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Biotechnol Biofuels. 2018 Jan 13;11:6. doi: 10.1186/s13068-017-1009-4. eCollection 2018.

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

Liu YJ#1, Liu S#1,2, Dong S1, Li R1,2, Feng Y1, Cui Q1.

Author information

1
1Shandong Provincial Key Laboratory of Energy Genetics, CAS Key Laboratory of Biofuels, Qingdao Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101 People's Republic of China.
2
2University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing, 100049 People's Republic of China.
#
Contributed equally

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.

KEYWORDS:

Activity; Cellulosome; Crystalline cellulose; Exocellulase; Lignocellulose; Substrate specificity

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