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.

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 

04 February, 2022

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

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.

Ref: "New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis" David Rodrigo-Frutos, Elena Jiménez-Ortega, David Piedrabuena, Mercedes Ramírez-Escudero, Noa Míguez, Francisco J. Plou, Julia Sanz-Aparicio & María Fernández-Lobato. Scientific Reports volume 11, Article number: 7158 (2021https://doi.org/10.1038/s41598-021-86568-6

02 February, 2021

Production and characterization of chitooligosaccharides

 Chitin-active enzymes are of great biotechnological interest due to the wide industrial application of chitinolytic materials. Non-stability and high cost are among limitations that hinder industrial application of soluble enzymes. Here we report the production and characterization of chitooligosaccharides (COS) using the fungal exo-chitinase Chit42 immobilized on magnetic nanoparticles and food-grade chitosan beads with an immobilization yield of about 60% using glutaraldehyde and genipin linkers. The immobilized enzyme gained operational stability with increasing temperature and acidic pH values, especially when using chitosan beads-genipin that retained more than 80% activity at pH 3. Biocatalysts generated COS from colloidal chitin and different chitosan types. The immobilized enzyme showed higher hydrolytic activity than free enzyme on chitosan, and produced COS mixtures with higher variability of size and acetylation degree. In addition, biocatalysts were reusable, easy to handle and to separate from the reaction mixture.

Ref.: " Production and characterization of chitooligosaccharides by the fungal chitinase Chit42 immobilized on magnetic nanoparticles and chitosan beads: selectivity, specificity and improved operational utility"  Peter E. Kidibule, Jessica Costa, Andrea Atrei, Francisco J. Plou, Maria Fernandez Lobato and Rebecca Pogni  RSC Adv., (2021), 11, 5529 https://doi.org/10.1039/d0ra10409d

04 December, 2020

Use of a β-galactosidase from Pantoea anthophila to produce galactooligosaccharides from a byproduct of the dairy industry

Milk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by β-galactosidases. B-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with β(1 → 6) and β(1 → 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD.

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

Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp


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.

Ref.:"Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp, both producers of honey sugarsMartin Garcia-Gonzalez, Marina Minguet-Lobato, Francisco J. Plou & Maria Fernandez-Lobato Microbial Cell Factories volume 19, Article number: 140 (2020) https://doi.org/10.1186/s12934-020-01397-y