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.     

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

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.     

Isothermal and non-isothermal bioreactors in the detoxification of waste waters polluted by aromatic compounds by means of immobilised laccase from Rhus vernicifera

Laccase from Rhus vernicifera was immobilised on a nylon membrane chemically grafted with glycidyl methacrylate (GMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GLU) were used as spacer and bifunctional coupling agent, respectively. Quinol was used as substrate.

To know how the immobilisation procedures affected the enzyme reaction rate the catalytic behaviour of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged from the experimental data: (i) the narrower pH-activity profile of the insoluble enzyme in comparison to that of the soluble counterpart; (ii) the increase of the affinity of the immobilised enzyme for its substrate.

The behaviour of the catalytic membrane was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increases with the increase of the temperature difference. These results have been discussed in the frame of reference of the process of thermodialysis driving thermodiffusive transmembrane substrate fluxes, which add to the diffusive ones.

The advantages of the catalytic process carried out under non-isothermal conditions have been thrown in relief through the evaluation of the reduction of the production times and of the percentage increases of the enzyme activity.     

Production of low-lactose milk by means of nonisothermal bioreactors

The effect of the immobilization time on the activity of immobilized beta-galactosidase from K. lactis was investigated. Six biocatalytic membranes, different only for the time of the enzyme immobilization, were obtained by using nylon membranes grafted with glycidyl methacrylate (GMA) and activated by hexamethylenediamine (HMDA) and glutaraldehyde (Glu), used as spacer and coupling agent, respectively. Comparison between the isothermal and nonisothermal yield of these biocatalytic membranes was carried out in the process of lactose hydrolysis in milk. All of the results, reported as a function of the immobilization time, have evidenced the influence of our variable parameter on the activity of the catalytic membranes. The membrane giving highest yield under isothermal and nonisothermal conditions was that obtained with 2 h of immobilization time. The industrial application of these membranes has been discussed in terms of percentage reduction of the production times.     

Influence of the spacer length on the activity of enzymes immobilised on nylon/polyGMA membranes: Part 2: Non-isothermal conditions

β-Galactosidase has been immobilised through spacers of different length on nylon membranes grafted with glycidyl methacrylate. Hexamethylendiamine, ethylendiamine or hydrazine have been separately used as spacers.

The behaviour of the catalytic membranes has been studied in a bioreactor operating under non-isothermal conditions as a function of the applied temperature difference ΔT.

Comparison of the enzyme reaction rates under isothermal and non-isothermal conditions resulted in percentage activity increases (PAI) and reduction of the production time (τ_r) proportional to the size of the applied ΔT. Both these parameters increased with the increase of the spacer length.

Results have been discussed in the frame of reference of the process of thermodialysis which reduces the limitations to the diffusion of substrate and reaction products across the catalytic membrane, limitations introduced by the grafting and immobilisation process.

The advantages of employing non-isothermal bioreactors in biotechnological productive processes have been outlined.     

Nonisothermal bioreactors in the treatment of vegetation waters from olive oil: laccase versus syringic acid as bioremediation model

Laccase from Trametes versicolor was immobilized by diazotization on a nylon membrane grafted with glycidil methacrylate, using phenylenediamine as spacer and coupling agent. The behavior of these enzyme derivatives was studied under isothermal and nonisothermal conditions by using syringic acid as substrate, in view of the employment of these membranes in processes of detoxification of vegetation waters from olive oil mills. The pH and temperature dependence of catalytic activity under isothermal conditions has shown that these membranes can be usefully employed under extreme pH and temperatures. When employed under nonisothermal conditions, the membranes exhibited an increase of catalytic activity linearly proportional to the applied transmembrane temperature difference. Percentage activity increases ranging from 62% to 18% were found in the range of syringic acid concentration from 0.02 to 0.8 mM, when a difference of 1 degrees C was applied across the catalytic membrane. Because the percentage activity increase is strictly related to the reduction of the production times, the technology of nonisothermal bioreactors has been demonstrated to be an useful tool also in the treatment of vegetation waters from olive oil mills.     

Modulation of immobilized enzyme activity by altering the hydrophobicity of nylon-grafted membranes: Part 2: Non-isothermal conditions

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 (M₁) was obtained by chemical grafting of methylmethacrylate (MAA); the other one (M₂) 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 M₂ was higher than that of membrane M₁. The 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 M₂ with respect to membrane M₁, the hydrophobicity being a prerequisite for the occurrence of the process of thermodialysis.     

Advantages in using non-isothermal bioreactors in bioremediation of water polluted by phenol by means of immobilized laccase from Rhus vernicifera

Laccase from Rhus vernicifera was immobilized on a polypropylene membrane chemically modified with chromic acid. Ethylenediamine and glutaraldehyde were used as spacer and bifunctional coupling agent, respectively. Phenol was used as substrate.To know how the immobilization procedures affected the enzyme reaction rate the catalytic behavior of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged: (i) the narrower pH–activity profile of the soluble enzyme in comparison to that of the insoluble counterpart and (ii) the increase in pH and thermal stability of the insoluble enzyme.The laccase catalytic behavior was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increased with the increase of the temperature difference.     

Biodegradation of bisphenols with immobilized laccase or tyrosinase on polyacrylonitrile beads

The biodegradation of waters polluted by some bisphenols, endowed with endocrine activity, has been studied by means of laccase or tyrosinase immobilized on polyacrylonitrile (PAN) beads. Bisphenol A (BPA), Bisphenol B (BPB), Bisphenol F (BPF) and Tetrachlorobisphenol A (TCBPA) have been used. The laccase-PAN beads system has been characterized as a function of pH, temperature and substrate concentration. The biochemical parameters so obtained have been compared with those of the free enzyme to evidence the modification induced by the immobilization process. Once characterized, the laccase-PAN beads have been employed in a fluidized bed reactor to determine for each of the four bisphenols the degradation rate constant (k); the τ(50), i.e., the time to obtain the 50% of degradation, and the removal efficiency (RE(90)) after 90 min of enzyme treatment. The same parameters have been measured for each of the four pollutants with the same fluidized bed bioreactor loaded with tyrosinase-PAN beads. The internal comparison, i.e., in each of the two catalytic systems, has shown that both enzymes exhibit a removal efficiency in the following order BPF>BPA>BPB>TCBPA. The external comparison, i.e., the comparison between the two catalytic system, has shown that the catalytic power of laccase were higher than that of tyrosinase. The operational stability of both catalytic systems resulted excellent, since they maintained more than 80% of the initial activity after 30 days of work.     

Advantages of using non-isothermal bioreactors for the enzymatic synthesis of antibiotics: the penicillin G acylase as enzyme model

A new hydrophobic and catalytic membrane was prepared by immobilizing Penicillin G acylase (PGA, EC.3.5.1.11) from E. coli on a nylon membrane, chemically grafted with butylmethacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (Glu) were used as a spacer and coupling agent, respectively. PGA was used for the enzymatic synthesis of cephalexin, using D(-)-phenylglycine methyl ester (PGME) and 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) as substrates. Several factors affecting this reaction, such as pH, temperature, and concentrations of substrates were investigated. The results indicated good enzyme-binding efficiency of the pre-treated membrane, and an increased stability of the immobilized PGA towards pH and temperature. Calculation of the activation energies showed that cephalexin production by the immobilized biocatalyst was limited by diffusion, resulting in a decrease of enzyme activity and substrate affinity. Temperature gradients were employed as a way to reduce the effects of diffusion limitation. Cephalexin was found to linearly increase with the applied temperature gradient. A temperature difference of about 3 degrees C across the catalytic membrane resulted into a cephalexin synthesis increase of 100% with a 50% reduction of the production times. The advantage of using non-isothermal bioreactors in biotechnological processes, including pharmaceutical applications, is also discussed.     

Reversibility of adenosine 3':5'-monophosphate-dependent protein kinase reactions.

Using a homogeneous enzyme from rabbit skeletal muscle, it has been demonstrated that the cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase reaction is reversible. In addition to the phosphorylated protein substrate, the reverse reaction requires Mg2+, ADP, and cyclic AMP when the holoenzyme is used as the source of enzyme. It is independent of cyclic AMP when the catalytic subunit of the protein kinase is used. The optimum pH for the reverse reaction with 32P-labeled casein as the substrate is 5.7, essentially the same as that for the forward reaction. Among the nucleotide subtrates tested, ADP serves as the best phosphoryl group acceptor. The Km of the enzyme for ADP is 3.3 mM and that for 32P-casein is 1.7 mg/ml. The equilibrium constant at 30 degrees is approximately 0.042 at a magnesium concentration of 10 mM and a pH of 6.9. This result indicates that the free energy of hydrolysis (deltaG0obs) of the phosphorylated protein substrate is relatively high, i.e. approximately -6.5 kcal/mol under these conditions.     

A novel packed-bed bioreactor operating under isothermal and non-isothermal conditions

A novel packed-bed bioreactor, operating under isothermal and non-isothermal conditions, has been constructed. The core of the apparatus consisted in a polypropylene ring filled with beta-galactosidase immobilized on beads of polyacrylic acid, grafted with dimethylaminoethyl methacrylate. Phenylendiamine and glutaraldehyde were used as spacer and coupling agent, respectively. Two lateral nylon membranes held the enzyme beads into the ring and allowed the occurrence of the process of thermodialysis when the bioreactor was operating under non-isothermal conditions. Comparison of the enzyme activity under isothermal and non-isothermal conditions has shown that in the presence of temperature gradients the rate of lactose hydrolysis was increased, with a reduction of the apparent Km value. Under non-isothermal conditions the percentage increases of enzyme activity were found to decrease with the increase of the substrate concentration. The results have been explained within the frame of reference of the process of thermodialysis.     

Influence of the spacer length on the activity of enzymes immobilised on nylon/polyGMA membranes: Part 1. Isothermal conditions

β-Galactosidase was immobilized on nylon/poly(glycidyl methacrylate) membranes through spacers of different length: hexamethylenediamine, ethylenediamine or hydrazine. The effect of the spacer length on the catalytic behavior of the three membranes was studied in isothermal bioreactors. The behavior of the soluble and insoluble enzymes was compared to know the effects of the immobilization process and of the spacer length.

The enzyme derivatives in comparison with the soluble enzyme exhibited shifts of the optimum pH values towards more acidic solutions. These shifts were found decreasing with the spacer length; while an opposite trend was observed when the optimum temperature values were considered. Also the values of the apparent K_m were found to decrease with the spacer length.

All these results indicated that a soluble enzyme could be considered as an enzyme immobilized on a solid support through a spacer of infinite length.     

Enzymatic activity under tangential flow conditions of photochemically grafted membranes containing immobilized catalase

Catalase has been immobilized within sandwich membranes prepared by the photoinduced grafting of an epoxy-diacrylate prepolymer onto commercial asymmetric cellulose membranes. The enzymatic activity of the membrane composite of hydrogen peroxide decomposition has been studied in a recirculation apparatus under tangential flow conditions without ultrafiltration. The enzymatic membranes were exposed to very low mechanical stresses and showed a very good catalytic performance and durability. Initial reaction rates, measured at 25 degrees C as a function of both substrate concentration and enzyme amount immobilized per unit membrane surface, indicate that the mechanism of action of catalase is not altered after immobilization, although substrate diffusion through the original thin layer of membranes may become rate controlling. (c) 1993 John Wiley & Sons, Inc.     

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

The catalytic behaviour under isothermal conditions of two different membranes loaded with β-galactosidase was investigated. One membrane (M₁) was constituted by a nylon sheet grafted with methylmethacrylate by means of chemical grafting. The other, (M₂), was prepared by a double chemical grafting: the first one with styrene (Sty) and the second one with methylmethacrylate. Membrane activity was characterized as a function of temperature, pH and substrate concentration. The role of Sty in increasing membrane hydrophobicity has been discussed. Membrane M₂ was found to be better suited for employment in non-isothermal bioreactors.     

Stereoselectivity in acid-catalyzed heteronucleophilic substitution of enantiomeric 3-O-methyloxazepam and 3-O-ethyloxazepam

Kinetics of acid-catalyzed heteronucleophilic substitution and racemization of enantiomeric MeOX in ethanol and enantiomeric EtOX in methanol were studied by quenching reaction products at various times by neutralization. Enantiomeric contents of remaining substrate and reaction product were determined by chiral stationary phase high-performance liquid chromatography. The experimental procedure allowed the determination of the stereoselectivity (i.e., the enantiomeric ratio of a substitution product formed from an enantiomerically pure substrate) involved in the heteronucleophilic substitution reactions. The stereoselectivity was found to vary between 58:42 and 87:13, depending on the acid concentration, substrate, solvent, and temperature. The enantiomeric purity of remaining substrates was identical to that of the starting substrate, indicating that the enantiomeric substrates did not undergo a ring-opening reaction. The results provided additional evidence supporting the mechanism proposed earlier in acid-catalyzed stereoselective heteronucleophilic and homonucleophilic substitutions and the resulting racemization of enantiomeric 3-alkoxy-1,4-benzodiazepines in alcoholic solvents. © 1995 Wiley-Liss, Inc.     

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

The behaviour of five different hydrophobic β-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.     

Growth factor stimulation of phospholipase C-gamma 1 activity. Comparative properties of control and activated enzymes.

We demonstrated previously tyrosine phosphorylation-dependent modulation of phospholipase C-gamma 1 (PLC-gamma 1) catalytic activity (Nishibe, S., Wahl, M. I., Hernandez-Sotomayor, S. M. T., Tonks, N. K., Rhee, S. G., and Carpenter, G. (1990) Science 250, 1253-1256). The increase in PLC-gamma 1 catalytic activity in A-431 cells occurs rapidly, with maximal activation 5 min after epidermal growth factor (EGF) stimulation. Certain other growth factors (fibroblast growth factor, platelet-derived growth factor) also stimulate PLC-gamma 1 catalytic activity, whereas insulin does not. A similar increase in PLC-gamma 1 specific activity (2-3-fold) was observed in both soluble (cytosol) and particulate (membrane) preparations from EGF-treated cells. Tyrosine-phosphorylated PLC-gamma 1 was detected in both cytosol and membrane fractions in lysates from EGF-treated A-431 cells, but the proportion of tyrosine-phosphorylated PLC-gamma 1 was higher in the cytosol (approximately 50%) than in the membrane (approximately 20%). Because a micellar concentration of the non-ionic detergent Triton X-100 allows detection of the tyrosine phosphorylation-dependent increase in PLC-gamma 1 catalytic activity in this assay, we evaluated the kinetic properties of PLC-gamma 1, immunoprecipitated from cytosol of control or EGF-treated cells, using substrate, phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2), solubilized in Triton X-100 at various molar ratios. The behavior of the control enzyme differed from the EGF-activated enzyme with respect to both Ks and Km. The control enzyme has a 7.5-fold higher Ks value than the activated enzyme (1.5 mM as compared with 0.22 mM). Activation by EGF is also a positive allosteric modifier of PLC-gamma 1-catalyzed PtdIns 4,5-P2 hydrolysis, i.e. the activated enzyme displayed apparent Michalis-Menton kinetics, with a Km of 0.6 mol fraction PtdIns 4,5-P2, whereas the control enzyme displayed sigmoidal kinetics with respect to PtdIns 4,5-P2 hydrolysis. At low substrate mol fractions (e.g. 0.07), the reaction velocity of the control enzyme was 4-fold lower than the activated enzyme. However, at a high substrate mol fraction (e.g. 0.33), the estimated maximal reaction velocities (Vmax) for both forms of PLC-gamma 1 were equivalent. PLC-gamma 1 activity from both control and EGF-treated cells was stimulated by increasing nanomolar Ca2+ concentrations. Although the catalytic activity of PLC-gamma 1 from EGF-treated cells was greater than control PLC-gamma 1 at every Ca2+ concentration tested, the relative stimulation of activity was markedly greater at Ca2+ concentrations above approximately 300 nM.     

Organization of enzymes on subcellular structures: relay at the surface

There is a large body of evidence that soluble cytoplasmic enzymes of eukaryotic cells, e.g., glycolytic enzymes and proteins of the translational machinery, are organized in some way in space and in time. The following features of such organization emerge from the experimental data: (1) metabolites are transferred between enzymes directly "from hand to hand" in short-living enzyme-enzyme complexes rather than by diffusion in aqueous media; (2) enzymes show a tendency to be absorbed on surfaces of subcellular structures, such as membranes, cytoskeleton and polyribosomes; (3) enzymes are desorbed from a surface of a subcellular structure after binding specific metabolites, i.e., substrates and/or products of the reactions catalyzed by these enzymes. These features are suggestive of a relay mechanism for the enzyme systems functioning in a cell; an enzyme adsorbed on a surface of a subcellular structure is desorbed after binding its substrate or in the course of the catalytic act. Within a complex with its product the enzyme diffuses into the environment, until it reaches the next enzyme adsorbed on the same surface; then a short-living enzyme-enzyme complex is formed, and a direct "from hand to hand" transfer of the metabolite takes place. As a result, the overall metabolic process appears to be localized near the surface. We termed this mechanism as a "relay at the surface".     

Kinetics of oligomycin inhibition and activation of Na+/K(+)-ATPase.

Oligomycin inhibition of the maximal hydrolysis activity of ox brain Na+/K(+)-ATPase was studied at varying NaCl concentrations and it was found that for a given amount of live enzyme, the observed inhibition of a particular total oligomycin concentration decreased as the amount of added, (heat-) denatured enzyme increased. In the present article we derive a scale factor for the oligomycin concentration, i.e., the fraction of the total concentration of oligomycin which is free in solution, as a function of the enzyme concentration used. This fraction decreased linearly with the protein concentration and may attain quite small values. We also study the Na(+)-dependence of the hydrolysis rate at saturating substrate concentrations ([Mg2+] = [ATP] = 3 mM), in the presence as well as the absence of KCl, at various concentrations of oligomycin. These data may be explained if it is assumed that the sole effect of oligomycin is to confer upon the enzyme an increased affinity for Na+, i.e., oligomycin merely enhances the inhibitory effect of Na+ on the (maximal) activity seen at high Na(+)-concentrations. The increased Na(+)-affinity in the presence of oligomycin should result in activation of the hydrolysis rate measured under conditions where Na(+)-activation is predominant, i.e., at low Na(+)-concentration and sub-saturating substrate concentrations. This prediction is verified for both Na(+)-ATPase and for Na+/K(+)-ATPase. This proposed action of oligomycin seems to be corroborated also by other evidence discussed in the text.