An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of β-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 β-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 BL21(DE3) pMD0107 strain producing CASA.

Фермент синтетаза цефалоспоринов-кислот продуцируется штаммом E. coli ВКПМ В-10182, обладает специфичностью к синтезу -лактамных антибиотиков, относящихся к классу цефалоспоринов-кислот (цефазолин, цефалотин, цефтезол и др.). Проведено сравнение ранее расшифрованной геномной последовательности штамма E. coli ВКПМ В-10182 с геномом родительского штамма E. coli ATCC 9637. Выявлены множественные мутации, свидетельствующие о долгой селекционной истории штамма, в том числе мутации в генах РНКаз и -лактамаз, которые могли способствовать повышению уровня синтеза фермента и снижению степени деградации синтезируемых цефалоспоринов-кислот. Методами биоинформатики идентифицирован ген CASA – прямой гомолог гена пенициллин G-ацилазы, что подтверждено результатами клонирования гена, его экспрессии и определения ферментативной активности в реакции синтеза цефазолина. Ген CASA выделен и клонирован в оригинальный вектор экспрессии, в результате чего получен эффективный штамм E. coli BL21(DE3)/pMD0107 – продуцент CASA.

Recombinant, as well as native alpha-amino acid ester hydrolase from Xanthomonas rubrilineans VKPM B-9915 (XrAEH, EC 3.1.1.43), was tested for synthesis of amino-beta-lactam antibiotic cephalexin. It was shown that the recombinant enzyme r-XrAEH produced by Escherichia coli VKPM B-11246 is more efficient in comparison with the native enzyme wt-XrAEH prepared from mutant strain Xanthomonas rubrilineans VKPM B-9915. When r-XrAEH was used as a biocatalyst, addition of ethylene glycol (33 vol %) to the reaction medium improved the yield from 70 to 95%. During synthesis of cephalexin under optimal conditions in the case of the native enzyme wt-XrAEH the cephalexin yield was 85%, in contrast to r-XrAEH where it was 95%. Furthermore, unlike native wt-XrAEH enzymes, preparations of recombinant r-XrAEH do not possess beta-lactamase side activity.

In order to use hydroxyapatite and other biocompatible calcium phosphates as carriers for local prolonged-release drug, it is necessary to have an active component capable of retaining the preparation deposited onto the carrier surface for a long time (at least, for a few days). The grafting of the layer of active functional groups onto the carrier surface is employed to affect kinetic adsorption-desorption characteristics of calcium phosphates. The method of chemical modification of biocompatible calcium phosphates that uses B(OC4H9)3, SiCl4, POCl3, PCl5, and SnCl4 is developed. The effect of the modification of calcium phosphate’s surface on the kinetics of hydrolytic desorption of octadecylamine and tetraethylenepentamine modeling the hydrophobic and hydrophilic drugs, respectively, as well as gentamicin, an antibiotic that is widely used in clinical practice to prevent and cure inflammation processes upon the damage of bony tissue, is studied. It is shown that the rate of desorption of these substances from calcium phosphates into the aqueous phase is significantly retarded when the carrier surface is preliminarily modified. By means of ESR and IR Fourier spectroscopy, the interaction of octadecylamine and tetraethylenepentamine with calcium phosphate’s surface is investigated. A conclusion about the existence of two mechanisms of adsorption of amines on modified calcium phosphates is drawn. The procedure for the preparation of biocompatible calcium phosphates with hemisorbed gentamicin is developed.

The methodology for the discovery of new biologically active betalactams is proposed. The one of the two ways proposed is specific modification, which is peculiar to any betalactam structure and involves introduction of substitutes changing particular physico-chemical properties of the natural or synthetic analogous. The principle of similarity is discussed as an alternative of the specific modification in the design of new biologically active compounds. The distinctive feature of this approach is usage as building blocks substitutes favourable for the well-known in medical practice betalactam antibiotics and their “implantation” into other (new) betalactam structures. The paths of new betalactam synthesis, including the methods of enzyme engineering are considered. The possibility of usage of enzyme engineering processes for production of not only new individual betalactams— hits, but also for synthesis of the complex betalactams—leads, is shown. More than 4500 new penicillins, cephalosporins and monobactams are constructed with accordance of the principle of similarity. More than halves of them can be produced by enzymatic synthesis or combination of chemical and enzymatic synthesis. The constructed compounds are enumerated in the tables, the request for the electronic version of which can be sent to the address: davidnys@writeme.com.

in the design of new biologically active compounds. The distinctive feature of this approach is usage as building blocks substitutes favourable for the well-known in medical practice betalactam antibiotics and their \implantation" into other (new) betalactam structures. The paths of new betalactam synthesis, including the methods of enzyme engineering are considered. The possibility of usage of enzyme engineering processes for production of not only new individual betalactams| hits, but also for synthesis of the complex betalactams|leads, is shown. More than 4500 new penicillins, cephalosporins and monobactams are constructed with accordance of the principle of similarity. More than halves of them can be produced by enzymatic synthesis or combination of chemical and enzymatic synthesis. The constructed compounds are enumerated in the tables, the request for the electronic version of which can be sent to the address: davidnys@writeme.com.

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 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.

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.

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.

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.

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.