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

Abstract

An extracellular β‐glucosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/21.html), 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.

A Lignocellulolytic Colletotrichum sp. OH with Broad-Spectrum Tolerance to Lignocellulosic Pretreatment Compounds and Derivatives and the Efficiency to Produce Hydrogen Peroxide and 5-Hydroxymethylfurfural Tolerant Cellulases

“…The enhancement of exoglucanase activities in the presence of rice husk was in line with the report of Liu et al [118] in utilizing rice straw as a sole carbon source for Aspergillus fumigatus Z5. The OH strain maintained a lower level of enzyme production in the presence of glucose as a carbon source, suggesting a weak glucose catabolic repression activity, and similar glucose induction was observed in Sporothrix schenckii as well [119]. Moreover, this study shows similar lignocellulolytic production titers to other endophytic strains reported in the literature, i.e., Acremonium sp.…”

Identification and molecular characterization of a psychrophilic GH1 β-glucosidase from the subtropical soil microorganism Exiguobacterium sp. GXG2

“…A total of 308,372 β-glucosidase protein sequences had been identified in the NCBI protein database as of September 20, 2018. Among these protein sequences, most of the β-glucosidases were came from bacteria (282,519) (Chan et al 2016; Chen et al 2017; Sun et al 2018), followed by fungi (11,863) (Guo et al 2015; Hernandez-Guzman et al 2016). The β-glucosidases have the characteristics of cold adaptation (Crespim et al 2016; Ueda et al 2010), heat resistance (Leis et al 2018), salt tolerance (Lee et al 2015), glucose tolerance (Chamoli et al 2016; Chan et al 2016), and organic solvent tolerance (Thomas et al 2018) in extreme environments.…”

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Similarly, β-glucosidases belonging to GH family 3 contain 600-900 amino acids in length with molecular weight of 65-90 kDa per subunit, but because these group of enzymes are usually glycosylated, their molecular mass ranges 110-130 kDa [44,157,158]. β-Glucosidase varies in quaternary structure arrangement, for example, monomeric [159], dimeric [160], trimeric [147], tetrameric [151] enzymes have been reported. Native molecular weight therefore may be higher than identified by SDS-PAGE [161].…”

“…For example, β-glucosidase has been purified up to 9.47 fold from culture supernatant of Tolypocladium cylindrosporum Syzx4 using ammonium sulfate (75% saturation), dialysis, (DEAE)-cellulose column, and Sephadex G-100 column [68]. β-Glucosidase has also been purified from Aureobasidium pullulans using ammonium sulfate precipitation, CM Bio-Gel A agaraose, and sephacryl S-200 gel filtration chromatography up to 129 fold [80] and that from the human pathogenic fungus Sporothrix schenckii, was purified to homogeneity using hydroxyapatite (HAp) adsorption chromatography and Sephacryl S200-HR size exclusion chromatography [81]. Affinity chromatography methods such as Immobilized metal-affinity chromatography (IMAC) has been utilized for purification of recombinant enzymes containing short affinity tag of polyhistdine residues which show strong interactions with transition metal ions such as Ni 2+ immobilized in matrix [82].…”