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Effect of Tris Buffer in the Intensity of the Multipoint Covalent Immobilization of Enzymes in Glyoxyl-Agarose Beads

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Abstract

Tris is an extensively used buffer that presents a primary amine group on its structure. In the present work trypsin, chymotrypsin and penicillin G acylase (PGA) were immobilized/stabilized on glyoxyl agarose in presence of different concentrations of Tris (from 0 to 20 mM). The effects of the presence of Tris during immobilization were studied analyzing the thermal stability of the obtained immobilized biocatalysts. The results indicate a reduction of the enzyme stability when immobilized in the presence of Tris. This effect can be observed in inactivations carried out at pH 5, 7, and 9 with all the enzymes assayed. The reduction of enzyme stability increased with the Tris concentration. Another interesting result is that the stability reduction was more noticeable for immobilized PGA than in the other immobilized enzymes, the biocatalysts prepared in presence of 20 mM Tris lost totally the activity at pH 7 just after 1 h of inactivation, while the reference at this time still kept around 61 % of the residual activity. These differences are most likely due to the homogeneous distribution of the Lys groups in PGA compared to trypsin and chymotrypsin (where almost 50% of Lys group are in a small percentage of the protein surface). The results suggest that Tris could be affecting the multipoint covalent immobilization in two different ways, on one hand, reducing the number of available glyoxyl groups of the support during immobilization, and on the other hand, generating some steric hindrances that difficult the formation of covalent bonds.

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Acknowledgements

RMS thanks to Ministerio de Educacion-Spanish Government for a FPU fellowship, and SAB and EHS thank Algerian Ministry of higher education and scientific research for their fellowships. The help and suggestions from Dr. Ángel Berenguer (Departamento de Química Inorgánica, Universidad de Alicante) are gratefully recognized.

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Funding

The research has been supported by Ministerio de Ciencia e Innovación-Spanish Government (project number CTQ2017-86170-R).

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Author notes

  1. Sabrina Ait Braham and Roberto Morellon-Sterling contributed equally to this work.

Authors and Affiliations

  1. Laboratoire de Biotechnologies Végétales et Ethnobotanique, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia, 06000, Bejaia, Algeria

    Sabrina Ait Braham & Ali Aksas

  2. Departamento de Biocatálisis, Instituto de Catálisis-CSIC, C/ Marie Curie 2, Campus UAM-CSI, Cantoblanco, 28049, Madrid, Spain

    Diandra de Andrades, El-Hocine Siar & Roberto Fernandez-Lafuente

  3. Biotechnology, Bioprocess, and Biocatalysis Group, Food Science and Technology Institute, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil

    Diandra de Andrades & Rafael C. Rodrigues

  4. Transformation and Food Product Elaboration Laboratory, Nutrition and Food Technology Institute (INATAA), University of Brothers Mentouri Constantine 1, Constantine, Algeria

    El-Hocine Siar

  5. Group of Plant Proteins, Department of Food and Health, Instituto de la Grasa-CSIC, Seville, Spain

    Justo Pedroche & Maria del Carmen Millán

  6. Center of Excellence in Bionanoscience Research, External Scientific Advisory Academics, King Abdulaziz University, Jeddah, 21589, Saudi Arabia

    Roberto Fernandez-Lafuente

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  1. Sabrina Ait Braham
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  2. Roberto Morellon-Sterling
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  3. Diandra de Andrades
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  4. Rafael C. Rodrigues
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  5. El-Hocine Siar
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  9. Roberto Fernandez-Lafuente
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Contributions

In this paper, Roberto Morellon-Sterling, El-Hocine Siar, Sabrina Ait Braham, and Diandra de Andrades prepared the biocatalysts and analyzed their performance, under the supervision of Rafa C. Rodrigues, Ali Aksas, and Roberto Fernandez-Lafuente. Justo Pedroche and Ma del Carmen Millán analyzed the amino-acid composition. Roberto Fernandez-Lafuente designed the experiments. All authors contributed to the writing of the paper.

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Correspondence to Roberto Fernandez-Lafuente.

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Braham, S.A., Morellon-Sterling, R., de Andrades, D. et al. Effect of Tris Buffer in the Intensity of the Multipoint Covalent Immobilization of Enzymes in Glyoxyl-Agarose Beads. Appl Biochem Biotechnol 193, 2843–2857 (2021). https://doi.org/10.1007/s12010-021-03570-4

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