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Abstract
BackgroundClostridium thermocellum is considered one of the most efficient natural cellulose degraders because of its cellulosomal system. As the major exoglucanase of cellulosome in C. thermocellum, Cel48S plays key roles and influences the activity and features of cellulosome to a great extent. Thus, it is of great importance to reveal the enzymatic features of Cel48S. However, Cel48S has not been well performed due to difficulties in purifying either recombinant or native Cel48S proteins.ResultsWe observed that the soluble fraction of the catalytic domain of Cel48S (Cel48S_CD) obtained by heterologous expression in Escherichia coli and denaturation-refolding treatment contained a large portion of incorrectly folded proteins with low activity. Using a previously developed seamless genome-editing system for C. thermocellum, we achieved direct purification of Cel48S_CD from the culture supernatant of C. thermocellum DSM1313 by inserting a sequence encoding 12 successive histidine residues and a TAA stop codon immediately behind the GH domain of Cel48S. Based on the fully active protein, biochemical and structural analyses were performed to reveal its innate characteristics. The native Cel48S_CD showed high activity of 117.61 ± 2.98 U/mg and apparent substrate preference for crystalline cellulose under the assay conditions. The crystal structure of the native GH48 protein revealed substrate-coupled changes in the residue conformation, indicating induced-fit effects between Cel48S_CD and substrates. Mass spectrum and crystal structural analyses suggested no significant posttranslational modification in the native Cel48S_CD protein.ConclusionOur results confirmed that the high activity and substrate specificity of Cel48S_CD from C. thermocellum were consistent with its importance in the cellulosome. The structure of the native Cel48S_CD protein revealed evidence of conformational changes during substrate binding. In addition, our study provided a reliable method for in situ purification of cellulosomal and other secretive proteins from C. thermocellum.
Highlights
Clostridium thermocellum is considered one of the most efficient natural cellulose degraders because of its cellulosomal system
The result indicated that the obtained rCel48S_CD had an activity of 17.54 U/mg using Avicel as the substrate, which was similar to or higher than that reported in the literature [16, 22]
The cellulase activity of the fraction-50 was only 8.45 ± 1.13 U/mg, which was 9.5% of that of the fraction-80 (89.02 ± 2.98 U/mg). Both circular dichroism (CD) spectra and tryptophan intrinsic fluorescence (TIF) analyses showed that fraction-50 and fraction-80 presented differences in their secondary structures (Fig. 1c, d), indicating that fraction-50 may be an incorrectly folded oligomer of rCel48S_CD
Summary
Clostridium thermocellum is considered one of the most efficient natural cellulose degraders because of its cellulosomal system. Olson et al investigated the contribution of Cel48S to the cellulolytic activity of C. thermocellum by gene deletion, and suggested that the lack of Cel48S could significantly reduce the hydrolysis rate of crystalline cellulose, thereby demonstrating the importance of the Cel48S enzyme in the cellulosome system [8, 15]. The role of Cel48S as an exoglucanase was further confirmed by biochemical and structural studies based on recombinant proteins those were heterologously expressed in E. coli [16,17,18,19,20]. Difficulties in expressing and purifying recombinant Cel48S protein have hindered further studies on the genetic modifications and site-directed mutations of Cel48S for higher activity or reduced feedback inhibition, i.e., features which have been proposed based on published crystal structures [19, 23, 24]
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