21 April, 2024
GLYCOENZ-GREEN meeting
In the GLYCOENZ-GREEN Project (PID2022-136367OB-C31/2/3) funded by the State Research Agency, we are studying the application of enzymes that act on carbohydrates for the production of bioactive substances (oligosaccharides and polyphenol derivatives). This week, we had a workshop at the Institute of Catalysis and Petrochemistry, along with colleagues from the Blas Cabrera Institute of Physical Chemistry and the Severo Ochoa Center for Molecular Biology, CBMSO. It was a very productive meeting where we strengthened collaborations, and where young students enthusiastically defended their work.
20 January, 2024
Publication in Microbial Cell Factories: three new chitinases from Mestchnikowia pulcherrima
Three new chitinases from M. pulcherrima, MpChit35, MpChit38 and MpChit41, were molecularly characterized and extracellularly expressed in Pichia pastoris.The three enzymes hydrolysed colloidal chitin with optimal activity at 45 ºC and pH 4.0-4.5, The partial separation and characterization of the complex COS mixtures produced from the hydrolysis of chitin and chitosan were achieved by a new anionic chromatography HPAEC-PAD method and mass spectrometry assays. An overview of the predicted structures of these proteins and their catalytic modes of action were also presented. Depicted their high sequence and structural homology, MpChit35 acted as an exo-chitinase producing di-acetyl-chitobiose from chitin while MpChit38 and MpChit41 both acted as endo-chitinases producing tri-acetyl-chitotriose as main final product.
Microbial Cell Factories 23, 31 (2024) https://doi.org/10.1186/s12934-024-02300-9
28 June, 2023
PARTICIPATION IN BIOTRANS 2023
Several members of GLICOENZ are participating the renowned Biotrans2023 congress held in La Rochelle, France. This congress offers an overview of the latest advancements in the fields of biocatalysis and biotransformations, bringing together innovative and interdisciplinary strategies to overcome scientific and technological challenges.
Marina Minguet, Fadia Cervantes, and Eglė Narmontaitė presented their work on hyaluronidases, tagatose, and fructooligosaccharides, respectively.
Marina Minguet-Lobato
Marina Minguet-Lobato, Fadia V. Cervantes, David Fernández-Polo, María Fernández-Lobato, Francisco J. Plou
06 April, 2022
Elimination of D-Glucose in carbohydrate syrups published in ACS Food Science & Technology
During the synthesis of many bioactive carbohydrates, D-glucose is released as a side-product of the transglycosylation process. It is desirable to remove it due to its caloric contribution and its effect on caries and diabetes.
04 February, 2022
The molecular machinery behind processive fungal chitinases
Ref.: “Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts dynamic enzymatic mechanism”. E. Jiménez-Ortega, P.E. Kidibule, M. Fernández-Lobato, J. Sanz-Aparicio. Computational and Structural Biotechnology Journal 19, 5466-5478 (2021) http://doi.org/10.1016/j.csbj.2021.09.027
30 September, 2021
Thesis on exopolisaccharides producing microorganisms
Ángel García Horstmann has presented his PhD Thesis entitled "Study of moderate halophilic microorganisms productors of exopolisaccharides from Castilla-La Mancha's inland salt mines", supervised by Adrián García de Marina Bay and María Fernández Lobato (Autonomous University of Madrid, UAM). Congratulations to Ángel and his supervisors!
09 September, 2021
Characterization of a GH10 xylanase active at extreme conditions
We carried out a comprehensive bioinformatics study of the GH10 family searching for enzymes able to replace the use of highly pollutant chemicals in the pulp and paper industry, . The phylogenetic analysis allowed the construction of a radial cladogram in which protein sequences putatively ascribed as thermophilic and alkaliphilic appeared grouped in a well-defined region of the cladogram, designated TAK Cluster. One among five TAK sequences selected for experimental analysis (Xyn11) showed extraordinary xylanolytic activity under simultaneous conditions of high temperature (90 °C) and alkalinity (pH 10.5). Addition of a carbohydrate binding domain (CBM2) at the C-terminus of the protein sequence further improved the activity of the enzyme at high pH. Xyn11 structure, which has been solved at 1.8 Å resolution by X-ray crystallography, reveals an unusually high number of hydrophobic, ionic and hydrogen bond atomic interactions that could account for the enzyme’s extremophilic nature.
Ref.: "Phylogenetic, functional and structural characterization of a GH10 xylanase active at extreme conditions of temperature and alkalinity". D.Talens-Perales, E. Jiménez-Ortega, P. Sánchez-Torres, J.Sanz-Aparicio, J. Polaina. RSC Advances, 19, 2676-2686 (2021) https://doi.org/10.1016/j.csbj.2021.05.004
12 July, 2021
Enzymatic synthesis of novel fructosylated compounds
The β-fructofuranosidase from the yeast Schwanniomyces occidentalis (Ffase) produces potential prebiotic fructooligosaccharides (FOS) by self-transfructosylation of sucrose, being one of the highest known producers of 6-kestose. The use of Green Solvents (GS) in biocatalysis has emerged as a sustainable alternative to conventional organic media for improving product yields and generating new molecules. In this work, the Ffase hydrolytic and transfructosylating activity was analysed using different GS, including biosolvents and ionic liquids. Among them, 11 were compatible for the net synthesis of FOS. Besides, two glycerol derivatives improved the yield of total FOS. Interestingly, polyols ethylene glycol and glycerol were found to be efficient alternative fructosyl-acceptors, both substantially decreasing the sucrose fructosylation. The main transfer product of the reaction with glycerol was a 62 g L−1isomeric mixture of 1-O and 2-O-β-d-fructofuranosylglycerol, representing 95% of all chemicals generated by transfructosylation. Unexpectedly, the non-terminal 2-Ofructo-conjugate was the major molecule catalysed during the process, while the 1-Oisomer was the minor one. This fact made Ffase the first known enzyme from yeast showing this catalytic ability. Thus, novel fructosylated compounds with potential applications in food, cosmetics, and pharmaceutical fields have been obtained in this work, increasing the biotechnological interest of Ffase with innocuous GS.
Ref.:"Enzymatic synthesis of novel fructosylated compounds by Ffase from Schwanniomyces occidentalisin green solvents".David Piedrabuena, Ángel Rumbero, Elísabet Pires, Alejandro Leal-Duaso ,Concepción Civera, María Fernández-Lobato and Maria J. Hernaiz . RSC Advances, 11, 39, 24312-24319 (2021), doi: 10.1039/d1ra01391b
30 April, 2021
Enzymatic Synthesis of Phloretin α‐Glucosides
Glycosylation of polyphenols may increase their aqueous solubility, stability, bioavailability and pharmacological activity. Herein, we used a mutant of sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum engineered to accept large polyphenols (variant TtSPP_R134A) to produce phloretin glucosides. The selective formation of a monoglucoside or a diglucoside can be kinetically controlled. MS and 2D-NMR determined that the monoglucoside was phloretin 4’-O-α-D-glucopyranoside and the diglucoside phloretin-4’-O-[α-D-glucopyranosyl-(1→3)-O-α-D-glucopyranoside], a novel compound. The molecular features that determine the specificity of this enzyme for 4’-OH phenolic group were analysed by induced-fit docking analysis of each putative derivative, using the crystal structure of TtSPP and changing the mutated residue. The mono- and diglucoside were, respectively, 71- and 1200-fold more soluble in water than phloretin at room temperature. Since phloretin attracts a great interest in dermocosmetic applications, we analyzed the percutaneous absorption of glucosides and the aglycon employing a pig skin model. Although the three compounds were detected in all skin layers (except the fluid receptor), the diglucoside was present mainly on superficial layers.
Ref: "Enzymatic Synthesis of Phloretin α‐Glucosides using a Sucrose Phosphorylase Mutant and its Effect on Solubility, Antioxidant Properties and Skin Absorption. Advanced Synthesis & Catalysis". J.L. Gonzalez-Alfonso, Z. Ubiparip, E. Jimenez-Ortega, A. Poveda, C. Alonso, L. Coderch, J. Jimenez-Barbero, J. Sanz-Aparicio, A. Ballesteros, T. Desmet, F.J. Plou. (2021). Volume363, Issue12, Pages 3079-3089 https://doi.org/10.1002/adsc.202100201
31 March, 2021
New insights into the molecular mechanism behind mannitol and erythritol fructosylation
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 30% and 48%, respectively. Moreover, it produced 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.