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Advisor(s)
Abstract(s)
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
Description
Áreas de pesquisa: Biochemistry & Molecular Biology ; Chemistry
Keywords
Carbohydrate-active enzymes (CAZymes) Plant biomass Gene synthesis PCR High-throughput (HTP) cloning HTP expression
Pedagogical Context
Citation
Cardoso V, Brás JLA, Costa IF, Ferreira LMA, Gama LT, Vincentelli R, Henrissat B, Fontes CMGA. 2022. Generation of a library of carbohydrate-active enzymes for plant biomass deconstruction. Internal Journal of Molecular Sciences, 23(7):4024. DOI 10.3390/ ijms23074024
Publisher
MDPI