Abstract
BackgroundThe ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate.ResultsThe TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs.ConclusionsWe hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.
Highlights
The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose
Preparation of Cel7A enzymes The Cel7A enzymes were purified from culture filtrates of T. atroviride IOC 4503, T. harzianum IOC 3844, and T. reesei QM9414 grown in submerged culture under cellulase inducing conditions
Extracellular protein production appeared to be slightly lower for T. atroviride than T. harzianum, and less protein was obtained than from T. reesei
Summary
The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Cellulolytic fungi are responsible for the majority of the degradation of terrestrial plants, which in turn accounts for most of the Earth’s biomass In many of these fungi, the major secreted enzymes are glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBH) [1]. Lignocellulosic biomass is by far the most abundant renewable carbon source available to humanity for transition from fossil-based to sustainable production of fuels and chemicals. As central as these enzymes are to biomass degradation, they have become the cornerstone of modern industrial enzyme formulations for biofuel processes [2]. GH7 CBHs are the target of intense structural, mechanistic, and engineering studies [3,4,5,6,7,8,9]
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