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

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

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 

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 $\sim$ 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.

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 (2021) https://doi.org/10.1038/s41598-021-86568-6

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

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

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 sugars" Martin 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

Bioconversion of cheese whey into D-Tagatose Syrup

 

We have developed a sustainable three-stage process for the revaluation of cheese whey permeate into D-tagatose, a rare sugar with functional properties used as sweetener. The experimental conditions (pH, temperature, cofactors, etc.) for each step were independently optimized. In the first step, concentrated whey containing 180–200 g/L of lactose was fully hydrolyzed by β-galactosidase from Bifidobacterium bifidum (Saphera®) in 3 h at 45 °C. Secondly, glucose was selectively removed by treatment with Pichia pastoris cells for 3 h at 30 °C. The best results were obtained with 350 mg of cells (previously grown for 16 h) per mL of solution. Finally, L-arabinose isomerase US100 from Bacillus stearothermophilus was employed to isomerize D-galactose into D-tagatose at pH 7.5 and 65 °C, in presence of 0.5 mM MnSO4. After 7 h, the concentration of D-tagatose was approximately 30 g/L (33.3% yield, referred to the initial D-galactose present in whey). The proposed integrated process takes place under mild conditions (neutral pH, moderate temperatures) in a short time (13 h), yielding a glucose-free syrup containing D-tagatose and galactose in a ratio 1:2.

Ref.:"A Three-Step Process for the Bioconversion of Whey Permeate into a Glucose-Free D-Tagatose Syrup" by Fadia V. Cervantes ,Sawssan Neifar ,Zoran Merdzo ,Javier Viña-Gonzalez ,Lucia Fernandez-Arrojo ,Antonio O. Ballesteros ,Maria Fernandez-Lobato ,Samir Bejar  and Francisco J. Plou.Catalysts 2020, 10(6), 647 https://doi.org/10.3390/catal10060647

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