28 Jul 2021

New article about theoretical three-dimensional zinc complexes

  

In collaboration with other colleages, a theoretical three-dimensional study of known zinc ionophores was performed. Aided by a computational energetic study, we proposed that some dietary flavonoids, glutathione and amino acids could form zinc complexes and facilitate the transport of zinc, with the possible biological implications and potential health benefits of these natural compounds. The study is based on obtaining a molecular conformational structure of the zinc complexes with the lowest possible energy content. The discovery of novel substances that act as zinc ionophores is an attractive research topic that offers exciting opportunities in medicinal chemistry. We propose that these novel complexes could be promising candidates for drug design to provide new solutions for conditions and diseases related to zinc deficiency or impairment derived from the dysregulation of this important metal.

Ref.: "Three-Dimensional Zinc Complexes with Glutathione, Amino Acids and Flavonoids" J. M. Pérez de la Lastra , C. Andrés-Juan , F.J. Plou and E. Pérez-Lebeña. Stresses (2021), 1(3), 123-141; doi: 10.3390/stresses1030011

12 Jul 2021

Fatty acid esters as anticancer agents published in IJMS

We have collaborated with the group of Prof. Maciej Guzik at Polish Academy of Sciences in this work on methods of obtaining various fatty acid esters (SFAEs) using combined biological, chemical and enzymatic methods. It presents how modification of SFAE’s hydrophobic chains can influence their cytotoxicity against human skin melanoma and prostate cancer cell lines. The compound’s cytotoxicity was determined by an MTT assay, which followed an assessment of SFAEs’ potential metastatic properties in concentrations below IC50 values. Despite relatively high IC50 values (63.3–1737.6 μM) of the newly synthesized SFAE, they can compete with other sugar esters already described in the literature. The chosen bioactives caused low polymerization of microtubules and the depolymerization of actin filaments in nontoxic levels, which suggest an apoptotic rather than metastatic process. Altogether, cancer cells showed no propensity for metastasis after treating them with SFAE. They confirmed that lactose-based compounds seem the most promising surfactants among tested sugar esters. This manuscript creates a benchmark for creation of novel anticancer agents based on 3-hydroxylated fatty acids of bacterial origin.



Ref.: "In Search of Effective Anticancer Agents—Novel Sugar Esters Based on Polyhydroxyalkanoate Monomers." JW. Snoch , D. Wnuk , T. Witko , J. Staroń , A. J. Bojarski , E. Jarek , F. J. Plou and Maciej Guzik.  International Journal of Molecular Sciences , 22, 7238 (2021), doi: 10.3390/ijms22137238


12 May 2021

Article on enzymatic synthesis of phloretin α‐glucosides

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Glycosylation of polyphenols may increase their aqueous solubility, stability, bioavailability and pharmacological activity. Herein, we used a mutant of sucrose phosphorylase 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 mono- and diglucoside were, respectively, 71- and 1200-fold more soluble in water than phloretin at room temperature. The α-glucosylation decreased the antioxidant capacity of phloretin, measured by DPPH and ABTS assays; however, this loss was moderate and the activity could be recovered upon deglycosylation in vivo. Since phloretin attracts a great interest in dermocosmetic applications, we analyzed the percutaneous absorption of glucosides and the aglycon employing a pig skin model. 

Ref.:" Enzymatic Synthesis of Phloretin α‐Glucosides using a Sucrose Phosphorylase Mutant and its Effect on Solubility, Antioxidant Properties and Skin Absorption." 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. Advanced Synthesis & Catalysis (2021). Volume 363, 3079-3089, doi: 10.1002/adsc.202100201

28 Apr 2021

The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA, Lipids and Proteins) and Induced Pathologies

 Living species are continuously subjected to all extrinsic forms of reactivants and others that are produced endogenously. There is extensive literature on the generation and effects of reactive oxygen species (ROS) in biological processes, both in terms of alteration and their role in cellular signaling and regulatory pathways. Cells produce ROS as a controlled physiological process, but increasing ROS becomes pathological and leads to oxidative stress and disease. The induction of oxidative stress is an imbalance between the production of radical species and the antioxidant defense systems, which can cause damage to cellular biomolecules, including lipids, proteins and DNA. Cellular and biochemical experiments have been complemented in various ways to explain the biological chemistry of ROS oxidants. However, it is often unclear how this translates into chemical reactions involving redox changes. This review addresses this question and includes a robust mechanistic explanation of the chemical reactions of ROS and oxidative stress. 

Ref: "The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA, Lipids and Proteins) and Induced Pathologies". Celia Andrés Juan , José Manuel P. L. , Francisco J. P. and Eduardo P.-L. International Journal of Molecular Sciences (2021), 22, 4642. https://doi.org/10.3390/ijms22094642

8 Mar 2021

Special Issue on Applied Biocatalysis in Europe


Applied Biocatalysis and Biotransformation, that is, the use of enzymes and whole-cell systems in manufacturing processes for synthetic purposes, has been experiencing a clear boom in recent years, which has led to the start of the so-called “fourth wave”. In fact, the latest advances in bioinformatics, system biology, process intensification, and, in particular, enzyme-directed evolution are widening the range of the efficacy of biocatalysts and accelerating the rate at which new enzymes are becoming available, even for activities not previously known. Traditionally, European scientists have been very actively involved in different aspects of Applied Biocatalysis. This Special Issue, edited by Profs. Andrés R.Alcántara and Francisco J. Plou, gather ten contributions from European research groups, in the form of either original research articles or up-to-date reviews, to exemplify recent advances in this area.

Andrés R. Alcántara and Francisco J. Plou. "Special Issue on “Applied Biocatalysis in Europe: A Sustainable Tool for Improving Life Quality”" Catalysts 2021, 11(3), 339; doi: 10.3390/catal11030339 

9 Oct 2020

"Science and Engineering applied to agrofood and biosystems" seminars


Francisco J. Plou will participate in the online seminars on "Science and Engineering applied to agrofood and biosystems" organized by University of Valladolid. He will deliver the speech "Transformation of residues rich in carbohydrates into high added-value products using enzymes" (14 October 2020, 16 h).

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10 Jun 2020

Tagatose production paper

We have developed a sustainable three-stage process for the bioconversion 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. 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 (w/w).

Ref: “A three-step process for the bioconversion of whey permeate into D-tagatose syrup free of glucose”. F.V. Cervantes, S. Neifar, Z. Merdzo, J. Viña-Gonzalez, L. Fernandez-Arrojo, A.O. Ballesteros, M. Fernandez-Lobato, S. Bejar and F.J. Plou. Catalysts 10(6), 647 (2020) https://doi.org/10.3390/catal10060647


25 Feb 2020

Visiting Iparlat factory

Last week Francisco Plou visited the new Iparlat factory in Corella (Navarra) trying to find synergies in the bioprocesses developed by the company for the production of vegetal beverages. The researcher Fernando López Gallego, from the CIC biomaGUNE, also participated in this visit. We really appreciate the hospitality of Alberto Larrauri, R&D manager of Iparlat, and look forward to a fruitful collaboration between the three partners.

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
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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