A. Bódalo

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.

Reduction of sulphate content in aqueous solutions by reverse osmosis using cellulose acetate membranes

Abstract The contamination of water by sulphate is a pollution problem that has increased in severity in recent decades. The behaviour of cellulose acetate membranes in the treatment by reverse osmosis of aqueous sulphate solutions is described. Experimental essays were performed in a commercial cross flow test module in which solutions of sulphate of con-centrations between 0.145 kg/m 3 and 25.455 kg/m 3 were treated. Recycling of both the concentrate and permeate was carried out in order to keep the concentration constant and to simulate a continuous process in a quasi-stationary state. A simulation model described for a semi-batch and unsteady-state system was applied to our experimental conditions (batch and steady-state mode) and verified. The model uses the solution diffusion approach to depict the mass transfer through the membrane, while the constants are determined experimentally. Good agreement between the experimental and model results was obtained, so that the model can be considered suitable for simulating of reverse osmosis treatment of aqueous sulphate solutions by cellulose acetate membranes in the concentrations range studied.

Analysis of diffusion effects on immobilized enzymes on porous supports with reversible Michaelis-Menten kinetics

Abstract A mathematical analysis for the evaluation of the effectiveness factor and concentration profiles for immobilized enzymes into porous spherical particles is presented. It has been solved for monosubstrate reactions following reversible Michaelis-Menten kinetics. It also allows the evaluation of simple Michaelis-Menten and product competitive inhibition kinetics. The mathematical model for such systems was solved by numerical calculus. It showed that the diffusion and concentration moduli were the parameters that affected the effectiveness factor to a greater extent. The influence of the modulus associated with the reaction reversibility degree was negligible. The model is operative at high diffusion modulus values at which previous reported models were not.

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.

Stabilization studies of L-aminoacylase-producing Pseudomonas sp. BA2 immobilized in calcium alginate gel

Abstract Pseudomonas sp. BA2 showing l -aminoacylase activity was immobilized by entrapment in calcium alginate gel. To enhance operational stability, the immobilized cells were treated with hardening reagents using four different methods: treatment of the cells with glutaraldehyde before immobilization, stabilization of the immobilized cell preparation with glutaraldehyde or glutaraldehyde and hexamethylenediamine, and finally, activation of the alginate solution with periodate followed by treatment with polyethyleneimine. Very active and stable immobilized cell preparations were obtained when Pseudomonas sp. BA2 cells immobilized in calcium alginate were treated with 20 m m glutaraldehyde. Compared with the untreated preparation, the enzymatic activity was markedly increased (132%) and the half-life period was increased by 4 h. Moreover, the suitability of the biocatalyst for reuse in batch processes was noticeably improved (only 60% of the initial l -aminoacylase activity was lost after four runs). When the immobilized and stabilized cell preparation was used for l -alanine production in a continuous stirred-tank reactor, substrate conversion at the reactor outlet was higher than that obtained with the untreated beads. The reactor production after 33 h was 61 of 17 m m l -alanine solution (9 g optically pure amino acid) more than double that obtained using the untreated immobilized cells, and significantly higher than those previously reported in the literature.

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+ .

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.

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.

Preliminary studies on the growth of Pseudomonas sp. BA2 and the production of L-aminoacylase

SummaryThe bacterial strain Pseudomonas sp. BA2 did not develop l-aminoacylase activity in the absence of N-acetyl-dl-alanine. The maximum growth rate and enzyme production were obtained when the acetylated amino acid was used as the sole carbon and nitrogen source. A maximum biomass of A660=1.543, after 24 h of cultivation, was obtained. The l-aminoacylase activity reached the maximum value (5.6 U ml−1 broth) in the stationary growth phase.