Purification,characterization, and inhibition sensitivity of peroxidase from wheat (Triticum aestivum ssp. vulgare)

The purification and characterization of peroxidase is currently growing interests since peroxidases have implications in various industrial and biochemical applications. In this study, wheat peroxidase was purified using (NH₄)₂SO₄ precipitation, CM-Sephadex cation exchange, and gel filtration chromatographies. Enzyme purity and molecular mass were checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Enzyme kinetics was studied using two substrates: guaiacol and hydrogen peroxide (H₂O₂). K_m and V_max values were calculated from Lineweaver-Burk graph for each substrate. Wheat peroxidase activity has been enhanced 284-fold. Wheat peroxidase had molecular mass of 38.8 kDa. K_m values for guaiacol and H₂O₂ were found as 2.467 mM and 7.307 mM, respectively. The pH and temperature optima were 5.5 and 40.0°C, respectively. Also, the enzyme was highly inhibited by citric acid and Cetyltrimethylammonium bromide.     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

Amorphophallus paeoniifolius corm: A potential source of peroxidase for wide applications

Peroxidases have wide applications in different areas including chemical synthesis, medicine, food industry, bioremediation of wastewater, biosensing, and biotechnology. Amorphophallus paeoniifolius (elephant foot yam) is an attractive source of enzymes; however, no effort has been made to assess peroxidase enzyme from this source. Therefore, the objectives of this study were to evaluate and compare peroxidases from corms of elephant foot yam and roots of Dacus carota (carrot) and Aramoracia rusticana (horseradish). Elephant foot yam corm peroxidase (ECP) demonstrated 4.5 times higher specific activity compared with carrot root peroxidase (CRP). ECP showed retention of high activity over a broad pH range and had higher temperature optima and thermal stability compared with CRP and horseradish peroxidase (HRP). The calculated K_M values of ECP, CRP, and HRP for the substrates guaiacol and hydrogen peroxide were 4.5 mM and 307.8 µM, 4.6 mM and 55.5 µM, and 3.5 mM and 413.0 µM, respectively. Peroxidases are used for the bioremediation of wastewater contaminated with hazardous aromatic compounds. Heavy metals such as cadmium (Cd²⁺), lead (Pb²⁺), and toxic compounds such as sodium azide (NaN₃) are often present in wastewater along with aromatic compounds. Results showed activation of ECP and inhibition of HRP at 250 and 500 µM concentrations of Cd²⁺ and Pb²⁺. Treatment with 1 mM NaN₃ led to 8.4%, 55.5%, and 11.0% inhibition in the activities of ECP, CRP, and HRP, respectively. Overall, our results suggest that elephant foot yam can be used as a rich and convenient source of peroxidase for various applications including wastewater remediation.     

Inhibitory effects of selected pesticides on peroxidases purified by affinity chromatography

The objective of this study was to determine the in vitro inhibition effects of seven commonly used pesticides including 2,4-d-acid dimethylamine, fenoxaprop-p-ethyl, glyphosate isopropylamine, haloxyfop-p-methyl, cypermethrin, λ-cyhalothrin, and dichlorvos on the peroxidase purified from turnip (Brassica rapa L.) and black radish (Raphanus sativus L.) using 4-amino benzohydrazide affinity column chromatography. The purification factors for the turnip and black radish peroxidases were found to be 263.29-fold (with a yield of 12.89%) and 36.20-fold (with a yield of 6.90%), respectively. Among these compounds, λ-cyhalothrin showed the strongest inhibitory effect against turnip peroxidase (K_i: 1.23 × 10^?2 ± 0.21 × 10^?2 mM) as noncompetitive inhibition. On the other hand, cypermethrin demonstrated the highest inhibition effect against black radish peroxidase (K_i: 2.14 × 10^?2 ± 0.08 × 10^?2 mM) as competitive inhibition.     

Partial characterization of a lipoxygenase from fusarium proliferatum

Summary

Biomass of the fungus Fusarium proliferatiim was produced and used to obtain a crude extract (FI) of lipoxygenase. The enzyme was further purified by ammonium sulfate precipitation at 40% of saturation (FII). The enzymatic extract (FII) showed its optimum activity at pH 6.0. The apparent K _m and V _max values for the lipoxygenase (FII) were calculated to be 5.15 × 10^?5 M and 1.61 μmol hydroperoxide/mg protein/min, respectively. Enzyme activity remained relatively stable at potassium cyanide concentrations as high as 60 mM. The presence of 5 mM ethylenediaminetetraacetate activated the enzyme by 50%, whereas the use of 1.2 mM hydroquinone resulted in a 2‐fold increase in lipoxygenase activity. The partially purified enzyme (FII) showed a three‐fold enhancement of activity towards linoleic acid compared to linolenic acid as well as mono‐, di‐ and trilinolein.     

Properties of thermostable extracellular FOS-producing fructofuranosidase from <Emphasis Type="Italic">Cryptococcus</Emphasis> sp.

The present work was devoted to investigations concerning the fructooligosaccharide producing activity of Cryptococcus sp. LEB-V2 (Laboratory of Bioprocess Engineering, Unicamp, Brazil) and its extracellular fructofuranosidase. After cell separation, the enzyme was purified by ethanol precipitation and anion exchange chromatography. The enzyme showed both fructofuranosidase (FA) and fructosyl transferase (FTA) activity. With sucrose as substrate, the data failed to fit the Michaelis–Menten behaviour, showing a substrate inhibitory model. The K _m, K _i and v _max values were shown to be 64 mM, 3 M and 159.6 μmol mL⁻¹ min⁻¹ for FA and 131 mM, 1.6 M and 377.8 μmol mL⁻¹ min⁻¹ for FTA, respectively. The optimum pH and temperature were found to be around 4.0 and 65 °C, while the best stability was achieved at pH 4.5 and temperatures below 60 °C, for both the FA and FTA. Despite the strong FA activity, the high transfructosylating activity allowed for good FOS production from sucrose (35% yield).     

PARTIAL PURIFICATION OF POLYPHENOL OXIDASE FROM PLUMS (Prunus domestica L., cv. STANLEY)

Polyphenol oxidase (PPO) was extracted and purified from Stanley plums (Prunus domestica L.) Crude PPO showed pH optima of 5.8 to 6.4 with different substrates. Heating for 5 min at 75C completely inactivated this enzyme. Plum PPO was stable at -20C for 16 weeks. K_mof this enzyme ranged from 17.5 mM with 4-methylcatechol to 31.2 mM with chlorogenic acid. The enzyme was purified 36-fold through (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose and Sephadex G-100. PAGE of crude and purified plum PPO showed 7 and 3 bands, respectively, when stained for activity with catechol. The molecular weight of 3 subunits of purified PPO was estimated in the range of 45–66 kD.     

Purification and characterisation of carrot (Daucus carota L) pectinesterase

A pectinesterase isoform with an alkaline isoelectric point of over 8.66 was detected in crude extracts of carrot. The enzyme was purified by ion exchange and molecular exclusion chromatography. The molecular weight of the isoform was 25 kDa, determined in native conditions by filtration through Sephadex G‐75 SF. The enzyme showed a high affinity for its substrate, with K_m and V_max values of 0.031 mg ml^?1 and 6.77 units respectively for apple pectin. The pectinesterase activity exhibited an optimum around pH 7.4 and was activated by metallic ions, with optimum activities at NaCl concentrations between 130 and 330 mM and at CaCl₂ concentrations between 15 and 50 mM . The enzyme was activated most by Ca²⁺ and exhibited a greater tolerance of high concentrations of Na⁺. Comparison of its heat stability with other pectinesterases of vegetable origin indicated that the purified isoform was very thermolabile, being rendered inactive by heating for 5 min at 70 °C. The enzyme was inhibited by high concentrations of polygalacturonic acid and competitively inhibited by D ‐galacturonic acid, with a K_i value of 1 mM . Copyright © 2003 Society of Chemical Industry     

Partial purification and characterisation of polyphenol oxidase and peroxidase from marula fruit (Sclerocarya birrea subsp. Caffra)

Marula fruit, native to sub-Saharan Africa, is of growing commercially importance. Polyphenol oxidase (PPO) and peroxidase from the fruit were partially purified by a combination of temperature induced phase separation in Triton X-114, DEAE-ion exchange and Sephadex G100 gel filtration. PPO activity was purified 58-fold with 75% recovery while the purification factor for peroxidase was 19% with 25% recovery. The enzymes were characterised for enzyme concentration–reaction rate relationship, thermal stability, pH activity and stability, molecular weight, isoelectric point (pI) and kinetic parameters. PPO and peroxidase shared the same molecular weight (71 kDa) and pI (5.43). Thermal deactivation curves were bi-phasic for both activities. Peroxidase displayed maximal activity at pH 4.0 with ABTS (2,2′-azino-(bis-3-ethylbenzthiazoline-6-sulfonic acid)) and a K_M of 1.77 mM for hydrogen peroxide. The pH optimum for PPO was 7.0 with catechol. Marula PPO had K_M values of 1.41, 1.43, 3.73 and 4.99 mM for catechin, 4-methylcatechol, 3,4-dihydroxyphenylpropanoic acid (DHPPA) and catechol, respectively.     

Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

Kinetic Properties of Peroxidase Enzyme from Chard (Beta vulgaris Subspecies cicla) Leaves

Peroxidases are heme containing enzymes that are produced by a number of organisms. A plant peroxidase was purified from leaves of chard (Beta vulgaris subspecies cicla) by ammonium sulphate precipitation, dialyses, and CM-sephadex cation exchange chromatography. Purification fold was approximately 36 with 14.4% yield. Optimum pH, optimum temperature, optimum ionic strength, and stable pH values of purified enzyme were determined for the guaiacol substrate. Michaelis constant (K_M) and maximum rate (V_max) values were determined from the Lineweaver–Burk graph for pyrogallol and guaiacol substrates. Phenolic and flavonoid contents in chard that connected with antioxidant properties were determined as 17.5 μg (GAE/mg extract) and 11.7 μg (QE/mg extract), respectively. In conclusion, chard is shown to be a novel rich source of phenolic antioxidants and has a high peroxidase activity.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Partial characterization of polyphenoloxidase from a hybridized wheat (Triticum aestivum L.)

Polyphenol oxidase was extracted and partially purified from wheat leaves by a procedure that included ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures led to 35.21-fold purification with 17.65% recovery. Optimum pH, temperature, and ionic strength were determined with six substrates. Some kinetic properties of the enzyme such as V _max, K _M, and k _cat were calculated for the substrates. The k _cat/K _M values of the PPO for catechol, catechin, pyrogallol, l-dopa, dopamine, and 4-methyl catechol were 31408, 31167, 28404, 15378, 4865, and 4967 mM/min, respectively. The best substrate of wheat PPO was found to be catechol. The native molecular weight of the PPO was estimated to be 243 kDa based on its mobility in gel filtration column. The inhibitory effects of glutathione, sodium azide, ascorbic acid, oxalic acid, l-cysteine, and thiourea on the reaction catalyzed by the enzyme were tested, and I ₅₀ values were estimated to be 8.0 mM, 10.12 mM, 11.18 mM, 77.33 mM, 183 mM, and 413 mM, and K _i constants were also calculated as 0.416 ± 0.244 mM, 0.317 ± 0.208 mM, 0.820 ± 0.111 mM, 13.80 ± 1.179 mM, 14.10 ± 5.069 mM, and 130 ± 62.45 mM, respectively, by means of Lineweaver–Burk graphs. The most effective inhibitor was glutathione. Glutathione, sodium azide, oxalic acid, and thiourea were competitive inhibitors, whereas ascorbic acid and l-cysteine were also noncompetitive inhibitors.     

Inhibitory effects of cinnamic acid and its derivatives on the diphenolase activity of mushroom (Agaricus bisporus) tyrosinase

The effects of cinnamic acid and its derivatives (2-hydroxycinnamic acid, 4-hydroxycinnamic acid and 4-methoxycinnamic acid) on the activity of mushroom tyrosinase have been studied. Results showed that cinnamic acid, 4-hydroxycinnamic acid and 4-methoxycinnamic acid strongly inhibited the diphenolase activity of mushroom tyrosinase and the inhibition was reversible. The IC₅₀ values were estimated to be 2.10, 0.50 and 0.42 mM, respectively. 2-Hydroxycinnamic acid had no inhibitory effect on the diphenolase activity of the enzyme. Kinetic analyses showed that the inhibition type of cinnamic acid and 4-methoxycinnamic acid was noncompetitive with the constants (K_I) determined to be 1.994 and 0.458 mM, respectively. The inhibition type of 4-hydroxycinnamic acid was competitive, with the inhibition constant (K_I) was 0.244 mM.     

Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry     

Partial characterization of peroxidase from the leaves of thymbra plant (Thymbra spicata L. var. spicata)

Peroxidase (EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase) catalyses the oxidation of various electron donor substrates (e.g. phenols, aromatic amines). In this study, the peroxidase was extracted from Thymbra spicata L. var. spicata and, then partially purified with (NH₄)₂SO₄ precipitation and dialysis. The substrate specificity of peroxidase was investigated using 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), o-dianisidine, o-phenylenediamine, catechol and guaiacol as substrates. Furthermore, the effects of buffer concentration, pH, temperature and thermal inactivation on enzyme activity were also studied. The results obtained have shown that (i) the best substrate is o-dianisidine, followed by ABTS, catechol, guaiacol and o-phenylenediamine, respectively; (ii) the best buffer concentration is 40 mM for o-dianisidine and catechol, 10 mM for ABTS and guaiacol, and 100 mM for o-phenylenediamine; (iii) optimum pH is 2.5 for ABTS and o-phenylenediamine, 6.0 for o-dianisidine, and 7.0 for catechol and guaiacol; (iv) optimum temperature is 20 °C for catechol, 40 °C for ABTS, guaiacol and o-dianisidine, and 50 °C for o-phenylenediamine; and (v) the enzyme activity in the thermal inactivation experiments was enhanced with increase in temperature with o-dianisidine as a substrate while its activity decreased with o-phenylenediamine.     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

α‐l‐Rhamnosidase from Aspergillus flavus MTCC‐9606 isolated from lemon fruit peel

An α‐l ‐rhamnosidase producing fungal strain has been isolated from decaying lemon fruit. The fungal strain has been identified as Aspergillus flavus. The α‐l ‐rhamnosidase has been purified from the culture filtrate of the fungal strain using ultra filtration and cation exchange chromatography on carboxy methyl (CM) cellulose. The molecular mass of the purified enzyme determined by SDS–PAGE analysis was 41 kDa. The K_m values of the enzyme using p‐nitrophenyl‐α‐l ‐rhamnopyranoside and naringin as the substrates were 1.89 and 1.6 mm respectively. The pH and temperature optima of the enzyme were 11.0 and 50 °C respectively. The effects of various chemical species present in grape fruit juice and wine on the activity of the enzyme have been determined.     

Purification and characterization of a novel thermostable phytase from the thermophilic Geobacillus sp. TF16

A novel phytase from thermophilic Geobacillus sp. TF16 was puri?ed approximately 5-fold using ammonium sulfate precipitation and ion exchange chromatography, and determined as a single band 106.04 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimum temperature and optimum pH were found to be 85°C and 4.0, respectively. The enzyme is highly thermostable and V_max and K_m values were calculated as 526.28 U/mg and 1.31 mM, respectively. It was also found that the enzyme exhibited a broad substrate selectivity and resistance toward proteases and effectively hydrolyzed soymilk phytate. These results suggest that this study provides an alternative phytase enzyme with enhanced properties.     

One-step purification of lactoperoxidase from bovine milk by affinity chromatography

Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC₅₀ of 0.848.10⁻⁵ M. The K_i for sulphanilamide was determined to be 3.57.10⁻⁵ M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-l-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and l-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS–PAGE and its R_z (A₄₁₂/A₂₈₀) value. The R_z value for the purified LPO was found to be 0.7. Maximum binding was achieved and K_m and V_max values were determined.