Browsing by Author "Krysteva M."
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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 Crystallization of type I chloramphenicol acetyltransferase: An approach based on the concept of ionic strength reducers(2000-01-01) Andreeva A.; Borissova B.; Mironova R.; Glykos N.; Kotsifaki D.; Ivanov I.; Krysteva M.; Kokkinidis M.Chloramphenicol acetyltransferase (CAT) is responsible for bacterial resistance to chloramphenicol. It catalyzes inactivation of the antibiotic by acetyl-group transfer from acetyl CoA to one or both hydroxyl groups of chloramphenicol. Type I CAT possesses some unique properties which are not observed in other CAT variants. Type I CAT overexpressed in Escherichia coli was purified and crystals with a resolution limit of 2.22 Å have been obtained using a novel procedure which is based on the concept of 'ionic strength reducers'. The crystals have the symmetry of space group P1 and unit-cell parameters a = 96.46, b = 113.86, c= 114.21 Å, α= 119.9, β= 94.1, γ = 98.6°. These dimensions are consistent with four to six trimers per unit cell, corresponding to a solvent fraction ranging from 65 to 47%.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 Immobilization of zinc phthalocyanines in silicate matrices and investigation of their photobactericidal effect on E. coli.(2006-01-01) Artarsky S.; Dimitrova S.; Bonnett R.; Krysteva M.The aim of the present investigation was to immobilize zinc phthalocyanines in a silicate matrix and to test the photobactericidal properties of the matrices so prepared toward Esherichia coli in model aqueous media. For the purpose, tetra tertiary butyl zinc phthalocyanine (TBZnPc) and zinc phthalocyanine tetrasulfonic acid (ZnPcTS) were used. The abilities of these two photosensitizers to generate singlet oxygen in solution were compared by following the rate of photobleaching of 1,3-diphenylisobenzofuran (DPBF) at 430 nm in dimethylformamide (DMF). The results of this study show clearly that, under the conditions used here, the TBZnPc is the more effective generator of singlet oxygen; with it the DPBF was virtually completely photobleached in 4 min, while with the ZnPcTS under the same conditions, it took 12 min to reach this point. Glass conjugates with the two phthalocyanines were obtained by the sol-gel technique and were characterized by a well-defined color due to the phthalocyanine incorporated in the silicate matrix. Glasses with an intense, but inhomogeneous, green color were obtained when the tetrasulfonic derivative of the zinc phthalocyanine was used, while blue glasses of evenly distributed coloration were formed from the tetra tertiary butyl derivative. The ZnPcTS conjugate demonstrates more effective singlet oxygen evolution than is the case with the TBZnPc conjugate. These results are the opposite of those obtained for the free phthalocyanines in solution. The structural formulae of the compounds show that TBZnPc has a more pronounced hydrophobic character than the sulfonic derivative. In our view, the relative reactivities of the conjugates can be explained by the tetrasulfonic derivative being situated mainly in the surface parts of the glass matrix where the hydrophilic character is prevailing, while the tertiary butyl derivative is mainly present in the internal parts of the matrix as a result of which it is less accessible and therefore less active. The results obtained on the effect of zinc phthalocyanine conjugates on E. coli show a trend similar to that observed with singlet oxygen evolution shown. Thus, for the ZnPcTS conjugate, the log kill is 1.32 and for the TBZnPc conjugate, it is 0.98, in each case after 120 min. The results obtained show that phthalocyanines can be immobilized successfully in a silicate matrix and used for photodisinfection of microbially polluted waters. The silicate matrix has some advantages in comparison with other organic matrices. It is insoluble in water, resistant towards microorganisms, easy to fabricate, and might be developed successfully for the photodisinfection of water, e.g., in swimming pools and in other open water reservoirs.Item Investigation of the Active Center of Porcine‐Pancreatic Amylase(1972-01-01) Elödi P.; Móra S.; Krysteva M.The effect of maltose was studied in porcine pancreatic amylase. At neutral pH 1% (29 mM) maltose produced with amylase a difference spectrum characteristic of the perturbation of tryptophan. The molar absorption difference at the maximum wavelength was Δ290= 1200. The difference spectrum appeared to be specific for maltose. Perturbation difference spectra measurements in 20% polyethylene glycol indicated that one tryptophyl side chain per mol amylase was involved in the interaction with maltose. The dissociation constant of the amylase · maltose complex calculated from the concentration dependence of the absorption difference at 290 nm was Ks= 13 mM. Maltose inhibited amylase activity competitively and an inhibition constant of Ki= 25 mM was obtained, a similar value to that found spectrophotometrically. It is assumed that the tryptophyl side chain interacting with maltose may be involved in the binding of substrate by pancreatic amylase. Copyright © 1972, Wiley Blackwell. All rights reservedItem Modelling of a continuous process for a conversion of d-sorbitol to L-sorbose by gluconobacter oxydans(1991-01-01) Tchaoushev S.; Nicolov N.; Krysteva M.A mathematical model of a multistage bioreactor for continuous conversion of D-sorbitol to L-sorbose has been developed. The model solution presents possibility for determining the biomass concentration along the reactor height and the required number of stages for achievement of a given conversion rate. The mathematical model was tested on the basis of experimental data obtained in a laboratory glass reactor. The experimental conversion rate of D-sorbitol to L-sorbose rose to approximately 37% in the seconnd stage of the laboratory bioreactor. The value was in good agreement with the numerical results from the solution of the model. On the basis of these investigations it may be assumed that full conversion of D-sorbitol to L-sorbose should be obtained in bioreactor with 5-6 stages. © 1991 Taylor & Francis Group, LLC.Item Modification of chitosan and possibilities of its application(1995-01-01) Krysteva M.; Naidenova E.; Andreeva A.; Huyen N.D.A modification of amino groups of chitosan were performed in two directions: treatment of vicinal amino and hydroxyl groups by periodate and subsequent interaction with urea and formaldehyde in order of covalent immobilization of enzymes; alkylation of chitosan amino groups for increasing the positive charge and utilization of polymer as an anion exchanger. The binding of enzymes: lipase, trypsin, penicillin amidase and glucose oxidase to the activated matrix was done at pH 4.0, 5.0 and 3.5 respectively. The immobilization of lipase was performed at pH 4.0 and 8.0. The immobilized enzymes were characterized by their pH optimum and relative enzyme activity. The alkylation of the amino groups has been achieved by varying the modifying agent (formaldehyde in the presence of formic acid) to the chitosan. The ion-exchange properties of the ionite obtained was verified by means of ovomucoid, a protein with improved acidic characteristics. © 1995 Taylor & Francis Group, LLC.Item Optical enzyme sensor for urea determination via immobilized pH indicator and urease onto transparent membranes.(2003-01-01) Krysteva M.; Al Hallak M.Transparent triacetylcellulose membranes with immobilized pH indicator (neutral red) as well as with simultaneously immobilized urease and neutral red were used as optical sensors for determination of urea concentrations in model solutions. Decomposition of urea with the enzyme urease is accompanied by evolution of ammonia. This leads to the changes of the neutral red absorption, which is proportional to the substrate (urea) within certain concentration limits in model solution. As a result of the investigation, standard curves were plotted for determination of urea over the range of 1 to 500 mM using immobilized indicator and free urease. Simultaneous immobilization of indicator and urease permitted determination of urea in the interval 50 to 500 mM. The membrane used contained 0.169 U urease activity on an area of 1.7 cm2. The standard curves were plotted using the linear region of the kinetic curves for the corresponding substrate concentrations. A possible scheme of the interaction between the activated triacetylcellulose membrane and the indicator and enzyme is proposed. The membranes obtained are suitable for repeated ecological applications where urea is to be determined.Item Studies on Tyrosine Environments of Chicken Ovomucoid: The Environment of the Most‐Exposed Tyrosine(1973-01-01) Krysteva M.; Mancheva I.; Dobrev I.The particular environments of tyrosine side chains were suggested to be responsible for the non‐denaturation acidic difference spectra of ovomucoid as well as of abnormal ionization of its phenolic groups. The micro environment of the most exposed tyrosine in ovomucoid was studied. The tyrosine was first labelled by means of tetranitromethane. Nitrotyrosine peptide was isolated. The study of the nine steps of nitrotyrosine peptide degradation results in the following amino acid sequence: Ala‐Gly‐Ala‐Lys‐Thr‐Gln‐NO2Tyr‐Gly‐Gly. The analysis of neutral sugars as well as glycosamine gave negative results. The presence of sialic acid however was detected. On the basis of evidence obtained, the existence of a glycosidic bond formed from the hydroxyl group of tyrosine and the glycosidic hydroxyl group of sialic acid was suggested. The spectral behaviour of the tyrosine studied was discussed in the light of above assumption. Copyright © 1973, Wiley Blackwell. All rights reserved