Purification and characterization of an intracellular ?-glucosidase from the methylotrophic yeast Pichia pastoris - [2]

Abstract

Pichia pastoris ?-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°C. The purified ?-glucosidase is effectively active on p-/o-nitrophenyl-?-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-?-D-glucopyranoside (4-MUG) with K m values of 0.12, 0.22, and 0.096 mM and V max values of 10.0, 11.7, and 6.2 ?mol/min per mg protein, respectively. It also exhibits different levels of activity against p-nitrophenyl-1-thio-?-D-glucopyranoside, cellobiose, gentiobiose, amygdalin, prunasin, salicin, and linamarin. The enzyme is competitively inhibited by gluconolactone, p-/o-nitrophenyl-?-D-fucopyranosides (p-/o-NPF), and glucose against p-NPG as substrate. o-NPF is the most effective inhibitor of the enzyme activity with K i 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 ?-glucosidases and the risk of high background ?-glucosidase activity is discussed.