22 juin 19381938/06/22 (N1102)-1938/06/22.Appartient à l’ensemble documentaire : Aquit

Qiu Cui (432104)

Renmin Li (417810)

Sheng Dong (1394983)

Shiyue Liu (4023818)

Ya-Jun Liu (1351770)

Yingang Feng (306604)

Biotechnology for Biofuels

Additional file 4. Kinetic analysis of ctCel48S_CD. a, Avicel hydrolysis over time. The glucose production was in a linear relationship with the reaction time for 6 h. b, Determination of kinetic parameters of ctCel48S_CD. The reaction lasted for 5 h with initial Avicel concentrations of 1–10 mg/mL. The linear or non-linear fit curves are shown in red, and the corresponding R2 values are given

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MOESM4 of Determination of the native features of the exoglucanase Cel48S from Clostridium thermocellum

Gallica, Bibliotheque Numerique

L'Athlète : journal hebdomadaire de tous les sports

Abstract Background Clostridium 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. Results We 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. Conclusion Our 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

Ya-Jun Liu

Shiyue Liu

Sheng Dong

Renmin Li

Yingang Feng

Qiu Cui

Directory of Open Access Journals

Determination of the native features of the exoglucanase Cel48S from Clostridium thermocellum

Additional file 3. The theoretical amino acid sequence of ctCel48S_CD with a His12-tag at the C-terminal (a) and the molecular weight analysis by HPLC-Q-TOF-MS (b). A linker composed of five glycine is shown in red, and the His12-tag is highlighted in yellow. The stop codon is indicated by a asterisk

MOESM3 of Determination of the native features of the exoglucanase Cel48S from Clostridium thermocellum

http://gallica.bnf.fr/ark:/12148/cb34474930f/date

L'Athlète : journal hebdomadaire de tous les sports

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Gallica, Bibliotheque Numerique

Directory of Open Access Journals

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