Activation Energy For Inactivation Research Articles

Increased stability of alcohol oxidase under high hydrostatic pressure

Alcohol oxidase (AOX), in the presence of oxygen, catalyzes the bioconversion of short-chained alcohols into their corresponding aldehydes and ketones. Alcohol oxidase can be used in the production of aldehydes that can serve as precursors of flavor compounds and fine chemicals. However due to AOX's poor stability, its practical industrial or analytical application is limited. Two kinetic inactivation regions suggest that one of AOX's isozymes is more resistant to thermal inactivation at atmospheric or high pressures. The resistant fraction was 4–34 times more stable than the labile fraction. High hydrostatic pressure (HHP) stabilized AOX against thermal inactivation. A 14-fold stabilization was observed at 49.4 °C for the labile fraction at 200 MPa relative to atmospheric pressure. The activation energy of inactivation at 40–160 MPa ranged from 95 to 184 kJ mol−1 for the resistant fraction and from 232 to 402 kJ mol−1 for the labile fraction. The activation volume of inactivation was independent of temperature and of approximately 20 cm3 mol−1.

Thermodynamic Properties of the β-glucosidase from Thermotoga maritima Extend the Upper Limit of Thermophilicity

Enzymes from thermophilic organisms are believed to be strong candidates for industrial applications due to their ability to withstand temperature-induced enzyme inactivation. The present study demonstrated molecular cloning, over-expression, purification and characterization of β-glucosidase from Thermotoga maritima. The bglA gene with a capacity to encode a 51 kDa enzyme was heterologously expressed in E. coli M15. The enzyme was produced @130 mgL(-1) in LB media and @440 mgL(-1) in Dubos salt medium accounting 40-47 % of total cellular soluble proteins when lactose was used as an inducer. The enzyme showed a peak activity between pH and temperature range of 5.0-7.0 and 80-100 °C, respectively. The activity was fairly stable up to 140 °C. The turnover rate (kcat) of the enzyme was 187.1±20 s(-1), whereas the Km and Vmax values were 0.56 mM and 238±2.4 IU mg(-1) protein, respectively. The enzyme was shown to have half-life of 136, 71 and 12.6 h at 80, 90 and 100 °C, respectively. Thermodynamics parameters including melting temperature (130 °C), activation energy for inactivation (36.92 kJmole(-1)), enthalpy (33.73 kJmole(-1)), Gibb's free energy (127.96 kJmole(-1)) and entropy (-246.46 Jmole(-1)K(-1)) have shown that the enzyme have enhanced hydrophobic interactions to prevent its thermal unfolding. These features endorse the industrial applications of the enzyme.

The catalytic properties and stability of β-galactosidases from fungi

The catalytic activity of β-galactosidases from fungi Penicillium canescens and Aspergillus oryzae is maximum in a weakly acidic medium and does not depend on the presence of magnesium cations in the reaction medium. The enzyme from Aspergillus oryzae fungi is more active, and that from Penicillium canescens is stabler. One of stability indications is the presence of an induction period in the kinetic curves of thermal inactivation. This period disappears at 54°C for the enzyme from Aspergillus oryzae and at 59°C for the enzyme from Penicillium canescens. The temperature dependences of the effective rate constants for the inactivation of the tetrameric enzyme from Penicillium canescens show that the main reason for enzyme inactivation is the dissociation of oligomeric forms below 66°C (Eact = 85 kJ/mol) and enzyme denaturation at higher temperatures (Eact = 480 kJ/mol). The dissociation stage is absent for monomeric β-galactosidase from Aspergillus oryzae fungi, and the activation energy of inactivation is 450 kJ/mol over the whole temperature range studied (53–60°C).

Kinetic study of a purified anionic peroxidase isolated from Eupatorium odoratum and its novel application as time temperature indicator for food materials

An anionic peroxidase isoenzyme having a pI of 3.5 was purified from Eupatorium odoratum, commonly called, ‘siam weed’, belonging to family, Asteraceae. The molecular weight of the enzyme was identified as 55kD. The specific activity of the crude extract was increased to 647U/mg from 62U/mg by ammonium sulfate precipitation. The enzyme was 114-fold purified by ion exchange chromatography and had a specific activity of 7094IU/mg. The specificity constant (kcat/Km) of the isozyme was 8.75×105s−1M−1 with ABTS and 6.9×105s−1M−1 with H2O2 as substrates. The enzyme was found to be very stable at room temperature (30±2°C) and retained more than 90% activity even after a period of 2months and was stable for more than 6months at 4±1°C without any additive, stabilizer or preservative. The activation energy for inactivation (Ea) of the isozyme was 120.14kJmol−1 and the half-life was found to be around 34h at 50°C. The purified Eupatorium peroxidase has an optimum pH of 4.5 and optimum temperature of 55°C. This isozyme was stable in metal ionic solutions and showed increased activity in presence of Hg2+, K+ and Ca2+. The enzyme can also be used as a low cost time-temperature indicator strip for which the preliminary works have already been carried out satisfactorily.

Thermal inactivation of susceptible and multiantimicrobial-resistant salmonella strains grown in the absence or presence of glucose.

The heat resistance of susceptible and multiantimicrobial-resistant Salmonella strains grown to stationary phase in glucose-free tryptic soy broth supplemented with 0.6% yeast extract (TSBYE-G; nonadapted), in regular (0.25% glucose) TSBYE, or in TSBYE-G with 1.00% added glucose (TSBYE+G; acid adapted) was determined at 55, 57, 59, and 61 degrees C. Cultures were heated in sterile 0.1% buffered peptone water (50 microl) in heat-sealed capillary tubes immersed in a thermostatically controlled circulating-water bath. Decimal reduction times (D values) were calculated from survival curves having r(2) values of >0.90 as a means of comparing thermal tolerance among variables. D(59 degrees C) values increased (P < 0.05) from 0.50 to 0.58 to 0.66 min for TSBYE-G, TSBYE, and TSBYE+G cultures, respectively. D(61 degrees C) values of antimicrobial-susceptible Salmonella strains increased (P < 0.05) from 0.14 to 0.19 as the glucose concentration increased from 0.00 to 1.00%, respectively, while D(61 degrees C) values of multiantimicrobial-resistant Salmonella strains did not differ (P > 0.05) between TSBYE-G and TSBYE+G cultures. When averaged across glucose levels and temperatures, there were no differences (P > 0.05) between the D values of susceptible and multiantimicrobial-resistant inocula. Collectively, D values ranged from 4.23 to 5.39, 1.47 to 1.81, 0.50 to 0.66, and 0.16 to 0.20 min for Salmonella strains inactivated at 55, 57, 59, and 61 degrees C, respectively. z(D) values were 1.20, 1.48, and 1.49 degrees C for Salmonella strains grown in TSBYE+G, TSBYE, and TSBYE-G, respectively, while the corresponding activation energies of inactivation were 497, 493, and 494 kJ/mol. Study results suggested a cross-protective effect of acid adaptation on thermal inactivation but no association between antimicrobial susceptibility and the ability of salmonellae to survive heat stress.

Thermal inactivation of pectin methylesterase, polygalacturonase, and peroxidase in tomato juice.

Thermal inactivation kinetics have been determined for pectin methylesterase (PME), polygalacturonase (PG), and peroxidase (POD) in tomato juice. Two parameters, the inactivation rate constant (k) at a reference temperature and the activation energy for inactivation (E(a)), were determined for each enzyme. For PME and PG, the k and E(a) values reported here do not agree with those in several previously published reports. These differences can be explained either by the differences in pH values used for inactivation determinations or by inadequacies in the heating methods used in some previous studies. POD showed simple first-order inactivation kinetics and was less thermally stable than either PME or PG. When different cultivars of tomatoes were evaluated, there was no difference in the thermal inactivation kinetics of these enzymes.

Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots.

A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.

Purification and partial characterization of three turnip (Brassicanapus L. var. esculenta D.C.) peroxidases.

Three turnip peroxidases (fractions C1, C2, and C3) were partially purified and characterized, to permit study of their feasibility for use in clinical and enzyme immunoassays. These fractions represented 20% of the initial activity, and fractions C1 and C2 were purified to homogeneity. The optimum pH was between 5.0 and 5.5, while optimum temperature ranged from 40 to 55 degrees C. The ABTS K(m) values for the two acidic fractions (C2 and C3) were 0.70 and 0.42 mM, respectively; about 5 times lower than that reported for the acidic commercial horseradish peroxidase (HRP). Fraction C3 had 4 times higher K(m) value than commercial cationic HRP. The molecular weights determined by SDS-PAGE ranged from 39.2 to 42.5 kDa. Activation energies for inactivation were 113 (C1), 130 (C2), and 172 kJ/mol (C3) which are higher or comparable to other peroxidase isoenzymes reported. Fractions C1 and C3 represent an alternative source of peroxidase because of their higher purification yield and specific activity, when compared to fraction C2.

Isolation and characterization of heat stable proteinases from Pseudomonas isolate AFT 21.

Pseudomonas strain AFT 21 produced three heat stable extracellular proteinases in milk and nutrient broth at 7 or 21 degrees C, but the proportions depended on medium and cultivation temperature. The three proteinases were EDTA- and o-phenanthroline-sensitive metalloenzymes and were not inhibited by N-ethylmaleimide or phosphoramidon. Proteinases I and II showed maximum activity at pH 7-7.5 and proteinase III at pH 8.5. All three enzymes showed maximum activity at 45-47.5 degrees C, but had relatively high (19-27% of maximum) activity at 4 degrees C. They were unstable at 55 degrees C in phosphate buffer, pH 6.6, or synthetic milk ultrafiltrate (SMUF) containing 12 mmol Ca2+, but were stabilized by short preheating at 100 degrees C. They were extremely heat stable in both phosphate buffer and SMUF, pH 6.6, at 70-150 degrees C. Their D-values at 140 degrees C were 69, 54 and 80 s respectively. The Z-values for Pseudomonas AFT 21 proteinase III in phosphate buffer and SMUF were 29.7 and 30.3 degrees C respectively; the corresponding activation energies for inactivation were 8.7 x 10(4) J mol-1 and 9.2 X 10(4) J mol-1.

Changes in the physicochemical characteristics of rabbit liver N-acetyltransferase during post-natal development

This study was undertaken to evaluate the characteristics of liver N-acetyltransferase, a drug conjugating enzyme, at different stages of development. This enzyme catalyzes the acetylation of isonicotinic acid hydrazide, various sulfonamides and other aromatic amines. Partially purified enzyme was prepared for these studies from the livers of 6–12-day-old, 3-week-old, and adult rabbits. One-week-old rabbits have less than 10% of the activity per g of liver found in mature animals. The relative activity increased to the adult level at about 4 weeks of age. Two distinguishing characteristics were noted in studies of enzyme from the youngest animals. These were: limiting K m for isonicotinic acid hydrazide, a kinetic parameter; and the activation energy for inactivation by heat, a physical characteristic. Enzyme from 3-week-old animals had the same limiting K m as that from adult animals. It is postulated that in addition to the different forms of N-acetyltransferase that exist in different tissues 1 there are different forms of the hepatic drug conjugating N-acetyltransferase that are specific for various developmental stages.

Thermal inactivation and reactivation of an enzyme in vivo. Pantothenate hydrolase of Pseudomonas fluorescens.

Thermal inactivation and reactivation of pantothenate hydrolase were studied in whole cells of Pseudomonas fluorescens. The enzyme is susceptible to thermal inactivation in whole cells at 37-40 degrees C, and is reactivated when the temperature is lowered again. Chloramphenicol does not prevent reactivation. The activation energy of enzyme inactivation in vivo is about 540kJ/mol. This activation energy is 220kJ/mol in vitro, but it is increased to 550-630kJ/mol by several metabolites, such as succinate, glyoxylate and oxalate. Generally, good carbon sources, causing rapid growth, protect the enzyme from thermal inactivation in vivo, and enable reactivation to occur at a fast rate. The enzyme is also inactivated below 35 degrees C, showing an activation energy of about 35kJ/mol. Good carbon sources prevent this inactivation as well, and cause slight reactivation. Glycine, although not utilized for growth, protects the enzyme well from this inactivation but not from inactivation at 37-40 degrees C, and prevents reactivation totally. From the activation energies of inactivation and the effects of the various carbon sources, it appears possible that changes in the concentrations of intracellular metabolites may be responsible for the changes in inactivation and reactivation.

Endothia parasitica Protease. Parameters Affecting Stability of the Rennin-like Enzyme

Endothia parasitica protease had maximum stability at pH 3.8 to 4.5. At 50C only 30% activity was lost after 30 minutes. At 24.1C, the enzyme was stable from pH 3.0 to 6.5 for 3 hours but was rapidly inactivated below pH 2.5 and above pH 7. The activation energy of inactivation, Ea, was 51,500 cal/mole at pH 5.5 to 6.5. Inactivation of the enzyme in this pH region resulted in decreased solubility of the protein but did not produce detectable ninhydrin-reactive groups. At pH 2.5, Ea was 14,100 cal/ mole and inactivation resulted in fragmentation of the molecule and the production of appreciable ninhydrin-reactive groups. Increase in ionic strength had a stabilizing effect on the enzyme at pH 6.5 but decreased stability of the enzyme at pH 2.5. The nature of the buffer affected stability of the enzyme (greater stability; Tris>acetate>phosphate). The effect of cations and anions on stability of the enzyme followed the order expected from previous data on the influence of ions on the general stability of proteins. Endothia parasitica protease was rapidly denatured by low concentrations of urea (2M) at pH 2.5 and 6.5, but was reasonably stable to 2M urea solutions at pH 3.5. There was no effect of 0.01M mercaptoethanol on stability of the enzyme at pH 2.5, 3.5, and 6.5.

D-Lacticodéshydrogénase de la levure anaérobie etude de l'inactivation irréversible par un chélateur

The kinetics of inactivation by Versene of d-lactate dehydrogenase from anaerobic yeast was studied. Inactivation is irreversible: a diminution of free Versene concentration down to 10 −14 M does not lead to any reactivation. The initial velocity of inactivation is proportional to Versene concentrations and to enzyme concentrations. The Michaelis constant (in the reaction d-lactate to pyruvate) of the partially inactivated enzyme is not modified. Activation energy of inactivation is low (⩽ 3kcal/mole). d-Lactate (but not l-) protects the enzyme from inactivation by decreasing the velocity of inactivation. Neither the acceptors nor flavine-adenine dinucleotide protect. Variation of initial inactivation velocity in relation to d-lactate concentration during the inactivation follows a hyperbolic kinetics: d-lactate concentration giving half protection is 7 · 10 −6 M, that is 300 times smaller than the Michaelis constant. Inactivation of d-lactate dehydrogenase by Versene is therefore due to the loss of a metal ion that is necessary for enzyme activity. A hypothesis is advanced that the fixation of the substrate takes place in the vicinity of the metal.