P. Canciglia

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.

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.

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.

Characterization of the activity of β-galactosidase immobilized on Teflon membranes preactivated with different monomers by γ-irradiation

The activity of β-galactosidase, immobilized by grafting technique on Teflon membranes preactivated with four different monomers, has been characterized from the biochemical and biophysical points of view. The monomers used were acrylic acid or acrylamide, or methacrylic acid and 2-hydroxyethyl methacrylate. When 2-hydroxyethyl methacrylate was used in the second grafting, the first three monomers have been used in the first modification step. The behavior of the free and immobilized enzyme has been analyzed as a function of temperature and pH. For each catalytic membrane, we have found general equations relating the absolute enzyme activity to pH and temperature. From these expressions, the experimental conditions giving the best yield of each catalytic membrane have been calculated. The kinetic parameters for the four membranes have also been determined. The advantages of using these membranes in nonisothermal bioreactors are also indicated.

β‐galactosidase immobilization on premodified Teflon membranes using γ‐radiation grafting

ABSTRACT: g-Radiations have been used to immobilize, by mutual grafting, the b-galactosidase enzyme and monomers of 2-hydroxyethyl methacrylate on Teflon (poly-tetrafluoroethylene) membrane previously grafted with acrylic acid monomers. Thisdouble grafting technique improved the catalytic activity of the membrane. Membraneactivity has been studied as a function of some of the most relevant parameters affectingthe grafting degree and of the amount of enzyme used for immobilization. Experimentalconditions producing the best membrane activity have been characterized. The advan-tages in using Teflon catalytic membrane in nonisothermal bioreactors, more efficientthan the analogous isothermal bioreactors, are also discussed. q 1998 John Wiley & Sons,Inc. J Appl Polym Sci 68: 625–636, 1998 Key words: radiation grafting; Teflon; immobilized enzymes; b-galactosidase; 2-hy-droxyethyl methacrylate; acrylic acid INTRODUCTION for the immunodiagnostic, and of microorganismsfor biomass conversion. All of these applicationsPolymer membranes are widely used as enzyme are performed under isothermal conditions.carriers. In many cases, modifications have been When biocatalysts are not immobilized, theintroduced on the polymeric matrices to increase grafted membranes have been used in water de-salination

Sulphate transport by thermodialysis in living cells: an experimental study

Abstract Experimental results are presented here which demonstrate the occurrence of direct coupling between fluxes of matter and of thermal energy crossing living biological membranes. Artificially produced heat fluxes, synergic or antagonistic to the naturally occurring flux of thermal energy of metabolic origin, do indeed modulate sulphate influx in algal cells of Valonia utricularis precisely in the way expected for thermally driven transport through porous membranes called thermodialysis. Physiological permeant fluxes are increased or decreased in proportion to the intensity of the artificially-produced trans-membrane gradients. These results lend strong support to our working hypothesis, according to which biochemical exergonic reactions are coupled with biological transmembrane matter transport by means of the flux of thermal energy produced by metabolic activity.