Synthesis of 6‐Kestose using an Efficient β‐Fructofuranosidase Engineered by Directed Evolution

Abreu, Miguel de; Álvaro-Benito, Miguel; Sanz‐Aparicio, Julia; Plou, Francisco J.; Fernández-Lobato, María; Alcalde, Miguel

doi:10.1002/adsc.201200769

Cited by 18 publications

(20 citation statements)

References 14 publications

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“…FOS are classified into three categories: 1 F-, 6 F-and 6 G-FOS (13)(14)(15). Among these FOS, only 1 F-FOS is commercially available.…”

Section: Introductionmentioning

confidence: 99%

Antineoplastic effect of a novel chemopreventive agent, neokestose, on the Caco-2 cell line via inhibition of expression of nuclear factor-κB and cyclooxygenase-2

Lee¹,

Chang²,

Wu³

et al. 2012

Molecular Medicine Reports

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Neokestose is a 6G-fructooligosaccharide (FOS) and an important prebiotic. When FOS are ingested by patients with colorectal cancer, they may come into contact with cancer cells prior to being fermented by bifidobacteria in the colon. In the present study, the effects of neokestose on cell proliferation, cell cycle and apoptosis of the colorectal cancer cell line Caco-2 were investigated to evaluate its anti-cancer effect. An MTT assay showed that neokestose-treated Caco-2 cells exhibited a significant and dose-dependent loss of viability. Flow cytometric analysis indicated that the sub-G1 population of Caco-2 cells was significantly increased following treatment with neokestose, and the percentage of Caco-2 cells in the stage of late apoptosis was also significantly increased in a dose-dependent manner. Western blot analysis showed that the overexpression of nuclear factor-κB, a central molecule responsible for the transition from inflammation to cancer, and cyclooxygenase-2, an important enzyme in colorectal tumorigenesis, in colorectal carcinoma cells was inhibited by neokestose. Accordingly, the present study provided in vitro evidence that neokestose may be used as a dietary chemopreventive agent, whose application is more rational than that of COX-2 inhibitors or aspirin for preventing colorectal cancer.

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“…FOS are classified into three categories: 1 F-, 6 F-and 6 G-FOS (13)(14)(15). Among these FOS, only 1 F-FOS is commercially available.…”

Section: Introductionmentioning

confidence: 99%

Antineoplastic effect of a novel chemopreventive agent, neokestose, on the Caco-2 cell line via inhibition of expression of nuclear factor-κB and cyclooxygenase-2

Lee¹,

Chang²,

Wu³

et al. 2012

Molecular Medicine Reports

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show abstract

“…Unless otherwise indicated, standard β-fructofuranosidase activity was assayed at 50 ºC for 20 min by measuring the amount of reducing sugars released from 20 g l −1 sucrose in 100 mM sodium acetate pH 5.5 using dinitrosalicylic acid (DNS) method adapted to 96-well microplates (50 µl final volume) as previously described 34 . Glucose (0.2—3.0 g l −1 ) was used for the calibration curve.…”

Section: Methodsmentioning

confidence: 99%

New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis

Rodrigo-Frutos

Jiménez-Ortega

Piedrabuena

et al. 2021

Sci Rep

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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 studied to explore their role in modulating this biocatalytic process. Interestingly, N254T variant enhanced the wild-type protein production of fructosyl-erythritol and FOS by $$\sim$$ ∼ 30% and 48%, respectively. Moreover, it produced neokestose, which represented $$\sim$$ ∼ 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.

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“…Standard β-fructofuranosidase hydrolytic activity assays. Unless otherwise indicated, standard βfructofuranosidase activity was assayed at 50 ºC for 20 min by measuring the amount of reducing sugars released from 20 g l − 1 sucrose in 100 mM sodium acetate pH 5.5 using dinitrosalicylic acid (DNS) method adapted to 96-well microplates (50 µl nal volume) as previously described 33 . Glucose (0.2-3.0 g l − 1 ) was used for the calibration curve.…”

Section: Methodsmentioning

confidence: 99%

New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis

Rodrigo-Frutos

Jiménez-Ortega

Piedrabuena

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

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.

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Synthesis of 6‐Kestose using an Efficient β‐Fructofuranosidase Engineered by Directed Evolution

Cited by 18 publications

References 14 publications

Antineoplastic effect of a novel chemopreventive agent, neokestose, on the Caco-2 cell line via inhibition of expression of nuclear factor-κB and cyclooxygenase-2

Antineoplastic effect of a novel chemopreventive agent, neokestose, on the Caco-2 cell line via inhibition of expression of nuclear factor-κB and cyclooxygenase-2

New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis

New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis

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