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Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production

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Abstract

Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α–(1–6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal sequence of the previously detected 60 kDa protein resides in this larger protein at residues 583–602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterologously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode synthesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements characteristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homologous human maltase-glucoamylase was obtained. Remarkably, the Xd-AlphaGlu C-terminal domain presents an unusually long 115-residue insertion that could be involved in this enzyme’s activity against long-size substrates such as maltoheptaose and soluble starch.

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Acknowledgments

Projects BIO2013-48779-C4-1/-3/-4 from the Spanish Ministry of Economy and Competitiveness supported this research. We thank Fundación Ramón Areces for the institutional grant to the Centro de Biología Molecular Severo Ochoa. M.G.P. was supported by a Spanish FPU fellowship from the Ministry of Economy and Competitiveness.

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Authors and Affiliations

  1. Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular (CSIC-UAM), Nicolás Cabrera 1. Universidad Autónoma Madrid, 28049, Madrid, Spain

    Patricia Gutiérrez-Alonso, María Gimeno-Pérez & María Fernández-Lobato

  2. Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006, Madrid, Spain

    Mercedes Ramírez-Escudero & Julia Sanz-Aparicio

  3. Instituto de Catálisis y Petroleoquímica, CSIC, Marie Curie 2, 28049, Madrid, Spain

    Francisco J. Plou

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  1. Patricia Gutiérrez-Alonso
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  2. María Gimeno-Pérez
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  3. Mercedes Ramírez-Escudero
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  6. María Fernández-Lobato
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Correspondence to María Fernández-Lobato.

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This study was funded by the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1/-3/-4).

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The authors declare that they have no competing interests.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Additional file 1

Table S1. Characteristics of the Xd-AlphaGlu introns. Figure S1. Positioning of peptide masses generated by the MALDI-TOF analysis of the protein Xd-AlphaGlu expressed in X. dendrorhous (a) and S. cerevisiae (b). The peptide m/z is indicated. (PDF 339 kb)

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Gutiérrez-Alonso, P., Gimeno-Pérez, M., Ramírez-Escudero, M. et al. Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production. Appl Microbiol Biotechnol 100, 3125–3135 (2016). https://doi.org/10.1007/s00253-015-7171-3

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