Las lipasas: enzimas con potencial para el desarrollo de biocatalizadores inmovilizados por adsorción interfacial

Las lipasas son enzimas con propiedades funcionales muy interesantes que permiten su utilización práctica en diversos campos de las industrias agroquímica, farmacéutica, de detergentes y alimentaria, así como en química fina. Entre las aplicaciones más importantes de estas moléculas se encuentran: la resolución de mezclas racémicas, la obtención de compuestos ópticamente puros y la bioconversión de principios activos. En este trabajo se presenta una amplia revisión del tema, que abarca desde aspectos estructurales y funcionales de las lipasas, hasta la inmovilización de estas enzimas mediante adsorción interfacial y su empleo en biotecnología. Palabras clave: activación interfacial, adsorción interfacial, bioconversión, esterasas. Abstract: Lipases are enzymes with very interesting functional properties that allow their practical use in different fields of Agro-Chemical, Pharmaceutical and Food industries, as well as in Fine Chemistry. Among the most relevant applications of these molecules are: racemic mixtures resolution, obtainment of optically pure compounds and bioconversion of active principles. In this work a broad review of this topic is presented. This includes since structural and functional features of lipases until the immobilization of these enzymes by interfacial adsorption and their employment in biotechnology. Key words: Bioconversion, esterases, interfacial activation, interfacial adsorptio

Lipolysis kinetics of milk-fat catalyzed by an enzymatic supplement under simulated gastrointestinal conditions

[EN] Pancreatic insufficiency is a clinical manifestation characterized by the in-ability of the pancreas to release enough pancreatic enzyme into the small intestine, necessary to digest intraluminal nutrients. The lack of digestive enzymes leads to the difficulty to absorb nutrients, which drives in infants, to malnutrition and lack of growth and development, due to the loss of calories. These patients generally need oral administration of enzymes to favor lipolysis and absorption of lipids from foods. However, there are a number of food related factors (matrix, type of fat, etc.) and digestive environment (intestinal pH, bile concentration, among others), which will influence the digestibility of nutrients. In this study, an "in vitro" digestion model was used to characterize the kinetics of the lipolysis of milk-fat catalyzed by an enzymatic supplement. Different intestinal conditions (pH (6, 7 and 8) and bile concentrations (1, 5 and 10 mml L-1)) were simulated, using a fixed concentration of supplement. Gastro-Intestinal conditions, significantly affected lipolysis. High pH and bile concentrations were translated into low values of the Michaelis-Menten constant and high values of the catalytic constant. The kinetic parameters obtained from this work allowed estimating the dose of enzymatic supplement required to optimize the lipolysis of milk-fat under different intestinal environments, sufficient and insufficient pancreatic conditions.Authors of this paper, on behalf of MyCyFAPP consortium, acknowledge the European Union and the Horizon 2020 Research and Innovation Framework Programme for funding the above-mentioned project under grant agreement number 643806.Peinado Pardo, I.; Larrea Santos, V.; Heredia Gutiérrez, AB.; Andrés Grau, AM. (2018). Lipolysis kinetics of milk-fat catalyzed by an enzymatic supplement under simulated gastrointestinal conditions. Food Bioscience. 23:1-8. https://doi.org/10.1016/j.fbio.2018.02.011S182

Identification of a potent and selective LAPTc inhibitor by RapidFire-Mass Spectrometry, with antichagasic activity

Background: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery.Methodology/Principal findings: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 &lt; 34 μM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 μM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of &gt;500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination.Conclusions/Significance: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.</p

Discovery of potent and selective inhibitors of the Escherichia coli M1-aminopeptidase via multicomponent solid-phase synthesis of tetrazole-peptidomimetics

The Escherichia coli neutral M1-aminopeptidase (ePepN) is a novel target identified for the development of antimicrobials. Here we describe a solid-phase multicomponent approach which enabled the discovery of potent ePepN inhibitors. The on-resin protocol, developed in the frame of the Distributed Drug Discovery (D3) program, comprises the implementation of parallel Ugi-azide four-component reactions with resin-bound amino acids, thus leading to the rapid preparation of a focused library of tetrazole-peptidomimetics (TPMs) suitable for biological screening. By dose-response studies, three compounds were identified as potent and selective ePepN inhibitors, as little inhibitory effect was exhibited for the porcine ortholog aminopeptidase. The study allowed for the identification of the key structural features required for a high ePepN inhibitory activity. The most potent and selective inhibitor (TPM 11) showed a non-competitive inhibition profile of ePepN. We predicted that both diastereomers of compound TPM 11 bind to a site distinct from that occupied by the substrate. Theoretical models suggested that TPM 11 has an alternative inhibition mechanism that doesn't involve Zn coordination. On the other hand, the activity landscape analysis provided a rationale for our findings. Of note, compound TMP 2 showed in vitro antibacterial activity against Escherichia coli. Furthermore, none of the three identified inhibitors is a potent haemolytic agent, and only two compounds showed moderate cytotoxic activity toward the murine myeloma P3X63Ag cells. These results point to promising compounds for the future development of rationally designed TPMs as antibacterial agents

Las lipasas: enzimas con potencial para el desarrollo de biocatalizadores inmovilizados por adsorción interfacial

Lipases are enzymes with very interesting functional properties that allow their practical use in different fields of Agro-Chemical, Pharmaceutical and Food industries, as well as in Fine Chemistry. Among the most relevant applications of these molecules are: racemic mixtures resolution, obtainment of optically pure compounds and bioconversion of active principles. In this work a broad review of this topic is presented. This includes since structural and functional features of lipases until the immobilization of these enzymes by interfacial adsorption and their employment in biotechnology.Las lipasas son enzimas con propiedades funcionales muy interesantes que permiten su utilización práctica en diversos campos de las industrias agroquímica, farmacéutica, de detergentes y alimentaria, así como en química fina. Entre las aplicaciones más importantes de estas moléculas se encuentran: la resolución de mezclas racémicas, la obtención de compuestos ópticamente puros y la bioconversión de principios activos. En este trabajo se presenta una amplia revisión del tema, que abarca desde aspectos estructurales y funcionales de las lipasas, hasta la inmovilización de estas enzimas mediante adsorción interfacial y su empleo en biotecnología

Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a <i>Leishmania major</i> M17-aminopeptidase

The Leishmania major leucyl-aminopeptidase (LAPLm), a member of the M17 family of proteases, is a potential drug target for treatment of leishmaniasis. To better characterize enzyme properties, recombinant LAPLm (rLAPLm) was expressed in Escherichia coli. A LAPLm gene was designed, codon-optimized for expression in E. coli, synthesized and cloned into the pET-15b vector. Production of rLAPLm in E. coli Lemo21(DE3), induced for 4 h at 37 °C with 400 μM IPTG and 250 μM l-rhamnose, yielded insoluble enzyme with a low proportion of soluble and active protein, only detected by an anti-His antibody-based western-blot. rLAPLm was purified in a single step by immobilized metal ion affinity chromatography. rLAPLm was obtained with a purity of ~10% and a volumetric yield of 2.5 mg per liter, sufficient for further characterization. The aminopeptidase exhibits optimal activity at pH 7.0 and a substrate preference for Leu-p-nitroanilide (appKM = 30 μM, appkcat = 14.7 s−1). Optimal temperature is 50 °C, and the enzyme is insensitive to 4 mM Co2+, Mg2+, Ca2+ and Ba2+. However, rLAPLm was activated by Zn2+, Mn2+ and Cd2+ but is insensitive towards the protease inhibitors PMSF, TLCK, E−64 and pepstatin A, being inhibited by EDTA and bestatin. Bestatin is a potent, non-competitive inhibitor of the enzyme with a Ki value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification

A Two-Step Purification Procedure of Phospholipases A2 from the Sea Anemone Condylactis gigantea

Submitted by EMERSON LEAL ([email protected]) on 2019-06-12T19:28:42Z No. of bitstreams: 1 Protocolo de purificación en dos etapas de fosfolipasas A2 a partir de la anémona marina Condylactis gigantea.pdf: 978834 bytes, checksum: 57fa15e40656a2df50e09018fe03ace6 (MD5)Approved for entry into archive by EMERSON LEAL ([email protected]) on 2019-06-12T20:08:42Z (GMT) No. of bitstreams: 1 Protocolo de purificación en dos etapas de fosfolipasas A2 a partir de la anémona marina Condylactis gigantea.pdf: 978834 bytes, checksum: 57fa15e40656a2df50e09018fe03ace6 (MD5)Made available in DSpace on 2019-06-12T20:08:42Z (GMT). No. of bitstreams: 1 Protocolo de purificación en dos etapas de fosfolipasas A2 a partir de la anémona marina Condylactis gigantea.pdf: 978834 bytes, checksum: 57fa15e40656a2df50e09018fe03ace6 (MD5) Previous issue date: 2017Universidad de Oriente. Facultad de Ciencias Naturales. Santiago de Cuba, Cuba.Universidad de La Habana. Facultad de Biología. Centro de Estudio de Proteínas. Ciudad Habana, Cuba.Instituto de Catálisis y Petroleoquímica. Madrid, España.Universidad de La Habana. Facultad de Biología. Centro de Estudio de Proteínas. Ciudad Habana, Cuba.Universidad de La Habana. Facultad de Biología. Centro de Estudio de Proteínas. Ciudad Habana, Cuba.Fundação Oswaldo Cruz. Centro de Estudos de Biomoléculas Aplicadas à Saúde. Universidade Federal de Rondônia. Departamento de Medicina. Porto Velho, RO, Brasil.Universidad de La Habana. Facultad de Biología. Centro de Estudio de Proteínas. Ciudad Habana, Cuba.El veneno de los celenterados marinos constituyen mezclas complejas de varios componentes, muchos de naturaleza proteica, y para los cuales se ha descrito la actividad fosfolipásica A2. El objetivo de la presente investigación fue purificar las fosfolipasas A2 (FLA2) procedentes de la anémona marina Condylactis gigantea a partir de un protocolo de dos etapas para su posterior caracterización. Se muestra la purificación de FLA2 en un soporte cromatográfico de afinidad con fosfatidilcolina de huevo inmovilizada covalentemente para la purificación de fosfolipasas A2, a los que se les comprobó cualitativamente la actividad fosfolipásica A2 por cromatografía en placa (TLC) utilizando sustrato marcado con fluorescencia. Dicho soporte cromatográfico permite que en un protocolo de purificación de solamente dos pasos se obtengan tres componentes proteicos de pesos moleculares entre 18000 y 14000, con al menos un componente que posee actividad fosfolipásica A2.Marine coelenterate venom is composed of complex mixtures of several substances, mainly of proteins, for which phospholipase A2 activity has been described. This research study aims to test a two-step purification procedure for phospholipases A2 (PLA2) to filter enzymes for further characterization. PLA2 purification from the sea anemone Condylactis gigantean is conducted through a chromatographic affinity support MANA - Sepharose CL 4B with covalently immobilized phosphatidylcholine egg. The phospholipase A2 activity was corroborated by using qualitative TLC and a fluorogenic substrate. By means of the above-mentioned support, purification of three protein-based components can be carried out. These components are produced with molecular weights between 18000 and 14000, and at least one component possessing phospholipase A2 activity

Extraction systems for isolating esterases having interfacial adsorption

Resumen: En el presente trabajo se optimizaron las condiciones de extracción de esterasas con actividad en interfaces, a partir de la anémona marina Stichodactyla helianthus y del camarón peneido Litopenaeus vannamei. Las esterasas interfaciales, cuya presencia en estas especies había sido informada previamente, presentan características funcionales que las hacen muy atractivas para su empleo industrial. Los homogenados de los animales se trataron con los detergentes Tritón X-100, Tween 20 y Tween 80 en dos concentraciones cada uno: la Concentración Micelar Crítica (CMC) y la mitad de ésta. Además se empleó NaCl 0,5 mol/L y n-butanol a las proporciones 5, 10 y 20%. Cada variante fue comparada con el método tradicional de extracción con agua destilada, que fue tomado como control. Los mejores resultados se obtuvieron empleando n-butanol al 20%, para recuperar las actividades esterasa y fosfolipasa, y al 10%, en el aislamiento de la actividad lipasa. La efectividad de este solvente en el aislamiento de estas enzimas con afinidad por las interfaces lípido/agua, pudiera estar dada por su capacidad para romper los agregados entre estas moléculas y causar la desorción de las mismas a los restos de membrana y tejidos presentes en la preparación. Palabras clave: activación interfacial, esterasas interfaciales, lipasas, Stichodactyla helianthus, Litopenaeus vannamei.interfacial activation, interfacial esterase, lipase, Stichodactyla helianthus, Litopenaeus vannamei. Abstract: Interfacial esterases present great functional versatility, making them very attractive molecules for industrial applications. The conditions for extracting interfacial esterases previously detected in the sea anemone Stichodactyla helianthus and the shrimp Litopenaeus vannamei were optimised in this work. Animal homogenates were treated with Triton X-100, Tween 20 and Tween 80 detergents at two different concentrations: critical micellar concentration (CMC) and half of that concentration; 0.5 mol/L NaCl and n-butanol at 5%, 10% and 20% v/v ratios were also tested. Each procedure was compared to the control extraction method using distilled water. The best results were obtained with 20% n-butanol for recovering esterase and phospholipase activity whilst 10% n-butanol extraction was the most effective for lipase activity isolation. This solvent’s suitability for isolating interface-activated enzymes could be explained by its ability to dissociate biomolecule aggregates and cause enzyme desorption from the membranes and tissues remaining in the preparation