[Status and prospects in the use of biocatalysis in the synthesis of beta-lactam antibiotics]. / Sostoianie i perspektivy ispol'zovaniia biokataliza dlia polucheniia betalaktamnykh antibiotikov.

The present world status of using biocatalysts for production of beta-lactam antibiotics and their semiproducts is analyzed and the biocatalytic and chemical methods for production of antibiotics are compared. The requirements to the quality of biocatalysts used in production of drugs are formulated and the criteria of the efficiency of biocatalytic production processes are presented. Interrelation between the requirements to the biocatalyst quality and the efficiency of the biocatalytic production processes is examined. The general principles of development of efficient enzymatic processes including biocatalytic processes in production of beta-lactam antibiotics as closed-loop and practically wasteless systems are discussed. The main advantages of the developed production processes are illustrated by an example of large-scale processes use in manufacturing of the basic products for synthesis of beta-lactam compounds.

[Kinetics and thermodynamics of the hydrolysis-synthesis reaction of acetyl-L-methionine catalyzed by acylase I from hog kidney]. / Kinetika i termodinamika reaktsii gidroliza-sinteza atsetil-L-metionina, kataliziruemoi atsilazoi I suinoi pochki.

The kinetics and thermodynamics of the equilibrium reaction of hydrolysis--synthesis of acetyl-L-methionine catalyzed by acylase I from hog kidney were studied. At high concentrations of the products (acetate ion and L-methionine) the acetyl-L-methionine hydrolysis does not proceed to completion but to the equilibrium position. The equilibrium constant of hydrolysis determined at the attained equilibrium in both directions, i.e. hydrolysis and synthesis, is equal to 3.6 +/- 0.4. Based on the initial rates of hydrolysis and synthesis, a kinetic pattern for the dependence of the reaction rate on concentration of the components of the system is proposed. Evidence for this kinetic pattern is supported by the Holden ratio and the coincidence of the kinetic parameters calculated from the total kinetic curves and the initial rates of hydrolysis and synthesis of acetyl-L-methionine.

[Substrate specificity of acylase I from pig kidney]. / Substratnaia spetsifichnost' atsilazy i svinoi pochki.

The substrate specificity of acylase I from pig kidney has been studied. For the N-acetyl derivatives of D,L-amino acids with an unbranched side chain the catalytic efficiency of enzymatic hydrolysis increases linearly with an increase of the substrate hydrophobicity. The tangent of the linear dependence of logarithm of the second order rate constant of enzymatic hydrolysis on the Hansch hydrophobicity constant is 0,7. This supports a simple "extraction" model of energy realization during the enzyme binding to the substrate in the course of the reaction. When the amino acids with a branched side chain are used, the efficiency of hydrolysis of N-acetyl derivatives decreases by one order of magnitude as compared to the amino acids with an unbranched side chain having the same number of carbon atoms. The N-acetyl derivatives of aromatic amino acids are hydrolyzed in a small degree. An analysis of the substrate specificity of pig kidney acylase I demonstrates that the enzyme has a narrow hydrophobic "cleft" with the length not less than four metrylene links.

[pH dependence of tryptophan ethyl ester hydrolysis]. / PH-zavisimost' nefermentativnogo gidroliza etilovogo efira triptofana.

The dependence of the rate of spontaneous hydrolysis of tryptophan ethyl ester within a wide range of pH (4,6-10,3) was studied. This dependence was found to differ from other dependences, i.e. within the pH range of 4,6-7,0 the value of the rate constant is practically independent on pH. In order to describe the dependence obtained a general pattern of hydrolysis was postulated and the kinetic parameters of individual elementary reactions were determined. The rate constants for the hydrolysis of the amino acid with a non-protonated amino group of ester was calculated using the literary values for the rate constants of carboxylic acid hydrolysis. The obtained values of the second order rate constants for the alkaline hydrolysis of non-protonated and protonated forms of tryptophane ester (k4 and k5) are 1,1 and 79 M-1 s-1, respectively; those for the hydrolysis of the protonated form of the substrate (k3) are 1,0.10(-5) M-1 s-1. The role of spontaneous (non-enzymatic) hydrolysis in stereoselective cleavage of amino acid esters is discussed.

[pH-dependence of tryptophan ethyl ester hydrolysis by alpha-chymotrypsin]. / pH-zavisimost' gidroliza etiolovogo efira triptofana, kataliziruemogo alpha-khimotripsinom.

The hydrolysis of L-tryptophane ethyl ester catalyzed by alpha-chymotrypsin and the effect of ethyl ster of D-tryptophane on the course of the reaction were studied. A kinetic pattern of a three-step enzymatic reaction based on the assumption that the enzyme complex with the protonated form of the substrate is the only reactive one, was proposed. It was shown that the limiting step of the enzymatic reaction consists in a formation of intermediate acyl enzyme. The pH-dependence of the bimolecular rate constant (kcat/Km) for the enzymatic hydrolysis of L-tryptophane ethyl ester is bell-shaped and is described by a pattern including ionization of two groups with pKa values of 7,0 +/- 0,1; and 7,5 +/- 0,1; the value of pKa equal to 7,5 +/- 0,1 corresponds to substrate ionization (pKas for the amino group of L-tryptophane ethyl ester is 7,6). The constants for the binding of protonated and non-protonated substrate forms by the enzyme were calculated from the step-wise dependence of the Km values of pH. An analysis of the bell-shaped dependence of the catalytic constant of enzymatic hydrolysis included determination of pK values of the ionogenic groups of the enzyme--substrate complex (pK'a = 6,8 +/- 0,1 and pK''a = 7,3 +/- 0,1).

[Study of E. coli penicillin amidase. The pH dependence of the equilibrium constant of ampicillin enzymatic hydrolysis]. / Izuchenie penitsillinamidazy iz E. coli. pH-zavisiomst' konstanty ravnovesiia fermentativnogo gidroliza ampitsillina.

The equilibrium parameters of the hydrolysis of ampicillin catalysed by penicillin amidase were determined within the pH range of 4.5 to 5.5. The values of the ionization constants of the carboxy group of D-(-)-ALPHA-AMINOPHENYLACETIC ACID (PK1=1.80) and amino group of 6-aminopenicillanic acid (pK2=4.60) were estimated and pH-dependence of the effective free energy of ampicillin hydrolysis was calculated. It was shown that the thermodynamic optimum of ampicillin synthesis was at 3.20 (the value of the effective free energy under the experimental conditions was 3.27 kcal/mole). The value of the "true", pH-independent free energy of hydrolysis (deltasigma) of the amide bond in the ampicillin molecule was determined to be equal to 9.72 kcal/mole. The thermodynamic parameters of ampicillin and benzylpenicillin hydrolysis were compared. The amino group in the alpha-position of phenylacetic acid was shown to have a significant effect on the values of "true" free energy of hydrolysis of the penicillin amide bond and free ionization energy in the system.

[Study of E. coli penicillin amidase. The pH-dependence of the enzymatic inactivation kinetics]. / Izuchenie penitsillinamidazy iz E. coli. pH-zavisimost' kinetiki inaktivatsii fermenta.

The pH-dependence of the inactivation rate constant of penicillin amidase at a temperature of 40 degrees C was studied. It was shown that in all cases the enzyme inactivation corresponded to the kinetics of the reaction of the 1st order. The pH-dependence profile was found to be bell-shaped, the effect of transfer from the highest to the lowest values of the inactivation rate constants increasing more than 100 times. On the basis of the data obtained and published earlier it was concluded that the enzyme inactivation proceeded in accordance with the scheme in which out of 3 equilibrium ionic forms of penicillin amidase, i.e. "acid", "neutral" and "alkaline" the neutral form of the active enzyme was most stable. Kinetic analysis of the scheme was carried out and it was shown that the dependence found was in accordance with the theoretical curve in which the pK values of the ionogenic groups controlling the interconvertions between the penicillin amidase forms were equal to 2.4 and 10.1 at a temperature of 40 degrees C. The value of the inactivation rate constant of the "acid" or "alkaline" form was equal to 5.95 min-1, while the "neutral" form of the enzyme was characterized by the inactivation rate constant equal to 5.1.10(-4) min-1. A mechanism for the enzyme inactivation was proposed. According to this mechanism, destruction of the salt bridge in the native structure of penicillin amidase resulted in production of extremely labile forms of the enzyme as compared to the native form.