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

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.Electronic supplementary materialThe online version of this article (10.1186/s13068-017-1009-4) contains supplementary material, which is available to authorized users.

Nanoscale resolution of microbial fiber degradation in action

“…S3). The structure highly resembles the previously reported crystallographic structure that lacks the linker and dockerin module 37 , although, in the current cryo-EM structure, an additional stretch of 10 amino-acids (MTYKVPGTPS) can be detected at the surface of the protein, which represents the major part of the linker between the catalytic module and the dockerin ( Fig. 2A, purple).…”

Cloning and characterization of an exocellulase and synergistic effect with recombinant endocellulase and beta-glucosidase from Bacillus subtilis 1AJ3

“…Cbh-A could not tolerate acidic or alkaline environment which led to enzyme denaturation and inactivation, while pH 6.0-7.4 would maintain its enzyme activity over 70%. Similar pH conditions for exocellulase was observed in Cel48S [20]. Cbh-A had the highest enzyme activity under 40-50 ℃, that it can combine with other enzymes with same temperatures.…”

“…It had also been found that most of the recombinant exocellulase studied were derived from fungal microorganisms, such as Thermobifida fusca [11,16], Thermomonospora fusca [17], Orpinomyces sp. [18], Irpex lacteus [19], but exocellulase from bacteria was rarely reported, such as Clostridium thermocellum [20]. In this study, we cloned an exocellulase Cbh-A from Bacillus subtilis 1AJ3 to found out its cellulose hydrolyzation ability.…”

Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30

“…The gene jgi|73493 encodes a putative calcium transporter with 10 transmembrane domains, and it was found as a hub in the coral1 module (Table S8 ). It has already been reported that metal ions, such as Ca 2+ and Mn 2+ , have a positive effect on the mycelial growth of T. reesei and cellulase production, and this molecular signaling mechanism is mediated by the Mn 2+ transporters TPHO84-1 and TPHO82-2, a Ca 2+ / Mn 2+ ATPase (TPMR1) and the components of the Ca 2+ /calmodulin signal transduction, including the TF Crz1 (Chen et al, 2016 , 2018 ). Therefore, it is worth investigating the role of this putative calcium transporter in the induction of the genes responsive to lignocellulose degradation, since it could transport cations that activate gene expression.…”