Mixed substrate kinetics

Kovarova-Kovar, K. and Egli, T. (1998). Growth kinetics of suspended microbial cells from single-substrate-controlled growth to mixed-substrate kinetics, Microbiol. Mol. Biol. Rev., 62, 646-666. 

Kovarova-Kovar K, Egh T (1998) Growth kinetics of suspended microbial cells from single-substrate-controUed growth to mixed-substrate kinetics. Microbiol Mol Biol Rev 62 646-666 Lageveen RG, Huisman GW, Preusting H, Ketelaar P, Eggink G, Witholt B (1988) Formation of polyesters by Pseudomonas oleovorans effect of substrates on formation and composition of poly-(/J)-3-hydroxyalkanoates and poly-(R)-3-hydroxyalkenoates. Appl Environ Microbiol 54 2924-2932... 

PCCase is a biotinylated protein that catalyzes a reaction required in the catabolism of amino acids and fatty acids of odd-numbered chain length, and in the catabolism and anabolism of branched-chain fatty acids. In order to characterize the structure of this enzyme from plants we undertook its purification. PCCase activity was purified from extracts of maize leaves by a four step scheme that included PEG precipitation, hydrophobic interaction chromatography, anion exchange chromatography and affinity chromatography. This purification scheme achieved a nearly 250-fold purification of PCCase activity. However, throughout this purification of PCCase, ACCase copurified. Indeed, SDS-PAGE analysis of the final purified PCCase preparation identified two biotinylated polypeptides of about 240 and 230 kDa. These polypeptides have previously been described as subunits of ACCase (7). Furthermore, mixed substrate kinetic studies (8) with the purified PCCase/ACCase preparation indicated that the carboxylation of propionyl-CoA and acetyl-CoA were carried out by the same enzyme. Furthermore, both PCCase and ACCase activities were similarly affected by a variety of inhibitors. 

Figure 3. Kinetics of conq)etitivc inhibition of Clostridium thermohydrosuljur-icum strain 39E purified amylopuUulanase activity with mixed substrates. The solid lines A and C indicate the theoretical plots for competitive inhibition at amylose ccmcentrations of 0.6 and 2.4 mg/ml, respectively. Lines B and D are the theoretical plots for the absence of inhibition at the same respective amylose ccmcentrations. PuUulan was used at concentrations of 0.4, 0.8, 1.2, 1.6, 2.0, 2.4 mg/ml. For clarity, only two sets of data points were used in the above plot. ( ) and (A) are the practical data points obtained at 0.6 and 2.4 mg/ml amylose concentrations. All reaction mixtures contained 5% (v/v) dimethyl sulfoxide for solubility of amylose. [S] = [A] + [P], where S is the total substrate ccmcentration. A and P are the concentrations of amylose and pullulan, respectively. (Reproduced with permissiem from Ref. 13. Copyright 1990 Academic Press, Inc.)...

The above formulation is somewhat overly general, since it includes the possibility of mixed diffusion-kinetic control of both the tip and substrate processes. In practice, at least one of those electrodes is held under diffusion control, so either Eq. (18a)... 

Keeping the factors such as pH, temperature and enzyme concentration at optimum levels, if the substrate concentration is increased, the velocity of the reaction recorded a rectangular hyperbola. At very low substrate concentration the initial reaction velocity (v) is nearly proportional to the substrate concentration (first order kinetics). However, if the substrate concentration is increased the rate of increase slows down (mixed order kinetics). With a further increase in the subshate concentration the reaction rate approaches a constant (zero order-reaction where velocity is independent of substrate concentration). 

B-Z reaction with mixed substrates Tartaric acid/acetone [Rastogi, R. P., Singh, H. J. and Singh, A. K., Kinetics of Physicochemical Oscillations, Preprints of Submitted Papers. Aachen Discussion Meeting of Deutsche Bunsengesellschaft fur Physicalisehe Chemie, (1979) 98-107], oxalic acid/acetone [Noszticzius, Mag. Kern. Foly 85 (1979) 330]. 

The effect of the addition of small quantities of sulphuric acid upon the reaction order with respect to substrate concentration was investigated. For toluene nitration in aqueous acetic acid, the addition of up to 0.218 mole l i of sulphuric acid increased the first-order rate constant greatly, but did not cause a reversion to zeroth order or mixed order kinetics. The same concentration of sulphuric acid disturbed the first-order nitration in acetonitrile of the more reactive... 

The process-engineering comparison between simple fermentation and a complex bioprocess such as that used for waste water treatment, shown in detail in Table 1.2, brings out the problems involved in quantifying practices used in complex cases multiple substrate kinetics operating in either sequential or parallel form mixed populations dependence on pH and temperature the influence of homogeneous or heterogeneous reactor operation in discontin-... 

The results of three sets of experiments with half cells on the basis of 1.5, 1.0 and 0.5 mm thick Coat-Mix substrates, re-oxidized for 15 minutes with an air flow rate of 1.2 l/min, show that the kinetics of the re-oxidation process is strongly determined by the temperature. 

Also the nearly congruent curves for the re-oxidation of 1.5 mm Coat-Mix substrates at 700 and 800 C (see Figs. 4 (a) and 5 (a)) can be explained by the relation between the two fundamental processes. At 800°C the oxidation kinetics is faster, but a smaller fraction of the substrate is supplied with oxygen. So the influences of both processes neutralize each other. The 1 mm substrate seems to be supplied with oxygen almost completely after a relatively short time, so the difference in oxidation kinetics between 700 and 800°C actually appears in the curves shown in the result section (see Figs. 4 (b) and 5 (b)). 

Noszticzius, Z. Horsthemke, W. McCormick, W. D. Swinney, H. L. 1990. Stirring Effects in the BZ Reaction with Oxalic Acid-Acetone Mixed Substrate in a Batch Reactor and in a CSTR, in Spatial Inhomogeneities and Transient Behavior in Chemical Kinetics (Gray, P. Nicolis, G. Baras, F. Borckmans, P. Scott, S. K., Eds.) Manchester University Press Manchester, U.K. pp. 647-652. 

Mixed Populations. Considerable literature has evolved on mixed populations kinetics. Virtually every possible biological interaction from the classical prey-predator model of Lotka (35) to various forms of commensalism, synergism, etc. have been modelled. Virtually all models end up exhibiting stable oscillations. Solutions are often expressed in triangular phase plane plots of the limiting substrate and the two species. Invariably the plots have a limit cycle as one of the stable steady state solutions. From this one gains the impression that oscillatory behavior is the norm rather than the exception in biological reactors. 

First, let us consider batch mixing processes, as exemplified by ordinaiy laboratory practice in solution kinetics. A portion of one solution (say, of the substrate) is added by pipet to a second solution (containing the reagent) in a flask, the flask is shaken to achieve homogeneity, and then samples are withdrawn at known times for analysis, or the solution is subjected to continuous observation as a function of time, for example, by spectrophotometry. For reactions on a time scale (measured by the half-life) of hours or even several minutes, the time consumed in these operations is a negligible portion of the reaction time, but as the half-life of the reaction decreases, it becomes necessary to consider these preliminary steps. Let us distinguish three stages ... 

Substrate and product inhibitions analyses involved considerations of competitive, uncompetitive, non-competitive and mixed inhibition models. The kinetic studies of the enantiomeric hydrolysis reaction in the membrane reactor included inhibition effects by substrate (ibuprofen ester) and product (2-ethoxyethanol) while varying substrate concentration (5-50 mmol-I ). The initial reaction rate obtained from experimental data was used in the primary (Hanes-Woolf plot) and secondary plots (1/Vmax versus inhibitor concentration), which gave estimates of substrate inhibition (K[s) and product inhibition constants (A jp). The inhibitor constant (K[s or K[v) is a measure of enzyme-inhibitor affinity. It is the dissociation constant of the enzyme-inhibitor complex. 

The performance data for plug versus mix reactor were obtained. The data were collected as the inverse of qx vs inverse of substrate concentration. Table E.1.1 shows the data based on obtained kinetic data. From the data plotted in Figure E.1.1, we can minimise the volume of the chemostat. A CSTR works better than a plug flow reactor for the production of biomass. Maximum qx is obtained with a substrate concentration in the leaving stream of 12g m 3. 

Fig. 2a-c. Kinetic zone diagram for the catalysis at redox modified electrodes a. The kinetic zones are characterized by capital letters R control by rate of mediation reaction, S control by rate of subtrate diffusion, E control by electron diffusion rate, combinations are mixed and borderline cases b. The kinetic parameters on the axes are given in the form of characteristic currents i, current due to exchange reaction, ig current due to electron diffusion, iji current due to substrate diffusion c. The signpost on the left indicates how a position in the diagram will move on changing experimental parameters c% bulk concentration of substrate c, Cq catalyst concentration in the film Dj, Dg diffusion coefficients of substrate and electrons k, rate constant of exchange reaction k distribution coefficient of substrate between film and solution d> film thickness (from ref. 

Acidity of the reaction mixes after incubation increased as the activity of the probe ro —se during determination of pectinesterase activity of the samples.1t was caused by the for—mation of carboxyl groups as a result of pectin ester bonds hydrolysis under pectinesterase ac—tion.That is why kinetic characteristics of substrate hydrolysis were measured according to the speed of pectin hydrolysis by continuously recorded titration of the free carboxyl groups (11). 

Enzyme linked electrochemical techniques can be carried out in two basic manners. In the first approach the enzyme is immobilized at the electrode. A second approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of product to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system which is amenable to in vivo analysis. 

The differences in the rate constant for the water reaction and the catalyzed reactions reside in the mole fraction of substrate present as near attack conformers (NACs).171 These results and knowledge of the importance of transition-state stabilization in other cases support a proposal that enzymes utilize both NAC and transition-state stabilization in the mix required for the most efficient catalysis. Using a combined QM/MM Monte Carlo/free-energy perturbation (MC/FEP) method, 82%, 57%, and 1% of chorismate conformers were found to be NAC structures (NACs) in water, methanol, and the gas phase, respectively.172 The fact that the reaction occurred faster in water than in methanol was attributed to greater stabilization of the TS in water by specific interactions with first-shell solvent molecules. The Claisen rearrangements of chorismate in water and at the active site of E. coli chorismate mutase have been compared.173 It follows that the efficiency of formation of NAC (7.8 kcal/mol) at the active site provides approximately 90% of the kinetic advantage of the enzymatic reaction as compared with the water reaction. 

A typical procedure for setting up a preparative incubation is as follows the substrate solution with a minimal amount of organic solvent or solvent mixture is mixed with buffer (with necessary additives) in an Erlenmeyer flask. Microsomes or S9 is then added to the mixture, followed by addition of NADPH (or UDPGA) stock solution. The flask is incubated at 37 °C in a water bath with gentle shaking. The reaction kinetics in a large-volume incubation will be different from the 0.2-1.OmL incubation, so progress of the reaction should be monitored closely with a short HPLC-UV-MS method. 

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