Performance of the continuous glucose isomerase reactor system for the production of fructose syrup

Glucose isomerase in the form of heat-treated whole-cell enzyme prepared from Streptomyces phaeochromogenus follows the reversible single-substrate reaction kinetics in isomerization of glucose to fructose. Based on the Kinetic constants determined and the mathematical model of the reactor system developed, the preformance of a plug-flow-type continuous-enzyme reactor system was studied experimentally and also simulated with the aid of a computer for the ultimate objective of optimization of the glucose isomerase reactor system. The enzyme decay function for both the enzyme storage and during the use in the continuous reactor, was found to follow the first-order decay kinetics. When the enzyme decay function is taken into consideration, the ideal homogeneous enzyme reactor kinetics provided a satisfactory working model without further complicatin of the mathematical model, and the results of computer simulation were found to be in good agreement with the experimental results. Under a given set of constraints the performance of the continuous glucose isomerase reactor system can be predicted by using the computer simulation method described in this paper. The important parameters studied for the optimization of reactor operation were enzyme loading, mean space time of the reactor, substrate feed concentration, enzyme decay constants, and the fractional conversion, in addition to the kinetic constants. All these parameters have significant effect on the productivity. Some unique properties of the glucose isomerization reaction and its effects on the performance of the continuous glucose isomerase reactor system have been studied and discussed. The reaction kinetics of glucose isomerase and the effects of both the enzyme loading and the changes in reaction rate within a continuous reactor on the productivity are all found to be of particular importance to this enzyme reactor system.     

New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates

Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox‐dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non‐invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H₂ and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase‐mediated reactions simultaneously. Using the proposed system, we confirmed that H₂ (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D₂/H₂O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (k_out) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.     

The influence of geometry on hydrodynamic and mass transfer characteristics in an external airlift reactor for the cultivation of filamentous fungi

The influences of geometric configuration, mycelial broth rheology and superficial gas velocity (U_sg) were investigated with respect to the following hydrodynamic parameters: gas holdup (), oxygen transfer coefficient (K_La) and mixing time (t_m). Increases in U_sg and height of gas separator (H_t) caused an increase in and K_La, and a decrease in t_m. Consequently, a diameter ratio (D_d/D_r) of 0.71 and H_t 0.20 m were found to be the best geometry and operation parameters to achieve high aeration and mixing efficiency for the high viscous broth system in the cultivation of filamentous fungi. An external airlift reactor (EALR) was developed and designed for the cultivation of filamentous fungi. The EALR with two spargers excels in reliability and high aeration and mass transfer coefficiency, resulting in a fast mycelial growth and high biomass productivity in the cultivation of the fungus Rhizopus oryzae.     

Potential of Thiobacillus ferrooxidans for waste gas purification. Part 2. Increase in continuous ferrous iron oxidation kinetics using immobilized cells

Thiobacillus ferrooxidans could be used to regenerate ferrous sulphate solution produced in a process to remove H₂S from waste gas if the reaction rate could be increased. The aim of the present study was to increase the volumetric productivity by using immobilized cells. Kinetic data of ferrous iron oxidation were determined in fixed-bed and fluidized-bed reactor configurations with different support materials in order to find the most practical system for scale-up. By using a fixed-bed reactor the iron oxidation rate can be increased to 3.6 g l^–1 h^–1, fivefold higher than suspended cells, and results in a bioreactor of reasonable size. With the kinetic data obtained, the biological reaction is no longer a limiting factor for industrial-scale application. Correspondence to: W. Schäfer-Treffenfeldt     

Kinetic Modeling of Amino Acid Oxidation Catalyzed by a New D‐Amino Acid Oxidase from Arthrobacter protophormiae

Amino acid oxidases, which enantiospecifically catalyze the oxidative deamination of either D‐ or L‐amino acids, belong to the class of oxidoreductases functioning with a tightly bound cofactor. This cofactor favors industrial applications of D‐amino acid oxidases (D‐AAO). Hence, the enzyme is very important for the industrial application in the purification and determination of certain amino acids. In developing the enzyme‐catalyzed reaction for large‐scale production, modeling of the reaction kinetics plays an important role. Therefore, the subject of this study was the kinetics of the oxidative deamination, a very complex reaction system, which is catalyzed by D‐AAO from Arthrobacter protophormiae using its natural substrate D‐methionine and the aromatic amino acid 3,4‐dihydroxyphenyl‐D‐alanine (D‐DOPA). The kinetic parameters determined by the measurement of the initial rate and nonlinear regression were verified in batch reactor experiments by comparing calculated and experimental concentration‐time curves. It was found that the enzyme is highly specific towards D‐methionine (K_m = 0.24 mM) and not as specific to D‐DOPA as a substrate (K_m = 9.33 mM). The enzyme activity towards D‐methionine ( = 3.01 U/mL) was approx. seven times higher than towards D‐DOPA ( = 20.01 U/mL). The enzyme exhibited no activity towards L‐methionine and L‐DOPA. Batch and repetitive batch experiments were performed with both substrates in the presence and in the absence of catalase for hydrogen peroxide decomposition. Their comparison made it possible to conclude that hydrogen peroxide has no negative influence on the enzyme activity.     

Optimization of glucose isomerase reactor: optimum operating temperature mode

The optimum temperature operation mode required to achieve high fructose productivity is studied for immobilized glucose isomerase (GI) packed bed reactor. In this study, the reactor design equation based on reversible Michaelis-Menten kinetics assumes both thermal enzyme deactivation and substrate protection. The optimization problem is formulated as a discretized constrained nonlinear programming problem (NLP). The formulation is expressed in terms of maximization of fructose productivity as the objective function subject to reactor design equation, kinetic parameter equations, substrate protection factor equation and feasibility constraints. The constraints are discretized along the reactor operating period by employing piecewise polynomial approximations. Approximately 7% improvement in terms of fructose productivity is achieved when running the reactor at the optimum decreasing temperature operation mode as compared to the constant optimum isothermal operation.     

Cephalexin synthesis by partially purified and immobilized enzymes

An enzyme which catalyzes the synthesis of cephalexin fromD -α phenylglycinemethylester (PGM) and 7-amino-3-desacetoxy-cephalosporanic acid (7-ADCA) was prepared from Xanthomonas citri (IFO 3835) and partially purified 30-fold by ammonium sulfate fractionation, DEAE-cellulose, and Sepharose-4B column chromatography. The K_m values for 7-ADCA, PGM, and cephalexin were determined as 11.1, 2.1, and 1.61 mM, respectively. The enzymatic cephalexin synthesis follows the reversible bi-uni reaction kinetics. The equilibrium constant is influenced by the initial mole ratios of 7-ADCA and PGM. The cephalexin hydrolysis is catalyzed by the same cephalexin synthesizing enzyme, but methanol does not participate in the hydrolytic reaction. The amount of enzyme in the reaction mixture affects the initial rate but does not influence the equilibrium product concentration. This cephalexin-synthesizing enzyme was immobilized onto several adsorbents. Among these, Kaolin and bentonite showed a higher retention of enzyme activity and stability for reuse. The immobilized-enzyme reaction kinetics were investigated and compared with those of the soluble enzyme. A rate expression for the enzymatic synthesis of cephalexin was derived. The results of computer simulation showed good agreement with the experimental results.     

Optimization of operating temperature for continuous glucose isomerase reactor system

The Optimal temperature control policy for an immobilized glucose isomerase reactor system was studied. This optimization study takes into consideration the enzyme deactivation during the continuous reactor operation. The Kinetic parameters including reduced Michaelis–Menten constant (K?_m), reduced maximum reaction rate (V?_m), equilibrium constant (K_e), and enzyme deactivation constant (k_d) and their functional relationships to temperature were determined experimentally. The optimization problem was formulated in terms of maximization of fructose productivity as the objective function. The optimization problem was solved by making use of a maximum principle and the control vector iteration method. Approximately optimal temperature control policy was employed as compared with the reactor operation at an optimum constant temperature.     

Reversible addition of bisulfite buffer to the cytidine ring system

The rate constants for the reversible addition of protons and sulfite to the 5,6 double bond of cytidine and 3-methylcytidine have been spectrophotometrically measured under conditions (25°C, μ = 1.0 ) where the deamination of 5,6-dihydrocytidine-6-sulfonate is minimal. Both the addition and the elimination of sulfite from the ring system are subject to general catalysis of proton transfer. For the reaction in either direction, plots of the pseudo-firstorder rate constants against increasing buffer concentration are biphasic and indicative of at least a two-step reaction pathway with both steps being subject to general acid-base catalysis. Kinetic hydrogen-deuterium isotope effects were measured for both buffer-catalyzed steps of sulfite elimination from 3-methyl-5,6-dihydrocytidine-6-sulfonate and sulfite addition to 3-methylcytidine. Both H₂O and D₂O were used as solvent. For both the addition and the elimination of SO₃²⁻ values of k₂^H/k₂^D were 6.3–7.1 and 2.3–2.6 at low and high imidazole buffer concentration, respectively. The large isotope effects values in the range of 6–7 can be attributed to rate-determining proton transfer to carbon-5 of the cytidine ring system. The smaller values are more likely caused by proton transfer to a electronegative atom such as the oxygen on carbon-2 of the cytidine ring. The equilibrium constants for bisulfite buffer addition to 3-methylcytidine and cytidine at 25°C, μ = 1.0 , pH 7.2, are 10.2 and 1.3 ⁻¹, respectively.     

Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcoreTM binding data. Analysis of the interaction of NC10 Fab with an anti-idiotype Fab′

The interaction of monovalent Fab fragments of NC10, an antiviral neuraminidase antibody, and the anti-idiotype antibody 3-2G12 has been used as a model system to demonstrate experimentally the influence of non-ideal binding effects on BIAcore^TM binding data. Because the association rate constant for these two molecules was found to be relatively high (about 5×10⁵ M ⁻¹ s⁻¹), mass transfer was recognised as a potential source of error in the analysis of the interaction kinetics. By manipulation of the flow rate and the surface density of the immobilised ligand, however, the magnitude to this error was minimised. In addition, the application of site-specific immobilisation procedures was found to improve considerably the correlation of experimental binding data to the ideal 1:1 kinetic model such that the discrepancy between experimental and fitted curves was within the noise range of the instrument. Experiments performed to measure the equilibrium constant (K_D) in solution resulted in a value of similar magnitude to those obtained from the ratio of the kinetic rate constants, even those measured with a heterogeneous ligand or with a significant mass transfer component. For this system, the experimental complexities introduced by covalent immobilisation did not lead to large errors in the K_D values obtained using the BIAcore © 1997 John Wiley & Sons, Ltd.     

Modeling and kinetic parameter estimation of alcohol dehydrogenase‐catalyzed hexanol oxidation in a microreactor

A mathematical model for hexanol oxidation catalyzed by NAD⁺‐dependent alcohol dehydrogenase from baker's yeast in a microreactor was developed and compared with the model when the reaction takes place in a macroscopic reactor. The enzyme kinetics was modeled as a pseudo‐homogeneous process with the double substrate Michaelis–Menten rate expression. In comparison with the kinetic parameters estimated in the cuvette, a 30‐fold higher maximum reaction rate and a relatively small change in the saturation constants are observed for the kinetic parameters estimated in the continuously operated tubular microreactor (V_m1=197.275 U/mg, K_m^hexanol=9.420 mmol/L, and K_m1^NAD+=0.187 mmol/L). Kinetic measurements performed in the microreactor, estimated from the initial reaction rate experiments at the residence time of 36 s, showed no product inhibition, which could be explained by hydrodynamic effects and the continuous removal of inhibiting products. The Fourier amplitude sensitivity test method was applied for global kinetic parameter analysis, which shows a significant increase in the sensitivity of K_m1^NAD+ in the microreactor. Independent experiments performed in the microreactor were used to validate and to verify the developed mathematical model.     

The specific interfacial area in external-loop airlift bioreactors

The specific interfacial areas in two external-loop airlift bioreactors of laboratory and pilot scale were determined, mainly by the chemical reaction method (sulphite oxidation). The parameter studied in water/salt and starch/salt solutions was greately affected by gas superficial velocity, A _D /A _R ratio, by H _R ?H _D /H _D ratio and η _ap , respectively. Correlations for the specific interfacial area in the two systems, considering the effects of the above-mentioned parameters, were proposed.     

Optimization of Operating Temperature for Continuous Immobilized Glucose Isomerase Reactor with Pseudo Linear Kinetics

In this work, the optimal operating temperature for the enzymatic isomerization of glucose to fructose using a continuous immobilized glucose isomerase packed bed reactor is studied. This optimization problem describing the performance of such reactor is based on reversible pseudo linear kinetics and is expressed in terms of a recycle ratio. The thermal deactivation of the enzyme as well as the substrate protection during the reactor operation is considered. The formulation of the problem is expressed in terms of maximization of the productivity of fructose. This constrained nonlinear optimization problem is solved using the disjoint policy of the calculus of variations. Accordingly, this method of solution transforms the nonlinear optimization problem into a system of two coupled nonlinear ordinary differential equations (ODEs) of the initial value type, one equation for the operating temperature profile and the other one for the enzyme activity. The ODE for the operating temperature profile is dependent on the recycle ratio, operating time period, and the reactor residence time as well as the kinetics of the reaction and enzyme deactivation. The optimal initial operating temperature is selected by solving the ODEs system by maximizing the fructose productivity. This results into an unconstrained one‐dimensional optimization problem with simple bounds on the operating temperature. Depending on the limits of the recycle ratio, which represents either a plug flow or a mixed flow reactor, it is found that the optimal temperature of operation is characterized by an increasing temperature profile. For higher residence time and low operating periods the residual enzyme activity in the mixed flow reactor is higher than that for the plug flow reactor, which in turn allows the mixed flow reactor to operate at lower temperature than that of the plug flow reactor. At long operating times and short residence time, the operating temperature profiles are almost the same for both reactors. This could be attributed to the effect of substrate protection on the enzyme stability, which is almost the same for both reactors. Improvement in the fructose productivity for both types of reactors is achieved when compared to the constant optimum temperature of operation. The improvement in the fructose productivity for the plug flow reactor is significant in comparison with the mixed flow reactor.     

Enzymatic modification of vegetable protein: Mechanism,kinetics, and production of soluble and partially soluble protein in a batch reactor

Enzymatic hydrolysis of insoluble soybean protein by a protease enzyme produced by Penicillium duponti K 1104, was investigated in a batch reactor. The reaction conditions were 30–55°C and pH 3.4–3.7. The mechanism of solubilization of the insoluble protein by the Penicillium duponti enzyme was deduced from a series of experiments. Kinetic models were developed that involved adsorption followed by peptic digestion of protein, inhibition of low-molecular-weight peptides, and enzyme deactivation. The uncoupled kinetic parameters were estimated using the Marquardt nonlinear parameter estimation algorithm. A bang–bang production of soluble and partially soluble protein is suggested for higher productivity. The essential amino acids pattern of the enzyme-Hydrolyzed soy protein was comparable with the unhydrolyzed protein isolate. Aggregation of the soluble protein for an extended time was observable. The low-molecular-weight soluble protein was incorporated into noncarbonated beverages. The amount of protein that could be incorporated into a can of 355 ml noncarbonated beverage, without observable changes in the optical density and also aggregation of the protein, was 2.5 g soluble protein. Beverages with caramel color showed excessive decrease in optical density and precipitation. The kinetics and diffusion in a multipore immobilized-enzyme recycle reactor will be considered in part II of this series.     

Production of precursors for anti-Alzheimer drugs: Asymmetric bioreduction in a packed-bed bioreactor using immobilized D. carota cells

(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (D_e) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate V_max?=?0.052?mmol?L^?1?min^?1, and constants of the Michaelis–Menten K_M?=?2.31?mmol?L^?1. Kinetics constants for immobilized cells, which are considered apparent values, are V_{max, app}?=?0.0407?mmol?L^?1 min^?1, K_{M, app}?=?3.0472?mmol?L^?1 for 2?mm bead diameter, and V_{max, app}?=?0.0453?mmol?L^?1 min^?1, K_{M, app}?=?4.9383?mmol?L^?1 for 3?mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97?×?10^?6?cm²?s^?1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee?>?99%) was produced with 75% conversion at 4-hr residence time.     

Glucose isomerase immobolized on porous glass

Partially purified glucose isomerase from a Streptomyces species was immobilized on porous glass particles and studied for various characteristics concerning its use as an industrial catalyst. The activities were investigated in relation to the reaction parameters and the enzyme deactivation was studied systematically under various reaction conditions. The half-life of the immobilized enzyme was found to exceed 200 days at 50°C. The rate equation of the reversible glucose ? fructose reaction was derived and the kinetic constants were determined. The rate equation was found to be in good agreement with experimental data for both forward and reverse reactions. The degree of diffusional effects was experimentally measured and theoretically analyzed.     

A study on intra-particle diffusion effects in enzymatic reactions: glucose-fructose isomerization

In this work different aspects of the glucose-fructose enzymatic isomerization, using immobilized glucose isomerase, are studied and quantified. Reaction temperatures range from 40?°C to 60?°C. Intra-particle effective diffusivities (D _e), determined after uptake experiments, are between 1.20?×?10^?6?cm²/s, at 40?°C, and 2.52?×?10^?6?cm²/s, at 60?°C. The estimated energy of activation for diffusion (E _aD) is 7.71?kcal/mol. No significant adsorption of the sugars on the support gel matrix is observed. Crushed particles (φ = 150–350?μ) are used during kinetic experiments. For this range of particle diameters, inherent kinetics is approached. A reversible Michaelis–Menten rate equation is fitted to the data, providing the following parameters at pH = 7.0: k ₀ = 2.15?×?10^?6?g/IU/s; E _a/R = 8998?K. Glucose (K _G) and fructose (K _F) affinity constants are essentially the same, ranging from 0.190?M, at 40?°C to 0.305?M, at 60?°C. The thermodynamic equilibrium constant is determined for the three temperatures, and the heat of reaction, estimated from a Van't Hoff plot, is ΔH = 1682?cal/mol. Independent experiments, where the reaction occurs in the presence of significant intra-particle mass transfer resistance, are used as validation tests.     

Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function

The kinetic and mechanistic details of the interaction between caldendrin, calmodulin and the B‐domain of AKAP79 were determined using a biosensor‐based approach. Caldendrin was found to compete with calmodulin for binding at AKAP79, indicating overlapping binding sites. Although the AKAP79 affinities were similar for caldendrin (K_D = 20 n m ) and calmodulin (K_D = 30 n m ), their interaction characteristics were different. The calmodulin interaction was well described by a reversible one‐step model, but was only detected in the presence of Ca²⁺. Caldendrin interacted with a higher level of complexity, deduced to be an induced fit mechanism with a slow relaxation back to the initial encounter complex. It interacted with AKAP79 also in the absence of Ca²⁺, but with different kinetic rate constants. The data are consistent with a similar initial Ca²⁺‐dependent binding step for the two proteins. For caldendrin, a second Ca²⁺‐independent rearrangement step follows, resulting in a stable complex. The study shows the importance of establishing the mechanism and kinetics of protein–protein interactions and that minor differences in the interaction of two homologous proteins can have major implications in their functional characteristics. These results are important for the further elucidation of the roles of caldendrin and calmodulin in synaptic function. Copyright © 2012 John Wiley & Sons, Ltd.     

A mathematical model for the synthesis of Gly-Phe by papain in an aqueous-organic system

The synthesis of the protected dipeptide BocGlyPheOMe, has been modellised when working in an aqueousorganic biphasic system, with papain as a catalyst. The mathematical model takes into account that one of the substrates, PheOMe, has parallel hydrolysis reactions and that the reaction only takes place in the aqueous phase while the whole reaction system is biphasic. The reaction system has been modellised when working in batch as well as when working in fed-batch mode, achieving a good prediction of the product evolution for both working strategies. When working in fed-batch mode, the extension of the undesired parallel reactions has been diminished, the model has been used for a computer aided optimisation of the addition sequence of PheOMe. The results obtained led to a process operation strategy with a compromise between yield and productivity.List of Symbols [i] concentration of any component i - [i] _aq concentration of i in the aqueous phase - [i] _bi concentration of i in the biphasic system - [E] ₀ initial concentration of enzyme - k _e, k_q first order kinetic constants - K _A, K_B equilibrium constants - r _m maximum rate of reaction This worked was financed by the Interministerial Commission for Science and Technology (CICYT)from the Spanish Government under projects number BIO/88-370 and SAF92-0261-CO2-02.