Purification and characterisation of beta-glucosidase from olive (Olea europaea L.)

Abstract

b-glucosidase is a glucosidase enzyme that acts uponb1->4bonds linking two glucose or glucose-substituted molecules (i.e.,the disaccharide cellobiose). It is an exocellulase with specificityfor a variety of beta-D-glycoside substrates. It catalyzes thehydrolysis of terminal non-reducing residues in beta-D-glucosideswith release of glucose. An olive(Olea europaeaL.)b-glucosi-dase was purified to apparent homogeneity by salting out withammonium sulfate and using specifically designed sepharose-4B-L-tyrosine-1-napthylamine hydrophobic interaction chromatogra-phy. The purification was 197.23 fold with an overall enzymeyield of 76.61%. The molecular mass of the protein was esti-mated as 65 kDa. The purifiedb-glucosidase was effectivelyactive on p-/o-nitrophenyl-b-D-glucopyranosides (p-/o-NPG)with K(m) values of 5.6 mM andV(max) values of 666 6667 U/mg. The enzyme was competitively inhibited by NaOH and sitricacid against p-NPG as substrate. The IC50 values of NaOH weredetermined as 68 160 mM while the enzyme was more tolerant tositric acid inhibition with IC50 values of 61 073 mM respectively,for p-NPG.