A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification, biochemical properties and industrial applications

A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg−1, with approximately 1840 fold purification. Analysis of the protein by SDS–PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC–MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a Km value of 1.73 mM at 37 °C. The energy of activation (Ea) calculated in the range of 35 to 60 °C, using Arrhenius equation, was determined to be 11.32 kcal mol−1. The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40 °C. Km and Ea for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol−1, respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60 °C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology.