Kinetic characterisation and thermal inactivation study of polyphenol oxidase and peroxidase from table grape (Crimson Seedless)

Polyphenol oxidase (PPO) and peroxidase (POD) were extracted from a table grape (Crimson Seedless) using Triton X-114 and characterized using spectrophotometric methods. Both PPO and POD were activated by acid shock. However, in the presence of the anionic detergent sodium dodecil sulphate (SDS), PPO was activated whereas POD was inactivated. The enzymes were kinetically characterized and both followed Michaelis–Menten kinetics, although with different values of their kinetic parameters. The V_m/K_m ratio showed that Crimson Seedless grape PPO presents a similar affinity for 4-tert-butyl-catechol (TBC) whether activated by acid shock (0.018 min⁻¹) or SDS (0.023 min⁻¹). With regards to POD, the K_m and V_m values for 2,2′-azinobis(3-ethylbenzothiazolinesulphonic acid) (ABTS) were 0.79 mM and 1.20 μM/min, respectively. In the case of H₂O₂, the K_m and V_m value were 0.4 mM and 0.93 μM/min, respectively. PPO and POD showed similar thermostability, losing >90% of relative activity after only 5 min of incubation at 78 °C and 75 °C, respectively. In addition, PPO´s activation energy was similar to that obtained for POD (295.5 kJ/mol and 271.9 kJ/mol, respectively).     

Polyphenol oxidase activity,phenolic acid composition and browning in cashew apple (Anacardium occidentale, L.) after processing

This study describes the extraction and characterisation of cashew apple polyphenol oxidase (PPO) and the effect of wounding on cashew apple phenolic acid composition, PPO activity and fruit browning. Purification factor was 59 at 95% (NH₄)₂SO₄ saturation. For PPO activity, the optimal substrate was catechol and the optimum pH was 6.5. PPO K_m and V_max values were 18.8 mM and 13.6 U min⁻¹ ml⁻¹, respectively. Ascorbic acid, citric acid, sodium sulphite and sodium metabisulphite decreased PPO activity, while sodium chloride increased PPO activity. Wounding at 2 °C and 27 °C for 24 h increased PPO activity but storage at 40 °C reduced PPO activity. Gallic acid, protocatechuic acid and cinnamic acid (free and conjugate) were identified in cashew apple juice. Cutting and subsequent storage at 40 °C hydrolysed cinnamic acid. 5-Hydroxymethylfurfural content in cashew apple juice increased after injury and storage at higher temperatures, indicating non-enzymatic browning.     

Quantification and characterisation of polyphenol oxidase from vanilla bean

Polyphenol oxidase (PPO) of Vanilla planifolia Andrews beans was extracted and purified through ammonium sulphate precipitation, dialysis, and gel filtration chromatography. PPO activity was measured by improved UV technique using 4-methylcatechol and catechol as substrates increasing substantial sensitivity of previous procedure. The optimum pH and temperature for PPO activity were found to be 3.0 and 3.4 and 37 °C, respectively. K_m and V_max values were found to be 10.6 mM/L and 13.9 OD₃₀₀ min⁻¹ for 4-methylcatechol and 85 mM/L and 107.2 OD₃₀₀ min⁻¹ for catechol. In an inhibition test, the most potent inhibitor was found to be 4-hexylresorcinol followed by ascorbic acid. The thermal inactivation curve was biphasic. Activation energy (E_a) and z values were calculated as 92.10 kJ mol⁻¹ and 21 °C, respectively.     

Properties of polyphenol oxidase from Anamur banana (Musa cavendishii)

Polyphenol oxidase (PPO) was extracted from Anamur banana, grown in Turkey, and its characteristics were studied. The optimum temperature for banana PPO activity was found to be 30 °C. The pH-activity optimum was 7.0. From the thermal inactivation studies, in the range 60–75 °C, the half-life values of the enzyme ranged from 7.3 to 85.6 min. The activation energy (E_a) and Z values were calculated to be 155 kJ mol⁻¹ and 14.2 °C, respectively. K_m and V_max values were 8.5 mM and 0.754 OD₄₁₀ min⁻¹, respectively. Of the inhibitors tested, ascorbic acid and sodium metabisulphite were the most effective.     

Purification and characterization of polyphenol oxidase from Ferula sp.

Polyphenol oxidase (PPO) of several Ferula sp. was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and gel filtration chromatography. Leaf and stem extracts were used for the determination of enzyme properties. Optimum conditions, for pH, temperature, and ionic strength were determined. The best substrates of PPO were catechol for leaf and (−) epicatechin for stem samples. Optimum pH and temperature were determined. K_M and V_max values were 2.34 × 10⁻³ M and 8541 EU/ml for catechol, and 2.89 × 10⁻³ M and 5308 EU/ml for (−) epicatechin. The most effective inhibitor was sodium diethyl dithiocarbamate for leaf samples and sodium metabisulphite for stem samples. Both inhibitors indicated competitive reactions. PPO showed irreversible denaturation after 40 min at 60 °C.     

Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.)

Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical characteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K_M and V_max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min⁻¹ for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min⁻¹ for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a temperature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione ≈ ascorbic acid > l-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa.     

Biochemical characterization and process stability of polyphenoloxidase extracted from Victoria grape (Vitis vinifera ssp. Sativa)

Polyphenol oxidase (PPO) was isolated from Victoria grapes (Vitis vinifera ssp. Sativa) grown in South Africa and its biochemical characteristics were studied. Optimum pH and temperature for grape PPO activity were pH 5.0 and T = 25 °C with 10 mM catechol in McIlvaine buffer as substrate. PPO showed activity using the following substances: catechol, 4 methyl catechol, d, l-DOPA, (+) catechin and chlorogenic acid. K_m and V_max values were 52.6 ± 0.00436 mM and 653 ± 24.0 OD_400 nm/min in the case of 10 mM catechol as a substrate. Eight inhibitors were tested in this study and the most effective inhibitors were found to be ascorbic acid, l-cysteine and sodium metabisulfite. Kinetic studies showed that the thermal inactivation of Victoria grape PPO followed first-order kinetics, with an activation energy, E_a = 225 ± 13.5 of kJ/mol. Both in semipurified extract and in grape juice, PPO showed a pronounced high pressure stability.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

Immobilization of α-amylase onto cyclic carbonate bearing hybrid material

Under ambient conditions, 4-((3-trimethoxysilyl)propoxy)methyl)1,3-dioxolan-2-one was synthesized from 3-glycidyloxypropyl trimethoxysilane. Methacrylate and cyclic carbonate functional hybrid matrix was prepared by sol-gel method. α-Amylase was covalently bounded onto the matrix via cyclic carbonate functionality. Immobilization yield was found as 34.4 ± 2.6 mg per gram of hybrid matrix. The maximum activity was observed at pH 6.5. Immobilization did not change the pH-dependency of the enzyme. The immobilized enzyme had a higher activity at elevated temperature (50–80° C) than the free one. Immobilized enzyme exhibited 80% activity after 20 runs and 69% after 30 runs. Free enzyme lost its activity completely within 15 days. Immobilized enzyme lost only 30% of its activity in 25 days. V_max values for the free and immobilized enzymes were calculated as 58 × 10⁻³ and 5.2 × 10⁻³ mg/ml min⁻¹, respectively.     

Partial purification and preparation of bovine lactoperoxidase and characterization of kinetic properties of its immobilized form incorporated into cross-linked alginate films

Lactoperoxidase (LPS), purified directly from bovine rennet whey by Toyopearl-SP cation-exchange chromatography and lyophilized by using dextran as supporting material, maintained almost 70 and 60% of its activity after almost 2 and 5 months storage at −18 °C, respectively. Incorporation of the prepared LPS into alginate films between 0.08 and 0.69 mg/cm² (516–4325 U/cm²) caused the immobilization of most of the enzyme and gave films with LPS activity between 0.05 and 2.8 U/cm², determined in the presence of 8 μM H₂O₂. Between 2 and 24 μM H₂O₂ concentrations, a two-fold increase in H₂O₂ concentration caused 1.5–2.5-fold increase in LPS activity of films incorporated with 0.24–0.28 mg/cm² (1200 U/cm²) LPS. The Q₁₀ and E_a of immobilized enzyme activity between 4 and 16 °C were 1.69 and 34.6 kJ/mol, respectively. However, in the 16–30 °C range, the temperature change had almost no effect on LPS activity of films. The optimal activity of immobilized LPS was observed at pH 6.0, but the enzyme maintained 30–85% of its activity between pH 3.0 and 7.0. The immobilized LPS also had a high stability between pH 4.0 and 6.0. The results of this study showed the good potential of LPS-incorporated alginate films in forming a natural antimicrobial mechanism in different foods.     

Purification and characterisation of a polyphenol oxidase from Boletus erythropus and investigation of its catalytic efficiency in selected organic solvents

Polyphenol oxidase (PPO) was purified from Boletus erythropus using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. Optimum pH and temperature were found to be 8.0 and 20 °C, respectively, using 4-methylcatechol as a substrate. The enzyme was extremely stable between pH 3.0 and 9.0 after 24 h incubation at 4 °C. B. erythropus PPO was also quite stable between 10 and 30 °C after 4 h incubation. The K_m and V_max values were calculated as 2.8 mM and 1430 U/mg protein by Lineweaver–Burk curve, respectively. The enzyme activity was inhibited by sodium metabisulfite, ascorbic acid, sodium azide and benzoic acid. It was seen that the mushroom PPO was an effective biocatalyst in selected organic solvents, such as dichloromethane, dichloroethane and toluene, when catechin was used as a substrate. All data support that B. erythropus has a highly active PPO, possessing similar biochemical and kinetic characteristics to other plant PPOs.     

Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l-cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K⁺, other metal ions such as Zn²⁺, Cu²⁺, Fe³⁺, Ca²⁺, Fe²⁺ and Na⁺ accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent K_m and V_max values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho-diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.     

Evaluation of the influence of arsenical livestock drinking waters on total arsenic levels in cow’s raw milk from Argentinean dairy farms

The concentrations of total arsenic in cow’s raw milk and in the livestock drinking water were determined and compared, in order to establish the influence of natural arsenic levels in groundwaters on the final presence of arsenic in milk production of the most important dairy region in Argentina. A dry ashing procedure was used for the mineralisation of the milk samples. The total arsenic concentrations were determined by flow injection hydride generation atomic absorption spectrometry (FI-HGAAS). The mineralised milk samples and well water samples were pre-reduced with concentrated HCl and KI–C₆H₈O₆ solutions. A volume of 500 μl of each solution of pre-treated sample was transported by a HCl 1.2 mol l⁻¹ carrier solution at a flow rate of 11 ml min⁻¹ and merged with a reducing NaBH₄ 0.2% (m/v) solution which flowed at 5.5 ml min⁻¹. The hydride generated in a reaction coil was transported to the detector with a N₂ flow of 100 ml min⁻¹. The recovery values of added concentrations at levels of 2.5 μg l⁻¹ and 5.0 μg l⁻¹ of arsenic in milk were 103 ± 8% and 102 ± 6% for n = 3, respectively. The accuracy of the method for the determination of total arsenic in water was checked by analysis of a certified sample NIST 1643d. Detection limits were 0.7 μg l⁻¹ and 0.6 μg l⁻¹ for milk and well water, respectively. The results showed a low biological transference level of arsenic to the cow milk from the drinking water ingestion.     

Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-β-GlcNAc) hydrolysis was five; the optimum temperature was 50 °C; the K_m was 0.18 mM, V_max was 37.6 μmol min⁻¹ mg⁻¹ and activation energy (E_a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30–45 °C for 40 min, but its activity decreased significantly when the temperature exceeded 50 °C. Heavy metal ions, Ag⁺ and Hg²⁺, at a concentration of 0.25 mM and Zn²⁺ and Cu²⁺, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The β-NAHA had only one active site for binding both pNP-β-GlcNAc and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-β-GalNAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon–Webb plots.     

Chitosan-tethered the silica particle from a layer-by-layer approach for pectinase immobilization

“Spherical polyelectrolyte brush” of core–shell structure were prepared by grafting poly (sodium 4-styrenesulphonate) (PSStNa) from SiO₂ nanoparticle via the surface-initiated atom transfer radical polymerization strategy. The colloidal stability was not impeded by the adsorbed proteins despite the fact that up to 316.8 mg of enzyme was adsorbed per gram of the carrier particles. The immobilized pectinase revealed acceptable pH stability over a broad experimental range of 3.0–4.5. The activity half lives for native and bound states of enzyme were found as 13.5 d and 30 d, respectively. The activity of immobilized pectinase adsorbed onto these particles was analyzed in terms of the Michaelis–Menten parameters. Kinetic parameters were calculated as 8.28 and 9.98 g pectin ml⁻¹ for K_m and 1.165 × 10⁻³ g pectin s⁻¹ g enzyme⁻¹ 1.124 × 10⁻³ g pectin s⁻¹ g particle⁻¹ for V_max in the case of free and immobilized enzymes, respectively. Enzyme activity was found to be approximately 49.7% for immobilized enzyme after storage for 1 month.     

Thermal inactivation of peroxidase during blanching of butternut squash

The kinetics of thermal inactivation of peroxidase in butternut squash during water blanching was studied. Thin slices of squash were immersed in water at 60-90 °C for 0-60 min. For treatments below 70 °C, a biphasic, first-order kinetics model was proposed for peroxidase inactivation that would correspond to heat-labile and heat-resistant fractions of the enzyme. Parameters of the first-order model for the heat-labile and heat-resistant fractions were k_L,ref=24.8733 min⁻¹, E_aL=14023 J/mol and k_R,ref=0.0729 min⁻¹, E_aR=15794 J/mol, respectively. For treatments above 70 °C, a monophasic first-order kinetics model was proposed. Parameters were k_ref=8.6425 min⁻¹, E_a=149900 J/mol. Decreases in ascorbic acid contents due to blanching were analyzed. Blanching processes at high temperature and short time resulted in higher ascorbic acid retention. A linear relationship between ascorbic acid retention and processing temperature was found.     

Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Amperometric detection of ascorbic acid in honey using ascorbate oxidase immobilised on amberlite IRA-743

A differential amperometric method for the specific determination of ascorbic acid in honey was developed by association of a flow injection analysis (FIA) system and a tubular reactor containing the ascorbate oxidase enzyme immobilised. A gold electrode modified by electrochemical deposition of palladium was employed as working electrode. Ascorbic acid was quantified in seven samples of commercial honeys using a potential of +0.60 V vs. Ag/AgCl_(sat). The linear dynamic range in ascorbic acid extends from 1 to 50 μmol L⁻¹, at pH 7.0. At flow rate of 1.5 mL min⁻¹ and injecting 250 μL sample volumes, a sampling frequency of 180 determinations per hour is afforded. The detection and quantification limit of this method are 0.14 and 0.49 μmol L⁻¹, respectively. The samples analyses were compared with the volumetric method, and showed an excellent correlation between the methods.     

Extraction and characterisation of pectin methylesterase from black carrot (Daucus carota L.)

This study was carried out to determine some of the biochemical properties of pectin methylesterase (PME) from black carrot. The enzyme showed very high activity in a broad pH range of 6.5–8.5, with the optimum pH occurring at 7.5. The optimum temperature for maximal PME activity was found to be 55 °C. NaCl enhanced PME activity, particularly at 0.2 M. K_m and V_max values for black carrot PME using apple pectin as substrate were found to be 2.14 mg/ml (r² = 0.988) and 3.75 units/ml, respectively. The enzyme was stable between the temperatures of 30–50 °C/5 min whereas it lost nearly all of its activity at 70 °C/5 min. E_a and Z values were found to be 196.8 kJmol⁻¹ (r² = 0.996) and 2.16 °C (r² = 0.995), respectively.