Protein Engineering of Endoglucanase CelR of Clostridium thermocellum for Enhanced Expression

Hafiz Muzzammel Rehman, Hira Nasir, Adnan Iqbal, Syed Zawar Shah, Ammara Ahad, Muhammad Umair Naseem, Muhammad Sajjad, Muhammad Waheed Akhtar, Sajjad Ahmed


Background: Enhanced production and improved properties of cellulases for a greater activity on plant biomass would rank amongst the top priorities for second-generation ethanol production. Based on the emergence of protein engineering as a cutting-edge technology for enhancing enzyme activity and expression level, the present study is aimed at the application of this technique to the major cellulosomal processing endoglucanase of C. thermocellum, CelR for refining enzyme characteristics. Methods: The full-length native enzyme gene (CelR) and a truncated version without the docking domains at C-terminus (CelR-CB) were PCR amplified using gene specific primers. The amplified PCR products were T/A cloned in the vector pTZ57 R/T and transformed in E. coli DH5α. The cellulase genes from the confirmed transformed plasmids were sub-cloned in T7 promoter-based expression vector pET-28a and expression analysis was done in E. coli (DE3) BL21 codon Plus. Results: An SDS PAGE analysis of both the CelR derivatives revealed that the truncated version i.e. CelR-CB showed a two-fold increase in expression level as compared to the full-length enzyme. Conclusion: The increased expression level of CelR in E. coli coupled with its increased production therefore makes it a promising method for augmenting the recombinant enzyme production for potential applications. 

Full Text:



Ben-Iwo J, Manovic V, Longhurst P. Biomass resources and biofuels potential for the production of transportation fuels in Nigeria. Renewable and Sustainable Energy Reviews, (2016); 63172-192.

Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol Mol Biol Rev, (2002); 66(3): 506-577.

Parsiegla G, Juy M, Reverbel‐Leroy C, Tardif C, Belaïch JP, et al. The crystal structure of the processive endocellulase CelF of Clostridium cellulolyticum in complex with a thiooligosaccharide inhibitor at 2.0 Å resolution. The EMBO Journal, (1998); 17(19): 5551-5562.

Himmel ME, Ding S-Y, Johnson DK, Adney WS, Nimlos MR, et al. Biomass recalcitrance: engineering plants and enzymes for biofuels production. science, (2007); 315(5813): 804-807.

Bayer EA, Chanzy H, Lamed R, Shoham Y. Cellulose, cellulases and cellulosomes. Current opinion in structural biology, (1998); 8(5): 548-557.

Coutinho P. Carbohydrate-active enzymes: an integrated database approach. 1999. In Gilbert HJ, Davies G, Henrissat H, Svensson B (eds), Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry,Cambridge, pp. 3–12.

Bhat M. Cellulases and related enzymes in biotechnology. Biotechnology advances, (2000); 18(5): 355-383.

Haki G, Rakshit S. Developments in industrially important thermostable enzymes: a review. Bioresource technology, (2003); 89(1): 17-34.

Okada H, Tada K, Sekiya T, Yokoyama K, Takahashi A, et al. Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414. Appl Environ Microbiol, (1998); 64(2): 555-563.

Nakatani F, Kawaguchi T, Takada G, Sumitani J-i, Moriyama Y, et al. Cloning and sequencing of an endoglucanase gene from Scopulariopsis brevicaulis TOF-1212, and its expression in Saccharomyces cerevisiae. Bioscience, biotechnology, and biochemistry, (2000); 64(6): 1238-1246.

Voorhorst W, Eggen R, Luesink EJ, De Vos W. Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli. Journal of Bacteriology, (1995); 177(24): 7105-7111.

Zverlov V, Mahr S, Riedel K, Bronnenmeier K. Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile ‘Anaerocellum thermophilum’with separate glycosyl hydrolase family 9 and 48 catalytic domains. Microbiology, (1998); 144(2): 457-465.

Guglielmi G, Béguin P. Cellulase and hemicellulase genes of Clostridium thermocellum from five independent collections contain few overlaps and are widely scattered across the chromosome. FEMS microbiology letters, (1998); 161(1): 209-215.

Johnson EA, Madia A, Demain AL. Chemically defined minimal medium for growth of the anaerobic cellulolytic thermophile Clostridium thermocellum. Applied and Environmental Microbiology, (1981); 41(4): 1060.

Mingardon F, Chanal A, López-Contreras AM, Dray C, Bayer EA, et al. Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting cellulolytic complexes. Appl Environ Microbiol, (2007); 73(12): 3822-3832.

Bayer EA, Belaich J-P, Shoham Y, Lamed R. The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annu Rev Microbiol, (2004); 58521-554.

Najmudin S, Guerreiro CI, Ferreira LM, Romão MJ, Fontes CM, et al. Overexpression, purification and crystallization of the two C-terminal domains of the bifunctional cellulase ctCel9D-Cel44A from clostridium thermocellum. Acta Crystallographica Section F: Structural Biology and Crystallization Communications, (2005); 61(12): 1043-1045.

Jiang X, Wang Y, Xu L, Chen G, Wang L. Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca. Biochemical and biophysical research communications, (2017); 491(1): 236-240.

Klein‐Marcuschamer D, Oleskowicz‐Popiel P, Simmons BA, Blanch HW. The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnology and bioengineering, (2012); 109(4): 1083-1087.

Wen F, Nair NU, Zhao H. Protein engineering in designing tailored enzymes and microorganisms for biofuels production. Current opinion in biotechnology, (2009); 20(4): 412-419.

Gold ND, Martin VJ. Global view of the Clostridium thermocellum cellulosome revealed by quantitative proteomic analysis. Journal of bacteriology, (2007); 189(19): 6787-6795.

Zverlov VV, Kellermann J, Schwarz WH. Functional subgenomics of Clostridium thermocellum cellulosomal genes: identification of the major catalytic components in the extracellular complex and detection of three new enzymes. Proteomics, (2005); 5(14): 3646-3653.

Ramchuran S, Karlsson EN, Velut S, de Maré L, Hagander P, et al. Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21 (DE3). Applied microbiology and biotechnology, (2002); 60(4): 408-416.

Sajjad M, Khan MIM, Akbar NS, Ahmad S, Ali I, et al. Enhanced expression and activity yields of Clostridium thermocellum xylanases without non-catalytic domains. Journal of biotechnology, (2010); 145(1): 38-42.


  • There are currently no refbacks.