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.
The production profile of cellulases of the mutant strain A19 from the filamentous fungus Stachybotrys microspora was studied in the presence of various carbon sources (glucose, lactose, cellulose, carboxymethylcellulose (CMC), and wheat bran) and a range of medium initial pH (5, 7, and 8). Two extracellular cellulases from the Stachybotrys strain (endoglucanases and β-glucosidases) were monitored by enzymatic assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and zymogram analysis. Glucose and lactose repressed CMCase time course production while they permitted a strong β-glucosidase one. On Avicel cellulose, CMC, and wheat bran, both activities were highly produced. Wheat bran (WB) is the best carbon source with an optimum of production at days 5 and 6. The production kinetics of both activities were shown to depend on the medium initial pH, with a preference for neutral or alkaline pH in the majority of conditions. The exception concerned the β-glucosidase which was much more produced at acidic pH, on glucose and cellulose. Most interestingly, a constitutive and conditional expression of an alkaline endoglucanase was revealed on the glucose-based medium at an initial pH of 8 units. The zymogram analysis confirmed such conclusions and highlighted that carbon sources and the pH of the culture medium directed a differential induction of various endoglucanases and β-glucosidases.
Stachybotrys microspora is a filamentous fungus characterized by the secretion of multiple hydrolytic activities (cellulolytic and non-cellulolytic enzymes). The production of these biocatalysts was studied under submerged culture using glucose, cellulose, and wheat bran as carbon sources. Endoglucanases, pectinases, xylanases, β-glucanases, chitinases, and proteases were induced on cellulose-based medium and repressed on glucose in both strains with higher amounts produced by the mutant. β-glucosidases were roughly equally produced by both strains under glucose and cellulose conditions. The yield of chitinases, β-glucanases, and proteases produced by Stachybotrys strains was as much higher than the commercialized lysing enzyme called "zymolyase," currently used in yeast DNA extraction. In this context, we showed that S. microspora hydrolases can be successfully applied in the extraction of yeast DNA.
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