Abstract
BackgroundBipolaris sorokiniana is a filamentous fungus that causes spot blotch disease in cereals like wheat and has severe economic consequences. However, information on the identities and role of the cell wall-degrading enzymes (CWDE) in B. sorokiniana is very limited. Several fungi produce CWDE like glycosyl hydrolases (GHs) that help in host cell invasion. To understand the role of these CWDE in B. sorokiniana, the first step is to identify and annotate all possible genes of the GH families like GH3, GH6, GH7, GH45 and AA9 and then characterize them biochemically.ResultsWe confirmed and annotated the homologs of GH3, GH6, GH7, GH45 and AA9 enzymes in the B. sorokiniana genome using the sequence and domain features of these families. Quantitative real-time PCR analyses of these homologs revealed that the transcripts of the BsGH7-3 (3rd homolog of the GH 7 family in B. sorokiniana) were most abundant. BsGH7-3, the gene of BsGH7-3, was thus cloned into pPICZαC Pichia pastoris vector and expressed in X33 P. pastoris host to be characterized. BsGH7-3 enzyme showed a temperature optimum of 60 °C and a pHopt of 8.1. BsGH7-3 was identified to be an endoglucanase based on its broad substrate specificity and structural comparisons with other such endoglucanases. BsGH7-3 has a very long half-life and retains 100% activity even in the presence of 4 M NaCl, 4 M KCl and 20% (v/v) ionic liquids. The enzyme activity is stimulated up to fivefold in the presence of Mn+2 and Fe+2 without any deleterious effects on enzyme thermostability.ConclusionsHere we reanalysed the B. sorokiniana genome and selected one GH7 enzyme for further characterization. The present work demonstrates that BsGH7-3 is an endoglucanase with a long half-life and no loss in activity in the presence of denaturants like salt and ionic liquids, and lays the foundation towards exploring the Bipolaris genome for other cell wall-degrading enzymes.
Highlights
Bipolaris sorokiniana is a filamentous fungus that causes spot blotch disease in cereals like wheat and has severe economic consequences
Glycoside hydrolase (GH) in B. sorokiniana: annotation and sequence characterization We performed a detailed genomic characterization of glycoside hydrolases (GH) families in B. sorokiniana genome and targeted five putative cellulases across GH families (GH3, GH6, GH7, GH45 and activity family 9 (AA9)) in this analysis. These five families comprise the minimum set of cellulolytic enzymes (EGs, CBHs and BGs) that are required for biomass hydrolysis and its identification is a step towards the search for all such cell wall-degrading enzymes in B. sorokiniana [46]
One additional homolog of the AA9 family was identified in this study (Fig. 1; Additional file 1: Figures S1, S2, Table S1)
Summary
Bipolaris sorokiniana is a filamentous fungus that causes spot blotch disease in cereals like wheat and has severe economic consequences. Several fungi produce CWDE like glycosyl hydrolases (GHs) that help in host cell invasion. One way to get around this limitation is to add the missing enzymes in the cellulase cocktail or to manipulate the hydrolytic efficiency of cellulolytic enzymes encoded in this model organism Another strategy could be to explore the fungal biodiversity for synergistic enzyme activities in order to supplement and increase the hydrolytic yield achieved by a T. reesei cocktail or, if possible, a new and more active cocktail based on enzymes from other organisms [3, 4]. Some of the CWDE-coding gene families have expanded during evolution among different groups of fungi [6, 7] These enzymes show preference for specific types of plant biomass [8, 9]. Cellulases can be classified into three major types, namely endoglucanases (EG), cellobiohydrolases (CBH) and β-glucosidases (BG), all of which work synergistically to efficiently degrade cellulose [10,11,12]
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