African Journal of
Biotechnology

  • Abbreviation: Afr. J. Biotechnol.
  • Language: English
  • ISSN: 1684-5315
  • DOI: 10.5897/AJB
  • Start Year: 2002
  • Published Articles: 12344

Full Length Research Paper

Production of truncated peptide (cellobiohydrolase Cel6A) by Trichoderma reesei expressed in Escherichia coli

Miriam Shirley Tellez Calzada
  • Miriam Shirley Tellez Calzada
  • Chemical and Biochemical Engineering Departament, Tecnológico Nacional de México (TecNM), Instituto Tecnológico de Durango (ITD), C.P. 34080, Durango, Dgo., Mexico.
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Juan Antonio Rojas Contreras
  • Juan Antonio Rojas Contreras
  • Chemical and Biochemical Engineering Departament, Tecnológico Nacional de México (TecNM), Instituto Tecnológico de Durango (ITD), C.P. 34080, Durango, Dgo., Mexico.
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Jesus Bernardo Paez Lerma
  • Jesus Bernardo Paez Lerma
  • Chemical and Biochemical Engineering Departament, Tecnológico Nacional de México (TecNM), Instituto Tecnológico de Durango (ITD), C.P. 34080, Durango, Dgo., Mexico.
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Nicolas Oscar Soto Cruz
  • Nicolas Oscar Soto Cruz
  • Chemical and Biochemical Engineering Departament, Tecnológico Nacional de México (TecNM), Instituto Tecnológico de Durango (ITD), C.P. 34080, Durango, Dgo., Mexico.
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Javier López Miranda
  • Javier López Miranda
  • Chemical and Biochemical Engineering Departament, Tecnológico Nacional de México (TecNM), Instituto Tecnológico de Durango (ITD), C.P. 34080, Durango, Dgo., Mexico.
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  •  Received: 23 November 2020
  •  Accepted: 23 April 2021
  •  Published: 31 May 2021

Abstract

Enzymatic cellulose hydrolysis is an important step for the production of second-generation biofuels. The filamentous fungus Trichoderma reesei is among the most important organisms for obtaining cellulolytic enzymes. The Cel6A (CBH II) cellulase from T. ressei plays an important role in cellulose hydrolysis and acts on the non-reducing end of cellulose, in contrast to Cel7B (CBH I), which acts on the reducing end of cellulose thus releasing cellobiose. Therefore, Cel6A deficiency becomes a limiting factor in cellulose saccharification. This work attempted to use codon optimization to enhance Cel6A expression in Escherichia coli. A plasmid expression vector, pUCITD04, was designed; this vector contains: the cel6a gene, regulatory regions (the promoter and terminator T7 sequences), the OmpT signal peptide that allows the secretion of proteins into the culture medium, and a 6His tail to allow purification of the protein by affinity chromatography. The protein expression experiment using a strain of E. coli transformed with pUCITD04 resulted in a 31 kDa polypeptide being secreted into the culture medium that did not possess enzymatic activity, meanwhile, the control strain transformed with the empty plasmid did not secrete any protein fragments, indicating that a truncated Cel6A was being produced by the experimental strain. This phenomenon has been reported during the production of recombinant cellulases in E. coli. In this research, we discuss probable causes of this phenomenon, as well as the drawbacks in the production of cellulases by E. coli, directing efforts to elucidate the causes of the production of truncated cellulases by this bacterial factory.

 

Key words: Gene construct, recombinant cellobiohydrolase, Escherichia coli.