S. Rossi

Influence of the microenvironment on the activity of enzymes immobilized on Teflon membranes grafted by γ-radiation

Abstract The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.

Non-isothermal cephalexin hydrolysis by penicillin G acylase immobilized on grafted nylon membranes

A new catalytic membrane has been prepared using a nylon membrane grafted by γ-radiation with methylmethacrylate (MMA) and using hexamethylenediamine (HMDA) as spacer. Penicillin G acylase (PGA) and cephalexin were employed as catalyst and substrate, respectively. Cephalexin hydrolysis was studied in bioreactors operated under isothermal and non-isothermal conditions. A hydrolysis increase was found when the temperature of the warm membrane surface was kept constant and the temperature of the other membrane surface was kept at a lower value. The hydrolysis increase was linearly proportional to the applied temperature difference. Cephalexin hydrolysis increased to about 10% when a temperature difference of 1°C was applied across the catalytic membrane. These results have been attributed to the non-isothermal cephalexin transport across the membrane, i.e., to the process of thermodialysis. In this way, the enzyme immobilized on and into the membrane reacts with a substrate concentration higher than that produced by simple diffusion under isothermal conditions.

A novel bioreactor operating under non-isothermal conditions

Activity of the β-galactosidase enzyme, immobilized on a nylon membrane, has been assessed in a bioreactor in which the two half-cells, maintained at different temperatures, are filled with substrate solutions coming from and ending in the same reservoir. The experimental results demonstrate that the presence of temperature gradients across the catalytic membrane increases the efficiency of the bioreactor, making it suitable for practical applications. The solution filling the apparatus during the treatment remains unchanged in all components, different from the substrate and its products, while the biocatalyst works at rates higher than those corresponding to comparable isothermal conditions. The production times in biotechnological processes employing immobilized enzymes are reduced proportionally with respect to the applied transmembrane temperature differences.

Urease immobilization on chemically grafted nylon membranes. Part 2. Non-isothermal characterization

Abstract The behaviour of an urease loaded membrane, obtained by chemically grafting butyl methacrylate (BMA) on a nylon sheet, has been studied under non-isothermal conditions. Hexamethylenediamine (HMDA) and glutaraldehyde were also used as spacer and binding agent, respectively. Results have shown that the catalytic activity of the membrane under non-isothermal conditions is increased when compared with the one found under comparable isothermal conditions. In addition, it has been found that the presence of temperature gradients decreases the apparent K m and increases the apparent V max with respect to the same values obtained under isothermal conditions. The percentage activity increases induced by the presence of a temperature gradient have been found to decrease with increasing average temperature and urea concentration. A parameter has also been identified correlating the percentage increase of enzyme activity under non-isothermal conditions with the hydrophobicity of the catalytic membrane, this parameter being the ratio between thermo-osmotic and hydraulic permeability. Results have been discussed in terms of reduction of diffusion limitations for substrate and products movement towards or away from the catalytic site by the process of thermodialysis. The usefulness of using non-isothermal bioreactors in industrial biotechnological processes and, in particular, in waste water treatment has been confirmed.

Effect of temperature gradients on the activity of enzymes immobilized on natural polymeric matrices

Abstract The invertase activity has been studied in the presence of a temperature gradient by interposing the enzyme onto a polymeric membrane between two solutions, kept at different temperatures. The enzyme activity has been determined as a function of average temperature (Tav) and of the applied temperature difference ΔT=Tw − Tc. It has been found that in the non-isothermal state the activity is higher than in comparable isothermal conditions, the increase being proportional to the applied transmembrane temperature difference. Mathematical relationships between the isothermal and non-isothermal enzyme activity have been derived. The rapid product removal from the enzyme complex by means of a process of matter transport associated to the flux of thermal energy is indicated as the way by which the temperature gradient affects the enzyme activity. Some practical applications are suggested.

Characterization of the activity of penicillin G acylase immobilized onto nylon membranes grafted with different acrylic monomers by means of γ-radiation

Penicillin G acylase (PGA) has been immobilized onto nylon membranes grafted with methylmethacrylate (MMA) or diethyleneglycoldimethacrylate (DGDA) monomers by means of γ-radiation. Hexamethylenediamine (HMDA) has been used as spacer between the grafted membranes and the enzyme. Glutaraldehyde (GA) was used as crosslinking to couple the enzyme to the HMDA. The catalytic membranes so prepared were studied as a function of pH and temperature of the solution containing the substrate. The membranes showing the best characteristics were the ones grafted with DGDA. The dependence of the behavior of these membranes on several experimental conditions was studied, i.e., the temperature and duration of the aminoalkylation process, spacer concentration, the glutaraldehyde concentration and the enzyme concentration. The experimental conditions giving the best performance of the catalytic membranes have been deduced. The time requested to obtain 50% of substrate conversion, i.e., hydrolysis of cephalexin, has been studied as a function of its initial concentration.

Isothermal and non-isothermal characterization of catalytic nylon membranes chemically grafted: dependence on the grafting percentage.

The behaviour of five different hydrophobic beta-galactosidase derivatives, obtained by grafting different amount of butylmethacrylate (BMA) on planar nylon membranes, has been studied under isothermal and non-isothermal conditions.Under isothermal conditions the effect of the grafting percentage on the enzyme activity has been studied as a function of pH, temperature and substrate concentration. Independently from the parameters under observation, the yield of the catalytic process reaches the maximum value at a grafting percentage value equal to 21%. The apparent K(m) values result linearly increasing with the increase of the grafting percentage, while the apparent V(max) exhibits a maximum value.Under non-isothermal conditions, a decrease of the apparent K(m) values and increase of the apparent V(max) has been found in respect to the same values obtained under isothermal conditions.The percentage activity increases induced by the presence of a temperature gradient have been found to decrease with the increase of the percentage of graft BMA.A parameter correlating the percentage increase of enzyme activity under non-isothermal conditions with the hydrophobicity of the catalytic membrane has also been identified. This parameter is the ratio between thermoosmotic and hydraulic permeability.Results have been discussed in terms of reduction of diffusion limitations for substrate and products movement towards or away from the catalytic site by the process of thermodialysis.The usefulness of using non-isothermal bioreactors in industrial biotechnological processes has been confirmed.

Modulation of immobilized enzyme activity by altering the hydrophobicity of nylon-grafted membranes: Part 1. Isothermal conditions

Abstract Lactose hydrolysis by β-galactosidase immobilized on two nylon membranes, differently grafted, has been studied in a bioreactor operating under isothermal and non-isothermal conditions. One membrane (M 1 ) was obtained by chemical grafting of methylmethacrylate (MAA); the other one (M 2 ) by a double chemical grafting: styrene (Sty) and MAA. Hexamethylenediamine was used as a spacer between the grafted membranes and the enzyme. Both membranes have been physically characterized studying their permeabilities in presence of pressure or temperature gradients. Under non-isothermal conditions, the increase in activity of membrane M 2 was higher than that of membrane M 1 . The α and β coefficients, giving the percentage of activity increase when a temperature difference of 1°C is applied across the catalytic membranes, have been calculated. Results have been discussed with reference to the greater hydrophobicity of membrane M 2 with respect to membrane M 1 , the hydrophobicity being a prerequisite for the occurrence of the process of thermodialysis.

Non-isothermal bioreactors utilizing catalytic Teflon membranes

Abstract A hydrophobic and catalytic membrane has been obtained by grafting with γ -radiations a Teflon membrane in presence of methylmethacrylate solution and by immobilizing on it, after treatment with glutaraldehyde, the β-Galactosidase enzyme. When employed in a non-isothermal bioreactor, the membrane showed an increase in catalytic activity proportional to the applied temperature gradients. The results have been explained in terms of distinct contributions from the process of thermodialysis and the conformational changes induced in the dynamic structure of the enzyme by the presence of heat flow. The increase of the yield of the process has been evaluated in terms of a coefficient α representing the percent increase of enzyme activity when a temperature difference of 1°C is applied across the membrane. The catalytic Teflon membrane used here gave values of 20%, which are comparable to those obtained with other membrane systems, making this kind of membrane useful for practical applications in industrial processes.

Enzyme reaction engineering: effect of methanol on the synthesis of antibiotics catalyzed by immobilized penicillin G acylase under isothermal and non-isothermal conditions.

The effect of methanol on the kinetically controlled synthesis of cephalexin by free and immobilized penicillin G acylase (PGA) was investigated. Catalytic and hydrophobic membranes were obtained by chemical grafting, activation, and PGA immobilization on hydrophobic nylon supports. Butyl methacrylate (BMA) was used as graft monomer. Increasing concentrations of methanol were found to cause a greater deleterious effect on the activity of free than on that of the immobilized enzyme. Methanol, however, improved the kinetic stability of cephalexin synthesized by free PGA, resulting in higher maximum yields. By contrast, immobilized PGA reached 100% yields even in the absence of the cosolvent. Cephalexin synthesis by the catalytic membrane was also performed in a non‐isothermal bioreactor. Under these conditions, a 94% increase of the synthetic activity and complete conversion of the limiting substrate to cephalexin were obtained. The addition of methanol reduced the non‐isothermal activity increase. The physical cause responsible for the non‐isothermal behavior of the hydrophobic catalytic membrane was identified in the process of thermodialysis.