Modification and Expression of Beta-1,4-Endoglucanase encoding sequences of fungal origin in Escherichia coli BL21

• Published in: Revista Colombiana de biotecnologia Vol. 22; no. 2; pp. 24 - 34
• Main Authors: Gutierrez Calle, Natalia, Restrepo Franco, Gloria Maria, Galeano Vanegas, Narmer Fernando
• Format: Journal Article
• Language: English
• Published: Bogota Universidad Nacional de Colombia 01-12-2020
• Subjects: Biofuels; Bioinformatics; Cellulolytic enzymes; Cellulose; Celulosa; Cloning; Degradation; E coli; Endoglucanase; Enzimas celulolíticas; Enzyme activity; Enzymes; Expresión heteróloga; Genes; Heterologous expression; Lignocellulolytic enzymes; Lignocellulose; Molecular docking; Production methods; Saccharification; Substrates; Three dimensional models; Colombia
• ISSN: 0123-3475; 1909-8758; 1909-8758
• DOI: 10.15446/rev.colomb.biote.v22n2.79448

Lignocellulose is the main and most abundant component of biomass. Annually, 200 million tons are generated in the world. Colombia has a high production of lignocellulosic residues that can be used in many industrial processes such as bioethanol production, promoting the bioeconomy. The objective of the present work was to express lignocellulolytic enzymes of eukaryotic origin in Escherichia coli BL21 (DE3). Initially, endoglucanase eukaryotic genes were selected and modified using bioinformatics methods for their production in E. coli BL21 (DE3) and saccharification of pure cellulose substrates. The gene selected for its modification and expression was eglB from the fungus Aspergillus nidulans. Subsequently the enzyme integrity was tested by 3D modeling and molecular docking, as well as the conformation of its active site and its affinity for substrates of interest. Finally, cloning of the modified gene in plasmid pET151 TOPO was made and transformed in the strain E. coli BL21 (DE3) where several lignocellulose degradation tests were carried out using semiquantitative methods for the enzyme activity in carboxymethylcellulose. The presence of the three genes of interest within the plasmid pET151 TOPO and within the transformed cells of E. coli TOP10 and E. coli BL21 (DE3) was verified by colony PCRs performed. The presence of this gen was corroborated by sequencing. Expression of the modified endoglucanase enzyme was achieved in E. coli BL21 (DE3) expression cells, in soluble and functional form, demonstrated by the hydrolysis of the CMC substrate.