title: Purification and characterization of an intracellular beta-glucosidase from the methylotrophic yeast Pichia pastoris.
abstract: Pichia pastoris beta-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate, gel filtration, and ion-exchange chromatography with Q-Sepharose and CM-Sepharose. The enzyme is a tetramer (275 kD) made up of four identical subunits (70 kD). The pH optimum is 7.3, and it is fairly stable in the pH range 5.5-9.5. The temperature optimum is 40 degrees C. The purified beta-glucosidase is effectively active on p-/o-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with Km values of 0.12, 0.22, and 0.096 mM and Vmax values of 10.0, 11.7, and 6.2 micromol/min per mg protein, respectively. It also exhibits different levels of activity against p-nitrophenyl-1-thio-beta-D-glucopyranoside, cellobiose, gentiobiose, amygdalin, prunasin, salicin, and linamarin. The enzyme is competitively inhibited by gluconolactone, p-/o-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), and glucose against p-NPG as substrate. o-NPF is the most effective inhibitor of the enzyme activity with Ki value of 0.41 mM. The enzyme is more tolerant to glucose inhibition with Ki value of 7.2 mM for p-NPG. Pichia pastoris has been employed as a host for the functional expression of heterologous beta-glucosidases and the risk of high background beta-glucosidase activity is discussed.
authors.affiliation: Balikesir University, Arts and Sciences Faculty, Department of Biology, Balikesir, 10100, Turkey. yturan3@yahoo.com
authors.forename: Y
authors.initials: Y
authors.lastname: Turan
authors.forename: M
authors.initials: M
authors.lastname: Zheng
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010843:Pichia / Q000201:enzymology*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D001617:beta-Glucosidase