Packed-bed Immobilized Enzyme Reactor Research Articles

Urea removal in rosé and red wines by immobilised acid urease in a packed bed reactor

The aim of this work was to develop and study the performance of a packed bed immobilized enzyme reactor for the effective removal of urea in high polyphenol wines. Purified acid urease from Lactobacillus fermentum was immobilized on Eupergit® C 250 L yielding a biocatalyst with enzyme loading and specific activity per g of dry support of 109.7 ± 2.4 mg and 677 ± 39 IU, respectively. Incubation of the developed biocatalyst at 20 °C in rosé and red wines resulted in a two-phase deactivation mechanism, with a residual asymptotic activity after about 170 h of 67% and 24%, respectively. The developed biocatalyst was used in a packed bed reactor with recycling to efficiently remove urea from rosé and red wines. A model for the bioreactor allowed to estimate the apparent pseudo-first order kinetic constant for urea hydrolysis, which for three repeated bioconversion cycles varied in the range 2.8–4.1 cm3 g−1 min−1 for the rosé wine and 1.1–2.7 cm3 g−1 min−1 for the red wine. Results of repeated bioconversion cycles allowed to conclude that enzyme deactivation by phenols is the main reason for the low urease activity observed in high polyphenol wines. This suggested the use of immobilization as a means to overcome the present limitations in the use of the free enzyme.

Effect of biocatalyst swelling on the operation of packed-bed immobilized enzyme bioreactor

A general mathematical model was developed for predicting the performance and simulation of a packed-bed immobilized enzyme reactor performing a reaction that follows Michaelis–Menten kinetics with competitive product inhibition. The performance of a packed-bed immobilized enzyme reactor was analyzed taking into account the effect of bed swelling on various diffusional phenomena such as axial dispersion, internal and external mass transfer limitations. The numerical solutions were compared with experimental data obtained for a packed-bed reactor operating with β-galactosidase entrapped in Ca-alginate-K-κ-carrageenan gels for lactose hydrolysis.

Determination of immobilized enzyme apparent kinetic parameters in packed-bed reactors: Presentation of a new methodology

A new explicit equation is proposed for the determination of immobilized enzyme apparent Michaelis–Menten kinetic parameters in packed-bed reactors. The mathematical analysis is based on recycling systems. The present methodology is easily applicable for a wide range of operating conditions. It is free from the probable limitations of the single-pass apparent immobilized enzyme kinetic parameter estimation technique that requires simultaneous measurement of column substrate inlet and effluent concentrations at a specified time. The experimental system is composed of a substrate reservoir and a circulating flow packed-bed immobilized enzyme reactor. The experimental data is gathered for glucose decomposition reaction in a recirculation system with immobilized glucose oxidase on a weak base ion exchanger resin. The apparent kinetic parameter, K ′ m values were calculated with a new explicit equation, proposed in this work, and they are found to be 0.48 × 10 −3 and 0.60 × 10 −3 mmol cm −3 for the recirculation rates of 75 and 25 cm 3 min −1, respectively.

Effects of simultaneous internal and external mass transfer and product inhibition on immobilized enzyme-catalyzed reactor

A mathematical model has been developed for predicting the performance and simulation of a packed bed immobilized enzyme reactor performing lactose hydrolysis, which follows Michaelis–Menten kinetics with competitive product (galactose) inhibition. The performance characteristics of a packed bed immobilized enzyme reactor have been analyzed taking into account the simultaneous effects of internal and external mass transfer limitations. The model design equations are then solved by the method of weighted residuals such as Galerkin's method and orthogonal collocation on finite elements. The effects of simultaneous internal and external mass transfer coupled with product inhibition have been studied and their effects were shown to reduce internal effectiveness factor. The effects of product inhibition have been investigated at different operating conditions correlated at different regimes using dimensionless β xo ( St, Bi, θ, ϕ). Product inhibition was shown to reduce substrate conversion, and to decrease effectiveness factor when β s > β xo; however, it increases internal effectiveness factor when β s < β xo. The effectiveness factor is found to be independent of product inhibition at crossover point at which β xo is defined. Effects of St and Bi have been investigated at different kinetic regimes and the results show their effects have a strong dependence on kinetic parameters θ, γ (i.e. K m/ K p) and β xo. The dimensionless residence time at crossover point, β xo, has been correlated with kinetic and mass transfer parameters.

Effects of internal mass transfer and product inhibition on a simulated immobilized enzyme-catalyzed reactor for lactose hydrolysis

A general mathematical model has been developed for predicting the performance and simulation of a packed-bed immobilized enzyme reactor performing lactose hydrolysis, which follows Michaelis–Menten kinetics with competitive product (galactose) inhibition. The performance characteristics of a packed-bed immobilized enzyme reactor have been analyzed taking into account the effects of various diffusional phenomena like axial dispersion, internal and external mass transfer limitations. The model design equations are then solved by the method of weighted residuals such as Galerkin's method and orthogonal collocation on finite elements. The effects of intraparticle diffusion resistances, external mass transfer and axial dispersion have been studied and their effects were shown to reduce internal effectiveness factor. The effects of product inhibition have been investigated at different operating conditions correlated at different regimes using dimensionless β xo ( St, θ, ϕ). Product inhibition was shown to reduce substrate conversion and to decrease internal effectiveness factor when β s > β xo, however it increases internal effectiveness factor when β s < β xo. The effectiveness factor is found to be independent of product inhibition at crossover point at which β xo is defined. Effects of St and Pe have been investigated at different kinetic regimes and the results show their effects have a strong dependence on kinetic parameters θγ (i.e. K m/ K p ) and β xo.

Effects of External Mass Transfer and Product Inhibition on a Simulated Immobilized Enzyme‐Catalyzed Reactor for Lactose Hydrolysis

AbstractA mathematical model has been developed for predicting the performance and simulation of a packed bed immobilized enzyme reactor performing lactose hydrolysis, which follows Michaelis‐Menten kinetics with competitive product (galactose) inhibition. The performance characteristics of a packed bed immobilized enzyme reactor have been analyzed taking into account the effects of various diffusional phenomena like axial dispersion and external mass transfer limitations. The model design equations are then solved by Galerkin's method and orthogonal collocation on finite elements. The effects of external mass transfer and axial dispersion have been studied and their effects were shown to reduce the external effectiveness factor. The effects of product inhibition have been investigated at different operating conditions correlated at different regimes using dimensionless moduli (St, γ, θ, Da)1). The product inhibition was shown to reduce the substrate conversion, and, additionally, to decrease the effectiveness factor when Da &gt; Daxo, however, it increases the effectiveness factor when Da &lt; Daxo. The effectiveness factor is found to be independent of the product inhibition at a crossover point at which Daxo is defined. Effects of St and Pe have been investigated at different kinetic regimes and the results show that their effects have a strong dependency on the kinetic parameters θ, γ (i.e., Km/Kp), and Daxo.

Determination of apparent kinetic parameters for competitive product inhibition in packed-bed immobilized enzyme reactors

In this study, a simple and effective technique for characterizing Michaelis–Menten type kinetics with competitive product inhibition in packed-bed re-circulated immobilized enzyme reactors is presented, where the use of nonlinear regression techniques for multi-parameter estimation are not required. In order to demonstrate the new technique introduced in this work, enzymatic conversion of lactose in a recycling packed-bed reactor is envisaged where β-galactosidase (lactase, EC 3.2.1.23) enzyme is immobilized on a weak base ion exchanger resin (Duolite A 568). For the experimental conditions used in this research, the total competitive inhibition by product (galactose) model is sufficient to represent the lactose hydrolysis kinetics in a packed-bed reactor.

Hydrolysis of lactose by beta-glycosidase CelB from hyperthermophilic archaeon Pyrococcus furiosus: comparison of hollow-fiber membrane and packed-bed immobilized enzyme reactors for continuous processing of ultrahigh temperature-treated skim milk.

Recombinant beta-glycosidase CelB from the hyperthermophilic archaeon Pyrococcusfuriosus was produced through expression of the plasmid-encoded gene in Escherichia coli. Bioreactor cultivations of E. coli in the presence of the inductor isopropyl-1-thio-beta-D-galactoside (0.1 mM) gave approx 100,000 U of enzyme activity/L of culture medium after 8 h of growth. A technical-grade enzyme for the hydrolysis of lactose was prepared by precipitating the mesophilic protein at 80 degrees C. A hollow-fiber membrane reactor was developed, and its performance during continuous processing of ultrahigh temperature-treated (UHT) skim milk at 70 degrees C was analyzed regarding long-term stability, productivity, and diffusional limitation thereof. CelB was covalently attached onto Eupergit C in yields of 80%, and a packed-bed immobilized enzyme reactor was used for the continuous hydrolysis of lactose in UHT skim milk at 70 degrees C. The packed-bed reactor was approximately 10-fold more stable and gave about the same productivity at 80% substrate conversion as the hollow-fiber reactor at 60% substrate conversion. The marked difference in the stability of free and immobilized CelB seems to reflect mainly binding of the soluble enzyme to the membrane surface of the hollow-fiber module. Under these bound conditions, CelB is essentially inactive. CelB is essentially inactive. Microbial contamination of the reactors did not occur during reaction times of up to 39 d, given that UHT skim milk and not pasteurized skim milk was used as the substrate.

A new method for determination of apparent kinetics parameters in recirculating packed-bed immobilized enzyme reactors

In this study, a simple and effective technique for characterizing Michaelis–Menten apparent kinetic parameters in packed-bed immobilized enzyme reactors is presented. The apparent kinetic parameters of immobilized glucose oxidase on weak base ion exchanger resin (Duolite A 568) were determined for different substrate flow rates in a recirculation system and compared with those for soluble glucose oxidase. It was observed that, for the experimental conditions, the immobilized enzyme K′ m values in a packed-bed reactor were less than the soluble enzyme K m value and were flow dependent. The value of K′ m decreased with increasing flow rate.

Electrostatic Effect on Apparent Kinetic Parameters in a Packed-Bed Immobilized Enzyme Reactor.

The effect of electrostatic field on apparent kinetic parameters in a packed-bed immobilized enzyme reactor was investigated using a mathematical model. The relationship between the dimensionless concentration of substrate consumed in the reactor and the logarithm of the unconverted fraction of substrate at the reactor outlet was nonlinear and changed remarkably according to the magnitude of electrostatic potential and the sign of charges that carry the support and substrate. For the opposite sign of these charges, the apparent Michaelis constant was less than its intrinsic value in the region of high substrate concentration, which reflects a promoting effect on the enzyme reaction by the electrostatic attraction.

A Computational Method for Determination of The Mass-Transfer Coefficient in Packed-Bed Immobilized Enzyme Reactors

A computational method was developed that determined the mass-transfer coefficient kL or the volumetric mass-transfer coefficient kLa in packed-bed immobilized enzyme (IME) reactors. To study the performance of this method, two experimental systems were considered where an enzyme was immobilized on a non-porous support surface (surface-IME system) or within a porous support (pore-IME system). The values of kL and kLa determined in these packed-bed IME reactor systems were successfully expressed in terms of the substrate concentration at the reactor inlet and the liquid flow rate. Furthermore, the correlations obtained for kL and kLa were used to calculate the unconverted fractions of substrate at the reactor outlet. Comparison showed that the calculated results were in satisfactory agreement with the experimental values.

Characteristics of apparent kinetic parameters in a packed-bed immobilized enzyme reactor

The design equation for a packed-bed immobilized enzyme reactor is expressed in terms of apparent kinetic parameters and the relationship between the exit concentration of substrate consumed and the logarithm of the exit unconverted fraction of substrate is studied. Agreement of calculated values with experimental data indicate that this relationship is not linear but is convex downward. Furthermore, simulation is made to characterize the apparent kinetic parameters. The results show that with increasing flow rates of a reaction mixture, the apparent kinetic parameters decrease rapidly towards their respective intrinsic values. As the entrance substrate concentration increases, on the other hand, these values initially increase, pass through maximums, and finally decrease towards their intrinsic values. These variations are found to occur remarkably with the apparent Michaelis constant.

Two-substrates packed-bed bioreactors: inhibition of enzyme and competitive binding of substrate and product to a ligand

An analytical model is developed to describe the performance of a packed-bed immobilized enzyme reactor in which parallel processes take place. In particular, two-substrate reaction, inhibition of the enzyme by one of the reaction products, and binding of one substrate and/or one product to an added ligand are taken into account. In addition, substrates and product diffusion into the porous catalyst are also considered. Using this model, numerical simulations were performed. The results point to the fact that, when all the above processes occur concomitantly, a variety of performance characteristics can be obtained, depending on the particular values of the related parameters. Moreover, under certain conditions, the reactor performance can be improved by controlled addition of ligand.

Coupling of kinetic processes and thermodynamic equilibria in two-substrate immobilized enzyme reactors

An analytical model has been developed which describes the performance of a packed-bed immobilized enzyme reactor. In particular, two-substrate, reaction kinetics, thermodynamic equilibrium of complexation between one of the substrates and a ligand, and substrate diffusion processes are considered. Numerical simulations were performed and the results suggest that, when substrate-ligand binding occurs either in the bulk phase alone or in both the bulk and the pore phases, distinct effects are to be expected.

Product inhibition in packed-bed immobilized enzyme reactors for complex processes: Modelling of two-substrate processes

An analytical model was developed for describing the performance of packed-bed enzymic reactors operating with two cosubstrates, and when one of the reaction products is inhibitory to the enzyme. To this aim, the compartmental analysis technique was used. The relevant equations obtained were solved numerically, and the effect of the main operational parameters on the reactor characteristics were studied.

Packed-Bed Bioreactors Under the Influence of Chemical Modulators

This report considers the behaviour of packed-bed immobilized enzyme reactors operating in the presence of substrate and/or product sequestrators. In some cases, enzyme inhibition by the reaction product and presence of chemical modulators are also considered. For each case, an appropriate analytical model is developed. Using numerical simulations, it is shown that reactor performance is impaired by substrate sequestration. This effect can be partially reversed when competitive sequestration by product or modulator is operational.In addition, a comparison is made between some of the predicted characteristics of the reactor and experimental data. It reveals the capabilities and limitations of the models employed.

Packed-bed immobilized enzyme reactors for complex processes

Utilization of enzymic reactors for biotechnological-biomedical applications is currently developing at a sustained pace.

Film diffusional influences on the kinetic parameters in packed‐bed immobilized enzyme reactors

A number of studies on packed-bed immobilized enzyme reactors have shown the significant influence of the external film mass transfer resistance on the apparent kinetic parameters. Some of the earlier mathematical models using approximation techniques have attempted to explain the linearity of the S(0)x vs ln (1 - x) plots observed experimentally for systems obeying Michaelis-Menten kinetics. However, there has been no critical examination of the bounds of validity of the approximations used. Further, the situations where the above linearity is not valid have been examined neither conceptually nor quantitatively. The work presented here overcomes these drawbacks of the earlier analyses by approaching the problem from a different angle. Methods of evaluation of the intrinsic kinetic parameters under different experimental situations have been outlined and illustrated with several examples.

Effect of the cascade operation of enzymes in packed bed immobilized enzyme reactors.

Effect of the cascade operation of enzymes in packed bed immobilized enzyme reactors.