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

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,...

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Published in:Protein expression and purification 2024-08, Vol.220, p.106490, Article 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: Article
Language:English
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
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Summary: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).
ISSN:1046-5928
1096-0279
1096-0279
DOI:10.1016/j.pep.2024.106490