Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction - Archive ouverte HAL Access content directly
Journal Articles International Journal of Molecular Sciences Year : 2022

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

(1) , (1) , (1) , (1) , (2) , (3) , (3, 4) , (2)
1
2
3
4

Abstract

In nature, the deconstruction of plant carbohydrates is carried out by carbohydrate-active enzymes (CAZymes). A high-throughput (HTP) strategy was used to isolate and clone 1476 genes obtained from a diverse library of recombinant CAZymes covering a variety of sequence-based families, enzyme classes, and source organisms. All genes were successfully isolated by either PCR (61%) or gene synthesis (GS) (39%) and were subsequently cloned into Escherichia coli expression vectors. Most proteins (79%) were obtained at a good yield during recombinant expression. A significantly lower number (p < 0.01) of proteins from eukaryotic (57.7%) and archaeal (53.3%) origin were soluble compared to bacteria (79.7%). Genes obtained by GS gave a significantly lower number (p = 0.04) of soluble proteins while the green fluorescent protein tag improved protein solubility (p = 0.05). Finally, a relationship between the amino acid composition and protein solubility was observed. Thus, a lower percentage of non-polar and higher percentage of negatively charged amino acids in a protein may be a good predictor for higher protein solubility in E. coli. The HTP approach presented here is a powerful tool for producing recombinant CAZymes that can be used for future studies of plant cell wall degradation. Successful production and expression of soluble recombinant proteins at a high rate opens new possibilities for the high-throughput production of targets from limitless sources.
Fichier principal
Vignette du fichier
cardoso_ijms-2021.pdf (1.7 Mo) Télécharger le fichier
Origin : Files produced by the author(s)

Dates and versions

hal-03781841 , version 1 (20-09-2022)

Licence

Attribution - CC BY 4.0

Identifiers

Cite

Vânia Cardoso, Joana Brás, Inês Costa, Luís Ferreira, Luís Gama, et al.. Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction. International Journal of Molecular Sciences, 2022, 23 (7), pp.4024. ⟨10.3390/ijms23074024⟩. ⟨hal-03781841⟩
29 View
9 Download

Altmetric

Share

Gmail Facebook Twitter LinkedIn More