[Cephalosporin-Acid Synthetase of Escherichia coli Strain VKPM B-10182: Genomic Context, Gene Identification, Producer Strain Production].

An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of ß-lactam antibiotics of the cephalosporin acids class (cefazolin, cefalotin, cefezole etc.). A comparison of the previously determined genomic sequence of E. coli VKPM B-10182 with a genome of the parent E. coli strain ATCC 9637 was performed. Multiple mutations indicating the long selection history of the strain were detected, including mutations in the genes of RNase and ß-lactamases that could enhance the level of enzyme synthesis and reduce the degree of degradation of the synthesized cephalosporin acids. The CASA gene--a direct homolog of the penicillin G-acylase gene--was identified by bioinformatics methods. The homology of the gene was confirmed by gene cloning and the expression and determination of its enzymatic activity in the reaction of cefazolin synthesis. The CASA gene was isolated and cloned into the original expression vector, resulting in an effective E. coli BL2l(DE3) pMD0107 strain producing CASA.

[The use of stock Sorbfil plates for the analysis of antibiotics and their stock drug forms]. / Ispol'zovanie gotovykh plastin "Sorbfil" dlia analiza antibiotikov i ikh gotovykh lekarstvennykh form.

Chromatographic plates Sorbfil (Krasnodar PKB Plastmash) of various modifications were shown to be useful in solving a wide range of analytical problems, as well as in chromatographic separation of substances belonging to different classes i.e. antibiotics, lipids and steroids. The Sorbfil plates were used to authenticate antibiotic-containing drugs, to determine the content of admixtures and to analyze antibiotic-containing dosage forms.

[A spectrophotometric method of analysis of fusidic acid]. / Spektrofotometricheskii metod opredeleniia fuzidievoi kisloty.

A spectrophotometric method for assay of fusidic acid is described. The method is based on reaction with a reagent consisting of acetic anhydride and concentrated sulfuric acid. Mathematical processing of the results of the main substance determination in fusidic acid preparations showed that the error did not exceed 2 per cent. Procedures for spectrophotometric assay of fusidic acid in control of the processes of its biosynthesis, isolation and purification were developed. The procedures provided control of the technological process of fusidic acid production.

[Determination of iodine-containing admixtures in semisynthetic penicillins]. / Opredelenie iodsoderzhashchikh primesei v nekotorykh polusinteticheskikh penitsillinakh.

Dependence of the values defining the content of iodine sorbing admixtures in semisynthetic penicillins on pH, reaction time and drug aliquots was studied. On the basis of this study a general approach to development of procedures for determining iodine sorbing admixtures in semisynthetic penicillins was suggested. Procedures for determination of iodine sorbing admixtures in carbenicillin, carfecillin, ampicillin, oxacillin, azlocillin and ampiox were developed.

[Penicillinase from B. licheniformis. Determination of the ionization constants of the enzymatic hydrolysis products of beta-lactam antibiotics]. / Penitsillinaza iz 8. licheniformis. Opredelenie konstant ionizatsii produktiv fermentativnogo gidroliza betalaktamnykh antibiotikov.

The level of transformation of beta-lactam antibiotics hydrolysed by penicillinase from B. licheniformis on determination with the method of pH-metric titration with sodium hydrate solutions depended on the electrochemical nature of the products formed. The data on the study of the pH dependence of the penicillinase-catalysed hydrolysis of beta-lactam antibiotics were used for estimation of the ionization constants of the products of the enzymatic hydrolysis of the beta-lactam ring in the molecules of azlocillin, carfecillin, benzylpenicillin, cephalothin and 7-PADCA. Methods for quantitative determination of the compounds were developed. The methods are based on penicillinase-catalysed enzymatic hydrolysis and pH-metric titration of the products with sodium hydrate solutions at pH 7.0 with regard to their dissociation levels.

[Extractive polarographic method of controlling the stereospecific hydrolysis of N-acylated derivatives of amino acid racemates]. / Ekstraktsionno-poliarimetricheskii metod kontrolia stereospetsificheskogo gidroliza N-atsilirovannykh proizvodnykh ratsematov aminokislot.

The equilibrium distribution of acetyl-D-phenylglycine prepared with stereospecific hydrolysis of acetyl-D,L-phenylglycine (Ac-D,L-PhG) was studied in a two-phase system of benzyl alcohol-1N HCl solution. It was found that the distribution coefficient of Ac-D-PhG did not practically depend on its concentration in the aqueous phase. This means that Ac-D-PhG does not form associates in the organic phase. The studies provided development of an extraction polarimetric method for quantitative determination of Ac-D-PhG. The method implies extraction of Ac-D-PhG with benzyl alcohol from a strong acid medium (1N HCl solution), polarimetric determination of the equilibrium concentration of Ac-D-PhG in the organic phase and subsequent calculation of the initial concentration of Ac-D-PhG in the aqueous phase with regard to its distribution coefficient.

[E. coli penicillin amidase. Methods for estimating the close ionization constants of ionogenic groups of the enzyme complex with substrates containing free amino groups]. / Penitsillinamidaza iz E coli. Metody rascheta blizkikh konstant ionizatsii ionogennykh grupp kompleksa fermenta s substratami, soderzhashchimi svobodnye aminogruppy.

The possible use of various procedures for estimation of the ionization constants of the Michaelis complex by the pH dependence of the maximum enzymatic reaction rate is discussed. It is shown that the procedures described in the literature for estimation of the close ionization constants of the enzyme-substrate complexes have limitations and in some cases cannot be used. The paper presents the methods for estimation of the constants and means for quantitative description of the bell-shaped pH dependence of the kinetic and equilibrium parameters of the biocatalytic reaction. The equations recommended in the paper were used in analysis of the pH dependences of the maximum rate of the reactions during the enzymatic synthesis of cefalexin catalysed with immobilized penicillinamidase (IPA) (CE 3.5. 1.11). The ionization constants of the enzyme-substrate complexes of IPA were compared during hydrolysis and synthesis of the compounds acylated with phenylacetic and aminophenylacetic acids. The effect of the nature of the leaving substrate group and added nucleophilic gent on the electrochemical state of the Michaelis complex is discussed.

[Penicillin amidase from E. coli. The kinetic and equilibrium parameters of the enzymatic hydrolysis of 7-phenylacetamidodesacetoxycephalosporanic acid catalyzed by an immobile enzyme]. / Penitsillinamidaza iz E. coli. Kineticheskie i ravnovesnye parametry fermentativnogo gidroliza 7-fenilatsetamidodezatsetoksitsefalosporanovoi kisloty, kataliziruemogo immobilizovannym fermentom.

The kinetics of 7-phenylacetamidodesacetoxycephalosporanic acid (7-PADCA) catalyzed by immobilized penicillinamidase was studied. The kinetic and equilibrium parameters of the reaction were determined by analysis of the kinetic curves of the reaction product accumulation. Inhibition of the enzymatic reaction by the substrate and hydrolysis products was studied. It was found that the Michaelis complex completely lost its activity after attachment of the substrate second molecule to it. The values of the Michaelis constants, catalytic constant and constants of inhibition by the substrate and reaction products were determined: Km = (9.3 +/- 1.1) . 10(-5) M, kcat = (65 +/- 5) c-1, Ks = (1.4 +/- 0.1) . 10(-2) M, K1 (FAA) = (2.5 +/- 0.3) . 10(-4) M, K1 (7-ADCA) = (1.4 +/- 0.1) . 10(-1) M. The diffusion effect in the kinetic reaction catalyzed by immobilized penicillinamidase is discussed. The values of the Thiele modulus and the actual value of Km were calculated.

[Equilibrium distribution of weak electrolytes in an organic solvent-water system]. / Ravnovesnoe raspredelenie slabykh élektrolitov v sisteme organicheskii rastvoritel'-voda.

Distribution of cephalotin, 2-tienylacetic acid, phenacetyl-D-(--)-alpha-aminophenylacetic acid, weak acids and D-(--)-alpha-amino-phenylacetic acid methyl ether, a weak base in two-phase systems containing two immiscible liquid phases was studied. The constants of the intraphase and interphase equilibrium necessary for complete quantitative description of the system of electrolyte aqueous solution-organic solvent were determined.

[Extraction and polarimetric method of determining phenacetyl-D(-)-alpha-aminophenylacetic acid]. / Ekstraktsionno-poliarimetricheskii metod opredeleniia fenatsetil-D(-)-al'fa-aminofeniluksusnoi kisloty.

Equilibrium distribution of phenacetyl-D-(--)-alpha-aminophenylacetic acid obtained on fermentative hydrolysis of D,L-aminophenylacetic acid in a two-phase system (chloroform-water) was studied within a wide range of the substance concentration in the organic phase. It was shown than in the organic phase the distributing substance formed associates. The empirical equation: y = A + Bx describing the dependence of the effective distribution of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid on its concentration in the organic phase was suggested. Coefficients A and B of the equation were determined and an equation for evaluating equilibrium concentrations in the equeous phase was suggested. On the basis of the studies an extraction-polarometric method for quantitative determination of phenacetyl-D-(--)-alpha-aminophenylacetic acid concentration was developed. The method consists of extraction of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid with chloroform, determination of the equilibrium concentration of the distributing substance in the organic phase by the polarimetric method and subsequent estimation of the equilibrium and initial concentrations of the electrolyte in the aqueous phase.

[Quantitative determination of the gentamicin and dimethyl sulfoxide in the "Gentaplast" film-forming aerosol preparation]. / Kolichestvennoe opredelenie gentamitsina i dimetilsul'foksida v plenkoobrazuiushchem aérozol'nom preparate "Gentaplast".

A method for quantitative determination of gentamicin and dimethylsulfoxide (DMSO) in "Gentaplast", a film-forming aerozol preparation was developed. Gentamicin was determined microbiologically after extraction. DMSO was determined by titration with the use of the Redox method. The results of determination of gentamicin and DMSO in "Gentaplast" were statistically treated.

[Determination of levomycetin in the aerosol preparation, "Levovinysol"]. / Opredelenie levomitsetina v aérozol'nom preparate "Levovinizol"

A spectrophotometric method for determination of levomycetin in "Levovinysol", an antiburn aerosol preparation was developed. The determination was performed after levomycetin extraction with a hydrochloric acid solution from the composition. The results of the determination were statistically treated.

[Determination of neomycin in the film-forming aerosol preparation "Neotizol'"]. / Opredelenie neomitsina v plenkoobrazuiushchem aérozol'nom preparate "Neotizol'"

A colorimetric method for guantitative determination of neomycin in the aerozol preparation "Neotizol" was developed. The determination was performed after neomycin isolation from the film-forming composition with the carboxylic ion exchange resins IRC-50 and KB-2 containing 2.5-3 per cent of divinylbenzol. The results of the colorimetric determination were compared with the data of the microbiological assay. The results of neomycin determination in the aerozol preparation "Neotizol" were treated statistically.