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     

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.     

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.     

Purification of a lipase from the hepatopancreas of oil sardine (Sardinella longiceps linnaeus) and its characteristics and properties

Lipase has been purified from the hepatopancreas of oil sardine (Sardinella longiceps) by defatting, water extraction, ammonium sulphate fractionation and chromatography on DEAE Sephadex and Sephadex G-100. The preparation was homogeneous on polyacrylamide disc gel electrophoresis and on gel filtration through Sephacryl S-200. The enzyme showed a molecular weight of 54000±57000 with 6.1% of carbohydrate. The pH and temperature optima of purified sardine lipase were 8 and 37°C respectively. Sardine lipase remained stable up to 45°C (15 min) and in the pH range 5 to 9.5. The K_m values obtained for the substrates tributyrin and triacetin were 4 × 10^?2 and 30 × 10^?2, respectively. The effect of halogens and various metal ions on sardine lipase activity, substrate specificity, amino acid and carbohydrate composition are also reported.     

Mechanism of Browning in Fresh Highbush Blueberry Fruit (Vaccinium corymbosum L). Partial Purification and Characterisation of Blueberry Polyphenol Oxidase

The most efficient method for polyphenol oxidase (PPO) extraction from Highbush blueberry fruits was the preparation of an acetone powder. No activity was detected after direct extraction with phosphate buffer (pH 6·5) and detergents such as Triton X-100. PPO has been purified 19-fold, by ultrafiltration, ammonium sulphate precipitation and hydrophobic chromatography. Native-PAGE of the purified fraction revealed the presence of two isoforms. PPO has an observed optimum pH at 4·0, followed by a shoulder at pH 5·0. Caffeic acid is the best substrate (100%), followed by chlorogenic acid (60%) and pyrocatechol (32·5%). No activity was detected towards catechin, protocatechuic acid, resorcinol and monophenols. © 1997 SCI.     

Purification and characterization of hydroperoxide lyase from amaranth tricolor (Amaranthus mangostanus L.) leaves

Hydroperoxide lyase (HPL) was extracted from amaranth tricolor leaves using Triton X-100, and purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography. The purified HPL preparation consisted of a single band and spot with a molecular mass of about 55 kDa as shown in SDS–PAGE and 2-D PAGE, respectively; the isoelectric point was found to be about 5.4. The maximum activity of the enzyme was observed at pH 6.0 and 25 °C, respectively. The HPL showed higher activity against 13-hydroperoxy-linolenic acid compared to 13-hydroperoxy-linoleic acid. K _m value for 13-hydroperoxy-linolenic acid was 62.7 μM, and the corresponding V _max was 178.5 μM min⁻¹. The activity of HPL was significantly inhibited by nordihydroguaiaretic acid, HgCl₂ and 2(E)-hexenal but not by EDTA and hexanal.     

Purification,characterisation and kinetics of a trypsin inhibitor from black gram (Vigna mungo)

A black gram (Vigna mungo) trypsin inhibitor (BGTI) was purified to homogeneity by ammonium sulphate precipitation, DEAE-cellulose chromatography and affinity chromatography. The homogeneity of the inhibitor was identified by polyacrylamide gel electrophoresis (PAGE), SDS–PAGE, immunodiffusion and immunoelectrophoresis. The isoelectric point of BGTI was 8.32. The molecular weight of the BGTI was found to be about 12500 by both gel filtration and SDS–PAGE. One mole of the BGTI was found to contain 75 amino acid residues, and it was identified as a glycoprotein, comprising about 18.2% carbohydrate. The BGTI was an uncompetitive inhibitor, stable in the pH range 2–10 and in the temperature range 30–80°C for 30 min.     

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     

Purification and some properties of polyphenoloxidase in eggplant (Solanum melongena)

Polyphenoloxidase (EC 1.10.3.1) in eggplant (Solatium melongena L) was purified by ammonium sulphate fractionation, DEAE-Cellulofine and DEAE-Toyopearl chromatography and Sephadex G-100 gel filtration. The enzyme was purified about 110-fold with a recovery of 5%. The purified enzyme more quickly oxidised chlorogenic acid (5-caffeoylquinic acid, IUPAC) than 10 other substrates used. The K_m value for the enzyme was found to be 0·50 mM with respect to chlorogenic acid; the optimum pH of the enzyme was about 4 with enzyme stability between pH 5 and 8. The enzyme was completely inactivated after heat treatment at 75°C for 30 min or 80°C for 5 min. Sodium metabisulphite, potassium cyanide and sodium fluoride markedly inhibited the enzyme activity.     

PURIFICATION AND CHARACTERIZATION OF AN α-AMYLASE INHIBITOR FROM RYE (SECALE CEREALE) FLOUR

An α-amylase inhibitor from rye (Secale cereale) flour has been purified to homogeneity by extraction with 70% ethanol, ammonium sulfate fractionation and column chromatography on DEAE- and CM-cellulose. The isoelectric point was pH 5.8, and the molecular weight 28,000 by polyacrylamide gel electrophoresis with different gel concentrations and 27,000 by sedimentation equilibrium centrifugation. Under denaturating conditions the molecular weight was about 14,000, indicating two subunits identical in size. The inhibitor was active towards human salivary and hog pancreatic α-amylases but inactive towards Bacillus subtilis and Aspergillus oryzae α-amylases. The pH optimum for the reaction between the rye inhibitor and human salivary α-amylase was 6.0. The inhibitor did not change activity when exposed to pH 2 (0.01M HCl), but prolonged digestion by trypsin destroyed the inhibitor. The rye α-amylase inhibitor lost about 80% of its activity after 10 min at 100°C.     

Enhanced Thermostability of Lactase (Escherichia coli) in Milk

Denaturation of lactase in phosphate buffer was a first-order process with a half-life of 1.12 min at 60°C. Denaturation in milk was a biphasic first-order process with a half-life of 115 min at 60°C. Electrophoresis, isoelectric focusing and kinetic analysis all indicated the presence of two isozymes. The minor isozyme exhibited 9% of the total activity and was less thermostable in milk than the major isozyme. In the temperature range 53.5–60°C, the rate of denaturation of the major isozyme in milk was more than 100 times less than the rate in buffer. Arrhenius plots of denaturation in milk and buffer were biphasic. Above 56°C, E_a denaturation in milk and buffer was 187 Kcals/mole and 19.8 Kcals/mole, respectively; below 56°C E_a denaturation in milk and buffer was 36.8 Kcals/mole and 158 Kcals/mole, respectively.     

PURIFICATION AND PHYSICOCHEMICAL PROPERTIES OF AN EXTRA CELLULAR AMYLASE FROM A STRAIN OF BACILLUS AMYLOLIQUEFACIENS ISOLATED FROM DRY ONION POWDER

An extracellular α-amylase from Bacillus amyloliquefaciens, isolated from dry onion powder, has been purified to homogeneity by ammonium sulfate fractionation, adsorption on starch, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100 column. The enzyme consisted of one polypeptide chain with a molecular weight of 60,000. The isoelectric point was pH 5.2, the pH optimum 5.5 and the temperature optimum ranging from 50°-70°C. Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The K_m for starch was 6.9 mg/ml. The enzyme was quite stable at 50°C, but lost about 85% of its activity at 60° after 30 min (pH 6.0).     

Purification and Characterization of Bacillus acidopullulyticus Pullulanase for Enzymatic Starch Modification

The pullulanase (EC 3.2.1.41) of Bacillus acidopullulyticus was purified using anion exchange chromatography and preparative isoelectric focusing. The purified preparation migrated as a single protein band upon SDS gel electrophoresis and its molecular weight was estimated to be 102,000. Two main activity bands having pl-values 5.0 and 5.2 were detected by analytical isoelectric focusing. The enzyme was purified 4-fold with the yield 38% by this two-step purification procedure. The purified pullulanase showed maximal activity at 50°C and pH 5 and was slightly activated by Ca₂₊. It was stable at 50°C but totally lost its activity at 60ºC in one hour. This purified pullulanase efficiently hydrolyzed the α-1,6-glucosidic bonds of pullulan and gelatinized starch.     

ISOLATION,PURIFICATION AND ELECTROPHORETIC PROPERTIES OF AN α-AMYLASE FROM MALTED BARLEY

An α-amylase component from malted barley was isolated and purified using aqueous extraction at pH 8·0, heat treatment of the extract at 70°C, specific precipitation with glycogen and ion exchange chromatography on carboxymethyl (CM) and diethylaminoethyl (DEAE) cellulose. The enzyme preparation was shown to be pure by disc electrophoresis at pH 8·9 and iso-electricfocusing on polyacrylamide gel in a pH 4–8 gradient.     

PURIFICATION AND CHARACTERIZATION OF INVERTASE FROM DATES (PHOENIX DACTYLIFERA L., VAR. ZAHDI)

Zahdi dates (Phoenix dactylifera) contain invertase at all development stages; the highest specific activity is present in the late yellow stage. The enzyme was purified to homogeneity, as determined by disc gel electrophoresis and isoelectric focusing, by a combination of techniques including ammonium sulfate precipitation, DEAE-cellulose chromatography and gel filtration on Sepharose 4B and Sephadex G-150 columns. A complex of invertase with a high molecular weight pectic substance of the date could not be dissociated by ammonium sulfate or DEAE-cellulose chromatography but the complex was dissociated by gel filtration on a Sepharose 4B column at pH 4.0 and ionic strength of 0.5 M. The enzyme contained 8.2% carbohydrate covalently linked probably via an amide linkage to aspartic acid. Molecular weight determination by exclusion gel chromatography and sedimentation equilibrium gave values of 130,000 and 97,100 ± 1,300, respectively. The enzyme is probably composed of two identical subunits as shown by SDS polyacrylamide gel electrophoresis. Amino acid analyses showed the enzyme to be low in sulfur-containing amino acids. Date invertase is an acid β-fructofuranosidase with a pH optimum between 3–4 and with a K_m and k_cat for sucrose of 6mM and 49 sec^-1, respectively. Activation energies for denaturation of enzyme and conversion of substrate to product were determined to be 48.7 and 17.6 kcal/mole, respectively. Chemical modification indicated that sulfhydryl groups are probably not essential for activity while carboxyl groups may be involved in the active site of the enzyme.     

Bovine pancreatic lipase.I.Isolation, homogeneity, and characterization.

Bovine pancreatic lipase was isolated in pure form by lyophilization of fresh bovine pancreas, extraction of the enzyme with sucrose solution, fractional precipitation with ammonium sulfate and acetone, followed by chromatography on Sephadex G-100. The specific activity of the purest lipase fraction was 1750 micromoles fatty acid, liberated in 30 min per milligram of protein, indicating a purification of approximately 473-fold, with an overall yield of about 42%. Homogeneity of the enzyme was confirmed by rechromatography on Sephadex G-100 as well as with the gel electrophoretic and ultracentrifugal techniques. The purified enzyme gave a typical protein ultraviolet absorption spectrum with maximum absorption at 276 nm and minimum at 252 nm. The purified enzyme exhibited a single pH optimum of 8.8 and an isoelectric point near pH 5.5. Its optimum temperature was 37 C, and its optimum substrate concentration was 10%. These properties resembled those of milk lipase.     

A Novel Type Mitochondrial Monoamine Oxidase from the Liver of Skipjack Tuna (Katsuwonus pelamis): Purification and Characterisation

A novel type of monoamine oxidase (EC. 1.4.3.4) present in the liver of skipjack tuna (Katsuwonus pelamis) was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by ammonium sulphate fractionation, followed by Sephadex G-200, butyl-toyopearl 650 M, phenyl-toyopearl 650 M and hydroxyapatite chromatography. The molecular weight of the enzyme was estimated to be about 110000 by gel filtration on Sephadex G-200. The enzyme was activated by Mn²⁺, but was inhibited by Cu²⁺, Zn²⁺ and p-chloromercuribenzoic acid (PCMB). Furthermore, the enzyme was completely inhibited by Hg²⁺ and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). © 1997 SCI.     

Isolation,purification and characterisation of polygalacturonase from ripened banana (Musa acuminata cv. Kadali)

Bananas are the most important fruit crop in the world. Short shelf life of the fruit is the major limiting factor in international trade, and this is due to the softening of the pulp during ripening. Fruit softening is an important aspect of ripening process in fleshy fruits and is caused by the cumulative action of a group of cell wall‐modifying enzymes. Polygalacturonase (PG) is the key enzyme involved in the fruit softening process in banana, and this study reports the isolation, purification and characterisation of polygalacturonase enzyme from ripened fruits of a delayed ripened banana cultivar found specifically in Kerala (Musa acuminata cv. Kadali). PG was purified by ammonium sulphate fractionation followed by DEAE cellulose ion exchange chromatography and gel filtration using Sephadex G 100. The purified protein showed two subunits on SDS‐PAGE and a single band on native PAGE. Enzyme showed maximum activity at pH 3.5 and 40 °C. Fe³⁺ enhanced the activity more, while Mg²⁺ and Ca²⁺ slightly stimulated the activity of purified enzyme. Km value for substrate polygalacturonic acid was 0.06%.     

Solubilization, partial purification and properties of lipoxygenase from apples

Summary The effectiveness of a number of solubilizing agents upon the extraction of apple lipoxygenase clearly demonstrated its membrane-bound nature. Triton X-100 (1% v/v) and sodium cholate (75 mM) proved most useful. Also, during subsequent purification procedures a suitable detergent (Triton X-100) had to be included to prevent aggregation phenomena, indicating the integral character of the enzyme.Partial purification of a lipoxygenase extract from apples was achieved by gel filtration on Sephadex G-50 and Sephacryl S-300 sf. Ion-exchange chromatography and affinity chromatography proved to be of little success.Some characteristics of the partially purified lipoxygenase were determined. The enzyme showed optimal activity at pH 6.9 and preferably peroxidized free linoleic acid. Apparent molecular weight (206,000 dalton) and Stokes' radius (51.5 Å) indicated a strong association with a substantial amount of Triton X-100.

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Solubilisierung, partielle Reinigung und Eigenschaften von Lipoxygenase aus Äpfeln

Zusammenfassung Die Wirksamkeit einer Anzahl lösender Agentien auf die Extraktion von Apfel-lipoxygenase zeigt deutlich ihre membrangebundene Natur. Es stellt sich heraus, daß Triton X-100 (1% v/v) und Natriumcholat (75 mM) am wirksamsten sind. Auch während der darauffolgenden Reinigung mußte ein geeignetes Detergens (Triton X-100) hinzugefügt werden, um Aggregations-Phänomene zu verhindern. Dieses zeigt den integralen Charaketer des Enzyms.Partielle Reinigung eines lipoxygenasehaltigen Extraktes aus Äpfeln wurde durch Gelfiltration über Sephadex G-50 and Sephacryl S-300 sf erreicht. Ionenaustausch-und Affinitäts-Chromatographie waren nicht sehr erfolgreich.Es wurden einige Eigenschaften der teilweise gereinigten Lipoxygenase untersucht. Das Enzym zeigte optimale Aktivität bei pH 6,9 and oxidierte vorwiegend freie Linolsäure. Das scheinbare Molekulargewicht (206000 Dalton) and der Stokes' Radius (51,5 Å) wiesen auf eine starke Bindung mit einer bedeutenden Menge von Triton X-100 hin.

     

Polyphenol Oxidase from Apple (Malus domestica Borkh. cv Bramley's Seedling): Purification Strategies and Characterization

ABSTRACT Polyphenol oxidase (PPO) was isolated from Bramley's Seedling apples with 75.7‐fold purification and 26.5% recovery by ammonium sulfate precipitation, phenyl sepharose chromatography, ion exchange chromatography, and hydroxyapatite chromatography. Molecular weight was estimated to be about 45 kDa by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS PAGE). Optimum PPO activity was at pH 6.5 and greater than 50% activity was retained during storage for 72 h at pH 5.5 to 6.5. Optimum temperature for activity was 30 °C and the enzyme had residual activity of greater than 50% during storage for 72 h at 20 °C to 30 °C and for 24 h at 40 °C to 50 °C. Of the substrates tested, activity was greatest with 4‐methylcatechol followed by catechol, pyrogallol, and (?)epicatechin. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid.