Kinetic analysis for suicide-substrate inactivation of microperoxidase-11: A modified model for bisubstrate enzymes in the presence of reversible inhibitors

Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H₂O₂. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism.The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor.Kinetic parameters of inactivation including the initial activity of MP-11, α₀, the apparent inactivation rate constant, k_i and the apparent inhibition rate constant for cysteine, k_I were obtained 0.282 ± 0.006 min^?1, 0.497 ± 0.013 min^?1 and 1.374 ± 0.007 min^?1 at [H₂O₂] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation.     

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.     

Bacteriophage phi11 lysin: Physicochemical characterization and comparison with phage phi80α lysin

Phage lytic enzymes are promising antimicrobial agents. Lysins of phages phi11 (LysPhi11) and phi80α (LysPhi80α) can lyse (destroy) cells of antibiotic-resistant strains of Staphylococcus aureus. Stability of enzymes is one of the parameters making their practical use possible. The objectives of the study were to investigate the stability of lysins of phages phi11 and phi80α in storage and functioning conditions, to identify optimum storage conditions and causes of inactivation. Stability of the recombinant LysPhi11 and LysPhi80α was studied using turbidimetry. CD-spectroscopy, dynamic light scattering, and electrophoresis were used to identify causes of inactivation. At 37 °C, pH 7.5 and concentration of NaCl not higher than 150 mM, LysPhi11 molecules contain a high percentage of random coils (43%). However, in spite of this the enzyme has high activity (0.4–0.8 OD_600 nm s⁻¹ mg⁻¹). In storage conditions (4 °C and 22 °C, pH 6.0–9.0, 10–500 mM NaCl) LysPhi11 is inactivated by a monomolecular mechanism. The optimum storage conditions for LysPhi11 (4 °C, pH 6.0–7.5, 10 mM NaCl) were selected under which the time of the enzyme half-inactivation is 120–160 days. LysPhi80α stability is insufficient: at 37 °C the enzyme loses half of its activity almost immediately; at 4 °C and 22 °C the time of half-inactivation of LysPhi80α varies in the range from several hours to 3 days. Despite the common properties in the manifestation of antistaphylococcal activity the kinetic behavior of the enzymes is different. LysPhi11 is a more promising candidate to be used as an antimicrobial agent.     

Effect of heat treatment on lactoperoxidase activity in caprine milk

Using isothermal conditions, inactivation of lactoperoxidase (LPO) in caprine milk was studied in a temperature range of 69–73 °C. In order to evaluate the effect of temperature on the reaction rate, the Arrhenius and thermal death time models were used for data analysis. Thermal inactivation of LPO can be accurately described by a first-order kinetic model, as indicated by the relationships obtained by plotting the retention values as a function of treatment time on a semi-logarithmic scale and confirmed by the high R²-values obtained. D- and k-values decreased and increased, respectively with increasing temperature, indicating a more rapid LPO inactivation at higher temperatures. The corresponding Z- and E_a-values calculated from the slope of the semi-logarithmic plots of D and k as a function of temperature were 9.45 °C and 225.98 kJ/mol, respectively.     

Characterization,purification, and temperature/pressure stability of polyphenol oxidase extracted from plums (Prunus domestica)

In this study, polyphenol oxidase (PPO) was extracted from Prunus domestica and partially purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and ion exchange chromatography. The final purification step revealed a 32.81-fold purification, and the molecular mass was estimated to be 65 kDa by SDS-PAGE. The purified PPO showed enzymatic activity mainly toward five substrates, namely catechol, catechin, 4-methyl catechol, chlorogenic acid, and L-3,4-dihydroxyphenylalanine, whereas it showed no activity toward caffeic acid, ferulic acid, p-coumaric acid, p-cresol, and l-tyrosine. The optimum pH and temperature values were 6.0 and 25 °C, respectively. The enzyme showed high stability in the pH range of 5.0–7.0 and in the temperature range of 25–65 °C. The most effective inhibitors of this enzyme were found to be ascorbic acid and l-cysteine. The thermal inactivation followed a first-order kinetic model, with activation energy of E_a 150.46 ± 1.29 kJ/mol. PPO extracted from plum showed stability at high pressure, with enzyme activation at 500 MPa.     

Efficient production of S-(+)-2-chlorophenylglycine by immobilized penicillin G acylase in a recirculating packed bed reactor

(S)-(+)-2-Chlorophenylglycine 1 is an important intermediate in the synthesis of Clopidogrel. A recirculating packed bed reactor (RPBR) was constructed for efficient production of (S)-1 by kinetic resolution of racemic N-phenylacetyl-2- chlorophenylglycine 2 using immobilized penicillin G acylase (PGA). The immobilized PGA exhibited maximum activity at 50 °C and pH 8.0 with (R,S)-2 as substrate. The kinetic constants (K_m and v_max) of immobilized PGA were calculated to be 20.61 mM and 83.2 mM/min/g, respectively. The substrate displayed inhibitory effect on immobilized PGA with inhibition constant of 221.23 mM. The immobilized PGA showed a strict enantiospecificity for substrate at different temperature, pH and substrate concentration examined. The performance and productivity of RPBR were evaluated by several critical parameters, including immobilized PGA load, substrate feeding rate, height to diameter ratio and so on. The kinetic resolution process shows higher initial reaction rate and conversion by recycling 100 mL of substrate solution (80 mM) through RPBRs packed with 6.0 g immobilized PGA with a feeding rate of 1.5 mL/min while the H/D ratio was 4.0. The immobilized PGA-catalyzed kinetic resolution of (R,S)-2 was successfully operated in the RPBR for 60 batches, with an average productivity of 1.2 g/L/h for (S)-1 in high optical purity (>97% enantiomeric excess) in semi-continuous operation. The residual (R)-2 can be easily racemized and then used as substrate.     

Effects of boldine on tyrosinase: Inhibition kinetics and computational simulation

Boldine is one of the most potent natural antioxidants and displays some important pharmacological activities, such as cytoprotective and anti-inflammatory activities. Based on its antioxidant properties, we studied the effects of boldine on l-DOPA oxidation by evaluating the inhibitory kinetics and a computational simulation between boldine and tyrosinase. Boldine reversibly inhibited tyrosinase from mushroom (Agaricus bisporus) in a mixed-type manner, with a K_i = 7.203 ± 0.933 mM. To gain insight into the inactivation process, we computed the kinetics via time-interval measurements and continuous substrate reactions. The results indicated that the inactivation induced by boldine was a first-order reaction with biphasic processes and that the substrate can promote the inactivation process. To gain further insight, we performed computational docking and molecular dynamics simulations, and the results showed that boldine can interact with several residues near the tyrosinase active site. Our study provides insight into the inhibition of tyrosinase in response to alkaloids. Based on its tyrosinase-inhibiting effect and low toxicity, boldine is a potential natural anti-pigmentation agent.     

Stability and structural changes of horseradish peroxidase: Microwave versus conventional heating treatment

Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4–6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30 W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.     

Effect of ultrasound combined with malic acid on the activity and conformation of mushroom (Agaricus bisporus) polyphenoloxidase

Polyphenoloxidase (PPO) plays an important role in the browning of vegetables, fruits and edible fungi. The effects of ultrasound, malic acid, and their combination on the activity and conformation of mushroom (Agaricus bisporus) PPO were studied. The activity of PPO decreased gradually with the increasing of malic acid concentrations (5–60 mM). Neither medium concentrations (10, 20, 30 mM) malic acid nor individual ultrasound (25 kHz, 55.48 W/cm²) treatment could remarkably inactivate PPO. However, the inactivation during their combination was more significant than the sum of ultrasound inactivation and malic acid inactivation. The inactivation kinetics of PPO followed a first-order kinetics under the combination of ultrasound and malic acid. The conformation of combination treated PPO was changed, which was reflected in the decrease of α-helix, increase of β-sheet contents and disruption of the tertiary structure. Results of molecular microstructure showed that ultrasound broke large molecular groups of PPO into small ones. Moreover, combined treatment disrupted the microstructure of PPO and molecules were connected together.     

Kinetic study on enzymatic esterification of tuna fish oil fatty acids with butanol

Docosahexaenoic acid (DHA) is an important polyunsatured fatty acid (PUFA) which can be purified from tuna fish oil fatty acids by selective enzymatic esterification. The present paper investigates the kinetic study for selective esterification of tuna fish oil fatty acids with butanol catalyzed by Rhizopus oryzae lipase (ROL) in biphasic solvent system. Under the most suitable reaction conditions, 76.2% esterification was achieved in 24 h. Different kinetic models for esterification given by Segel [1], Oliveira et al. [2], Gogoi et al. [3], and Kraai et al. [4] were tested for fitting the esterification data and the model given by Oliveira et al. [2] was found to be most suitable. The model given by Prazeres et al. [5] for hydrolysis was also tested for esterification and the model with second order product inhibition was found to provide better match between the predicted and experimental values than that of model by Oliveira et al. [2]. The kinetic model was fitted using MATLAB^® to determine the best kinetic parameters. The average value of kinetic constants using the model given by Prazeres et al. were estimated as K_m = 23.6 μmoles FFA/ml, K_i1 = 4.6 × 10⁻⁵ μmoles FFA/mg enzyme h, K_i2 = 0.0062 μmoles FFA/mg enzyme h and K₂ = 149.5 μmoles FFA/mg enzyme h.     

Characterization of the lipase immobilized on Mg–Al hydrotalcite for biodiesel

Immobilization of Saccharomyces cerevisiae lipase by physical adsorption on Mg–Al hydrotalcite with a Mg/Al molar ratio of 4.0 led to a markedly improved performance of the enzyme. The immobilized lipase retained activity over wider ranges of temperature and pH than those of the free lipase. The immobilized lipase retained more than 95% relative activity at 50 °C, while the free lipase retained about 88%. The kinetic constants of the immobilized and free lipases were also determined. The apparent activation energies (E_a) of the free and immobilized lipases were estimated to be 6.96 and 2.42 kJ mol^?1, while the apparent inactivation energies (E_d) of free and immobilized lipases were 6.51 and 6.27 kJ mol^?1, respectively. So the stability of the immobilized lipase was higher than that of free lipase. The water content of the oil must be kept below 2.0 wt% and free fatty acid content of the oil must be kept below 3.5 mg KOH g [oil]^?1 in order to get the best conversion. This immobilization method was found to be satisfactory to produce a stable and functioning biocatalyst which could maintain high reactivity for repeating 10 batches with ester conversion above 81.3%.     

Laboratory evolution of laccase for substrate specificity

A laccase, CotA, from Bacillus subtilis was engineered using a combination of rational and directed evolution approaches. CotA is a generalist, an enzyme with broad specificity, and it was optimized to be a specialist, an enzyme with narrowed specificity. Wild-type CotA oxidizes ABTS (ABTS = diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and SGZ (SGZ = 4-hydroxy-3,5-dimethoxy-benzaldehyde azine), and it was engineered for increased specificity for ABTS. Based on the ABTS-bound crystal structure of CotA, 19 amino acids are within 6 Å of ABTS, and they were simultaneously randomized. A mutant was identified that was 132 times more specific for ABTS. Unexpectedly, the variant was found to acquire enhanced thermal stability. The half-life for the heat inactivation (t_1/2) at 80 °C was increased by 62 min for the mutant. Laccases have several applications in biotechnology, which include pulp bleaching, biosensors, bioremediation, and biofuel cells. The substrate specificity of CotA is moldable and the enzyme can be tailored to oxidize a variety of target molecules for specific purposes.     

The effect of PI3 kinase inhibitor LY294002 on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

AimsWe examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K⁺ (Kv) channels.Main methodsElectrophysiological recordings were performed in freshly isolated rabbit coronary arterial smooth muscle cells.Key findingsThe Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a K_d value of 1.48 μM. Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83 ± 0.01 μM^? 1 s^? 1 and 2.59 ± 0.14 s^? 1, respectively. Application of LY294002 had no significant impact on the steady-state activation or inactivation curves. In the presence of LY294002, the recovery time constant from inactivation was increased, and Kv channel inhibition increased under train pulses (1 or 2 Hz). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10 μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002.SignificanceBased on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition.     

Synthesis and biological evaluation of novel hydroxybenzaldehyde-based kojic acid analogues as inhibitors of mushroom tyrosinase

Two series of novel kojic acid analogues (4a–j) and (5a–d) were designed and synthesized, and their mushroom tyrosinase inhibitory activities was evaluated. The result indicated that all the synthesized derivatives exhibited excellent tyrosinase inhibitory properties having IC₅₀ values in the range of 1.35 ± 2.15–17.50 ± 2.75 μM, whereas standard inhibitor kojic acid have IC₅₀ values 20.00 ± 1.08 μM. Specifically, 5-phenyl-3-[5-hydroxy-4-pyrone-2-yl-methylmercap-to]-4-(2,4-dihydroxyl-benzylamino)-1,2,4-triazole (4f) exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value of 1.35 ± 2.15 μM. The kinetic studies of the compound (4f) demonstrated that the inhibitory effects of the compound on the tyrosinase were belonging to competitive inhibitors. Meanwhile, the structure-activity relationship was discussed.     

Thermodynamic and kinetic studies for environmentaly friendly Ni(II) biosorption using waste pomace of olive oil factory

The objective of this study is to assess the environmentaly friendly Ni(II) adsorption from synthetic wastewater using waste pomace of olive oil factory (WPOOF). Batch kinetic studies were performed in order to investigate the adsorbent and adsorbate dose, solution pH, agitating speed and temperature. The maximum Ni(II) adsorption was obtained at pH 4.0. The equilibrium nature of Ni(II) adsorption at different temperature was described by the Freundlich, Langmuir and Temkin isotherms. The equilibrium data fit well the Temkin and Langmuir isotherm. The monolayer adsorption capacities of WPOOF as obtained from Langmuir isotherm at 60 °C was found to be 14.80 mg/g. The adsorption mechanism was examined by the FTIR technique. The results of the thermodynamic investigations indicated that the adsorption reactions were spontaneous (ΔG < 0), slightly endothermic (ΔH > 0) and irreversible (ΔS > 0). The pseudo first-order, pseudo second-order, Elovich and intraparticle diffusion kinetic models were used to describe the kinetic data.     

Characterization of catechol derivative removal by lignin peroxidase in aqueous mixture

The use of lignin peroxidase (LIP) as an alternative method for the removal of four catechols (1,2-dihydroxybenzene): catechol (CAT), 4-chlorocatechol (4-CC), 4,5-dichlorocatechol (4,5-DCC) and 4-methylcatechol (4-MC) typical pollutants in wastewater derived from oil and paper industries, was evaluated. The removal of 2 mM catecholic substrates by 1 μM LIP after 1 h was in the following order: 4,5-DCC (95%) > 4-CC(90%) > CAT(55%) > 4-MC(43%). Except for 4-MC, all reactions were accompanied by the formation of insoluble products, leading to LIP precipitation. LIP was exposed to soluble or insoluble product-dependent inactivation, depending on the substrates tested, immediately at the start of the reactions. Despite immediate enzyme inactivation, removal of catecholic substrates continued, resulting in oligomeric product formation. Major oxidation products analyzed were compatible with dimeric, trimeric and tetrameric structures. Ether linkages and a benzoquinone structure were detected in two purified oligochlorocatechols.Catechol derivatives removal initiated by LIP, seems to be different for each catecholic substrate in terms of substrate consumption and transformation, and of enzyme activity.     

Stability and activity of Dictyoglomus thermophilum GH11 xylanase and its disulphide mutant at high pressure and temperature

The functional properties of extremophilic Dictyoglomus thermophilum xylanase (XYNB) and the N-terminal disulphide-bridge mutant (XYNB-DS) were studied at high pressure and temperature. The enzymes were quite stable even at the pressure of 500 MPa at 80 °C. The half-life of inactivation in these conditions was over 30 h. The inactivation at 80 °C in atmospheric pressure was only 3-times slower. The increase of pressure up to 500 MPa at 80 °C decreased only slightly the enzyme's stability, whereas in 500 MPa the increase of temperature from 22 to 80 °C decreased significantly more the enzyme's stability. While the high temperature (80–100 °C) decreased the enzyme reaction with short xylooligosaccharides (xylotetraose and xylotriose), the high pressure (100–300 MPa) had an opposite effect. The temperature of 100 °C strongly increased the K_m but did not affect the k_cat to the same extent, thus indicating that the interaction of the substrate with the active site suffers before the catalytic reaction begins to decrease as the temperature rises. Circular dichroism spectroscopy showed the high structural stability of XYNB and XYNB-DS at 93 °C.     

Effect of spermidine and its metabolites on the activity of pea seedlings diamine oxidase and the problems of biosensing of biogenic amines with this enzyme

Spermidine is one of the several biogenic amines, produced during the microbial decarboxylation of proteins. Individual biogenic amines in the formed mixtures are frequently analyzed with oxygen sensor based biosensors, as their content serves as a good biomarker for the determination of food quality. In these biosensors, diamine oxidase from pea seedlings (PSAO), catalyzing the oxidation of various biogenic amines by dissolved oxygen is commonly used for the bio-recognition of amines. However, in the presence of spermidine and/or its metabolite 1,3-diaminopropane, the activity of PSAO and the sensitivity of PSAO-based biosensors decrease due to inhibition. The inhibition constant of soluble spermidine, acting as an inhibiting substrate toward PSAO, was found to be (40 ± 15) mM in freshly prepared solution and (0.28 ± 0.05) mM in solution, incubated 30 days at room temperature. The inhibition constant of 1,3-diaminopropane, acting as a competitive inhibitor, was (0.43 ± 0.12) mM as determined through the oxidation reaction of cadaverine. The metabolic half-life of soluble spermidine was 7 days at room temperature and 186 days at 4 °C. The kinetic measurements were carried out with an oxygen sensor; the composition of the solution of degraded spermidine was analyzed with MS.     

Ultrasonic pretreatment for lipase-catalyzed synthesis of 4-methoxy cinnamoyl glycerol

4-Methoxy cinnamoyl glycerol (4MCG) is a very promising UV filters material in personal care products. In order to effectively improve the yield of 4MCG, a systematic study on ultrasonic pretreatment enzymatic esterification for 4MCG products was carried out. An ultrasonic frequency of 35 kHz, ultrasonic power of 150 W and ultrasound irradiation time of 1.5 h was determined to guarantee satisfactory degree of esterification and lipase activity. The optimum production was achieved in organic solvent system at 65 °C with 4MCA to glycerol molar ratio of 1:5, enzyme amount of 15 mg/mL, resulting in a monoester yield of above 66% and 55% after 48 h and 24 h of reaction under ultrasonic pretreatment, respectively. The experimental kinetic data were studied. The reactions were modeled by a system of sequential first-order rate expressions, kinetic parameters were estimated from experimental data fit to the model equations. Results show that the monoester yield in the ultrasonic pretreatment process (24 h) were above 1.5-fold as that in mechanical stirring process without essential damaging to lipase activity. The enzymatic method using ultrasonic pretreatment was obviously superior to the mechanical stirring for enzymatic method and chemical method in terms of conversion rate and the monoester yield. These results are of great significance for applying ultrasonic pretreatment method to prepare 4MCG.     

Virus inactivation in a factor VIII/VWF concentrate treated using a solvent/detergent procedure based on polysorbate 20

Treatment with solvent/detergent is a widely used method for ensuring the virus safety of plasma products. In the present study, virus inactivation by a novel solvent/detergent combination, i.e. TnBP (tri-n-butyl phosphate) and polysorbate 20 during the manufacture of the factor VIII/VWF concentrate Optivate^® has been investigated. The inactivation of most enveloped viruses was rapid, i.e. >5 log in 2 min, although the inactivation of vaccinia virus was slower, i.e. 4 log in 1 h. Virus inactivation was effective over a wide range of conditions, i.e. solvent/detergent concentration, protein concentration and temperature, irrespective of whether tested individually or in combination. This confirms the effectiveness and robustness of this alternative version of the solvent/detergent procedure, and allows appropriate control limits to be set for this manufacturing step. Polysorbate 20 provides an alternative to the non-ionic detergents currently in use with the solvent/detergent procedure.