Participation in the IV Spanish Biocatalysis Conference

The GLICOENZ consortium has participated in the IV Spanish Biocatalysis Conference, organized by CICbiomaGUNE and SEBIOT at the Aquarium of Donostia (September 5-6th, 2024). We presented our latest results on glycosylation of bioactive polyphenols:

"Molecular basis of specificity of a sucrose phosphorylase mutant in the glycosylation of polyphenols". M. Ruiz-Nuñez, J.L. Gonzalez-Alfonso, M. Moreno-Garcia, C. Uceda, M. Fernandez-Lobato, F.J. Plou and J. Sanz-Aparicio.

"Glucosylation of polyphenols with high yield and regioselectivity using a sucrose phosphorylase mutant". J.L. Gonzalez-Alfonso, D. Rodriguez-García, M. Moreno-García, C. Uceda-Domínguez, C. Alonso, A. Poveda, Z. Ubiparip, A.O. Ballesteros, T. Desmet, J. Jiménez-Barbero, L. Coderch, and F.J. Plou

Potential of alpha-glucosidase from Sch. occidentalis published in Appl. Microbiol. Biotechnol.

The α-glucosidase from Schwanniomyces occidentalis (GAM1p) was expressed in Komagataella phaffii to about 70 mg/L, and its transferase activity studied in detail. Several isomaltooligosaccharides (IMOS) were formed using 200 g/L maltose. The major production of IMOS (81.3 g/L) was obtained when 98% maltose was hydrolysed, of which 34.8 g/L corresponded to isomaltose, 26.9 g/L to isomaltotriose, and 19.6 g/L to panose. In addition, the potential of this enzyme to glycosylate 12 possible hydroxylated acceptors, including eight sugars and four phenolic compounds, was evaluated. Among them, only sucrose, xylose, and piceid (a monoglucosylated derivative of resveratrol) were glucosylated, and the main synthesised products were purified and characterised by MS and NMR. 

Ref: “Insights into the transglucosylation activity of α-glucosidase from Schwanniomyces occidentalis”. Zoran Merdzo, Egle Narmontaite, Jose L. Gonzalez-Alfonso, Ana Poveda, Jesus Jimenez-Barbero, Francisco J. Plou, María Fernández-Lobato. Applied Microbiology and Biotechnology 108, 443 (2024). https://doi.org/10.1007/s00253-024-13262-8

GLICOENZ at the IX National Congress of Industrial Microbiology and Microbial Biotechnology.

The GLICOENZ consortium has been widely represented in the IX National Congress of Industrial Microbiology and Microbial Biotechnology. 

Laura Barahona, Noa Miguez, Egle Narmontaite, María Martínez-Ranz, Mercedes Moreno presented their works on different topics of glycoenzymes and their applications.

 

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.

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

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.

                               Eglė Narmontaitė

Characterization of a novel invertase from Trichoderma sp., a producer of a wide range of fructooligosaccharides.

Eglė Narmontaitė, Francisco J. Plou, María Fernández-Lobato

                             Fadia V. Cervantes

Fully biocatalytic synthesis of D-tagatose from whey permeate as a raw material.

Fadia V. Cervantes, Sawssan Neifar, Zoran Merdzo, Antonio O. Ballesteros, Maria Fernandez-Lobato, Samir Bejar, and Francisco J. Plou

                                                                                                                                                                                                                                                                                                                                                    

                       Marina Minguet-Lobato

New Hyaluronic Acid Degrading Enzymes from Fungi

Marina Minguet-Lobato, Fadia V. Cervantes, David Fernández-Polo, María Fernández-Lobato, Francisco J. Plou

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.

In this work, we have investigated the use of immobilized Komagataella phaffii (formerly Pichia pastoris) for elimination of D-glucose and D-fructose in several sugar syrups. K. phaffii cells were immobilized in calcium alginate beads to facilitate the separation of the yeast cells from the reaction medium and reuse of the biocatalyst. The immobilized yeasts were successfully reutilized for at least 20 cycles  to remove D-glucose and D-fructose in a FOS syrup, without affecting the concentration of oligosaccharides. Excellent selectivity was also found for elimination of D-glucose in IMOS syrups. 

The methodology is versatile and easy to scale-up, as demonstrated in the removal of D-glucose and D-fructose  for the purification of heteroglucooligosaccharides synthesized by Metschnikowia reukaufii α-glucosidase. In addition, D-glucose was selectively removed by K. phaffii beads in the presence of D-galactose for at least 20 cycles of 150 min and applied to GOS purification.

Ref.: "Reuse of Immobilized Komagataella phaffii Cells for the Elimination of d-Glucose in Syrups of Bioactive Carbohydrates". Fadia V. Cervantes, David Fernandez-Polo, Zoran Merdzo, Noa Miguez, Martin Garcia-Gonzalez, Antonio O. Ballesteros, Maria Fernandez-Lobato, and Francisco J. Plou. ACS Food Science & Technology (2022), https://doi.org/10.1021/acsfoodscitech.2c00008 

The molecular machinery behind processive fungal chitinases

 Chitinases degrade chitin, one of the most widespread polysaccharides in nature, into low molecular weight chitooligomers (COS), which have a broad range of pharmaceutical and medicinal applications. We disclosed a detailed picture of the molecular events behind substrate/products binding in a fungal chitinase, giving full insight into its donor and acceptor subsites. Our crystallographic analysis revealed a previously unobserved dynamic on-off ligand binding process associated with motion of its insertion CID domain. This might represent a molecular mechanism complementary to the crucial role ascribed to aromatics at the catalytic site in processivity, which is an essential property modulating the bioconversion of chitin.  Furthermore, our analysis elucidates the implication of some highly flexible residues in activity and suggested new targets to address engineering of these biotechnologically important enzymes.

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

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!

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