Suicide-peroxide inactivation of horseradish peroxidase in the presence of sodium n-dodecyl sulphate: a study of the enzyme deactivation kinetics

In the presence of the anionic surfactant sodium n-dodecyl sulphate (SDS), horseradish peroxidase (HRP) undergoes a deactivation process. Suicide inactivation of horseradish peroxidase by hydrogen peroxide(3 mM) was monitored by the absorbance change in product formation in the catalytic reaction cycle. The progress curve of the catalytic reaction cycle was obtained at 27degrees C and phosphate buffer 2.5 mM (pH = 7.0). The corresponding kinetic parameters i.e., intact enzyme activity (alpha i); the apparent rate constant of suicide inactivation by peroxide (ki); and the apparent rate constants of enzyme deactivation by surfactant (kd) were evaluated from the obtained kinetic equations. The experimental data are accounted for by the equations used in this investigation. Addition of SDS to the reaction mixture intensified the inactivation process. The deactivation ability of denaturant could be resolved from the observed inactivation effect of the suicide substrate by applying the proposed model. The results indicate that the deactivation and the inactivation processes are independent of each other.     

Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus

The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.     

Reusable ω-transaminase sol–gel catalyst for the preparation of amine enantiomers

Heterogeneous ω-transaminase sol–gel catalysts were prepared and characterized in terms of immobilization degree, loading capacity and catalytic behavior in the kinetic resolution of racemic 1-phenylethylamine (a model compound) with sodium pyruvate in phosphate buffer (pH 7.5). The catalyst obtained when ω-transaminase from Arthrobacter sp. was encapsulated from the aqueous solution of the enzyme, isopropyl alcohol and polyvinyl alcohol in the sol–gel matrices, consisting of the 1:5 mixture of tetramethoxysilane and methyltrialkoxysilane, proved to be optimal including the reuse and storage stabilities of the catalyst. The optimized immobilizate was shown to perform well in the kinetic resolution of four structurally different aromatic primary amines in aqueous DMSO (10, v/v-%). The enzyme preparation showed synthetic potential by enabling the catalyst reuse in five consecutive preparative scale kinetic resolutions using 100 mM 1-phenylethylamine in aqueous DMSO (10, v/v-%). It was typical to fresh catalyst preparations that the kinetic resolution tended to exceed 50% before the reaction stopped leaving the (S)-amine unreacted while thereafter in reuse the reactions stopped at 50% conversion as expectable to highly enantioselective reactions.     

Kinetic modelling of the thermal inactivation of an industrial β-galactosidase from Kluyveromyces fragilis

The thermal and operational inactivation of a commercial β-galactosidase from Kluyveromyces fragilis (Lactozym, from Novozymes) was studied in several buffered solutions usually employed to study the activity of this enzyme. Some previous experiments have been done to understand the effect of the composition of the buffers on the enzyme stability. Afterwards, data obtained at temperatures from 25 to 50 °C were fitted by several kinetic models. Discrimination among the kinetic models was performed considering the temperature as a constant or as a variable. When using a 50 mM phosphate buffer pH 7.5, first-order reaction model is able to fit inactivation data, while a more complex model, involving two consecutive first-order reactions, was chosen for the lacteous buffer BM, with and without lactose. In both cases, the final form of the enzyme was totally inactive. Both lactose and mercaptoethanol have proved to be stabilisers of the enzyme, increasing half-life four to five times or twice, respectively. The intermediate forms of the enzyme during the inactivation process proved to have an activity, which, surprisingly, was higher at higher temperatures when lactose was present.     

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.     

Effect of disruption by sonication under different conditions on the activity of glucose dehydrogenase from resting spores of Bacillus subtilis

The activity of glucose dehydrogenase present in resting spores of Bacillus subtilis varied strikingly with the conditions for disrupting the spores by sonic treatment, namely, the time and strength of sonication, and the type and pH of the solution used for suspending the spores. When the resting spores were sonicated for 30 min at a current of 1.45 A in 100 mM phosphate buffer in the range of pH 6.0 to 6.6 or in deionized water, the enzyme activity of the former suspension was approximately 10 times higher than that of the latter suspension. However, the enzyme activity of the latter was markedly stimulated in the presence of sodium chloride. The glucose dehydrogenase from resting spores disrupted in 100 mM phosphate buffer (pH 6.6) was a salt-independent, active enzyme with a molecular weight of about 120,000, whereas the enzyme from resting spores disrupted in deionized water was a salt-dependent, inactive one with a molecular weight of about 55,000. A high concentration of dipicolinic acid strongly inhibited activation by a salt of inactive glucose dehydrogenase from resting spores in deionized water, suggesting one of its several important roles in vivo.     

Kinetic properties of Helicobacter pylori urease compared with jack bean urease

The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.     

Optimization of the hide powder azure assay for quantitating the protease of Pseudomonas fluorescens

The extracellular protease of Pseudomonas fluorescens NC 3 was optimally active at 40°C in a reaction mixture containing: 50 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer (pH 6.6), 0.5 mM CaCl₂, and 25 mg hide powder azure in 5 ml total volume. Divalent cation chelators, i.e., EDTA, o-phenanthroline, citrate or phosphate, inhibited the enzyme. Protease production by P. fluorescens NC 3 was initiated during late-logarithmic-growth phase in a sodium caseinate medium and reached its maximum at the onset of the stationary phase.     

Mechanistic studies on carboxypeptidase A from goat pancreas: role of arginine residue at the active site

Chemical modification of carboxypeptidase Ag1 from goat pancreas with phenylglyoxal or ninhydrin led to a loss of enzymatic activity. The inactivation by phenylglyoxal in 200 mM N-ethylmorpholine, 200 mM sodium chloride buffer, pH 8.0, or in 300 mM borate buffer, pH 8.0, followed pseudo-first-order kinetics at all concentrations of the modifier. The reaction order with respect to phenylglyoxal was 1.68 and 0.81 in 200 mM N-ethylmorpholine, 200 mM NaCl buffer and 300 mM borate buffer, pH 8.0, respectively, indicating modification of single arginine residue per mole of enzyme. The kinetic data were supported by amino acid analysis of modified enzyme, which also showed the modification of single arginine residue per mole of the enzyme. The modified enzyme had an absorption maximum at 250 nm, and quantification of the increase in absorbance showed modification of single arginine residue. Modification of arginine residue was protected by beta-phenylpropionic acid, thus suggesting involvement of an arginine residue at or near the active site of the enzyme.     

Effect of several anions on the activity of mitochondrial malate dehydrogenase from pig heart

Mitochondrial malate dehydrogenase (mMDH) shows a complex dependence upon ionic environment that includes kinetic and structural effects. We measured mMDH activity in several buffers (phosphate, MOPS, and MES) at pH 6.5 and 7.5, and in the presence of a number of anions, at highly diluted enzyme concentrations where mMDH showed significant loss of activity. Under these conditions, mMDH activity shows a non-linear dependence on enzyme concentration, in agreement with the existence of a dimer–monomer equilibrium, where only the dimeric form is active. According to this hypothesis, the dissociation constant of mMDH dimer has been determined to be 5.4 nM in the MES buffer at pH 6.5. Either the presence of a small anion like phosphate, or an increase of the pH from 6.5 to 7.5 shifts the equilibrium in favor of the dimeric form with the two effects appearing to be additive. To extend the study, we analysed the effect of a number of anions on the mMDH activity in 50 mM MOPS buffer at pH 7.5. All the anions had a dual effect: at low concentrations, they increased the activity of mMDH, while at high concentrations, they inhibited it. A more accurate analysis of the data revealed that the activation capacity of all the anions tested was similar, although they differed in their inhibitory influence. To show these differences more clearly, the experiment was repeated in 50 mM phosphate buffer at pH 7.5, under conditions where almost all activations were due to the buffer. The analysis of the results obtained under these conditions revealed the following sequence of inhibition potency: phosphate相似文献   

Possibility of association and activation of glucose dehydrogenase during germination and outgrowth of Bacillus subtilis spores

Both a salt-dependent form and an active form of glucose dehydrogenase [EC 1.1.1.47] were isolated from germinated spores of Bacillus subtilis disrupted in deionized water and 100 mM phosphate buffer (pH 6.6), and most of the enzyme isolated from young vegetative cells was the active form regardless of the conditions for breakage by sonication. The molecular weight of the salt-dependent form of the enzyme was about 55,000 and that of the active form was about 120,000. From the above results and the results on the glucose dehydrogenase extracted from resting spores disrupted in deionized water and 100 mM phosphate buffer (pH 6.6) reported in a previous paper, we propose that glucose dehydrogenase is present in resting spores as a monomeric form and is activated with association in vivo during germination and outgrowth.     

Developing procedures for the large-scale purification of human serum butyrylcholinesterase

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2× LD₅₀ of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05–0.2 M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.     

Influence of salts on the activity and the subunit structure of ornithine decarboxylase from rat liver

The influence of salts on the subunit structure and the kinetics of purified rat ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) was examined. Salts were found to cause subunit dissociation of the enzyme, producing the monomeric form of molecular weight 55 000 in the presence of 0.25 M NaCl/10 mM sodium phosphate buffer (pH 7.0): the molecular weight was estimated to be 150 000 in 10 mM and 250 000 in 1 mM sodium phosphate buffer. Inclusion of NaCl in kinetic assays of rat ornithine decarboxylase had little effect on maximal velocity. However, the Km value for L-ornithine was dramatically increased with increasing sodium chloride concentration: the presence of 0.25 M NaCl resulted in a 10-fold increase of the Km. Thus, the presence of salts caused dramatic changes both in the subunit structure and in the catalytic property of the enzyme, although a direct correlation between both the changes was not evidenced.     

Suicide-peroxide inactivation of microperoxidase-11: a kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H2O2. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H2O2] > [AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, alphai, and the apparent inactivation rate constant, ki, were obtained as 0.282 +/- 0.006 min(-1) and 0.497 +/- 0.013(-1) min at [H2O2] = 1.0 mM, 27 degrees C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).     

Study of the Stability Of Vaccinium myrtillus Peroxidase in Reverse Micellar Systems

The stability of a cationic peroxidase isolated and purified from a cell suspension of Vaccinium myrtillus, microencapsulated in reverse micelles of sodium dioctylsulfosuccinate (AOT) was evaluated. By using a central composite design (CCD), some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH and buffer molarity, were analysed. The response surface curves showed that 50 mM AOT, 500 mM water, 80 mM buffer and pH 7.6 were the best conditions for enzyme stability. The effect of carbohydrates and polyols on enzyme stability was also evaluated. At 20 mM, carbohydrates like arabinose, and trehalose increased the enzymatic stability by a factor of 4.4 and 2.3, respectively, but melezitose had no effect. From the three polyols tested, inositol and sorbitol increased the peroxidase stability by a factor of 3.8 and 1.8, respectively, while mannitol had no effect.     

Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase: kinetics and operational stability

Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase was studied. The K(m)-value for lactose, obtained by a traditional enzymatic assay, was 0.066 mM at pH 6.4 and 38 degrees C. The effect of oxygen on the enzymatic rate of reaction as well as the operational stability of the enzyme was studied by performing reactions at constant pH and temperature in a stirred tank reactor. Catalase was included in all reactions to avoid inhibition and deactivation of the oxidase by hydrogen peroxide. At pH 6.4 and 38 degrees C, K(m) for oxygen was 0.97 mM, while the catalytical rate constant, k(cat), was 94 s(-1). Furthermore, we found that the operational stability of the oxidase was dependent on the type of base used for neutralization of the acid produced. Thus, when 2 M NaOH was used for neutralization of a reaction medium containing 50 mM phosphate buffer, significant deactivation of the oxidase was observed. Also, we found that the oxidase was protected against deactivation by base at high lactose concentrations. A simple model is proposed to explain the obtained results.     

Study of the Stability Of Vaccinium myrtillus Peroxidase in Reverse Micellar Systems

The stability of a cationic peroxidase isolated and purified from a cell suspension of Vaccinium myrtillus , microencapsulated in reverse micelles of sodium dioctylsulfosuccinate (AOT) was evaluated. By using a central composite design (CCD), some relevant parameters for the enzymatic activity, such as surfactant and water concentration, pH and buffer molarity, were analysed. The response surface curves showed that 50 mM AOT, 500 mM water, 80 mM buffer and pH 7.6 were the best conditions for enzyme stability. The effect of carbohydrates and polyols on enzyme stability was also evaluated. At 20 mM, carbohydrates like arabinose, and trehalose increased the enzymatic stability by a factor of 4.4 and 2.3, respectively, but melezitose had no effect. From the three polyols tested, inositol and sorbitol increased the peroxidase stability by a factor of 3.8 and 1.8, respectively, while mannitol had no effect.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.     

Selective removal of histone H1 from nucleosomes at low ionic strength

The method for removal of histone H 1 from chromatin by treatment with ion-exchange resin AG 50 WX 2 in the presence of 100 mM NaCl and 50 mM phosphate buffer (Thoma and Koller, 1977, Cell, 12, 101–107) results in production not only of H1-depleted chromatin but also free DNA. We have now modified this procedure so that the nucleosome is treated with the cation exchange resin in two steps, first in 50 mM sodium phosphate buffer and then in 50 mM sodium phosphate and 50 mM NaCl whereby histone H 1 is selectively removed without a release of free DNA at low resin concentrations.Abbreviations NaP Sodium phosphate buffer of molarities and pH as stated in the text - SDS Sodium dodecyl sulfate     

Collagen degradation by superoxide anion in pulse and gamma radiolysis

Delipidated collagen fibrils reconstituted from acid-soluble calf skin collagen, suspended in 50 mM phosphate buffer, pH 7.4, containing 100 mM sodium formate, were submitted to pulse radiolysis in Febetron devices or to gamma radiolysis in a 60Co irradiator. A collagen degradation process was found. The kinetics of this degradation was followed by evaluation of the amount of 4-hydroxyproline present in the small peptides liberated during the irradiation period. The yield of 4-hydroxyproline small peptides was low (0.1 mol/100 eV for an initial collagen concentration 3.2 microM). It increased linearly with the dose of irradiation and the concentration of collagen in suspension. The kinetic competition between O2-. dismutation and O2-. reaction with collagen was studied by pulse radiolysis at several concentrations of collagen. A value of the kinetic constant of k(O2-. + collagen) = 4.8 . 10(6) mol-1.l.s-1 was determined.