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Stabilization of Glycosylated β-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues

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Abstract

Many industrial enzymes can be highly glycosylated, including the β-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized β-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of β-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of β-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

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Acknowledgments

The authors thank the support from Research Institute of Food Science (CIAL), Embrapa Instrumentation of São Carlos, Foundation for Research Support of Espírito Santo (FAPES), and Federal University of Espírito Santo (UFES).

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Authors and Affiliations

  1. Departamento de Engenharias e Tecnologia, Universidade Federal do Espírito Santo, Rodovia BR 101 Norte Km 60, São Mateus, ES, Brazil

    Laura Marina Pinotti

  2. Programa de Pós-Graduação em Engenharia Química, Departamento de Engenharia Química, Universidade Federal de São Carlos, Rodovia Washington Luís, Km 235, São Carlos, SP, Brazil

    Paulo Waldir Tardioli & Cristiane Sanchez Farinas

  3. Embrapa Instrumentação, Rua XV de Novembro, São Carlos, SP, 1452, Brazil

    Cristiane Sanchez Farinas

  4. Departamento de Biotecnologia y Microbiologia de los Alimentos, Instituto de Investigación en Ciências de los Alimentos, C/Nicolás Cabrera 9, Campus UAM Cantoblanco, Madrid, Spain

    Gloria Fernández-Lorente & Benevides C. Pessela

  5. Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica - CSIC, Campus UAM Cantoblanco, 28049, Madrid, Spain

    Alejandro H. Orrego & Jose M. Guisan

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  1. Laura Marina Pinotti
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Correspondence to Laura Marina Pinotti or Jose M. Guisan.

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Pinotti, L.M., Tardioli, P.W., Farinas, C.S. et al. Stabilization of Glycosylated β-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues. Appl Biochem Biotechnol 192, 325–337 (2020). https://doi.org/10.1007/s12010-020-03321-x

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