Inhibition of hexokinase by polyphenol derivatives and the Warburg effect, published in Chemistry Select

Contrary to differentiated cells, cancer cells predominantly convert glucose to lactate even under conditions of adequate oxygen supply (“Warburg effect”). The initial enzyme implicated in this route is hexokinase, which transforms D-glucose into D-glucose-6-phosphate. We proposed the use of different polyphenols (resveratrol, epigallocatechin gallate, pterostilbene, phloretin) and their derivatives (α-glucosides and acylated α-glucosides) to inhibit this enzyme. For this study, we used Saccharomyces cerevisiae hexokinase, whose two isoforms show high resemblance at the active site with human hexokinase HK2. Remarkably, most of the assayed compounds inhibited the enzyme more than 50 % in the standard assay. Among them, phloretin 4’-O-(6’’-O-octanoyl)-α-D-glucopyranoside showed the highest inhibition and was studied in depth to determine the inhibition pattern and inhibition constant. Computational models of inhibition were carried out with the three molecules displaying the highest inhibition, and correlated adequately with the observed inhibitory effects on the enzyme. The inhibitory effect of several of the assayed polyphenols on hexokinase and their lack of toxicity renders them promising candidates as adjuvant drugs for cancer therapy.

Ref.: “Inhibition of Yeast Hexokinase by Acyl Glucosides of Phloretin and its Implication in the Warburg Effect”. Fadia V. Cervantes, Lucía Fernandez-Arrojo, Cristina Coscolin, Alicia Berrojo, José L. Gonzalez-Alfonso, José M. Perez de la Lastra, Manuel Ferrer, Celia M. Curieses-Andres, Celia Andres-Juan, Antonio O. Ballesteros, Eduardo Perez-Lebeña, and Francisco J. Plou. Chemistry Select (2024) https://doi.org/10.1002/slct.202401086

Collaboration with TVE's science outreach program Órbita Laika

Our collaboration with TVE's science outreach program Órbita Laika continues. This time, we assisted them with a demonstration on the enzyme lactase and lactose-free products.

It is a magnificent program, and it is a pleasure to collaborate with them from the Institute of Catalysis and Petrochemistry (CSIC) through their demonstration director Daniel Erice.

 

IX National Congress of Industrial Microbiology and Microbial Biotechnology

The Applied Biocatalysis Group has been present in the IX National Congress of Industrial Microbiology and Microbial Biotechnology. Noa Miguez has presented her work on xylooligosaccharides production and Mercedes Moreno on the enzymatic modification of the flavonoid baicalein. Good job!

Publication in Biocatalysis and Biotransformation: acylation of the flavonoid puerarin

This study was performed in collaboration with CIATEJ (Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C, Guadalajara, Jal, Mexico). The objective was to investigate the transesterification of isoflavones puerarin and its fructosylated derivative to enhance their lipophilicity for specific pharmacological applications. The process achieved high yields (80-100%) in the isoflavones conversion when lipases from Candida antarctica (CALB) and Thermomyces lanuginosus (TL IM) were used. The acyl derivatives presented improved lipophilicity and antioxidant activity.

Ref: A. Campos; L. Casas-Godoy; J.-G. Torres-Tolentino; G. Sandoval; L. Hernández; F. Plou; J. Arrizon. Lipase-catalyzed synthesis of laurate esters from puerarin and its β-D-fructofuranosyl-(2→6)-puerarin derivative.  Biocatalysis and Biotransformation (2024), https://doi.org/10.1080/10242422.2024.2332294

A strategy for the enzymatic acylation of polyphenols

In collaboration with Ghent University, CICbioGUNE and the Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), we have developed an efficient strategy for the enzymatic acylation of polyphenols. It is based on a first α-glucosylation step catalyzed by a sucrose phosphorylase, followed by acylation using a lipase. The method was applied to phloretin, a bioactive dihydrochalcone mainly present in apples. Phloretin underwent initial glucosylation at the 4′-OH position, followed by subsequent (and quantitative) acylation with C8, C12, and C16 acyl chains employing an immobilized lipase from Thermomyces lanuginosus. Interestingly, C12 acyl-α-glucoside displayed an enhanced (3-fold) transdermal absorption (using pig skin biopsies) compared to phloretin and its α-glucoside.

Ref.: J.L. Gonzalez-Alfonso, C. Alonso, A. Poveda, Z. Ubiparip, A.O. Ballesteros, T. Desmet, J. Jiménez-Barbero, L. Coderch, and F.J. Plou. Strategy for the enzymatic acylation of the apple flavonoid phloretin based on prior α-glucosylation. J. Agric. Food Chem. (2024), https://doi.org/10.1021/acs.jafc.3c09261

Publication in Microbial Cell Factories: three new chitinases from Mestchnikowia pulcherrima

In collaboration with the group of Prof. Fernández-Lobato at CBMSO (UAM-CSIC), we have investigated three new chitinases from M. pulcherrima, MpChit35, MpChit38 and MpChit41. They were molecularly characterized and extracellularly expressed in Pichia pastoris.The three enzymes hydrolysed colloidal chitin with optimal activity at 45 ºC and pH 4.0-4.5, The partial separation and characterization of the complex COS mixtures produced from the hydrolysis of chitin and chitosan were achieved by a new anionic chromatography HPAEC-PAD method and mass spectrometry assays. An overview of the predicted structures of these proteins and their catalytic modes of action were also presented. Depicted their high sequence and structural homology, MpChit35 acted as an exo-chitinase producing di-acetyl-chitobiose from chitin while MpChit38 and MpChit41 both acted as endo-chitinases producing tri-acetyl-chitotriose as main final product.

Microbial Cell Factories 23, 31 (2024)  https://doi.org/10.1186/s12934-024-02300-9

Our research group in the magazine from the Spanish Society for Microbiology

In the December issue of the SEM@foro magazine from the Spanish Society for Microbiology (SEM), there is a special issue from the Specialized Group on Industrial Microbiology and Microbial Biotechnology, in which our Applied Biocatalysis Group is featured. You can access the full article by clicking here. 

UAM Master of Biotechnology (2023/2024)

 

Our group has participated again in the Master of Biotechnology of Autonomous University of Madrid (2023/2024). 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. We organized a variety of enzymatic workshops: immobilization, lactose removal, chromatography and directed evolution. Excellent teamwork with the Directed Evolution Group of Prof. Miguel Alcalde.

Visit of UCM Master in Industrial and Environmental Biotechnology students

Today the students of the Master in Industrial and Environmental Biotechnology of the Complutense University of Madrid (with their professor Isabel de la Mata from the Faculty of Biology) visited our institute ICP-CSIC. We delivered two conferences on enzymes and their applications, and directed evolution. After that, they visited the laboratories of Applied Biocatalysis and Directed Evolution. Overall, the visit was productive and successful.

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)