Simulation of a reactor for glucose isomerization to fructose by immobilized glucose isomerase with continuous enzyme renewal

Two different dispositions of laboratory-scaled columns have been tested to simulate the isomerization of glucose to fructose in a mobile bed reactor where exhausted immobilized glucose isomerase is continuously renewed. If the simulation columns working at 65°C are arranged in parallel and connected to a section for final enzyme exploitation at 75°C, a syrup with constant composition can be produced, at relatively constant total throughput, by feeding the individual columns at flow rate decreasing according to the enzyme decay profile and following a programmed disphased mode of operation.This revised version was published online in October 2005 with corrections to the Cover Date.     

Design of immobilized enzyme reactors for the continuous production of fructose syrup from whey permeate

Biocatalyst inactivation is inherent to continuous operation of immobilized enzyme reactors, meaning that a strategy must exist to ensure a production of uniform quality and constant throughput. Flow rate can be profiled to compensate for enzyme inactivation maintaining substrate conversion constant. Throughput can be maintained within specified margins of variation by using several reactors operating in parallel but displaced in time. Enzyme inactivation has been usually modeled under non-reactive conditions, leaving aside the effect of substrate and products on enzyme stability. Results are presented for the design of enzyme reactors under the above operational strategy, considering first-order biocatalyst inactivation kinetics modulated by substrate and products. The continuous production of hydrolyzed-isomerized whey permeate with immobilized lactase and glucose isomerase in sequential packed-bed reactors is used as a case study. Kinetic and inactivation parameters for immobilized lactase have been determined by the authors; those for glucose isomerase were taken from the literature. Except for lactose, all other substrates and products were positive modulators of enzyme stability. Reactor design was done by iteration since it depends on enzyme inactivation kinetics. Reactor performance was determined based on a preliminary design considering non-modulated first-order inactivation kinetics and confronted to such pattern. The new pattern of inactivation was then used to redesign the reactor and the process repeated until reactor performance (considering modulation) matched the assumed pattern of inactivation. Convergence was very fast and only two iterations were needed.     

Optimum design of a series of CSTRs performing reversible Michaelis-Menten kinetics: a rigorous mathematical study

The optimum design of a given number of CSTRs in series performing reversible Michaelis-Menten kinetics in the liquid phase assuming constant activity of the enzyme is studied. In this study, the presence of product in the feed stream to the first reactor, as well as the effect of the product intermediate concentrations in the downstream reactors on the reaction rate are investigated. For a given number of N CSTRs required to perform a certain degree of substrate conversion and under steady state operation and constant volumetric flow rate, the reactor optimization problem is posed as a constrained nonlinear programming problem (NLP). The reactor optimization is based on the minimum overall residence time (volume) of N reactors in series. When all the reactors in series operate isothermally, the constrained NLP is solved as an unconstrained NLP. And an analytical expression for the optimum overall residence time is obtained. Also, the necessary and sufficient conditions for the minimum overall residence time of N CSTRs are derived analytically. In the presence of product in the feed stream, the reversible Michaelis-Menten kinetics shows competitive product inhibition. And this is, because of the increase in the apparent rate constant K^' _m that results in a reduction of the overall reaction rate. The optimum total residence time is found to increase as the ratio (‚₀) of product to substrate concentrations in the feed stream increases. The isomerization of glucose to fructose, which follows a reversible Michaelis-Menten kinetics, is chosen as a model for the numerical examples.     

An annular bound-enzyme reactor

A column reactor with an annular cross section was formed by rolling up DEAE cellulose paper and a screening spacer. Glucoamylase was attached by ion adsorption. For the spacer used, pressure drop was very low, suggesting that this form may be useful with feed streams that are not completely particle-free. Tests of this reactor at the high substrate concentrations characteristic of commercial reactors showed very little diffusional resistance, exhibiting zero-order behavior over most of the concentration range. At low concentrations, the reactor had an apparent “half-order” behavior caused by diffusional limitation in the paper. In this range, flow rate influenced the reaction rate, showing that mass transfer in the main stream also is a contributing factor in this range. Because of the high concentrations and the low Michaelis constant (0.0011 M) the reactor does not show first-order behavior, even at very high conversions. The design of a plant-scale reactor was formulated from these data. The increase in the quantity of enzyme necessary to compensate for the effects of diffusion was only a few percent. Two reactors were formed with sheets nonporous to the enzyme, binding the enzyme with cyanogen bromide after forming the reactor. The amount of enzyme bound was about one monolayer, and there appeared to be no diffusional limitations, even at low substrate concentrations.     

Countercurrent multistage fluidized bed reactor for immobilized biocatalysts: I. Modeling and simulation

For the application of immobilized enzymes, fixed bed reactors are used almost exclusively. Fixed bed reactors have specific disadvantages, especially for processes with a deactivating catalyst. Therefore, we have studied a novel reactor type with continuous transport of the immobilized biocatalyst. Flow of biocatalyst is countercurrent to the substrate solution. Because of a stagewise reactor design, back-mixing of biocatalyst is very limited and transport is nearly plug flow. The reactor operates at a constant flow rate and conversion, due to constant holdup of catalytic activity. The reactor performance is compared with a configuration of fixed bed reactors. For reactions in the first-order regime, enzyme requirements in this new reactor are slightly less than for fixed bed processes. The multistage fluidized bed appears to be an attractive reactor design to use biocatalyst to a low residual activity. However, nonuniformity of the particles might affect plug flow transport of the biocatalyst. A laboratory scale reactor and experiments are described in Part II(1) of this series. Hydrodynamic design aspects of a multistage fluidized bed are discussed in more detail in Part III.(2).     

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.     

Optimization of a reactor assembly for the production of 6-APA from penicillin-V

The design and operation of an industrial penicillin-V deacylation reactor is simulated, using a kinetic expression and mass transport parameters for the immobilized enzyme particles which were determined experimentally in a previous study. It is desirable to use a series of equalsized plug flow reactors with pH control at the entrance to each reactor, and with a possibility of recycling reactant in each reactor. These measures are necessary to avoid a steep pH profile through the reactor; the deacylation reaction is accompanied by an increase of acidity of the reaction medium, and H(+) is a strong inhibitor and may deactivate the enzyme. The optimization study which is carried out at a fixed penicillin conversion of x = 0.99 shows that it is uneconomical to use penicillin feed concentrations above 150mM-175mM, and that the buffer concentration in the reaction medium should not be less than 50mM-75mM. Increasing the number of reactors from 4 to 8 or 10 leads to higher productivity of 6-APA, and a moderate recycle in the first couple of reactors diminishes the sharp decrease in pH which will be found in a straight plug flow reactor operation of the equipment. Higher pumping costs and lower productivity are unavoidable drawbacks of an operation mode where the separation costs for the product mixture are desired to be low.     

Development of a Polyaniline-coated Monolith Reactor for the Synthesis of Cephalexin Using Penicillin G Acylase Aggregates

A monolith reactor for the synthesis of cephalexin was developed using capillary columns. The micro channel in the monolith reactor was coated with polyaniline (PANI), and penicillin G acylase was aggregated with PANI using 0.5% of glutaraldehyde as a cross-linker. The developed monolith reactor exhibited many advantages over other enzyme reactors such as batch and continuous reactors. It showed fast enzyme reaction rates owing to the decrease in external mass transfer and internal diffusion limitations. The reactor can easily be scaled up by bundling together multiple monolith reactors, enabling a corresponding increase in feed rate. Furthermore, the monolith reactor showed good operational stability, with 95% of its original activity maintained after 48 h of continuous operation. The PANI coating on the surface of the capillary column increased the enzyme immobilization capacity and conversion was increased from 15.4% to 70.6% after PANI coating. The conversion ratio increased to approximately 70.6% with an increase in residence time and reactor length.     

Hydrolysis of particulate tributyrin in a fluidized lipase reactor.

Pancreatic lipase has been immobilized onto stainless steel beads by adsorption followed by crosslinking, and onto polyacrylamide by covalent bonding. The activities of the two types of immobilized enzyme toward the particulate substrate, tributyrin emulsion droplets, were determined experimentally, and rate constants based on Michaelis-Menten kinetics were calculated. The activity of the stainless steel-lipase was determined for various flow conditions and for various support sizes by the use of a differential fluidized bed recycle reactor. The rate constants calculated indicate that the experimental reaction rate is free from mass transfer influences, since the observed Michaelis constant does not vary with the fluidization velocity or with the support particle size. In addition, the Michaelis constant of the stainless steel-lipase was found to be equal to that of the free enzyme, suggesting that adsorption and subsequent crosslinking does not alter the enzyme-substrate affinity. The emulsion substrate mass transfer rates, calculated from the filtration theory, indicate that each substrate particle which contact the immobilized enzyme is hydrolyzed to a significant extent. The experimentally determined kinetic rate constants may be used directly to predict the size of integral fluidized bed reactors.     

Solubilization and concentration of carbon dioxide: novel spray reactors with immobilized carbonic anhydrase

Novel spray reactors are described that employ immobilized biocatalyst (carbonic anhydrase), enabling concentration and solubilization of emitted CO(2) by allowing catalytic contact with water spray. The reactors were fed with simulated emission gas. The performance of the reactors was investigated with respect to operation variable: emission flow rate; gas composition in the emission stream; water flow rate; area-to-volume ratio of immobilized reactor core; and the enzyme load within the core. The reactors were also investigated for pressure drop and extractability of CO(2) from the emission with single vs. multiple reactors (of combined equal volume). The biotechnological process of solubilization and concentration of CO(2) from emission exhausts or streams occurring in the spray reactors could be coupled for further biochemical/chemical conversion of the concentrated CO(2).     

Optimal design for CSTR's in series performing enzymatic lactose hydrolysis

Analytical expressions are derived for the optimal design (based on minimum overall reactors volume) of a series of N CSTR's performing enzymatic lactose hydrolysis. It is assumed that lactose hydrolysis obeys Michaelis-Menten kinetics with competitive product (galactose) inhibition and no enzyme deactivation occurs. The optimum design of a cascade of ideally mixed reactors are compared with equal size reactors and with plug flow reactor required for a given overall degree of lactose conversion. The effect of operating parameters such as temperature, lactose initial (feed) concentration and conversion, enzyme and product initial concentration on the optimal overall holding time are also investigated. Optimization results for a series of N CSTR's up to five are obtained and compared with plug flow reactor.     

The production of 6-aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase

A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two-stage plug-flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two- and three-stage PFRs were simulated by computer. In the case of the two-stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree-stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.     

Reduced fouling and enhanced microbial inactivation during online sterilization of cheese whey using UV coil reactors in series

The effectiveness of coil UV reactor series for the online sterilization of cheese whey was compared to those of the single conventional and coil reactors at various flow rates (5–70 mL/min). The residence time varied from 168 to 12 min and from 48 to 24 min for the single and the series reactors, respectively. Hundred percent destruction efficiency could not be achieved in the single reactors whereas in the coil reactor series the destruction efficiency reached 100% at the flow rates of 35 and 40 mL/min. The rate of microbial destruction was described by polynomial equation for the single coil reactor and by exponential equations for the single conventional reactor and the coil reactor series. The temperature of the effluent decreased with the increase in flow rate in all the reactors. The maximum effluent temperatures in the single conventional reactor, single coil reactor and coil reactor series were 45.8, 46.1, and 36.4 °C (Δt = 20.8, 21.1, 11.4 °C), respectively. The flow in all the reactors was laminar (R _e = 1.39–20.10) and the Dean number was in the range of 1.09–15.41 in the coil reactors. Visual observation revealed less fouling on the UV lamps of coil reactors than on that of the conventional reactor due to the impact of Dean flow. The total operating time during which 100% destruction efficiency is achieved prior to the advent of fouling was 240 min in the coil reactor series compared to only 45 min in the conventional reactor.     

Kinetcs and decay of fumarase activity of immobilized Brevibacterium ammoniagenes cells for continuous production of L-malic acid

The kinetics of the reversible fumarase reaction of immobilized Brevibacterium ammoniagenes cells and the decay behavior of enzyme activity were investigated in a plug flow system. The time course of the reaction in the immobilized cell column was well explained by the time-conversion equation including the apparent kinetic constants of the immobilized cell enzyme. The decay rate of fumarase activity was faster in the upper sections of the column (inlet side of the substrate solution) compared with the lower sections when 1M sodium fumarate (pH 7.0) was continuously passed through the column at 37°C. It was shown that the decay rate of the fumarase activity in the immobilized cell column depends on the flow rate of the substrate solution. The effect of flow rate on the decay rate of enzyme activity was considered to be related to the rate of contamination of enzyme with poisonous substances derived from the substrate solution or to the rate of leakage of enzyme stabilizers and/or enzyme itself from the immobilized cells.     

Thermal stability of microsomal glucose-6-phosphatase

The thermal stability of glucose-6-phosphatase in rat liver microsomes was examined in untreated and cholate-treated microsomes. Activity of the enzyme was measured with both glucose-6-P and mannose-6-P as substrates. Heat treatment did not cause glucose-6-phosphatase activity to decline to zero with a single rate constant in untreated microsomes. Instead, heat treatment produced an enzyme with a small residual activity that was stable. The residual level of activity was not stimulated by addition of detergent. In untreated microsomes the energies of activation for the processes of decay were different for glucose-6-phosphatase and mannose-6-phosphatase activities, suggesting that the rate-limiting steps for the hydrolysis of these compounds were different. Treatment of microsomes with detergent increased the rate constants for the thermal decay of glucose-6-phosphatase by about 150 times, and, in contrast to untreated microsomes, glucose-6-phosphatase and mannose-6-phosphatase decayed to zero with a single rate constant in cholate-treated microsomes. Also, rate constants for thermal inactivation of glucose-6-phosphatase and mannose-6-phosphatase were the same in cholate-treated microsomes. Removal of cholate increased the stability of glucose-6-phosphatase but did not regenerate the form of the enzyme present in untreated microsomes. The data for the stability of glucose-6-phosphatase under different conditions provide evidence that the enzyme can exist in at least five different stable states that are enzymatically active.     

Quenching of protein fluorescence by transient intermediates in the liver alcohol dehydrogenase reaction.

The addition of saturating concentrations of NAD-+ and alcohol to liver alcohol dehydrogenase in a stopped flow fluorimeter results in a triphasic quenching of enzyme fluorescence. A rapid quenching occurs with a rate constant of 300 to 500 s-minus 1, followed by a slower reaction at 50 to 100 s-minus 1, and ultimately followed by a very slow reaction. The addition of NAD-+ to enzyme in the absence of substrate causes a rapid quenching of enzyme fluorescence at 300 to 500 s-minus 1, with the same amplitude as the rapid phase in the presence of substrate. These studies demonstrate that NAD-+ binding to liver alcohol dehydrogenase causes a conformational change at a rate compatible with the previously reported rate constant for proton release, indicating that proton release is probably coupled to the conformational change.     

Optimization of batch processes involving simultaneous enzymatic and microbial reactions

Two general models for batch simultaneous enzymatic and microbial reaction (SEMR) processes are presented, the second derived from and simpler than the first and accounting for enzyme denaturation. Using the second model and parameter values from the literature, simulation was used to examine a range of enzyme addition rate strategies (in which the rate was a linear function of time) for a relatively fast ethanol fermentation and for a longer duration citric acid fermentation, both using cellulose as the substrate. For the ethanol process it is optimal (for a specific objective function which accounts for product value and enzyme cost) to add all the enzyme at the beginning of the process. But for the citric acid process a linearly decreasing enzyme addition rate, coupled with the addition of a small fraction of the enzyme at time zero, is better than pure batch operation or operation with the best constant enzyme feed rate.     

On-line sterilization of cheese whey using ultraviolet radiation

The effectiveness of ultraviolet radiation for on-line sterilization of cheese whey was investigated. The effects of flow rate and residence time on the performance of three UV reactors having different gap sizes (18, 13, and 6 mm) were studied. Six flow rates and six residence times were tested with the three UV reactors. The cheese whey used in this study had a very high turbidity (4317 NTU), very poor transmittance in the UV radiation germicidal range ( approximately 0%), and high percentage of large solid particles ( approximately 20% > 100 microm). Although the cheese whey physical characteristics showed low probability of sterilization using UV radiation, the study showed that UV radiation can be used on-line to sterilize cheese whey if the proper reactor gap size and the appropriate residence time are used. There were combined effects of the flow rate and gap size. The cell removal efficiency increased with increases in residence time and decreases in the UV reactor gap size. Removal efficiency of 100% was not achieved in this study with the first UV reactor (18-mm gap size), whereas 100% removal efficiency was achieved with the second (13-mm gap size) and third (6-mm gap size) UV reactors at residence times of 2.0 and 0.5 h, respectively. The microbial decay rates achieved in this study were 4.94, 7.62, and 20.9 h(-)(1) using the first, second, and third UV reactor, respectively. Residence times of 3.3, 2.1, and 0.8 h would be required to completely destruct a microbial population of 5.95 x 10(6) cells/mL using the first, second, and third UV reactors, respectively. Although cheese whey sterilization using UV radiation seems to be a good alternative to pasteurization, increases in cheese whey temperature resulted in lamp fouling. If online sterilization is to be used, the fouling problem should be investigated and a maintenance scheme for the UV reactor should be developed.     

Determination of the turn-off reaction for the hormone-activated adenylate cyclase.

Previous work suggested that hormonal activation of adenylate cyclase involves the introduction of GTP to the regulatory site, and subsequent hydrolysis of the bound GTP terminates the activation. In many tissues the turn-off GTPase reaction cannot be readily measured because of a high background of nonspecific GTP hydrolysis. To circumvent this problem a general assay for the turn-off reaction has now been developed. The adenylate cyclase is first activated by hormone and GTP and the introduction of GTP is then stopped either by addition of an excess of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) or by addition of a receptor blocking agent. The decay of adenylate cyclase activity brought on by these inhibitors is used to calculate the rate constant of the turn-off reaction. In turkey erythrocyte and rat parotid membranes the rate constant of the decay process as determined with GDP beta S is similar to that determined with the beta-adrenergic blocker propranolol. The rate constants (min-1 at 30 degrees C) for various adenylate cyclase preparations are 10 for turkey erythrocyte, 7.5 for rat parotid, and 6.2 for the rat liver enzyme. The finding of similar rate constants in the various preparations indicates that GTP hydrolysis at the regulatory site is a general mechanism for terminating the activation of adenylate cyclase.