1 Jul 2019

INNOVA Prize to José Luis Gonzalez-Alfonso

Our PhD student José Luis Gonzalez-Alfonso has been awarded with the INNOVA Prize auspiced by the Spanish Platform SUSCHEM (Sustainable Chemistry) to the best collaborative work between public and private institutions. This work was performed in collaboration with Professor Yoshihiko Hirose (Enzyme Techno, Japan). The ceremony took place in Madrid on June 26th 2019.
The awarded work was: ''Enzymatic synthesis of a novel pterostilbene α-glucoside by the combination of cyclodextrin glucanotransferase and amyloglucosidase''. 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. Molecules 25;23(6) (2018). doi:10.3390/molecules23061271
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30 Jun 2019

Production and Surfactant Properties of Tert-Butyl α-D-Glucopyranosides Catalyzed by Cyclodextrin Glucanotransferase

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). In particular, CGTases from Thermoanaerobacter sp. and Thermoanaerobacterium thermosulfurigenes EM1 gave rise to the appearance of at least two glycosylation products, which 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% (13 g/L of tert-butyl-α-D-glucoside and 4 g/L of tert-butyl-α-D-maltoside). 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.

Chemical structure of the synthesized glycosides: (1) tert-butyl-O-α-D-glucopyranoside and (2) tert-butyl-O-α-D-maltoside. 

Variation of surface tension vs. concentration for tert-butyl and octyl glucosides.

''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

27 Jun 2019

Oral presentation at SECAT19 Congress

Francisco J. Plou presented in the Spanish Congress of Catalysis (Cordoba, 24-26 June 2019) the communication "Continuous reactor for the production of fructose syrups using the glucose isomerase from  Caldicoprobacter algeriensis immobilized in amino carriers".

This work was the result of a collaboration between the Laboratory of Microbial Biotechnology and Engineering Enzymes (Centre of Biotechnology of Sfax, University of Sfax, Tunisia), the Laboratory of Cellular and Molecular Biology (Faculty of Biological Sciences, University of Sciences and Technology of Houari Boumediene, Algeria) and the Instituto de Catálisis y Petroleoquímica (CSIC, Madrid, Spain).

28 Mar 2019

Participation in the Second Mediterranean Congress of Biotechnology (MCB2)

Francisco J. Plou has recently participated in the Second Mediterranean Congress of Biotechnology (MCB2, http://www.mcb2019.com), held from 16 to 20 March 2019 in Hammamet (Tunisia), as representative of Biocatalysis in the Spanish Society of Biotechnology (SEBIOT). Francisco Plou pronounced a conference on “Novel trends in the enzymatic transformation of carbohydrates into value-added products for the food and nutraceutical industries”.

19 Mar 2019

Article on the effect of α-glucosylation of epigallocatechin gallate published in Frontiers in Nutrition

(–)-Epigallocatechin gallate (EGCG), the predominant catechin (≥50%) in green tea (Camellia sinensis), displays several bioactive properties but its stability and bioavailability are low. In this work, the properties of two a-glucosyl derivatives of EGCG (3′- and 7-O-α-D-glucopyranoside), obtained by enzymatic synthesis, were assessed. The α-glucosylation enhanced the pH and thermal stability of EGCG. The analysis of scavenging activity toward ABTS·+ radicals showed that the alpha-glucosylation at C-7 of A-ring caused a higher loss of antioxidant activity compared with the sugar conjugation at C-3′ of B-ring. The 3′-glucoside also showed higher potential to alleviate intracelular reactive oxygen species (ROS) levels and to boost REDOX activity. The toxicity of EGCG and its monoglucosides was tested in human SH-S5Y5 neurons, RAW 264.7 macrophages, MRC5 fibroblasts, and HT-29 colon cancer cells. Interestingly, the 3′-O-α-D-glucoside increased the viability of neural cells in vitro (2.75-fold at 100μM) in the presence of H2O2, whilst EGCG gave rise only to a 1.7-fold enhancement. In conclusion, the α-glucoside of EGCG at C-3′ has a great potential for nutraceutical, cosmetic and biomedical applications.
Cell viability assays in presence of EGCG and its a-glucosides 1 and 2 on: (A) SH-SY5Y neuronal cells; (B) RAW 264.7 macrophages; (C) MRC5 fibroblasts; (D) HT-29 colon cancer cells. The values are referred to the control (cells containing 1% DMSO). The data is expressed as mean ± SD (n = 8, *p < 0.05 vs. control group; #p < 0.05 vs. EGCG (100μM) group).

''Effect of a-glucosylation on the stability, antioxidant properties, toxicity and neuroprotective activity of (-)-epigallocatechin gallate''. Jose L. Gonzalez-Alfonso, P. Peñalver, A.O. Ballesteros, J.C. Morales and F.J. Plou. Frontiers in Nutrition, (2019) doi: 10.3389/fnut.2019.00030

21 Jan 2019

Special recognition to Francisco J. Plou

During the Congress on "Enzymes in the Food Industry" (ENZAL 2019, Santiago de Chile, January 10-11 2019), Francisco J. Plou received a special recognition for his contribution to biocatalysis. Prof. Andrés Illanes, from the Pontificia Universidad Católica de Valparaíso (PUCV, Chile) and Prof. Agustín López-Munguía, from the Institute of Biotechnology at UNAM (Mexico) also received this distinction.

20 Jan 2019

The ABG, present in a Congress and a Practical Workshop on "Enzymes in the Food Industry" (ENZAL 2019, Santiago de Chile)

Our group has participated in a Practical Workshop and a Congress on "Enzymes in the Food Industry" (ENZAL 2019) held in Santiago de Chile, from January 8th to 11th 2019. Francisco Plou supervised a practical class on elimination of lactose with enzymes. Furthermore, he delivered a conference on "Enzymatic synthesis of bioactive oligosaccharides: production of chitooligosaccharides".

13 Dec 2018

Master in Design and Manufacture in Pharmaceutical Industry

The ABG group has participated in the Master 2018/2019 in Design and Manufacture in Pharmaceutical Industry organized by the Universidad de Navarra. Francisco Plou delivered a class on "Application of Biocatalysis in Pharmaceutical Processes", focusing in the increasing contribution of enzymes to the production of pharmaceuticals.

28 Nov 2018

UIMP-CSIC Course on Analysis and Characterization of Materials

Our group has collaborated in the first edition of the Universitary Course on Techniques for the Analysis and Characterization of Materials, organized by our institute (ICP-CSIC)  in collaboration with the UIMP and CSIC Foundation. We were in charge of teaching theoretical and practical lessons on High Performance Liquid Chromatography (HPLC).

26 Nov 2018

One Day Symposium: Research in Carbohydrates

The Applied Biocatalysis Group has participated in the "One Day Symposium: Research in Carbohydrates" organized by the Group of Carbohydrates at the Spanish Royal Society of Chemistry. The Symposium took place at the Faculty of Chemistry of University of Sevilla. The title of the conference delivered by Francisco Plou was “Recent examples of the enzymatic synthesis of bioactive oligosaccharides and polyphenol glycosides”

15 Nov 2018

Master of Biotechnology (UAM) 2018

Students from the International Master of Biotechnology at Autonomous University of Madrid (UAM) have visited our laboratories to perform a series of experiments related with biocatalysis, including the determination of enzyme activity and immobilization. They have also visited the laboratory of directed evolution of enzymes, whose supervisor is Dr. Miguel Alcalde. Our participation in the teaching of this Official Master started in 2008, and during this ten years we have tried to bring the biocatalysis closer to the students.

14 Nov 2018

Week of Science and Technology 2018 at CSIC

Our group has participated in the Week of Science and Technology 2018 at CSIC. Several hundreds of High School students have visited the Institute of Catalysis to perform different activities related with Catalysis and Biocatalysis. In our laboratory the students have learned basic concepts on biochemistry and enzymology using lactose-free milk.

5 Nov 2018

Article on glucosylation of hesperetin.

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.

''Optimization of Regioselective Glucosylation of Hesperetin Catalyzed by Cyclodextrin Glucanotransferase'' 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. Molecules, 23, 2285, (2018) doi:10.3390/molecules23112285 

4 Nov 2018

Immobilization 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.

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

17 Oct 2018

Glucosylation of pterostilbene published in Molecules

In this paper was performed the synthesis of a novel α-glucosylated derivative of pterostilbene by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp.
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 glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙+ radicals. 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.

''Enzymatic synthesis of a novel pterostilbene α-glucoside by the combination of cyclodextrin glucanotransferase and amyloglucosidase''. 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. Molecules 25;23(6) (2018)doi:10.3390/molecules23061271 

16 Oct 2018

Our group visited CPhI Worldwide in Madrid

The ABG group visited the pharmaceutical exhibition CPhI Worldwide in Madrid on October 10th 2018. It was an opportunity to connect with pharma professionals and meet companies from around the world.

1 Oct 2018

Glycosylation of hydroxytyrosol published in ChemCatChem

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)

26 Jul 2018

Article on glucosylation of epigallocatechin gallate

In this work, the synthesis of various α-glucosyl derivatives of (‒)-epigallocatechin gallate (EGCG), the predominant catechin in green tea, was performed in water at 50 °C by a transglycosylation reaction catalyzed by cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. The molecular weight of reaction products was determined by HPLC-MS. Using hydrolyzed potato starch as glucosyl donor, two main monoglucosides were obtained with conversion yields of 58% and 13%, respectively.

“Efficient α-glucosylation of epigallocatechin gallate catalyzed by cyclodextrin glucanotransferase from Thermoanaerobacter sp.”. J.L. Gonzalez-Alfonso, L. Leemans, A. Poveda, J. Jimenez-Barbero, A.O. Ballesteros and F.J. Plou. Journal of Agricultural and Food Chemistry 66, 7402–7408 (2018), doi:10.1021/acs.jafc.8b02143

28 Jun 2018

II Spanish Meeting on Biocatalysis (JEB 2018)

 The Group has participated actively in the II Spanish Meeting on Biocatalysis (JEB 2018) held in Oviedo (25-26 June 2018). We have contributed in the Scientific Committee, and with an oral presentation by Jose-Luis González, a flash presentation by Fadia Cervantes, and two posters by Noa Míguez and David Fernandez. One of the sessions of the meeting was devoted to Prof. Antonio Ballesteros, pioneer in the development of Biocatalyst in Spain.