M.F. Máximo

A comparison of different methods of β-galactosidase immobilization

Abstract β-Galactosidase was immobilized in a range of supports showing suitable physico-chemical characteristics for use in fluidized bed reactors. Uncoated porous glass, alginate and κ-carrageenan beads and chromosorb-W were used as carriers. The intrinsic kinetic constants (V max and K M ) and coupling parameters for the immobilization were calculated. The highest immobilized protein percentages and activity yields were obtained when β-galactosidase was attached through its amine groups to aldehyde-glass. The final choice of derivative for use in fluidized bed reactors should be based not only on the enzymatic activity shown by the derivatives but also on the hydrodynamic behaviour of the supports.

Fluidized bed reactors operating with immobilized enzyme systems: Design model and its experimental verification

Abstract A general model for the design of fluidized bed reactors operating with immobilized enzymes in spherical porous particles is presented. It was solved for monosubstrate reactions following reversible Michaelis-Menten kinetics, as well as competitive inhibition by the product. The general model also allows the evaluation of irreversible and/or non-inhibited kinetics. To check the model, experimental conversions obtained in a fluidized bed reactor were compared to those predicted theoretically. The chemical process used for the validation of the model was the hydrolysis of o -nitrophenyl-β, d -galactopyranoside catalyzed by β-galactosidase covalently immobilized in Chromosorb-W. The model was tested in 160 different experimental conditions.

Ultrafiltration membrane reactors for enzymatic resolution of amino acids: design model and optimization.

In this paper the possibility of continuous resolution of DL-phenylalanine, catalyzed by L-aminoacylase in a ultrafiltration membrane reactor (UFMR) is presented. A simple design model, based on previous kinetic studies, has been demonstrated to be capable of describing the behavior of the experimental system. The model has been used to determine the optimal experimental conditions to carry out the asymmetrical hydrolysis of N-acetyl-DL-phenylalanine.

Kinetic calculations in the enzymatic resolution of dl-amino acids

Abstract The literature contains several papers describing the kinetic mechanism of the optical resolution of N- acetyl- dl -amino acids catalyzed by l -aminoacylase. Most authors propose a reversible Michaelis–Menten kinetic reaction scheme inhibited by substrate and products. Such studies are mostly based on initial rate measurements. In this paper, an alternative method is presented to determine both the reaction scheme and the value of the involved constants. The method is based on measuring product concentration for different initial substrate concentrations up until the time when equilibrium is reached and on the numerical integration of the rate equation. The optical resolution of N -acetyl- dl -phenylalanine and N- acetyl- dl -valine catalyzed by l -aminoacylase were used as model systems to check the validity of the proposed method, and a slightly substrate-inhibited reversible Michaelis–Menten reaction scheme was demonstrated. In disagreement with other previously published studies, no product inhibition was detected. Previous experiments were performed to determine the most suitable enzyme concentration and the optimal concentration of the activator, Co 2+ .

Continuous tank reactors in series: an improved alternative in the removal of phenolic compounds with immobilized peroxidase

Immobilized derivatives of soybean peroxidase, covalently bound to a glass support, were used in a continuous stirred tank reactor in series, in order to study the removal of two phenolic compounds: phenol and 4-chlorophenol. The use of two reactors in series, rather than one continuous tank, improved the removal efficiencies of phenol and 4-chlorophenol. The distribution of different amounts of enzyme between the two tanks showed that the relative distributions influenced the removal efficiency reached and the degree of the enzyme deactivation. The highest removal percentages were reached at the outlet of the second tank for a distribution of 50% of the enzyme in each tank. However, with a distribution of 75% in the first tank and 25% in the second, the elimination percentage in the second tank was slightly lower than in the previous case, and the effects of deactivation of the enzyme in the first tank were less pronounced. In all the distributions assayed it was observed that the first tank acts as a filter for the second one, which receives a feed with a smaller load of phenolic compounds, thus diminishing enzyme deactivation in the second tank.

Immobilization of β-Galactosidase by physical adsorption on Chromosorb-W

β-Galactosidase was immobilized by physical adsorption on Chromosorb-W. 94.8 % of the initial protein was adsorbed at 4oC, pH=5.5 and ionic strength=0.31. The specific activity of the immobilized derivative was 105.3 U/mg and KM for o-nitrophenyl- β-D-galactopyranoside was 0.12 mM.

Screening of three commercial plant peroxidases for the removal of phenolic compounds in membrane bioreactors

A comparative study of three plant peroxidases, horseradish (HRP), soybean (SBP) and artichoke (AKPC), was carried out to select the most appropriate one for 4-chlorophenol treatment in an ultrafiltration membrane reactor. Soybean peroxidase showed the highest enzymatic activity, followed by HRP and AKPC. The same tendency was observed in a discontinuous tank reactor, where SBP attained more than 90% of 4-chlorophenol removal within the pH range tested. The optimum temperature was 30 °C, with SBP showing highest thermostability. With the ultrafiltration membrane reactor, SBP attained the highest operational stability, with 4-chlorophenol conversions of around 90% in the permeate stream for up to 200 minutes. Finally, permeate samples were analysed and no significant amount of enzyme was detected, so the observed loss of activity, less pronounced with SBP, was attributed to enzyme adsorption on the polymeric products deposited on the membrane surface. Soybean peroxidase was selected as the most appropriate peroxidase for future research.

Two-parameter model for evaluating effectiveness factor for immobilized enzymes with reversible Michaelis–Menten kinetics

Abstract A two-parameter mathematical model was developed to calculate the effectiveness factor for immobilized enzymes in porous spherical particles. The model was resolved for reversible Michaelis–Menten kinetics, including simple Michaelis–Menten and product competitive inhibition kinetics. Since only two dimensionless moduli are involved in the model, the effectiveness factor for the three kinetic equations considered can be estimated by using only one generalized graph.