title: Purification, recovery, and characterization of chick pea (Cicer arietinum) β-galactosidase in single step by three phase partitioning as a rapid and easy technique.
abstract: In this study chick pea β-galactosidase was first time purified and recovered in single step by three phase partitioning (TPP). Optimal purification parameters for TPP were 60% ammonium sulfate saturation (w/v) with 1:0.5 (v/v) ratio of crude extract:t-butanol at pH 6.8, which gave 10.1 purification fold with 133% recovery of β-galactosidase. SDS-PAGE analysis showed that protein has two subunits with molecular masses of 48 and 38kDa, respectively. Characterization of enzyme showed that optimal pH of purified enzyme was 2.8 and optimal temperature was 50°C. Enzyme was further characterized by the Arrhenius activation energy and Michael-Menten kinetic constants. Activation energy (Ea) was calculated by using Arrhenius equation and determined to be 15.52kcalmol(-1). Km value of purified enzyme was estimated for the o-nitrophenyl β-d galactopyranoside (ONPG) substrate as 1.09mM, while its maximum velocity, Vm was 0.90U/mL/min at 37°C. TPP improved substrate affinity of enzyme by the increased flexibility during the partitioning. TPP is simple, easy and economic technique for purification and recovery of β-galactosidase from chick pea, and has a big potential use for industrial applications.
authors: Kocaeli University, Faculty of Arts and Sciences, Department of Chemistry, Section of Biochemistry, Umuttepe Campus, 41380 İzmit-Kocaeli, Turkey. Electronic address: yavci@kocaeli.edu.tr.
authors: Yonca Avci
authors: YA
authors: Duman
authors: Erdem
authors: E
authors: Kaya
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D029863:Cicer / Q000737:chemistry / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry / Q000201:enzymology; D013696:Temperature; D001616:beta-Galactosidase / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D020002:tert-Butyl Alcohol / Q000737:chemistry
chemical_list: D010940:Plant Proteins; D001616:beta-Galactosidase; D020002:tert-Butyl Alcohol; D000645:Ammonium Sulfate
keywords: Characterization; Purification; Recovery; Three phase partitioning; β-Galactosidase