Abstract
The present study reports the isolation and analysis of two novel GH1 β-glucosidases from the alkalophilic fungus Stachybotrys microspora, using PCR and Nested-PCR. Three major gene fragments were obtained by PCR: the first two are very similar and constitute a novel gene, which was named Smbgl1A, and the third PCR fragment is part of a different gene, named Smbgl1B. The truncated gene sequences were completely filled using the recent partial whole genome sequencing data of S. microspora (data not yet published).Moreover, we investigated the relative effects of glucose in comparison to cellulose rather than evaluate their absolute effects.In fact, RT-PCR analysis showed that while Smbgl1A was expressed when the fungus was grown in the presence of cellulose but not when grown with glucose, Smbgl1B was equally expressed under both conditions.The putative catalytic residues and the conserved glycone binding sites were identified. Zymogram analysis showed the intracellular production of β-glucosidases in S. microspora. The predicted secondary structure exhibited a classical (β/α)8 barrel fold, showing that both SmBGL1A and SmBGL1B belong to the GH1 family.Phylogenetic studies showed that SmBGL1A and SmBGL1B belong to the same branch as β-glucosidases from Stachybotrys chlorohalonata and Stachybotrys chartarum. However, SmBGL1A and SmBGL1B form two distinct clades.
Highlights
We investigated the relative effects of glucose in comparison to cellulose rather than evaluate their absolute effects
Several known structures of β-glucosidases and those presenting significant homologies with SmBGL1A and SmBGL1B were submitted to a multiple alignment (Fig. 3a), and the findings revealed that the acid/base and catalytic nucleophile were conserved in all GH1 enzymes
Genomic sequence analysis shows a codon bias toward G/C at the third position, and primary and secondary structure analyses suggest that SmBGL1A and SmBGL1B are intracellular enzymes and have the classical (β/α) 8 barrel of the GH1 family
Summary
We investigated the relative effects of glucose in comparison to cellulose rather than evaluate their absolute effects. Introduction β-glucosidases are a heterogeneous group of hydrolytic enzymes that are widely distributed in eukaryotic and prokaryotic organisms These enzymes hydrolyze glycosidic bonds to liberate non-reducing terminal glucosyl residues from glycosides and from short oligosaccharides. Β-glucosidases play a variety of pivotal functions in several biological processes, including the degradation of cellulosic biomass and the modification of secondary metabolites. They play important roles in the regulation of cellulase genes for they represent key enzymes in the synthesis of sophorose, which is an efficient inducer of the cellulolytic system of Trichoderma reesei (Singhania et al, 2013)
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