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Synthesis, properties, and application in peptide chemistry of a magnetically separable and reusable biocatalyst

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Abstract

Enzyme-catalyzed chemical processes are selective, very productive, and generate little waste. Nevertheless, they may be optimized using enzymes bound to solid supports, which are particularly important for protease-mediated reactions since proteases undergo fast autolysis in solution. Magnetic nanoparticles are suitable supports for this purpose owing to their high specific surface area and to be easily separated from reaction media. Here we describe the immobilization of bovine α-chymotrypsin (αCT) on silica-coated superparamagnetic nanoparticles (Fe3O4@silica) and the characterization of the enzyme-nanoparticle hybrid (Fe3O4@silica-αCT) in terms of protein content, properties, recovery from reaction media, application, and reuse in enzyme-catalyzed peptide synthesis. The results revealed that (i) full acid hydrolysis of the immobilized protease followed by amino acid analysis of the hydrolyzate is a reliable method to determine immobilization yield; (ii) despite showing lower amidase activity and a lower K cat/K m value for a specific substrate than free αCT, the immobilized enzyme is chemically and thermally more stable, magnetically recoverable from reaction media, and can be consecutively reused for ten cycles to catalyze the amide bond hydrolysis and ester hydrolysis of the protected dipeptide Z-Ala-Phe-OMe. Altogether, these properties indicate the potential of Fe3O4@silica-αCT to act as an efficient, suitably stable, and reusable catalyst in amino acid, peptide, and protein chemistry as well as in proteomic studies.

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Acknowledgments

This work was supported by grants from FAPESP to MTM (2008/11695-1; 2012/09068-4) and fellowships from CNPq and FAPESP to VAU and NJSC, respectively. We also thank Prof. Dr. Pedro K. Kiyohara (IF-USP) for the TEM images. MTM and LMR are members of the NAPCatSinQ-USP.

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Authors and Affiliations

  1. Department of Biochemistry, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes, 748, PO Box 26077, São Paulo, SP, 05513-970, Brazil

    Cleber W. Liria, Vitor A. Ungaro, Raphaella M. Fernandes, Sandro R. Marana & M. Teresa Machini

  2. Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, PO Box 26077, São Paulo, SP, 05513-970, Brazil

    Natália J. S. Costa & Liane M. Rossi

Authors
  1. Cleber W. Liria
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  2. Vitor A. Ungaro
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  3. Raphaella M. Fernandes
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  4. Natália J. S. Costa
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  5. Sandro R. Marana
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  6. Liane M. Rossi
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  7. M. Teresa Machini
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Correspondence to M. Teresa Machini.

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Guest Editors: Carlos Lodeiro Espiño, José Luis Capelo Martinez

This article is part of the topical collection on Composite Nanoparticles

Cleber W. Liria, Vitor A. Ungaro, and Raphaella M. Fernandes have contributed equally to this study.

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Liria, C.W., Ungaro, V.A., Fernandes, R.M. et al. Synthesis, properties, and application in peptide chemistry of a magnetically separable and reusable biocatalyst. J Nanopart Res 16, 2612 (2014). https://doi.org/10.1007/s11051-014-2612-y

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  • DOI: https://doi.org/10.1007/s11051-014-2612-y

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