title: Purification of beta-galactosidase from Erythrina indica: involvement of tryptophan in active site.
abstract: beta-Galactosidase (EC: 3.2.1.23), one of the glycosidases detected in Erythrina indica seeds, was purified to 135 fold. Amongst the four major glycosidases detected beta-galactosidase was found to be least glycosylated, and was not retained by Con-A CL Seralose affinity matrix. A homogenous preparation of the enzyme was obtained by ion-exchange chromatography, followed by gel filtration. The enzyme was found to be a dimmer with a molecular weight of 74 kDa and 78 kDa, by gel filtration and SDS-PAGE, respectively. The optimum pH and optimum temperature for enzyme activity were 4.4 and 50 degrees C, respectively. The enzyme showed a K(m) value of 2.6 mM and V(max) of 3.86 U/mg for p-nitrophenyl-beta-D-galactopyranoside as substrate and was inhibited by Zn(2+) and Hg(2+). The enzyme activity was regulated by feed back inhibition as it was found to be inhibited by beta-D-galactose. Chemical modification studies revealed involvement of tryptophan and histidine for enzyme activity. Involvement of tryptophan was also supported by fluorescence studies and one tryptophan was found to be present in the active site of beta-galactosidase. Circular dichroism studies revealed 37% alpha helix, 27% beta sheet and 38% random coil in the secondary structure of the purified enzyme.
authors.affiliation: Division of Biochemistry, Department of Chemistry, University of Pune, Pune-411007, Maharashtra, India.
authors.forename: Rakesh M
authors.initials: RM
authors.lastname: Kestwal
authors.forename: Shobhana V
authors.initials: SV
authors.lastname: Bhide
mesh_terms: D001665:Binding Sites; D001974:Bromosuccinimide / Q000737:chemistry / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004338:Drug Combinations; D004895:Erythrina / Q000201:enzymology*; D008970:Molecular Weight; D009821:Oils; D010636:Phenols; D012639:Seeds / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D004338:Drug Combinations; D009821:Oils; C027222:P & S Liquid; D010636:Phenols; D014364:Tryptophan; D001616:beta-Galactosidase; D001974:Bromosuccinimide