Initial Substrate Concentration Research Articles

Purification and Production Optimization of Alpha Amylases from Bacillus Species

Amylases are the digestive enzymes belong to the family of glycoside hydrolase capable of hydrolysing starch and polysaccharides. These are the recent breakthroughs in food, feed, pulp and pharma industries. Our aim of this study is to purify amylases from Bacillus subtilis and Bacillus megaterium and optimize the growth conditions to achieve the cost-effective production of amylases from bacillus species. Extensive research on amylases from different sources is essential in producing enzymes in large scale and in optimizing the growth conditions and minimize the production cost to meet the increased demand of these industrially important enzymes. Amylases were produced in the current study using submerged fermentation and purified the extracellular enzymes from bacillus species using ultracentrifugation, Ammonium sulphate precipitation and gel filtration chromatography. There was no effect on amylase production from bacillus species when the initial substrate concentration is increased from 1% to 5%. Increase in inoculum size from 1% to 5% and the addition of yeast extract to broth had significantly increased the amylase production. The study observed significantly higher levels of amylase production using rice starch when compared with wheat starch also higher levels of amylase production observed with B. megaterium when compared with B. subtilis. The study concludes 1% rice starch and 5% B. megaterium inoculum can be used for cost effective production of amylases. In the current study, cost effective amylase production was evaluated and able to optimize the yield of extracellular amylases from Bacillus megaterium to a concentration level of 120 IU/ml of fermentation broth.

Removal of Safranin O from Water by UV/TiO2/IO3− Advanced Oxidation Process: Parametric Study and Impact of Inorganic Ions and Humic Acid

In the present paper, the influence of iodate (IO3−) ions on the photocatalytic process using titanium dioxide (UV/TiO2) was systematically examined. The innovative studied system (UV/TiO2/IO3−) demonstrated high performance in the elimination of safranin O (SO), a model dye, from water due to the implication of iodine radicals (IO3•, IO2•, IO•, etc.). The degradation was assessed by monitoring the change in initial substrate concentration (5–30 mg∙L−1), TiO2 loading (0.01–3 g∙L−1), IO3− concentration, liquid temperature, and initial pH. Further enhancement of oxyanion amount was beneficial for the initial rate of degradation (r0) over the range 0.1–50 mM, such that r0 rose from 0.724 to 1.12 mg∙L−1∙min−1. However, a concentration of 100 mM IO3− slowed the removal kinetics. Low pH values were found to be favorable for pollutant removal. Furthermore, a variety of inorganic and organic substances was employed to clarify the effect of the UV/TiO2/IO3− process in natural waters. The findings revealed a negative impact of chloride excess in seawater and a decline in SO degradation in mineral water as a result of HCO3− presence at more than 1 mM. At high concentrations of natural organic matter, the heterogeneity and the strong adsorption of humic acid on the TiO2 surface were found to be harmful for SO decay.

A Review on Green and Efficient Synthesis of 5-Hydroxymethylfurfural (HMF) and 2,5-Furandicarboxylic Acid (FDCA) from Sustainable Biomass

The present work gives an overview of the approaches for green synthesis of 5-hydroxymethylfurfural (HMF) and 2,5 furan dicarboxylic acid (FDCA) focusing on reduction in the byproduct formation such as levulinic acid and humins. It was elucidated that the use of highly polar and aqueous solvents, high Brønsted acidity, high pH, reaction time, and temperature increases the levulinic acid formation in the reaction. In contrast, humin formation is increased by high basicity, temperature, and initial substrate concentration. Specific catalytic and solvent systems for minimizing the byproduct formation during HMF synthesis are also discussed along with options for use of microwave, ultrasound, and continuous processes. In terms of intensification, safety, and ease of scale-up, the use of ultrasound for HMF synthesis is promising; however, microreactors are currently a better choice for higher substrate concentration usage and higher yields. Effect of parameters like reaction time, temperature, oxidizing source, solvent, and base on FDCA synthesis is also analyzed. Depending on the reaction conditions and the reactor type, the optimum temperature typically should be between 80 and 160 °C, moderate bases, such as K2CO3 and KHCO3, are preferred to maintain a preferred pH of 7−11 and O2 is an ideal oxidizing agent. The reaction pathway for FDCA synthesis from HMF was also studied and the effect of the base and solvent elucidated. Finally, one-pot synthesis of FDCA from HMF and the strategies necessary to optimize the FDCA yield has been discussed. It was demonstrated that one-pot two-step FDCA synthesis from sugars provides enhanced FDCA yield, selectivity, and lower byproduct formation, compared to one-pot one-step synthesis.

Efficient conversion of rebaudioside C to steviol by Paenarthrobacter ilicis CR5301.

To improve the conversion efficiency of rebaudioside C, this study screened the Paenarthrobacter ilicis CR5301 from soil samples and identified it by 16S rRNA. The conversion experiment proved that P. ilicis CR5301 was capable of converting rebaudioside C. The effects of initial pH, temperature, inoculation amount, and substrate concentration on rebaudioside C conversion rate were investigated. The results showed that the conversion rate of rebaudioside C reached up to 100% when CR5301 was incubated in a conversion medium with an initial pH of 7.0 for 8 h at 28°C and 270 rpm. The conversion time was reduced by at least 16 h compared with previous studies. The conversion product was analyzed and identified as steviol by high performance liquid chromatography, ultra performance liquid chromatography-triple-time of flight mass spectrometer, and Fourier transform infrared spectroscopy methods. In addition, stevioside, rebaudioside A, dulcoside A, and some unknown components in steviol glycosides byproduct were all efficiently converted to steviol. These findings provide an efficient approach to the conversion of rebaudioside C and byproduct to steviol to simplify the subsequent industrial process and improve the reuse value of steviol glycosides.

Unstructured kinetic models with time-averaged growth approach in the biodegradation of triclosan by activated sludge culture

The concentration of triclosan in wastewater is expected to rise dramatically as a consequence of the COVID-19 pandemic. A modeling analysis of the growth kinetics of microbial culture during triclosan degradation was necessary in order to establish effective wastewater treatment. The kinetic parameters are used by engineers to aid in the design and process control of biological processes. Studies were conducted using triclosan-acclimated culture to examine biomass growth and associated substrate degradation at different initial substrate concentrations (0.35–4.9 mg L−1) to this end. Substrate inhibition was calculated from experimental growth parameters using unstructured kinetic models. Unlike other model studies, a time-averaged specific bacterial growth rate in the log phase was considered for kinetic models in this study. Overestimation of the conventional log phase calculation for unstructured kinetic model constants was eliminated when the slowdown growth part of the log growth phase was taken into account. The Haldane Model was more accurate to fitting experimental data in an excellent manner. In this case, the time-averaged specific growth rate, saturation constant, and inhibition constant were 0.56 h−1, 12.77 mg L−1, 0.52 mg L−1, respectively. A yield coefficient of 0.404 mgX.mgS−1 was calculated. The critical triclosan concentration was 2.57 mg L−1. Wastewater treatment plants can be more sensitive to the value of the critical triclosan concentration. The value for time-averaged critical specific growth rate was 0.051 h−1. Pre-or post-treatment requirements can be estimated using time-averaged critical growth rate values as a benchmarking tool in biological treatment systems.

CATALYTIC CONVERSION OF FRUCTOSE INTO 5-HYDROXYMETHYLFURFURAL BY USING Cr-MODIFIED KUNIPIA-F CLAY

Catalytic conversion of fructose into 5-hydroxymethylfurfural (HMF) is investigated in detail using chromium-exchanged Kunipia-F clay (K550-Cr-calcined at 550°C) using dimethylsulfoxide (DMSO) as the solvent. The as-synthesized catalyst is characterized using XRD, ICE-AES, BET, SEM, FTIR, and EDX. The clay catalysts are initially screened for fructose conversion and HMF yield and then K550-Cr, which shows the superior activity amongst the catalysts understudy, is chosen for further experiments. Various reaction parameters like reaction temperature and time, catalyst dosage, initial substrate concentration, and catalyst reusability are studied in detail. The maximum HMF yield obtained is about 89% at milder reaction conditions (130°C for 3 h). The most interesting result is that the catalyst can be recovered easily without any loss and reused four times without loss in activity (HMF yield after five cycles is 88%).

Kinetic resolution of sulfoxides with high enantioselectivity using a new homologue of methionine sulfoxide reductase B.

Optically pure sulfoxides are noteworthy compounds that find wide applications in various industrial fields. Here, we report a methionine sulfoxide reductase B (MsrB) homologue that exhibits high enantioselectivity and broad substrate scope for the kinetic resolution of racemic (rac) sulfoxides. This MsrB homologue, named liMsrB, was identified from Limnohabitans sp. 103DPR2 and showed good activity together with enantioselectivity towards a series of aromatic, heteroaromatic, alkyl and thioalkyl sulfoxides. Chiral sulfoxides in the S configuration were prepared in approximately 50% yield and 92-99% enantiomeric excess through kinetic resolution at an initial substrate concentration of up to 90 mM (11.2 g L-1). This study presents an efficient route for the enzymatic preparation of (S)-sulfoxides through kinetic resolution.

Biodegradation and power production kinetics in microbial fuel cell during rice mill wastewater treatment

The influence of the initial substrate concentration of rice mill wastewater on substrate degradation rate and power production in a dual-chambered MFC was explored over a wide range of chemical oxygen demand (COD: 500–6000 mg/L) and lignin (77.5–930 mg/L) levels. Within a limited concentration range, increasing COD concentration increased degradation rates and electricity generation, but both processes were hindered at high substrate concentrations. A maximum power density of 4.3 W/m3 was attained in a MFC operated with an initial COD concentration of 2480 ± 17 mg/L which can be attributed to the low ohmic resistance (23.8 Ω) due to better solution conductivity. Different kinetic models were used to examine inhibition kinetics from an electrical perspective. The relationship between power density and COD concentration was best described by Han–Levenspiel model. The maximum power density, half-saturation constant, and critical inhibitory concentration were predicted to be 4.56 W/m3, 799.6 mg/L, and 7627 mg/L, respectively. A maximum lignin degradation rate of 0.063 ± 0.007 kg/m3∙d was achieved at a lignin concentration of 387.5 ± 2.8 mg/L which accounts for 83.17 ± 0.26 % removal efficiency, suggesting the possibility of lignin degradation in MFC even at higher concentrations. The detection of low molecular weight degradation by-products of lignin in the effluent sample further confirmed the ability of exoelectrogens to degrade lignin.

Kinetic modelling of yeast growth and pollutant removal in secondary effluent

In recent years, there has been significant interest in yeast-based wastewater treatment due to its high pollutant removal rate and ability to perform in non-sterile environments. In this work, a kinetic model was developed to predict yeast growth and substrate consumption during wastewater treatment. To determine the biological constants for use in the kinetic models, a unique approach is presented. Candida utilis was cultivated in synthetic wastewater, using eight different ratios of carbon, nitrogen and phosphorus to determine its growth rate, and the removal rates of carbon, nitrogen, and phosphorus. The concentrations of C, N and P were chosen within the range of secondary effluent wastewater. In all experiments, carbon was found to be the limiting substrate, and 100 % TOC removal was achieved in all cases. Candida utilis reduced the COD concentration by up to 99 % in <24 h. In the model, both yeast growth and substrate consumption were satisfactorily described by Monod kinetics. The apparent half-saturation coefficients for carbon, nitrogen, and phosphorus, determined via the optimization of the model, were found to the function of initial substrate concentration. The maximum specific growth rate found was 0.59 h−1. This model was used on different initial concentrations of substrates, and predicted data with an R2 above 80 %. Both model and experimental results suggest that Candida utilis can be used in the tertiary treatment of wastewater. The simple approach described here can be applied to find biological coefficients for other microorganisms.

Ultrasound-assisted synthesis of gold nanoparticles supported on β-cyclodextrin for catalytic reduction of nitrophenols

Nitrophenols with high toxicity are well known as one of the main pollutants in water environment while aminophenols, products of the reduction, are important intermediates in the fabrication of several drugs. Thus, the reduction of nitrophenols is considerably attractive in wastewater treatment and pharmaceutical fields. In this work, gold nanoparticles (AuNPs) catalyst was straightforwardly fabricated using β-cyclodextrin as a reducing and stabilizing agent under ultrasound irradiation. The synthetic conditions were explored via the changes in concentration of β-cyclodextrin, environmental pH, temperature and time. The synthesized AuNPs under the optimal condition showed a size distribution in range of 3–21 nm with a mean size of 12 nm. The dynamic lighting scatter and zeta potential data showed the stable colloidal solutions and X-ray diffraction and selected area electron diffraction patterns confirmed crystalline structure of AuNPs. The influence of reagents and the nanoparticles on kinetic data of the reduction was investigated. The kinetic data showed that the performance of 2-nitrophenol reduction is better than that of 4-nitrophenol while rate constants of the reduction is linear to both initial concentrations of nitrophenol substrates and catalyst amount.

Concept of an Enzymatic Reactive Extraction Centrifuge

Biocatalytic processes often provide an ecological alternative to many chemical processes. However, further improvements in terms of the economic efficiency are required. In order to achieve that, the concept of process integration is a promising option. Applying this within a biocatalytic process, a highly integrated apparatus working as a reactive extraction centrifuge was developed and operated. For this purpose, a commercially available extraction centrifuge was modified to implement a biocatalytic reaction. The novel apparatus was used within a multi-enzyme cascade for the production of a natural flavor and fragrance, namely cinnamic ester. The characterization of the reactive extraction centrifuge and the suitable operation conditions for the inlet streams and the rotational speed for a stable operation were determined. Furthermore, different initial substrate concentrations were applied to prove the reaction. The results provide a successful proof of concept for the novel reactive extraction centrifuge.

Cyclic voltammetric response of homogeneous catalysis of electrochemical reaction. Part 3: A theoretical and numerical approach for one-electron two-step reaction scheme

• Homogeneous molecular catalysis of electrochemical reactions for the one-electron two-step reaction scheme is discussed. • The exact expression of species concentrations, current-voltage and turnover frequency is derived. • The effect of the intrinsic and operational parameters on current and turnover frequency is analysed. A theoretical model for homogeneous redox catalysis of electrochemical reactions for the one-electron two-step reaction scheme is discussed. This model is described by the system of nonlinear time dependent partial differential equations for which the nonlinear term is related to a homogeneous reaction. The species concentration, current-voltage and turnover frequency curves for the transient condition have been calculated by solving the equations using a new analytical method. The current response and turnover frequency predicted under steady-state conditions when t → ∞ are in good agreement with earlier results, proving the validity of the mathematical analysis. The effect of two rate constants and initial concentration of substrate on current and turn over frequency is discussed. Our analytical results for concentration are compared with numerical (MATLAB) results, and good agreement is noted.

Optimized cell growth and poly(3-hydroxybutyrate) synthesis from saponified spent coffee grounds oil

Spent coffee ground (SCG) oil is an ideal substrate for the biosynthesis of polyhydroxyalkanoates (PHAs) by Cupriavidus necator. The immiscibility of lipids with water limits their bioavailability, but this can be resolved by saponifying the oil with potassium hydroxide to form water-soluble fatty acid potassium salts and glycerol. Total saponification was achieved with 0.5 mol/L of KOH at 50 °C for 90 min. The relationship between the initial carbon substrate concentration (C0) and the specific growth rate (µ) of C. necator DSM 545 was evaluated in shake flask cultivations; crude and saponified SCG oils were supplied at matching initial carbon concentrations (C0 = 2.9–23.0 g/L). The Han-Levenspiel model provided the closest fit to the experimental data and accurately described complete growth inhibition at 32.9 g/L (C0 = 19.1 g/L) saponified SCG oil. Peak µ-values of 0.139 h−1 and 0.145 h−1 were obtained with 11.99 g/L crude and 17.40 g/L saponified SCG oil, respectively. Further improvement to biomass production was achieved by mixing the crude and saponified substrates together in a carbon ratio of 75:25% (w/w), respectively. In bioreactors, C. necator initially grew faster on the mixed substrates (µ = 0.35 h−1) than on the crude SCG oil (µ = 0.23 h−1). After harvesting, cells grown on crude SCG oil obtained a total biomass concentration of 7.8 g/L and contained 77.8% (w/w) PHA, whereas cells grown on the mixed substrates produced 8.5 g/L of total biomass and accumulated 84.4% (w/w) of PHA.Key points• The bioavailability of plant oil substrates can be improved via saponification.• Cell growth and inhibition were accurately described by the Han-Levenpsiel model.• Mixing crude and saponified oils enable variation of free fatty acid content.

Nitrification upon Nitrogen Starvation and Recovery: Effect of Stress Period, Substrate Concentration and pH on Ammonia Oxidizers’ Performance

Nitrification has been widely applied in wastewater treatment, however gaining more insight into the nitrifiers’ physiology and stress response is necessary for the optimization of nutrient removal and design of advanced processes. Since nitrification initiates with ammonia oxidation performed by ammonia-oxidizing bacteria (AOB), the purpose of this study was to investigate the effects of short-term ammonia starvation on nitrogen uptake and transformation efficiency, as well as the performance of starved nitrifiers under various initial substrate concentrations and pH values. Ammonium deprivation for 3 days resulted in fast ammonium/ammonia accumulation upon nitrogen availability, with a maximum uptake rate of 3.87 mmol gprotein−1 min−1. Furthermore, a delay in the production of nitrate was observed with increasing starvation periods, resulting in slower recovery and lower nitrification rate compared to non-starved cells. The maximum accumulation capacity observed was 8.51% (w/w) independently of the external nitrogen concentration, at a range of 250–750 mg N L−1, while pH significantly affected ammonia oxidizers’ response, with alkaline values enhancing nitrogen uptake. In total, ammonia accumulation after short-term starvation might serve as an important strategy that helps AOB restore their activity, while concurrently it could be applied in wastewater treatment for effective nitrogen removal and subsequent biomass utilization.

Modeling of 1,2-Dibromoethane Biodegradation in Constant Electric Field

This study proposes a mathematical modeling approach for evaluating the effect of applying a permanent electric field on the biodegradation of 1,2-dibromoethane by bacterial cells of Bradyrhizobium japonicum 273. Two models for inhibited microbial growth including product inhibition were composed—one using the Monod–Yerusalimsky approach and another one—the Levenspiel kinetic equation. The models were used to process own experimental data obtained without an electric field and ones obtained at the application of an electric field. The experiments were carried out at an optimum anode potential of 0.8 V vs. the standard hydrogen electrode (SHE). Three initial concentrations of substrate were tested: 0.05, 0.1, and 0.15 g dm−3. The modeling takes into account the product inhibition on microbial growth assuming 2-bromoethanol as the first biodegradation product. It was found that the positive effect of the electric field is the enhancement of microbial growth, expressed by the increase in the maximum specific growth rate and the increase in the inhibition constant when the model of Monod–Yerusalimsky is applied. The main effect of the electric field is in the increase in the rate constant of 2-bromoethanol removal by electrochemical oxidation, enabling the enhancement the microbial growth and substrate conversion to the product. The obtained results show that the application of a permanent electric field leads to a higher electrochemical oxidation rate (with a rate constant up to 60% higher than for the control experiments) and complete substrate and 2-bromoethanol biodegradation. The model of Levenspiel is not so sensitive to the effects of the electric field on product inhibition.

Conversion of golden oyster mushroom, Pleurotus citrinopileatus to sugar syrup using enzymatic hydrolysis as a substrate for novel bacterial cellulose (Nata) fermentation.

Enzymatic hydrolysis of the golden oyster mushroom (Pleurotus citrinopileatus) generated a new bacterial cellulose (BC). The sugar syrup obtained from the hydrolysis of mushroom powder by commercial enzymes gave maximum total soluble solids (TSS) content at 8.83 ± 0.29°Brix, while 8.82 ± 0.06mg GAE/g substrate of total phenolic content(TPC) was obtained when using initial substrate and enzyme concentrations at 125g/L and 5.0%, respectively. Glutamic acid, aspartic acid, alanine and valine were determined as the main amino acids found in P. citrinopileatus hydrolysis at 524.74 ± 0.03, 247.09 ± 0.04, 176.82 ± 0.07 and 174.57 ± 0.01mg/100g sample, respectively. Thin-layer chromatography revealed that the obtained sugar syrup was glucose. The hydrolyzed mushroom fermented with Komagataeibacter xylinus AGR 60 at 30 ± 2°C for 9days produced optimal conditions at 4.0°Brix of the initial mushroom syrup and 12.0% (v/v) of the starter culture. Maximum BC thickness was 0.88 ± 0.03cm with 7.90 ± 0.07g dry weight, equivalent to 39.50 ± 0.35g/L and 4.39 ± 0.04g/L/day for BC production (P) and BC production rate (R p), respectively. The obtained BC was characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy, small-angle X-ray scattering and wide-angle X-ray diffraction. These showed the structure and functional properties as a natural source of fiber from the fermentation of a novel substrate.

Modelling of anaerobic digester for the conversion of organic waste into hydrogen & methane

Multistage Anaerobic digestion (AD) nowadays plays a beneficial role compared to single-stage AD to achieve a high rate of waste conversion with higher energy recovery. Hence, obtaining a stable operation with optimal operating conditions is of paramount importance in the modeling study. In the current study, modeling of two stages continuous stirred anaerobic digesters was considered using mass balances over the substrates (organic waste) and biomass in the respective reactor. All the simulation work was performed using MATLAB, from which steady-state concentration values were taken for the calculation of Hydrogen flow rate (QH2) and Methane flow rate (QCH4). The operating volumes of both the reactors were computed by looking at the effect on the production rates (QH2 &QCH4) as a function of dilution rate of first (D1) and second (D2) reactors for constant initial substrate concentration (S0). At last, the effect of Initial substrate concentration on both QH2 &QCH4 was studied by considering different S0 values as 4 g/L, 8 g/L, and 12 g/L. From our modeling result, it was found out that for Maximum Hydrogen production, operating D1 (in day-1) was 0.8, 0.9 & 1 and the Methane production rate was Maximum at operating D2 (inday-1) of 0.25, for S0 values of 4 g/L, 8 g/L, and 12 g/L respectively.

Production of Blending Quality Bioethanol from Broken Rice: Optimization of Process Parameters and Kinetic Modeling.

The enzymatic and bio-enzymatic saccharification of waste broken rice (79.8% of starch) was successfully carried out to produce a reducible sugar solution (160g/L). Bioethanol of concentration 71.2g/L (9.0% v/v) was prepared by fermentation of reducing sugar solution, using the commercially available waste brewer's yeast (Saccharomyces cerevisiae). The fermentation process parameters were optimized through response surface methodology (RSM) and hybrid artificial neural network-genetic algorithm (ANN-GA) for optimizing the ethanol concentration. The hybrid ANN-GA model predicted a maximum concentration of 71.9g/L with a deviation of only 0.97% from the experimental value (71.2g/L). Four different kinetic models were attempted to fit the experimental time evolution of concentrations with the kinetic parameters estimated by the Levenberg-Marquardt optimization technique. The 4th order Runge-Kutta algorithm was implemented through a C program module. The accuracy of each model was checked against coefficient of determination R2, adjusted R2, the absolute mean deviation (AMD), and root mean square deviation (RMSD). The Andrew-Levenspiel kinetics produced the best performance criteria at two initial substrate concentrations of 160 and 170g/L. Finally, the FTIR analysis of 781.2g/L (98.5% v/v) bioethanol (concentrated by two-stage vacuum distillation followed by treatment with 3A molecular sieve) showed a favorable blending possibility with the commercial gasoline (petrol) as a green fuel.

Cooperative catalytic nanokinetics

• Theoretical framework for cooperative kinetics on nanoclusters with several adsorbed molecules. • Development of kinetic equations for common reaction mechanisms. • Analysis of selectivity in consecutive and parallel reactions. • Comparison of theory with experimental data on chemoselective hydrogenation. Cooperative kinetics of heterogeneous catalytic reactions on nanoclusters or in a nanoconfined space is considered for the Eley-Rideal and Langmuir-Hinshelwood types of mechanisms as well as for a reaction mechanism with two kinetically significant steps, by assuming few (minimum two) molecules of different type per a single metal cluster/catalytic ensemble. The treatment accounted for the lateral interactions between adsorbed species by introducing kinetic and adsorption constants dependence on the presence of adsorbed species of the same or another type. Kinetic behavior for the Eley-Rideal type of mechanism with the product adsorption was different from the classical mean –field approximation, resulting in S-shape behavior and cooperativity, while the Langmuir-Hinshelwood mechanism essentially gives the same behavior for the case of two adsorbed species of different type on a two-site ensemble. Differences in the kinetic behavior for the bimolecular mechanism compared to the conventional treatment could be seen for larger ensembles, when at least three species are adsorbed per cluster or located in a nanoconfined space. For parallel reactions of the Eley-Rideal type, it was demonstrated that the cooperative behavior of two adsorbed molecules of a reactant per cluster results in selectivity dependence on conversion for reactions of the same order contrary to the mean field approach. In the case of consecutive reactions with such cooperative behavior, selectivity towards the intermediate product depends on the initial substrate concentration in contrast to the mean field approximation.

IFIT: An automated web tool for determining enzyme-kinetic parameters based on the high-curvature region of progress curves.

The area where progress curve exhibits maximum curvature contains the most information about kinetic parameters. To determine these parameters more accurately from progress curves, we propose an iterative approach that calculates the area of maximum curvature based on an estimate of kinetic parameters and then recalculates the parameters based on time-concentration data points within this area. Based on this algorithm, we developed a computer script called iFIT as a free web application at http://www.i-fit.si. The benefits of working with iFIT are that it decreases the importance of initial substrate concentration and the impact of certain side reactions on the final calculated kinetic parameters.