Publications by authors named "Marco Filice"

29 Publications

  • Page 1 of 1

Enzyme Conformation Influences the Performance of Lipase-powered Nanomotors.

Angew Chem Int Ed Engl 2020 11 16;59(47):21080-21087. Epub 2020 Sep 16.

Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Baldiri i Reixac 10-12, 08028, Barcelona, Spain.

Enzyme-powered micro/nanomotors have myriads of potential applications in various areas. To efficiently reach those applications, it is necessary and critical to understand the fundamental aspects affecting the motion dynamics. Herein, we explored the impact of enzyme orientation on the performance of lipase-powered nanomotors by tuning the lipase immobilization strategies. The influence of the lipase orientation and lid conformation on substrate binding and catalysis was analyzed using molecular dynamics simulations. Besides, the motion performance indicates that the hydrophobic binding (via OTES) represents the best orienting strategy, providing 48.4 % and 95.4 % increase in diffusion coefficient compared to hydrophilic binding (via APTES) and Brownian motion (no fuel), respectively (with C of 100 mm). This work provides vital evidence for the importance of immobilization strategy and corresponding enzyme orientation for the catalytic activity and in turn, the motion performance of nanomotors, and is thus helpful to future applications.
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http://dx.doi.org/10.1002/anie.202008339DOI Listing
November 2020

Role of Folic Acid in the Therapeutic Action of Nanostructured Porous Silica Functionalized with Organotin(IV) Compounds Against Different Cancer Cell Lines.

Pharmaceutics 2020 Jun 3;12(6). Epub 2020 Jun 3.

COMET-NANO Group, Departamento de Biología y Geología, Física y Química Inorgánica, ESCET, Universidad Rey Juan Carlos, 28933, Móstoles, Spain.

The synthesis, characterization and cytotoxic activity against different cancer cell lines of various mesoporous silica-based materials containing folate targeting moieties and a cytotoxic fragment based on a triphenyltin(IV) derivative have been studied. Two different mesoporous nanostructured silica systems have been used: firstly, micronic silica particles of the MSU-2 type and, secondly, mesoporous silica nanoparticles (MSNs) of about 80 nm. Both series of materials have been characterized by different methods, such as powder X-ray diffraction, X-ray fluorescence, absorption spectroscopy and microscopy. In addition, these systems have been tested against four different cancer cell lines, namely, OVCAR-3, DLD-1, A2780 and A431, in order to observe if the size of the silica-based systems and the quantity of incorporated folic acid influence their cytotoxic action. The results show that the materials are more active when the quantity of folic acid is higher, especially in those cells that overexpress folate receptors such as OVCAR-3 and DLD-1. In addition, the study of the potential modulation of the soluble folate receptor alpha (FOLR1) by treatment with the synthesized materials has been carried out using OVCAR-3, DLD-1, A2780 and A431 tumour cell lines. The results show that a relatively high concentration of folic acid functionalization of the nanostructured silica together with the incorporation of the cytotoxic tin fragment leads to an increase in the quantity of the soluble FOLR1 secreted by the tumour cells. In addition, the studies reported here show that this increase of the soluble FOLR1 occurs presumably by cutting the glycosyl-phosphatidylinositol anchor of membrane FR-α and by the release of intracellular FR-α. This study validates the potential use of a combination of mesoporous silica materials co-functionalized with folate targeting molecules and an organotin(IV) drug as a strategy for the therapeutic treatment of several cancer cells overexpressing folate receptors.
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http://dx.doi.org/10.3390/pharmaceutics12060512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355810PMC
June 2020

Fine Modulation of the Catalytic Properties of Lipase Driven by Different Immobilization Strategies for the Selective Hydrolysis of Fish Oil.

Molecules 2020 Jan 27;25(3). Epub 2020 Jan 27.

'Nanobiotechnology for Life Sciences' Group, Department of Chemistry in Pharmaceutical Sciences Dept., Faculty of Pharmacy, Universidad Complutense de Madrid (UCM), Plaza Ramón y Cajal s/n, E-28040 Madrid, Spain.

Functional properties of each enzyme strictly depend on immobilization protocol used for linking enzyme and carrier. Different strategies were applied to prepare the immobilized derivatives of lipase (RML) and chemically aminated RML (NH-RML). Both RML and NH-RML forms were covalently immobilized on glyoxyl sepharose (Gx-RML and Gx-NH-RML), glyoxyl sepharose dithiothreitol (Gx-DTT-RML and Gx-DTT-NH-RML), activated sepharose with cyanogen bromide (CNBr-RML and CNBr-NH-RML) and heterofunctional epoxy support partially modified with iminodiacetic acid (epoxy-IDA-RML and epoxy-IDA-NH-RML). Immobilization varied from 11% up to 88% yields producing specific activities ranging from 0.5 up to 1.9 UI/mg. Great improvement in thermal stability for Gx-DTT-NH-RML and epoxy-IDA-NH-RML derivatives was obtained by retaining 49% and 37% of their initial activities at 70 °C, respectively. The regioselectivity of each derivative was also examined in hydrolysis of fish oil at three different conditions. All the derivatives were selective between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. The highest selectivity (32.9 folds) was observed for epoxy-IDA-NH-RML derivative in the hydrolysis reaction performed at pH 5 and 4 °C. Recyclability study showed good capability of the immobilized biocatalysts to be used repeatedly, retaining 50-91% of their initial activities after five cycles of the reaction.
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http://dx.doi.org/10.3390/molecules25030545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037125PMC
January 2020

Multifunctional Silica-Based Nanoparticles with Controlled Release of Organotin Metallodrug for Targeted Theranosis of Breast Cancer.

Cancers (Basel) 2020 Jan 12;12(1). Epub 2020 Jan 12.

Nanobiotechnology for Life Sciences Group, Department of Chemistry in Pharmaceutical Sciences, Faculty of Pharmacy, Universidad Complutense de Madrid (UCM), Plaza Ramón y Cajal s/n, E-28040 Madrid, Spain.

Three different multifunctional nanosystems based on the tethering onto mesoporous silica nanoparticles (MSN) of different fragments such as an organotin-based cytotoxic compound PhSn{SCHCHCHSi(OMe)} (MSN-AP-Sn), a folate fragment (MSN-AP-FA-Sn), and an enzyme-responsive peptide able to release the metallodrug only inside cancer cells (MSN-AP-FA-PEP-S-Sn), have been synthesized and fully characterized by applying physico-chemical techniques. After that, an in vitro deep determination of the therapeutic potential of the achieved multifunctional nanovectors was carried out. The results showed a high cytotoxic potential of the MSN-AP-FA-PEP-S-Sn material against triple negative breast cancer cell line (MDA-MB-231). Moreover, a dose-dependent metallodrug-related inhibitory effect on the migration mechanism of MDA-MB-231 tumor cells was shown. Subsequently, the organotin-functionalized nanosystems have been further modified with the NIR imaging agent Alexa Fluor 647 to give three different theranostic silica-based nanoplatforms, namely, MSN-AP-Sn-AX (AX-1), MSN-AP-FA-Sn-AX (AX-2), and MSN-AP-FA-PEP-S-Sn-AX (AX-3). Their in vivo potential as theranostic markers was further evaluated in a xenograft mouse model of human breast adenocarcinoma. Owing to the combination of the receptor-mediated site targeting and the specific fine-tuned release mechanism of the organotin metallodrug, the nanotheranostic drug MSN-AP-FA-PEP-S-Sn-AX (AX-3) has shown targeted diagnostic ability in combination with enhanced therapeutic activity by promoting the inhibition of tumor growth with reduced hepatic and renal toxicity upon the repeated administration of the multifunctional nanodrug.
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http://dx.doi.org/10.3390/cancers12010187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017138PMC
January 2020

Tailor-made PEG coated iron oxide nanoparticles as contrast agents for long lasting magnetic resonance molecular imaging of solid cancers.

Mater Sci Eng C Mater Biol Appl 2020 Feb 11;107:110262. Epub 2019 Oct 11.

Nanobiotechnology for Life Sciences Group, Department of Chemistry in Pharmaceutical Sciences, Faculty of Pharmacy, Complutense University of Madrid (UCM), Plaza Ramón y Cajal, 28040, Madrid, Spain; Department of Energy, Environment and Health, Institute of Materials Science of Madrid, ICMM-CSIC, Sor Juana Inés de La Cruz 3, Cantoblanco, 28049, Madrid, Spain. Electronic address:

Magnetic resonance imaging (MRI) is the most powerful technique for non-invasive diagnosis of human diseases and disorders. Properly designed contrast agents can be accumulated in the damaged zone and be internalized by cells, becoming interesting cellular MRI probes for disease tracking and monitoring. However, this approach is sometimes limited by the relaxation rates of contrast agents currently in clinical use, which show neither optimal pharmacokinetic parameters nor toxicity. In this work, a suitable contrast agent candidate, based on iron oxide nanoparticles (IONPs) coated with polyethyleneglycol, was finely designed, prepared and fully characterized under a physical, chemical and biological point of view. To stand out the real potential of our study, all the experiments were performed in comparison with Ferumoxytol, a FDA approved IONPs. IONPs with a core size of 15 nm and coated with polyethyleneglycol of 5 kDa (OD15-P5) resulted the best ones, being able to be uptaken by both tumoral cells and macrophages and showing no toxicity for in vitro and in vivo experiments. In vitro and in vivo MRI results for OD15-P5 showed r relaxivity values higher than Ferumoxitol. Furthermore, the injected OD15-P5 were completely retained at the tumor site for up to 24 h showing high potential as MRI contrast agents for real time long-lasting monitoring of the tumor evolution.
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http://dx.doi.org/10.1016/j.msec.2019.110262DOI Listing
February 2020

miR-1 induces endothelial dysfunction in rat pulmonary arteries.

J Physiol Biochem 2019 Nov 20;75(4):519-529. Epub 2019 Aug 20.

Departament of Pharmacology and Toxicology. School of Medicine, Universidad Complutense de Madrid, 28040, Madrid, Spain.

Endothelial dysfunction plays a central role in the pathophysiology of pulmonary arterial hypertension (PAH). MicroRNAs (miRNAs) are small single-strand and non-coding RNAs that negatively regulate gene function by binding to the 3'-untranslated region (3'-UTR) of specific mRNAs. microRNA-1 (miR-1) is upregulated in plasma from idiopathic PAH patients and in lungs from an experimental model of PAH. However, the role of miRNA-1 on endothelial dysfunction is unknown. The aim of this study was to analyze the effects of miR-1 on endothelial function in rat pulmonary arteries (PA). Endothelial function was studied in PA from PAH or healthy animals and mounted in a wire myograph. Some PA from control animals were transfected with miR-1 or scramble miR. Superoxide anion production by miR-1 was quantified by dihydroethidium (DHE) fluorescence in rat PA smooth muscle cells (PASMC). Bioinformatic analysis identified superoxide dismutase-1 (SOD1), connexin-43 (Cx43), caveolin 2 (CAV2) and Krüppel-like factor 4 (KLF4) as potential targets of miR-1. The expression of SOD1, Cx43, CAV2, and KLF4 was determined by qRT-PCR and western blot in PASMC. PA incubated with miR-1 presented decreased endothelium-dependent relaxation to acetylcholine. We also found an increase in the production of O and decreased expression of SOD1, Cx43, CAV2, and KLF4 in PASMC induced by miR-1, which may contribute to endothelial dysfunction. In conclusion, these data indicate that miR-1 induces endothelial dysfunction, suggesting a pathophysiological role in PAH.
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http://dx.doi.org/10.1007/s13105-019-00696-2DOI Listing
November 2019

Modulation of the Catalytic Properties of Lipase B from by Immobilization on Tailor-Made Magnetic Iron Oxide Nanoparticles: The Key Role of Nanocarrier Surface Engineering.

Polymers (Basel) 2018 Jun 5;10(6). Epub 2018 Jun 5.

Department of Biomaterials and Bioinspired Material, Materials Science Institute of Madrid (ICMM-CSIC), Sor Juana Inés de la Cruz 3, Cantoblanco, 28049 Madrid, Spain.

The immobilization of biocatalysts on magnetic nanomaterial surface is a very attractive alternative to achieve enzyme nanoderivatives with highly improved properties. The combination between the careful tailoring of nanocarrier surfaces and the site-specific chemical modification of biomacromolecules is a crucial parameter to finely modulate the catalytic behavior of the biocatalyst. In this work, a useful strategy to immobilize chemically aminated lipase B from on magnetic iron oxide nanoparticles (IONPs) by covalent multipoint attachment or hydrophobic physical adsorption upon previous tailored engineering of nanocarriers with poly-carboxylic groups (citric acid or succinic anhydride, [email protected] and [email protected] respectively) or hydrophobic layer (oleic acid, [email protected]) is described. After full characterization, the nanocatalysts have been assessed in the enantioselective kinetic resolution of racemic methyl mandelate. Depending on the immobilization strategy, each enzymatic nanoderivative permitted to selectively improve a specific property of the biocatalyst. In general, all the immobilization protocols permitted loading from good to high lipase amount (149 < immobilized lipase < 234 mg/g). The hydrophobic [email protected] was the most active nanocatalyst, whereas the covalent [email protected] and [email protected] were revealed to be the most thermostable and also the most enantioselective ones in the kinetic resolution reaction (almost 90% ee R-enantiomer). A strategy to maintain all these properties in long-time storage (up to 1 month) by freeze-drying was also optimized. Therefore, the nanocarrier surface engineering is demonstrated to be a key-parameter in the design and preparation of lipase libraries with enhanced catalytic properties.
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http://dx.doi.org/10.3390/polym10060615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404122PMC
June 2018

Non-Invasive Detection of Extracellular Matrix Metalloproteinase Inducer EMMPRIN, a New Therapeutic Target against Atherosclerosis, Inhibited by Endothelial Nitric Oxide.

Int J Mol Sci 2018 Oct 19;19(10). Epub 2018 Oct 19.

Cardiology Department, University Francisco de Vitoria/Hospital Ramón y Cajal Research Unit (IRYCIS), CIBERCV, 28223 Madrid, Spain.

Lack of endothelial nitric oxide causes endothelial dysfunction and circulating monocyte infiltration, contributing to systemic atheroma plaque formation in arterial territories. Among the different inflammatory products, macrophage-derived foam cells and smooth muscle cells synthesize matrix metalloproteinases (MMPs), playing a pivotal role in early plaque formation and enlargement. We found increased levels of MMP-9 and MMP-13 in human endarterectomies with advanced atherosclerosis, together with significant amounts of extracellular matrix (ECM) metalloproteinase inducer EMMPRIN. To test whether the absence of NO may aggravate atherosclerosis through EMMPRIN activation, double NOS3/apoE knockout (KO) mice expressed high levels of EMMPRIN in carotid plaques, suggesting that targeting extracellular matrix degradation may represent a new mechanism by which endothelial NO prevents atherosclerosis. Based on our previous experience, by using gadolinium-enriched paramagnetic fluorescence micellar nanoparticles conjugated with AP9 (NAP9), an EMMPRIN-specific binding peptide, magnetic resonance sequences allowed non-invasive visualization of carotid EMMPRIN in NOS3/apoE over apoE control mice, in which atheroma plaques were significantly reduced. Taken together, these results point to EMMPRIN as a new therapeutic target of NO-mediated protection against atherosclerosis, and NAP9 as a non-invasive molecular tool to target atherosclerosis.
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http://dx.doi.org/10.3390/ijms19103248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214015PMC
October 2018

Hybrid Decorated [email protected] Janus Nanoparticles as a Flexible Platform for Targeted Multimodal Molecular Bioimaging of Cancer.

ACS Appl Mater Interfaces 2018 Sep 7;10(37):31032-31043. Epub 2018 Sep 7.

Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) , Melchor Fernández Almagro, 3 , 28029 Madrid , Spain.

In the recent years, targeted cancer theranosis, the concomitant therapeutic treatment and selective visualization of cancerous tissue, has become a powerful strategy to improve patient prognosis. In this context, targeted multimodal molecular imaging, the combination of different imaging modalities overcoming their individual limitations, has attracted great attention. Due to their unique properties, advanced nanomaterials have taken center stage in the development of theranostics. In this work, we report a novel Janus nanoplatform by combining an FeO NPs/mesoporous silica [email protected] face together with an Au nanoparticle face. Due to its anisotropy, this hybrid nanomaterial enabled the orthogonal site-selective modification of each face permitting the incorporation of a targeting peptide for cancer detection (cRGD) and a fluorescent dye. Due to the intrinsic characteristics of this Janus nanoplatform together with those selectively generated on their surfaces, the resulting hybrid nanocarrier successfully promoted the in vivo tumor-targeted multimodal imaging by magnetic resonance (FeO core), computed tomography (AuNP face), and fluorescent tracking (fluorescent dye loading) in a fibrosarcoma-bearing mouse model. The achieved results endorse these hybrid Janus nanoparticles as a powerful and flexible platform with integrated imaging and carrier functionalities to be equipped with therapeutic features to generate an advanced multifunctional nanocarrier for targeted cancer theranosis.
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http://dx.doi.org/10.1021/acsami.8b10452DOI Listing
September 2018

Applications of Nanomaterials Based on Magnetite and Mesoporous Silica on the Selective Detection of Zinc Ion in Live Cell Imaging.

Nanomaterials (Basel) 2018 Jun 14;8(6). Epub 2018 Jun 14.

Departamento de Biología y Geología, Física y Química Inorgánica, ESCET, Universidad Rey Juan Carlos, Calle Tulipán s/n, E-28933 Móstoles, Madrid, Spain.

Functionalized magnetite nanoparticles (FMNPs) and functionalized mesoporous silica nanoparticles (FMSNs) were synthesized by the conjugation of magnetite and mesoporous silica with the small and fluorogenic benzothiazole ligand, that is, 2(2-hydroxyphenyl)benzothiazole (). The synthesized fluorescent nanoparticles were characterized by FTIR, XRD, XRF, C CP MAS NMR, BET, and TEM. The photophysical behavior of FMNPs and FMSNs in ethanol was studied using fluorescence spectroscopy. The modification of magnetite and silica scaffolds with the highly fluorescent benzothiazole ligand enabled the nanoparticles to be used as selective and sensitive optical probes for zinc ion detection. Moreover, the presence of in FMNPs and FMSNs induced efficient cell viability and zinc ion uptake, with desirable signaling in the normal human kidney epithelial (Hek293) cell line. The significant viability of FMNPs and FMSNs (80% and 92%, respectively) indicates a potential applicability of these nanoparticles as in vitro imaging agents. The calculated limit of detections (LODs) were found to be 2.53 × 10 and 2.55 × 10 M for Fe₃O₄[email protected] and MSN-Et₃N-IPTMS-hpbtz-f1, respectively. FMSNs showed more pronounced zinc signaling relative to FMNPs, as a result of the more efficient penetration into the cells.
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http://dx.doi.org/10.3390/nano8060434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027406PMC
June 2018

Recent advances in the preparation and application of multifunctional iron oxide and liposome-based nanosystems for multimodal diagnosis and therapy.

Interface Focus 2016 Dec;6(6):20160055

Advanced Imaging Unit, Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), CIBER de Enfermedades Respiratorias, C/Melchor Fernández-Almagro 3 , 28029 Madrid , Spain.

Nowadays, thanks to the successful discoveries in the biomedical field achieved in the last two decades, a deeper understanding about the complexity of mechanistic aspects of different pathological processes has been obtained. As a consequence, even the standard therapeutic protocols have undergone a vast redesign. In fact, the awareness about the necessity to progress towards a combined multitherapy in order to potentially increase the final healing chances has become a reality. One of the crucial elements of this novel approach is that large amounts of detailed information are highly needed and imaging techniques represent one of the most powerful tools to visualize and monitor the pathological state of the patient. To this scope, due to their unique features, nanostructured materials have emerged as attractive elements for the development of multifunctional tools for diagnosis and therapy. Hence, in this review, the most recent and relevant advances achieved by applying multifunctional nanostructures in multimodal theranosis of different diseases will be discussed. In more detail, the preparation and application of single multifunctional nano-radiotracers based on iron oxides and enabling PET/MRI dual imaging will be firstly detailed. After that, especially considering their highly promising clinical potential, the preparation and application of multifunctional liposomes useful for multimodal imaging and therapy will be reviewed. In both cases, a special focus will be set on the application of such a multifunctional nanocarriers in cancer as well as cardiovascular diseases.
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http://dx.doi.org/10.1098/rsfs.2016.0055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5071816PMC
December 2016

Palladium nanoparticles enzyme aggregate (PANEA) as efficient catalyst for Suzuki-Miyaura reaction in aqueous media.

Enzyme Microb Technol 2016 Dec 1;95:242-247. Epub 2016 Feb 1.

Departamento de Biocatálisis, Instituto de Catálisis (CSIC), c/Marie Curie 2, Cantoblanco, Campus UAM, 28049 Madrid, Spain. Electronic address:

Palladium nanoparticles enzyme aggregate (PANEA) were prepared from Candida antarctica B lipase and palladium salt by precipitation and subsequent in situ Pd nanoparticle formation. This heterogeneous catalyst was successfully used for the Suzuki-Miyaura cross-coupling reaction between bromobenzene with different phenylboronic acid derivatives under mild reaction conditions and using low Pd amount. The nanocatalyst exhibited the highest catalytic activity in a mixture of methanol/water (1:1), obtaining good to excellent product yields from the cross-coupling reaction. A variety of functional groups were accepted and the catalyst was recycled 4 times without activity loss.
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http://dx.doi.org/10.1016/j.enzmictec.2016.01.014DOI Listing
December 2016

Immobilization of Trypsin in Lignocellulosic Waste Material to Produce Peptides with Bioactive Potential from Whey Protein.

Materials (Basel) 2016 May 12;9(5). Epub 2016 May 12.

Faculdade de Ciências Farmacêuticas, UNESP-Univ. Estadual Paulista, 14800-903, Departamento de Alimentos e Nutrição, Araraquara-SP, Brazil.

In this study, trypsin (Enzyme Comission 3.4.21.4) was immobilized in a low cost, lignocellulosic support (corn cob powder-CCP) with the goal of obtaining peptides with bioactive potential from cheese whey. The pretreated support was activated with glyoxyl groups, glutaraldehyde and IDA-glyoxyl. The immobilization yields of the derivatives were higher than 83%, and the retention of catalytic activity was higher than 74%. The trypsin-glyoxyl-CCP derivative was thermally stable at 65 °C, a value that was 1090-fold higher than that obtained with the free enzyme. The trypsin-IDA-glyoxyl-CCP and trypsin-glutaraldehyde-CCP derivatives had thermal stabilities that were 883- and five-fold higher, respectively, then those obtained with the free enzyme. In the batch experiments, trypsin-IDA-glyoxyl-CCP retained 91% of its activity and had a degree of hydrolysis of 12.49%, while the values for trypsin-glyoxyl-CCP were 87% and 15.46%, respectively. The stabilized derivative trypsin-glyoxyl-CCP was also tested in an upflow packed-bed reactor. The hydrodynamic characterization of this reactor was a plug flow pattern, and the kinetics of this system provided a relative activity of 3.04 ± 0.01 U·g and an average degree of hydrolysis of 23%, which were suitable for the production of potentially bioactive peptides.
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http://dx.doi.org/10.3390/ma9050357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503075PMC
May 2016

Biosynthesis of Metal Nanoparticles: Novel Efficient Heterogeneous Nanocatalysts.

Nanomaterials (Basel) 2016 May 5;6(5). Epub 2016 May 5.

Advanced Imaging Unit, Spanish National Research Center for Cardiovascular Disease (CNIC), 28049 Madrid, Spain.

This review compiles the most recent advances described in literature on the preparation of noble metal nanoparticles induced by biological entities. The use of different free or substituted carbohydrates, peptides, proteins, microorganisms or plants have been successfully applied as a new green concept in the development of innovative strategies to prepare these nanoparticles as different nanostructures with different forms and sizes. As a second part of this review, the application of their synthetic ability as new heterogonous catalysts has been described in C-C bond-forming reactions (as Suzuki, Heck, cycloaddition or multicomponent), oxidations and dynamic kinetic resolutions.
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http://dx.doi.org/10.3390/nano6050084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302502PMC
May 2016

New emerging bio-catalysts design in biotransformations.

Biotechnol Adv 2015 Sep-Oct;33(5):605-13. Epub 2015 Jan 2.

Departamento of Biocatalisis, Instituto de Catalisis (CSIC), Marie Curie 2, Cantoblanco 28049, Madrid, Spain.

The development of new and successful biotransformation processes of key interest in medicinal and pharmaceutical chemistry involves creating new biocatalysts with improved or even new activities and selectivities. This review emphasizes the new emerging developed strategies to achieve this goal, site-selective chemical modification of enzymes using tailor-made peptides, specific insertion of metals or organometallic complexes into proteins producing bio-catalysts with multiple activities and computational design for creating evolved artificial enzymes with non-natural synthetic catalytic activities.
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http://dx.doi.org/10.1016/j.biotechadv.2014.12.010DOI Listing
March 2016

Useful oriented immobilization of antibodies on chimeric magnetic particles: direct correlation of biomacromolecule orientation with biological activity by AFM studies.

Langmuir 2014 Dec 4;30(49):15022-30. Epub 2014 Dec 4.

Departamento de Biomateriales y Materiales Bioinspirados, Instituto de Ciencia de Materiales de Madrid (ICMM-CSIC), Campus UAM Cantoblanco , 28049 Madrid, Spain.

The preparation and performance of a suitable chimeric biosensor based on antibodies (Abs) immobilized on lipase-coated magnetic particles by means of a standing orienting strategy are presented. This novel system is based on hydrophobic magnetic particles coated with modified lipase molecules able to orient and further immobilize different Abs in a covalent way without any previous site-selective chemical modification of biomacromolecules. Different key parameters attending the process were studied and optimized. The optimal preparation was performed using a controlled loading (1 nmol Ab g(-1) chimeric support) at pH 9 and a short reaction time to recover a biological activity of about 80%. AFM microscopy was used to study and confirm the Abs-oriented immobilization on lipase-coated magnetic particles and the final achievement of a highly active and recyclable chimeric immune sensor. This direct technique was demonstrated to be a powerful alternative to the indirect immunoactivity assay methods for the study of biomacromolecule-oriented immobilizations.
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http://dx.doi.org/10.1021/la502972vDOI Listing
December 2014

Improving lipase activity by immobilization and post-immobilization strategies.

Methods Mol Biol 2013 ;1051:255-73

Institute of Catalysis, CSIC, CAMPUS UAM-Cantoblanco, Madrid, Spain.

One important parameter for the application of lipase catalysts in chemical industries is the specific activity displayed towards natural or unnatural substrates. Different strategies to enhance the lipase activity have been described. The immobilization of lipases on hydrophobic supports by interfacial adsorption at low ionic strength permitted the hyper-activation of these enzymes by fixing the open conformation of the lipase on the hydrophobic support. Improvements of activity from 1.2- up to 20-fold with respect to the initial one have been observed for lipases from different sources. A second strategy was based on the presence of additives, in particular surfactants or ionic liquids, with hydrophobic character to enhance the activity of lipases immobilized on macroporous supports up to eightfold and even more than 100-fold in some cases for soluble lipases. Finally, a third strategy to improve the activity in immobilized lipases was based on a site-directed chemical modification of the protein by glycosylation on the enzyme N-terminal group or on a unique reactive cysteine of the enzyme by disulfide exchange using different tailor-made disulfide activated activated polymers.
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http://dx.doi.org/10.1007/978-1-62703-550-7_17DOI Listing
February 2014

Synthesis of ascorbyl oleate by transesterification of olive oil with ascorbic acid in polar organic media catalyzed by immobilized lipases.

Chem Phys Lipids 2013 Sep 23;174:48-54. Epub 2013 Jul 23.

Instituto de Catalisis, ICP-CSIC, Campus UAM, 28049 Madrid, Spain; Instituto de Investigación en Ciencias de la Alimentación (CIAL), Campus UAM, 28049 Madrid, Spain.

The reaction of transesterification between oils (e.g., olive oil) and ascorbic acid in polar anhydrous media (e.g., tert-amyl alcohol) catalyzed by immobilized lipases for the preparation of natural liposoluble antioxidants (e.g., ascorbyl oleate) was studied. Three commercial lipases were tested: Candida antarctica B lipase (CALB), Thermomyces lanuginosus lipase (TLL) and Rhizomucor miehei lipase (RML). Each lipase was immobilized by three different protocols: hydrophobic adsorption, anionic exchange and multipoint covalent attachment. The highest synthetic yields were obtained with CALB adsorbed on hydrophobic supports (e.g., the commercial derivative Novozym 435). The rates and yields of the synthesis of ascorbyl oleate were higher when using the solvent dried with molecular sieves, at high temperatures (e.g. 45°C) and with a small excess of oil (2 mol of oil per mol of ascorbic acid). The coating of CALB derivatives with polyethyleneimine (PEI) improved its catalytic behavior and allowed the achievement of yields of up to 80% of ascorbyl oleate in less than 24h. CALB adsorbed on a hydrophobic support and coated with PEI was 2-fold more stable than a non-coated derivative and one hundred-fold more stable than the best TLL derivative. The best CALB derivative exhibited a half-life of 3 days at 75°C in fully anhydrous media, and this derivative maintained full activity after 28 days at 45°C in dried tert-amyl alcohol.
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http://dx.doi.org/10.1016/j.chemphyslip.2013.06.003DOI Listing
September 2013

Synthesis of heterogeneous enzyme-metal nanoparticle biohybrids in aqueous media and their applications in C-C bond formation and tandem catalysis.

Chem Commun (Camb) 2013 Aug 24;49(61):6876-8. Epub 2013 Jun 24.

Departamento de Biocatálisis, Instituto de Catálisis (ICP-CSIC), Marie Curie 2, Cantoblanco, Campus UAM, 28049 Madrid, Spain.

The straightforward synthesis of novel enzyme-metalNP nanobiohybrids in aqueous medium was developed. These new nanobiohybrids were excellent multivalent catalysts combining both activities in various sets of synthetic reactions even at ultra-low concentrations (ppb amount).
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http://dx.doi.org/10.1039/c3cc42475hDOI Listing
August 2013

Preparation of lipase-coated, stabilized, hydrophobic magnetic particles for reversible conjugation of biomacromolecules.

Biomacromolecules 2013 Mar 18;14(3):602-7. Epub 2013 Feb 18.

Instituto de Catálisis, CSIC, C/Marie Curie 2, Campus UAM, 28049 Madrid, Spain.

This Communication presents the development of a novel strategy for the easy conjugation of biomolecules to hydrophobic magnetic microparticles via reversible coating with previously functionalized lipase molecules. First, the ability of lipase to be strongly adsorbed onto hydrophobic surfaces was exploited for the stabilization of microparticles in aqueous medium by the creation of a hydrophilic surface. Second, the surface amino acids of lipase can be tailored to suit biomolecule conjugation. This approach has been demonstrated by amino-epoxy activation of lipase, enabling the conjugation of different biomolecules to the magnetic particle's surface. For example, it was possible to immobilize 70% of Escherichia coli proteins on the recovered particles. Furthermore, this strategy could be extended to other lipase chemical modification protocols, enabling fine control of biomolecule coupling. These conjugation techniques constitute a modular methodology that also permits the recycling of the magnetic carrier following use.
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http://dx.doi.org/10.1021/bm400032qDOI Listing
March 2013

Regioselective monodeprotection of peracetylated carbohydrates.

Nat Protoc 2012 Oct 6;7(10):1783-96. Epub 2012 Sep 6.

Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Madrid, Spain.

This protocol describes the regioselective deprotection of single hydroxyls in peracetylated monosaccharides and disaccharides by enzymatic or chemoenzymatic strategies. The introduction of a one-pot enzymatic step by using immobilized biocatalysts obviates the requirement to carry out tedious workups and time-consuming purifications. By using this straightforward protocol, different per-O-acetylated glycopyranosides (mono- or disaccharides, 1-substituted or glycals) can be transformed into a whole set of differentially monodeprotected 1-alcohols, 3-alcohols, 4-alcohols and 6-alcohols in high yields. These tailor-made glycosyl acceptors can then be used for stereoselective glycosylation for oligosaccharide and glycoderivative synthesis. They have been successfully used as building blocks to synthesize tailor-made di- and trisaccharides involved in the structure of lacto-N-neo-tetraose and precursors of the tumor-associated carbohydrate antigen T and the antitumoral drug peracetylated β-naphtyl-lactosamine. We are able to prepare a purified monoprotected carbohydrate in between 1 and 4 d. With this protocol, the small library of monodeprotected products can be synthesized in 1-2 weeks.
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http://dx.doi.org/10.1038/nprot.2012.098DOI Listing
October 2012

Semisynthetic peptide-lipase conjugates for improved biotransformations.

Chem Commun (Camb) 2012 Sep 2;48(72):9053-5. Epub 2012 Aug 2.

Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Marie Curie 2, Cantoblanco, CampusUAM, 28049 Madrid, Spain.

An efficient chemoselective method for the creation of semisynthetic lipases by site-specific incorporation of tailor-made peptides on the lipase-lid site was developed. These new enzymes showed excellent improved specificity and regio- or enantioselectivity in different biotransformations.
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http://dx.doi.org/10.1039/c2cc34816kDOI Listing
September 2012

A novel halophilic lipase, LipBL, showing high efficiency in the production of eicosapentaenoic acid (EPA).

PLoS One 2011 10;6(8):e23325. Epub 2011 Aug 10.

Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Seville, Spain.

Background: Among extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity.

Methods And Findings: A lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C β-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80°C and the pH optimum determined at 25°C was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested.

Conclusions: In this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023325PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3154438PMC
December 2011

trans,trans-2,4-Hexadiene incorporation on enzymes for site-specific immobilization and fluorescent labeling.

Org Biomol Chem 2011 Aug 22;9(15):5535-40. Epub 2011 Jun 22.

Departamento de Biocatálisis, Instituto de Catálisis (CSIC), c/marie curie 2, Cantoblanco, Campus UAM, 28049 Madrid, Spain.

Lipase B from Candida antarctica (CAL-B) has been site-directedly modified by the introduction of a trans,trans-hexadiene moiety onto lipase molecules, identified by MALDI-TOF. This modification on CAL-B permitted its immobilization on Q-Sepharose supports in excellent yields (>95%) when native lipase was not immobilized at pH 7 and 25 °C. After the entire modification procedure, the catalytic activity of the protein on the solid support was surprisingly increased 2-fold. A tailor-made maleimide-fluorophore derivative was specifically covalently linked to the protein in high yield via a selective Diels-Alder reaction in aqueous media. Furthermore, the NBD-labeled-CAL-B was also immobilized on the ionic support, retaining around 80% of the specific activity. The preparation of this labeled-CAL-B was also possible by a Diels-Alder reaction on solid phase in excellent yields.
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http://dx.doi.org/10.1039/c1ob05401eDOI Listing
August 2011

Hydrolysis of fish oil by hyperactivated Rhizomucor miehei lipase immobilized by multipoint anion exchange.

Biotechnol Prog 2011 Jul 13;27(4):961-8. Epub 2011 May 13.

Dept. of Biocatalysis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain.

Rhizomucor miehei lipase (RML) is greatly hyperactivated (around 20- to 25-fold toward small substrates) in the presence of sucrose laurate. Hyperactivation appears to be an intramolecular process because it is very similar for soluble enzymes and covalently immobilized derivatives. The hyperactivated enzyme was immobilized (in the presence of sucrose laurate) on cyanogen bromide-activated Sepharose (very mild covalent immobilization through the amino terminal residue), on glyoxyl Sepharose (intense multipoint covalent immobilization through the region with the highest amount of Lys residues), and on different anion exchangers (by multipoint anionic exchange through the region with the highest density of negative charges). Covalent immobilization does not promote the fixation of the hyperactivated enzyme, but immobilization on Sepharose Q retains the hyperactivated enzyme even in the absence of a detergent. The hydrolysis of fish oils by these hyperactivated enzyme derivatives was sevenfold faster than by covalently immobilized derivatives and three and a half times faster than by the enzyme hyperactivated on octyl-Sepharose. The open structure of the hyperactivated lipase is fairly exposed to the medium, and no steric hindrance should interfere with the hydrolysis of large substrates. These new hyperactivated derivatives seem to be more suitable for hydrolysis of oils by RML immobilized inside porous supports. In addition, the hyperactivated derivatives are fairly stable against heat and organic cosolvents.
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http://dx.doi.org/10.1002/btpr.635DOI Listing
July 2011

Purification, immobilization, and characterization of a specific lipase from Staphylococcus warneri EX17 by enzyme fractionating via adsorption on different hydrophobic supports.

Biotechnol Prog 2011 May-Jun;27(3):717-23. Epub 2011 Apr 20.

Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Cantoblanco, Madrid 28049, Spain.

Staphylococcus warneri strain EX17 produces three lipases with different molecular weights of 28, 30, and 45 kDa. The 45 kDa fraction (SWL-45) has been purified from crude protein extracts by one chromatographic step based on the selective adsorption of this lipase by interfacial activation on different hydrophobic supports at low ionic strength. The adsorption of SWL-45 on octyl-Sepharose increased the enzyme activity by 60%, but the other lipases were also adsorbed on this support. Using butyl-Toyopearl, which is a lesser hydrophobic support, the purification factor was close to 20, and the only protein band detected on the sodium dodecyl sulfate-polyacrylamide electrophoresis analysis gel was that corresponding to the SWL-45, which could be easily desorbed from the support by incubation with triton X-100, producing a purified enzyme. SWL-45 was immobilized under very mild conditions on cyanogen bromide Sepharose, showing similar activities and stability as for its soluble form but without intermolecular interaction. The effects of different detergents over the activity of the immobilized SWL-45 were analyzed, which was hyperactivated by factors of 1.3 and 2.5 with 0.01% Tween 80 and 0.1% Triton X-100, respectively, while ionic detergents produced detrimental effects on the enzyme activity even at very low concentrations. Optimal reaction conditions and the effect of other additives on the enzyme activity were also investigated.
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http://dx.doi.org/10.1002/btpr.601DOI Listing
November 2011

Kinetically controlled synthesis of monoglyceryl esters from chiral and prochiral acids methyl esters catalyzed by immobilized Rhizomucor miehei lipase.

Bioresour Technol 2011 Jan 31;102(2):507-12. Epub 2010 Aug 31.

Departamento de Biocatálisis, Instituto de Catálisis (CSIC), Spain.

Partial acylation of only one primary hydroxyl group of glycerol generates a chiral center at position 2. Rhizomucor miehei lipase (RML) catalyzes the kinetically controlled transesterification of different aromatic carboxylic acids methyl esters with glycerol. High synthetic yields of glyceryl esters (around 70-80%) were obtained even in the presence of significant concentrations of water (from 5% to 20%). After a long incubation of the reaction mixture in the presence of the biocatalyst only pure free acid was obtained. Other lipases (from Geobacillus thermocatenulatus and from Thermomyces lanuginose) also catalyzed similar kinetically controlled transesterifications although less efficiently. RML immobilized on Sepharose-Q showed a high activity and specificity, compared to the immobilization by other techniques, only producing monoglyceryl esters with all substrates. In particular, monoglyceryl-phenylmalonate product was synthesized in 82% overall yield and >99% diastereomeric excess at pH 7.0 and 37°C and 90% glycerol.
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http://dx.doi.org/10.1016/j.biortech.2010.08.095DOI Listing
January 2011

Single-step purification of different lipases from Staphylococcus warneri.

J Chromatogr A 2010 Jan 20;1217(4):473-8. Epub 2009 Nov 20.

Departamento de Biocatalisis, Instituto de Catalisis (CSIC), Madrid, Spain.

Three different lipases from the extract crude of Staphylococcus warneri have been purified by specific lipase-lipase interactions using different lipases (TLL, RML, PFL, BTL2) covalently attached to a solid support as adsorption matrix. BTL2 immobilized on glyoxyl-DTT adsorbed selectivity only a 30 kDa lipase from the crude, which was desorbed by adding 0.1% triton X-100. Using glyoxyl-PFL as matrix, two new lipases (28 and 40 kDa) were adsorbed, and completely pure 40 kDa lipase was obtained after desorption using 0.01% triton, whereas 28 kDa lipase was desorbed after the incubation of the lipase matrix with 3% detergent. When using other matrixes as glyoxyl-TLL or glyoxyl-RML, different lipases were adsorbed. This methodology could be a very efficient and useful method to purify several lipases from crude extracts from different sources.
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http://dx.doi.org/10.1016/j.chroma.2009.11.055DOI Listing
January 2010

Improved reactivation of immobilized-stabilized lipase from Thermomyces lanuginosus by its coating with highly hydrophilic polymers.

J Biotechnol 2009 Oct 6;144(2):113-9. Epub 2009 Sep 6.

Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM, Cantoblanco, Madrid, Spain.

Immobilized-stabilized aminated lipase from Thermomyces lanuginosus (TLL-A) is not easily reactivated after inactivation by incubation in the presence of organic solvents or chaotropic reagents. To improve the recovered activity of this biocatalyst, immobilized TLL-A has been submitted to different modifications. The best results were obtained when the enzyme was coated with a very hydrophilic and inert polymer: dextran modified with glycine (Dx-Gly). This modification did not reduce enzymatic activity while it increased the stability of this already very stable preparation, in thermal and organic solvent induced inactivation (by a 4-fold factor). Simple incubation in aqueous medium at pH 7 and 25 degrees C permitted to fully recover the activity of the immobilized and modified TLL-A enzyme inactivated by incubation in organic solvents or saturated guanidine during 3 cycles, while the non-modified enzyme only recover some activity. When the inactivation was caused by exposition at high temperatures, the reactivation was higher using the modified biocatalyst, but was far for complete (40% after 3 inactivation-reactivation cycles). The determination of the TLL-A activity in the presence of detergents (that helps the opening of active site of the lipase) allowed, in this case, to significantly improve the results, now near to 90% of the initial activity was recovered (using the non-modified enzyme the recovered activity was around 60%). This very hydrophilic and inert polymer, coating the enzyme surface, seems to help the correct positioning of the hydrophilic and hydrophobic groups of the enzyme, and that way improve both the stability and possibility of reactivation of the enzyme.
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http://dx.doi.org/10.1016/j.jbiotec.2009.09.002DOI Listing
October 2009