An optimal way to design an enzymatic process for the production of betalactam antibiotics based on thermodynamic and kinetic studies is described. The study was performed on model reactions involving synthesis of cephalosporin-acids (cephalotin, cefazolin, cefoxitin) using immobilised cephalosporin-acid synthetase from Escherichia coli as biocatalyst, and aminocephalosporins (cephalexin) using immobilised cells of Xanthomonas rubrilineans containing the aminocephalosporin synthetase. The possibility of direct synthesis of cephalotin and cefoxitin was shown, the main equilibrium parameters were determined and the operation conditions were evaluated. The maximum key amino acid conversion to product of approximately 90% for cefoxitin and cephalotin was achieved using initial concentrations of the corresponding key amino acids of 0.05 u M and, respectively, 2-fold and 4-fold molar excess of the carboxylic acids. Cefazolin and cephalexin production by enzymatic synthesis with using of corresponding biocatalyst with a mechanism of action involving the acylenzyme intermediate was shown possible. The kinetic parameters of the process were estimated and the relationship between the maximum antibiotic yield and the initial concentrations of the substrate and nucleophile in the kinetically controlled synthesis was determined. The technologies for cefazolin and cephalexin enzymatic synthesis were designed and the cefazolin technology was optimised. Maximum yields of cefazolin and cephalexin of more than 90% were predicted by the kinetic model using 4-6-fold molar excess of the acylating agents and maximum yields of approximately 85% were achieved in experiments.

The methology of the development of new biologically active betalactams is proposed. One of the two ways proposed is specific modification which is peculiar to certain betalactam structure and involves introduction of substitutes changing particular physico-chemical properties of the natural or synthetic analogous. General guidelines for modification of the biologically active compounds are given. The space and depth of the necessary physico-chemical investigation are determined. The alternative way of the new biologically active compounds development is the principle of similarity. The distinctive feature of this approach is the use as building blocks of the substitutes already used in the well-known betalactam antibiotics and "implantation" of this substitutes into other (new) betalactam structures. The ways of the new betalactams synthesis including the methods of enzyme engineering are considered. The possibility to use enzyme engineering processes for production of not only new individual betalactams--hits, but also for synthesis of the groups of betalactams--leads, is shown. More than 6000 new betalactam structures were constructed on the base of the principle of similarity. At least 700 of this compounds demonstrates not only antimicrobial activity but other types of biological activity due to the implementation of additional pharmaceutical units other than betalactams. The constructed compounds are summarized in the tables, the request for the electronic version of the tables can be sent by the address: valan@orc.ru.

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.

Enzymatic synthesis of cephalexin and cefaclor with the use of immobilized aminocephalosporin synthetase from Xanthomonas sp. as a biocatalyst was studied. The employment of a mathematical model based on the acyl-enzyme mechanism of the biocatalyst action was shown possible for the quantitative description of the antibiotic syntheses. A relationship providing determination of the complex of the physico-chemical investigations required for the enzymatic synthesis design was suggested. Kinetic and thermodynamic parameters of the processes were evaluated and the ratios of the maximum conversion of the key amino acids and the initial concentrations of the substrate and nucleophile were calculated. The regions of the model fit to the experimental data within a wide range of the substrate and nucleophile concentrations were defined. The technology for the enzymatic synthesis of aminocephalosporins exemplified by cephalexin and cefaclor was designed.

Production of cefazolin by acyl transfer enzymatic synthesis with immobilised cefazolin synthetase from Escherichia coli as a biocatalyst acting in accordance with the mechanism including formation of the acyl-enzyme complex was shown possible. The process kinetic parameters and the ratio of the maximum conversion of the key amino acid and the initial concentrations of the substrate and nucleophile were determined. Correlation of the calculated and experimental data on the cefazolin yield in the enzymatic synthesis was good. The main physico-chemical properties of the substrates and the reaction products i.e. dissociation constants and solubility were investigated. The complex of the physico-chemical studies makes it possible to design a highly efficient technological process for production of cefazolin including not only the stage of the enzymatic synthesis but also the stage of separation of the reaction mixture components.

Stability of 7-aminocephalosporanic acid (7-ACA) and desacetyl-7-ACA in aqueous solutions at the pH value higher than 6 and wide ranges of the temperature was studied. The kinetic parameters of the hydrolysis of the ester link in the molecule of 7-ACA and the openings of the betalactam link in the molecules of 7-ACA and desacetyl-7-ACA were estimated. It was shown that all the destruction processes were describable by the 1st order equations. A procedure for the calculation of the velocity constants of the above mentioned three processes by changes in the concentration of 7-ACA and desacetyl-7-ACA in the solutions is described.

The paper presents the results of the studies carried out by the authors within 20 years on development of processes for production of beta-lactam antibiotics with using biocatalysis. The proposed general principles for the development of efficient biocatalytic technologies are discussed in regard to production of the key compounds and synthesis of beta-lactams. The paper includes 4 parts concerned with comparison of the biocatalytic and chemical processes for production of beta-lactam antibiotics, requirements to the quality of the biocatalysts used and criteria for estimation of the efficiency of the stage of the technological biocatalyst production. The criteria provided determination of the optimal biocatalyst for production of the key compounds in the synthesis of beta-lactams. A retrospective analysis of the biocatalytic processes for production of 6-amino-penicillanic acid is presented and the impact of the activity and the form of the biocatalyst on the process efficiency is substantiated. Various schemes of the enzymatic synthesis of beta-lactam antibiotics and the approaches to the improvement of the technological processes including those with the use of immobilized microbial cultures at the stage of the production of the initial biosynthetic antibiotics are described. The prospects for the improvement of the processes for production of drugs with the use of biocatalysis are indicated and the main trends of the research required for the large scale use of the immobilized enzymes, microbial cells, oligo-enzymatic and poly-enzymatic systems in transformation and synthesis of organic compounds are defined.

The kinetics of splitting out the substitute at C3 in the molecule of beta-lactam antibiotics such as cefazolin, cefaclor, cefazedone and others as well as that of splitting out the chlorine atom in the side radical of the cefazedone molecule was studied within wide ranges of pH and temperature. The destruction processes for all the investigated compounds were shown to be described by the 1st order equations. The values of the activation energy, pre-exponential factors and reaction order by the hydrogen ions were measured. This provided the mathematical description of the destruction processes within wide ranges of pH and temperature. An unusual mechanism of synchronous splitting out the substitute at C3 and one of the chlorine atoms in the side radical of the cefazedone molecule was observed.

A new approach to preparation of highly efficient biocatalysts is described. It implies cell modification directed to changing the cell wall permeability for providing free access of the substrate to the intracellular enzymes and removing ballast substances from the cells during the immobilization with simultaneous preservation of the structure intactness and if possible the biochemical composition of the structures including the required enzyme. Possible realization of such a way is illustrated with an example on development of BC for transformation of betalactam antibiotics based on E. coli cells and penicillin amidase.

The phenomenon of coagulation autolysis was observed in two model microorganisms, i.e., a bacterial culture and an imperfect fungus. It was characterized by impairment of the cell membranes, followed by condensation and dehydration of the cytoplasm and long-term preservation of the cells in the form of coagulated cytoplasm. In this respect, it was similar to coagulation necrosis of human tissues. The autolysis in the microorganisms was accompanied by increase of their coagulase activity, the substrate specificity of the enzyme rather broad. The coagulase activity of the microorganisms was detected during the culture period between the lag-phase and the exponential growth phase, i.e., the phase of their active growth. It served as a signal to induce biosynthesis of peptidohydrolase and cleavage of proteins. We believe that the phenomenon of coagulation autolysis in these microorganisms is rather typical and can be considered as an adaptative reaction, inducing a cascade of events from synthesis of coagulase to overproduction of peptidohydrolases with proteolytic activity.

Methods for development of bioengineering systems of different types useful in synthesis and transformation of antibiotics are discussed. It was shown that in development of monoenzymatic biocatalysts on the basis of immobilized cells and in preparation of immobilized cultures producing secondary metabolites with enzymological engineering directed action on the cells could be provided which made it possible to establish highly efficient bioengineering systems. Various means for providing the directed action and method for estimation of the carrier-culture interation are proposed. The prospects of using the second generation biocatalysts in improvement of the processes for production of antibiotics are described.