Product Inhibition Effects Research Articles

Engineered cytidine triphosphate synthetase with reduced product inhibition.

Cytidine triphosphate (CTP) synthetase (CTPS) (EC number 6.3.4.2) is a key enzyme involved in de novo synthesis of CTP. It catalyzes the rate-limiting step of the process due to the product inhibition effects on the enzyme. In this study, a novel CTPS from Corynebacterium glutamicum ATCC 13032 (CgCTPS) was cloned, expressed and characterized. A series of mutagenesis in its N-terminal ammonia ligase (ALase) domain was performed in order to reduce CTP product inhibition. All single mutation variants (D160E, E162A, E168K) lowered product inhibition by lowering the enzyme's binding affinity for CTP. The homology model of CgCTPS showed that D160E mutant caused steric hindrance for the pyrimidine ring of CTP stacking, E162A disrupted the hydrogen bond between CTP ribose and side chain and D168K caused minor localized structure perturbations of CTP binding pocket. The triple mutant of CTPS (D160E-E162A-E168K) with halved Km, doubled Vmax and the 23.5-fold increased IC50 for CTP shows a potential for use in industrial-scale CTP production by its better performance in enzyme kinetics and product inhibition.

Factors limiting the enzymatic hydrolysis of wheat gluten.

The enzymatic hydrolysis of wheat gluten for the production of seasonings using mixtures of endo- and exopeptidases results in yields typically below 40%. Possible limiting parameters, such as an increasing product inhibition, autopeptidolysis of the enzymes, and lack of cleavage sites, were studied using novel peptidases from Flammulina velutipes or the commercial Flavourzyme preparation. Seven intermittent electrodialysis steps (10 g/L gluten and 10 kaU/mL) for the in situ removal of amino acids minimized the product inhibition. During 16 h, hydrolysis progressed nearly linearly. Compared to the batch control, a 3-fold yield of amino acids released was obtained indicating that an integrated product removal alleviates the problem of product inhibition. Autopeptidolysis, as shown using sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme activity assays, was suppressed with increasing concentrations of competing gluten substrate. Peptidases of F. velutipes showed product inhibition only, whereas a combined effect of product inhibition and lack of cleavage sites was observed for Flavourzyme.

Extractive Fermentation of Ethanol from Fresh Cassava Roots Using Vacuum Fractionation Technique

Fresh cassava roots were used as a raw material for ethanol fermentation. Conventional batch mode (CF), simultaneous saccharification and fermentation (SSF) and simultaneous liquefaction, saccharification and fermentation (SLSF) were investigated with various enzyme systems. The ethanol concentrations obtained in batch fermentations ranged from 8-12 wt%. In addition, vacuum fractionating technique was successfully introduced to simultaneously remove high purity ethanol from fermentation broth in batch mode. The distilled ethanol concentration was approximately 86 wt% whilst its concentration in the bioreactor was kept lower than 2 wt%. As a result, the product inhibition effect to yeast cells was reduced.

Strategic manipulation of an industrial biocatalyst – evolution of a cephalosporin C acylase

Semi-synthetic cephalosporins are synthesized from the 7-amino cephalosporanic acid (7-ACA) nucleus produced from the antibiotic cephalosporin C (CephC). In recent years, a single-step enzymatic process in which CephC is directly converted into 7-ACA by a cephalosporin C acylase (CA) has attracted industrial interest because of the prospects of simplifying the process and reducing costs. CAs are members of the glutaryl acylase family that specifically use CephC as their substrate; however, known natural glutaryl acylases show very low activity on the antibiotic. We previously enhanced the catalytic efficiency on CephC of a glutaryl acylase from Pseudomonas N176 (named VAC) by a protein engineering approach, and solved the structures of the VAC, thus providing insight into the substrate binding and catalytic activity of CAs. However, the properties of such enzymes are not sufficient to encourage 7-ACA manufacturers to shift to single-step enzymatic conversion of CephC. Here, we combine structural knowledge, semi-rational design, computational approaches and evolution analysis to isolate VAC variants with altered substrate specificity (i.e. with a > 11,000-fold increase in specificity constant for CephC versus glutaryl-7-amino cephalosporanic acid, compared to wild-type) and with the highest kinetic efficiency so far obtained for a CA. Indeed, the H57βS-H70βS-L154βY VAC variant shows the highest conversion of CephC into 7-ACA under conditions resembling those used at industrial level because of its high kinetic efficiency and the absence of substrate or product inhibition effects, and may be suitable for industrial application of the mono-step process for CephC conversion.

Bench-scale biosynthesis of isonicotinic acid from 4-cyanopyridine by Pseudomonas putida

AbstractPseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L−1 of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L−1 h−1. P. putida shows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production reported so far.

Production of Ethanol from Sweet Sorghum Juice Using VHG Technology: A Simulation Case Study

The aims of this study were to develop the kinetic model and determine kinetic parameters describing ethanol production from sweet sorghum juice using very high gravity technology in the batch fermentation of Saccharomyces cerevisiae NP01. The obtained experimental data were tested with four different types of model, based on the experimental data, accounting for the substrate limitation, substrate inhibition, product inhibition, and the combination of those three effects, respectively. The optimization technique to find kinetic parameters was non-linear regression using Marquardt method performed through numerical procedure. The chosen model with its kinetic parameters obtained in the batch mode was validated and tested against the other independent experimental data in the small batch-scale and large-scale fermenter, in order to investigate the applicability and scale-up effect of the model, respectively. Then, the obtained model with its parameters was applied in the simulations of the continuous and fed-batch operations to examine the concentration profiles of fermentation components with the variations in operating parameters such as the dilution rate, feed-flow rate, start-up time, and feed concentration. The results indicated that the kinetic model (the substrate limitation with substrate and product inhibition effects) was suitable to describe ethanol fermentation. In the continuous mode, using the dilution rate of 0.01h(-1), the maximum ethanol concentration obtained was, approximately, 90g/l whereas the simulated results from the fed-batch operation revealed that the maximum ethanol concentration at quasi-steady state condition was, approximately, 96g/l. The start-up time of 21h was the fastest time to reach the steady-state and quasi-steady state for both the continuous and fed-batch modes, respectively.

Inhibitory Effect of the Byproduct Glycerol Resulting from the Phase Behavior on the Transesterification Reaction Kinetics

The effect of the phase behavior of the multicomponent transesterification reaction between soybean oil triglycerides and methanol catalyzed by homogeneous potassium hydroxide over a wide range of methanol/triglyceride molar ratios was studied. It has been known that increasing the molar ratio results in increased methyl ester yields; this is normally attributed to the reaction kinetics. In the subsequent investigation, however, it is shown that the cause of the incomplete yields at low molar ratios results from inhibitory effects caused by the byproduct glycerol. As it has previously been shown that the methanol phase acts as the primary reaction volume, it is thought that, as the glycerol concentration in the methanol phase increases as the reaction progresses, triglycerides become excluded from the methanol phase and the reaction drastically slows, hence, byproduct inhibition. At increased methanol/triglyceride molar ratios, the excess methanol acts to dilute the concentration of glycerol and, subsequently, reduce the inhibitory effects, resulting in increased yields. A kinetic model incorporating the effects of product inhibition is therefore proposed.

Lipase-catalyzed transesterification synthesis of citronellyl acetate in a solvent-free system and its reaction kinetics

The lipase-catalyzed citronellol synthesis of citronellyl acetate via transesterification was investigated in a solvent-free system. After screening several lipases, the lipase from Pseudomonas fluorescens was identified as the optimal enzyme for the system. The optimal reaction temperature was 40 °C. The external diffusion limitation could be greatly reduced by increasing the agitation speed to 200 rpm. A linear relationship between the initial reaction rate and an enzyme load of up to 6 mg mL−1 demonstrated that the internal diffusion limitations could be minimized. Substrate inhibition was absent when the substrate concentration was below 500 mmol L−1, but the experimental results indicated that the product inhibition effect should be considered. The results from the reaction kinetics analysis showed that the reaction obeys the ping-pong bi–bi mechanism that is inhibited by citronellyl acetate. Matlab was used to simulate the model parameters. The experimental values could be satisfactorily fitted to the simulated values with a relative error of 11.98 %.

The cofactor preference of glucose‐6‐phosphate dehydrogenase from Escherichia coli– modeling the physiological production of reduced cofactors

In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of cofactor preference has yet to be reported. In this work, the specificity constants for NADP and NAD for G6PDH were determined using a pure enzyme preparation. Absence of the phosphate group on the cofactor leads to a 410-fold reduction in the performance of the enzyme. Furthermore, the contribution of the phosphate group to binding of the transition state to the active site was calculated to be 3.6 kcal·mol(-1). In order to estimate the main kinetic parameters for NAD(P) and NAD(P)H, we used the classical initial-rates approach, together with an analysis of reaction time courses. To achieve this, we developed a new analytical solution to the integrated Michaelis-Menten equation by including the effect of competitive product inhibition using the ω-function. With reference to relevant kinetic parameters and intracellular metabolite concentrations reported by others, we modeled the sensitivity of reduced cofactor production by G6PDH as a function of the redox ratios of NAD/NADH (rR(NAD)) and NADP/NADPH (rR(NADP)). Our analysis shows that NADPH production sharply increases within the range of thermodynamically feasible values of rR(NADP), but NADH production remains low within the range feasible for rR(NAD). Nevertheless, we show that certain combinations of rR(NADP) and rR(NAD) sustain greater levels of NADH production over NADPH.

Corrigendum: Reactions of ZnR2 Compounds with Dibenzoyl: Characterisation of the Alkyl-Transfer Products and a Striking Product-Inhibition Effect

Corrigendum: Reactions of ZnR2 Compounds with Dibenzoyl: Characterisation of the Alkyl-Transfer Products and a Striking Product-Inhibition Effect

Deuterium Exchange Study for Hydrogenation of D5-1-Pentene (4,4,5,5,5) Over Conventional Cobalt Fischer–Tropsch Catalyst

The hydrogen–deuterium exchange reaction was performed for hydrogenation of D5-1-pentene (4,4,5,5,5) under realistic cobalt Fischer–Tropsch synthesis conditions. In the presence of CO, the added deutero-1-pentene did not show any significant H/D exchange but a step-wise H/D exchange occurs when CO was replaced with N2. The inhibition effects of CO and other FT products on H/D exchange of D5-1-pentene and a pressure dependency effect on H/D exchange are observed.

NMR for direct determination of Km and Vmax of enzyme reactions based on the Lambert W function-analysis of progress curves

1H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, Km and Vmax, were directly determined from progress curves at only one concentration of the substrate. For comparison with the classical Michaelis–Menten analysis, the reaction progress was also monitored at various initial concentrations of 3.5 to 41.8mM. Using the Lambert W function the parameters Km and Vmax were fitted to obtain the experimental progress curve and resulted in Km=28mM and Vmax=13μM/s. The result is almost identical to an initial rate analysis that, however, costs much more time and experimental effort. The effect of product inhibition was also investigated. Furthermore, we analyzed a much more complex reaction, the conversion of farnesyl diphosphate into (+)-germacrene D by the enzyme germacrene D synthase, yielding Km=379μM and kcat=0.04s−1. The reaction involves an amphiphilic substrate forming micelles and a water insoluble product; using proper controls, the conversion can well be analyzed by the progress curve approach using the Lambert W function.

Stimulation and inhibition effects of algae-lytic products from Bacillus cereus strain L7 on Anabaena flos-aquae

Alga-bacterium relationships between a Bacillus cereus strain L7 and Anabaena flos-aquae were studied based on the effects of the algicidal substances on algal growth indicators such as enzyme activity and membrane lipid peroxidation. When exposed to algae-lytic products at a concentration of 0.05 mg mL−1, chlorophyll a (Chla), protein and phycobiliprotein contents increased significantly (p < 0.05); superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities and malondialdehyde (MDA) concentrations increased slightly to stimulate the algae growth. When exposed to algae-lytic products at a concentration of 0.5 mg mL−1, algae growth and composition were inhibited. Chla, protein and phycobiliprotein concentrations decreased significantly (p < 0.05 for protein, p < 0.01 for Chla and phycobiliprotein). MDA concentrations increased significantly (p < 0.05). POD and CAT activities increased by approximately six and three times, respectively, in 24 h compared with the control, then decreased to the initial level in 4 days. Algae-lytic products have not only inhibition but also stimulation effects on A. flos-aquae. Such effects are associated with antioxidative/oxidative reactions as indicated by the biomarkers SOD, POD, CAT, and MDA.

Reactions of ZnR2 Compounds with Dibenzoyl: Characterisation of the Alkyl-Transfer Products and a Striking Product-Inhibition Effect

The first systematic theoretical and experimental studies of reaction systems involving ZnR(2) (R=Me, Et or tBu) with dibenzoyl (dbz) as a non-innocent ligand revealed that the character of the metal-bonded R group as well as the ratio of the reagents and the reaction temperature significantly modulate the reaction outcome. DFT calculations showed four stable minima for initial complexes formed between ZnR(2) and dbz and the most stable structure proved to be the 2:1 adduct; among the 1:1 adducts three structural isomers were found of which the most stable complex had the monodentate coordination mode and the chelate complex with the s-cis conformation of the dbz unit appeared to be the least stable form. Interestingly, the reaction involving ZnMe(2) did not lead to any alkylation product, whereas the employment of ZntBu(2) resulted in full conversion of dbz to the O-alkylated product [tBuZn{PhC(O)C(OtBu)Ph}] already at -20 °C. A more complicated system was revealed for the reaction of dbz with ZnEt(2). Treatment of a solution of dbz in toluene with one equivalent of ZnEt(2) at room temperature afforded a mixture of the O- and C-alkylated products [EtZn{PhC(O)C(OEt)Ph}] and [EtZn{OC(Ph)C(O)(Et)Ph}], respectively. The formation of the C-alkylated product was suppressed by decreasing the initial reaction temperature to -20 °C. Moreover, in the case of the dbz/ZnEt(2) system monitoring of the dbz conversion over the entire reaction course revealed a product inhibition effect, which highlights possible participation of multiple equilibria of different zinc alkoxide/ZnEt(2) aggregates. Diffusion NMR studies indicated that dbz forms an adduct with the O-alkylated product, which is a competent species for executing the inhibition of the alkylation event.

Theoretical approach to modelling and analysis of the bioprocess with product inhibition and impulse effect

Abstract This work presents the first mathematical model of a bioprocess with product inhibition and impulse effect. To begin with, an exemplary mathematical bioprocess model with product inhibition and impulse effect is formulated. Then, according to the model, the analysis of bioprocess stability is presented. The article expresses the product oscillation period, which provides the precise feeding time frame for the regulator bioprocess to achieve an equivalent stable output as that of a bioprocess with impulse effect in the same production environment. Moreover, in this work, the optimization of the production process with respect to the tunable parameters is investigated, and analytical expressions of their optimal values are provided. Numerical simulations using biological data are presented to illustrate the main results.

Modeling of an integrated fermentation/membrane extraction process for the production of 2-phenylethanol and 2-phenylethylacetate

An unstructured model for an integrated fermentation/membrane extraction process for the production of the aroma compounds 2-phenylethanol and 2-phenylethylacetate by Kluyveromyces marxianus CBS 600 was developed. The extent to which this model, based only on data from the conventional fermentation and separation processes, provided an estimation of the integrated process was evaluated. The effect of product inhibition on specific growth rate and on biomass yield by both aroma compounds was approximated by multivariate regression. Simulations of the respective submodels for fermentation and the separation process matched well with experimental results. With respect to the in situ product removal (ISPR) process, the effect of reduced product inhibition due to product removal on specific growth rate and biomass yield was predicted adequately by the model simulations. Overall product yields were increased considerably in this process (4.0g/L 2-PE+2-PEA vs. 1.4g/L in conventional fermentation) and were even higher than predicted by the model. To describe the effect of product concentration on product formation itself, the model was extended using results from the conventional and the ISPR process, thus agreement between model and experimental data improved notably. Therefore, this model can be a useful tool for the development and optimization of an efficient integrated bioprocess.

A Kinetic Study on Sesame Cake Protein Hydrolysis by Alcalase

In the present study, the hydrolysis of sesame cake protein was performed by Alcalase, a bacterial protease produced by Bacillus licheniformis, to investigate the reaction kinetics of sesame cake hydrolysis and to determine decay and product inhibition effects for Alcalase. The reactions were carried out for 10 min in 0.1 L of aqueous solutions containing 10, 15, 20, 25, and 30 g protein/L at various temperature and pH values. To determine decay and product inhibition effects for Alcalase, a series of inhibition experiments were conducted with the addition of various amounts of hydrolysate. The reaction kinetics was investigated by initial rate approach. The initial reaction rates were determined from the slopes of the linear models that fitted to the experimental data. The kinetic parameters, K(m) and V(max), were estimated as 41.17 g/L and 9.24 meqv/L x min. The Lineweaver-Burk plots showed that the type of inhibition for Alcalase determined as uncompetitive, and the inhibition constant, K(i), was estimated as 38.24% (hydrolysate/substrate mixture). Practical Application: Plant proteins are increasingly being used as an alternative to proteins from animal sources to perform functional roles in food formulation. Knowledge of the kinetics of the hydrolysis reaction is essential for the optimization of enzymatic protein hydrolysis and for increasing the utilization of plant proteins in food products. Therefore, in the present study, the hydrolysis of sesame cake protein was performed by Alcalase, a bacterial protease produced by B. licheniformis, to investigate the reaction kinetics of sesame cake hydrolysis and to determine decay and product inhibition effects for Alcalase.

An improved nitrilase-mediated bioprocess for synthesis of nicotinic acid from 3-cyanopyridine with hyperinduced Nocardia globerula NHB-2

Nitrilase of Nocardia globerula NHB-2 was induced by short-chain aliphatic nitriles (valeronitrile > isobutyronitrile > butyronitrile > propionitrile) and exhibited activity towards aromatic nitriles (benzonitrile > 3-cyanopyridine > 4-cyanopyridine > m-tolunitrile > p-tolunitrile). Hyperinduction of nitrilase (6.67 U mg (DCW) (-1), 18.7 U mL(-1)) was achieved in short incubation time (30 h, 30°C) through multiple feeding of isobutyronitrile in the growth medium. The nitrilase of this organism exhibits both substrate and product inhibition effects. In a fed batch reaction at 1 L scale using hyperinduced resting cells corresponding to 10 U mL(-1) nitrilase activity (1.5 mg(DCW) mL(-1)), a total of 123.11 g nicotinic acid was produced at a rate of 24 g h(-1) g (DCW) (-1).

Kinetic analysis and mathematical modeling of growth and lactic acid production of Lactobacillus casei var. rhamnosus in milk whey

Lactobacillus casei is a lactic acid bacterium (LAB) that colonizes diverse ecological niches and that has found broad commercial application. The aim of this study was to characterize the kinetics of biomass production, lactic acid production, and substrate consumption of Lactobacillus casei var. rhamnosus cultured in deproteinized milk whey. Batch culture experiments were performed in an instrumented, 2-L, stirred tank bioreactor using different inoculum concentrations (0.5 to 1.0g/L) and lactose levels (35 to 70g/L). The time series of experimental data corresponding to biomass growth, lactose consumption, and lactic acid formation were differentiated to calculate the corresponding kinetic rates. Strong exponentially dependent product inhibition effects were evident at low lactic acid concentrations, and lactic acid production rate was partially associated with biomass growth. A mathematical model is presented that reproduces the experimental lactose, biomass, and lactic acid concentration profiles.

Intramolecular Hydroamination/Cyclization of Aminoalkenes Catalyzed by Ln[N(SiMe3)2]3 Grafted onto Periodic Mesoporous Silicas

Homoleptic rare-earth metal silylamide complexes Ln[N(SiMe(3))(2)](3) (Ln = Y, La, Nd) were grafted onto a series of partially dehydroxylated periodic mesoporous silica (PMS) supports, SBA-15(-500) (d(p) = 7.9 nm), SBA-15LP(-500) (d(p) = 16.6 nm), and MCM-41(-500) (d(p) = 4.1 nm). The hybrid materials Ln[N(SiMe(3))(2)](3)@PMS efficiently catalyze the intramolecular hydroamination/cyclization reaction of 2,2-dimethyl-4-penten-1-amine. Under the prevailing slurry conditions the metal size (Y > La > Nd), the pore size, and the particle morphology affect the catalytic performance. Material Y[N(SiMe(3))(2)](3)@SBA-15LP(-500) displayed the highest activity (TOF = up to 420 h(-1) at 60 °C), with the extralarge pores minimizing restrictive product inhibition and substrate diffusion effects. The catalytic activity of Y[N(SiMe(3))(2)](3)@SBA-15LP(-500) is found to be much higher than that of the molecular counterpart (TOF = up to 54 h(-1)), and its recyclability is demonstrated.