Effects of water activity and aqueous solvent ordering on thermal stability of lysozyme, alpha-chymotrypsinogen A, and alcohol dehydrogenase.

Effects of water activity (aW) and solvent ordering were separately analyzed on the thermal unfolding of lysozyme and alpha-chymotrypsinogen A, and also on the thermal deactivation of yeast alcohol dehydrogenase (YADH) in aqueous solutions with various additives. With the coexistence of additives, water activity was the determinant of the extent of the change in the thermal stability of proteins while solvent ordering was the determinant of the direction of the change. The parameter alpha, determined from the activity coefficient of water, representing the deviation of aW from that of the ideal solution, was useful as a quantitative index of the solvent ordering showing good correlations with the unfolding temperature and enthalpy of lysozyme and alpha-chymotrypsinogen A and also with the thermal deactivation rate constant of YADH at a constant aW. Solvent ordering seemed to affect the thermal stability of proteins mainly through its effect on the intramolecular hydrophobic interaction among amino acid residues in a protein molecule but the contribution of the electrostatic interaction including hydrogen bonding through the change in permittivity of solution was also suggested.

Determination of log P for pressurized carbon dioxide and its characterization as a medium for enzyme reaction.

The log P value of pressurized CO(2) at 50 degrees C was determined from the solubility of 1-octanol in CO(2) and compared with other solvent parameters such as permittivity, epsilon, and polarity parameter, E(T)(30). The log P indicated that pressurized CO(2) is rather hydrophilic although it seems hydrophobic being judged from epsilon(r) and E(T)(30). With a change in pressure from 3 to 11.8 MPa, the log P changed from 0.9 to 2.0 while epsilon(r) and E(T)(30) changed only slightly. The log P was linearly correlated to the logarithm of the solubility of water among organic solvents. Pressurized CO(2) was located close to the linear correlation line among the solvents at high pressure (>11 MPa) but its location deviated to the hydrophilic side with a decrease in pressure. Lipase-catalyzed esterification of stearic acid with ethanol and hydrolysis of ethyl stearate were carried out in pressurized CO(2), benzene (log P = 2.0), and n-hexane (log P = 3.5). In spite of the lowest log P value for CO(2), the reaction rate in CO(2) was the highest among solvents tested in pressure range over 10 MPa. The reaction rate was strongly dependent on pressure of CO(2).

Scaling analysis of the concentration dependence on elasticity of agarose gel.

Since the critical exponent of the elastic modulus is related to the spatial dimension and the critical exponent of the correlation length, depending on the characteristics of elasticity, we experimentally evaluated both the elastic modulus of a sol-gel transition system and also the correlation length. We could determine the correlation length of agarose gel by the dynamic light scattering method; it was well described by the power law as a function of the deviation from the sol-gel transition point. Three scaling laws between the critical exponent of the correlation length (v) and that of the elastic shear modulus (t) were compared, and the critical exponent of the elastic modulus was described by the equation of de Gennes expression (t=1+v(d-2), where d is the spatial dimension). This result suggests that agarose fibers are stiff enough to show scalar elasticity.

Influence of water activity and aqueous solvent ordering on enzyme kinetics of alcohol dehydrogenase, lysozyme, and beta-galactosidase.

Effects of the water activity (a(w)) and the solvent ordering, as determined by the activity coefficient of water, were investigated on the enzyme kinetics of alcohol dehydrogenase, lysozyme, and beta-galactosidase in various aqueous solutions. The water activity and the solvent ordering were adjusted by addition of electrolytes (NaCl, KCl, CsCl, etc.) or nonelectrolytes (sugars, alcohols, urea, etc.) at various concentrations. Although the enzyme kinetics were strongly dependent on a(w), a(w) was not a complete determinant of the enzyme behavior in aqueous solutions. Enzyme kinetics were also dependent on the solvent ordering. At a fixed a(w), all the enzyme kinetic parameters tested had a good correlation with the solvent ordering parameter as represented by the parameter alpha, an index of the deviation of the water state from the ideal solution, determined from the activity coefficient of water in solutions. Solvent ordering was expected to affect the enzyme kinetics through its effect on the hydrophobic interaction between the enzyme and the substrate and also on the thermal fluctuation.

Conductance and relaxations of gelatin films in glassy and rubbery states.

The dielectric constant, epsilon', and the dielectric loss, epsilon'', for gelatin films were measured in the glassy and rubbery states over a frequency range from 20 Hz to 10 MHz; epsilon' and epsilon'' were transformed into M* formalism (M* = 1/(epsilon' - i epsilon'') = M' + iM''; i, the imaginary unit). The peak of epsilon'' was masked probably due to dc conduction, but the peak of M'', e.g. the conductivity relaxation, for the gelatin used was observed. By fitting the M'' data to the Havriliak-Negami type equation, the relaxation time, tauHN, was evaluated. The value of the activation energy, Etau, evaluated from an Arrhenius plot of 1/tauHN, agreed well with that of Esigma evaluated from the DC conductivity sigma0 both in the glassy and rubbery states, indicating that the conductivity relaxation observed for the gelatin films was ascribed to ionic conduction. The value of the activation energy in the glassy state was larger than that in the rubbery state.

Continuous production of L-carnitine with NADH regeneration by a nanofiltration membrane reactor with coimmobilized L-carnitine dehydrogenase and glucose dehydrogenase.

L-carnitine dehydrogenase (CDH) was partially purified from Pseudomonas putida IAM12014 for the stereospecific reduction of 3-dehydrocarnitine to L-carnitine. CDH and glucose dehydrogenase (GDH) were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of L-carnitine from 3-dehydrocarnitine with NADH regeneration. In the NFMBR, NAD was partially immobilized through rejection by the nanofiltration membrane and effectively regenerated by the conjugation reaction of CDH and GDH. Since 3-dehydrocarnitine was unstable at neutral pH, it was maintained under acidic conditions (pH 0.7) and supplied to the NFMBR separately from the other substrates, glucose and coenzyme NAD. As 50 mM 3-dehydrocarnitine in HCl solution, 0.05 mM NAD, and 100 mM glucose in 0.5 M Tris buffer (pH 8) were continuously supplied to the NFMBR with immobilized CDH (200 U/ml) and GDH (200 U/ml) at the retention time of 80 min and temperature of 25 degrees C, the maximum conversion, reactor productivity, and NAD regeneration number were 78%, 113 g/l/d, and 780, respectively. The half-life of the NFMBR was longer than 500 h.

Prevention of Initial Supercooling in Progressive Freeze-concentration.

A physical method is proposed that uses a cooling plate with many small holes to prevent initial supercooling in progressive freeze-concentration, and thus avoid serious contamination of the ice produced. The higher chance for ice nucleation of the water molecules in the holes due to the temperature gradient in the cooling plate resulted in the initial supercooling being completely prevented. Accordingly, the purity of the ice initially formed was substantially improved when compared with that by the standard vessel without holes in the cooling plate.

Effects of Substrate Solubility in Interesterification with Triolein by Immobilized Lipase in Supercritical Carbon Dioxide.

The interesterification reaction by immobilized lipase between triolein and behenic acid (BA) or ethyl behenate (EB) was investigated in supercritical carbon dioxide (SCCO2) to produce 1,3-dibehenoyl-2-oleoyl glycerol (BOB). The incorporation rate of behenoyl group to triolein was much higher with EB as a substrate than with BA. The solubility in SCCO2 was found to be significantly higher for EB than BA, which seemed to cause the higher formation rate of the behenoyl-enzyme complex in the former case to give the higher production rate of the final interesterification product, BOB.

Water permeability of plasma membranes of cultured rice, grape, and CH27 cells measured dielectrically.

The capacitance of suspensions of cultured rice cells (Oryza sativa L. ssp. japonica), grape cells (Vitis sp.), and CH27 cells originated from murine B-cell lymphoma was measured in the frequency range of 0.2 to 10 MHz. The relationship between the increase in capacitance caused by the presence of cells at 0.4 MHz, delta C, and the cell density was linear. Measurement of capacitance was useful in measurement of transitional changes in cell volume under external osmotic stress when sucrose was added. From the course of volume changes with such stress, the water permeabilities of the plasma membrane, Lp, were measured to be 0.015, 0.020, and 0.090 pm/(s.Pa) at 25 degrees C, for rice cells, grape cells, and CH27 cells, respectively. The smaller Lp for plant cells seemed to explain why preservation of plant cells by freezing is more difficult than for animal cells. From the temperature dependence of Lp, the apparent activation energies were calculated to be 12.0 +/- 2.9 and 13.0 +/- 5.2 kcal/mol for rice cells and CH27 cells, respectively.

Continuous Production of l-Alanine with NADH Regeneration by a Nanofiltration Membrane Reactor.

A conjugated enzyme system, alanine dehydrogenase (AIDH) for stereospecific reduction of pyruvate to l-alanine and glucose dehydrogenase (GDH) for regeneration of NADH, were coimmobilized in a nanofiltration membrane bioreactor (NFMBR) for the continuous production of l-alanine from pyruvate with NADH regeneration. Since pyruvate was proved to be unstable at neutral pH, it was kept under acidic conditions and supplied to NFMBR separately from the other substrates. As 0.2 m pyruvate in HCl solution (pH 4), 10 mm NAD, 0.2 m glucose, and 0.2 m NH4Cl in 0.5 m Tris buffer (pH 8) were continuously supplied to NFMBR with immobilized AIDH (100 U/ml) and GDH (140 U/ml) at the retention time of 80 min, the maximum conversion, reactor productivity, and NAD regeneration number were 100%, 320 g/liter/d, and 20,000, respectively. To avoid the effect of pyruvate instability, a consecutive reaction system, lactate dehydrogenase (l-LDH) and AIDH, was also used. In this system, the l-LDH provides pyruvate, the substrate for the AIDH reaction, from l-lactate regenerating NADH simultaneously, so the pyruvate could be consumed as soon as it was produced. As 0.2 m l-lactate, 10 mm NAD, 0.2 m NH4Cl in 0.5 m Tris buffer (pH 8) were continuously supplied to NFMBR with immobilized l-LDH (100 U/ml) and AIDH (100 U/ml) at the retention time of 160 min, the maximum conversion, reactor productivity, and the NAD regeneration number were 100%, 160 g/Iiter/d, and 20,000, respectively.

Dielectric Relaxation of Aqueous Solution with Low-molecular-weight Nonelectrolytes and Its Relationship with Solution Structure.

Dielectric relaxation of water molecules was measured in the frequency range from 0.2 to 20 GHz for aqueous solutions of urea, formamide, alcohols, and saccharides. The relaxation behavior was described well by the Debye equation with a single relaxation process in most cases. The static permittivity of solution and relaxation time of water in solution changed linearly with solute concentration. The relaxation time shift of water molecules through the existence of solute was correlated well to the first virial coefficient of the activity coefficient of water suggesting the close relationship between dielectric relaxation time and aqueous solution structure.

Electrochemical bioreactor with immobilized glucose-6-phosphate dehydrogenase on the rotating graphite disc electrode modified with phenazine methosulfate.

Physical adsorption, covalent binding through the carbodiimide reaction between the surface carboxyl group and the amino group of the protein, and the crosslinking method with bovine serum albumin by glutaraldehyde were applied for the immobilization of glucose-6-phosphate dehydrogenase (G6PDH) on a graphite electrode. Among those, the crosslinking method was employed for its highest apparent enzyme activity per unit surface area. Phenazine methosulfate (PMS), as a mediator for the electrochemical oxidation of NADH, was also immobilized on the graphite surface through adsorption. The conjugation reaction of G6PDH and the electrochemical oxidation of NADH were confirmed by cyclic voltammetry and the constant potential electrochemical reaction. An electrochemical bioreactor system was established by using a rotating disc graphite electrode with G6PDH immobilized. The coenzyme, NAD, was effectively recycled between the electrochemical and the enzymatic reactions.

Measurement of the Thermal Conductivity of Unfrozen and Frozen Food Materials by a Steady State Method with Coaxial Dual-cylinder Apparatus.

Coaxial dual-cylinder apparatus was used to measure the effective thermal conductivity of aqueous solutions of glucose, sucrose, gelatin and egg albumin over a temperature range from -20° to 20°C by the steady state method. The accuracy of the apparatus was confirmed by testing with water and ice. The effective thermal conductivity decreased with an increase in the total solid content in both the frozen and unfrozen states. In the unfrozen state, the effective thermal conductivity was slightly dependent on temperature. In the frozen state, however, the effective thermal conductivity was strongly dependent on temperature; lower temperatures gave higher effective thermal conductivity, reflecting the increase in the ice fration. For the unfrozen samples, the intrinsic thermal conductivity of each solid component was calculated by heat transfer models. All the models tested, series, parallel and Maxwell-Eucken, were equally applicable to describe the heat conduction in the unfrozen state. In the frozen state, however, the strong temperature dependency of the effective thermal conductivity suggests that the effect of the temperature dependency of the ice fraction should be incorporated into theoretical models.

Electrochemical bioreactor with regeneration of NAD+ by rotating graphite disk electrode with PMS adsorbed.

Kinetic constants were compared among p-quinone, 2,6-dichlorophenolindophenol, phenazine methosulfate (PMS), methylene blue, and FAD in the oxidation of NADH. Among those, PMS was selected for its highest rate constant as a mediator for the electrochemical oxidation of NAD. The PMS could be stably immobilized on a graphite electrode surface by adsorption. The PMS adsorbed and that in the solution showed distinctly separated peaks in the cyclic voltammogram. The immobilized PMS functioned as an immobilized mediator to reduce the overpotential in the electrochemical oxidation of NAD so that the electrode could be used as an NAD regenerator. For the construction of an electrochemical bioreactor, a specially designed rotating disk graphite electrode was used. In spite of its extraordinarily large surface area, the behavior of the rotating disc electrode was described well by the Levich law. The NAD oxidation system of the rotating graphite disk electrode with PMS adsorbed was combined with glucose-6-phosphate dehydrogenase reaction, which reduced NAD with the consumption of glucose-6-phosphate. The electrochemical bioreactor system worked well with recycling of NAD at a high current efficiency.

Dynamic affinity between dissociable coenzyme and immobilized enzyme in an affinity chromatographic reactor with single enzyme.

The affinity chromatographic reactor (ACR) is a bioreactor which utilizes the dynamic interaction or the dynamic affinity between a free coenzyme and immobilized enzymes for the highly efficient regeneration of dissociable coenzymes. Dynamic affinity between free NAD and immobilized alcohol dehydrogenase (ADH) in ACR was investigated by three different methods. ADH catalyzed both oxidation and reduction of NAD, consuming propionaldehyde and ethanol. The theoretical model under consideration elucidated a criterion for the expression of the dynamic affinity as a relationship among the affinity constants and the concentrations of a coenzyme and immobilized enzyme. This criterion was confirmed experimentally by the measurements of the retention time of NAD and the half-life period of the reactor activity after one-shot pulse injection of NAD to ACR. In the stability measurement of the immobilized enzyme, it became clear that ADH was more stable at the higher concentration in immobilization. Although the present case of coenzyme cycling by a single enzyme is very special, with limited chance for the direct application, the results obtained here provide a theoretical basis for ACR with multienzymes-which is of more general use.

Freezing and Ice Structure Formed in Protein Gels.

Ice structure was photographically analyzed for frozen soy protein curd and egg albumin gel frozen under various conditions. Dendritic ice structure was observed growing from the cooling plate parallel to the direction of the heat flux. The change in the ice structure size was analyzed at different locations from the cooling plate in the plane perpendicular to the direction of heat flux. In accordance with the theoretical relationship proposed by us before, the mean ice structure size was inversely proportional to the moving speed of the freezing front and the proportionality constant was not very much different from the diffusion coefficient of water, showing the important role of the molcular diffusion mechanism in the process of ice crystal growth. For the freezing accompanied with supercooling, the ice structure became very small, reflecting the very rapid moving speed of the freezing front when supercooling ceased. The theoretical model by us had advantages over the models proposed in the literature for its simple theoretical basis and wider applicability.

Modeling of hollow-fiber capillary reactor for the production of L-Alanine with coenzyme regeneration.

Continuous production of L-alanine with conjugated enzyme systems of alanine dehydrogenase (AlaDH) and lactate dehydrogenase (LDH) or alcohol dehydrogenase (ADH) was carried out with NAD regeneration in an ultrafiltration hollow-fiber capillary reactor (HFCR) which was proposed as a test bioreactor with very small scale. In the AlaDH/LDH system, pyruvate is the intermediate product for L-alanine so that an optimal point existed in pyruvate concentration for the production rate of L-alanine. NAD cycling number of 4850 and L-alanine productivity of 61.7 mmol/L h were obtained at the best condition. In the AlaDH/ADH system, however, the substrate inhibition in the AlaDH reaction by pyruvate should be considered and the best results of NAD cycling number and (L)-alanine productivity were 2700 and 13.5 mmol/L h, respectively. In consideration of concentration distribution and mixing in the axial direction on an HFCR, performance of the reactor was theoretically analyzed with a multistage stirred tank reactor model combined with the kinetic model based on all the elementary reactions involved. Although quantitative discrepancy existed in some cases, the present theoretical model could explain experimental results and is expected to be generally applicable to standard hollow fiber reactors.

Formation of propylene oxide by Nocardia corallina immobilized in liquid paraffin.

Nocardia corallina B276 cells were immobilized by emulsification with liquid paraffin and an antifoam agent at room temperature. The immobilized cells were studied for their ability to carry out the formation of propylene oxide from propylene and oxygen. The evaluations were done with the cells in a bubble-type reactor with a continuous gas feed of 5% propylene and 11.6-95% oxygen, with the balance nitrogen. By using liquid paraffin and antifoam, both the epoxidation activity and the stability were improved, especially for the P-1-200 strain, over that for nonimmobilized cells. The N. corallina cells showed an apparent preference for a hydrophobic, as compared to a hydrophilic, environment. The propylene-oxide-forming activity of the immobilized cells was higher at 40 than at 30 degrees C reactor temperature and with 20% (versus 95%) oxygen in the feed. The stability was markedly better at 30 degrees C and with 20% oxygen. High gas flowrates gave increased apparent activity probably because of less resistance to substrate mass transfer. The effects of pH were minor. The role of glucose as the energy source for regeneration of cofactors for the monooxygenase system also is discussed.