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Research Article
María Fernández-Lobato
Centro de Biología Molecular Severo Ochoa
Corresponding Author
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The β-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructo-conjugates. In this work, the structural determinants of Ffase involved in the transfructosylating reaction of the alditols mannitol and erythritol have been studied in detail. Complexes with fructosyl-erythritol or sucrose were analyzed by crystallography and the effect of mutational changes in positions Gln-176, Gln-228, and Asn-254 revised to explore their role in modulating this biocatalytic process. Interestingly, N254T variant enhanced the wild-type protein production of fructosyl-erythritol and FOS by `30% and 48%, respectively. Moreover, it showed a shift towards neokestose, which represented ~27% of total FOS, and yielded 31.8 g l-1 blastose by using glucose as exclusive fructosyl-acceptor. Noteworthy, N254D and Q176E replacements turned the specificity of Ffase transferase activity towards the synthesis of the fructosylated polyols at the expense of FOS production, but without increasing the total reaction efficiency. The results presented here highlight the relevance of the pair Gln-228/Asn-254 for Ffase donor-sucrose binding and opens new windows of opportunity for optimizing the generation of fructosyl-derivatives by this enzyme enhancing its biotechnological applicability.
Keywords
β-fructofuranosidase, alditols, transfructosylation, fructooligosaccharides, X-ray crystallography
Figure 1
Figure 2
Figure 3
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Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- SupMataterialSCR.docx
Supplementary information: Table S1. Crystallographic statistics; Figure S1. Schematic view of the reactions catalyzed by Ffase on sucrose; Figure S2. HPLC chromatograms from transfructosylating reactions produced by N254T (a) or wild-type (b) Ffase variants; Figure S3. Mass spectrum of the reaction mixture obtained with the Ffase-N254T variant in reactions containing sucrose and erythritol; Figure S4. SDS-PAGE analysis of the purified Ffase variants.
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See the published version of this preprint at Scientific Reports.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
María Fernández-Lobato
Centro de Biología Molecular Severo Ochoa
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Scientific Reports.
Version 1
Posted 16 Dec, 2020
No community comments so far
Editorial decision: Major revision
On 04 Jan, 2021
Reviews received
Received 22 Dec, 2020
Reviewers agreed
On 15 Dec, 2020
Reviewers invited
Invitations sent on 15 Dec, 2020
Editor assigned
On 15 Dec, 2020
Editor invited
On 07 Dec, 2020
Submission checks complete
On 07 Dec, 2020
First submitted
On 01 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Scientific Reports.
The β-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructo-conjugates. In this work, the structural determinants of Ffase involved in the transfructosylating reaction of the alditols mannitol and erythritol have been studied in detail. Complexes with fructosyl-erythritol or sucrose were analyzed by crystallography and the effect of mutational changes in positions Gln-176, Gln-228, and Asn-254 revised to explore their role in modulating this biocatalytic process. Interestingly, N254T variant enhanced the wild-type protein production of fructosyl-erythritol and FOS by `30% and 48%, respectively. Moreover, it showed a shift towards neokestose, which represented ~27% of total FOS, and yielded 31.8 g l-1 blastose by using glucose as exclusive fructosyl-acceptor. Noteworthy, N254D and Q176E replacements turned the specificity of Ffase transferase activity towards the synthesis of the fructosylated polyols at the expense of FOS production, but without increasing the total reaction efficiency. The results presented here highlight the relevance of the pair Gln-228/Asn-254 for Ffase donor-sucrose binding and opens new windows of opportunity for optimizing the generation of fructosyl-derivatives by this enzyme enhancing its biotechnological applicability.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- SupMataterialSCR.docx
Supplementary information: Table S1. Crystallographic statistics; Figure S1. Schematic view of the reactions catalyzed by Ffase on sucrose; Figure S2. HPLC chromatograms from transfructosylating reactions produced by N254T (a) or wild-type (b) Ffase variants; Figure S3. Mass spectrum of the reaction mixture obtained with the Ffase-N254T variant in reactions containing sucrose and erythritol; Figure S4. SDS-PAGE analysis of the purified Ffase variants.
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