Purification and characterization of an extracellular β-glucosidase fromSporothrix schenckii.
An extracellular β-glucosidase (E.C. 3.2.1.21), induced by cellulose in the mycelial form of human pathogen fungusSporothrix 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.KmandVmaxvalues 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-1whenp-nitrophenyl β-D-glucopyranoside (p-NPG) was used. The purified β-glucosidase was active against cellobioside, laminarin, 4-MUG, andp-NPG and slightly active against 4-methylumbelliferyl β-D-cellobioside andp-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 functionin vivoas a mechanism involved in the degradation of cellulolytic biomass in the saprophytic stage ofS. schenckiiremains to be determined.
Departamento de Biología División de Ciencias Naturales y Exactas Universidad de Guanajuato México.
Alicia
A
Hernández-Guzmán
Departamento de Biología División de Ciencias Naturales y Exactas Universidad de Guanajuato México.
Alberto
A
Flores-Martínez
Departamento de Biología División de Ciencias Naturales y Exactas Universidad de Guanajuato México.
Patricia
P
Ponce-Noyola
Departamento de Biología División de Ciencias Naturales y Exactas Universidad de Guanajuato México.
Julio C
JC
Villagómez-Castro
Sporothrix schenckii; biochemical characterization; purification; β‐glucosidase