3 Dec 2019

Article 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. 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. The acceptor capacity of the different polyphenols seems mediated by their ability to make flexible polar links with the protein.

Reference: “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

22 Nov 2019

Participation in the Week of Science 2019

The Applied Biocatalysis Group has participated in the activities of the Institute of Catalysis and Petrochemistry during the Week of Science 2019. Students from different high schools performed experiments in which the concepts of biocatalysis, enzymes, specificity, etc. were addressed.

14 Nov 2019

Conference devoted to the Periodic Table of the Elements

During the VIII Symposium of Scientific Theatre celebrated in Medellín (Badajoz) from 14-17 November 2019, Francisco J. Plou has pronounced a conference on the relationships between the Periodic Table of the Elements and the Art (literature, music, movies, architecture, etc.). The symposium commemorates the 150 anniversary of the Periodic Table developed by Dmitri Mendeleev, basis of our current table.

13 Nov 2019

Master of Biotechnology UAM 2019

Our group has been participating since 2008 in the teaching of the Master of Biotechnology of Autonomous University of Madrid. This year the students of Biocatalysis/Nanobiotechnology subject visited the laboratories of biotransformations and directed evolution to take a closer look at the work of researchers and perform some experiments.

29 Oct 2019

Production of High Fructose Syrup (HFS) with immobilized glucose isomerase



We have published an article in collaboration with University of Sfax (Tunisia) and University of Sciences and Technology of Houari Boumediene (Algeria). The glucose isomerase (GICA) from Caldicoprobacter algeriensis 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

25 Oct 2019

Conference in Universidad de Vigo Symposium

Francisco J. Plou delivered a conference on "Enzymatic biotransformations of carbohydrates into bioactive compounds" during the one-day symposium "Xornada Enzimas: Ciencia e Tecnoloxía 2019” held in Orense (University of Vigo). Several speakers from the academic and industrial areas explained the current possibilities of using enzymes in the food sector.

2 Jul 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

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
More information

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