Peroxidase activity of germinating Sorghum bicolor grains: effect of some cations

We investigated some properties of the major ionic peroxidase (POD) from germinating Sorghum bicolor var Fara Fara. Peroxidase activity increased eightfold during the first 36 h of germination and declined thereafter to four times the value in the dry grain after 144 h. Gel filtration followed by ion exchange chromatography resolved the major peroxidase into two forms. The predominant POD had an optimum temperature of 55 °C, two pH optima at 5.0 and 7.0, with a sharp decline in activity below pH 4.5, an apparent activation energy of 463 kJ mol^?1, an apparent molecular weight of 63.1 kDa and was relatively heat‐stable up to 70 °C. The enzyme was strongly inhibited by dithiothreitol and sodium metabisulphite. Calcium chloride and magnesium chloride below 2 mM enhanced POD activity without any adverse effect on germination, while ammonium chloride and ferric chloride inhibited the enzymatic activity of both forms. At 5 mM , magnesium chloride inhibited POD activity by 50% with only a 14% reduction in germination, while calcium chloride achieved the same effect at 10 mM . These results are pertinent to controlling the undesirable activity of peroxidase in a typical malting or brewing process. © 2002 Society of Chemical Industry     

Characterization of Soluble and Bound Peroxidases in Green Asparagus

Soluble and ionically bound peroxidases were extracted from green asparagus with 0.05M sodium phosphate (pH 7.0) and the same buffer containing 1.0M NaCl, respectively. The two forms of peroxidase have been purified 237 and 53 fold, respectively, through ammonium sulphate fractionation, and successive chromatography on Sephacryl S-200 and ConA Sepharose 4B columns. Eleven isoenzymes with different pI values were detected from the soluble form using isoelectric focusing and eight from the ionically bound form. The two forms of perooxidase showed a similar optimum pH range of 4.2–5.0 using three kinds of hydrogen donor with different buffers. The optimum temperature of the two peroxidase forms at pH 4.5 was around 50°C. Heat inactivation of both forms at 70° and 90°C was observed to be biphasic.     

Characterisation of an acidic peroxidase from papaya (Carica papaya L. cv Tainung No. 2) latex and its application in the determination of micromolar hydrogen peroxide in milk

An acidic peroxidase isoform, POD-A, with a molecular mass of 69.4 kDa and an isoelectric point of 3.5 was purified from papaya latex. Using o-phenylenediamine (OPD) as a hydrogen donor (citrate–phosphate as pH buffer), the optimum pH for the function of POD-A was 4.6, and the optimum temperature was 50 °C. The peroxidase activity of POD-A toward hydrogen donors was both pH- and concentration-dependent. Under optimal conditions, POD-A catalysed the oxidation of OPD at higher rates than pyrogallol, catechol, quercetin and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The chemical modification reagents N-bromosuccinimide and sodium azide significantly inhibited POD-A activity. The results of kinetic studies indicated that POD-A followed a ping-pong mechanism and had a K_m value of 2.8 mM for OPD. Using CPC silica-immobilised POD-A for the determination of micromolar H₂O₂ in milk, the lower limit of determination was 0.1 μM, and the recoveries of added H₂O₂ were 96–109%.     

Partial Purification and Characterization of Asparagus Lipoxygenase

Lipoxygenase (LOX) from fresh asparagus was partially purified by extraction of acetone-washed asparagus powder with pH 4.5 potassium phosphate, ammonium sulfate fractionation and carboxymethyl-cellulose (CMC) chromatography. Asparagus LOX purified by ammonium sulfate fractionation had a pH activity optimum of 5.5–6.0 and was stable at pH 4.5–8.0 when stored at 2°C. Asparagus LOX was active on monolinolein as well as linoleic acid, but activity was very low on di- or tri-linolein. The CMC fractions with greatest LOX activity were nearly free of peroxidase activity while the protein fractions which did not bind with CMC at pH 5 were peroxidase active. The LOX activity in the purified asparagus extract was 90% inhibited by 1 mM cyanide when preincubated for 30 min at 25°C.     

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.     

沙田柚皮过氧化物酶的纯化及酶学性质的研究

为了获得一种富含过氧化物酶（POD）的植物,本研究利用循环催化流动分析法（RCFA）初步筛选了15种植物样品,对样品中POD活性最高的沙田柚皮粗酶液进行了硫酸铵盐析和Sephadex G-200凝胶层析纯化,并对已纯化的POD进行了酶学性质研究。得到的结果表明:每克沙田柚皮中POD可达15.08U,纯化后的比活力可达313.29U/mg,纯化倍率为26.22倍,POD最终产率为43.20%;优化得到的POD最适温度和pH分别为55℃和5.5,在最适条件下测得该酶以H2O2为底物的米氏常数Km为0.099mmol/L,以愈创木酚（GA）为底物的米氏常数Km为0.864mmol/L。      

Heat resistance of Escherichia coli O157:H7 in cook‐in‐bag ground beef as affected by pH and acidulant†

Summary The heat resistance of a four‐strain mixture of Escherichia coli O157:H7 was tested. The temperature range was 55–62.5 °C and the substrate was beef at pH 4.5 or 5.5, adjusted with either acetic or lactic acid. Inoculated meat, packaged in bags, was completely immersed in a circulating water bath and cooked to an internal temperature of 55, 58, 60, or 62.5 °C in 1 h, and then held for pre‐determined lengths of time. The surviving cell population was enumerated by spiral plating meat samples on tryptic soy agar overlaid with Sorbitol MacConkey agar. Regardless of the acidulant used to modify the pH, the D ‐values at all temperatures were significantly lower (P < 0.05) in ground beef at pH 4.5 as compared with the beef at pH 5.5. At the same pH levels, acetic acid rendered E. coli O157:H7 more sensitive to the lethal effect of heat. The analysis of covariance showed evidence of a significant acidulant and pH interaction on the slopes of the survivor curves at 55 °C. Based on the thermal‐death–time values, contaminated ground beef (pH 5.5/lactic acid) should be heated to an internal temperature of 55 °C for at least 116.3 min and beef (pH 4.5/acetic acid) for 64.8 min to achieve a 4‐log reduction of the pathogen. The heating time at 62.5 °C, to achieve the same level of reduction, was 4.4 and 2.6 min, respectively. Thermal‐death–time values from this study will assist the retail food processors in designing acceptance limits on critical control points that ensure safety of beef originally contaminated with E. coli O157:H7.     

Impact of Supercritical Carbon Dioxide and High Pressure on Lipoxygenase and Peroxidase Activity

The effects of supercritical carbon dioxide (ScCO2) treatment and high hydrostatic pressure treatment on the activities of lipoxygenase (LOX) and peroxidase (POD) were studied. Hydrostatic pressure treatment (240 MPa, 55 °C, 15 min) of LOX and POD in 30% sucrose solutions without buffer led to approximately 80% and approximately 50% residual activity, respectively. Application of ScCO2 (35.2 MPa, 40 °C, 15 min for LOX and 62.1 MPa, 55 °C, 15 min for POD) achieved approximately 35% LOX and approximately 65% POD inactivity in 30 % sucrose solutions. Total inactivation of LOX (10.3 MPa, 50 °C and 15 min) and of POD (62.1 MPa, 55 °C and 15 min) could be achieved through ScCO2 treatment of unbuffered solution. Increasing the concentration of sucrose and buffering (pH range 4 to 9) of enzyme solutions resulted in increased resistance of the enzymes to ScCO₂ treatment.     

Impact of pH and Total Soluble Solids on Enzyme Inactivation Kinetics during High Pressure Processing of Mango (Mangifera indica) Pulp

This study was undertaken with an aim to enhance the enzyme inactivation during high pressure processing (HPP) with pH and total soluble solids (TSS) as additional hurdles. Impact of mango pulp pH (3.5, 4.0, 4.5) and TSS (15, 20, 25 °Brix) variations on the inactivation of pectin methylesterase (PME), polyphenol oxidase (PPO), and peroxidase (POD) enzymes were studied during HPP at 400 to 600 MPa pressure (P), 40 to 70 °C temperature (T), and 6‐ to 20‐min pressure‐hold time (t). The enzyme inactivation (%) was modeled using second order polynomial equations with a good fit that revealed that all the enzymes were significantly affected by HPP. Response surface and contour models predicted the kinetic behavior of mango pulp enzymes adequately as indicated by the small error between predicted and experimental data. The predicted kinetics indicated that for a fixed P and T, higher pulse pressure effect and increased isobaric inactivation rates were possible at lower levels of pH and TSS. In contrast, at a fixed pH or TSS level, an increase in P or T led to enhanced inactivation rates, irrespective of the type of enzyme. PPO and POD were found to have similar barosensitivity, whereas PME was found to be most resistant to HPP. Furthermore, simultaneous variation in pH and TSS levels of mango pulp resulted in higher enzyme inactivation at lower pH and TSS during HPP, where the effect of pH was found to be predominant than TSS within the experimental domain.     

Isolation,purification and properties of the peroxidase from the hull of Glycine max var HH2

Soybean hull peroxidase (EC 1.11.1.7), an acidic peroxidase isolated from soybean (Glycine max var HH2) hulls was purified to electrophoretic homogeneity by a combination of ammonium sulphate fractionation, DEAE‐Sephadex A‐50 chromatography, concanavalin A‐Sepharose 4B affinity chromatography and Bio‐Gel P‐60 gel filtration. The specific activity of purified peroxidase was about 57‐fold higher than that of crude extract. The yield was about 16.4%. The molecular weight of the enzyme was estimated to be 38 000 by SDS‐polyacrylamide gel electrophoresis. The peroxidase was a glycoprotein containing about 18.7% carbohydrate, approximately one‐quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.9. The enzyme was most active at pH 4.6 and 45°C, and was stable in the pH range 2.5–11.5. The enzyme could tolerate heating for 10 min at 75°C without being inactivated, and at 85°C, it took 40 min to inactivate the enzyme 50%, confirming that the peroxidase was a novel thermostable enzyme. Fe ²⁺, Fe³⁺, Sn²⁺, CN ⁻ and N₃⁻ inhibited enzyme activity, while Hg²⁺, Ag⁺, Pb ²⁺, Cr³⁺, EDTA and SDS were not significantly inhibitory. © 1999 Society of Chemical Industry     

Characterisation of cellulose-hydrolysing enzymes from the fungus Bipolaris sorokiniana

Cellulose-hydrolysing enzymes from the phytopathogenic fungus Bipolaris sorokiniana were partially purified and characterised. The enzyme production was variable according to the carbon source. β-Glucosidase and cellobiohydrolase activities were higher by growing the fungus on cellulose than on other carbon sources. Carboxymethyl cellulase production was stimulated by other carbohydrates, mainly lactose. Partial enzyme purification was carried out by liquid chromatography on Sepharose CL4B. The purification was about 17-fold, with a yield of 41% as judged by assay with p-nitrophenyl-β-D -glucopyranoside as substrate. The optimum pH and temperature were 5.0 and 55–60 °C respectively. The enzyme was stable at 28 and 37 °C but lost about 50% of its initial activity after 120 min at 55 °C. Saccharification of cellulosic materials such as crystalline cellulose, filter paper and wheat straws was carried out using the partially purified enzyme, resulting in the production of reducing sugars. © 1999 Society of Chemical Industry     

Purification and Characterization of α-Amylases of an Alkalopsychrotrophic Micrococcus sp

Two amylases of an alkalopsychrotrophic Micrococcus were purified by chromatographies of DEAE-Toyopearl, Butyl-Toyopearl and Shodex WS-2003. Molecular weights and pI values of the purified enzymes, I and II, were 185000 and 125000 by SDS-PAGE and 4.8 and 4.3 by isoelectric focusing, respectively. Enzyme I had not only amylase but also pullulanase activity. In the presence of Ca²⁺ ions, other properties of both enzymes were very similar: optimum temperature 55–60°C, optimum pH 7.5–8.0 k_m value for maltopentaose 0.09 mM. Both amylases were completely inactivated after incubation with EDTA at 30°C and thereafter, could be reactivated by an addition of CaCl₂. In the absence of Ca²⁺ ions, amylase I became thermoresistant, while the thermostability of amylase II decreased. Neither amylase activity of enzyme I nor enzyme II was inhibited by pullulan.     

Purification and Characterization of Proteases from Digestive Tract of Grass Shrimp (Penaeus monodon)

Proteases in grass shrimp (Penaeus monodon) digestive tract were extracted. Four fractions, A, B, C and D, demonstrated caseinolytic activity and were purified to electrophoretic homogeneity. A, C and D were trypsin-like, while B was a chymotrypsin-like protease. Optimal temperature for proteases A, B and C were 65°C, and that for D was 55°C for hydrolysis of casein. Optimal pH of proteases A and C was 8.0, and that of D was 7.0 for hydrolysis of p-toluenesulfonyl-L-arginine methyl ester. Optimal pH of protease B for hydrolysis of N-benzoyl-L-tyrosine ethyl ester was 8.0. Inactivation of 50 % enzyme activity in 5 min occurred at 67°C for protease B and 50°C for proteases A, C and D.     

Effect of combined high pressure and thermal treatment on kiwifruit peroxidase

The effects of high pressure and heat treatments on peroxidase (POD) activity in kiwifruit were investigated. Pressure levels ranging from 200 to 600 MPa and temperatures varying from 10 to 50 °C were applied for up to 30 min. Assays were carried out on crude peroxidase in kiwifruit juice and on partially purified peroxidase in a model system. Pressures higher than 400 MPa could be combined with mild heat (?50 °C) to accelerate enzyme inactivation. Prolongation of the exposure time had no great effect after the first 15 min. The slope of POD in kiwifruit juice at 30 °C was slightly decreased compared with that in a model system. Furthermore, the optimum pH for POD was 6.0–8.5. The presence of POD isoenzymes and their difference in resistance to pressure were thought to be responsible for the final residual activity observed in this study.     

Improved extraction of disulphide‐rich bioactive proteins from soya hulls: characterisation of a novel aspartic proteinase

Soya hull, an underutilised coproduct of soya processing, was investigated as a source of disulphide‐rich bioactive proteins. A Viscozyme L‐assisted extraction method was developed to improve the yield of extracted proteins. The extracted proteins were identified by MALDI TOF–TOF MS, and the most abundant disulphide‐rich protein among identified proteins was purified and the enzymatic properties were evaluated. The results indicated that the Viscozyme L‐assisted extraction method extracted significantly (P < 0.05) more proteins (39.01%) than did the aqueous method (4.52%). The yield of the purified aspartic proteinase nepenthesin‐1‐like [Glycine max] (GmAPN1K) (the most abundant disulphide‐rich protein) is 570 mg Kg^?1. The specific activity of GmAPN1K was 62.1 U mg^?1 at pH 3.0 and 37 °C. The enzyme was optimally active at pH 3.0 and 55 °C. It was stable within pH range 3.0–10.0 and up to 55 °C, respectively, and was specifically inhibited by pepstatin A.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF PEACH PECTINESTERASE

Pectinesterase (EC 3.1.1.11) was extracted from peaches (Prunus persica) and partially purified by preparative free solution isoelectric focusing. On SDS-PAGE gels, protein bands at 36.3 and 33.9 kilodaltons represented the major bands; minor bands were observed at 108.4, 40.7, and 17.0 kilodaltons. The pH optimum for pectinesterase activity in the partially purified extract was 8.0. The enzyme was stable at 30°C for 30 min between pH values of 5 and 8. Peach pectinesterase is stable when heated at 55°C for 5 min in 0.1 M NaCl, 50 mM sodium phosphate, pH 7, buffer. However, residual activity decreased to 23% of 65°C for 5 min and was inactivated at 70°C for 5 min. The energy of activation of peach pectinesterase was determined to be 34, 600 J/mol °K. The Q₁₀ between 30°C and 60°C was estimated to be 1.5–1.6.     

Partial purification and characterization of peroxidase from olives (<Emphasis Type="Italic">Olea europaea</Emphasis> cv. Koroneiki)

The enzyme peroxidase (POD) activity was extracted from olives (Olea europaea cv. Koroneiki) and was partially purified by ammonium sulfate fractionation and gel permeation chromatography (Sephacryl S 300). Further characterization of the POD was performed using the ammonium sulfate purified fraction. POD showed a molecular mass of 44 ± 2 kDa and it expressed catalytic activity with 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), N,N-dimethyl-p-phenylenediamine (DMPD) and some olive fruit phenols. However, the enzyme was found ineffective as regards the oxidation of oleuropein, the major polyphenol of olives, as well as with coumaric, ferulic, ascorbic and p-hydroxy benzoic acids. pH optimum of the peroxidase-catalyzed oxidation depended on the substrate used and it varied from 4.0 to 6.0. Olive peroxidase shows high thermal stability. Oleuropein, the major polyphenol of olives, drastically inhibited ABTS peroxidation by the POD preparation with an IC₅₀ value of 47 μM. The presence of POD enzyme activity in virgin olive oil samples was also confirmed.     

Thermal Inactivation of Asparagus Lipoxygenase and Peroxidase

Thermal stability of lipoxygenase (LOX) and peroxidase (POD) in fresh asparagus tips and partially purified asparagus LOX and POD were compared. In all cases, heating at 50, 60 and 70°C resulted in higher percentages of residual LOX activity than POD activity. Inactivation of LOX followed first order kinetics while inactivation of POD followed a biphasic curve. Activation energies for thermal denaturation of the partially purified enzymes were 47.5 kcal/mol for LOX and 41.9 kcal/mol for POD.     

Purification,properties and heat inactivation of pectin methylesterase from apple (cv Golden Delicious)

Pectin methylesterase from apple (cv Golden Delicious) was extracted and purified by affinity chromatography on a CNBr‐Sepharose®‐PMEI column. A single pectin methylesterase peak was observed. Isoelectric points were higher than 9. Kinetic parameters of the enzyme were determined as K_m = 0.098 mg ml⁻¹ and V_max = 3.86 µmol min⁻¹ ml⁻¹ of enzyme. The optimum pH of the enzyme was above 7.5 and its optimum temperature was 63 °C. The purified PME required the presence of NaCl for optimum activity, and the sodium chloride optimum concentration increased with decreasing pH (from 0.13 M at pH 7 to 0.75 M at pH 4). The heat stability of purified PME was investigated without and with glycerol (50%), and thermal resistance parameters (D and Z values) were calculated showing that glycerol improved the heat resistance of apple PME. © 2000 Society of Chemical Industry     

Purification and characterization of cathepsin B from the gut of the sea cucumber (<Emphasis Type="Italic">Stichopus japonicas</Emphasis>)

Cathepsin B from the gut of sea cucumber (Stichopus japonicas) was purified 81-fold with a 3% recovery by ammonium sulfate fractionation and a series chromatography on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-Gel 3000 SWxl. The purified protein appeared as a single band on Native-PAGE but showed 2 bands of 23 and 26 kDa on SDS-PAGE. The optimum activity was found at pH 5.5 and 45°C. The enzyme was stable at pH 4.5–6.0 and the thermal stability was up to 50oC. The enzyme was strongly inhibited by E-64, iodoacetic acid, and antipain, demonstrating it is a cysteine protease containing sulfhydryl groups. Cu²⁺, Ni²⁺, and Zn²⁺ could strongly inhibit the enzyme activity. The amino acid sequences of the purified enzyme were acquired by mass spectrometer, which did not show any homology with previously described cathepsins, suggesting it may be a novel member.