Purification, recovery, and characterization of chick pea (Cicer arietinum) β-galactosidase in single step by three phase partitioning as a rapid and easy technique

摘要

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 38 kDa, 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 (E_a) was calculated by using Arrhenius equation and determined to be 15.52 kcal mol~(-1). K_m value of purified enzyme was estimated for the o-nitrophenyl β-d galactopyranoside (ONPG) substrate as 1.09 mM, while its maximum velocity, V_m was 0.90 U/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.