Purification and Characterization of an Intracellular beta-Glucosidase from the Methylotrophic Yeast Pichia pastoris

---

Y. Turan^1* and M. Zheng²

¹Balikesir University, Arts and Sciences Faculty, Department of Biology, 10100 Balikesir, Turkey; fax: (90-266) 245-6366; E-mail: yturan3@yahoo.com

²Department of Molecular Physiology and Cell Biology, University of Virginia, 22903-0208 VA, USA; fax: (1-434) 982-1616; E-mail: meiyingzheng@yahoo.com

^* To whom correspondence should be addressed.

Received August 31, 2004; Revision received September 5, 2005

---

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°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 K_m values of 0.12, 0.22, and 0.096 mM and V_max values of 10.0, 11.7, and 6.2 µmol/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 K_i value of 0.41 mM. The enzyme is more tolerant to glucose inhibition with K_i 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.

---

KEY WORDS: beta-glucosidase, Pichia pastoris, yeast enzyme, purification

---