Purification and biochemical characterization of a transglucosilating β-glucosidase of Stachybotrys strain

Abstract

The filamentous fungus Stachybotrys sp has been shown to possess a rich beta-glucosidase system composed of five beta-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second beta-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly beta-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50 degrees C. The kinetic parameters, K (m) and V (max), on para-nitro-phenyl-beta-D: -glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 +/- 0.11 mM and 211 +/- 0.08 micromol min(-1) ml(-1). One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl beta-glucosidase activity towards salicin, methylumbellypheryl-beta-D: -glucoside (MU-Glc), and p-NPG, it showed a true beta-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.

In situ demonstration and quantitative analysis of the intrinsic properties of glycoside hydrolases

“…3 (the gel image on the left), there are at least two components in each type of glycoside hydrolase in the extracellular crude samples of the four filamentous fungi. These results are consistent with the previous reports [39][40][41]. Thus, when the native electrophoresis is carried out with the same amount of sample analyzed under the same conditions, and the postelectrophoresis gel strips are soaked simultaneously in the same substrate solution corresponding to each enzyme at different pHs, the changes in concentration of the substrate or product (reflected by each band's gray values) are expected to be indicators of the pH dependence of the corresponding enzyme component, as the gray values were in positive linear correlation with relative enzyme activities.…”

Expression and characterization of a novel highly glucose-tolerant β-glucosidase from a soil metagenome

“…9), indicating that Unbgl1A possesses a transglucosylation activity that transfers a glucose unit to the receptor sugar (in this case, it is cellobiose). This result is consistent with the reported enzymes from Stachybotrys microbispora and zygomycete R. miehei [18,21]. Krisch and other groups showed that after 35% (350 mg/ml) cellobiose was mixed with b-glucosidases for 72 h, there was 86 mg/ml cellotriose generated [18,21,22].…”

Constitutive over-expression of pectinases in Penicillium occitanis CT1 mutant is transcriptionally regulated

“…The reaction procedure was modified in our laboratory by Saibi et al (2007). One unit of enzymatic activity is the amount of enzyme required to release 1 lmol p-nitrophenol per min.…”