Kinetic and Thermodynamic Approach to Design of Processes for Enzymatic Synthesis of Betalactams

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 λM 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.     

Kinetic and Thermodynamic Approach to Design of Processes for Enzymatic Synthesis of Betalactams

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 &#117 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.     

Integrated reactor concepts for the enzymatic kinetic synthesis of cephalexin

Integrated process concepts for enzymatic cephalexin synthesis were investigated by our group, and this article focuses on the integration of reactions and product removal during the reactions. The last step in cephalexin production is the enzymatic kinetic coupling of activated phenylglycine (phenylglycine amide or phenylglycine methyl ester) and 7-aminodeacetoxycephalosporanic acid (7-ADCA). The traditional production of 7-ADCA takes place via a chemical ring expansion step and an enzymatic hydrolysis step starting from penicillin G. However, 7-ADCA can also be produced by the enzymatic hydrolysis of adipyl-7-ADCA. In this work, this reaction was combined with the enzymatic synthesis reaction and performed simultaneously (i.e., one-pot synthesis). Furthermore, in situ product removal by adsorption and complexation were investigated as means of preventing enzymatic hydrolysis of cephalexin. We found that adipyl-7-ADCA hydrolysis and cephalexin synthesis could be performed simultaneously. The maximum yield on conversion (reaction) of the combined process was very similar to the yield of the separate processes performed under the same reaction conditions with the enzyme concentrations adjusted correctly. This implied that the number of reaction steps in the cephalexin process could be reduced significantly. The removal of cephalexin by adsorption was not specific enough to be applied in situ. The adsorbents also bound the substrates and therewith caused lower yields. Complexation with beta-naphthol proved to be an effective removal technique; however, it also showed a drawback in that the activity of the cephalexin-synthesizing enzyme was influenced negatively. Complexation with beta-naphthol rendered a 50% higher cephalexin yield and considerably less byproduct formation (reduction of 40%) as compared to cephalexin synthesis only. If adipyl-7-ADCA hydrolysis and cephalexin synthesis were performed simultaneously and in combination with complexation with beta-naphthol, higher cephalexin concentrations also were found. In conclusion, a highly integrated process (two reactions simultaneously combined with in situ product removal) was shown possible, although further optimization is necessary.     

Penicillin acylase-catalyzed synthesis of cefazolin in water–solvent mixtures: enhancement effect of ethyl acetate and carbon tetrachloride on the synthetic yield

The effects of organic solvents on the penicillin acylase-catalyzed, kinetically controlled synthesis of cefazolin have been examined in various water–solvent mixtures. In the presence of water-miscible solvents, the initial rate and maximum yield of cefazolin (CEZ) synthesis reaction were found to be reduced. The extent of inhibition was increased with increasing hydrophobicity of the solvent in the reaction mixtures. Enzymatic synthesis of cefazolin was also carried out in the water–solvent biphasic systems. Among the water-immiscible solvents tested, ethyl acetate (EtOAc) and carbon tetrachloride (CCl₄) were found to markedly improve the yield of cefazolin in the two-phase reaction system. Our study showed that the enhancement effect of EtOAc and CCl₄ on the synthetic yield was mainly caused by a reduction of the hydrolysis of acyl donor and product in the two-phase system rather than extraction of the product into the solvent phase.     

Fractal kinetic analysis of the enzymatic saccharification of CO2 laser pretreated corn stover

The enzymatic hydrolyses of laser pretreated corn stover as a novel pretreatment method were examined to establish a simplified kinetic model for the complicated hydrolysis process. The time dependence of the total reducing sugars amount was closely related to the amounts of cellulosic materials and amounts of cellulase. The evaluated model fitted very well with the experimental data of enzymatic hydrolysis of laser pretreated corn stover under different conditions, including cellulase loading, nature of substrate, substrate loading in the reaction medium. The results indicated that the complex kinetics of cellulase enzymatic saccharification could be assessed with the fractal kinetic model. The cellulase enzymatic reaction process was effectively predicted and controlled with the kinetic model. The result showed that the model could effectively reflect dynamic process of enzyme hydrolysis.     

Effect of polaymines on yeast cell-free protein synthesizing system. I. Influence of spermine and spermidine on aminoacyl-tRNA transfer reaction.

Spermine and spermidine added to a Saccharomyces cerevisiae cell-free protein synthesizing system increased phenylalanine polymerization reaction several-fold at suboptimal concentration of Mg2+ and approximately two-fold at optimal amounts of Mg2+. The addition of polyamines greatly stimulated the enzymatic and nonenzymatic binding of phenylalanyl-tRNA and N-acetylphenylalanyl-tRNA to ribosomes. The binding of the acetylated derivative was higher than phenylalanyl-tRNA, however, as it was shown the former was bound exclusively to the A site of the ribosome. Contrary to the binding process, the puromycin reaction was not stimulated by spermine added at a concentration which enhanced the polyphenylalanine synthesis. These results indicate that polyamines have not only a sparing effect on the Mg2+ requirement for yeast protein synthesis in vitro and suggest that one of the possible sites of polyamines action might be the binding of aminoacyl-tRNA to ribosomes.     

Development of a novel chemiluminescence method for the determination of cefazolin sodium in injectable powder and human urine based on a luminol‐Cu(III) complex reaction in alkaline medium

A novel chemiluminescence (CL) method was developed for the determination of cefazolin sodium based on the CL reaction between the [Cu(HIO₆)₂]^5‐Cu(III) complex and luminol in alkaline solution. Results showed that CL emission of Cu(III) complex–luminol in alkaline medium was significantly different from that in acidic medium. A possible mechanism of the enhanced effect of cefazolin on CL emission of the [Cu(HIO₆)₂]^5‐‐ luminol system was proposed. The effect of the reaction conditions on CL emissions was examined. Under optimized conditions, a good linear relationship was obtained between CL intensity and concentrations of cefazolin sodium in the range of 2.0 x 10^‐8 to 2.0 x 10^‐6 g/mL with a correlation coefficient of R² = 0.9978. The limit of detection was 4.58 x 10^‐9 g/mL. The proposed method was applied for the determination of cefazolin sodium in real samples with recoveries of 82.0‐109% with an RSD of 0.7‐2.1%. The proposed method was successfully used for the determination of cefazolin sodium in injectable powder preparations and human urine with satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.     

Chemiluminescence flow-injection analysis of beta-lactam antibiotics using the luminol-permanganate reaction.

A new flow injection chemiluminescence (CL) method has been developed for the determination of six beta-lactam antibiotics, including amoxicillin, cefadroxil, cefoperazone sodium, cefazolin sodium, cefradine and ceftriaxone sodium. When the antibiotic was injected into a stream of KMnO4 with alkaline luminol, a strong CL signal was produced. The method allows the measurements of 0.1-50.0 mg/L amoxicillin, 0.1-80.0 mg/L cefadroxil, 1.0-30.0 mg/L cefoperazone sodium, 1.0-30.0 mg/L cefazolin sodium, 3.0-50.0 mg/L cefradine and 3.0-50.0 mg/L ceftriaxone sodium. The detection limits are 0.05 mg/L for amoxycillin, 0.05 mg/L for cefadroxil, 0.4 mg/L for cefoperazonum sodium, 0.4 mg/L for cefazolin sodium, 0.8 mg/L for cefradine and 0.8 mg/L for ceftriaxone sodium. The relative standard deviations in 11 repeated measurements are 0.6%, 0.8%, 1.5%, 1.2%, 0.4% and 0.3% for 3.0 mg/L amoxicillin, 1.0 mg/L cefadroxil, 10.0 mg/L cefoperazone sodium, 10.0 mg/L cefazolin sodium, 10.0 mg/L cefradine and 10.0 mg/L ceftriaxone sodium, respectively. The method was successfully applied to the determination of amoxicillin in pharmaceutical preparations. A possible CL reaction mechanism is also discussed.     

Haloperoxidases: Enzymatic Synthesis of alpha,beta-Halohydrins from Gaseous Alkenes

The enzymatic synthesis of alpha,beta-halohydrins from gaseous alkenes is described. The enzymatic reaction required an alkene, a halide ion, dilute hydrogen peroxide, and a haloperoxidase enzyme. A wide range of gaseous alkenes were suitable for this reaction, including those containing isolated, conjugated, and cumulative carbon-carbon double bonds. Chlorohydrins, bromohydrins, and iodohydrins could be formed. The combining of this enzymatic synthesis with a previously described enzymatic synthesis of epoxides from alpha,beta-halohydrins provides an alternate pathway, other than the well-known enzymatic direct epoxidation pathway, from alkene to an epoxide.     

Enzymatic synthesis of cefazolin using immobilized recombinant cephalosporin-acid synthetase as the biocatalyst

Bioprocess and Biosystems Engineering - A method for the synthesis of β-lactam antibiotic cefazolin (CEZ) by enzymatic acylation of...     

Electron spectroscopy and the electron structure of molecules

The experimental-theoretical approach to the study of physico-chemical, in particular, spectroscopic and photochemical properties of groups of related molecules is considered. The problem of physically correct spectral decompositions of complex electronic spectra into bands corresponding to separate electronic transitions and of the information obtained therefrom is discussed. Concrete examples of revealing photo- and enzymatic reaction mechanisms by combined spectroscopic and theoretical studies of molecular electronic structure and spectra are given. Special attention is paid to the connection of electronic structure of molecules with their physico-chemical, in particular, spectroscopic properties.     

Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis.

An enzymatic system for poly gamma-glutamate (PGA) synthesis in Bacillus subtilis, the PgsBCA system, was investigated. The gene-disruption experiment showed that the enzymatic system was the sole machinery of PGA synthesis in B. subtilis. We succeeded in achieving the enzymatic synthesis of elongated PGAs with the cell membrane of the Escherichia coli clone producing PgsBCA in the presence of ATP and D-glutamate. The enzyme preparation solubilized from the membrane with 8 mM Chaps catalyzed ADP-forming ATP hydrolysis only in the presence of glutamate; the D-enantiomer was the best cosubstrate, followed by the L-enantiomer. Each component of the system, PgsB, PgsC, and PgsA, was translated in vitro and the glutamate-dependent ATPase reaction was kinetically analyzed. The PGA synthetase complex, PgsBCA, was suggested to be an atypical amide ligase.     

S-腺苷甲硫氨酸的研究进展

S-腺苷甲硫氨酸(SAM)是甲硫氨酸和三磷酸腺苷相结合的代谢物,广泛存在于动植物和微生物体内,参与40多种生化反应,主要作为三种代谢途径(转甲基、转硫基、转氨丙基)的前体,临床上被广泛用于治疗肝病、抑郁症、关节炎等。SAM的制备方法主要有化学合成法、酶促合成法、发酵法三种。化学合成的SAM是消旋体,需进行光学拆分,且存在产率低、原料L-高半胱氨酸价格昂贵和环境污染等问题。酶促合成法合成的SAM纯度高,但原料ATP成本太高。发酵法已成为目前生产SAM最常用的方法,欧洲利用发酵法生产SAM已实现了产业化,但国内的起步较晚,目前还处于实验室研究阶段。因此,应加强发酵法生产SAM的产业化关键技术研究。     

Inactivation of alpha-ketoglutarate dehydrogenase during enzyme-catalyzed reaction

Alpha-Ketoglutarate dehydrogenase is inactivated during the enzymatic reaction. This inactivation is revealed both in a model system with an artificial electron acceptor and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither the substrate depletion and the product accumulation nor the changing of the alpha-ketoglutarate dehydrogenase oligomeric structure induces the inactivation. There are two independent mechanisms of alpha-ketoglutarate dehydrogenase inactivation in the course of the enzymatic reaction which are consistent with the two stages of the process. The first one corresponds to an essential decrease in activity during several minutes from the beginning of the reaction. This process can be reversed, occurs only during catalysis and manifests itself in the same degree both in the model system and during the functioning of the alpha-ketoglutarate dehydrogenase complex. The second stage is slow, irreversible and more apparent in the model system; it coincides with the inactivation due to the alpha-ketoglutarate dehydrogenase preincubation with hexacyanoferrate alone. The data obtained provide evidence that irreversible inactivation of alpha-ketoglutarate dehydrogenase during the enzymatic reaction is caused by the oxidant.     

pH-imprinted lipase catalyzed synthesis of dextran fatty acid ester

The application of enzymatic catalysis for the synthesis of polysaccharide-based surfactants was investigated. The polysaccharide dextran, a neutral bacterial polysaccharide consisting of -1,6 linked glucose units, was chemically modified by the attachment of hydrophobic groups through a transesterification reaction with a vinyl decanoate. A screening of commercially available lipases and protease for the synthesis of amphiphilic polysaccharides in DMSO suggested that lipase AY from Candida rugosa modified dextran T-40 with vinyl decanoate at the highest conversion. A pH-adjustment in a phosphate buffer at pH 7.5 prior to use is crucial to make this enzyme active in DMSO. The effect of enzyme concentration and mole ratio of fatty ester to dextran T-40 on the conversion and the rate of reaction were studied. Finally, investigation of the kinetics and regioselectivity of lipase AY-catalyzed modification offer a possibility to regulate the position and the extent of hydrophobic group attached to dextran. These two properties are fundamental for controlling the physico-chemical properties of the final polymeric surfactants.     

Enzymatic synthesis of N-10-undecenoyl-phenylalanine catalysed by aminoacylases from Streptomyces ambofaciens

Due to its physico-chemical and biological activities, N-10-undecenoyl-phenylalanine (C11’F) is one of the most interesting lipoaminoacids used in cosmetic and pharmaceutical industries. Its production is currently based on the Schotten-Baumann chemical reaction, which shows some environmental issues in terms of effluents. As a possible biocatalytic alternative, this study presents the evaluation of the reactional and process conditions allowing the production of C11’F using aminoacylases from Streptomyces ambofaciens culture. These aminoacylases showed the best activity at 45 °C and pH between 7 and 8 with a moderate thermal stability. The influence of substrates concentrations on the kinetic parameters of C11’F synthesis showed a more important impact of the phenylalanine concentration as compared to the 10-undecenoic acid concentration. As a reactional product, C11’F appeared to have an inhibitory effect on the enzymatic N-acylation. Cobalt addition allowed an eleven-fold increase of the reaction rate. Batch reactors were used with free aminoacylases without impact on the final C11’F concentration. For the first time, enzymatically produced C11’F was finally purified at the gram scale as a 99% purity white powder. The evaluation of the biological activity on melanocytes cultures showed the presence of skin lightening activity similar to the one obtained with the chemically produced C11’F.     

A simple approach to detect active-site-directed enzyme-enzyme interactions. The aldolase/glycerol-phosphate-dehydrogenase enzyme system

A novel approach has been elaborated to identify the mechanism of intermediate transfer in interacting enzyme systems. The aldolase/glycerol-3-phosphate-dehydrogenase enzyme system was investigated since complex formation between these two enzymes had been demonstrated. The kinetics of dihydroxyacetone phosphate conversion catalyzed by the dehydrogenase in the absence and presence of aldolase was analyzed. It was found that the second-order rate constant (kcat/Km) of the enzymatic reaction decreases due to the formation of a heterologous complex. The decrease could be attributed to an increase of the Km value since kcat did not change in the presence of aldolase. In contrast, an apparent increase in the second-order rate constant of dihydroxyacetone phosphate conversion by the dehydrogenase was observed if the triose phosphate was produced by aldolase from fructose 1,6-bisphosphate (consecutive reaction). Moreover, no effect of dihydroxyacetone phosphate on the dissociation constant of the heterologous enzyme complex could be detected by physico-chemical methods. The results suggest that the endogenous dihydroxyacetone phosphate produced by aldolase complexed with dehydrogenase is more accessible for the dehydrogenase than the exogenous one, the binding of which is impeded due to steric hindrance by bound aldolase.     

Influence of supramolecular structure on the enzyme mechanisms of rat liver lysyl-tRNA synthetase-catalyzed reactions. Synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate

Lysyl-tRNA synthetase, dissociated from the multienzyme complexes of aminoacyl-tRNA synthetases from rat liver, was previously found to be 6-fold more active than the synthetase complex in the enzymatic synthesis of P1,P4-bis(5'-adenosyl)tetraphosphate. The bi-substrate and product inhibition kinetics of the reaction are analyzed. Free lysyl-tRNA synthetase exhibits distinctly different kinetic patterns from those of an 18 S synthetase complex containing lysyl-tRNA synthetase. The 18 S synthetase complex shows kinetic patterns which are consistent with an ordered Bi Uni Uni Bi ping-pong mechanism. Free lysyl-tRNA synthetase shows kinetic patterns consistent with a random mechanism. The differences in the enzymatic properties are attributed to the organization of the supramolecular structure of the synthetase complex. The results suggest that association of the synthetases may affect the mechanisms of the synthesis of AppppA.     

Metabolic modeling of polyhydroxybutyrate biosynthesis

A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium.