Purification and characterization of two beta-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 microenvironment. About 12% molar yield of octyl-glucoside 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.
Wallecha
Anu
A
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New Delhi 110016, India.
Mishra
Saroj
S
D000596:Amino Acids / Q000032:analysis; D001592:Benzyl Alcohols / Q000378:metabolism; D002475:Cellobiose / Q000378:metabolism; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D005656:Fungal Proteins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D005942:Gluconates / Q000378:metabolism / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D005960:Glucosides; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007783:Lactones; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D020539:Sequence Analysis, Protein; D012997:Solvents / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*
D000596:Amino Acids; D001592:Benzyl Alcohols; D005656:Fungal Proteins; D005942:Gluconates; D005960:Glucosides; D007783:Lactones; D008670:Metals; D012997:Solvents; D002475:Cellobiose; C005696:salicin; D001617:beta-Glucosidase; D005947:Glucose; C010730:beta-glucono-1,5-lactone