Purification of beta-glucosidase from olive (Olea europaea L.) fruit tissue with specifically designed hydrophobic interaction chromatography and characterization of the purified enzyme
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An olive (Olea europaea L) beta-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 beta-glucosidase was effectively active on p-/o-nitrophenyl-beta-b-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 delta-gluconolactone and glucose against p-NPG as substrate. The K-i and IC50 values of delta-gluconolactone were determined as 0.016 mM and 0.23 mM while the enzyme was more tolerant to glucose inhibition with Ki and IC50 values of 6.4 mM and 105.5 mM, respectively, for p-NPG. The effect of various metal ions on the purified beta-glucosidase was investigated. Of the ions tested, only the Fe2+ increased the activity while Cd2+ Pb2+ Cu2+, Ni+, and Ag+ exhibited different levels of inhibitory effects with Ki and IC50 values of 4.29 x 10(-4) and 0.38 x 10(-4), 1.26 x 10(-2) and 5.3 x 10(-3), 2.26 x 10(-4) and 6.1 x 10(-4), 1.04 x 10(-4) and 0.63 x 10(-4), 3.21 x 10(-3) and 3.34 x 10(-3) mM, respectively.