Purification and characterization of two β-glucosidases from a thermo-tolerant yeast Pichia etchellsii

The thermo-tolerant yeast Pichia etchellsii produced two cell-wall-bound inducible beta-glucosidases, BGLI (molecular mass 186 kDa) and BGLII (molecular mass 340 kDa), which were purified by a simple, three-step method, comprising ammonium sulfate precipitation, ion-exchange and hydroxyapatite chromatography. The two enzymes exhibited a similar pH and temperature optima, inhibitory effect by glucose and gluconolactone, and stability in the pH range of 3.0-9.0. Placed in family 3 of glycosylhydrolase families, BGLI was more active on salicin, p-nitrophenyl beta-D-glucopyranoside and alkyl beta-D-glucosides whereas BGLII was most active on cellobiose. k(cat) and K-M values were determined for a number of substrates and, for BGLI, it was established that the deglycosylation step was equally effective on aryl- and alkyl- glucosides while the glycosylation step varied depending on the substrate used. This information was used to synthesize alkyl-glucosides (up to a chain length of C-10) using dimethyl sulfoxide stabilized single-phase reaction microenvirontrient. About 12% molar yield of octylglucoside was calculated based on a simple spectrophotometric method developed for its estimation. Further, detailed comparison of properties of the enzymes indicated these to be different from the previously cloned beta-glucosidases from this yeast. (C) 2003 Elsevier Science B.V. All rights reserved.