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

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 o...

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Published in:Applied microbiology and biotechnology 2019-01, Vol.103 (1), p.279-289
Main Authors: Rodrigo-Frutos, David, Piedrabuena, David, Sanz-Aparicio, Julia, Fernández-Lobato, María
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creator Rodrigo-Frutos, David
Piedrabuena, David
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description 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.
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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.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>30357454</pmid><doi>10.1007/s00253-018-9446-y</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0003-0778-7443</orcidid><oa>free_for_read</oa></addata></record>
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subjects Baking yeast
Biomedical and Life Sciences
Biotechnologically Relevant Enzymes and Proteins
Biotechnology
Cultures (Biology)
Enzymes
Fructooligosaccharides
Glucose
Health promotion
Invertase
Life Sciences
Microbial Genetics and Genomics
Microbiological research
Microbiology
Oligosaccharides
Prebiotics
Probiotics
Proteins
Saccharomyces cerevisiae
Substitutes
Sucrose
Sugar
Yeast
Yeasts
Yeasts (Fungi)
title Yeast cultures expressing the Ffase from Schwanniomyces occidentalis, a simple system to produce the potential prebiotic sugar 6-kestose
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