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

Foundation Ramón Areces Grant

One of the members of GLICOENZ consortium, Prof. María Fernández Lobato, has obtained a grant  by Foundation Ramón Areces  in the XIX Call of Research Grants in Life and Materials Sciences. The project will be focused on bioactive properties of oligosaccharides and glycoderivatives, including biomedical applications.

Enzymatic glucosylation of hesperetin with CGTase.

The regioselective α -glucosylation of hesperetin was achieved by a transglycosylation reaction catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. using soluble starch as glucosyl donor. By combining mass spectrometry (ESI-TOF) and 2D-NMR analysis, the main monoglucosylated derivative was fully characterized (hesperetin 7-O-α-D-glucopyranoside).
In order to increase the yield of monoglucoside, several reaction parameters were optimized: Nature and percentage of cosolvent, composition of the aqueous phase, glucosyl donor, temperature, and the concentrations of hesperetin and soluble starch. Under the optimal conditions, which included the presence of 30% of bis(2-methoxyethyl) ether as cosolvent, the maximum concentration of monoglucoside was approximately 2 mM, obtained after 24 h of reaction. To our knowledge, this is the first report of direct glucosylation of hesperetin employing free enzymes instead of whole cells.

Ref. J.L. González-Alfonso, N. Míguez, J. D. Padilla, L. Leemans, A. Poveda, J. Jimnez-Barbero, A. O. Ballesteros, G. Sandoval and F. J. Plou. ''Optimization of Regioselective Glucosylation of Hesperetin Catalyzed by Cyclodextrin Glucanotransferase'' . Molecules, 23, 2285, (2018) doi: 10.3390/molecules23112885

Immobilization techniques of the β-fructofuranosidase from Xanthophyllomyces dendrorhous published in Catalysts.

The β-fructofuranosidase (Xd-INV) from the basidiomycota yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) is unique in its ability to synthesize neo- fructooligosaccharides (neo-FOS). In order to facilitate its industrial application, the recombinant enzyme expressed in Pichia pastoris(pXd-INV) was immobilized by entrapment in polyvinyl alcohol (PVA) hydrogels. The encapsulation efficiency exceeded 80%. The PVA lenticular particles of immobilized pXd-INV were stable up to approximately 40 °C. Using 600 g/L sucrose, the immobilized biocatalyst synthesized 18.9% (w/w) FOS (59.1 g/L of neokestose, 30.2 g/L of 1-kestose, 11.6 g/L of neonystose and 12.6 g/L of blastose). The operational stability of PVA-immobilized biocatalyst was assayed in a batch reactor at 30 °C. The enzyme preserved its initial activity during at least 7 cycles of 26 h.

Ref.: N. Miguez, M. Gimeno-Perez, D. Fernandez-Polo, F.V. Cervantes, A.O. Ballesteros, M. Fernandez-Lobato, M.H. Ribeiro, F.J Plou. "Immobilization of the β-fructofuranosidase from Xanthophyllomyces dendrorhous by entrapment in polyvinyl alcohol and its application to neo-fructooligosaccharides production". Catalysts 8(5), 201 (2018). doi:10.3390/catal8050201

A simple system to produce 6-kestose

The β-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces potential prebiotic fructooligosaccharides with health-promoting properties, making it of biotechnological interest. Ffase is one of the highest and more selective known producers of 6-kestose by transfructosylation of sucrose. In this work, production of 6-kestose was simplified by directly using cultures of S. occidentalis and Saccharomyces cerevisiae expressing both the wild-type enzyme and a mutated Ffase variant including the Ser196Leu substitution (Ffase-Leu196). Best results were obtained using yeast cultures supplemented with sucrose and expressing the Ffase-Leu196, which after only 4 h produced ~ 116 g/L of 6-kestose, twice the amount obtained with the corresponding purified enzyme. The Ser196Leu substitution skewed production of 6-kestose and neofructooligosaccharides resulting in an increase of ~ 2.2- and 1.5-fold, respectively. Modeling neokestose and blastose into the Ffase-active site revealed the molecular basis explaining the peculiar specificity of this enzyme.

Ref.: D. Rodrigo-Frutos, D. Piedrabuena, J. Sanz-Aparicio, M. Fernández-Lobato. "Yeast cultures expressing the Ffase from Schwanniomyces occidentalis, a simple system to produce the potential prebiotic sugar 6-kestose". Applied Microbiology and Biotechnology (2018), doi:10.1007/s00253-018-9446-y

Enzymatic fructosylation of hydroxytyrosol: reaction and mechanism

We have investigated the ability of the β-fructofuranosidase pXd-INV from the yeast Xanthophyllomyces dendrorhous to glycosylate the olive biophenol hydroxytyrosol (HT). Two fructosylated derivatives (Fru-HT1 and Fru-HT2) were synthesized.MS and 2D-NMR analyses showed that the major product (Fru-HT1) was fructosylated at the primary OH of HT. The structure of the complexes with the substrates and the product analyzed by crystallography led to the understanding of the molecular determinants regulating the enzymatic mechanism. Product-soaked crystals revealed that the minor derivative (Fru-HT2) was fructosylated at the phenolic p-OH group.  One of the studied mutants (N342Q) was notably more specific for the fructosylation at the phenolic OH than the wild-type.



Reference:“Fructosylation of hydroxytyrosol by the β-fructofuranosidase from Xanthophyllomyces dendrorhous: Insights into the molecular basis of the enzyme specificity”. N. Míguez, M. Ramírez-Escudero, M. Gimeno-Pérez, A. Poveda, J. Jiménez-Barbero, A. O. Ballesteros, M. Fernández-Lobato, J. Sanz-Aparicio* and F. J. Plou* ChemCatChem, http://dx.doi.org/10.1002/cctc.201801171 (2018)

Enzymatic synthesis of a novel pterostilbene alpha-D-glucoside

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

Ref: J.L. González-Alfonso, D. Rodrigo-Frutos, E. Belmonte-Reche, P. Peñalver, A. Poveda, J. Jimenez-Barbero, A.O. Ballesteros, Y. Hirose, J. Polaina, J.C. Morales, M. Fernández-Lobato and F. J. Plou. Enzymatic synthesis of a novel pterostilbene α-glucoside by the combination of cyclodextrin glucanotransferase and amyloglucosidase. Molecules 23(6), 1271 (2018). https://doi.org/10.3390/molecules23061271

Workshop April 25th 2018

A Workshop of the GLICOENZ Consorptium was held on April 25th 2018 at the Faculty of Sciences of the Autonomous University of Madrid. The students of the four laboratories involved in GLICOENZ presented their main results. These presentations served as a basis for planning  new experiments and initiating new sinergies.

Recombinant chitinase Chit42 from Trichoderma harzianum for the production of chitooligosaccharides

Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-D-glucosamine (GlcNAc) units. Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates.

Reference: “Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity”. P.E. Kidibule,  P. Santos-Moriano, E. Jiménez-Ortega, M. Ramírez-Escudero, M.C. Limón, M. Remacha, F.J. Plou, J. Sanz-Aparicio,  M. Fernández-Lobato. Microbial Cell Factories 17:47 (2018). doi:10.1186/s12934-018-0895-x

Production of deacetylated chitooligosaccharides

Several commercial enzymes were screened for chitosanolytic activity. The hydrolysis of different chitosans was followed by size exclusion chromatography (SEC-ELSD), mass spectrometry (ESI-Q-TOF), and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Neutrase 0.8L converted 10 g/L of various chitosans into mostly deacetylated oligosaccharides, yielding approximately 2.5 g/L of chitobiose, 4.5 g/L of chitotriose and 3 g/L of chitotetraose. In collaboration with the Institute of Parasitology and Biomedicine "Lopez-Neyra" (CSIC), the synthesized COS were tested in vitro for their neuroprotective and anti-inflammatory activities, and compared with other functional ingredients, namely fructooligosaccharides.

Reference: “Enzymatic production of fully deacetylated chitooligosaccharides and their neuroprotective and anti-inflammatory properties”. P. Santos-Moriano, L. Fernandez-Arrojo, M. Mengibar, E. Belmonte-Reche, P. Peñalver, F.N. Acosta, P. Kidibule, A.O. Ballesteros, J.C. Morales, M. Fernandez-Lobato and F.J. Plou. Biocatalysis and Biotransformation (2017), doi: 10.1080/10242422.2017.1295231