & Conclusions E. coli \beta -galactosidase is characterized as a 410 kD protein consisting of 3 x 120 kD trimeric subunits complexed to a 50 kD peptide with a specific activity of 170 U/mg protein and 17% yield with the applied procedure. The Km app is 1 x 10-3 M for the substrate ONPG being slightly lower than the data in literature On further investigation of thermodynamics of the reaction for the substrate lactose, E. coli CSH-36 may serve as a new source for in vivo and in vitro hydrolysis of lactose.

Keywords: lactose intolerance \beta -Galactosidase, E. coli, purification, kinetics.

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