Monitoring meeting of LIFE CYCLOPS project

 

The Monitoring meeting of LIFE CYCLOPS project ("Polyphenols recovery from olive oil and wine industries) was held in Cabra (Cordoba) on March 2nd 2023. The meeting was  was well-organized and productive, with everyone actively engaging in discussions and sharing their perspectives. The agenda was thoroughly covered, and all necessary decisions were made. The meeting had a positive atmosphere, with participants demonstrating respect and cooperation towards one another. There was a sense of accomplishment and satisfaction among the attendees as they left the meeting, having achieved the desired outcomes. Overall, it was a successful and fruitful meeting.

Applied Biocatalysis Group (Picture 2022)

 

Bioconversion of xylan into a xylobiose derivative with antioxidant properties

Acidic xylooligosaccharides (XOS), also called aldouronics, are hetero-oligomers of xylose randomly branched with 4-O-methyl-D-glucuronic acid residues linked by α(1 → 2) bonds, which display bioactive properties. We have developed a simple and integrated method for the production of acidic XOS by enzymatic hydrolysis of a glucurono-xylan from beechwood. The acidic fraction contained a major product, characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and mono- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) as 2′-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). Starting from 2 g of beechwood xylan, 1.5 g of total XOS were obtained, from which 225 mg (11% yield) corresponded to the aldouronic X2_MeGlcA. The acidic XOS exhibited higher antioxidant activity (measured by the ABTS·+ discoloration assay) than xylan, whilst neutral XOS displayed no antioxidant activity. This work demonstrates that it is possible to obtain a safe and natural antioxidant by enzymatic biotransformation of hardwood hemicellulose.

“Enzymatic bioconversion of beechwood xylan into the antioxidant 2’-O-α-(4-O-methyl-D-glucuronosyl)-xylobiose”. N. Miguez, D. Fernández-Polo, P. Santos-Moriano, B. Rodríguez-Colinas, A. Poveda, J. Jiménez-Barbero, A.O. Ballesteros, F.J. Plou. Biomass Conversion and Biorefinery, published online in September 7th 2022 https://doi.org/10.1007/s13399-022-03240-3.

The ABG Group at the Spanish Congress of Chemistry

José Luis González-Alfonso and David Fernández-Polo have presented their works on enzymatic glucosylation of hydroxytyrosol and hydrolysis of hyaluronic acid, respectively, in the Congress of the Spanish Royal Society of Chemistry (BienalRSEQ2022). Good work!

Elimination of D-Glucose in carbohydrate syrups published in ACS Food Science & Technology

During the synthesis of many bioactive carbohydrates, D-glucose is released as a side-product of the transglycosylation process. It is desirable to remove it due to its caloric contribution and its effect on caries and diabetes.

In this work, we have investigated the use of immobilized Komagataella phaffii (formerly Pichia pastoris) for elimination of D-glucose and D-fructose in several sugar syrups. K. phaffii cells were immobilized in calcium alginate beads to facilitate the separation of the yeast cells from the reaction medium and reuse of the biocatalyst. The immobilized yeasts were successfully reutilized for at least 20 cycles  to remove D-glucose and D-fructose in a FOS syrup, without affecting the concentration of oligosaccharides. Excellent selectivity was also found for elimination of D-glucose in IMOS syrups. 

The methodology is versatile and easy to scale-up, as demonstrated in the removal of D-glucose and D-fructose  for the purification of heteroglucooligosaccharides synthesized by Metschnikowia reukaufii α-glucosidase. In addition, D-glucose was selectively removed by K. phaffii beads in the presence of D-galactose for at least 20 cycles of 150 min and applied to GOS purification.

Ref.: "Reuse of Immobilized Komagataella phaffii Cells for the Elimination of d-Glucose in Syrups of Bioactive Carbohydrates". Fadia V. Cervantes, David Fernandez-Polo, Zoran Merdzo, Noa Miguez, Martin Garcia-Gonzalez, Antonio O. Ballesteros, Maria Fernandez-Lobato, and Francisco J. Plou. ACS Food Science & Technology (2022), https://doi.org/10.1021/acsfoodscitech.2c00008 

Participation of the ABG group in the III Spanish Conference on Biocatalysis

 The Applied Biocatalysis Group has been present in the III Spanish Conference on Biocatalysis (JEB 2021) that was held in Murcia between Thursday 25 and Saturday 27 November 2021. This congress was organized by the Spanish Biotechnology Society (SEBIOT) and the University of Murcia, giving continuity to the editions held in Madrid in 2015, and in Oviedo in 2018. Fadia Cervantes presented her work on glucose isomerase immobilization, José Luis González-Alfonso on phloretin glycosylation and Marina Minguet on the discovery of novel chitinolytic enzymes. We also contributed to other works in collaboration with several GLICOENZ members.

Article published in the José Luis Garcı́a Fierro Festschrift special issue of I&EC Research

 

The journal Industrial & Engineering Chemistry Research is publishing a collection of papers honoring Professor José Luis García Fierro of Institute of Catalysis and Petrochemistry, CSIC (Spain) to recognize his contributions to applied chemistry, mainly in catalysis.

We have had the honour to participate in this issue with a work that describes a fast, simple, and easily scalable process for polyglucosylation of the flavonoid rutin. This can be used as a strategy to enhance its aqueous solubility and bioavailability. We isolated and chemically characterized six glucosides. The addition of amyloglucosidase STA1 allowed us to increase the monoglucoside yield up to 2-fold. Glucosylated derivatives of rutin also possess a great potential to improve the dissolution and absorption of poorly water-soluble pharmaceuticals.

“Polyglucosylation of rutin catalyzed by cyclodextrin glucanotransferase from Geobacillus sp.: Optimization and chemical characterization of products”. J.L. González-Alfonso, A. Poveda, M. Arribas, Y. Hirose, M. Fernández-Lobato, A.O. Ballesteros, J. Jiménez-Barbero & F.J. Plou. Industrial and Engineering Chemistry Research (2021). https://doi.org/10.1021/acs.iecr.1c03070

Participation in the book "CSIC Scientific Challenges: Towards 2030"

 

Our group has participated in the book "CSIC Scientific Challenges: Towards 2030", which provides an insight into the research currently being accomplished at the CSIC, and at the same time, build a global vision of what will be the key scientific challenges over the next decade. We contributed to the Challenge 7, "Catalysis for industrial production and of energy resources", coordinated by Prof. José C. Conesa (ICP-CSIC)

M. López Granados, V. Cortés, F.J. Plou, M.A. Peña, J.M. Coronado, A. Iglesias, P. Ávila, M.A. Bañares, A. Serrano, A. Martínez, M. Boronat, M.A.Centeno, A. Rodríguez González-Elipe and J. Pérez Torrente. "Catalysis for industrial production and of energy sources".

In: "CSIC Scientific Challenges: Towards 2030" (J.M. de Lucas & V. Moreno-Arribas, Coor.).Editorial CSIC (2021), Volume 8: Clean, safe and efficient energy, pp 175-205. 

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

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." W. 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

Applied Biocatalysis Group (Picture 2021)

 

Article on enzymatic synthesis of phloretin α‐glucosides

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

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

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 

"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).

More information

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

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.

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

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.

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.