RESUMO
The filamentous fungus Stachybotrys microspora possess a rich ß-glucosidase system composed of five ß-glucosidases. Three of them were already purified to homogeneity and characterized. In order to isolate the ß-glucosidase genes from S. microspora and study their regulation, a PCR strategy using consensus primers was used as a first step. This approach enabled the isolation of three different fragments of family 3 ß-glucosidase gene. A representative genomic library was constructed and probed with one amplified fragment gene belonging to family 3 of ß-glucosidase. After two rounds of hybridization, seven clones were obtained and the analysis of DNA plasmids leads to the isolation of one clone (CF3) with the largest insert of 7 kb. The regulatory region shows multiple TC-rich elements characteristic of constitutive promoter, explaining the expression of this gene under glucose condition, as shown by zymogram and RT-PCR analysis. The tertiary structure of the deduced amino acid sequence of Smbgl3 was predicted and has shown three conserved domains: an (α/ß)8 triose phosphate isomerase (TIM) barrel, (α/ß)5 sandwich, and fibronectin type III domain involved in protein thermostability. Zymogram analysis highlighted such thermostable character of this novel ß-glucosidase.
