Here, we characterize two novel GH5 endoglucanases (GH5CelA and GH5CelB) from an uncultured bacterium identified in termite gut microbiomes. Both genes were codon-optimized, synthetized, cloned, and expressed as recombinant proteins in Escherichia coli for subsequent purification. Both enzymes showed activity on the pNPC and barley β-glucan substrates, whereas GH5CelB also showed low activity on carboxymethyl cellulose. The optimum conditions for both enzymes were an acid pH (5) and moderate temperature (35 to 50 °C). The enzymes differed in the kinetic profiles and patterns of the generated hydrolysis products. A structural-based modeling analysis indicated that both enzymes possess a typical (β/α)8-barrel fold characteristic of GH5 family, with some differential features in the active site cleft. Also, GH5CelB presents a putative secondary binding site. Furthermore, adjacent to the active site of GH5CelA and GH5CelB, a whole subdomain rarely found in GH5 family may participate in substrate binding and thermal stability.
Therefore, GH5CelA may be a good candidate for the production of cello-oligosaccharides of different degrees of polymerization applicable for feed and food industries, including prebiotics. On the other hand, GH5CelB could be useful in an enzymatic cocktail for the production of lignocellulosic bioethanol, because of the production of glucose as a hydrolysis product.
• Synthetic metagenomics is a powerful approach for discovering novel enzymes.
• Two novel GH5 endoglucanases from nonculturable microorganisms were characterized.
• Structural differences between them and other GH5 endoglucanases were observed.
• The enzymes may be good candidates for feed, food, and/or bioethanol industries.
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MPS, EC, and PMT acknowledge CONICET as career research members. EBG and RMDV belong to the National Institute Agriculture Technology (INTA). The authors are grateful to Dr. Julia Sabio y García for linguistic improvement of the manuscript.
This study was funded by grants from the Instituto Nacional de Tecnología Agropecuaria (INTA) (PNAIyAV-1130034) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) Proyectos de Investigación Científica y Tecnológica (PICT) 2013 No.1454 (Argentina) and 2018 No. 4149.
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Guerrero, E.B., de Villegas, R.M.D., Soria, M.A. et al. Characterization of two GH5 endoglucanases from termite microbiome using synthetic metagenomics. Appl Microbiol Biotechnol 104, 8351–8366 (2020). https://doi.org/10.1007/s00253-020-10831-5
- Biochemical characterization
- Synthetic metagenomics