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

Article on beta-fructofuranosidases and polyphenols published in Scientific Reports

Enzymatic glycosylation of polyphenols is a tool to improve their physicochemical properties and bioavailability. On the other hand, glycosidic enzymes can be inhibited by phenolic compounds. In this work, we studied the specificity of various phenolics (hydroquinone, hydroxytyrosol, epigallocatechin gallate, catechol and p-nitrophenol) as fructosyl acceptors or inhibitors of the β-fructofuranosidase from Xanthophyllomyces dendrorhous (pXd-INV). Only hydroquinone and hydroxytyrosol gave rise to the formation of glycosylated products. For the rest, an inhibitory effect on both the hydrolytic (H) and transglycosylation (T) activity of pXd-INV was observed. To disclose the binding mode of each compound and elucidate the molecular features determining its acceptor or inhibitor behaviour, ternary complexes of the inactive mutant pXd-INV-D80A with fructose and the different polyphenols were analyzed by X-ray crystallography. All the compounds bind by stacking against Trp105 and locate one of their phenolic hydroxyls making a polar linkage to the fructose O2 at 3.6–3.8 Å from the C2, which could enable the ulterior nucleophilic attack leading to transfructosylation. The acceptor capacity of the different polyphenols seems mediated by their ability to make flexible polar links with the protein.

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

Production of isomelezitose by Metschnikowia reukaufii cell extracts

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

Production of High Fructose Syrup (HFS) with immobilized glucose isomerase published in Food Chemistry

In collaboration with University of Sfax (Tunisia) and University of Sciences and Technology of Houari Boumediene (Algeria), we have investigated the glucose isomerase (GICA) from Caldicoprobacter algeriensis. The enzyme was immobilized by ionic adsorption on polymethacrylate carriers. The Sepabeads EC-HA yielded the highest recovery of activity (89%). The adsorbed enzyme displayed higher relative activity at acidic pH, greater thermostability, and better storage stability, compared to the free form. Moreover, the immobilized enzyme showed an excellent operational stability, in 15 successive 3h reaction cycles at 85°C under a batch reactor, preserving 83% of its initial activity. Interestingly, a continuous process for High Fructose Syrup (HFS) production was established with the adsorbed GICA using a packed bed reactor during eleven days at 70°C. HPAEC-PAD analysis showed a maximum bioconversion rate of 49% after 48h of operation.

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

Enzymatic synthesis and surfactant properties of tert-butyl α-D-glucopyranosides

While testing the ability of cyclodextrin glucanotransferases (CGTases) to glucosylate a series of flavonoids in the presence of organic cosolvents, we found out that this enzyme was able to glycosylate a tertiary alcohol (tert-butyl alcohol). Two glycosylation products were characterized by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as tert-butyl-α-D-glucoside (major product) and tert-butyl-α-D-maltoside (minor product). Using partially hydrolyzed starch as glucose donor, the yield of transglucosylation was approximately 44%. The synthesized tert-butyl-α-D-glucoside exhibited the typical surfactant behavior (critical micellar concentration, 4.0–4.5 mM) and its properties compared well with those of the related octyl-α-D-glucoside. To the best of our knowledge, this is the first description of an enzymatic α-glucosylation of a tertiary alcohol.

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