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Applied Microbiology and Biotechnology

, Volume 103, Issue 1, pp 279–289 | Cite as

Yeast cultures expressing the Ffase from Schwanniomyces occidentalis, a simple system to produce the potential prebiotic sugar 6-kestose

  • David Rodrigo-Frutos
  • David Piedrabuena
  • Julia Sanz-Aparicio
  • María Fernández-LobatoEmail author
Biotechnologically relevant enzymes and proteins

Abstract

The β-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces potential prebiotic fructooligosaccharides with health-promoting properties, making it of biotechnological interest. Ffase is one of the highest and more selective known producers of 6-kestose by transfructosylation of sucrose. In this work, production of 6-kestose was simplified by directly using cultures of S. occidentalis and Saccharomyces cerevisiae expressing both the wild-type enzyme and a mutated Ffase variant including the Ser196Leu substitution (Ffase-Leu196). Best results were obtained using yeast cultures supplemented with sucrose and expressing the Ffase-Leu196, which after only 4 h produced ~ 116 g/L of 6-kestose, twice the amount obtained with the corresponding purified enzyme. 6-Kestose represented ~ 70% of the products synthesized. In addition, a small amount of 1-kestose and the neofructoligosaccharides neokestose and blastose were also produced. The Ser196Leu substitution skewed production of 6-kestose and neofructooligosaccharides resulting in an increase of ~ 2.2- and 1.5-fold, respectively, without affecting production of 1-kestose. Supplementing yeast cultures with glucose clearly showed that blastose originates from direct fructosylation of glucose, a property that has not been described for other similar proteins from yeasts. Modeling neokestose and blastose into the Ffase-active site revealed the molecular basis explaining the peculiar specificity of this enzyme.

Keywords

Schwanniomyces occidentalis β-Fructofuranosidase Prebiotic sugars 6-Kestose Blastose Yeast cultures 

Notes

Acknowledgements

We thank Mrs. Asunción Martín-Redondo and María Gimeno-Pérez for their technical support and Mr. Tom Halmos for reading and correcting this manuscript.

Funding

This work was supported by the Spanish Ministry of Economy and Competitiveness: BIO2016-76601-C3-2/-3, and by institutional grants from Fundación Ramón Areces and Banco de Santander to the Centro de Biología Molecular Severo Ochoa. Besides, funding has been received from the European Union’s Horizon 2020 research and innovation program [Blue Growth: Unlocking the potential of Seas and Oceans] under grant agreement No [634486; INMARE]. D.P. was supported by the Spanish Ministry of Education’s University Personnel Training Plan ref. FPU014/01004.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Molecular Biology, Center of Molecular Biology Severo Ochoa (CBMSO; CSIC-UAM)University Autónoma from MadridMadridSpain
  2. 2.Department of Crystallography and Structural BiologyInstitute of Physical Chemistry-Rocasolano (CSIC)MadridSpain

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