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- Title
Purification and characterization of an extracellular β-glucosidase from Sporothrix schenckii.
- Authors
Hernández-Guzmán, Alicia; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Villagómez-Castro, Julio C.
- Abstract
An extracellular β-glucosidase (), induced by cellulose in the mycelial form of human pathogen fungus Sporothrix schenckii, was purified to homogeneity using hydroxyapatite ( HAp) adsorption chromatography in batch and Sephacryl S200- HR size exclusion chromatography. The molecular mass of the purified enzyme was estimated to be 197 kDa by size exclusion chromatography with a subunit of 96.8 kDa determined by SDS/ PAGE. The β-glucosidase exhibited optimum catalytic activity at pH 5.5/45 °C and was relatively stable for up to 24 h at 45 °C. Isoelectric focusing displayed an enzyme with a pI value of 4.0. Its activity was inhibited by Fe2+ but not by any other ions or chelating agents. Km and Vmax values of the purified enzyme were 0.012 m m and 2.56 nmol·min−1·mg−1, respectively, using 4-methylumbelliferyl β-D-glucopyranoside (4- MUG) as the substrate and 44.14 m m and 22.49 nmol·min−1·mg−1 when p-nitrophenyl β-D-glucopyranoside ( p- NPG) was used. The purified β-glucosidase was active against cellobioside, laminarin, 4- MUG, and p- NPG and slightly active against 4-methylumbelliferyl β-D-cellobioside and p-nitrophenyl β-D-cellobioside but did not hydrolyze 4-methylumbelliferyl β-D-xyloside, 4-methylumbelliferyl β-D-galactopyranoside nor 4-methylumbelliferyl α-D-glucopyranoside. In addition, the enzyme showed transglycosylation activity when it was incubated along with different oligosaccharides. Whether the transglycosylation and cellulase activities function in vivo as a mechanism involved in the degradation of cellulolytic biomass in the saprophytic stage of S. schenckii remains to be determined.
- Subjects
GLUCOSIDASES; CELLULOSE; HYDROXYAPATITE; ADSORPTION chromatography; GLUCOPYRANOSIDE
- Publication
FEBS Open Bio, 2016, Vol 6, Issue 11, p1067
- ISSN
2211-5463
- Publication type
Article
- DOI
10.1002/2211-5463.12108