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PubMed1995

A new approach to determine the effect of mismatches on kinetic parameters in DNA hybridization using an optical biosensor.

We have demonstrated a simple yet direct method for determining the kinetic parameters in DNA-DNA interactions using biosensor technology based on the surface plasmon resonance phenomenon; a technique that does not require complex DNA labeling. To determine the effect of mismatches on the kinetics involved in DNA-DNA interactions, DNA hybridization kinetics were monitored in real time using synthetic oligonucleotides less than 20 bases in length which contained either a complementary sequence or mismatched bases. Upon analysis of the kinetic parameters obtained in oligonucleotide hybridization, we found that they were significantly affected by the presence of mismatches as well as by their number and location in a DNA duplex. In addition, the presented biosensor method is sensitive enough to detect kinetic effects caused by the presence of a single-mismatched base pair. Our findings strongly suggest that analysis of kinetic parameters involved in DNA-DNA interactions is advantageous for detecting the presence of mismatch base pairs in a DNA duplex.

Base Sequence↗

PubMed1995

Ligand binding kinetics of IL-2 and IL-15 to heteromers formed by extracellular domains of the three IL-2 receptor subunits.

Studies on the binding of IL-2 to its receptor (IL-2R) have generally been limited to receptors expressed on cell surfaces. This has hampered detailed kinetic and mechanistic studies at the molecular level. We have prepared the soluble extracellular domains of all three receptor subunits (called alpha, beta and gamma) by recombinant techniques and have used these to perform detailed kinetic studies of their binding properties using the technique of surface plasmon resonance. We describe a novel approach whereby the receptors are assembled on an antibody surface, being held by an epitope engineered into the C-terminus of each of these domains. Thus the receptors are oriented naturally leading to homogeneous ligand binding kinetics. We have characterized the interactions of the heteromeric complexes of these subunits with mouse and human IL-2 and their analogs, as well as the recently discovered cytokine, IL-15. We have also studied the extracellular domains of the mouse receptor subunits for the first time and have used these as well as mouse-human hybrid receptors to probe the mechanism of assembly of these complexes. We show that no additional proteins are required to reproduce the properties of these complexes in vitro. In addition, kinetic studies with site-specific analogs of IL-2 and the mouse-human receptor hybrids clearly indicate that the extracellular domains of alpha and beta can together readily bind ligand with kinetic properties distinct from those of the constituent subunits. In contrast, a complex containing ligand and the extracellular domains of beta and gamma was comparatively difficult to assemble and required prolonged exposure to IL-2. Our method enabled us to calculate the stoichiometry of these complexes and to determine that anchoring these subunits is necessary to efficiently drive complex formation. The kinetic and equilibrium differences between the mouse and human receptor complexes, and between IL-2 and IL-15 binding to these receptors clarify the roles of the alpha and gamma subunits in the differential response of cells to different cytokines that may be present simultaneously in the environment.

Amino Acid Sequence↗

PubMed1993

The kinetic properties of switch antimetabolites.

BACKGROUND: Maintaining homeostasis is the biological function of negative feedback, a process that plays a well-understood role in the biochemistry of antimetabolite drugs. An equally important property of living systems--the ability to respond to external stimuli by switching rapidly from one state to another--is mediated by positive feedback. Kinetic analysis of multi-enzyme biochemical pathways has shown that pathways containing positive feedback coupled with negative feedback may act as biochemical switching systems in which multiple steady states are possible. PURPOSE: A computer model was used to study the kinetic effects of antimetabolites that inhibit biochemical pathways at positive feedback sites and to determine whether the kinetics of such systems differed from those of classical antimetabolites. METHODS: Kinetics were simulated by microcomputer to model the effects of inhibitors on a simplified metabolic pathway. RESULTS: Antimetabolite drugs that act at positive feedback sites are predicted to display highly nonclassical properties. Three nonclassical properties are kinetically possible. First, the drugs may switch off the pathway at substoichiometric concentrations; classical antimetabolites require stoichiometric levels and sometimes much more. Second, instead of demonstrating classical continuous inhibition, antimetabolite drugs that act at positive feedback sites may give "all-or-none" dose-response curves with discontinuity at a specific value. Inhibitor concentrations below this value would have no overall effect on the system, while inhibitor concentrations at or above this value would give an abrupt transition to an inhibited steady state. Third, the inhibited system may show hysteresis and remain switched off after the inhibitor is removed. CONCLUSIONS: These findings suggest that antimetabolites acting at switch points could have kinetic properties very different from those of classical antimetabolites and could provide a noncytotoxic method of switching off pathways in neoplastic cells, perhaps leading to cell stasis. IMPLICATIONS: Experimental validation of these conclusions will require identification of the positive feedback sites of metabolic and signaling pathways and exploration of the effects of inhibitors of these sites. The properties of "switch antimetabolites" should prompt a search for new kinds of targets for drug design.

Antimetabolites, Antineoplastic↗

PubMed2006

Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion.

The tethered particle motion (TPM) allows the direct detection of activity of a variety of biomolecules at the single molecule level. First pioneered for RNA polymerase, it has recently been applied also to other enzymes. In this work we employ TPM for a systematic investigation of the kinetics of DNA looping by wild-type Lac repressor (wt-LacI) and by hinge mutants Q60G and Q60 + 1. We implement a novel method for TPM data analysis to reliably measure the kinetics of loop formation and disruption and to quantify the effects of the protein hinge flexibility and of DNA loop strain on such kinetics. We demonstrate that the flexibility of the protein hinge has a profound effect on the lifetime of the looped state. Our measurements also show that the DNA bending energy plays a minor role on loop disruption kinetics, while a strong effect is seen on the kinetics of loop formation. These observations substantiate the growing number of theoretical studies aimed at characterizing the effects of DNA flexibility, tension and torsion on the kinetics of protein binding and dissociation, strengthening the idea that these mechanical factors in vivo may play an important role in the modulation of gene expression regulation.

Bacterial Proteins↗

PubMed2001

Quantitative studies of bone using (18)F-fluoride and (99m)Tc-methylene diphosphonate: evaluation of renal and whole-blood kinetics.

We report a study of the renal and whole-blood kinetics of (18)F-fluoride and (99m)Tc-methylene diphosphonate ((99m)Tc-MDP) and their effect on the evaluation of the skeletal kinetics of the two bone tracers. Data were obtained during an investigation of postmenopausal women taking hormone replacement therapy who were compared with untreated, age-matched controls. After intravenous injection of 18F-fluoride (1 MBq), (99m)Tc-MDP (1 MBq), (51)Cr-ethylenediaminetetraacetic acid (51Cr-EDTA) (3 MBq) and (125)I-human serum albumin ((125)I-HSA) (0.25 MBq), multiple blood samples and urine collections were taken between 0 and 4 h after injection. (51)Cr-EDTA data were used to evaluate the glomerular filtration rate (GFR) and the completeness of each timed urine collection. (125)I-HSA data were used to evaluate the plasma volume and the red cell uptake of the other three tracers. At 4 h, the cumulative urine excretions (and standard deviations, SDs) were: (99m)Tc-MDP, 58.2% (4.8%); (18)F-fluoride, 36.1% (5.7%); (51)Cr-EDTA, 81.5% (4.5%). Plots of the renal clearance of (18)F-fluoride against urine volume showed that urine flow rates greater than 5 ml.min-1 were necessary to ensure a constant renal clearance of (18)F and hence stable conditions for the evaluation of bone tracer kinetics. In contrast, a low urine flow rate had no effect on the renal kinetics of (99m)Tc-MDP. For MDP, the evaluation of skeletal kinetics requires data on protein binding so that calculations can be performed for free MDP. In the present study, protein binding of MDP was evaluated from the ratio of total (99m)Tc-MDP renal clearance to GFR based on the principle that free (99m)Tc-MDP is a GFR tracer. Between 0 and 4 h after injection, the fractional protein binding of MDP increased linearly with time, changing from 21+/-5% immediately after injection to 58+/-5% at 4 h. Although red cell uptake of (99m)Tc-MDP was negligible, for (18)F-fluoride around 30% of circulating tracer was transported in red cells. In view of the data showing the rapid transport of (18)F-fluoride across the red cell membrane, bone kinetic data for (18)F are more accurately reported as whole-blood clearance rather than plasma clearance.

Aged↗

PubMed1999

Predicting protein decomposition: the case of aspartic-acid racemization kinetics.

The increase in proportion of the non-biological (D-) isomer of aspartic acid (Asp) relative to the L-isomer has been widely used in archaeology and geochemistry as a tool for dating. the method has proved controversial, particularly when used for bones. The non-linear kinetics of Asp racemization have prompted a number of suggestions as to the underlying mechanism(s) and have led to the use of mathematical transformations which linearize the increase in D-Asp with respect to time. Using one example, a suggestion that the initial rapid phase of Asp racemization is due to a contribution from asparagine (Asn), we demonstrate how a simple model of the degradation and racemization of Asn can be used to predict the observed kinetics. A more complex model of peptide bound Asx (Asn + Asp) racemization, which occurs via the formation of a cyclic succinimide (Asu), can be used to correctly predict Asx racemization kinetics in proteins at high temperatures (95-140 degrees C). The model fails to predict racemization kinetics in dentine collagen at 37 degrees C. The reason for this is that Asu formation is highly conformation dependent and is predicted to occur extremely slowly in triple helical collagen. As conformation strongly influences the rate of Asu formation and hence Asx racemization, the use of extrapolation from high temperatures to estimate racemization kinetics of Asx in proteins below their denaturation temperature is called into question. In the case of archaeological bone, we argue that the D:L ratio of Asx reflects the proportion of non-helical to helical collagen, overlain by the effects of leaching of more soluble (and conformationally unconstrained) peptides. Thus, racemization kinetics in bone are potentially unpredictable, and the proposed use of Asx racemization to estimate the extent of DNA depurination in archaeological bones is challenged.

Aspartic Acid↗

PubMed2005

Direct reconstruction of kinetic parameter images from dynamic PET data.

Our goal in this paper is the estimation of kinetic model parameters for each voxel corresponding to a dense three-dimensional (3-D) positron emission tomography (PET) image. Typically, the activity images are first reconstructed from PET sinogram frames at each measurement time, and then the kinetic parameters are estimated by fitting a model to the reconstructed time-activity response of each voxel. However, this "indirect" approach to kinetic parameter estimation tends to reduce signal-to-noise ratio (SNR) because of the requirement that the sinogram data be divided into individual time frames. In 1985, Carson and Lange proposed, but did not implement, a method based on the expectation-maximization (EM) algorithm for direct parametric reconstruction. The approach is "direct" because it estimates the optimal kinetic parameters directly from the sinogram data, without an intermediate reconstruction step. However, direct voxel-wise parametric reconstruction remained a challenge due to the unsolved complexities of inversion and spatial regularization. In this paper, we demonstrate and evaluate a new and efficient method for direct voxel-wise reconstruction of kinetic parameter images using all frames of the PET data. The direct parametric image reconstruction is formulated in a Bayesian framework, and uses the parametric iterative coordinate descent (PICD) algorithm to solve the resulting optimization problem. The PICD algorithm is computationally efficient and is implemented with spatial regularization in the domain of the physiologically relevant parameters. Our experimental simulations of a rat head imaged in a working small animal scanner indicate that direct parametric reconstruction can substantially reduce root-mean-squared error (RMSE) in the estimation of kinetic parameters, as compared to indirect methods, without appreciably increasing computation.

Algorithms↗

PubMed2003

Heat-induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first-order kinetics.

The heat-induced denaturation kinetics of two different sources of ovalbumin at pH 7 was studied by chromatography and differential scanning calorimetry. The kinetics was found to be independent of protein concentration and salt concentration, but was strongly dependent on temperature. For highly pure ovalbumin, the decrease in nondenatured native protein showed first-order dependence. The activation energy obtained with different techniques varied between 430 and 490 kJ*mole(-1). First-order behavior was studied in detail using differential scanning calorimetry. The calorimetric traces were irreversible and highly scan rate-dependent. The shape of the thermograms as well as the scan rate dependence can be explained by assuming that the thermal denaturation takes place according to a simplified kinetic process where N is the native state, D is denatured (or another final state) and k a first-order kinetic constant that changes with temperature, according to the Arrhenius equation. A kinetic model for the temperature-induced denaturation and aggregation of ovalbumin is presented. Commercially obtained ovalbumin was found to contain an intermediate-stable fraction (IS) of about 20% that was unable to form aggregates. The denaturation of this fraction did not satisfy first-order kinetics.

Animals↗

PubMed1980

Analysis of the proliferation kinetics of Burkitt's lymphoma cells.

The in vitro proliferation kinetics of a cell line derived from a patient with American Burkitt's lymphoma were investigated at three different growth phases: lag (day 1), exponential (day 3) and plateau (day 5). The growth curve, labeling and mitotic indices, percentage labeled mitosis (PLM) curves and DNA content distributions were determined. The data obtained have been analysed by the previously developed discrete-time kinetic (DTK) model by which a time course of DNA distributions during a 10-day growth period was characterized in terms of other cell kinetic parameters. The mean cell cycle times, initially estimated from PLM curves on days 1, 3 and 5, were further analysed by the DTK model of DNA distributions and subsequently the mean cell cycle times with respect to DNA distributions during the entire growth period were determined. The doubling times were 39.6, 31.2 and 67.2 hr, respectively, at days 1, 3 and 5. The mean cell cycle time increased from 23.0 to 37.7 hr from day 3 to day 5 mainly due to an elongation of the G1 and G2 phases. A slight increase in the cell loss rate from 0.0077 to 0.0081 fraction/hr was accompanied by a decrease in the cell production rate from 0.0299 to 0.0184 fraction/hr. This calculated cell loss rate correlated significantly with the number of dead cells determined by trypan blue exclusion. Analysis of the number of dead cells in relation to the cell cycle stage revealed that a majority of cell death occurred in G1 (r + 0.908; P < 0.0001). There was a good correlation between the in vitro proliferation kinetics at plateau phase of this Burkitt's lymphoma derived cell line and the in vivo proliferation kinetics of African Burkitt's lymphoma (Iversen et al., 1974), suggesting the potential utility of information obtained by in vitro kinetic studies.

Burkitt Lymphoma↗

PubMed1980

The number of functional active sites per molecule of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, as determined by a kinetic method.

1. A kinetic approach to the determination of the number of functional active sites per molecule of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, is described. 2. Time courses for formation and breakdown of a cob(II)alamin intermediate during reaction of the enzyme, fully saturated with adenosylcobalamin, with L-2-aminopropanol as substrate, were followed using a stopped-flow spectrophotometer under two conditions: (a) enzyme concentration much greater than that of substrate, (b) substrate concentration much greater than that of enzyme. 3. Results were analysed in terms of a three-step mechanism involving binding of substrate (k+1 step), cob(II)alamin formation (k+2 step) and cob(II)alamin breakdown (k+3 step). the kinetic scheme was shown to be sufficient to account for the observed time courses and rate constants of 80 s-1 (k+2) and 1.5 s-1 (k+3) were determined. 4. The number of active sites per enzyme molecule (n) was calculated from the kinetic data in three ways: (a) calculation from amplitude of absorbance measurement, (b) calculation from measurements of the values of rate constants and (c) analysis by computation of the kinetic data using the computer program FACSIMILE. A value for n close to 6 was calculated by each of these methods. This value is in disagreement with the literature value of about two sites per molecule but is consistent with the I6II6 subunit structure of the enzyme. 5. Kinetic analysis of data from experiments in which the adenosylcobalamin concentration was varied while substrate and enzyme concentrations remained constant showed that all the active sites function with identical rate constants. 6. The principle and mathematical basis of the kinetic method for determining the value of n is given as an Appendix.

Ammonia-Lyases↗

PubMed1982

A coupled diffusion and barrier model for the recombination kinetics of myoglobin with carbon monoxide.

The rebinding kinetics of myoglobin (Mb) with carbon monoxide (CO) after photodissociation have been analyzed using a single energy barrier coupled by a two-dimensional channel to the solvent. The total binding rate is written as the product of the barrier rate coefficient and the probability of the ligand being at the barrier. Assuming that the photodissociated ligands are initially localized near the barrier, the probability then decays as 1 t as the ligands migrate away toward the solvent. The time-dependent rate coefficient yields power-law kinetics as observed in the MbCO recombination kinetics. At a given temperature two parameters are sufficient to simulate power-law kinetics of arbitrary slope. Using a model of diffusion from a region of lower to higher diffusivity, four parameters can simulate an initial power law followed by a final slow exponential process. Estimates of the diffusion coefficients inside the protein are two to three orders of magnitude slower than the ligand diffusivity in the external solvent. A single Arrhenius rate coefficient for the barrier can be used for the entire range from 60 K to 320 K. The barrier diffusion model implies a certain shape of the kinetics which is in good agreement with the observed recombination kinetics of many heme protein ligand systems.

Calorimetry↗

PubMed1983

Isoleucyl-tRNA synthetase from Baker's yeast. Catalytic mechanism, 2',3'-specificity and fidelity in aminoacylation of tRNAIle with isoleucine and valine investigated with initial-rate kinetics using analogs of tRNA, ATP and amino acids.

The aminoacylation of three modified tRNAIle species with isoleucine and with valine by isoleucyl-tRNA synthetase has been investigated by initial rate kinetics. For aminoacylation of tRNAIle-C-C-3'dA with isoleucine, a bi-bi uni-uni ping-pong mechanism has been found by bisubstrate kinetics and inhibition by products and by 3'dATP; for aminoacylation with valine a bi-uni uni-bi ping-pong mechanism. For isoleucylation of tRNAIle-C-C-A(3'NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni-bi uni-uni-uni ping-pong mechanism; for valylation of this tRNA a bi-bi uni-uni ping-pong mechanism is observed by bisubstrate kinetics and product inhibition. tRNAIle-C-C-2'dA was aminoacylated under modified conditions with isoleucine in a bi-bi uni-uni ping-pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P[NH]P as product analog and by isoleucinol. Aminoacylation with valine is achieved in a rapid-equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3'dATP and valinol. All six reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle-C-C-A. The Km values of the three substrates and the kcat values of the six reactions are given. For aminoacylation at the terminal 2'OH group of the tRNA differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine have been calculated which result in discrimination factors of 181 and 167. For aminoacylation at the terminal 3'-OH group a difference of only 4.43 kJ and a low discrimination factor of only 6 is observed. Thus maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2'-OH group and conclusions drawn from experiments with modified tRNAs concerning 2',3'-specificity have led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNAs. The stability of Ile-tRNAIle-C-C-2'dA and Val-tRNAIle-C-C-2'dA, the lesser stability of Val-tRNAVal-C-C-2'dA and the instability of Thr-tRNAVal-C-C-2'dA are consistent with postulations for a 'pre-transfer' proofreading step for isoleucyl-tRNA synthetase and a 'post-transfer' hydrolytic editing step for valyl-tRNA synthetase at the terminal 3'OH group of the tRNA.

Amino Acyl-tRNA Synthetases↗

PubMed1983

Steady-state kinetics of coupled two-enzyme reactor with recycling of poly(ethylene glycol)-bound NAD.

The kinetic properties of a continuous enzyme reactor containing rabbit muscle lactate dehydrogenase, horse liver alcohol dehydrogenase and poly(ethylene glycol)-bound NAD (PEG-NAD) were investigated experimentally and theoretically. The enzymes and PEG-NAD were retained in the reactor with an ultrafiltration membrane, and the substrates (pyruvate and ethanol) were fed continuously. The reactions of the dehydrogenases were coupled by the recycling of the cofactor. The steady-state concentration of L-lactate, one of the products, was measured under different experimental conditions and compared with the corresponding theoretical value. The theoretical value was calculated based on a simplified ordered bi-bi mechanism for the individual enzyme reactions, of which kinetic constants were determined by independent kinetic studies. Differences were found between the kinetic constants of the enzymes for NAD(H) and PEG-NAD(H). The steady-state values obtained by continuous operation were lower than those calculated, possibly due to the simplification made for the kinetic model; but there was general agreement between them in the dependence on the experimental conditions. The steady-state behavior of the enzyme reactor was explained semi-quantitatively by the simple kinetic model.

Alcohol Dehydrogenase↗

PubMed1994

Kinetics of the two forms of acetyl-CoA carboxylase from Pisum sativum. Correlation of the substrate specificity of the enzymes and sensitivity towards aryloxyphenoxypropionate herbicides.

Steady-state kinetics of the 220-kDa form of acetyl-CoA carboxylase (ACC220), as purified from mature pea seeds, have been investigated with respect to the substrate specificity and inhibition by quizalofop, a herbicide of the aryloxyphenoxypropionate type. The enzyme showed a dual specificity, being able to carboxylate propionyl-CoA at a maximal rate approximately 20% that measured in the presence of the acetyl-CoA substrate. These two reactions occur at separate sites on the enzyme. One site binds either acetyl-CoA (Km = 226 microM) or propionyl-CoA (Km = 38 microM) and is strongly inhibited by quizalofop (Ki = 25 microM and 9.3 microM for the acetyl-CoA and propionyl-CoA substrates, respectively). The other is specific for acetyl-CoA (Km = 11 microM) and is much less inhibited by quizalofop (Ki = 256 microM). Owing to the existence of these two catalytically different sites, the enzyme obeyed Michaelis-Menten kinetics with propionyl-CoA, but exhibited kinetic co-operativity in the presence of acetyl-CoA. Also, kinetics of propionyl-CoA carboxylase activity of ACC220 exhibited hyperbolic inhibition in the presence of quizalofop, but co-operative inhibition when following the ACC activity of the enzyme. The results suggest that the higher the substrate specificity, the lower the quizalofop sensitivity of the active site. Similar kinetic behaviour was observed with ACC220 purified from pea leaves. Also, the apparent correlation between the substrate specificity and the sensitivity of ACC towards quizalofop was confirmed by kinetic analyses of the low-molecular-mass form of ACC present in chloroplasts of young pea leaves. This enzyme was insensitive to quizalofop inhibition and was not able to carboxylate propionyl-CoA. No other propionyl-CoA carboxylase activity, different from that catalysed by ACC220, could be detected from either reproductive or vegetative organs of pea plants at any stage of development.

Acetyl Coenzyme A↗

PubMed1996

Kinetic analysis of synthetic analogues of linear-epitope peptides of glycoprotein D of herpes simplex virus type 1 by surface plasmon resonance.

The interaction between mAb A16 and glycoprotein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface-plasmon-resonance biosensor. mAb A16 belongs to group VII antibodies, which recognize residues 11-19 of gD. In a previous study, three critical residues, Asp13, Arg16 and Phe17, of this epitope were identified by screening a phage display library that contained a random 15-amino-acid insert with the antibody. The contribution to binding of these residues in the motif DXXRF was further analyzed by an amino-acid-replacement study of the epitope gD-(9-19)-peptide and of a gD-(9-19)-peptide mimotope, previously obtained by screening the phage display library. Amino acid residues of the motif were replaced by a neutral amino acid residue, an amino acid residue with opposite charge and a corresponding D-amino acid residue. Kinetic parameters of peptide analogues were determined with a surface plasmon-resonance biosensor. The kinetic parameters of the peptide analogues were compared with the kinetic parameters of the interaction between mAb A16 and the epitope gD-(9-19)-peptide. The minimal size of the gD epitope for mAb A16 was also determined in this study. The kinetic constants of the resulting gD-(11-17)-peptide were found to be similar to those of entire gD. The kinetic analysis precisely defined the epitope on gD for mAb A16 to residues 11-17, identified Arg16 as an essential residue and suggested that Asp13 and Phe17 are mainly involved in stabilization of the secondary structure of the peptide.

Amino Acid Sequence↗

PubMed2005

Kinetics of intra- and intermolecular zymogen activation with formation of an enzyme-zymogen complex.

A mathematical description was made of an autocatalytic zymogen activation mechanism involving both intra- and intermolecular routes. The reversible formation of an active intermediary enzyme-zymogen complex was included in the intermolecular activation route, thus allowing a Michaelis-Menten constant to be defined for the activation of the zymogen towards the active enzyme. Time-concentration equations describing the evolution of the species involved in the system were obtained. In addition, we have derived the corresponding kinetic equations for particular cases of the general model studied. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. The validity of the results obtained were checked by using simulated progress curves of the species involved. The model is generally good enough to be applied to the experimental kinetic study of the activation of different zymogens of physiological interest. The system is illustrated by following the transformation kinetics of pepsinogen into pepsin.

Animals↗

PubMed2004

Growth kinetics of aerobic granules developed in sequencing batch reactors.

AIMS: This paper attempts to develop a kinetic model to describe the growth of aerobic granules developed under different operation conditions. METHODS AND RESULTS: A series of experiments were conducted by using four-column sequencing batch reactors to study the formation of aerobic granules under different conditions, e.g. organic loading rates, hydrodynamic shear forces and substrate N/COD ratios. A simple kinetic model based on the Linear Phenomenological Equation was successfully derived to describe the growth of aerobic granules. It was found that the growth of aerobic granules in terms of equilibrium size and size-dependent growth rate were inversely related to shear force imposed to microbial community, while a high organic loading favoured the growth of aerobic granules, leading to a large size granule. The effect of substrate N/COD ratio on the growth kinetics of aerobic granules was realized through change in microbial populations, and enriched nitrifying population in aerobic granules developed at high substrate N/COD ratio resulted in a low overall growth rate of aerobic granules. CONCLUSIONS: The proposed model can provide good prediction for the growth of aerobic granules indicated by the correlation coefficient >0.95. SIGNIFICANCE AND IMPACT OF THE STUDY: The kinetic model proposed could offer a useful tool for studying the growth kinetics of cell-to-cell immobilization process. The study confirmed that the growth of aerobic granules and biofilms are subject to a similar kinetic pattern. This work would also be helpful for better understanding the mechanism of aerobic granulation.

Aerobiosis↗

PubMed2005

Morphology and kinetics studies on cephalosporin C production by Cephalosporium acremonium M25 in a 30-l bioreactor using a mixture of inocula.

AIMS: In this study, the relationship between morphology and cephalosporin C (CPC) production in a 30-l bioreactor culture of Cephalosporium acremonium M25 using a 3:7 seed mixture was investigated. In addition, the kinetic model was established and applied. METHODS AND RESULTS: CPC production was performed in a 30-l bioreactor using a 3:7 seed mixture. It was recognized that a 3:7 seed mixture was able to reduce lag phase and enhance CPC production. The maximum CPC production and cell mass were 1.96 and 81.5 g l-1 respectively. Through a morphology study by observation using image analysis, it was concluded that changes of morphological features predicted the progressive production of CPC and that a morphology study could be useful in monitoring the CPC fermentation by C. acremonium M25. In the kinetics study, a kinetic model of CPC fermentation was developed and applied. The proposed model could adequately describe the fermentation of C. acremonium M25 in a 30-l bioreactor. CONCLUSIONS: CPC productivity was improved by using a 3:7 seed mixture in a 30-1 bioreactor. The changes in morphological features showed a very similar tendency with CPC production. A kinetic model of CPC fermentation was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study suggest that the use of a 3:7 seed mixture inocula has considerable possibilities for improving CPC productivity if applied to industrial scale fermentations. Through morphology and kinetics study, the kinetic model to describe the morphological differentiation and CPC production by C. acremonium M25 was established.

Acremonium↗