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Liu et al. Biotechnol Biofuels (2018) 11:6

https://doi.org/10.1186/s13068-017-1009-4


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

Ya?Jun Liu1*† , Shiyue Liu1,2†, Sheng Dong1 , Renmin Li1,2, Yingang Feng1* and Qiu Cui1* 

Abstract

Background: Clostridium thermocellum is considered one of the most efcient natural cellulose degraders because of its cellulosomal system. As the major exoglucanase of cellulosome in C. thermocellum, Cel48S plays key roles and infu? ences 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 difculties 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 heter? ologous 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 purifcation 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 char? acteristics. 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 substratecoupled changes in the residue conformation, indicating induced-ft efects between Cel48S_CD and substrates. Mass spectrum and crystal structural analyses suggested no signifcant posttranslational modifcation in the native Cel48S_CD protein.

Conclusion: Our results confrmed that the high activity and substrate specifcity 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 purifcation of cellulosomal and other secretive proteins from C. thermocellum.

Keywords: Activity, Cellulosome, Crystalline cellulose, Exocellulase, Lignocellulose, Substrate specifcity

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SDS?PAGE and protein analyses

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check protein purity and composition as previously described [32] using QuickRun bufer (MDbio) and protein standards ranging from 10 to 245  kDa or from 14 to 116  kDa (New England BioLabs). Protein quantifcation was performed using the Bradford method before further analyses [33]. Protein molecular weight was determined using a highperformance liquid chromatography (HPLC) system (Agilent 1290) coupled to a Q-TOF–MS (Agilent 6530). Te HPLC was equipped with a Zorbax 300SB-C8 column (4.6 × 250 mm, Agilent). Samples were run at a fow rate of 1 mL/min with 6-μL injection volume. Te mobile phases contained 0.1% (vol/vol) formic acid in water (A) or in acetonitrile (B). A four-step linear gradient of 5% B from 0 to 5 min, 5–50% B from 5 to 10 min, 50–90% B from 10 to 12 min, and 90% B from 12 to 15 min was  used. Te scan range was m/z 800–1800. Te deconvolution analysis was displayed by the software Deconvolute (MS):protein (Agilent).

 

 



























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