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.     

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.     

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.     

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.     

Effect of temperature and pH on 31P nuclear magnetic resonances of phospholipids in cholate micelles

Accurate and precise determination of phospholipid composition by ³¹P NMR spectroscopy requires correct assignments and adequate spectral resolution. Because temperature and pH may affect chemical shifts (δ), our first aim was to establish the temperature coefficient (Δδ/ΔT) of common phospholipid classes when using sodium cholate as detergent. This parameter can then be used to aid in resonance assignments. The second goal was to investigate the pH dependence of δ so that, in addition to temperature, pH control can be used to minimize spectral overlap. For phosphatidylcholine, sphingomyelin, dihydrosphingomyelin and phosphatidylglycerol, δ values were invariant with pH and temperature. Whereas the Δδ/ΔT for phosphatidylinositol was 4 × 10⁻³ ppm/°C, regardless of pH, these coefficients were highly pH-dependent for phosphatidic acid, phosphatidylethanolamine and phosphatidylserine, exhibiting maximal variations with the deprotonation of the headgroup, particularly for phosphatidic acid. These trends indicate the importance of H-bonding on δ and Δδ/ΔT for phospholipid resonances.     

How are neuronal thermosensitivity and lack of thermoreception related in the duck's hypothalamus? A tentative answer

1. 1.|In 15 conscious Pekin ducks, 40 “warm sensitive” hypothalamic neurons were identified according to their discharge rates at 40°C T_hy (F₄₀), local temperature coefficients (Δ/ΔT) and Q₁₀.

2. 2.|Q₁₀ and either F₄₀ or ΔF/ΔT were little or not related.

3. 3.|A positive correlation between F₄₀ and ΔF/ΔT was observed which was particularly close (r = 0.94 and 0.96) when the neurons were classified according to their Q₁₀ of <2 and >2.

4. 4.|The results suggest that neurons with positive temperature coefficients in the duck's hypothalamus mostly exhibit linear to exponential temperature-discharge relationships.

5. 5.|This is an contrast to observations on mammalian hypothalamic thermosensitive neurons and may relate to the absence of the thermosensory function in the duck's rostral brainstem.

The combined effects of ambient temperature and enforced sustained swimming activity on body temperatures of arctic charr (Salvelinus alpinus L.)

1. 1.|The difference between tissue temperatures and ambient water temperatures (ΔT) of the ectothermic Arctic charr (Salvelinus alpinus L.) ranged between 0.2 and 0.6°C.

2. 2.|For fish held at 5.7°C there were no significant differences in ΔT of exercising fish and those of controls.

3. 3.|By contrast, for fish held at 1.7°C sustained exercise led to a significant increase in ΔT of all body compartments compared with fish held in standing water (controls).

4. 4.|It is suggested that Arctic charr are capable of a limited control of metabolic heat exchange between body compartments and surrounding water when subjected to sustained exercise and ambient temperatures <2°C.

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.     

The physiological ecology of haemocyanin in some selected crabs. II. The characteristics of haemocyanin in relation to terrestrialness

The haemocyanins of five crabs ranging in habit from aquatic to terrestrial have been investigated.

The mean P₅₀ values of the respiratory pigments were determined at 0 mm Hg CO₂ and 28 °C (the average environmental temperature of all the species). Comparison of these data adjusted to the individual mean physiological pH indicate an increase in P₅₀ with terrestrialization, perhaps related to the greater abundance of oxygen in the aerial than in some the aquatic habits, and the progressive elaboration of lung breathing with terrestrialization.

The Bohr shifts (Δ log P₅₀/ΔpH) were determined (using different P_CO2 values to vary pH) and were found to decrease with terrestrialization, perhaps in adaptation to an associated rise in internal P_CO2 (6–8-fold between the aquatic Callinectes sapidus Rathbun and the terrestrial Cardisoma guanhumi Latreille and probably resulting from progressive gill reduction.

The temperature shifts (ΔH cal/mol) of the haemoeyanins were found and it is suggested that they diminish with increasing evironmental temperature and temperature fluctuation accompanying terrestrialization.     

The influence of temperature on catalytic efficiency of pyruvate kinase of crickets (Orthoptera: gryllidae)

Investigations have been conducted to determine the chemical nature of immediate temperature-regulatory mechanisms for enzyme activity, such as positive or negative temperature modulation and an adaptation-temperature dependence of the free energy of activation ΔG^≠. Three species of crickets have been selected for experiments in consideration of their different natural temperature demands: Gryllus campestris, Gryllus bimaculatus, and Acheta domesticus. Discontinuous Arrhenius plots (Fig. 1) show that all pyruvate kinases can exist in at least two temperature-dependent conformational states. Sizes of ΔH^≠-and ΔG^≠-values are correlated with the species' adaptation temperature (Table 1). Decreased barriers of ΔG^≠ after cold adaptation in G. campestris and A. domesticus are not sufficient for complete temperature compensation of the catalytic efficiency. Maximum enzyme-substrate affinity closely corresponds to the acclimation temperature of the crickets (Fig.2); K_m-values for PEP, however, are hardly influenced by experimental temperatures within the normal temperature range of the species. Data on enzyme function appear to corroborate the idea that optimal catalytic properties will be set according to the highest temperatures experienced respectively.     

Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity

The influence of the reaction conditions on the enantioselectivity of reactions catalysed by lipases or hydroxynitrile lyases (HNLs) in organic solvents was investigated. The lipases catalysed kinetic resolution of chiral secondary alcohols or chiral carboxylic acids and the HNLs catalysed asymmetric addition of hydrogen cyanide to aldehydes.

The temperature effects on enantioselectivity were studied in detail. From measurements of the enantiomeric ratio (E) at different temperatures the activation parameters ΔΔH^# and ΔΔS^# were determined. In the lipase-catalysed reactions the enthalpic and entropic effects on E always counteracted, while in a few of the HNL-catalysed reactions, ΔΔH^# and ΔΔS^# had opposite signs and therefore the effects cooperated to give high E values (−RTlnE = ΔΔG^# = ΔΔH^# − TΔΔS^#). In all the HNL-catalysed reactions and most of the lipase-catalysed ones, the enantioselectivity increased with decreasing reaction temperature. However, in one of the lipase-catalysed reactions, the enantioselectivity decreased with decreasing temperature. The theoretical background of these observations was discussed.

In the HNL-catalysed reactions, the enantioselectivity increased with increasing water content up to water saturation, while in the lipase-catalysed reactions the opposite trend was found in one case and in the others no significant effect was observed. Solvent mixtures of diisopropylether and hexane were used to obtain solvents with different log P values. The log P value of the solvent did not influence the enantioselectivity in the HNL-catalysed reactions, while the enantioselectivity increased with increasing log P value in two of the lipase-catalysed reactions. The reaction temperature was shown to be a very useful way to influence enzyme selectivity and the effects obtained could be rationalised. The influence of the reaction medium (solvent and water activity) is much more difficult to rationalise and predict.     

Transfer of light-induced electron-spin polarization from the intermediary acceptor to the prereduced primary acceptor in the reaction center of photosynthetic bacteria

In reaction centers and chromatophores of photosynthetic bacteria strong light-induced emissive ESR signals have been found, not only after a flash, but also under continuous illumination. The signal, with g = 2.0048 and ΔH_pp = 7.6 G, is only present under reducing conditions in material in which the primary acceptor, ubiquinone, U and its associated high-spin ferrous ion are magnetically uncoupled. Its amplitude under continuous illumination is strongly dependent on light intensity and on microwave power.

The emissive signal is attributed to the prereduced primary acceptor, U⁻, which becomes polarized through transfer of spin polarization by a magnetic exchange interaction with the photoreduced, spin polarized intermediary acceptor, I⁻. A kinetic model is presented which explains the observed dependence of emissivity on light intensity and microwave power. Applying this analysis to the light saturation data, a value of the exchange rate between I⁻ and U⁻ of 4 · 10⁸ s⁻¹ is derived, corresponding to an exchange interaction of 3–5 G.     

Cytochrome oxidase from Pseudomonas aeruginosa. II. Reaction with copper protein

The reaction between a cytochrome oxidase and a copper protein from Pseudomonas aeruginosa has been studied by a rapid mixing technique. The data support the view that a complex is formed rapidly between the two proteins and is followed by a transfer of electrons in either direction. Reduced copper protein donates an electron to the heme c moiety of the oxidase with an apparent first-order rate constant of about 30 s⁻¹ while the transfer in the reverse direction proceeds with a constant of about 120 s⁻¹. The reaction between the copper protein and the heme c of the oxidase is followed by a much slower internal reaction involving electron transfer between the heme c and heme d.

The kinetic data have been analyzed in terms of the thermodynamics of the interactions. This analysis indicates that the copper protein has a ΔE of about 0.038 V more positive than the heme c component, a value that compares favorably with that of 0.040 V obtained by equilibrium methods. The value of ΔE obtained by the kinetic method for the internal reaction is less precise but is reasonably close to that of 0.070 V determined by an equilibrium technique.     

Mus81 functions in the quality control of replication forks at the rDNA and is involved in the maintenance of rDNA repeat number in Saccharomyces cerevisiae

Previous studies in yeast have suggested that the SGS1 DNA helicase or the Mus81-Mms4 structure-specific endonuclease is required to suppress the accumulation of lethal recombination intermediates during DNA replication. However, the structure of these intermediates and their mechanism of the suppression are unknown. To examine this reaction, we have isolated and characterized a temperature-sensitive (ts) allele of MUS81. At the non-permissive temperature, sgs1Δ mus81^ts cells arrest at G₂/M phase after going through S-phase. Bulk DNA replication appears complete but is defective since the Rad53 checkpoint kinase is strongly phosphorylated under these conditions. In addition, the induction of Rad53 hyper-phosphorylation by MMS was deficient at permissive temperature. Analysis of rDNA replication intermediates at the non-permissive temperature revealed elevated pausing of replication forks at the RFB in the sgs1Δ mus81^ts mutant and a novel linear structure that was dependent on RAD52. Pulsed-field gel electrophoresis of the mus81Δ mutant revealed an expansion of the rDNA locus depending on RAD52, in addition to fragmentation of Chr XII in the sgs1Δ mus81^ts mutant at permissive temperature. This is the first evidence that Mus81 functions in quality control of replication forks and that it is involved in the maintenance of rDNA repeats in vivo.     

Determination of the thermodynamics of carbonic anhydrase acid-unfolding by titration calorimetry

The enthalpy of unfolding (Δ_uH) of carbonic anhydrase II was determined by titrating the protein with acid and measuring the heat using isothermal titration calorimetry (ITC) in the temperature range of 5 to 59 °C. By combining the ITC results with our previous findings by differential scanning calorimetry (DSC) in the temperature range of 39 to 72 °C, the Δ_uH dependence over a wide temperature range was obtained. The temperature dependence of the enthalpy displays significant curvature indicating that the heat capacity of unfolding (Δ_uC_p) is dependent on temperature. The T-derivative of Δ_uC_p was equal to 100 ± 30 J/(mol × K²), with the result that the Δ_uC_p is equal to 15.8 kJ/(mol × K) at 5 °C, 19.0 kJ/(mol × K) at 37 °C and 21.8 kJ/(mol × K) at 64 °C. The enthalpy of unfolding is zero at 17 °C. At lower temperatures, the Δ_uH becomes exothermic.

This method of determining protein unfolding thermodynamics using acid-ITC, significantly widens the accessible T-range, provides direct estimate of the thermodynamic parameters at physiological temperature, and gives further insight into the third T-derivative of the Gibbs free energy of unfolding.     

Magnetic field-stimulated luminescence and a matrix model for energy transfer. A new method for determining the redox state of the first quinone acceptor in the reaction center of whole cells of Rhodospirillum rubrum

In whole cells of Rhodospirillum rubrum the light-induced absorbance difference spectrum of the reduction of the first quinone electron acceptor Q₁ was determined in order to relate the emission yield ф and the magnetic field-induced emission increase Δф to the redox state of Q₁. It was found that Δф/ф² is a linear function of the number of reaction centers, in which Q₁ is reduced, independent of the fraction of reaction centers in the oxidized state. The emission yield is a hyperbolic function of the fraction of reaction centers closed, either by reduction of the acceptor Q₁ or by oxidation of the primary electron donor P. Apparently, in whole cells of R. rubrum a matrix model for energy transfer between various photosynthetic units can be applied. A model is presented, which is a generalization of theoretical considerations reported before (Duysens, L.N.M. (1978) in Chlorophyll Organization and Energy Transfer in Photosynthesis, Ciba Found. Symp. 61 (New Series), pp. 323–340, Elsevier/North-Holland, Amsterdam) and which is in excellent agreement with the experiments. From simultaneous measurements of Δф and ф the redox state of the reaction center can relatively easily be determined. So far, this is the only method for simultaneously measuring the fractions P⁺ and Q⁻₁ in intact cells under steady-state conditions.     

Differential scanning calorimetry and X-ray diffraction study of tendon collagen thermal denaturation

Differential scanning calorimetry, high and small angle X-ray diffraction analyses have been carried out on air-dried and rehydrated rat tail tendon collagen in order to test the reversibility of collagen thermal denaturation. The mean enthalpy values calculated for the denaturation process of air-dried and rehydrated samples are ΔH_D = 9.0 ± 0.8 cal/g and ΔH_D = 11.9 ±0.7 cal/g respectively, while the denaturation temperatures are T_D = 112 ± 1°C and T_D = 51 ± 1°C. Partial reversibility of the coiled coil—random coil process can be obtained by storing the samples in air or more rapidly by equilibration in water. After denaturation air-dried collagen fibres recover not only their molecular structure but also their characteristic fibrillar structure. The latter does not greatly influence the mean experimental enthalpy values.     

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.     

Ring rotation and anomalous metal atom motion in octamethyl ferrocene and spin-lattice relaxation in octamethyl ferrocenium hexafluorophosphate

The dynamics of the metal atom motion in sym octamethyl ferrocene (OMF) has been elucidated over the temperature range 85≤T≤350 K by ⁵⁷Fe Mössbauer effect spectroscopy, and shows a marked increase in the mean-square-amplitude of vibration at 348 K, some 80° below the melting point of the neat solid. Differential scanning calorimetry shows an endothermic peak at about the same temperature, and ΔH is 1.50 kJ mol⁻¹ and ΔS is 4.31 J mol⁻¹ K⁻¹. Corresponding data for OMF⁺PF₆⁻ can be fitted by a relaxation algorithm and confirm the intra-molecular nature of the transition. The spin-lattice relaxation over the above temperature range is fast compared to the characteristic Mössbauer time scale and can be accounted for by a Raman process in the high temperature limit. The transition at 348 K is associated with the onset of ring rotation/libration in the neat solid.     

Cephalexin synthesis by immobilised penicillin G acylase under non-isothermal conditions: reduction of diffusion limitation

The effect of thermodialysis on the enzymatic kinetic synthesis of the antibiotic cephalexin was investigated. As reference points, two existing models for an immobilised enzyme (Assemblase^®) and for the free enzyme were used. For Assemblase^®, it is known that diffusion limitation occurs and that therefore considerably more of the undesired side-product phenylglycine is formed.

The enzyme was immobilised on a membrane, and under isothermal conditions (293 K) the course of the reaction resembled that of the Assemblase^® enzyme. However, if a temperature gradient was applied across the membrane, with an average temperature of 293 K for the enzyme, than the course of the reaction changed. For large temperature gradients (30° and more), the course of the reaction resembled that of free enzyme. Thermodialysis enhances mass transfer across the membrane and therewith reduces diffusion limitations in the immobilised enzyme on the membrane.

The stability of the immobilised enzyme is such that the reactor can be re-used repeatedly. This, together with the positive effect of the temperature gradient on the course of the reaction, makes thermodialysis an interesting new technique that has potential to be applied on a larger scale if the membrane surface area per volume of reactor can be improved.