Purification and characterization of an extracellular β-glucosidase from Sporothrix schenckii

Alicia Hernández-Guzmán, Alberto Flores-Martínez, Patricia Ponce-Noyola, Julio C Villagómez-Castro

FEBS Open Bio. 2016 Oct 6;6(11):1067-1077.

PMID: 27833847

Abstract:

An extracellular β-glucosidase (E.C. 3.2.1.21), 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 mm and 2.56 nmol·min-1·mg-1, respectively, using 4-methylumbelliferyl β-D-glucopyranoside (4-MUG) as the substrate and 44.14 mm 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP18997574	4-Methylumbelliferyl β-D-glucopyranoside		18997-57-4	Price