Activation Energy Of Thermal Inactivation Research Articles

Engineering enhanced thermostability into the Geobacillus pallidus nitrile hydratase.

Nitrile hydratases (NHases) are important biocatalysts for the enzymatic conversion of nitriles to industrially-important amides such as acrylamide and nicotinamide. Although thermostability in this enzyme class is generally low, there is not sufficient understanding of its basis for rational enzyme design. The gene expressing the Co-type NHase from the moderate thermophile, Geobacillus pallidus RAPc8 (NRRL B-59396), was subjected to random mutagenesis. Four mutants were selected that were 3 to 15-fold more thermostable than the wild-type NHase, resulting in a 3.4–7.6 ​kJ/mol increase in the activation energy of thermal inactivation at 63 ​°C. High resolution X-ray crystal structures (1.15–1.80 ​Å) were obtained of the wild-type and four mutant enzymes. Mutant 9E, with a resolution of 1.15 ​Å, is the highest resolution crystal structure obtained for a nitrile hydratase to date. Structural comparisons between the wild-type and mutant enzymes illustrated the importance of salt bridges and hydrogen bonds in enhancing NHase thermostability. These additional interactions variously improved thermostability by increased intra- and inter-subunit interactions, preventing cooperative unfolding of α-helices and stabilising loop regions. Some hydrogen bonds were mediated via a water molecule, specifically highlighting the significance of structured water molecules in protein thermostability. Although knowledge of the mutant structures makes it possible to rationalize their behaviour, it would have been challenging to predict in advance that these mutants would be stabilising.

Improved thermal stability of phytase from Yersinia intermedia by physical adsorption immobilization on amino-multiwalled carbon nanotubes.

Phytase is used in poultry diets to hydrolyze and release of phytate-bound phosphorus. Immobilization on nanomaterials optimizes enzyme's thermal stability and reusability. This study aimed to immobilize the recombinant phytase from Yersinia intermedia on the surface of amino-multi-walled carbon nanotubes (amino-MWCNTs) by physical adsorption. For this, zeta potential measurement, FTIR spectroscopic analysis, scanning electron microscope (SEM), kinetic as well as thermodynamic parameters were used to characterize immobilized phytase on amino-MWCNTs. According to results, the optimum temperature of the immobilized phytase increased from 50 to 70°C and also thermal and pH stability improved considerably. Moreover, immobilization led to an increase in the value of Km and kcat from 0.13 to 0.33mM and 2220 to 2776s-1, respectively. In addition, the changes in activation energy of thermal inactivation (ΔE#a (D)), the free energy of thermal inactivation (ΔG#D) and the enthalpy of thermal inactivation (ΔH#D) for immobilized phytase increased by +11.05, +24.7 and +11.4kj/mole, respectively, while the value of the change in the entropy of thermal inactivation (ΔS#D) decreased by -0.04 kj/mole.K. Overall, our results showed that adsorption immobilization of phytase on amino-MWCNTs increases thermal, pH and storage stability as well as some of kinetic parameters.

Catalytic power of enzymes decreases with temperature: New insights for understanding soil C cycling and microbial ecology under warming.

Most current models of soil C dynamics predict that climate warming will accelerate soil C mineralization, resulting in a long-term CO2 release and positive feedback to global warming. However, ecosystem warming experiments show that CO2 loss from warmed soils declines to control levels within a few years. Here, we explore the temperature dependence of enzymatic conversion of polymerized soil organic C (SOC) into assimilable compounds, which is presumed the rate-limiting step of SOC mineralization. Combining literature review, modelling and enzyme assays, we studied the effect of temperature on activity of enzymes considering their thermal inactivation and catalytic activity. We defined the catalytic power of enzymes (Epower ) as the cumulative amount of degraded substrate by one unit of enzyme until its complete inactivation. We show a universal pattern of enzyme's thermodynamic properties: activation energy of catalytic activity (EAcat )<activation energy of thermal inactivation (EAinact ). By investing in stable enzymes (high EAinact ) having high catalytic activity (low EAcat ), microorganisms may maximize the Epower of their enzymes. The counterpart of such EAs' hierarchical pattern is the higher relative temperature sensitivity of enzyme inactivation than catalysis, resulting in a reduction in Epower under warming. Our findings could explain the decrease with temperature in soil enzyme pools, microbial biomass (MB) and carbon use efficiency (CUE) reported in some warming experiments and studies monitoring the seasonal variation in soil enzymes. They also suggest that a decrease in soil enzyme pools due to their faster inactivation under warming contributes to the observed attenuation of warming effect on soil C mineralization. This testable theory predicts that the ultimate response of SOC degradation to warming can be positive or negative depending on the relative temperature response of Epower and microbial production of enzymes.

Kinetics of Denaturation and Effects of Surfactants and Polyethylene Glycol on Soybean Esterase (Glycine max L) Stability

AbstractThe objective of this work was to evaluate the kinetics and thermodynamics parameters and the effects of anionic, cationic and nonionic surfactants and polyethylene glycol on the activity and stability of a crude esterase extracted from soybeans (Glycine max L.). The activation energy for thermal inactivation was calculated from the Arrhenius plot was found to be 59.4 kJ mol−1 and the ΔH* 56.82 kJ mol−1 at 40 °C, which was the optimum temperature for enzyme activity. The ΔS* and ΔG* of the enzyme were found to be 61.67 kJ mol−1 and 15.50 J mol−1 K−1, respectively, at the optimum temperature. The activity was only enhanced by the cationic surfactants cetyltrimethylammonium bromide and tetradecylmethylammonium bromide at a concentration of 3.0 mM. The anionic surfactant showed a positive effect on enzyme activity at the concentrations of 1.5 and 3.0 mM. Aqueous PEG (polyethylene glycols) solutions activated the esterase, and maximum activation (170 %) occurred with the addition of 6 kDa PEG. PEG with molecular weights of 0.4 and 10 kDa enhanced enzyme stability at 40 °C.

Characteristics and Applicability of Phytase of the Yeast Pichia anomala in Synthesizing Haloperoxidase.

The phytase of the yeast Pichia anomala is a histidine acid phosphatase based on signature sequences and catalytic amino acids identified by site-directed mutagenesis. Among modulators, N-bromosuccinimide and butanedione inhibit phytase, while Ca(2+) and Ni(2+) stimulate slightly. Vanadate exhibits competitive inhibition of phytase, making it bifunctional to act as haloperoxidase. Molecular docking supports vanadate to share its binding site with phytate. The T 1/2, activation energy (E a ), temperature quotient (Q 10), activation energy of thermal inactivation (Ed), and enthalpy (ΔH d (0) ) of the enzyme are 4.0 min (80 °C), 27.72 kJ mol(-1), 2.1, 410.62 kJ mol(-1), and ∼407.8 kJ mol(-1) (65-80 °C), respectively. The free energy of the process (ΔG d (o) ) increases from 49.56 to 71.58 kJ mol(-1) with rise in temperature, while entropy of inactivation (ΔS d (0) ) remains constant at ∼1.36 kJ mol(-1) K(-1). The supplementation of whole wheat dough with rPPHY resulted in 72.5 % reduction in phytic acid content of bread. These characteristics confirm that the phytase has adequate thermostability for its applicability as a food and feed additive.

A differential behavior of α-amylase, in terms of catalytic activity and thermal stability, in response to higher concentration CaCl2

A differential relationship was observed between thermal stability and catalytic activity of α-amylase in the presence of different concentrations of CaCl2. The enzyme displays optimum catalytic activity in the presence of 1.0–2.0mM CaCl2. Further addition of CaCl2 leads to inhibition of the enzyme, however, at the same time the enzyme gains an additional resistance against thermal denaturation. It was evident that the enzyme is thermodynamically more stable (compared to the active enzyme) in the presence of inhibitory concentration of CaCl2. For example, the thermal transition temperature (Tm) of optimally active α-amylase was found to be 64±1°C, whereas, for the less active enzyme (in the presence 10mM CaCl2) the value was determined to be 71±1°C. Similarly, the activation energy of thermal inactivation (Ea) was found to be 228±12kJ/mol and 291±15kJ/mol for the optimally active enzyme and the enzyme in the presence of 10mM CaCl2, respectively. Biophysical analysis of different states of the enzymes in response to variable calcium level indicates no significant change in the secondary structure in response to different concentration of CaCl2, however the less active but thermodynamically stable enzyme (in the presence of higher concentration of CaCl2) was shown to have relatively more compact structure. The results suggest that the enzyme has separate catalytic and structure stabilizing domains and they significantly vary in their functional attributes in response to calcium level.

Thermal inactivation of alkali phosphatases under various conditions

The thermal inactivation of alkali phosphatases from bacteria Escherichia coli (ECAP), bovine intestines (bovine IAP), and chicken intestines (chicken IAP) was studied in different buffer solutions and in the solid state. The conclusion was made that these enzymes had maximum stability in the solid state, and, in a carbonate buffer solution, their activity decreased most rapidly. It was found that the bacterial enzyme was more stable than animal phosphatases. It was noted that, for ECAP, four intermediate stages preceded the loss of enzyme activity, and, for bovine and chicken IAPs, three intermediate stages were observed. The activation energy of thermal inactivation of ECAP over the range 25–70°C was determined to be 80 kJ/mol; it corresponded to the dissociation of active dimers into inactive monomers. Higher activation energies (∼200 kJ/mol) observed at the initial stage of thermal inactivation of animal phosphatases resulted from the simultaneous loss of enzyme activity caused by dimer dissociation and denaturation. It was shown that the activation energy of denaturation of monomeric animal alkali phosphatases ranged from 330 to 380 kJ/mol depending on buffer media. It was concluded that the inactivation of solid samples of alkali phosphatases at 95°C was accompanied by an about twofold decrease in the content of β structures in protein molecules.

Isolation and Characterization of Superoxide Dismutase from Wheat Seedlings

Two major superoxide dismutases (SODs; SODs I and II) were found in the crude enzyme extract of wheat seedlings after heat treatment, ammonium sulfate fractionation, anionic exchange chromatography, and gel permeation chromatography. The purification fold for SODs I and II were 154 and 98, and the yields were 11 and 2.4%, respectively. SOD I was further characterized. It was found that SOD I from wheat seedlings is a homodimer, with a subunit molecular mass of 23 kDa. Isoelectric focusing electrophoresis (IEF) and zymogram staining results indicated that the isoelectric point of SOD I is 3.95. It belongs to the MnSOD category due to the fact that it was insensitive to KCN or hydrogen peroxide inhibitor. This MnSOD from wheat seedlings was found to be stable over pH 7-9, with an optimum pH of 8, but was sensitive to extreme pH, particularly to acidic pH. It was stable over a wide range of temperatures (5-50 degrees C). Thermal inactivation of wheat seedling MnSOD followed first-order reaction kinetics, and the temperature dependence of rate constants was in agreement with the Arrhenius equation. The activation energy for thermal inactivation of wheat seedling MnSOD in the temperature range of 50-70 degrees C was found to be 150 kJ/mol. HgCl2 and SDS at a concentration of 1.0 mM significantly inhibited enzyme activity. Chemical modification agents, including diethyl pyrocarbonate (2.5 mM) and Woodward's reagent K (50 mM), significantly inhibited the activity of wheat seedling SOD, implying that imidazole groups from histidine and carboxyl groups from aspartic acid and glutamic acid are probably located at or near the active site of the enzyme.

Expression of the melA gene from Rhizobium etli CFN42 in Escherichia coli and characterization of the encoded tyrosinase

The gene melA from the nitrogen-fixing bacterium Rhizobium etli CFN42 was amplified using PCR, cloned in the expression vector pTtrc99A to obtain plasmid pTrc melA, and transformed into E. coli strain W3110. The resulting recombinant strain W3110/pTrc melA synthesized a dark pigment when growing in solid or liquid media containing l-tyrosine and copper. This pigment was identified as melanin by comparing it with analytical grade melanin using a spectrophotometric assay. Melanin was synthesized when recombinant E. coli cells were incubated at 30 °C; however, at 37 °C significantly less polymer was produced. The recombinant tyrosinase expressed intracellularly in E. coli was purified 40-fold with a 25% yield from a cell extract by ammonium sulfate precipitation and ion exchange chromatography. With the partially purified tyrosinase, the K m for l-dopa and l-tyrosine were determined as 2.44 and 0.19 mM, respectively. Temperature and pH for maximum activity were 50 °C and 6.5–7.5, respectively. Activation energy for thermal inactivation (50.77 kJ/mol; using l-dopa as substrate at pH 7) and half-life values indicate a higher thermal stability of R. etli tyrosinase in comparison with mushroom tyrosinase. Interestingly, for a bacterial tyrosinase, MelA showed an unusually higher activity for l-tyrosine than for l-dopa.

Characterization of tyrosinase for the treatment of aqueous phenols

Mushroom tyrosinase (polyphenol oxidase, EC 1.14.18.1) was investigated as an alternative to peroxidase enzymes for the catalytic removal of phenolic compounds from wastewaters. Maximum catalytic activity was observed at pH 7 and more than 50% of optimum activity was observed at pHs ranging between 5 and 8. Tyrosinase was unstable under acidic conditions and at elevated temperatures. The activation energy for thermal inactivation of tyrosinase at pH 7 was determined to be 1.85 kJ mol −1 using l-tyrosine as a substrate. Phenol was successfully transformed by tyrosinase over wide ranges of pH 5–8 and initial phenol concentration (0.5–10 mM, 47–940 mg/l). Several chlorinated phenols were also successfully transformed. Polyethylene glycol and chitosan did not protect tyrosinase from inactivation during the treatment of phenol; however, chitosan induced the precipitation of reaction products arising from phenol transformation.

A comparison of thermal characteristics and kinetic parameters of trehalases from a thermophilic and a mesophilic fungus

Trehalases from a thermophilic fungus Thermomyces lanuginosus ( M r 145 kDa) and a mesophilic fungus Neurospora crassa ( M r 437 kDa) were purified to compare their thermal characteristics and kinetic constants. Both trehalases were maximally active at 50°C, had an acidic pH optimum and were glycoproteins (20% and 43%, w/w, carbohydrate content for T. lanuginosus and N. crassa, respectively). At their temperature optimum, their K m was similar (0.57 and 0.52 mM trehalose, for T. lanuginosus and N. crassa, respectively) but the V max of N. crassa enzyme was nine times higher than of T. lanuginosus enzyme. The catalytic efficiency, k cat / K m , for N. crassa trehalase was one order of magnitude higher (6.2×10 6 M −1 s −1) than of T. lanuginosus trehalase (4×10 5 M −1 s −1). At their T opt (50°C), trehalase from both sources exhibited similar thermostability ( t 1/2 >6 h). The energy of activation, E a , for T. lanuginosus trehalase was 15.12 kcal mol −1 and for N. crassa trehalase it was 9.62 kcal mol −1. The activation energy for thermal inactivation for the N. crassa enzyme (92 kcal mol −1) was two-fold higher than for the T. lanuginosus enzyme (46 kcal mol −1). The present study shows that the trehalase of N. crassa is not only more stable but also a better catalyst than the T. lanuginosus enzyme.

PARTIAL PURIFICATION AND CHARACTERIZATION OF SWEET CORN GERM LIPOXYGENASE

Lipoxygenase (LPO) extracted from the germ fraction of sweet corn (Zea mays L. var. Jubilee) was purified by acetone powder preparation ; extraction with 0.1 M Tris-HCl, pH 8 ; 40-60% fractionation with ammonium sulfate ; and conventional column chromatography on Sephacryl S-300 HR and Fast Protein Liquid Chromatography (FPLC) on a Mono Q column. A 124-fold purification was achieved with 26.3% recovery. Further purification was achieved by FPLC chromatofocusing on a Mono P column and size exclusion chromatography on Superose-12, with a resultant 436-fold purification and 16. 8% recovery. The apparent molecular weight and isoelectric point (pI) determined by FPLC on Superose-12 and Mono P columns were 90,500 and 5.06, respectively. Lipoxygenase-catalyzed formation of conjugated dienes was inhibited by both synthetic (BHA, BHT) and natural phenolic antioxidants (quercetin, chlorogenic acid) at a concentration of0.2 mM with 57.2, 16.3, 61.4 and 32.3 inhibition, respectively. The activation energy for thermal inactivation of sweet corn germ lipoxygenase from the ammonium sulfate preparation at 55- 70C was 56.3 kcal/mol.

Thermal inactivation kinetics of penicillin G acylase obtained from a mutant derivative of Escherichia coli ATCC 11105.

Thermal inactivation kinetics of native and glutaraldehyde cross-linked forms of penicillin G acylase obtained from a mutant derivative of Escherichia coli ATCC 11105 were studied. Apparent activation energies for thermal inactivation of both native and cross-linked forms of enzyme were calculated to be [57.71 +/- 8.46] and [67.11 +/- 13.83] kcal mol-1 respectively. This slight increase in activation energy suggested that glutaraldehyde cross-linking did not markedly protect against thermal activation. Cross-linked enzyme did, however, have a significantly improved half-life at temperatures between 40 degrees C and 50 degrees C.

Potentiation of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase by photodynamic treatment. A possible model for the synergistic interaction between photodynamic therapy and hyperthermia

Thermal inactivation of glyceraldehyde-3-phosphate dehydrogenase appeared to be caused by a conformational mechanism, without involvement of covalent reactions. On the other hand, photodynamic inactivation of the enzyme (induced by illumination in the presence of Photofrin II) was caused by photo-oxidation of the essential thiol group in the active centre. A short photodynamic treatment of the enzyme, leading to only a limited inactivation, caused a pronounced potentiation of subsequent thermal inactivation, as measured over the temperature range 40-50 degrees C. Analysis of the experimental results according to the Arrhenius equation revealed that both the activation energy of thermal inactivation and the frequency factor (the proportionality constant) were significantly decreased by the preceding photodynamic treatment. The experimental results indicate a mechanism in which limited photodynamic treatment induced a conformational change of the protein molecule. This conformational change did not contribute to photodynamic enzyme inhibition, but was responsible for the decreased frequency factor and activation energy of subsequent thermal inactivation of the enzyme. The opposing effects of decreased activation energy and decreased frequency factor resulted in potentiation of thermal inactivation of the enzyme over the temperature range 40-50 degrees C. With other proteins, different results were obtained. With amylase the combined photodynamic and thermal effects were not synergistic, but additive, and photodynamic treatment had no effect on the frequency factor and the activation energy of thermal inactivation. With respect to myoglobin denaturation, the photodynamic and thermal effects were antagonistic over the whole practically applicable temperature range. Limited photodynamic treatment protected the protein against heat-induced precipitation, concomitantly increasing both the frequency factor and the activation energy of the process. These results offer a model for one of the possible mechanisms of synergistic interaction between photodynamic therapy and hyperthermia in cancer treatment.

Purification and characterization of Paecilomyces lilacinus dextranase

Two dextranase isoenzymes [endo-(1,6)-α- d-glucan-6-glucanohydrolase, EC 3.2.1.11] have been isolated from a crude enzyme powder prepared from the culture supernatant of Paecilomyces lilacinus. Purification was achieved by means of a two-stage ion-exchange chromatography on DEAE-cellulose. Dextranase I was recovered with a 35.3-fold increase in specific activity and a yield of 16%; dextranase II was purified 19-fold with a yield of 4%. The characteristics of the isoenzymes were very similar; both exhibited maximum hydrolytic activity at pH 4.5 and 55°C. Activation energies for thermal inactivation were 402 and 330 kJ mol −1 for dextranase I and II, respectively. The dextranases were not inhibited by EDTA or N-ethylmaleimide.

Studies on inactivation of the semi-alkaline proteinase from Aspergillus melleus.

The thermal inactivationoof the semi-alkaline proteinase from Aspergillus melleus, proceeded following the first order kinetics. The activation energy for the thermal inactivation was calculated to be 53 kcal per mol. Autolysis takes place directly proportional with the inactivation and the enzyme was degraded into oligopeptide and or amino acid. It was found that calcium ion, ammonium sulfate, glycerol and saccharose have a stabilizing effect on the semi-alkaline proteinase against the thermal inactivation. In the enzyme, 4-6 mol of calcium ion per mol of protein were found to bound, and by the loss of calcium ion, the enzyme became extremely unstable. In the presence of denaturing agents such as urea, guanidine-HCl and alcohols, the inactivation of the enzyme was accelerated and proceeded with first order kinetics. Activation energy for thermal inactivation decreased in the presence of urea and guanidine-HCl, but unchanged in the presence of alcohols. From these results, it was concluded that thermal inactivation of the semi-alkaline proteinase proceeded in two steps, that is, denaturation with conformational change and followed degradation with the residual proteinase. The rate limiting step was the denaturation.

Thermal inactivation of pantothenase from Pseudomonas fluorescens

Protection of pantothenase (pantothenate amidohydrolase, EC 3.5.1.22) against thermal inactivation by ligands was studied. The protective properties of 162 different small-molecule compounds were tested to find the most effective compounds. The best protective results were obtained with oxamate, 3-oxoglutarate, 2-oxomalonate, oxalate and oxaloacetate, in that order. The protection constants (π) of certain metabolic intermediates were estimated using differential thermal inactivation. Generally they were observed to be lower than the corresponding inhibition constants. The ΔH of the oxalate binding derived from π values was −165 kJ/mol. Normally, the activation energy of thermal inactivation of pantothenase in the absence of protective compounds is 220 kJ/mol, and the protective ligands enhance the measured activation energies of inactivation.

Conformations of lysine-sensitive aspartokinase

1. 1.|The technique of differential thermal and proteolytic inactivation has been employed as a conformational probe for the lysine-sensitive aspartokinase (EC 2.7.2.4) of Escherichia coli B. 2. 2.| l-Amino acid inhibitors of this enzyme each induce a characteristic enzyme conformation. This is evidenced by rates of thermal and proteolytic inactivation and Arrhenius activation energies for thermal inactivation which are characteristic of the amino acid present. 3. 3.|Phenylalanine and leucine binding are mutually exclusive as evidenced by competitive behavior in thermal inactivation experiments, suggesting a hydrophobic amino acid binding site with broad specificity. 4. 4.|The phenylalanine-dependent conformation and the leucine-dependent conformation differ considerably. In comparison with the native enzyme, the former is more labile to proteolysis by trypsin whereas the latter is more stable. First-order rate constants for thermal inactivation of the phenylalanine- and leucine-dependent conformations are, respectively, about one-half and one-tenth that of the native enzyme. 5. 5.|Items 3 and 4 taken together suggest that the conformations are ligand induced and do not arise via ligand stabilization of spontaneously arising conformers.

Activation energy for thermal inactivation and Km of carbonic anhydrase from the cotton plant

A low-activation energy (35 000 cal.) for thermal inactivation and a large K m value (11·10 −3 moles) for substrate affinity were found from carbonic anhydrase of the cotton plant, which was reported to be heat-tolerant but low in the rate of CO 2-fixation.

Initial kinetics of degradation of MS2 ribonucleic acid by ribonuclease, heat and alkali and the presence of configurational restraints in this ribonucleic acid

The initial kinetics of degradation of MS2 RNA by ribonuclease, heat and alkali were followed by the exponential decline in RNA infectivity. The turnover number of pancreatic ribonuclease is independent of RNA concentration between 10 11 and 10 15 RNA molecules per ml. Inactivation at high pH is almost proportional to the hydroxyl ion concentration between pH 11 and 12.3. The activation energy for thermal inactivation at neutral pH is 22 kcal./mole. MS2 RNA possesses no covalent configurational restraints, as shown by the correspondence between the decline in infectivity produced by ribonuclease and the production of RNA fragments, by the coincident sedimentation of the RNA infective material and the bulk RNA under a variety of conditions, and by the presence of only one component when the RNA is sedimented under denaturing conditions. The RNA chain does form intramolecular hydrogen bonds, which under certain conditions give rise to a multiplicity of discrete, infective components.