Kinetics Of Product Formation Research Articles

Kinetic studies of recombinant human interferon-gamma expression in continuous cultures of E. coli

A series of continuous cultures was performed to understand the product formation kinetics of recombinant human interferon gamma (rhIFN-gamma) in Escherichia coli at different dilution rates ranging from 0.1 to 0.3 h(-1) in different media. A T7 promoter-based vector was used for expression of IFN-gamma in E. coli BL21 (DE3) cells. The recombinant protein was produced as inclusion bodies, thus allowing a rapid buildup of rhIFN-gamma inside the cell, with the specific product yield (Y(p/X)) reaching a maximum value of 182 mg g(-1) dry cell weight (DCW). In all the media tested, the specific product formation rate (q(p)) was found to be strongly correlated with the specific growth rate (mu), demonstrating the growth-associated nature of product formation. The q(p) values show no significant decline with time postinduction, even though the recombinant protein has been over produced inside the cell. The maximum q(p) level of 75.5 mg g(-1) h(-1) was achieved at the first hour of induction at the dilution rate of 0.3 h(-1). Also, this correlation between q(p) and mu was not critically dependent on media composition, which would made it possible to grow cells in defined media in the growth phase and then push up the specific growth rate just before induction by pulse addition of glucose and yeast extract. This would ensure the twin objectives of high biomass and high specific productivities, leading to high volumetric product concentration.

Asymmetric Amino Acid Activation by Class II Histidyl-tRNA Synthetase from Escherichia coli

Aminoacyl-tRNA synthetases (ARSs) join amino acids to their cognate tRNAs to initiate protein synthesis. Class II ARS possess a unique catalytic domain fold, possess active site signature sequences, and are dimers or tetramers. The dimeric class I enzymes, notably TyrRS, exhibit half-of-sites reactivity, but its mechanistic basis is unclear. In class II histidyl-tRNA synthetase (HisRS), amino acid activation occurs at different rates in the two active sites when tRNA is absent, but half-of-sites reactivity has not been observed. To investigate the mechanistic basis of the asymmetry, and explore the relationship between adenylate formation and conformational events in HisRS, a fluorescently labeled version of the enzyme was developed by conjugating 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) to a cysteine introduced at residue 212, located in the insertion domain. The binding of the substrates histidine, ATP, and 5'-O-[N-(l-histidyl)sulfamoyl]adenosine to MDCC-HisRS produced fluorescence quenches on the order of 6-15%, allowing equilibrium dissociation constants to be measured. The rates of adenylate formation measured by rapid quench and domain closure as measured by stopped-flow fluorescence were similar and asymmetric with respect to the two active sites of the dimer, indicating that conformational change may be rate-limiting for product formation. Fluorescence resonance energy transfer experiments employing differential labeling of the two monomers in the dimer suggested that rigid body rotation of the insertion domain accompanies adenylate formation. The results support an alternating site model for catalysis in HisRS that may prove to be common to other class II aminoacyl-tRNA synthetases.

Simultaneous saccharification and fermentation of wheat bran flour into ethanol using coculture of amylotic Aspergillus niger and thermotolerant Kluyveromyces marxianus

Studies on simultaneous saccharification and fermentation (SSF) of wheat bran flour, a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation. Experiments based on central composite design (CCD) were conducted to maximize the glucose yield and to study the effects of substrate concentration, pH, temperature, and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungal α-amylase and the above parameters were optimized using response surface methodology (RSM). The optimum values of substrate concentration, pH, temperature, and enzyme concentration were found to be 200 g/L, 5.5, 65°C and 7.5 IU, respectively, in the starch saccharification step. The effects of pH, temperature and substrate concentration on ethanol concentration, biomass and reducing sugar concentration were also investigated. The optimum temperature and pH were found to be 30°C and 5.5, respectively. The wheat bran flour solution equivalent to 6% (w/V) initial starch concentration gave the highest ethanol concentration of 23.1 g/L after 48 h of fermentation at optimum conditions of pH and temperature. The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model. Simultaneous saccharificiation and fermentation of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A. niger and nonamylolytic sugar fermenting K. marxianus.

Enantioselective whole-cell biotransformation of acetophenone to S-phenylethanol by Rhodotorula glutinis. Part II. Aqueous–organic systems: Emulsion and membrane bioreactors

This paper reports the development and performance of a membrane bioreactor for biotransformations (MBB) using organic solvent nanofiltration membranes for the bioreduction of acetophenone to S-phenylethanol by whole cells of Rhodotorula glutinis. It follows a previous paper dealing with the kinetics of this reaction in aqueous media [R. Valadez-Blanco, A.G. Livingston, Enantioselective whole-cell biotransformation of acetophenone to S-phenylethanol by Rhodotorula glutinis. Part I. Product formation kinetics and feeding strategies in aqueous media, Biochem. Eng. J. 46 (2009) 44–53]. A number of organic solvents were tested, with dibutyl ether found to be the best solvent in terms of toxicity and organic/aqueous partition coefficients. Despite the fact that most of the MBBs were mass-transfer limited, their reaction rates were comparable to those of the aqueous systems, with prolonged biocatalytic activity due to in situ product extraction. The use of direct injection of substrate and glucose to the aqueous phase, together with high biomass concentrations, resulted in the highest overall product formation and acceptable reaction rates. This strategy also avoided the need for additional cumbersome downstream processing to recover the product from the aqueous phase. In contrast, direct-contact biphasic reactors proved to be unsuitable for this biotransformation due to poor product partition or high volatility of the solvents.

Enantioselective whole-cell biotransformation of acetophenone to S-phenylethanol by Rhodotorula glutinis: Part I. Product formation kinetics and feeding strategies in aqueous media

This work explores product-formation kinetics of the reduction of acetophenone to ( S)-phenylethanol by Rhodotorula glutinis in aqueous media. Different feeding strategies were investigated to improve biotransformation productivity. An empirical kinetic model was formulated considering two different reaction periods: Phase-1, a very fast chemical-shock reaction in the first 5 min; Phase-2, a subsequent slower reaction which, depending on the product level, could be subinhibitory or inhibitory. The kinetics of the biotransformation metabolism were modulated by the levels of glucose and acetic acid in the bioreactor. In addition, two substrate feeding regimes were compared: intermittent and continuous. Although similar overall productivities were found, the intermittent method clearly holds greater potential for enhanced space-time yields as it takes advantage of the Phase-1 high reaction rates – additions of substrate pulses produce reaction rates of the same order of magnitude as those achieved in Phase-1. However, product accumulation to a “toxic limit” leads to the total cessation of biocatalytic activity, thus highlighting the importance of in-situ product removal during the biotransformation. Strategies for in-situ product removal are addressed in Part II of this study.

A simple kinetic model for growth and biosynthesis of polyhydroxyalkanoate in Bacillus flexus

Polyhydroxyalkanoate (PHA), which is produced by several bacteria, is a biodegradable polymer that has many industrial and medical applications. This study deals with development of a simple kinetic model and modification of the logistic equation that can provide an adequate description of PHA formation process by Bacillus flexus. The parameters studied were kinetics of microbial growth, substrate consumption, and product formation. The microbial growth was described by simplification of Monod's model. A simplified Luedeking-Piret type model could be employed to represent the product kinetics. The kinetic constants were evaluated on the basis of non-linear regression and the differential equations were solved using Runge-Kutta algorithm and MATLAB software. A good agreement was found between the experimental and predicted values, which indicated that the model differential equations could describe the PHA formation and fermentation process. In this study, a modification of the logistic equation has also been attempted for describing the growth of B. flexus.

Estimating the kinetic parameters of activated sludge storage using weighted non-linear least-squares and accelerating genetic algorithm

In this study, weighted non-linear least-squares analysis and accelerating genetic algorithm are integrated to estimate the kinetic parameters of substrate consumption and storage product formation of activated sludge. A storage product formation equation is developed and used to construct the objective function for the determination of its production kinetics. The weighted least-squares analysis is employed to calculate the differences in the storage product concentration between the model predictions and the experimental data as the sum of squared weighted errors. The kinetic parameters for the substrate consumption and the storage product formation are estimated to be the maximum heterotrophic growth rate of 0.12 1/h, the yield coefficient of 0.44 mg COD X/mg COD S (COD, chemical oxygen demand) and the substrate half saturation constant of 16.9 mg/L, respectively, by minimizing the objective function using a real-coding-based accelerating genetic algorithm. Also, the fraction of substrate electrons diverted to the storage product formation is estimated to be 0.43 mg COD STO/mg COD S. The validity of our approach is confirmed by the results of independent tests and the kinetic parameter values reported in literature, suggesting that this approach could be useful to evaluate the product formation kinetics of mixed cultures like activated sludge. More importantly, as this integrated approach could estimate the kinetic parameters rapidly and accurately, it could be applied to other biological processes.

Anaerobic batch degradation of cattail by rumen cultures

The anaerobic batch digestion of cattail with two substrate concentrations of 10 g and 30 g Volatile Solids (VS)/l was investigated in this study. The hydrolysis of cattail was not significantly affected by the substrate concentrations, and it was able to be described by a first-order kinetic model with hydrolysis constants of 0.298 d−1 and 0.294 d−1, respectively. The main aqueous products from cattail degradation were Volatile Fatty Acids (VFA), in which acetate, propionate and butyrate were dominant. The molar ratio of acetate to propionate (3.3) at 10 g VS/l was larger than that (2.3) at 30 g VS/l, when the acidogenesis phase was at the stationary period. The lag period of the methanogenic phase at 10 g VS/l was shorter than that at 30 g VS/l. A modified Gompertz equation could be adopted to describe the kinetics of products formation.

A model forL-methionine production describing oxygen-productivity relationship

BACKGROUND: The state-time profile of cell mass, substrate and methionine concentrations of a methionine synthesis process shows strongly nonlinear features. A mathematical representation of this process was developed that conformed to systems analysis required for monitoring and controlling methionine production. The specific growth rate was defined by an exponential term to describe the lag phase in growth, extended before the onset of methionine production and substrate inhibition observed for this process. A switching function was used to describe the relation between methionine synthesis and dissolved oxygen concentration. In addition, the product formation kinetics of this model described the reutilization of methionine feedback regulation whenever the residual substrate concentration dropped below a critical value. RESULTS: The parameters for the model were determined from experimental data using a nonlinear regression technique. A complete nonlinear systems analysis of the model proved that using this model, the system was controllable and observable. The model prediction of methionine production in controlled and uncontrolled environments was satisfactory. Six statistical measures were employed to validate model prediction and its adequacy was shown through simulations. CONCLUSIONS: The proposed model for methionine production possesses the correct system architecture for application in process control. It predicts satisfactorily the relationship between methionine synthesis and dissolved oxygen, and time profiles of state variables. Copyright © 2008 Society of Chemical Industry

Heterogeneous reactivity of pyrene and 1-nitropyrene with NO 2: Kinetics, product yields and mechanism

The heterogeneous reactivity of nitrogen dioxide with pyrene and 1-nitropyrene (1NP) adsorbed on silica particles has been investigated using a fast-flow-tube in the absence of light. Reactants and products were extracted from particles using pressurised fluid extraction (PFE) and concentration measurements were performed using gas chromatography/mass spectrometry (GC/MS). The pseudo-first order rate constants were obtained from the fit of the experimental decay of particulate polycyclic compound concentrations versus reaction time. Experiments were performed at three different NO 2 concentrations and second order rate constants were calculated considering the oxidant concentration. The following rate constant values were obtained at room temperature: k(NO 2 + pyrene) = (9.3 ± 2.3) × 10 −17 cm 3 molecule −1 s −1 and k(NO 2 + 1NP) = (6.2 ± 1.5) × 10 −18 cm 3 molecule −1 s −1, showing that the reactivity of 1NP was slower by a factor of 15 than that of pyrene. 1NP was identified as the only NO 2-initiated oxidation product of pyrene and all the three dinitropyrenes were identified in the case of the 1NP reaction. The product quantification allowed showing that the kinetics of oxidation product formation was equal to that measured for parent compounds degradation, within uncertainties, confirming the validity of the reaction kinetics measurements.

Effects of glucose and nitrogen source concentration on batch fermentation kinetics ofLactococcus lactisunder hemin-stimulated respirative condition

Analytical solutions to the ordinary differential equations governing the kinetics of cell growth, substrate utilization, and product formation of batch fermentation processes were derived and used to study the kinetics of the hemin-stimulated respiratory cultivation of Lactococcus lactis at varied initial glucose concentrations and nitrogen source concentrations. Studies revealed that initial glucose concentration varying in the range of 60 to 90 g/L had no significant substrate inhibitive effect. Furthermore, elevating the concentration of complex nitrogen sources while maintaining glucose concentration at 60% led to a high final biomass concentration of 6.6 g/L, substantially higher than that obtained with the basic medium, which was 4.1 g/L.

Simpler noninstrumented batch and semicontinuous cultures provide mammalian cell kinetic data comparable to continuous and perfusion cultures

Perfusion culture optimization in multiple noninstrumented small-scale flasks allows reduced expense and time associated with process development. These cultures normally use a different process mode because at small scales it is not practical to retain the cells for medium perfusion. In this work, the kinetics of growth, nutrient consumption, metabolite, and product formation were compared in spinner cultures operated in batch, semicontinuous, chemostat, and perfusion modes. Fed-batch was also included to provide an added comparison. Using logistic fitting for more reliable specific rate estimates in transient conditions, the growth phase of batch cultures predicted similar kinetics to fed-batch and continuous processes. For daily medium exchange rates up to 50%, the semicontinuous mode also predicted the perfusion process kinetics. Differences between the chemostat and semicontinuous culture results were only observed at higher exchange rates with the greatest daily culture perturbation. Overall, the batch or semicontinuous cultures were shown to readily provide results similar to the far more complex to operate chemostat or perfusion cultures.

Mechanisms of Human Immunodeficiency Virus Type 1 Concerted Integration Related to Strand Transfer Inhibition and Drug Resistance

The "strand transfer inhibitors" of human immunodeficiency virus type-1 (HIV-1) integrase (IN), so named because of their pronounced selectivity for inhibiting strand transfer over 3' OH processing, block virus replication in vivo and ex vivo and prevent concerted integration in vitro. We explored the kinetics of product formation and strand transfer inhibition within reconstituted synaptic complexes capable of concerted integration. Synaptic complexes were formed with viral DNA donors containing either two blunt ends, two 3'-OH-processed ends, or one of each. We determined that one blunt end within a synaptic complex is a sufficient condition for low-nanomolar-range strand transfer inhibition with naphthyridine carboxamide inhibitors L-870,810 and L-870,812. We further explored the catalytic properties and drug resistance profiles of a set of clinically relevant strand transfer inhibitor-resistant HIV-1 IN mutants. The diketo acids and naphthyridine carboxamides, mechanistically similar but structurally distinct strand transfer inhibitors, each select for a distinct set of drug resistance mutations ex vivo. The S153Y and N155S IN resistance mutants were selected with the diketo acid L-841,411, and the N155H mutant was selected with L-810,812. Each mutant exhibited some degree of catalytic impairment relative to the activity of wild type IN, although the N155H mutant displayed near-wild-type IN activities. The resistance profiles indicated that the S153Y mutation potentiates susceptibility to L-870,810 and L-870,812, while the N155S mutation confers resistance to L-870,810 and L-870,812. The N155H mutation confers resistance to L-870,810 and potentiates susceptibility to L-841,411. This study illuminates the interrelated mechanisms of concerted integration, strand transfer inhibition, and resistance to strand transfer inhibitors.

Examination of the influence of molecular weight on polymer laser ablation: polystyrene at 248 nm

Following previous studies on the influence of the polymer molecular weight (MW) on the ablation of poly(methyl methacrylate) (PMMA) at 248 nm, this work extends the examination to the ablation of polystyrene (PS) at 248 nm. The ablation threshold and the etching rates are found to be nearly independent of MW. Optical microscopy demonstrates an excellent crater morphology, few small bubbles are formed on the surface of the low MW. Examination of the formation kinetics of products in the irradiation of samples doped with the photoreactive iodophenanthrene demonstrates that high temperatures develop upon irradiation, suggesting that thermal mechanism dominates in the ablation of PS at 248 nm. In similarity to the etching rates, the attained temperatures are largely independent of the PS MW. The factors for the weak dependence of the process on PS MW are discussed.

Elucidation of 2-hydroxybiphenyl effect on dibenzothiophene desulfurization by Microbacterium sp. strain ZD-M2

The effect of 2-hydroxybiphenyl (2-HBP), the end product of dibenzothiophene (DBT) desulfurization via 4S pathway, on cell growth and desulfurization activity was investigated by Microbacterium sp. The experimental results indicate that 2-HBP would inhibit the desulfurization activity. Providing 2-HBP was added in the reaction media, the DBT degradation rate decreased along with the increase of 2-HBP addition. By contrast, cell growth would be promoted in the addition of 2-HBP at a low concentration (<0.1mM). At high concentration of 2-HBP, the inhibition on the cell growth occurred. Meanwhile, the inhibitory effect of 2-HBP on DBT desulfurization activity was tested both in the oil/aqueous two-phase system and the aqueous system. A mathematical model was developed to explain the product formation kinetics with DBT as the sole sulfur source. The predicted results were close to the experimental data, it elucidated that along with the 2-HBP accumulation, the inhibitory effect of 2-HBP on DBT desulfurization and cell growth was enhanced.

Kinetics of growth and leukotoxin production by Mannheimia haemolytica in continuous culture

The growth and product formation kinetics of the bovine pathogen Mannheimia (Pasteurella) haemolytica strain OVI-1 in continuous culture were investigated. The leukotoxin (LKT) concentration and yield on biomass could substantially be enhanced by supplementation of a carbon-limited medium with an amino acid mixture or a mixture of cysteine and glutamine. Acetic acid was a major product, increasing to 1.66 g l(-1) in carbon-limited chemostat culture at intermediate dilution rates and accounting for more than 80% of the glucose carbon, whereas in amino acid-limited cultures high acetic acid concentrations were produced at low dilution rates, suggesting a carbon-overflow metabolism. The maintenance coefficients of carbon-limited and carbon-sufficient cultures were 0.07 and 0.88 mmol glucose g(-1) h(-1), respectively. LKT production was partially growth-associated and the LKT concentration was maximised to 0.15 g l(-1) and acetic acid production minimised by using a carbon-limited medium and a low dilution rate.

Skeletal isomerization of 1-butene: A thorough kinetic study over ZSM-22

Zeolites and mesoporous materials are important catalysts not only for oil-refining processes, but nowadays even for the production of fine and specialty chemicals. Few thorough studies on the important factors in chemical reaction engineering: kinetics, deactivation, mass and heat transfer and flow modeling have been carried out. In the present work, a comprehensive analysis of 1-butene skeletal isomerization over ZSM-22 was conducted. Kinetics of the main product formation, i.e. isobutene, as well as side-products, i.e. propene and pentene, was modeled using Langmuir–Hinshelwood approach. The axial-dispersion model was used for describing the reactor. The behavior of the main products and byproducts was successfully explained. Uniform coking was applied to describe the deactivation behavior of the reaction and the formation of byproducts.

Product formation kinetics in genetically modified E. coli bacteria: inclusion body formation

A data-driven model is presented that can serve two important purposes. First, the specific growth rate and the specific product formation rate are determined as a function of time and thus the dependency of the specific product formation rate from the specific biomass growth rate. The results appear in form of trained artificial neural networks from which concrete values can easily be computed. The second purpose is using these results for online estimation of current values for the most important state variables of the fermentation process. One only needs online data of the total carbon dioxide production rate (tCPR) produced and an initial value x of the biomass, i.e., the size of the inoculum, for model evaluation. Hence, given the inoculum size and online values of tCPR, the model can directly be employed as a softsensor for the actual value of the biomass, the product mass as well as the specific biomass growth rate and the specific product formation rate. In this paper the method is applied to fermentation experiments on the laboratory scale with an E. coli strain producing a recombinant protein that appears in form of inclusion bodies within the cells' cytoplasm.

Cytochrome b5 Increases the Rate of Product Formation by Cytochrome P450 2B4 and Competes with Cytochrome P450 Reductase for a Binding Site on Cytochrome P450 2B4

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios <or=1:1, the rate of metabolism of cyclohexane and benzphetamine is enhanced, whereas at higher cyt b(5):cyt P450 molar ratios, cyt b(5) progressively inhibits both NADPH consumption and the rate of metabolism. It is proposed that the ability of cyt b(5) to enhance substrate metabolism by cyt P450 is related to its ability to increase the rate of catalysis and that the inhibitory properties of cyt b(5) are because of its ability to occupy the reductase-binding site on cyt P450 2B4, thereby preventing reduction of ferric cyt P450 and initiation of the catalytic cycle. It is proposed that cyt b(5) and CPR compete for a binding site on cyt P450 2B4.

Formation Kinetic Study of Thermal Products of Tocopherols

The kinetic analyses for thermal products of alpha-, gamma- and delta-tocopherols during heating as functions of temperature and time were studied. Alpha-, gamma- and delta-tocopherols dissolved in glycerol were heated at 100～200℃ for 5～60 min. The thermal products were separated by hexane extraction and analyzed by HPLC using a reversed phase μ-Bondapak C₁?-column with two kinds of elution solvents in a gradient mode. The formation kinetics of thermal products of tocopherols followed a first-order kinetic model. The formation rate of thermal products of tocopherols was dependent on heating temperatures and heating times. The activation energy and enthalpy for the thermal products of γ-and δ-tocopherols were higher than those for α-tocopherol as in the case of the oxidative degradation kinetics of tocopherol. The magnitude order of the activation energy was γ-＞δ-＞α-tocopherol.