RESUMEN
We demonstrated that a change in the catalytic activity of fungal lipases synthesized by Rhizopus microsporus, Penicillium sp. and Oospora lactis and their ability to absorb on different sorbents depended on the nature of groups on the solid phase surface in the model systems water: lipid and water: solid phase. Thus, the stability of Penicillium sp. lipases increased 85% in the presence ofsorsilen or DEAE-cellulose, and 55% of their initial activity respectively was preserved. In the presence of silica gel and CM-cellulose, a decreased rate of lipid hydrolysis by Pseudomonas sp. enzymes was observed in water medium, and the hydrolysis rate increased by 2.4 and 1.5 times respectively in the presence of aminoaerosil and polykefamid. In an aqueous-alcohol medium, aminoaerosil and polykefamid decreased the rate of substrate hydrolysis by more than 30 times. The addition of aerosil to aqueous and aqueous-alcohol media resulted in an increase in the hydrolysis rate by 1.2-1.3 times. Sorsilen stabilized Penicillium sp. lipase activity at 40, 45, 50 and 55 degrees C. Either stabilization or inactivation of lipases was observed depending on the pH of the medium and the nature of chemical groups localized on the surface of solid phase. The synthetizing activity of lipases also changed depending on the conditions.
Asunto(s)
Lipasa/metabolismo , Penicillium/enzimología , Rhizopus/enzimología , Catálisis , Hidrólisis , Lipasa/química , Lipasa/genética , Lípidos/química , Penicillium/genética , Transición de Fase , Rhizopus/genética , Especificidad por Sustrato , Agua/química , Agua/metabolismoRESUMEN
Abstract-A simple and efficient method of preparing highly purified extracellular proteinases of B. subtilis B-1 (SKB 256) has been developed. A sorbent based on sorsilen impregnated with hemoglobin or cytochrome c has been synthesized for this purpose. A significant difference between the efficiency of hemoglobin and cytochrome c as biospecific ligands has been observed: the enzyme yield amounted to 40.6 and 65.6% of the total amount of enzyme adsorbed, respectively. The culture was shown to contain two major proteinase forms with different molecular masses that could be separated by chromatography on a Sephadex G-50 but gave only one band with MW 27 kDa upon denaturing electrophoresis in 12.5% PAG in the presence of 0.1% SDS. The influence of eluent pH, ionic strength and ethanol concentration on the sorption of the proteinases on the biospecific sorbent, as well as on the desorption from it, has been investigated. Positive influence of 20% ethanol on proteinase desorption has been demonstrated.
Asunto(s)
Bacillus subtilis/enzimología , Cromatografía de Afinidad , Líquido Extracelular/enzimología , Péptido Hidrolasas/aislamiento & purificación , Adsorción , Bacillus subtilis/química , Cromatografía de Afinidad/métodos , Cromatografía en Gel , Citocromos c/química , Citocromos c/metabolismo , Electroforesis en Gel de Poliacrilamida , Etanol/química , Líquido Extracelular/química , Hemoglobinas/química , Hemoglobinas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Peso Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Polímeros/químicaRESUMEN
The behavior of intact and immobilized invertase in aqueous and water-organic media has been studied. In a water-organic medium, the transferase properties of the enzyme changed: pH optima of intact and immobilized invertase undergo shifts of 0.5 units: the temperature optimum decreases (50 degrees C and 25 degrees C in aqueous and water-organic media, respectively). The extent of conversion of isoamyl alcohol in water-organic medium shows a dependence on several factors (the enzyme and substrate concentrations; amount of the organic phase; and duration of the enzyme incubation with the substrate). Optimum parameters of isoamyl alcohol conversion were used for transforming fusel alcohols into alkyl fructosides. The results of this applied research have important practical implications (conversion of fusel oils of alcoholic beverages).
Asunto(s)
Enzimas Inmovilizadas/química , Pentanoles/química , Saccharomyces cerevisiae/enzimología , beta-Fructofuranosidasa/química , Estabilidad de Enzimas , Solventes/química , Especificidad por Sustrato , Agua/químicaRESUMEN
The transferase reaction between phospholipids and inositol catalyzed by phospholipase D on phase interface in water-organic solvent systems was studied. Optimal conditions for phosphatidylinositol synthesis in water-organic solvent heterogeneous system were determined. The rapid separation of the hydrophobic components, phospholipids, from water-soluble products, alcohols, was observed in the systems with organic solvents. Displacement of myo-inositol from phosphatidylinositol by methanol, alcohol substrate, added to the reaction medium was shown in hexane-water system. Myo-inositol was isolated from the mixture of its isomers by two-stage transferase reaction catalyzed by phospholipase D.
Asunto(s)
Enzimas/metabolismo , Fermentación , Inositol/aislamiento & purificación , SolucionesRESUMEN
The optimum conditions for extraction of catalytic active conjugates of alpha-amylase and antibodies for staphylococcal toxin (ST) have been created. The optimum correlation of antibodies and enzyme for the effective use of the extracted conjugate during enzyme immunoassay for ST has been established. The covalent immobilization of antibodies on the micropore polyamide membranes has been carried out. Apart from this, the extracted conjugated were purified by TSK-gel HN-55 chromatography. The optimum concentration of the conjugates has been defined.
Asunto(s)
Toxinas Bacterianas/análisis , Staphylococcus/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Toxinas Bacterianas/inmunología , Cromatografía en Gel , Técnicas para Inmunoenzimas , Membranas Artificiales , Neumonía Estafilocócica/diagnóstico , Staphylococcus/química , alfa-Amilasas/inmunologíaRESUMEN
Phospholipid composition of synovial fluid was studied in patients with reactive, rheumatoid and juvenile chronic forms of arthritis as compared with that of normal synovial fluid. The most pronounced alterations of the synovial fluid phospholipid composition (additional phospholipid fractions, increase in content of lyso-derivatives) were found in the patients with rheumatoid and juvenile chronic arthritis, which appear to occur due to activation of endogenous phospholipases A2, C and lysophospholipase A1.
Asunto(s)
Artritis/metabolismo , Fosfolípidos/metabolismo , Líquido Sinovial/metabolismo , Adolescente , Artritis/enzimología , Niño , Preescolar , Activación Enzimática , Humanos , Fosfolipasas A/metabolismo , Líquido Sinovial/enzimología , Fosfolipasas de Tipo C/metabolismoRESUMEN
A polyamide with the covalently coupled phosphatidyl ethanolamine was used for affinity adsorption of an alkaline lipase from Pseudomonas aeruginosa. The immobilization resulted in increase of the enzyme specific activity. Some properties of native and adsorbed enzyme were compared. The temperature optima, heat and pH stability, KM and Vmax values were determined for both native and immobilized enzymes.
Asunto(s)
Enzimas Inmovilizadas , Lipasa/metabolismo , Pseudomonas aeruginosa/enzimología , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Fosfatidiletanolaminas/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
The enzymic hydrolysis of fish with lipases from various sources was studied. The lipase from the fungus Rhizopus microsporus preferentially removes saturated fatty acids, while lipase from the pyloric caeca of salmon unsaturated fatty acids upon hydrolysis of fish fats. The enzymes can be used to obtain fatty products enriched with eicosanopentaenoic acid, mono- and diacylglycerols by enzymic hydrolysis of the ivasi fat.
Asunto(s)
Aceites de Pescado/metabolismo , Lipasa/metabolismo , Animales , Diglicéridos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Glicéridos/metabolismo , Hidrólisis , Rhizopus/enzimología , Triglicéridos/metabolismoRESUMEN
A method of affinity chromatography was developed for purification of phospholipase A2(PL-A2) from the Central Asian cobra venon. The enzyme was covalently coupled to a polyamide sorbent with phosphatidilethanolamine (PEA) and cytotoxin (CT). The effect of CA2+ concentration and the ion strength of the solution on the enzyme adsorption was studied. The most efficient coupling of the enzyme to the sorbent was observed at pH 8--9 in case of the Ca2+ absence and a low ion strength of the solution. For desorption of the enzyme Triton X-100 at a concentration of 0.5% should be introduced in the eluting solution. The affinity adsorption chromatography enabled the isolation of two forms of phospholipase A2 with different affinity for PEA and CT. The total yield of the enzyme was 91% at a purification degree of 5.5 and 3.5, respectively. The introduction of the second ligand (CT) in the composition of the sorbent with the phospholipid ligand allowed the authors to increase its capacity and affinity for the phospholipase A2 from the snake venom.
Asunto(s)
Venenos Elapídicos/análisis , Fosfolipasas A/aislamiento & purificación , Fosfolipasas/aislamiento & purificación , Cromatografía de Afinidad , Fosfolipasas A2RESUMEN
A procedure for the purification of isoenzyme I of phospholipase C from Cl. perfringens was developed. The isoenzyme was purified to homogeneity (data from disc electrophoresis) using affinity chromatography on polycephamide and gel filtration through Ultrogel AcA-54, the enzyme yield being 41%. Some properties of the purified isoenzyme I (pH and temperature optima, stability, effect of metal ions and detergents, substrate dependence) were investigated. No significant differences between the properties of the unfractionated enzyme and isoenzyme I were established.
Asunto(s)
Clostridium perfringens/enzimología , Isoenzimas/aislamiento & purificación , Fosfolipasas de Tipo C/aislamiento & purificación , Cationes Bivalentes , Cromatografía de Afinidad , Cromatografía en Gel , Concentración de Iones de Hidrógeno , Hidrólisis , Isoenzimas/metabolismo , Fosfatidilcolinas/metabolismo , Tensoactivos , Fosfolipasas de Tipo C/metabolismoRESUMEN
The properties of membrane-bound mitochondrial phospholipase D were investigated. The enzyme was shown to catalyze the hydrolysis of endogenous mitochondrial phospholipids, particularly phosphatidylethanolamine. The phospholipase activity was maximal at pH 5.0 and 7.0 was stimulated by Ca2+ and sodium oleate and was inhibited by SDS. The conditions for solubilization of the mitochondrial enzyme and its adsorption on a biospecific adsorbent were elaborated. The adsorption resulted in a 17-fold purification of the enzyme with a 33% yield. The adsorbed enzyme, similar to the membrane-bound one, was activated by Ca2+ and sodium oleate but, in contrast to the latter, was able to catalyze the hydrolysis of an exogenous substrate.
Asunto(s)
Mitocondrias Hepáticas/enzimología , Fosfolipasa D/metabolismo , Fosfolipasas/metabolismo , Animales , Catálisis , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Ratas , Especificidad por SustratoRESUMEN
Incubation of liver mitochondria at 37 degrees C causes changes in the phospholipid composition, such as the decrease in the levels of major phospholipids (e.g. phosphatidylcholine, phosphatidylethanolamine, cardiolipin) and their lysoderivatives as well as an increase in the levels of phosphatidic and lysophosphatidic acids. Similar changes in the phospholipid composition are observed upon heat incubation of mitochondrial fragments ("ghosts", inner and outer mitochondrial membranes). Ca2+ accelerate the heat-induced changes in the phospholipid levels resulting from heat incubation, whereas EGTA, in contrast, decelerates them. The role of an endogenous system of lipolytic enzymes in the observed conversions of mitochondrial phospholipids is discussed.
Asunto(s)
Calor , Mitocondrias Hepáticas/metabolismo , Fosfolípidos/metabolismo , Animales , Mitocondrias Hepáticas/enzimología , Fosfolipasas/metabolismo , Ratas , Especificidad por SustratoRESUMEN
Changes in the amount of phospholipids and lysophospholipids of mitochondria and their fragments have been studied under long-term heat incubation. A discrepancy is found between a decrease in the content of phospholipids as a result of their hydrolysis by mitochondrial phospholipase A2 and accumulation of the corresponding lysoderivatives. Data are presented which show that all this is a result of lysoderivatives splitting by lysophospholipase A. The activity of this enzyme is observed in incubation of intact mitochondria, their "ghosts" as well as fractions of the external and internal mitochondrial membranes. It is shown that lysophospholipase A is able to hydrolyze both endogenic and exogenic substrates. The enzyme is active at pH 6.0, lysocardiolipin being the most preferable substrate.
Asunto(s)
Membranas Intracelulares/enzimología , Lisofosfolipasa/metabolismo , Lípidos de la Membrana/metabolismo , Mitocondrias Hepáticas/enzimología , Fosfolipasas/metabolismo , Fosfolípidos/metabolismo , Animales , Concentración de Iones de Hidrógeno , Ratas , Especificidad por Sustrato , TemperaturaRESUMEN
The catalytic properties of membrane-bound phospholipase A2 from inner and outer mitochondrial membranes were studied. Differences were found in the properties of phospholipase A2 during the hydrolysis of both endogenous and exogenous substrates, i.e. the dependence of the hydrolysis rate on pH, temperature, bivalent metal ion concentrations and EDTA. It was demonstrated that purification and adsorption immobilization of the inner mitochondrial membrane enzyme on biospecific adsorbent cause changes in the enzyme catalytic properties. The role of phospholipase A2 microenvironment in the manifestation of the enzyme activity and catalytic properties is discussed.
Asunto(s)
Membranas Intracelulares/enzimología , Mitocondrias Hepáticas/enzimología , Fosfolipasas A/aislamiento & purificación , Fosfolipasas/aislamiento & purificación , Animales , Cationes Bivalentes/farmacología , Ácido Edético/farmacología , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Lípidos de la Membrana/análisis , Fosfolipasas A/análisis , Fosfolipasas A2 , Fosfolípidos/análisis , RatasRESUMEN
The effect of the immobilization technique and the ligand nature on catalytic properties of phospholipase A2 from the cobra venom was studied. Preparations of phospholipase A2 adsorbed on and covalently bound to polyamide sorbents were obtained. The enzyme was coupled to polyamide beads modified with glutaraldehyde. In this case only 9% of the enzyme activity was retained. The enzyme adsorbed on polyamide modified with phosphatidylethanolamine retained up to 20% of the initial activity. The binding selectivity of phospholipase A2 was maximum in case of the sorbent with a binary ligand, e. g. phosphatidylethanolamine+cytotoxin, the sorbent capacity for the bound enzyme increased 2-3 times (460-600 units/g sorbent. The specific activity of the adsorbed phospholipase A2 was 17-40 units/g sorbent in contrast to 8.6 units/g sorbent for the covalently bound enzyme. Immobilization of the enzyme on polyamide sorbents resulted in changes of the pH-optimum, sensitivity to Ca2+ ions and the character of the enzyme-substrate interactions. Heart stability of the adsorbed phospholipase A2 was lower than that of the covalently bound enzyme. However, the adsorbed enzyme can be used, for example, in affinity chromatography due to its higher specific activity, selectivity and reversibility of the sorption.
Asunto(s)
Venenos Elapídicos , Enzimas Inmovilizadas , Fosfolipasas A/aislamiento & purificación , Fosfolipasas/aislamiento & purificación , Adsorción , Animales , Calcio/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Ligandos , Nylons , Fosfolipasas A2 , Solubilidad , TemperaturaRESUMEN
Using biospecific chromatography on polylysocephamide, a toxic phospholipase possessing a presynaptic effect on neuromuscular preparations was isolated from the venom of the giant hornet Vespa orientalis. The enzyme was shown to possess a high hydrolytic activity towards 1-acyllysophosphatidylcholine within a narrow pH range (pH optimum 7.5). The enzyme activity was suppressed by detergents of various chemical composition. Lysophospholipase caused an intensive hemolysis of washed human erythrocytes. The catalytic and hemolytic functions of the enzyme were sensitive to metal ions, however, in a different degree. Ca2+ and Mn2+ activated, while Cu2+ and Zn2+ inhibited the enzyme. Mg2+ and Sr2+ had no effect on the enzyme activity.
Asunto(s)
Venenos de Abeja/análisis , Lisofosfolipasa/aislamiento & purificación , Fosfolipasas/aislamiento & purificación , Venenos de Avispas/análisis , Catálisis , Detergentes , Estabilidad de Enzimas , Hemólisis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Técnicas In Vitro , Cinética , Lisofosfolipasa/antagonistas & inhibidores , Lisofosfolipasa/farmacología , TemperaturaRESUMEN
Phospholipid composition was studied in membranes of various tissues as well as of mitochondria and erythrocytes of rats with alcohol intoxication. A new phospholipid--phosphatidyl ethanol, which was absent in tissues of control animals, was detected in liver, heart, kidney, small intestine tissues as well as in erythrocytes and liver mitochondria of rats consuming ethanol independently on the alcohol administration procedure used. Studies of rat liver mitochondria and mitochondrial "ghosts" showed that formation of phosphatidyl ethanol and phosphatidic acid was catalyzed by membranal endogenous phospholipase D. Phosphatidyl ethanol appears to be of importance in membranotropic effect of ethanol--one of main factors participating in alcohol pathology development. This phospholipid was found also in erythrocytes and blood of patients with chronic alcoholism, which were subjected to alcohol provocation before examination. In erythrocytes of the other group of patients with chronic alcoholism, not provocated with alcohol, the phosphatidyl ethanol fraction was not found but content of phosphatidyl ethanolamine was distinctly increased. In patients with delirium tremens phosphatidyl ethanol was not observed, content of phosphatidyl ethanolamine was not increased, while amount of phosphatidic acid was considerably elevated.
Asunto(s)
Intoxicación Alcohólica/metabolismo , Glicerofosfolípidos , Mitocondrias Hepáticas/metabolismo , Ácidos Fosfatidicos/biosíntesis , Adulto , Animales , Cromatografía en Capa Delgada , Eritrocitos/metabolismo , Humanos , Masculino , Lípidos de la Membrana/metabolismo , Persona de Mediana Edad , Ácidos Fosfatidicos/sangre , Fosfolípidos/metabolismo , RatasRESUMEN
Phospholipid composition was studied in liver mitochondrial membranes of rats consuming ethanol for a long time. A new phospholipid phosphatidyl ethanol was found in the membranes, which appears to be formed due to the action of endogenous phospholipase D. The phospholipase D activity was measured in mitochondria using direct evaluation. Administration of ethanol led to alteration in the ratio of mitochondrial phospholipids.
Asunto(s)
Etanol/toxicidad , Membranas Intracelulares/metabolismo , Lípidos de la Membrana/metabolismo , Mitocondrias Hepáticas/metabolismo , Fosfolípidos/metabolismo , Animales , Membranas Intracelulares/efectos de los fármacos , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , RatasRESUMEN
Methods of biospecific adsorption chromatography of phospholipase A2 obtained from porcine pancreas and Naja naja oxiana, Vipera ursini renardi, Vespa orientalis venoms were developed. Granulated polyamide with covalently linked phosphatidylethanolamine were used as an affinity adsorbent. Chemical inertness of linked phosphatidylethanolamine to the hydrolytic action of phospholipase A2 and its high affinity for biospecific complexes are shown. Forms of phospholipase A2 different in their affinity for an immobilized substrate was isolated by biospecific adsorption chromatography. The role of hydrophobic and electrostatic interactions in formation of enzyme-ligand complexes was studied.
Asunto(s)
Cromatografía de Afinidad/métodos , Fosfolipasas A/aislamiento & purificación , Fosfolipasas/aislamiento & purificación , Adsorción , Animales , Caprolactama/farmacología , Venenos Elapídicos , Concentración de Iones de Hidrógeno , Ligandos , Páncreas/enzimología , Fosfatidiletanolaminas/farmacología , Fosfolipasas A2 , Porcinos , Venenos de VíborasRESUMEN
Catalytic activity and stability of glucoamylases immobilized by different methods (adsorption, covalent binding) are studied comparatively. The highest stability is shown to be obtained under covalent binding. The binding efficiency and immobilized glucoamylase properties depend on the nature of insoluble carrier and a purification degree of the enzyme preparations. The choice of the cross-linking agent promoting a binding between the enzyme and the carrier is very significant. The activity and stability of immobilized glucoamylases obtained when using different cross-linking agents rise in such a sequence: 2,4-toluylenediisocyanate, cyanurochloride, glutaric dialdehyde, gossypol. Catalytic properties and stability are determined for soluble and immobilized glucoamylase forms from different sources.