Non-competitive Product Inhibition Research Articles

Variants at position 603 of the CGTase from Bacillus circulans STB01 for reducing product inhibition

Inhibition of cyclodextrin glycosyltransferases (CGTases) by their product cyclodextrins limits the efficiency of cyclodextrin production. In an effort to produce variants with good activity but reduced product inhibition, six mutants were constructed at position 603 of the CGTase from Bacillus circulans STB01, which exhibits mixed-type product inhibition. In a kinetic analysis, N603I showed reduced noncompetitive inhibition while N603K, N603H and N603R showed increased noncompetitive inhibition. Unexpectedly, N603E and N603D exhibited reductions in both competitive and noncompetitive product inhibition. Noncompetitive product inhibition is closely related to the interaction between the cyclodextrin and the enzyme in maltose binding site 2 (MBS2). Structural models led to the suggestion that there is increased interaction between maltose binding sites 1 and 2 in mutants N603E and N603D, which may have led to the unexpected results. N603D exhibited a 23.9% greater cyclodextrin yield per gram of enzyme than the wild-type, suggesting it has potential for industrial use. Further reductions in product inhibition may be gained through studies of maltose binding site interactions.

Studi Kinetika Konsumsi Glukosa oleh Aspergillus Niger dalam Produksi Bioethanol dari Lignoselulosa

Global warming resulted from CO2 level increase in the atmosphere has caused elevation of earth temperature and uncertain climate changes. To prevent the rise of CO2 in the atmosphere can be done by using biomass fuel such as bioethanol. The raw materials of bioethanol can be derived from oil palm empty fruit bunch. Enzymatic hydrolysis utilizes cellulase-producing fungus and in this research, Aspergillus niger was chosen. The glucose produced is consumed by A niger as carbon source and this is undesirable, therefore it should be minimized as low as possible. Knowing the rate of glucose consumption is important to have a model of the hydrolysis reaction rate which will be helpful in the design process on an industrial scale hydrolysis reactor. This study aimed to determine the equations that can be used to approximate the growth rate of A. niger, glucose consumption, the formation of citric acid, and the kinetic parameters used to modeling the kinetics of glucose consumption by A. niger. Kinetics of glucose consumption by A. niger was studied in batch system with variation of initial glucose concentration of 30, 50, 70 g/l. The growth rate of A. niger, glucose consumption, and the formation of citric acid were modeled using 3 equations; i.e. Monod with non-competitive product inhibition, Luedeking-Piret, and Luedeking-Piret growth associated product formation, respectively. The values of kinetic parameters such as μmax, Ks, Kp, were 0.65 hour-1, 157.5 g/l, 0.3 g/l, for initial glucose concentration of 30, 50, 70 g/l, respectively. The values of α (kinetic parameter for growth associated product formation and α would be equal to Yp/x) and Yx/s were 0.4903, 0.8531, 0.9863; 0.5124, 0.2704, 0.2381, for initial glucose concentration of 30, 50, 70 g/l, respectively. Higher initial glucose concentration would increase α but it lowered Yx/s.

Kinetic analysis of two-phase enzymatic hydrolysis of hemicellulose of xylan type

We present a coupled experimental and theoretical framework for quantifying the kinetics of two-phase enzymatic hydrolysis of hemicellulose. For xylan loading of 1–5mg/ml, the nature of inhibition by the product xylose (non-competitive), the kinetic constants (Km=3.93mg/ml, Vmax=0.0252mg/ml/min) and the xylose inhibition constant (Kx=0.122mg/ml) are experimentally determined. Our multi-step two-phase kinetic model incorporating enzyme adsorption to the solid substrate and non-competitive product inhibition is simulated using our kinetic data and validated against our experimentally measured temporal dynamics of xylose and reducing sugars. Further experiments show that higher substrate loading reduces the specific adsorption of the endoxylanase to the solid xylan and the enzyme’s solid–liquid distribution ratio, which decelerates the solid hydrolysis and accelerates the liquid phase reactions. Thus, the xylose yield increases with substrate loading, which increases product inhibition and decreases reducing sugar yields. An operating cost analysis gives 3mg/ml as the optimal substrate loading.

Theoretical analysis of intrinsic reaction kinetics and the behavior of immobilized enzymes system for steady-state conditions

Mathematical modeling of immobilized enzymes under different kinetics mechanism viz. simple Michaelis–Menten, uncompetitive substrate inhibition, total competitive product inhibition, total non-competitive product inhibition and reversible Michaelis–Menten reaction are discussed. These five kinetic models are based on reaction diffusion equations containing non-linear terms related to Michaelis–Menten kinetics of the enzymatic reaction. Modified Adomian decomposition method is employed to derive the general analytical expressions of substrate and product concentration for all these five mechanisms for all possible values of the parameters ΦS (Thiele modulus for substrate), ΦP (Thiele modulus for product) and α (dimensionless inhibition degree). Also we have presented the general analytical expressions for the mean integrated effectiveness factor for all values of parameters. Analytical results are compared with the numerical results and also with the limiting case results, which are found to be good in agreement.

Mechanistic Study of Manganese-Substituted Glycerol Dehydrogenase Using a Kinetic and Thermodynamic Analysis

Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.

Inhibitory effects of acetate and ethanol on biohydrogen production of Ethanoligenens harbinese B49

Inhibitory effects of acetate and ethanol on biohydrogen production from glucose by Ethanoligenens harbinese B49 were investigated in this study. In batch test, sodium acetate (0, 10, 20, 50, 100 and 150 mmol/l) and ethanol (0, 20, 40, 80, 100 and 200 mmol/l) were added respectively. Their inhibitory effects on glucose degradation, cell growth, distribution of liquid products and hydrogen production were discussed. Compared with ethanol, acetate exhibited more significant inhibition on growth and hydrogen producing performance of E. harbinese B49. The inhibitory effects of acetate and ethanol were compared and analyzed on the basis of a noncompetitive product inhibition model. For acetate addition, the maximum specific hydrogen production rate r max = 722 ml/gVSS/h, inhibition constant K C = 55 mmol/l and the exponent of inhibition n = 0.6 were estimated, whereas the maximum hydrogen yield r max = 2.2 mol H 2/mol glucose, K C = 57 mmol/l and n = 0.8 were calculated from kinetic analysis. For ethanol addition, the maximum specific rate r max = 729 ml/g VSS/h, K C = 139 mmol/l and n = 0.8 were estimated, whereas the maximum hydrogen yield r max = 2.2 mol H 2/mol glucose, K C = 153 mmol/l and n = 0.9 were calculated. In addition, deducing from dose-response curves, the C I ,50 values of ethanol and acetate were 154 and 62 mmol/l, respectively. Acetate has a strong inhibitory effect on hydrogen production with ethanol-type fermentation. Thus, hydrogen production can be improved by optimizing the fermentation strategy through removing the acetate as soon as it was produced.

A fundamental analysis of continuous flow bioreactor and membrane reactor models with non-competitive product inhibition. III. Linear inhibition

The steady-state production of a product produced through the growth of microorganisms in a continuous flow bioreactor is presented. A generalised reactor model is used in which both the classic well-stirred bioreactor and the idealised membrane bioreactor are considered as special cases. The reaction is assumed to be governed by Monod growth kinetics subject to non-competitive product inhibition. Inhibition is modelled as a decreasing linear function of the product concentration with a finite cut-off. This reaction scheme is well documented in the literature, although a stability analysis of the governing equations has not previously been presented. The steady-state solutions for the models have been obtained, and the stability has been determined as a function of the residence time. The key dimensionless parameter (γ) that controls the degree of non-competitive product inhibition is obtained by scaling of the equations, and its effect on the reactor performance is quantified in the limit when product inhibition is ‘small’ and ‘large’. The parameter γ is the reciprocal of a scaled inhibition constant (Pm) that depends upon the substrate and product yield factors and the Monod constant (). © 2011 Curtin University of Technology and John Wiley & Sons, Ltd.

A fundamental analysis of continuous flow bioreactor and membrane reactor models with noncompetitive product inhibition

AbstractWe analyze the steady‐state production of a product produced through the growth of microorganisms in both a continuous flow bioreactor and in an idealized continuous flow membrane reactor. The reaction is assumed to be governed by Monod growth kinetics subject to noncompetitive product inhibition. Although this reaction scheme is often mentioned in textbooks, a stability analysis does not appear in the literature.The steady‐state solutions of the model are found and their stability determined as a function of the residence time. The performance of the reactor at large residence times is obtained. Knowledge of the steady‐state solutions and their asymptotic limits may be useful to estimate parameter values from experimental data. The key dimensionless parameter that controls the degree of noncompetitive product inhibition is identified and we quantify the effect that this has on the reactor performance in the limit when product inhibition is 'small' and 'large'. Copyright © 2009 Curtin University of Technology and John Wiley & Sons, Ltd.

Biohydrogen production with mixed anaerobic cultures in the presence of high-concentration acetate

Inhibition of acetate addition on hydrogen production from sucrose-rich synthetic wastewater by mixed anaerobic culture was investigated in this study. Experimental results showed that the added acetate had a significant influence on both substrate degradation and hydrogen production during the fermentation process. The distribution of aqueous products was also influenced by the acetate addition. Modified logistic equations were able to simulate the acidogenesis process well, while a noncompetitive product inhibition model was successfully used to describe the inhibitory effects of acetate addition on both substrate degradation and hydrogen production. From kinetic analysis, the maximum specific rate rmax=584.9mg/gVSS/h, inhibition constant KC=8.27g/l and the exponent of inhibition n=1.53 were estimated for sucrose degradation, whereas rmax=221.7ml/gVSS/h, KC=9.44g/l and n=1.52 were calculated for hydrogen production. In addition, the CI,50 values of added acetate on the specific hydrogen production rate and hydrogen yield were 11.05 and 31.90g/l, respectively. The fermentation patterns in the reactor were substantially changed because of the acetate addition, especially at higher dosages.

Structure of BeF3−-Modified Response Regulator PleD: Implications for Diguanylate Cyclase Activation, Catalysis, and Feedback Inhibition

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger involved in the regulation of cell surface-associated traits and persistence. We have determined the crystal structure of PleD from Caulobacter crescentus, a response regulator with a diguanylate cyclase (DGC) domain, in its activated form. The BeF(3)(-) modification of its receiver domain causes rearrangement with respect to an adaptor domain, which, in turn, promotes dimer formation, allowing for the efficient encounter of two symmetric catalytic domains. The substrate analog GTPalphaS and two putative cations are bound to the active sites in a manner similar to adenylate cyclases, suggesting an analogous two-metal catalytic mechanism. An allosteric c-di-GMP-binding mode that crosslinks DGC and an adaptor domain had been identified before. Here, a second mode is observed that crosslinks the DGC domains within a PleD dimer. Both modes cause noncompetitive product inhibition by domain immobilization.

Kinetics of Aspergillus niger Cellulase Inhibition by Reducing Sugar Produced by the Hydrolysis of Carboxymethylcellulose

Hydrolysis of two types of cellulose, namely, totally amorphous Carboxymethylcellulose (CMC) and highly crystalline wood shavings, by Aspergillus niger cellulase was studied in a stirred batch reactor at 50oC. Two kinetic models were devised to predict the rate of reducing sugar production assuming competitive and non-competitive product inhibition of the enzyme. The applicability of the models was tested by comparing experimental results with those predicted by the models. Both models followed the experimental data fairly well, giving standard deviation of 13.3 and 7.9 for the competitive and the non-competitive inhibition models, respectively. However the non-competitive inhibition model showed more realistic behaviour than the non-competitive model and represented better the experimental data. The significance of crystallinity was determined by comparing the production of reducing sugar from wood shavings to the production from CMC. At same initial substrate concentration, it was found that the production of reducing sugar ceased at much shorter times for wood shavings in comparison to that for CMC.

Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end complex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406-415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.

Allosteric Control of Cyclic di-GMP Signaling

Cyclic di-guanosine monophosphate is a bacterial second messenger that has been implicated in biofilm formation, antibiotic resistance, and persistence of pathogenic bacteria in their animal host. Although the enzymes responsible for the regulation of cellular levels of c-di-GMP, diguanylate cyclases (DGC) and phosphodiesterases, have been identified recently, little information is available on the molecular mechanisms involved in controlling the activity of these key enzymes or on the specific interactions of c-di-GMP with effector proteins. By using a combination of genetic, biochemical, and modeling techniques we demonstrate that an allosteric binding site for c-di-GMP (I-site) is responsible for non-competitive product inhibition of DGCs. The I-site was mapped in both multi- and single domain DGC proteins and is fully contained within the GGDEF domain itself. In vivo selection experiments and kinetic analysis of the evolved I-site mutants led to the definition of an RXXD motif as the core c-di-GMP binding site. Based on these results and based on the observation that the I-site is conserved in a majority of known and potential DGC proteins, we propose that product inhibition of DGCs is of fundamental importance for c-di-GMP signaling and cellular homeostasis. The definition of the I-site binding pocket provides an entry point into unraveling the molecular mechanisms of ligand-protein interactions involved in c-di-GMP signaling and makes DGCs a valuable target for drug design to develop new strategies against biofilm-related diseases.

Structural basis of activity and allosteric control of diguanylate cyclase

Recent discoveries suggest that a novel second messenger, bis-(3'-->5')-cyclic di-GMP (c-diGMP), is extensively used by bacteria to control multicellular behavior. Condensation of two GTP to the dinucleotide is catalyzed by the widely distributed diguanylate cyclase (DGC or GGDEF) domain that occurs in various combinations with sensory and/or regulatory modules. The crystal structure of the unorthodox response regulator PleD from Caulobacter crescentus, which consists of two CheY-like receiver domains and a DGC domain, has been solved in complex with the product c-diGMP. PleD forms a dimer with the CheY-like domains (the stem) mediating weak monomer-monomer interactions. The fold of the DGC domain is similar to adenylate cyclase, but the nucleotide-binding mode is substantially different. The guanine base is H-bonded to Asn-335 and Asp-344, whereas the ribosyl and alpha-phosphate moieties extend over the beta2-beta3-hairpin that carries the GGEEF signature motif. In the crystal, c-diGMP molecules are crosslinking active sites of adjacent dimers. It is inferred that, in solution, the two DGC domains of a dimer align in a two-fold symmetric way to catalyze c-diGMP synthesis. Two mutually intercalated c-diGMP molecules are found tightly bound at the stem-DGC interface. This allosteric site explains the observed noncompetitive product inhibition. We propose that product inhibition is due to domain immobilization and sets an upper limit for the concentration of this second messenger in the cell.

Isomerase Activity of the C-terminal Fructose-6-phosphate Binding Domain of Glucosamine-6-phosphate Synthase fromEscherichia coli

The isomerase activity of the C-terminal fructose-6P binding domain (residues 241-608) of glucosamine-6-phosphate synthase from Escherichia coli has been studied. The equilibrium constant of the C-terminal domain k(eq) ([glucose-6P]/[fructose-6-P]) = 5.0. A non-competitive product inhibition of the isomerase activity by the reaction product glucose-6-P has been detected. The existence of more than one binding and reaction sites for the substrate fructose-6P on the molecule of glucosamine-6-phosphate synthase can be expected. The fructose-6P binding domain possibly includes a regulatory site, different from the catalytic center of the enzyme.

The role of benzoate in anaerobic degradation of terephthalate.

The effects of acetate, benzoate, and periods without substrate on the anaerobic degradation of terephthalate (1, 4-benzene-dicarboxylate) by a syntrophic methanogenic culture were studied. The culture had been enriched on terephthalate and was capable of benzoate degradation without a lag phase. When incubated with a mixture of benzoate and terephthalate, subsequent degradation with preference for benzoate was observed. Both benzoate and acetate inhibited the anaerobic degradation of terephthalate. The observed inhibition is partially irreversible, resulting in a decrease (or even a complete loss) of the terephthalate-degrading activity after complete degradation of benzoate or acetate. Irreversible inhibition was characteristic for terephthalate degradation only because the inhibition of benzoate degradation by acetate could well be described by reversible noncompetitive product inhibition. Terephthalate degradation was furthermore irreversibly inhibited by periods without substrate of only a few hours. The inhibition of terephthalate degradation due to periods without substrate could be overcome through incubation of the culture with a mixture of benzoate and terephthalate. In this case no influence of a period without substrate was observed. Based on these observations it is postulated that decarboxylation of terephthalate, resulting in the formation of benzoate, is strictly dependent on the concomitant fermentation of benzoate. In the presence of higher concentrations of benzoate, however, benzoate is the favored substrate over terephthalate, and the culture loses its ability to degrade terephthalate. In order to overcome the inhibition of terephthalate degradation by benzoate and acetate, a two-stage reactor system is suggested for the treatment of wastewater generated during terephthalic acid production.

Kinetic studies and medium improvement of growth and vitamin B 12 production by Acetobacterium sp. in batch culture

Kinetics of inhibition of the growth of Acetobacterium sp. by methanol (substrate) and acetate (inhibitory end-product) were analyzed using a second-order substrate inhibition model coupled with a non-competitive product inhibition model. The analysis revealed that the substrate saturation constant K s was 83.1 mM methanol; the substrate inhibition constant K i was 277.8 mM methanol; the inhibition constant K p was 0.835 mM undissociated acetic acid (239 mM of total acetic acid at pH 7.2); the exponent of inhibition n was 0.72; and μ max was 0.175 h −1. The growth characteristics of Acetobacterium sp. in a defined medium containing methanol and CO 2 were investigated and this medium was modified in order to optimize growth of the bacterium and production of vitamin B 12 in batch culture. The composition of the modified medium was as follows (per liter of deionized water): methanol, 8 g; NaHCO 3, 15.0 g; KH 2PO 4, 0.685 g; NH 4Cl, 1.0 g; MgSO 4·7H 2O, 0.1 g; CoCl 2·6H 2O, 0.02 g; 5,6-dimethylbenzimidazole, 0.02 g; yeast extract, 2.0 g; l-cysteine-HCl·H 2O, 0.5 g; trace element solution, 80 ml; vitamin solution, 40 ml; titanium (III) citrate, 0.015 mM. The maximum cell concentration of Acetobacterium sp. doubled (1.3 g/ l) when the acetogen was grown in the modified medium compared with that in the basal medium (0.63 g/ l).

Degradation kinetics of acetate and propionate by immobilized anaerobic mixed cultures

The paper presents a short review of kinetics describing the anaerobic degradation of acetate and propionate by mixed cultures of bacteria. The authors discuss their measurements in lab scale extended fixed bed reactors containing porous polyurethane particles of different sizes for the immobilization of anaerobic mixed cultures. It can be demonstrated that the influences of diffusion within the particles can be neglected for the reactors under study. The results for both acetate and propionate degradation can be fitted by Haldane kinetics using the unionized fatty acid as the actual substrate. Product inhibition of propionate degradation by acetate is shown and described as a non-competitive product inhibition.

Product Inhibition and Dose‐Dependent Bioavailability of Propranolol in the Isolated Perfused Rat Liver Preparation

We investigated in the isolated perfused rat liver (IPRL) whether product inhibition of metabolism contributes to the dose‐dependent bioavailability of propranolol, a drug with a high, but saturable, hepatic first‐pass effect. (±)‐Propranolol was infused in the IPRL, using a recirculating design, for three 36‐min periods (n= 9). Mean steady‐state reservoir, i.e. hepatic inflow concentrations (Cin), were 4.97, 10.4, and 20.4 μM, respectively. Mean reservoir concentrations of the metabolites 4′‐hydroxypropranolol, 5′‐hydroxypropranolol, N‐desisopropylpropranolol, and naphthoxylactic acid (NLA), a major side‐chain‐oxidation metabolite, increased disproportionately with propranolol dose, but their production rate did not reach steady state. In separate experiments (n= 4), perfusate containing 7.1, 12.8, and 21.6 μM (±)‐propranolol, corresponding to administration rates of 114, 205, and 346 nmol/min, respectively, was passed through the liver for 30 min each using a single‐pass design. The bioavailability (hepatic outflow concentration/Cin) of propranolol increased withCinfrom 0.012 to 0.150 to 0.288 in the recirculating IPRL. In the single‐pass IPRL the increase (0.0077 in 0.0669 to 0.136) was significantly less (P< 0.001). The greater bioavailability of propranolol in recirculating experiments was attributed to product inhibition since metabolites do not accumulate with the single‐pass design. NLA did not appear to be the inhibiting metabolite because in further single‐pass experiments with propranololCinof 21.6 μM the presence of NLA (21.6 μM) in perfusate had no effect on propranolol bioavailability (n= 7) compared with control experiments (n= 5). These data suggest that, with the recirculating IPRL, dose‐dependent bioavailability of propranolol is due to competitive inhibition of propranolol metabolism by propranolol metabolites, which is distinct from the noncompetitive product inhibition that has been reported to accompany chronic propranolol administration.

Non-competitive product inhibition in lactic acid fermentation from glucose

A kinetic study regarding product inhibition in lactic acid fermentation by Streptococcus faecalis, which produces l-lactic acid, was performed in a chemostat at various feed concentrations of glucose (10, 20, and 30 g/l) at pH 7.0. Steady-state kinetic constants for the specific consumption rate of glucose and the specific production rate of lactic acid were determined at a residual glucose concentration below 2 g/l, which was accomplished in a chemostat. All the parameters, the specific growth rate, the specific consumption rate of glucose, and the specific production rate of lactic acid, were definitely related to non-competitive inhibition with regard to the concentration of the product, lactic acid.