title: A β-galactosidase from chick pea (Cicer arietinum) seeds: its purification, biochemical properties and industrial applications.
abstract: 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 K(m) value of 1.73 mM at 37°C. The energy of activation (E(a)) 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. K(m) and E(a) for lactose hydrolysis was found to be 10mM 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.
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India.
authors.forename: Devesh
authors.initials: D
authors.lastname: Kishore
authors.forename: Arvind M
authors.initials: AM
authors.lastname: Kayastha
mesh_terms: D000595:Amino Acid Sequence; D029863:Cicer / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012639:Seeds / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D001616:beta-Galactosidase; D005690:Galactose