Journal Article

Heterologous expression and structure prediction of a xylanase identified from a compost metagenomic library

Silvério, Sara C., Sousa, Joana, Sousa, Sérgio F., Costa, Ângela M. A., Rodrigues, Lígia R., Gomes, Joana S., Santos, Andréia O., Franco-Duarte, Ricardo, Linhares, João M. M., Santos-Pereira, Cátia

2024

Overview

Xylanases are key biocatalysts in the degradation of the β‐1,4‐glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-β-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. Key points • A GH10 endo-1,4-β-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB’s in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis Graphical abstract

• Publisher: Springer Berlin Heidelberg,Springer,Springer Nature B.V
• Subject: active sites / Amino acids / Biocatalysts / Biodegradation / Biomedical and Life Sciences / Bioprocessing / Biotechnologically Relevant Enzymes and Proteins / Biotechnology / Catalysis / catalytic activity / Cloning, Molecular / Composting / Composts / E coli / Endo-1,4-beta Xylanases - chemistry / Endo-1,4-beta Xylanases - genetics / Endo-1,4-beta Xylanases - isolation & purification / Endo-1,4-beta Xylanases - metabolism / endo-1,4-β-Xylanase / Enzymatic activity / Enzyme activity / Enzymes / Escherichia coli / Escherichia coli - genetics / Escherichia coli - metabolism / family / Fermentation / Gene Expression / Gene Library / genomic libraries / Genomics / Glycosidases / Glycosyl hydrolase / Hemicellulose / heterologous gene expression / High temperature / Identification and classification / Industrial applications / Libraries / Life Sciences / Matlab / Metagenome / Metagenomics / Methods / Microbial Genetics and Genomics / Microbiology / Molecular Docking Simulation / Phylogeny / Physiological aspects / prediction / Soil Microbiology / submerged fermentation / Substrates / Xylan / Xylanase / Xylanases / Xylans - metabolism