02 February, 2021
04 December, 2020
Use of a β-galactosidase from Pantoea anthophila to produce galactooligosaccharides from a byproduct of the dairy industry
Ref.:"Synthesis of β(1 → 3) and β(1 → 6) galactooligosaccharides from lactose and whey using a recombinant β-galactosidase from Pantoea anthophila" Claudia V.Yañez-Ñeco, Fadia V.Cervantes, LorenaAmaya-Delgado, Antonio O.Ballesteros, Francisco J.Plou, Javier Arrizon. Electronic Journal of Biotechnology, Volume 49, January 2021, Pages 14-21. https://doi.org/10.1016/j.ejbt.2020.10.004
11 July, 2020
α-Glucosidases are widely distributed enzymes with a varied substrate specificity that are traditionally used in biotechnological industries based on oligo- and polysaccharides as starting materials. According to amino acid sequence homology, α-glucosidases are included into two major families, GH13 and GH31. The members of family GH13 contain several α-glucosidases with confirmed hydrolytic activity on sucrose. Previously, a sucrose splitting activity from the nectar colonizing yeast Metschnikowia reukaufii which produced rare sugars with α-(1→1), α-(1→3) and α-(1→6) glycosidic linkages from sucrose was described.
In this study, genes codifying for α-glucosidases from the nectaries yeast M. gruessii and M. reukaufii were characterised and heterologously expressed in Escherichia coli for the first time. Recombinant proteins (Mg-αGlu and Mr-αGlu) were purified and biochemically analysed. Both enzymes mainly displayed hydrolytic activity towards sucrose, maltose and p-nitrophenyl-α-D-glucopyranoside. Structural analysis of these proteins allowed the identification of common features from the α-amylase family, in particular from glycoside hydrolases that belong to family GH13. The three acidic residues comprising the catalytic triad were identified and their relevance for the protein hydrolytic mechanism confirmed by site-directed mutagenesis. Recombinant enzymes produced oligosaccharides naturally present in honey employing sucrose as initial substrate and gave rise to mixtures with the same products profile (isomelezitose, trehalulose, erlose, melezitose, theanderose and esculose) previously obtained with M. reukaufii cell extracts. Furthermore, the same enzymatic activity was detected with its orthologous Mg-αGlu from M. gruessii. Interestingly, the isomelezitose amounts obtained in reactions mediated by the recombinant proteins, ~ 170 g/L, were the highest reported so far.
09 June, 2020
13 April, 2020
Selective Synthesis of Galactooligosaccharides with β-Galactosidase from Bifidobacterium bifidum (Saphera)
The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions for the operation of this enzyme, measured with o-nitrophenyl-β-d-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the property of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS). The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40–50% lactose conversion. Using high-performance anion-exchange chromatography with pulsed amperometric detection, semipreparative high-performance liquid chromatography-hydrophilic interaction liquid chromatography, mass spectrometry, and 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme. The main identified products were Gal-β(1→3)-Gal-β(1→4)-Glc (3′-O-β-galactosyl-lactose), Gal-β(1→6)-Glc (allolactose), Gal-β(1→3)-Glc (3-galactosyl-glucose), Gal-β(1→3)-Gal (3-galactobiose), and the tetrasaccharide Gal-β(1→3)-Gal-β(1→3)-Gal-β(1→4)-Glc. In general, B. bifidum β-galactosidase showed a tendency to form β(1→3) linkages followed by β(1→6) and more scarcely β(1→4).
Ref.:"Selective Synthesis of Galactooligosaccharides Containing β(1→3) Linkages with β-Galactosidase from Bifidobacterium bifidum (Saphera)." Vera Füreder, Barbara Rodriguez-Colinas, Fadia V. Cervantes, Lucia Fernandez-Arrojo, Ana Poveda, Jesus Jimenez-Barbero, Antonio O. Ballesteros, and Francisco J. Plou. Journal of Agricultural and Food Chemistry 2020, 68, 17, 4930–4938 https://doi.org/10.1021/acs.jafc.0c00997
03 December, 2019
Ref.: “Deciphering the molecular specificity of phenolic compounds as inhibitors or glycosyl acceptors of β fructofuranosidase from Xanthophyllomyces dendrorhous”. M. Ramirez-Escudero, N. Miguez, M. Gimeno-Perez, A.O. Ballesteros, M. Fernandez-Lobato, F.J. Plou* and J. Sanz-Aparicio* Scientific Reports 9, 17441 (2019), doi:10.1038/s41598-019-53948-y
17 November, 2019
Metschnikowia reukaufii is a widespread yeast able to grow in the floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l−1, which led us into the search for enzymatic activities involved in this sugar transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload‐sucrose reactions. These products were purified and structurally characterized by MS‐ESI and NMR . The reaction mixture included new sugars showing a great variety of glycosidic bonds including α‐(1→1), α‐(1→3) and α‐(1→6) . The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l−1, the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l−1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. The sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide. as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.
Ref.: M. Garcia-Gonzalez, F.V. Cervantes, F.J. Plou, M. Remacha, A. Poveda, J. Jimenez-Barbero and M. Fernandez-Lobato. “Efficient production of isomelezitose by a novel glucosyltransferase activity in Metschnikowia reukaufii cell extracts”. Microbial Biotechnology (2019), doi:10.1111/1751-7915.13490
12 November, 2019
Production of High Fructose Syrup (HFS) with immobilized glucose isomerase published in Food Chemistry
Reference: "Immobilization of the glucose isomerase from Caldicoprobacter algeriensis on Sepabeads EC-HA and its efficient application in continuous High Fructose Syrup production using packed bed reactor". S. Neifar, F.V. Cervantes, H. BenHlima, A. Bouanane-Darenfed, A.O. Ballesteros, F.J. Plou, S. Bejar. . Food Chemistry (2019), doi: 10.1016/j.foodchem.2019.125710
04 July, 2019
Reference; ''Production and Surfactant Properties of Tert-Butyl α-D-Glucopyranosides Catalyzed by Cyclodextrin Glucanotransferase'' H. Garcia-Arellano, J. L. Gonzalez-Alfonso, C. Ubilla, F. Comelles, M. Alcalde, M. Bernabé, J-L Parra, A. O. Ballesteros and F. J. Plou. Catalysts (2019), https://doi.org/10.3390/catal9070575