RESUMEN
A method for the synthesis of ß-lactam antibiotic cefazolin (CEZ) by enzymatic acylation of 7-amino-3-(5-methyl-l,3,4-thiadiazol-2-yl)thiomethyl-3-cephem-4-carboxylic acid (TDA) using immobilized cephalosporin-acid synthetase (IECASA) from recombinant E. coli strain VKPM B-12316 has been developed. A stepwise pH gradient designed on the basis of investigations on the solubility of components was applied for synthesis. This helped in avoiding the precipitation of TDA in the reaction when its initial concentration was high (150-200 mM). Thus, under optimal conditions a high yield of CEZ (relative to TDA) of 92-95% was obtained. Where the final reaction mixture contained 65-85 mg/mL of CEZ, 4-5 mg/mL of unreacted TDA, and 40-60 mg/mL of the by-product, 1(H)-tetrazolylacetic acid (TzAA). Testing of optimized CEZ synthesis using IECASA in a batch reactor has proved sufficiently high operational stability of the biocatalyst, with its residual activity after the 25th cycle accounting for about 83 ± 2% of its starting value. The half-inactivation period of IECASA was estimated as 85 cycles of CEZ synthesis.
Asunto(s)
Aciltransferasas/química , Biocatálisis , Cefazolina/síntesis química , Enzimas Inmovilizadas/química , Acilación , Cefazolina/química , Escherichia coli/enzimología , Proteínas Recombinantes/químicaRESUMEN
The modified asparaginase Was79 was derived from the recombinant wild-type L-asparaginase of Wolinella succinogenes. The Was79 contains the amino acid substitutions V23Q and K24T responsible for the resistance to trypsinolysis and the N-terminal heparin-binding peptide KRKKKGKGLGKKR responsible for the binding to heparin and tumor K562 cells in vitro. When tested on a mouse model of Fischer lymphadenosis L5178Y, therapeutic efficacy of Was79 was significantly higher than that of reference enzymes at all single therapeutic doses used (125-8000 IU/kg). At Was79 single doses of 500-8000 IU/kg, the complete remission rate of 100 % was observed. The Was79 variant can be expressed intracellularly in E. coli as a less immunogenic formyl-methionine-free form at high per cell production levels.
Asunto(s)
Antineoplásicos/administración & dosificación , Asparaginasa/genética , Asparaginasa/metabolismo , Heparina/metabolismo , Leucemia L5178/tratamiento farmacológico , Wolinella/enzimología , Sustitución de Aminoácidos , Animales , Antineoplásicos/farmacología , Asparaginasa/administración & dosificación , Asparaginasa/farmacología , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Células K562 , Ratones , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Wolinella/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fungal strains degrading plant biomass available from the Russian National Collection of Industrial Microorganisms (VKPM) have been screened for the xyloglucanase activity. Under conditions of submerged cultivation, the thermophilic strains Sporotrichum thermophile VKPM F-972, Myceliophthora thermophila VKPM F-244, and Sporotrichum pruinosum VKPM F-235 produced extracellular xyloglucanases with optimal activity at 60°C, pH 5.0. 88-100% of the initial enzyme activity was retained after l-h incubation at 50°C; 79-84% of the activity was retained after l-h incubation at 60°C. S. thermophile VKPM F-972, M. thermophila VKPM F-244, and S. pruinosum VKPM F-235 strains may be used as the gene sources for construction of highly active producers of the thermostable xyloglucanases.
Asunto(s)
Proteínas Fúngicas/química , Glicósido Hidrolasas/química , Calor , Sordariales/enzimología , Sporothrix/enzimología , Estabilidad de Enzimas , Proteínas Fúngicas/metabolismo , Glicósido Hidrolasas/metabolismo , Federación de RusiaRESUMEN
Cephalosporin acid synthetase (CASA) is responsible for specific to synthesis of cephalosporin-acids, its expression in Escherichia coli cells is accompanied by accumulation of unprocessed insoluble precursor. In order to optimize conditions of recombinant CASA production we have studied the effects of several parameters of strain cultivation, including growth media composition, temperature, and inoculation dose. Also plasmids for production of CASA variants with the signal sequence of Erwinia carotovora L-asparaginase (ansCASA) and "leaderless" CASA were created in search of more efficient expression constructs. Removal of the N-terminal secretion signal sequence reduced the production of functionally active CASA more than 10-fold and inhibited strain growth. Insertion of the L-asparaginase signal sequence increased the specific enzyme activity in the resultant recombinant strain. The ansCASA producing strain was used to develop the method of immobilization of the recombinant enzyme on an epoxy-activated macroporous acrylic support. The resultant biocatalyst performed effective synthesis of cefazolin from 3-[(5-methyl-1,3,4-thiadiazol-2-il)-thiomethyl]-7- aminocephalosporanic acid (MMTD-7-ACA) and methyl ester of 1(H)-tetrazolilacetic acid (ÐETzAA), under mild conditions a transformation level of MMTD-7-ACA to cefazolin of 95% is reached.
Asunto(s)
Asparaginasa/metabolismo , Proteínas Bacterianas/metabolismo , Cefazolina/metabolismo , Proteínas Inmovilizadas/metabolismo , Complejos Multienzimáticos/metabolismo , Acrilatos/química , Asparaginasa/genética , Proteínas Bacterianas/genética , Biocatálisis , Clonación Molecular , Medios de Cultivo/química , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Ingeniería Genética , Proteínas Inmovilizadas/genética , Complejos Multienzimáticos/genética , Pectobacterium carotovorum/química , Pectobacterium carotovorum/enzimología , Plásmidos/química , Plásmidos/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Alpha-amino acid ester hydrolase (EC 3.1.1.43, AEH) is a promising biocatalyst for the production of semi-synthetic ß-lactam antibiotics, penicillins and cephalosporins. The AEH gene from Xanthomonas rubrilineans (XrAEH) was recently cloned in this laboratory. The three-dimensional structure of XrAEH was simulated using the homology modeling method for rational design experiments. The analysis of the active site was performed, and its structure was specified. The key amino acid residues in the active site - the catalytic triad (Ser175, His341 and Asp308), oxyanion hole (Tyr83 and Tyr176), and carboxylate cluster (carboxylate groups of Asp209, Glu310 and Asp311) - were identified. It was shown that the optimal configuration of residues in the active site occurs with a negative net charge -1 in the carboxylate cluster. Docking of different substrates in the AEH active site was carried out, which allowed us to obtain structures of XrAEH complexes with the ampicillin, amoxicillin, cephalexin, D-phenylglycine, and 4-hydroxy-D-phenylglycine methyl ester. Modeling of XrAEH enzyme complexes with various substrates was used to show the structures for whose synthesis this enzyme will show the highest efficiency.
RESUMEN
The article deals with experimental determination of ionization constants and solubility for the compounds (target products, initial ß-lactams, acylating agents and by-products) involved in enzymatic synthesis of some therapeutically used aminopenicillins and aminocephalosporins, namely ampicillin, amoxicillin, cephalexin, cephadroxil, cephaloglycin, cefaclor, cefprozil, cefatrizine. Methodology of investigations and the evaluation of experimental data for the determination of ionization constants and solubility of the different type electrolytes are presented. Applications of the methods based on acid-base potentiometric titration and on determination of solubility-pH dependence of assayed substances are discussed. The original data on ionization constants and solubility of amoxicillin, cefprozil, cefatrizine, cephadroxil and initial ß-lactams for production of cefaclor, cefprozil and cefatrizine, as well as solubility of by-product D-(-)-p-hydroxyphenylglycine are presented. Experimentally determined parameters and constants available in the literature for all abovementioned aminopenicillins and aminocephalosporins are collected. These data might be used for choice of the conditions of both processes: the enzymatic synthesis and the isolation of the product from reaction mixture.
Asunto(s)
Cefalosporinas/química , Ácido Penicilánico/análogos & derivados , Penicilinas/química , Aminoácidos/química , Química Farmacéutica/métodos , Diseño de Fármacos , Electrólitos , Concentración de Iones de Hidrógeno , Iones , Modelos Químicos , Modelos Teóricos , Ácido Penicilánico/química , Solubilidad , Temperatura , Termodinámica , beta-Lactamas/químicaRESUMEN
The cell walls of Streptomyces antibioticus 39 contain a glycosylated poly(glycerol phosphate), in which the repeating monomeric unit is O-alpha-D-galactopyranosyl-(1--3)-O-2-acetamido-2-deoxy-beta-D-galactopyranosyl-(1--1)-glycerol monophosphate. The localization of the phosphodiester linkages between hydroxyl groups at positions 2 and 3 of the adjacent glycerol residues and the structure of the glycoside were established by 13C nuclear-magnetic-resonance spectroscopy. The spectral data are also in accordance with the results of the methylation analysis and enzyme degradation of the glycoside.