Browsing by Author "Yotova L."
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Item Amperometric biosensor with “liquid” enzyme membrane(1994-01-01) Neikov A.; Entchcva E.; Krysteva M.; Yotova L.The advantages of the “liquid” membranes for biosensors with enzyme, covalently bound to soluble dextran, are discussed in this article. A three layer steady state model is used to compare biosensors with homogeneous (liquid) and nonhomogeneous enzyme membranes with respect to their technical characteristics: sensitivity and response time. Theoretical analysis shows that the “liquid” membrane sensor is by 10 % more sensitive and by 20 %-120 % faster than the one with a nonhomogeneous enzyme membrane. The experiments were made with a new prototype of oxygen sensor, more suitable to fix an uniform enzyme (glucose oxydase + catalase) layer to the fronthead. The experimental results confirm the theoretical conclusions. © 1994 Taylor & Francis Group, LLC.Item Application of three-stage approach to isolation and purification of Pseudozyma antarctica lipase B(2018-01-01) Borisov B.; Manova D.; Yaneva S.; Danalev D.; Yotova L.Lipases are serine hydrolases defined as triacyl glycerol acyl hydrolases (EU 3.1.1.3). The unique characteristics and biotechnological potential of lipases have led to their use in food, detergents, pharmaceuticals, etc. industry. Lipolytic enzymes can be isolated from plants, animals, bacteria, fungi and yeasts. In this work we report the synthesis, isolation and purification of an extracellular lipase from Pseudozyma antarctica NBIMCC 8340 using a three-step process. The lipase activity is analyzed by potentiometric and colorimetric method at every stage of the isolation. In order to optimize the synthesis and isolation conditions two separate experiments of fermentation process, test and real one, are realized. The isolation and separation of the lipase in our case are carried out by fractional precipitation with (NH4)2SO4, followed by purification through ultrafiltration using micro concentration tubes Sartorius VIVASPIN 6. The resulting isolate of P. antarctica lipase B is detected by SDS PAGE and analyzed for lipase activity. Preparative isolation of the enzyme in native PAGE is further conducted as a third step aiming to obtain a high purity lipase. Thus a highly purified CalB of a specific activity of 92 U/mg is obtained.Item Co-immobilization of peroxidase and tyrosinase onto hybrid membranes obtained by the sol-gel method for the construction of an optical Biosensor(2013-09-04) Yotova L.; Yaneva S.; Marinkova D.; Serfaty S.The sol-gel method was used to synthesise two groups of hybrid membranes from silica precursors: ETMS (ethyltrimethoxysilane) and MTES (methyltriethoxysilane), and cellulose derivatives. Peroxidase isolated from horseradish and tyrosinase isolated from mushrooms was immobilized by covalent immobilization. The kinetic parameters of the isolated enzymes, pH and temperature optimum were studied. The results of this study showed a higher relative and residual activity of the enzyme when immobilized on membranes containing cellulose acetate propionate with high molecular weight CAP/H. Membranes with the best parameters were used as carriers of the multienzyme peroxidase-tyrosinase system. An optical biosensor with an oxygen electrode was constructed. Potentially, the sensor could be used for monitoring of the environment, detecting phenols in wastewater, food stuff, and in medicine.Item Coimmobilization of acetylcholinesterase and choline oxidase on new nanohybrid membranes obtained by sol gel technology(2012-06-01) Yotova L.; Medhat N.In this study, a multi-enzyme optical biosensor was constructed. Therefore, the first step was synthesis of different kinds of SiO2 hybrid membranes suitable for optical biosensors using sol-gel techniques. The aim of this study was to perform separate covalent immobilization and coimmobilization of the enzymes acetylcholinesterase and choline oxidase and to determine the catalytic properties of the new biocatalyst procedures on new nanohybrid membranes. The catalytic properties of the two enzymes were studied separately and simultaneously. Also, determination of the most suitable membrane for its subsequent application in the optical biosensors is important. This multienzyme membrane will be applied in the design of a fiber optic biosensor for pesticide detection. © 2012 American Scientific Publishers.Item Determination of N-methyl carbamates in a liver sample using an optical biosensor(2019-01-01) Yaneva S.; Stoykova I.; Danalev D.; Yotova L.N-Methyl carbamates are insecticides that inhibit acetylcholinesterase (AChE) causing same symptomatology in acute and chronic exposures. Traces of them could be found in animal tissues, milk, honey, eggs, etc. because of some foods treatment during growth. There is an increasing interest in fast screening methods for detection of different pollutant groups in the foods. Biosensors are a promising alternative of the existing chromatographic methods such as HPLC, GC, etc. They are fast, easy to use and provide fully acceptable values for the monitoring methods like sensitivity, LOD, LOQ, etc. In order to construct an optical biosensor, AChE is immobilized as a target enzyme for N-methyl carbamates on the surface of new membranes synthesized by a sol-gel technology. The designed biosensensor is tested for determination of methomyl, aldicarb, carbofuran, propoxur in liver samples. An appropriate method for sample preparation is also developed. Further, the new method is validated in accordance with document SANTE/11945/2015 covering the required criteria for N-Methyl carbamates determination. The biosensor could detect levels lower than 10 μg/kg which is the maximum residue limit (MRL) for pesticides in foods. Km of 2,2x10-3 M is calculated for acetylcholine using a 4 month life-time of a biosensor.Item Determination of organophosphorus pesticides in liver samples using an optical biosensor(2019-01-01) Yaneva S.; Stoykova I.; Danalev D.; Yotova L.Biosensors are a promising alternative of the existing chromatographic methods as GC, HPLC, etc. They are fast, easy to use and provide fully acceptable values of monitoring like sensitivity, LOD, LOQ, etc. In order to construct an optical biosensor, AChE is immobilized as a target enzyme for organophosphorus pesticides (OPPs) onto new membranes synthesized by a sol-gel technology. The designed biosensensor is tested for determination of chlorpyrifos-methyl, parathion, pirimiphos-methyl and diazinon in liver samples. An appropriate method for sample preparation is also developed. The new method is validated in accordance with document SANTE/11945/2015 covering the required criteria for pesticides determination. The biosensor developed can detect levels lower than 30μg/ kg. The latter is the maximum residue limit (MRL) for pesticides in food. Km of 2,2x10-3M for acetylcholine and 4 months life-time of the biosensor are obtained.Item Effects of the flow type on the immobilization of Horseradish peroxidase on polymers(2018-01-01) Yaneva S.; Velinov T.; Yotova L.This paper presents the investigation of Horseradish peroxidase (HPR) immobilization on poly-(acrylonitrile- co-acrylamide) and polyamide membranes using three different regimes of a fluid movement: a turbulent, a laminar and an intermediate one. The turbulent movement of the liquid is realized through a magnetic stirrer application. The laminar flow is achieved in a microfluidic system, while the attached amount of enzymes is monitored by a quartz crystal microbalance (QCM). The third type of movement, a fluid movement in an inclined pipe with a varied angle of inclination, is realized in a laboratory made rig. We find that the type of flow influences the immobilized enzyme amount, the time of the immobilization and the structure of the immobilized film. The main conclusion is that the laminar flow favours the process of covalent immobilization most probably because of the orientation of the molecules and their movement parallel to the carrier surface.Item Hydrolysis of wine proteins by means of pepsin, immobilized to ultra-fil-tration membranes(1993-01-01) Karasavova M.; Krysteva M.; Shopova B.; Yotova L.Pepsin is covalentlyimmobilized via hydroxymethyl groups to ultrafiltration membrane, composed of acrylonitrile and acrylamide copolymer. The properties of the immobolized enzyme showed high relative activity of 75%, pH optimum at pH 2.3 and temperature optimum at 50°C. Isolated wine proteins were treated with immobilized pepsin. This treatment shows not the production of small fragments in limited proteolysis conditions. The bound pepsin to ultra-filtration membrane by hydroxymethyl groups has considerable adventages for protein hydrolysis in comparison with the method used till now: one step activation of membrane with formaldehyde; high relative activity of immobilized enzyme; controlled degree of hydrolysis; no introduction of additional substances in hydrolysates. © 1993 Taylor & Francis Group, LLC.Item Influence of Ni2+ on urease activity produced by biofilms of Arthrobacter oxydans 1388(2014-01-01) Marinkova D.; Yotova L.; Ringeard J.; Griesmar P.New TiO2-based hybrid materials composed of an organic polymer, cellulose acetate butyrate and copolymer of acrylonitrile acrylamide (AN + AA) were prepared. The effectiveness of immobilization of microbial strain Arthrobacter oxydans 1388 on the newly synthesized hybrid membranes was investigated by biochemical methods. The obtained results revealed that the matrix more suitable for biofilm formation was composed of organic polymers without a metal component in the membrane composition. The influence of Ni2+ on urease activity produced by biofilms was investigated. The experimental results demonstrated that 2 mg L -1 concentration of Ni2+ in the nutrient medium is more appropriate for biofilm proliferation. © 2014 The Author(s). Published by Taylor & Francis.Item Investigation of different enzyme activities from Pergularia tomentosa L. and Ecballium elaterium L.(2016-01-01) Imen L.; Yotova L.Plant enzymes are derived from a variety of plants and are effective within a broad pH range. They have a greater activity and a higher stability than those of similar animal or microbial based enzymes. They are breaking down more proteins, fats, and carbohydrates in the broadest range of conditions than other sources. Many enzymes like proteinases, beta-amylase, lipoxygenase, L-asparaginase, polyphenol oxidase, tyrosinase, lipase are synthesized in plants. The present study is undertaken to assess the presence of the enzymes pointed above and their quantities in aqueous/ethanol extracts and in latex of two plants applied in medicine. The results obtained reveal presence of ß-amylase, proteolytic, pepsinogene, lipoxygenase, L-asparaginase, polyphenol oxidase, tyrosinase and lipase in aqueous extracts. The latex shows no presence of lipoxygenase but a good milk clotting activity is observed in case of Pergularia tomentosa L. However, Ecballium elaterium L. shows the same activities at a low concentration but no milk clotting activity.Item Investigation of formation, development and application of arthrobacter oxydans 1388 biofilm(2009-01-01) Yotova L.; Marinkova D.; Mironova V.; Dulgerova R.Biofilms are microbial communities attached to different kinds of surfaces or associated with interfaces. These microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. Bioremediation is an innovation technology, which controls the pollutants, using biological systems for degradation or transformation of different toxic compounds Taking in consideration the efficiency of the bioremediation by means of different microbial strains the aim of this study is to investigate the creation and application of biofilms for wastewater treatment. The dynamics of the formation of a biofilm from cells of strain Artrobacter oxydans 1388 on newly synthesized different carriers was investigated by scanning electron microscopy (SEM). Extracellular polymeric substances (EPS) are biopolymers of microbial origin in which biofilm microorganisms are embedded. Exopolysaccharides and proteins are one of the main factors in biofilm formation, evidence for microenviromental changes of microbial cells. In this study the biofilm growth and characteristics are researched by biochemical methods of investigation of extacellular polymeric substances. The microbial cells are immobilized by two methods—covalently binding and adhesion to the different polymeric matrixes. Matrixes were obtained on the base of copolymer of acrylonitrile with acrylamide and mixed with cellulose acetate butyrate. In this case they were with high mechanical stability. Two methods of immobilization were applied. The obtained results demonstrated that better and more effective was the covalent binding. © 2009 Taylor and Francis Group, LLC.Item Investigation on isolated and purified lipoxygenase from avocado in the presence of linolenic acid(2015-01-01) Manovski L.; Semedzieva V.; Yotova L.Lipoxygenase (LOX) is an enzyme that is found in many plants and animals, which catalyses the oxygenation of polyunsaturated fatty acids (PUFA) to form fatty acid hydroperoxides. Linoleic and linolenic acid are the major polyunsaturated fatty acids in plant tissues, and insertion of the oxygen takes place at either the 9th or the 13th position to generate the corresponding 9- or 13-hydroperoxides. Characterization of avocado LOX offers the potential of increasing scientific knowledge that can aid in the establishment of optimum processing and storage conditions at which the detrimental effects of this enzyme are minimized, preventing product organoleptic changes and nutritional quality losses. The determination of other catalytic properties of avocado LOX such as the ability to co-oxidize carotenoids may also help to promote the use of the avocado enzyme as a bleaching agent in the food industry. In the laboratory, lipoxygenase from avocado was isolated, purified and characterized. After processing of the results from the experiments, the value for the enzyme activity is 60.7287 U/ml in the presence of a substrate at concentration of 3 mM. pH and temperature optimums are 6,5 and 40 oC. The kinetic parameters obtained in coordinates Lineweaver-Burk at different concentrations of substrate, have values as follows: 0.0248.10-6 M; Vmax = 0.745. 10-6 M/mg min.Item Kinetics of the biodegradation of phenol in wastewaters from the chemical industry by covalently immobilized Trichosporon cutaneum cells(2009-03-01) Yotova L.; Tzibranska I.; Tileva F.; Markx G.; Georgieva N.A simple method for the preparation of the biocatalyst with whole cells is presented, and the applicability of the technique for biodegradation of phenol in wastewater from the chemical industries using the basidomycetes yeast Trichosporon cutaneum is explored. Kinetic studies of the influence of other compounds contained in wastewater as naphthalene, benzene, toluene and pyridine indicate that apart from oil fraction, which is removed, the phenol concentration is the only major factor limiting the growth of immobilized cells. Mathematical models are applied to describe the kinetic behavior of immobilized yeast cells. From the analysis of the experimental curves was shown that the obtained values for the apparent rate parameters vary depending on the substrate concentration (μmaxapp from 0.35 to 0.09 h-1 and K sapp from 0.037 to 0.4 g dm-3). The inhibitory effect of the phenol on the obtained yield coefficients was investigated too. It has been shown that covalent immobilization of T. cutaneum whole cells to plastic carrier beads is possible, and that cell viability and phenol degrading activity are maintained after the chemical modification of cell walls during the binding procedure. The results obtained indicate a possible future application of immobilized T. cutaneum for destroying phenol in industrial wastewaters. © 2008 Society for Industrial Microbiology.Item Lc-ms quantification of thyroid hormones, their metabolites, amino acids and neurotransmitters in liver tissue(2009-01-01) Zanov K.; Yotova L.; Klapa M.Thyroid hormones are involved in the regulation of various biological processes: growth and development, carbohydrate metabolism, oxygen consumption, protein synthesis and fetal development. The actions of thyroid hormones affect the levels of wide range of different enzymes (mainly oxidoreductases) that are involved in the metabolism in liver, heart, and brain. These enzymes influence metabolic reactions involving carbohydrates, steroids, lipids and vital signaling proteins. Moreover, there is sufficient information in the literature about non-genomic action of thyroid hormones. Apart from the thyroid hormones T4 and T3 other iodothyronine metabolites also exhibit influence over the metabolisms of the tissues. Reverse T3, similarly to T4 has an inhibiting effect over the deiodinase involved in the synthesis of T3. Diiodothyronines have been reported to influence the activity of different enzymes: oxidative in the mitochondria, deiodinase in the pituitary glands, and glucose 6-phosphate dehydrogenase in the liver. We have developed an analytical method for simultaneous quantification of thyroid hormones, their metabolites, amino acids and organic acids present in rat liver. The analysis is carried out with the aid of a liquid chromatograph with an ion-trap mass detector. Two main emphasizes in our work were sample preparation and the choice of internal standard. This was inevitable, due to the fact that our goal was to conduct a high-throughput analysis of the small metabolites and the relatively high- molecular thyroid hormones. © 2009 Taylor and Francis Group, LLC.Item Production and chracterization of lipase from pseudozyma antarctica NBIMCC 8340(2015-01-01) Borisov B.; Manova D.; Marinkova D.; Yotova L.; Danalev D.Lipases are serine hydrolases, defined as triacyl-glycerol acylhydrolases. They catalyze both hydrolytic and synthetic reactions showing substrate, regio- and stereo selectivity. The psyhrophilic strain Pseudozyma antarctica, isolated in the cold conditions of the Antarctica, can produce a thermostable extracellular lipase. In this work we report the synthesis of an extracellular lipase from Pseudozyma antarctica. The strain was cultured in flasks and various parameters were monitored during the proliferation process. Several cultivation procedures were done in order to find optimal conditions. Further, activity of the crude lipase, its temperature and pH optimum were determined using a potentiometric method. We found temperature optimum at 60°C and pH optimum at pH 8.0. The determined specific activity of the crude lipase was 97,2 U/mg. Activity and protein content of the ``crude`` post cultivation liquid were also determined.Item Simultaneous immobilization of uricase and peroxidase to copolymer of acrylonitrile with acrylamide(2002-01-01) Ivanov I.; Yotova L.A method for individual and simultaneous covalent immobilization of uricase and peroxidase to copolymer of acrylonitrile with acrylamide was presented. The immobilized enzymes revealed slight change in their pH optima and an appreciable increase in their temperature optima, Km and activation energy compared to data for the soluble enzymes. A substrate inhibition of the soluble and immobilized peroxidase at high substrate concentrations was established. A little glass column packed with immobilized enzymes was used for manual uric acid determination in blood sera. The method was characterized by high analytical precision and reproducibility. The developed method is economical (the enzyme-carrier conjugate may be used more than 200 times) and easier to perform than the manual methods utilizing soluble enzymes. The established manual method for uric acid determination in biological fluids is suitable for clinical laboratory diagnostics and for work in small clinical laboratories. © 2002 Taylor and Francis Group, LLC.Item Synthesis and investigation of the properties of hybrid materials for enzyme immobilization(2018-01-01) Yaneva S.; Semerdzhieva V.; Raykova R.; Marinkova D.; Chernev G.; Iliev I.; Yotova L.The chemical nature of carriers for enzyme immobilization plays an important role for retention of the enzyme activity and stability. Two new materials are synthesized for enzyme immobilization based on trimethoxy silane/cellulose acetate butyrate/poly (amido amine) dendrimers (TMOS/CAB/PAMAM) and methyltriethoxy silane/cellulose acetate propionate/ poly (amido amine) dendrimers (MTES/CAP/PAMAM). The synthesis is carried out via the sol-gel method, which allows the preparation of porous glasses through hydrolysis and poly-condensation at a low temperature using high purity initial materials. The PAMAM dendrimers are mono-dispersive, well defined and have a developed three dimensional structure of a high functional groups concentration. The obtained materials are used to investigate the properties of immobilized enzymes such as lipoxygenase and laccase. These enzymes are widely used in industry as bleaching agents. There is also data on application of laccase and lipoxygenase in preparation of biosensors for toxic pollutants determination. Biosensors with immobilized laccase are used to determine phenolic compounds, whereas immobilized lipoxygenase is applied to biosensors formulation for determining Aflatoxin B1 presence.Item Valorization of a plant β-amylase: Immobilization and dataset on the kinetic process(2018-02-01) Lahmar I.; Radeva G.; Marinkova D.; Velitchkova M.; Belghith H.; Ben Abdallah F.; Yotova L.; Belghith K.The data presented in this article are related to the research article titled “Immobilization nd topochemical mechanism of a new β-amylase extracted from Pergularia tomentosa” (Lahmar et al., 2017) [1]. This article documented information on the determination of the molecular weight of the β-amylase, the method of its immobilization and a comparison of the kinetic mechanism between the free and the immobilized forms by a mathematical method. Fresh Pergularia tomentosa was collected from Tunisia and a special method for β-amylase extraction was followed (Yotova et al., 2000) [2]. Public dissemination of this dataset will allow further analyses of the data.