Stability and catalytic activity of alpha-amylase from barley malt at different pressure-temperature conditions

The impact of high hydrostatic pressure and temperature on the stability and catalytic activity of alpha-amylase from barley malt has been investigated. Inactivation experiments with alpha-amylase in the presence and absence of calcium ions have been carried out under combined pressure-temperature treatments in the range of 0.1-800 MPa and 30-75 degrees C. A stabilizing effect of Ca(2+) ions on the enzyme was found at all pressure-temperature combinations investigated. Kinetic analysis showed deviations of simple first-order reactions which were attributed to the presence of isoenzyme fractions. Polynomial models were used to describe the pressure-temperature dependence of the inactivation rate constants. Derived from that, pressure-temperature isokinetic diagrams were constructed, indicating synergistic and antagonistic effects of pressure and temperature on the inactivation of alpha-amylase. Pressure up to 200 MPa significantly stabilized the enzyme against temperature-induced inactivation. On the other hand, pressure also hampers the catalytic activity of alpha-amylase and a progressive deceleration of the conversion rate was detected at all temperatures investigated. However, for the overall reaction of blocked p-nitrophenyl maltoheptaoside cleavage and simultaneous occurring enzyme inactivation in ACES buffer (0.1 M, pH 5.6, 3.8 mM CaCl(2)), a maximum of substrate cleavage was identified at 152 MPa and 64 degrees C, yielding approximately 25% higher substrate conversion after 30 min, as compared to the maximum at ambient pressure and 59 degrees C.     

Comparative kinetic characteristics of catalase of Penicillium species molds

Extracellular catalases produced by fungi of the genus Penicillium: P. piceum, P. varians and P. kapuscinskii were purified by consecutive filtration of culture liquids. The maximum reaction rate of H2O2 decomposition, the Michaelis constants and specific catalytic activities of isolated catalases were determined. The operational stability was characterized by effective rate of catalase inactivation during enzymatic reaction (kin at 30 degrees C). The thermal stability was determined by the rate of enzyme thermal inactivation at 45 degrees C (k*[symbol: see text]H, s-1). Catalase from P. piceum displayed the maximum activity, which was higher than the activity of catalase from bovine liver. The operational stability of catalase from P. piceum was twofold to threefold higher than the stability of catalase from bovine liver. The physicochemical characteristics of catalases of fungi are better than the characteristics of catalase from bovine liver and intracellular catalase of yeast C. boidinii.     

Kinetic characteristics of extracellular catalase from Penicillium piceum F-648 and variants of fungi,adapted to hydrogen peroxide

A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H2O2 was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H2O2 degradation (Vmax),Vmax/KM ratio, and the catalase inactivation rate constant in the enzymatic reaction (kin, s-1) were estimated in phosphate buffer (pH 7.4) at 30 degrees C. The effective constant representing the rate of catalase thermal inactivation (kin*, s-1) was determined at 45 degrees C. In all samples, the specific activity and KM for catalase were maximum at a protein concentration in culture liquid filtrates of 2.5-3.5 x 10(-4) mg/ml. The effective constants describing the rate of H2O2 degradation (k, s-1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H2O2. Catalases from variants 5 and 3 with high and low affinities for H2O2, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H2O2 can be used to obtain fungal variants producing extracellular catalases with improved properties.     

Some properties of the extracellular protease produced by the psychotrophic bacterium Pseudomonas fluorescens strain AR-11.

The major extracellular protease from Pseudomonas fluorescens strain AR-11 has been partially purified by a factor of 300 by a combination of DEAE-cellulose ion-exchange chromatography and gel filtration. The enzyme had a molecular weight of 38 400 and exhibited optimum activity with isoelectrically precipitated casein substrate at pH 6.5 with Km - 0.13 mM. The protease was strongly inhibited by a number of heavy metal ions at the 10 mM level and also inhibited by thiol agents, while 10 mM EDTA led to slight activation. Optimum activity was retained, amounting to 33% of the maximum activity at 4 degrees C and 72% at 20 degrees C. Heat inactivation studies in which the isolated protease was heated at high temperature before subsequent incubation at 35 degrees C with substrate showed that for 50% inactivation 25 s heating at 130 degrees C or 17 s at 140 degrees C of 8.5 s at 150 degrees C was requried. The combination of high stability to heat treatments and retention of considerable activity at low incubation temperatures indicates that such a protease might have considerable significance in the processing and subsequent storage of food and other products.     

Inactivation of purified phenylalanine hydroxylase by dithiothreitol.

Purified rat liver phenylalanine hydroxylase is inactivated in vitro by ascorbate and thiol compounds, dithiothreitol being the most effective inhibitor, with a second order rate constant for the inactivation of 0.066 +/- 0.002 mM-1.min-1 at 20 degrees C and pH 7.2. Anaerobic conditions and catalase protected the enzyme from inactivation by dithiothreitol. This suggests that hydrogen peroxide, produced by oxidation of the thiol, is involved in the inactivation. The substrate, L-phenylalanine, also partially protected the enzyme from this inactivation. It is shown that incubation of the enzyme with dithiothreitol at aerobic conditions, followed by gel filtration, causes the release of iron from the active site. The inactivation by dithiothreitol was reversed by incubation of the iron-depleted enzyme with Fe(II).     

Peroxidase activity of succinylated catalase

The catalase succinylation by succinic anhydride excess results in an almost complete dissociation of the enzyme into subunits possessing no catalase activity. The catalase subunits show the peroxidatic activity on o-dianisidine oxidation. The oxidation kinetics of this substrate by the succinylated enzyme was studied at various temperatures. The activation energy for this process is 10.1 kcal/mole. Within the temperature range of 31-65.5 degrees, the succinylated enzyme thermostability was studied by monitoring the peroxidatic activity decrease upon o-dianisidine oxidation. The activation energy for the succinylated catalase thermoinactivation equals to 15.5 kcal/mole. The peroxidatic activity of catalase subunits obtained by enzyme succinylation and acidic solution treatment was compared to that of horseradish peroxidase in the oxidation of the same substrate, i.e., o-dianisidine.     

Activity of magnetite-immobilized catalase in hydrogen peroxide decomposition

The present work analyzes the activity in decomposition of H₂O₂ using magnetite-immobilized catalase. The support of catalase is a glutaraldehyde-treated magnetite (Fe₃O₄). The data obtained in the H₂O₂ decomposition are analyzed. The fitting of the initial rate of the H₂O₂ decomposition versus hydrogen peroxide concentration data is discussed using a specific program for enzyme kinetics modeling (Leonora). The free catalase from Aspergillus niger (3.5 or 10 U/mL) does not show substrate inactivation up to 0.4 M H₂O₂. The immobilized catalase at low catalyst concentration shows substrate inhibition. Using 1 mg/mL of supported catalase the predicted maximum activity is higher than in the case of the free catalase at similar catalase concentration, although the optimum temperature is lower (40 °C versus 60 °C).     

Effects of temperature on diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart. Aspects of the kinetics, stability, and quarternary structure of the enzyme.

A temperature-dependent conformational change of the active DPN-linked isocitrate dehydrogenase was observed. When initial reaction kinetic data were examined between 35 and 5 degrees, the Hill number (n) varied from 2 at higher to n approaching unity at lower temperatures, with an inflection point at 17 degrees. The presence of manganous isocitrate in the incubation media shifted the transition temperature for enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoate) from 8-16 degrees. These temperature-dependent transitions were paralleled by progressive changes in sedimentation velocities from s20, w of 10.4 at 25 degrees to 7.3 at 10 degrees as measured by active band centrifugation. The linear Arrhenius plot for apparent V max and the constancy of S0.5 for the substrate manganous isocitrate between 35 and 5 degrees suggest that this temperature-dependent conformational change may not be solely related to manganous isocitrate. Further indications of equilibria between different species of enzyme in solution and effects of substrates and cofactors on conformation came from studies of specific activity of enzyme diluted into buffers at 3 and 25 degrees. Dilution to concentrations between 10 and 25 mum enzyme resulted in relatively rapid protein concentration-dependent inactivation which could be prevented and fully reversed by manganous isocitrate. No further substantial inactivation was found subsequent to this phase at 25 degrees. Lowering the temperature of the dilution buffer to 3 degrees favored formation of enzyme species exhibiting a further time and pH-dependent loss of activity which became independent of protein concentration below 7 mum enzyme. The rate of cold inactivation was reduced by raising the ionic strength of the buffer and its progress could be arrested by manganous isocitrate; however, the substrate did not restore the original activity.     

Factors affecting the cold inactivation of an acetyl-coenzyme-A hydrolase purified from the supernatant fraction of rat liver

An extramitochondrial acetyl-coenzyme-A hydrolase from rat liver is shown to be a cold-labile oligomeric enzyme that undergoes a reversible conformational transition between a dimeric and a tetrameric form in the presence of adenosine 5'-triphosphate or adenosine 5'-diphosphate at 25-37 degrees C, and between a dimeric and a monomeric form at low temperature. The enzymatically active dimer is fairly stable at 25-37 degrees C, but much less stable at low temperature, dissociating into monomer with no activity. At 37 degrees C and low concentrations of enzyme protein (less than or equal to 14 micrograms/ml), the activity decreased rapidly and only 10% of the initial activity remaining after 60 min. Addition of bovine serum albumin or immunoglobulin G to the medium completely prevented inactivation of the dimeric enzyme at low concentration at 37 degrees C, but had little effect on cold inactivation of the enzyme. Cold inactivation of the dimeric enzyme was partially prevented by the presence of various CoA derivatives. The order of potency was acetyl-CoA (substrate) greater than or equal to butyryl-CoA greater than octanoyl-CoA greater than CoA (product) greater than acetoacetyl-CoA. Another enzyme product, acetate, had little effect on cold inactivation. Polyols, such as sucrose, glycerol, and ethylene glycol, and high concentrations of NaCl, KCl, pyrophosphate and phosphate also greatly prevented cold inactivation. Cold inactivation was scarcely affected by pH within the pH range at which the enzyme was stable at 37 degrees C.     

Thermal inactivation and product inhibition of Aspergillus terreus CECT 2663 alpha-L-rhamnosidase and their role on hydrolysis of naringin solutions

The kinetics of thermal inactivation of A. terreus alpha-rhamnosidase was studied using the substrate p-nitrophenyl alpha-L-rhamnoside between 50 degrees C and 70 degrees C. Up to 60 degrees C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60 degrees C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60 degrees C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.     

The thermal stability of a castor bean seed acid phosphatase

The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45 degrees C using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55 degrees C. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60 degrees C. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60 degrees C, with the reaction products inorganic phosphate-P (10 mM) and p-nitrophenol-p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (Tm) value of 75 degrees C and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.     

Effect of Mg2+ on the thermal inactivation and unfolding of creatine kinase

The effect of Mg2+ on the thermal inactivation and unfolding of rabbit muscle creatine kinase has been studied for various temperatures and Mg2+ concentrations. Increasing the Mg2+ concentration in the denatured system significantly enhanced the inactivation and unfolding of creatine kinase during thermal denaturation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47 degrees C the increased free Mg2+ concentration caused the creatine kinase inactivation rate to increase. Increasing the temperature strengthened the effect of Mg2+ on the thermal inactivation. Control experiments showed that treating native creatine kinase with different concentrations of Mg2+ did not change the enzymatic activity. The fluorescence emission spectra showed that the emission maximum for creatine kinase red-shifted from 335 to 337 nm during thermal denaturation at 47 degrees C for 10 min, while the presence of 3 mM Mg2+ caused the enzyme emission maximum to red-shift from 335 to 342.5 nm for the same thermal denaturation conditions. In addition, Mg2+ also enhanced the unfolding of the equilibrium state and decreased the time required to reach the equilibrium state of creatine kinase at 47 degrees C. The potential biological significance of these results are discussed.     

Effect of temperature on kinetics and differential mobility of empty and loaded nucleoside transporter of human erythrocytes

The transmembrane equilibration of [3H]uridine was measured in human erythrocytes as a function of temperature using rapid kinetic techniques. Arrhenius plots of the maximum velocity of equilibrium exchange were continuous between 5 and 30 degrees C (Ea = 17-20 kcal/mol), but the increase in velocity with increase in temperature leveled off above 30 degrees C. This leveling off did not reflect heat inactivation of the carrier since transport activity was stable for 3 h at 37 degrees C. Transmembrane equilibration of uridine in equilibrium exchange and zero-trans modes at 5, 15, 25, and 35 degrees C conformed to appropriate integrated rate equations derived for the simple transporter. The nucleoside transporter exhibited directional symmetry, but the loaded carrier moved on the average 5 times more rapidly than the empty carrier at 15, 25, and 35 degrees C, but 25-40 times faster at 5 degrees C. This marked shift in differential mobility of loaded and empty carrier between 15 and 5 degrees C was entirely attributable to an impairment of mobility of empty carrier. The Michaelis-Menten constant for equilibrium exchange increased about 3-fold with increase in temperature between 5 and 35 degrees C. The van't Hoff plot of the values was approximately linear and yielded an estimate of the enthalpy of carrier:substrate dissociation of 7.8 kcal/mol.     

Purification and characterization of a cold-adapted isocitrate lyase and a malate synthase from Colwellia maris, a psychrophilic bacterium.

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20 degrees C and was rapidly inactivated at the temperatures above 30 degrees C, but the optimum temperature for the activity of MS was 45 degrees C. NaCl was found to protect ICL from heat inactivation above 30 degrees C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The Km for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the Km for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.     

Heat inactivation of catalase from Staphylococcus aureus MF-31.

The effects of heat on catalase from Staphylococcus aureus lysates were examined. Catalase activity increased with increasing concentrations of potassium phosphate buffer, when heated at temperatures between 50 and 65 degrees C for 10 min. Inactivation of catalase by NaCl during heating was demonstrated. Extended heating of S. aureus cells at 52 degrees C resulted in a slight decrease in catalase activity of the resultant lysates. This decrease was more pronounced in the presence of salt. Heating at 62 degrees C caused a decrease in catalase activity, but not complete inactivation. These results implicate the combined effects of heat, and NaCl in the inactivation of catalase from S. aureus. The findings are consistent with the hypothesis that H2O2 may accumulate as a result of decreased catalase activity and be responsible for the decreased colony-forming ability of stressed S. aureus.     

Heat inactivation of catalase from Staphylococcus aureus MF-31.

The effects of heat on catalase from Staphylococcus aureus lysates were examined. Catalase activity increased with increasing concentrations of potassium phosphate buffer, when heated at temperatures between 50 and 65 degrees C for 10 min. Inactivation of catalase by NaCl during heating was demonstrated. Extended heating of S. aureus cells at 52 degrees C resulted in a slight decrease in catalase activity of the resultant lysates. This decrease was more pronounced in the presence of salt. Heating at 62 degrees C caused a decrease in catalase activity, but not complete inactivation. These results implicate the combined effects of heat, and NaCl in the inactivation of catalase from S. aureus. The findings are consistent with the hypothesis that H2O2 may accumulate as a result of decreased catalase activity and be responsible for the decreased colony-forming ability of stressed S. aureus.     

Influence of temperature and pH on xylitol production from xylose by Debaryomyces hansenii

The production of xylitol from concentrated synthetic xylose solutions (S(o) = 130-135 g/L) by Debaryomyces hansenii was investigated at different pH and temperature values. At optimum starting pH (pH(o) = 5.5), T = 24 degrees C, and relatively low starting biomass levels (0.5-0.6 g(x)/L), 88% of xylose was utilized for xylitol production, the rest being preferentially fermented to ethanol (10%). Under these conditions, nearly 70% of initial carbon was recovered as xylitol, corresponding to final xylitol concentration of 91.9 g(P)/L, product yield on substrate of 0.81 g(P)/g(S), and maximum volumetric and specific productivities of 1.86 g(P)/L x h and 1.43 g(P)/g(x) x h, respectively. At higher and lower pH(o) values, respiration also became important, consuming up to 32% of xylose, while negligible amounts were utilized for cell growth (0.8-1.8%). The same approach extended to the effect of temperature on the metabolism of this yeast at pH(o) = 5.5 and higher biomass levels (1.4-3.0 g(x)/L) revealed that, at temperatures ranging from 32-37 degrees C, xylose was nearly completely consumed to produce xylitol, reaching a maximum volumetric productivity of 4.67 g(P)/L x h at 35 degrees C. Similarly, both respiration and ethanol fermentation became significant either at higher or at lower temperatures. Finally, to elucidate the kinetic mechanisms of both xylitol production and thermal inactivation of the system, the related thermodynamic parameters were estimated from the experimental data with the Arrhenius model: activation enthalpy and entropy were 57.7 kJ/mol and -0.152 kJ/mol x K for xylitol production and 187.3 kJ/mol and 0.054 kJ/mol x K for thermal inactivation, respectively.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Heat-induced temperature sensitivity of outgrowing Bacillus cereus spores.

Inactivation of Bacillus cereus spores during cooling (10 degrees C/h) from 90 degrees C occurred in two phases. One phase occurred during cooling from 90 to 80 degrees C; the second occurred during cooling from 46 to 38 degrees C. In contrast, no inactivation occurred when spores were cooled from a maximum temperature of 80 degrees C. Inactivation of spores at a constant temperature of 45 degrees C was induced by initial heat treatments from 80 to 90 degrees C. The higher temperatures accelerated the rate of inactivation. Germination of spores was required for 45 degrees C inactivation to occur; however, faster germination was not the cause of accelerated inactivation of spores receiving higher initial heat treatments. Repair of possible injury was not observed in Trypticase soy broth (BBL Microbiology Systems), peptone, beef extract, starch, or L-alanine at 30 or 35 degrees C. Microscopic evaluation of spores outgrowing at 45 degrees C revealed that when inactivation occurred, outgrowth halted at the swelling stage. Inhibition of protein synthesis by chloramphenicol at the optimum temperature also stopped outgrowth at swelling; thus protein synthesis may play a role in the 45 degree C inactivation mechanism.     

Characterization of Trametes versicolor laccase for the transformation of aqueous phenol

Laccase (oxygen oxidoreductase, EC 1.10.3.2) from Trametes versicolor was thoroughly characterized in terms of its catalytic stability and its effectiveness as a biocatalyst under various reaction conditions when using phenol as a model substrate. This enzyme demonstrated high or moderate degrees of stability at pHs from 5 to 8 at 25 degrees C and at temperatures from 10 to 30 degrees C at pH 6. Exponential decay expressions were successfully used to model laccase inactivation when incubated under various conditions of pH and temperature. Phenol transformation was optimum at pH 6, but significant transformation was observed over a pH range of 4-7, provided that sufficient laccase was present in the reacting solution. Partial inactivation of laccase was observed during the oxidation of phenol, even under conditions of optimal stability (pH 6 and 25 degrees C).