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Enhanced xyloglucan-specific endo-β-1,4-glucanase efficiency in an engineered CBM44-XegA chimera

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Abstract

Xyloglucan-specific endo-β-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for β-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s−1; XegA, 144.31 s−1) and reduced KM (chimera, 1.30 mg mL−1; XegA, 1.50 mg mL−1) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.

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Acknowledgments

This work was supported by research grants FAPESP 2010/18850-2, CNPq 307795/2009-8 (RJW), FAPESP 2013/13309-0 and 2014/07135-1 (MTM), FAPESP 2012/20549-4 and 2013/18910-3 (ARLD), FAPESP stipends, FAPESP 2010/07133-8 (LR), and CAPES stipend (GPF).

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Departamento de Bioquímica e Imunologia, FMRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

    Gilvan P. Furtado & Lucas F. Ribeiro

  2. Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Giuseppe Maximo Scolfaro, 10000, 13083-970, Campinas, SP, Brazil

    Camila R. Santos, Rosa L. Cordeiro & Mário T. Murakami

  3. Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisas em Energia e Materiais, Campinas, SP, Brazil

    André R. L. Damásio & Richard J. Ward

  4. Departamento de Biologia, FFCLRP-USP, Universidade de São Paulo, Ribeirão Preto, SP, Brazil

    Maria de Lourdes T. M. Polizeli

  5. Computational Modelling Group: Simulation and Evolution of Biomolecules, Fundação Oswaldo Cruz—Fiocruz-CE, Fortaleza, CE, Brazil

    Luiz A. B. de Moraes & Marcos R. Lourenzoni

  6. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, 14040-901, Ribeirão Preto, SP, Brazil

    Richard J. Ward

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  1. Gilvan P. Furtado
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  2. Camila R. Santos
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  3. Rosa L. Cordeiro
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  4. Lucas F. Ribeiro
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  5. Luiz A. B. de Moraes
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  6. André R. L. Damásio
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  7. Maria de Lourdes T. M. Polizeli
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  8. Marcos R. Lourenzoni
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  9. Mário T. Murakami
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  10. Richard J. Ward
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Corresponding authors

Correspondence to Mário T. Murakami or Richard J. Ward.

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Gilvan P. Furtado and Camila R. Santos have contributed equally to this work.

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Furtado, G.P., Santos, C.R., Cordeiro, R.L. et al. Enhanced xyloglucan-specific endo-β-1,4-glucanase efficiency in an engineered CBM44-XegA chimera. Appl Microbiol Biotechnol 99, 5095–5107 (2015). https://doi.org/10.1007/s00253-014-6324-0

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