Azadeh Kheirolomoom

The combined effects of pH and temperature on penicillin G decomposition and its stability modeling

Abstract The stability of penicillin G was analyzed as a function of temperature, pH, and the combined effects of pH and temperature were investigated by proposing a second-order polynomial model for penicillin G decomposition reaction rate constant. The modeling results obtained for the first-order decomposition reaction of penicillin G are in good agreement with the experimental data obtained at the pH range of 1.8–10.0 and temperature range of 0–52°C. The stability analysis shows that penicillin G is more stable at the pH range of 5.0–8.0 than out of it and lower temperatures for subsequent isolation. The maximum stability of penicillin G is at about pH 6.0 and its instability its much higher at acidic than basic pH values. The stability of penicillin G decreases by increase in temperature for all pH values. The stability results as well as the proposed second-order model for penicillin G decomposition reaction rate constant can be used for investigating penicillin G losses during its isolation process.

Influence of External Mass Transfer Limitation on Apparent Kinetic Parameters of Penicillin G Acylase Immobilized on NonPorous Ultrafine Silica Particles.

Immobilization of enzymes on nonporous supports provides a suitable model for investigating the effect of external mass transfer limitation on the reaction rate in the absence of internal diffusional resistance. In this study, deacylation of penicillin G was investigated using penicillin acylase immobilized on ultrafine silica particles. Kinetic studies were performed within the low-substrate-concentration region, where the external mass transfer limitation becomes significant. To predict the apparent kinetic parameters and the overall effectiveness factor, knowledge of the external mass transfer coefficient, k(L)a, is necessary. Although various correlations exist for estimation of k(L)a, in this study, an optimization scheme was utilized to obtain this coefficient. Using the optimum values of k(L)a, the initial reaction rates were predicted and found to be in good agreement with the experimental data.

Clarification of penicillin G acylase reaction mechanism

Abstract The kinetics of the enzymic reaction of penicillin G acylase from a mutant of Escherichia coli ATCC 11105 in forward and reverse directions were studied and the kinetic constants determined. Results show that the enzyme is inhibited by excess substrate, penicillin G (Pen G), and by both products. The non-competitive inhibition by 6-aminopenicillanic acid (6-APA) and competitive inhibition by phenylacetic acid were observed for the ordered uni bi deacylation reaction in the forward direction. The optimum pH value for the reverse acylation reaction was 5.7. The bi uni mechanism for the reverse reaction was investigated and the inhibitory effects of the substrates, 6-APA and phenyl acetic acid, and the product, Pen G, were studied. Result shows that Pen G is the mixed-type inhibitor for the reverse reaction.

The stability analysis and modeling of pH- and ionic strength inactivation of penicillin G acylase obtained from various species of Escherichia coli

Abstract A modified model was proposed for the pH-inactivation rate constant of penicillin G acylase obtained from various species of Escherichia coli. A new approach was applied and a new parameter called steric factor defined to obtain the more precise description of the pH-inactivation rate constant. The parameter is used as the measure of the conformational rigidity of the enzyme with respect to pH. The proposed model accuracy was verified and compared with the reported models. The model can be used for pH-inactivation studies of other biocatalysts by determining its parameters. Also, the effect of the ionic strength on the stability of the enzymatic solution of penicillin G acylase has been studied in NaCl and KCl solution. The inactivation reaction of the enzyme with respect to the ionic strength of the solution obeys first order irreversible mechanism. The rate constant of this reaction was determined as a function of the solution ionic strength and a mathematical description was developed. The results show the role of the ionic strength of the non-inhibitory salt solution on the stability of penicillin G acylase, especially for long-time storage and operation.

Application of an optimization algorithm for estimating intrinsic kinetic parameters of immobilized enzymes

A simple optimization methodology is applied to estimate the intrinsic kinetic parameters for both reversible and irreversible unireactant immobilized enzyme systems that follow the Michaelis-Menten mechanism. The method utilizes a direct-search optimization algorithm along with the numerical solution of the governing differential equations. The usefulness and validity of the method is demonstrated by comparing the predicted values of the intrinsic kinetic constants using the proposed method with a series of experimental values reported in the literature for different immobilized enzyme systems with irreversible and reversible reactions.

Incorporation and activation of a membrane-bound enzyme in bilayers of liposomes.

SummaryThe effects of lipid composition and fluidity of lipid bilayers on incorporation and activation of membrane-bound d-fructose dehydrogenase are described in this study. The incorporation of the enzyme into bilayers of small unilamellar vesicles (SUV) made of several phospholipids resulted in enzyme activation with magnitudes higher than that observed in the presence of Triton X-100, indicating that this higher activation is due to lipid-protein interaction. The activity was highest in the presence of SUV formed by the addition of 10% dl-α-dipalmitoylphosphatidylethanolamine to l-α-dimyristoylphosphatidylcholine, which resulted in eightfold higher activation compared with that of the enzyme in its free state. This activation did not appear to be due to the degree of incorporation of the enzyme, indicating that incorporation is distinct from the activation event. Thus, it is probably the lipid environment that leads to higher activation of the enzyme. A break in the Arrhenius plot of the activity of the membrane-bound enzyme at temperatures close to the phase transition of the phospholipid implies that changes in the physical state of the lipid bilayer influence the enzyme activity. Furthermore, immobilization of d-fructose dehydrogenase, previously adsorbed to SUV, on urethane prepolymer also resulted in about eightfold higher activation than that of the free enzyme.

Effects of arachidonic acid concentration on prostaglandin biosynthesis and feasibility of semibatch processes

The reaction characteristics of prostaglandin E2 biosynthesis by PGH-synthase and PGE2 isomerase and the substrate dependency of this biosynthesis were studied. The activity of PG-synthases was blocked by the inhibitory action of one or more byproducts, probably resulting from the action of PGH-synthase. This inhibitory action then appeared to be partly reversible, indicating that the substrate and the inhibitor compete for the catalytic sites. According to these findings, the feasibility of a successful semibatch biosynthesis was investigated. A combination of the substrate concentration reducing procedure and the semibatch process resulted in an about 3.5-fold higher increase in the total amount of PGE2 formed in comparison with the batch results obtained at the substrate concentration of 1.0 mg/cm3. Since the cost of enzyme is a governing factor in this biosynthesis, development of semibatch biosynthesis of PGE2 becomes a matter of economic importance.

Effects of reconstitution of membrane-bound lipoprotein lipase and dispersity of substrate on its reaction characteristics

Abstract Reaction characteristics of a membrane-bound lipoprotein lipase acting on a hydrophobic substrate were investigated in aggregated structures—lipid bilayers of liposomes and mixed micelles of Triton X-100. The enzyme activity was enhanced with increases in Triton X-100 and phospholipid concentrations in micellar and liposomal structures. This higher activity was found to be due to both the solubilization state of the hydrophobic substrate and the hydrophobic interactions of the enzyme with either phospholipid or Triton X-100 molecules as a result of its incorporation into the aggregated systems. The enzyme reconstituted into lipid bilayers of liposomes prepared from 15 mM DMPC in the presence of 0.05% Triton X-100 showed a further 1.5-fold higher activity in comparison with the activity without reconstitution in micelles of 1.0% Triton X-100. These results indicate the necessity of the bilayer structure to retain the membrane-bound enzyme in an active conformation.

Substrate Specificity and Positional Preference of a Lipoprotein Lipase

Abstract The substrate specificity and positional preference of a membrane-bound lipoprotein lipase with and without reconstitution in liposomes were studied. The enzyme showed the same preference for the acyl groups in the 1- and 3-positions of triglycerides, while its activity toward the 2-position was one-third of that toward the 1- or 3-positions. The substrate specificity of the enzyme toward fatty acids of different chain length and degree of unsaturation was in the order of C:14>C:16>C:18:1≥C:18. The enzyme with and without reconstitution showed the same positional preference and substrate specificity.

Reaction mechanism of prostaglandin E2 biosynthesis and its modeling

Abstract The reaction mechanism of PGE 2 biosynthesis was investigated by a detailed examination of the cyclo-oxygenase and PGE 2 -isomerase activities in acetone-pentane powder (microsomal fraction of ram seminal vesicular glands). Two main types of inactivating process were recognized in the reaction system. One type was due to irreversible inactivation caused by the oxidizing agent [O] · X released through the reduction of PGG 2 to PGH 2 , while the other type was due to reversible inhibition which was supposed to be derived from the precursor arachidonic acid (AA). This inhibitor was found to block the activities of both cyclooxygenase and PGE 2 -isomerase, and to compete with the substrates AA and PGH 2 . Although no significant substrate inhibition was observed, arachidonic acid was slightly inhibitory toward PGE 2 -isomerase.