A new mechanism and kinetic model for the enzymatic synthesis of short-chain fructooligosaccharides from sucrose

A kinetic model based on a ping-pong mechanism was developed under the steady-state hypothesis to account for the short-chain fructooligosaccharides (sc-FOS) synthesis using the commercial cellulolytic enzyme preparation, Rohapect CM. This new mechanism takes into account the interactions between the enzyme species and potential substrates (sucrose and sc-FOS) as a single complex reaction, allowing a better understanding of the reaction kinetics.The initial reaction rate laws appropriately describe the kinetic profiles of the examined substrates. Whereas sucrose exhibited Michaelis–Menten behavior with substrate inhibition, 1-kestose and nystose followed Michaelis–Menten and sigmoid enzyme kinetics. In addition, the enzyme was competitively inhibited by glucose and exhibited significant hydrolytic activity in the presence of nystose.The overall model was simultaneously fitted to experimental data from three initial sucrose concentrations (0.5, 1.5 and 2.1 M) using a multi-response regression with kinetic parameters that have biochemical relevance and are independent of the enzyme concentration. According to the model, sucrose acts almost exclusively as a fructosyl donor substrate. The mathematical development described herein is expected to be suitable for modeling similar enzymatic reaction systems.     

Kinetic model for synthesis of fructosyl-stevioside using suspended β-fructofuranosidase

The synthesis experiments of fructosyl-stevioside were conducted under the various conditions of the initial concentrations of the substrates and the enzyme. The transfructosylation of stevioside with sucrose and the hydrolyses of sucrose and fructosyl-stevioside simultaneously occurred. The fructosyl-stevioside synthesis was inhibited by the side products, glucose and fructose. A kinetic model was constructed by considering the Ping-Pong Bi Bi mechanism for the transfructosylation, the apparent Ordered Uni Bi mechanism for the hydrolysis and the competitive inhibition by the side products. The model constants were estimated by fitting the model equations with the experimental results for the sucrose hydrolysis and the fructosyl-stevioside synthesis. The model can predict not only the appropriate conditions to efficiently synthesize the fructosyl-stevioside, but also the reaction time giving the maximum conversion.     

Kinetic studies on dextransucrase from the cariogenic oral bacterium Streptococcus mutans

The kinetic mechanism of dextransucrase was studied using the Streptococcus mutans enzyme purified by affinity chromatography to a specific activity of 36.9 mumol/min/mg of enzyme. In addition to dextran synthesis, the enzyme catalyzed sucrose hydrolysis and isotope exchange between fructose and sucrose. The rates of sucrose hydrolysis and dextran synthesis were partitioned as a function of dextran concentration such that exclusive sucrose hydrolysis was observed in the absence of dextran and exclusive dextran synthesis at high dextran concentrations. An analogous situation was observed with fructose-dependent partitioning of sucrose hydrolysis and fructose exchange. Steady state dextran synthesis and fructose isotope exchange kinetics were simplified by assay at dextran or fructose concentrations high enough to eliminate significant contributions from sucrose hydrolysis. This limited dextran synthesis assays to dextran concentrations above apparent saturation. The limitation was diminished by establishing conditions in which the enzyme does not distinguish between dextran as a substrate and product which allowed initial discrimination among mechanisms on the basis of the presence or absence of dextran substrate inhibition. No inhibition was observed, which excluded ping-pong and all but three common sequential mechanisms. Patterns of initial velocity fructose production inhibition and fructose isotope exchange at equilibrium were consistent with dextran synthesis proceeding by a rapid equilibrium random mechanism. A nonsequential segment was apparent in the exchange reaction between fructose and sucrose assayed in the absence of dextran. However, the absence of detectable glucosyl exchange between dextrans and the lack of steady state dextran substrate inhibition indicate that glucosyl transfer to dextran must occur almost exclusively through the sequential route. A review of the kinetic constants from steady state dextran synthesis, fructose product inhibition, and fructose isotope exchange showed a consistency in constants derived from each reaction and revealed that dextran binding increases the affinity of sucrose and fructose for dextransucrase.     

Mechanism of chain initiation by dextransucrase: Absence of terminal ‘sucrose’ linkage in newly synthesized dextran chains

Dextran was synthesized using dextransucrase from Streptococus sanguis 10558 and (F)-[¹⁴C]sucrose as substrate to test the possibility that sucrose may be the initial acceptor for glucose. If sucrose is the initial acceptor, then dextran chains should have [¹⁴C] fructose in a terminal ‘sucrose’ linkage which can be cleaved under mild conditions. Although incorporation of [¹⁴C]fructose into dextran was observed, the label was not released by mild hydrolysis, indicating that sucrose is not the initiator for dextran synthesis. Incorporation of [¹⁴C]fructose into dextran might represent its ability to act as an acceptor, as suggested by the isolation of leucrose as a by-product in the reaction.     

Kinetic studies of mold α-galactosidase on PNPG hydrolysis

The kinetic properties of α-galactosidase of Mortierella vinacea were investigated in detail using PNPG (p-nitrophenyl-α-D -galactopyranoside) as a substrate. Consequently, the enzyme was markedly inhibited not only by the substrate, but also by the galactose hydrolized. The initial rate of reaction at sufficiently high substrate concentrations, however, did not fall to zero and did approach a finite value. Galactose behaved as a mixed inhibitor and was neither totally competitive nor totally noncompetitive. A rate equation was obtained from a generalized equation derived from a kinetic model which took both the inhibitions into consideration. The constants used in the equation were appropriately estimated. The calculated rate agreed fairly well with the observed initial rate. Moreover, the PNPG hydrolysis progressing in a batch system was found to be approximately representable by simple first order kinetics in which the rate constant was dependent on the initial substrate concentration.     

Kinetic modeling of a multiple immobilized enzyme system. I. Development and testing of the model

A kinetic model for the reaction sequence catalyzed by coimmobilized invertase and glucose oxidase with a sucrose substrate in a tubular reactor has been developed. The computerized mathematical model employs and orthogonal collection technique for solving oxidase were coimmobilized in poly(2-hydroxyethlmethacrylate) gels and used in a continuous flow packed-bed tubular reactor system. In addition to describing the development of the kinetic model, this article compares experimentally determined reactor effluent concentrations for various sucrose feed solutions to those predicted by the model. Variations between experimental and predicted reactor effluent concentrations were found to be on the micromolar level for sucrose feed concentrations as low as 1.38mM.     

The complete pathway for ERK2-catalyzed reaction. Evidence for an iso random Bi Bi mechanism

In the present study, the enzymatic mechanism of ERK2 is re-examined by a combination of steady-state kinetic studies in the absence and presence of viscosogenic agents. Kinetic studies carried out in various concentrations of sucrose revealed that both k(cat) and k(cat)/K(m) for either ATP or EtsDelta138 were highly sensitive to solvent viscosity, suggesting that the rapid equilibrium assumption is not valid for the phosphorylation of protein substrate by ERK2. Furthermore, the kinetic analysis with the minimal random Bi Bi reaction mechanism is shown to be inconsistent with the principle of the detailed balance. This inconsistent calculation strongly suggests that there is isomerization of the enzyme-substrate ternary complex. The viscosity-dependent steady-state kinetic data are combined to establish a kinetic mechanism for the ERK2-catalyzed reaction that predicts initial reaction velocities under varying concentrations of ATP and substrate. These results complement previous structure-function studies of mitogen-activated protein kinases and provide important insight for mechanistic interpretation of the kinase functions.     

The regulation of turgor pressure during sucrose mobilisation and salt accumulation by excised storage-root tissue of red beet

The changes in turgor pressure that accompany the mobilisation of sucrose and accumulation of salts by excised disks of storage-root tissue of red beet (Beta vulgaris L.) have been investigated. Disks were washed in solutions containing mannitol until all of their sucrose had disappeared and then were transferred to solutions containing 5 mol·m^-3 KCl+5 mol·m^-3 NaCl in addition to the mannitol. Changes in solute contents, osmotic pressure and turgor pressure (measured with a pressure probe) were followed. As sucrose disappeared from the tissue, reducing sugars were accumulated. For disks in 200 mol·m^-3 mannitol, the final reducing-sugar concentration equalled the initial sucrose concentration so there was no change in osmotic pressure or turgor pressure. At lower mannitol concentrations, there was a decrease in tissue osmotic pressure which was caused by a turgor-driven leakage of solutes. At concentrations of mannitol greater than 200 mol·m^-3, osmotic pressure and turgor pressure increased because reducing-sugar accumulation exceeded the initial sucrose concentration. When salts were provided they were absorbed by the tissue and reducing-sugar concentrations fell. This indicated that salts were replacing sugars in the vacuole and releasing them for metabolism. The changes in salf and sugar concentrations were not equal because there was an increase in osmotic pressure and turgor pressure. The amount of salt absorbed was not affected by the external mannitol concentration, indicating that turgor pressure did not affect this process. The implications of the results for the control of turgor pressure during the mobilisation of vacuolar sucrose are discussed.To whom correspondence should be addressed.     

Inhibierungserscheinungen bei der alkoholischen Gärung,II. Einfluß der Substratkonzentration auf die spezifische Ethanolbildungsgeschwindigkeit von Saccharomyees cerevisiae

The influence of sucrose concentration on the specific ethanol production rate was studied during batch processes using the yeast strain Saccharomyces cerevisiae Hansen Sc 5. From experimental data a model could be derived for the simulataneous effect of substrate and product inhibition. It was found that both the decreases of fermentation activity of the cells caused by sucrose and ethanol have an additional relation to each other. This model also takes into consideration the fact that the maximum ethanol concentration P′ can't be realized at high substrate concentrations in a batch process. Compared to it sucrose concentrations below 100 g/l did not inhibit the ethanol production by the strain used in this investigation.     

A comprehensive kinetic model of laccase‐catalyzed oxidation of aqueous phenol

A comprehensive model was developed to describe the kinetics of the laccase‐catalyzed oxidation of phenol that incorporates enzyme kinetics, enzyme inactivation, variable reaction stoichiometry between substrate and oxygen, and oxygen mass‐transfer. The model was calibrated and validated against data obtained from experiments conducted in an open system, which allowed oxygen to transfer from air to the reacting mixture and phenol conversion to approach completion. Inactivation of laccase was observed over the course of the reaction and was found to be dependent on the rate of substrate transformation. A single kinetic expression was sufficient to describe laccase inactivation arising from interaction with reacting species over time. Excellent agreement was found between model predictions of phenol and oxygen concentrations and experimental data over time for a wide range of initial substrate concentrations and enzyme activities. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Determination of effective diffusion coefficient of substrate in gel particles with immobilized biocatalyst

A method of determining of the effective diffusion coefficient of substrate in a particle, where the diffusion and consumption of substrate by biocatalytic reaction are present simultaneously, was designed and experimentally verified. The method is based on measuring the overall rate of heterogeneous biocatalytic reaction in particles of varying diameter. The effective diffusion coefficient, D_e, was determined by fitting the measured reaction rates with the solution of the reaction-diffusion equation. The method is tailored for cases where the enzyme reaction is governed by the Michaelis-Menten kinetics. The value of K_m required for the solution of the mathematical model was adopted from the measurement of the kinetics of free cells, whereas the rate parameter, k₂, was optimized together with D_e. As an experimental model, the sucrose hydrolysis catalyzed by Ca-alginate-entrapped yeast cells was examined. The particle diameter varied in the range of 1.2–3.9 mm and the initial reaction rates were measured in a batch-stirred reactor at a sucrose concentration of 100 m . The D_e of sucrose at 30°C was found to be 2.9 · 10⁻¹⁰ m²s⁻¹.     

Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments.

Viscosogenic agents were used to test the diffusion limits of the reaction catalyzed by the catalytic subunit of the cAMP-dependent protein kinase. The effects of glycerol and sucrose on the maximum rate (kcat) and the apparent second-order rate constants (kcat/Kpeptide) for the phosphorylation of four peptidic substrates were measured at their pH optima. The agents were found to have moderate to no effect on kcat/Kpeptide for good and poor substrates, respectively. Conversely, kcat was highly sensitive to solvent viscosity for three of the four peptides at high concentrations of ATP. Taken together, these data indicate that enzymatic phosphorylation by the catalytic subunit proceeds with rapid or near rapid equilibrium binding of substrates and that all steps following the central substrate complex (i.e., chemical and conformational events) are fast relative to the rate-determining dissociation of product, ADP, when ATP levels are high. Under saturating concentrations of peptide I, LRRASLG, an unproductive form of the enzyme is populated. The observed phosphorylation rate from this complex is involved in rate limitation owing to a slow step separating unproductive and productive enzyme forms. The data are used to establish a kinetic mechanism for the catalytic subunit that predicts initial reaction velocities under varying concentrations of ATP and substrate.     

Allosteric properties, substrate specificity, and subsite affinities of honeybee alpha-glucosidase I

The substrate specificity of honeybee alpha-glucosidase I, a monomeric enzyme was kinetically investigated. Unusual kinetic features were observed in the cleavage reactions of sucrose, maltose, p-nitrophenyl alpha-glucoside, phenyl alpha-glucoside, turanose, and maltodextrin (DP = 13). At relatively high substrate concentrations, the velocities of liberation of fructose from sucrose, glucose from maltose, p-nitrophenol from p-nitrophenyl alpha-glucoside, and phenol from phenyl alpha-glucoside were accelerated, and so the Lineweaver-Burk plots were convex, indicating negative kinetic cooperativity: the Hill coefficients were calculated to be 0.50, 0.64, 0.50, and 0.67 for sucrose, maltose, p-nitrophenyl alpha-glucoside, and phenyl alpha-glucoside, respectively. For the degradation of turanose and maltodextrin, the enzyme showed a sigmoidal curve in v versus s plots and thus catalyzed the reaction with positive kinetic cooperativity. The Lineweaver-Burk plots were concave and the Hill coefficients were 1.2 and 1.5 for turanose and maltodextrin, respectively. These unique properties cannot be interpreted by the reaction mechanism that Huber and Thompson proposed: (1973) Biochemistry 12, 4011-4020. The rate parameters for the hydrolysis of sucrose, maltose, p-nitrophenyl alpha-glucoside and phenyl alpha-glucoside were estimated by extrapolating the linear part of the Lineweaver-Burk plots at low substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mathematical modeling of asymmetric reduction of ethyl 4-chloro acetoacetate by bakers’ yeast

A mathematic model was developed to simulate the asymmetric reduction of ethyl 4-chloro acetoacetate (ECA) by bakers’ yeast. The model of the process considered the kinetics of enzymatic reaction, the effect of substrate inhibition and the spontaneous degradation of the substrate. The reaction kinetics of the ECA degradation was determined empirically. The inhibition by the substrate was analyzed and the apparent kinetic constants of the overall enzymatic reaction, of the S-enzymes and of the R-enzymes, were estimated individually. The system of equations was solved numerically using the Runge–Kutta method. The close correlation between the predicted and experimental results concerning product formation, reaction yield and optical purity of product under various substrate concentrations, implied the reliability of the established model.     

Switching the mode of sucrose utilization by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H⁺ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.     

Degradation of ferrous(II) cyanide complex ions by Pseudomonas fluorescens

Degradation of ferrous(II) cyanide complex (ferrocyanide) ions by free cells of P. fluorescens in the presence of glucose and dissolved oxygen was investigated as a function of initial pH, initial ferrocyanide and glucose concentrations and aeration rate in a batch fermenter. The microorganism used the ferrocyanide ions as the sole source of nitrogen. The ferrocyanide biodegradation rate was 30.7 mg g⁻¹ h⁻¹ under the conditions of initial pH: 5, stirring rate: 150 rpm, aeration rate: 0.15 vvm, initial ferrous(II) cyanide complex ion and glucose concentrations: 100 mg l⁻¹ and 0.465 g l⁻¹, respectively. The culture utilized glucose as the main substrate following the non-competitive toxic component inhibition model in the presence of 100 mg l⁻¹ initial ferrous(II) cyanide complex ion concentration. The inhibition of ferrous(II) cyanide complex ions as a secondary substrate began at very low concentrations. A mathematical model, based on non-competitive substrate inhibition was used to describe the inhibitory effect of ferrous(II) cyanide complex ions on the growth of microorganism and the best fitted model parameters were determined by non-linear regression techniques.     

Effect of high sucrose concentrations on mitochondria: Analysis of mitochondrial populations by density-gradient centrifugation after fixation with glutaraldehyde

It has been shown previously that intact rat liver mitochondria can be separated into two populations (designated B2 and B3) with mean buoyant densities of 1·184 and 1·216 respectively, by isopycnic sucrose density gradient centrifugation. A comparison has been made of some properties of these mitochondrial fractions from density gradients with non-fractionated mitochondria. Use was made of density gradient centrifugation for analysis of preparations fixed with appropriate concentrations of glutaraldehyde. The permeability of the membranes of non-fractionated mitochondria to sucrose was increased by exposure to hypoosmotic sucrose solutions. The B3 mitochondria differed from the non-fractionated mitochondria in their response to changes in osmotic pressure of the suspending medium while the B2 mitochondria showed essentially identical behaviour with the controls. However, under conditions of energized swelling the B2 mitochondria were markedly different to the controls. This difference, which is attributed to reduced permeability of the mitochondrial membranes to metabolites brought about by exposure to the high concentrations of sucrose encountered in the density gradient, was reversed by incubation in hypo-osmotic sucrose solutions in the presence of oxidizable substrate and permeant ions.Died December, 1969.     

Sucrose Synthetase of Sweet Potato Roots

The effect of concentration of each substrate in the reaction catalyzed by sucrose synthetase isolated from sweet potato roots was determined. For the sucrose synthesizing reaction, UDP-glucose(ADP-glucose)+fructose→sucrose+UDP(ADP), the substrate saturation curves for UDP-glucose, ADP-glucose and fructose were hyperbolic in shape and the reaction was strongly inhibited by UDP competitively. On the other hand, the substrates for the reversal of sucrose synthetase reaction, sucrose+UDP(ADP)→UDP-glucose(ADP-glucose)+fructose, exhibited a sigmoidal shaped saturation curve which was deviated from the Michaelis-Menten equation. The plot of data according to the empirical Hill equation gives a values greater than 1.0 for every substrate examined in the latter case. In view of these experimental data, the major role of sucrose synthetase is postulated in that this enzyme is involved in the breakdown of sucrose in sweet potato root tissues instead of the sucrose synthesizing reaction. The molecular weight of the enzyme was determined to be about 540,000 by the Sephadex gel filtration chromatography.     

NMR for direct determination of K(m) and V(max) of enzyme reactions based on the Lambert W function-analysis of progress curves

(1)H NMR spectroscopy was used to follow the cleavage of sucrose by invertase. The parameters of the enzyme's kinetics, K(m) and V(max), 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 K(m) and V(max) were fitted to obtain the experimental progress curve and resulted in K(m)=28mM and V(max)=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 K(m)=379μM and k(cat)=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.     

Biochemical properties of inulosucrase from Leuconostoc citreum CW28 used for inulin synthesis

Biochemical properties of inulosucrase from Leuconostoc citreum CW28, a potential biocatalyst for inulin synthesis, were determined in order to select optimal reaction conditions. The hydrolysis reaction was about 3.5 times more efficient than the transferase reaction. It was found that high sucrose concentrations (≈250 g L^-1) were required for maximum fructose transferase yields. High molecular weight inulin distributions were obtained with cell associated inulosucrase, while lower size products were associated to the activity of the free enzyme in solution. When using whole cells, mannitol was found as a by-product of the reaction resulting from the reduction of fructose released by sucrose hydrolysis. A 30 L pilot plant synthesis with 250 g L^-1 of sucrose was carried out using the cell associated inulosucrase resulting in 76% of the substrate being transformed to inulin.