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Identification and Characterization of a Glycoside Hydrolase Family 9 Member from the Digestive Gland of the Snail Achatina fulica

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Abstract

Biomass-degrading enzymes can aid the development of new technologies to increase the production efficiency of green fuels like ethanol. This study reports the identification and characterization of a novel glycoside hydrolase family 9 (GH9) member from the digestive gland of the snail Achatina fulica, an endo-β-1,4-glucanase named AfEG66. The enzyme has a high level of identity with other mollusk endoglucanases and harbors a family 2 carbohydrate-binding module (CBM2) linked to a GH9 domain. Recombinant AfEG66 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography followed by ion exchange chromatography. The enzyme was active against barley β-glucan, lichenan, and showed a specific activity of 41.56 ± 1.21 IU/mg towards carboxymethyl cellulose. AfEG66 showed higher activities at pH 6.0–6.5 and 45 °C, being stable at temperatures equal or below 37 °C. The enzyme activity on cello-oligosaccharides with different glucose units was evaluated by thin-layer chromatography and showed the hydrolysis of substrates containing at least three glucose residues. A structural model was obtained to perform docking assays that clarified the hydrolytic properties of this enzyme. The results presented here show that A. fulica has an endogenous cellulase gene that encodes a GH9 endoglucanase associated to a putative CBM2 with β-1,4 glycoside hydrolase activity.

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Data Availability

The accession codes are: AfEG66 cloned cDNA: KY373245. Accession codes used for phylogenetic and sequence analysis: WP_026418585.1, ABD24276.1, XP_016907626.1, XP_005111869.2, WP_054493315.1, AMH40360.1, WP_026587001.1, BAU45388.1, XP_013069170.1, WP_027630841.1, ADB12483.1, AGS32241.1, BAF38757.1, XP_011423159.1, BAM14716.1, AFQ98380.1, XP_009049941.1, CAD54730.1, AMH40372.1, KOX76353.1, BAH22180.1, BAH85844.1, XP_012526654.1, BAA33708.1, ADB82658.1, AAF80585.1, ANR02619.1, BAA34050.1, WP_003517595.1, WP_052391031.1, AGP76433.1, WP_011292599.1 and XP_013419079.1.

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Not applicable for that section.

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Funding

This work was supported by the Instituto Nacional de Metrologia, Qualidade e Tecnologia – INMETRO and by the research foundations Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, and Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq).

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  1. Norton Heise and Ednildo de Alcantara Machado contributed equally to this work.

Authors and Affiliations

  1. Diretoria de Metrologia Aplicada as Ciencias da Vida, Instituto Nacional de Metrologia, Qualiodade e Tecnologia (NMETRO), Duque de Caxias, Rio de Janeiro, Brazil

    Youssef Bacila Sade, Camila Silva Gonçalves, Sandra Mara Naressi Scapin, Guilherme Luiz Pinheiro & Roberto Becht Flatschart

  2. Laboratório de Bioquímica de Insetos e Parasitos (Labip) do Instituto de Biofísica Carlos Chagas Filho, Universidade Federal Do Rio de Janeiro, RJ, 21941-902, Rio de Janeiro, Brazil

    Youssef Bacila Sade, Wanderley de Souza, Norton Heise & Ednildo de Alcantara Machado

  3. Laboratório de Ultraestrutura Celular Hertha Meyer do Instituto de Biofísica Carlos Chagas Filho, Centro Nacional de Biologia Estrutural E Bioimagem and Instituto Nacional de Ciência E Tecnologia Em Biologia Estrutural E Bioimagem, Universidade Federal do Rio de Janeiro, RJ, Rio de Janeiro, Brazil

    Wanderley de Souza

  4. Instituto Nacional de Ciência e Tecnologia Em Entomologia Molecular, RJ, Rio de Janeiro, Brazil

    Ednildo de Alcantara Machado

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  1. Youssef Bacila Sade
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Contributions

Y.B.S., G.L.P., and R.B.F. conceived and designed the experiment(s), Y.B.S, C.S.G, and S.M.N.S. conducted the experiments. W.S., N.H., and E.A.M. analyzed the results and performed a critical revision of the manuscript.

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Correspondence to Ednildo de Alcantara Machado.

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Sade, Y.B., Gonçalves, C.S., Scapin, S.M.N. et al. Identification and Characterization of a Glycoside Hydrolase Family 9 Member from the Digestive Gland of the Snail Achatina fulica. Bioenerg. Res. 15, 466–478 (2022). https://doi.org/10.1007/s12155-021-10303-2

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