Expression and characterization of a novel β-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater

• Published in: Protein expression and purification Vol. 220; p. 106490
• Main Authors: Ríos-Alvarado, Joel, Avitia-Rodríguez, Olga Noelia, Urtiz-Estrada, Norma, Zazueta-Álvarez, David Enrique, López-Miranda, Javier, Vázquez-Ortega, Perla Guadalupe, Rojas-Contreras, Juan Antonio
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2024
• Subjects: Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Bacillus subtilis - isolation & purification; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Cellulase - biosynthesis; Cellulase - chemistry; Cellulase - genetics; Cellulase - isolation & purification; Cellulase - metabolism; Cellulases; Cloning, Molecular; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Modified plasmid; Recombinant protein; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Wastewater - chemistry; Wastewater - microbiology; β-1,4- endoglucanase; β-1,4- endoglucanase; Recombinant protein; Cellulases; Modified plasmid
• ISSN: 1046-5928; 1096-0279; 1096-0279
• DOI: 10.1016/j.pep.2024.106490

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by β-1,4 bonds. The enzyme β-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A β-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a β-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa β-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated β-1,4-endoglucanase had higher activity and stability. •A strain of Bacillus subtilis with endoglucanase activity was isolated from the wastewater of a pulp and paper mill.•The endoglucanase gene was inserted into a modified plasmid (E. coli) and into pYD1 (S. cerevisiae).•The endoglucanase in pYD1 is expressed with good activity in E. coli, being the first report of this finding.•A truncated recombinant endoglucanase is obtained and is very stable at pH (2-10) and temperature (30–55 °C).