title: Purification of beta-glucosidase from olive (Olea europaea L.) fruit tissue with specifically designed hydrophobic interaction chromatography and characterization of the purified enzyme.
abstract: An olive (Olea europaea L.) β-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate and using specifically designed sepharose-4B-L-tyrosine-1-napthylamine hydrophobic interaction chromatography. The purification was 155 fold with an overall enzyme yield of 54%. The molecular mass of the protein was estimated as ca. 65 kDa. The purified β-glucosidase was effectively active on p-/o-nitrophenyl-β-D-glucopyranosides (p-/o-NPG) with K(m) values of 2.22 and 14.11 mM and V(max) values of 370.4 and 48.5 U/mg, respectively. The enzyme was competitively inhibited by δ-gluconolactone and glucose against p-NPG as substrate. The K(i) and IC(50) values of δ-gluconolactone were determined as 0.016 mM and 0.23 mM while the enzyme was more tolerant to glucose inhibition with K(i) and IC(50) values of 6.4 mM and 105.5 mM, respectively, for p-NPG. The effect of various metal ions on the purified β-glucosidase was investigated. Of the ions tested, only the Fe(2+) increased the activity while Cd(2+) Pb(2+) Cu(2+), Ni(+), and Ag(+) exhibited different levels of inhibitory effects with K(i) and IC(50) values of 4.29×10(-4) and 0.38×10(-4), 1.26×10(-2) and 5.3×10(-3), 2.26×10(-4) and 6.1×10(-4), 1.04×10(-4) and 0.63×10(-4), 3.21×10(-3) and 3.34×10(-3) mM, respectively.
authors.affiliation: Department of Biology, Faculty of Art and Science, Balikesir University, Balikesir, Turkey. hatbertuk@yahoo.com
authors.forename: Hatibe Ertürk
authors.initials: HE
authors.lastname: Kara
authors.forename: Selma
authors.initials: S
authors.lastname: Sinan
authors.forename: Yusuf
authors.initials: Y
authors.lastname: Turan
mesh_terms: D000645:Ammonium Sulfate; D002853:Chromatography, Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005638:Fruit / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D019216:Metals, Heavy / Q000494:pharmacology; D031658:Olea / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013696:Temperature; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D004791:Enzyme Inhibitors; D019216:Metals, Heavy; D010940:Plant Proteins; D001617:beta-Glucosidase; D000645:Ammonium Sulfate