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.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).     

Properties of polyphenol oxidase in mango (Mangifera indica) kernel

Polyphenol oxidase (PPO) activity of filtered extract of ground mango kernel suspension (400 g litre⁻¹) was studied spectrophotometrically at 420 nm using catechol as substrate. The enzyme was most active at pH 6·0 and 25°C. Activity was reduced by 50% at pH values of 5·0 and 7·1, and also at temperatures of 14°C and 30°C. The calculated activation energy and the Michaelis constant (K_m) were 21·4 kcal mol⁻¹ °C⁻¹ and 24·6 mM , respectively. The V_max value was 2·14 units g⁻¹ mango kernel. The time to heat inactivate PPO decreased rapidly to < 10 min with increasing temperature of ⩾ 70°C at 50% activity. © 1998 SCI.     

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.     

Candida albicans phosphatidylinositol synthase has common features with both Saccharomyces cerevisiae and mammalian phosphatidylinositol synthases

Phosphatidylinositol (PI) synthase (cytidine 5′-diphospho (CDP)-1,2-diacyl-sn-glycerol:myo-inositol 3phosphatidyltransferase, EC 2.7.8.11) was isolated from the microsomal cell fraction of Candida albicans. The Triton X-100 extracted enzyme was enriched 140-fold by affinity chromatography on CDP-diacylglycerol–Sepharose. The enzyme had a pH optimum at 9·5 in glycine/NaOH buffer. It had an absolute requirement for Mg²⁺ or Mn²⁺ and was inhibited by Ca²⁺ and Zn²⁺. Maximal activity was at 0·2–0·6 mm-CDP-diacylglycerol, higher concentrations inhibited the enzyme. With 2′-deoxy-CDP-diacylglycerol as the lipid substrate, optimal activity was at 0·7 mm. The K_m for myo-inositol was determined to be 0·55 mm. The optimal temperature for the PI synthase reaction was 55°C. The C. albicans PI synthase shows differences to the Saccharomyces cerevisiae enzyme, such as activation by bivalent cations, inhibition by nucleotides, temperature optimum and activation energy, but also to the human PI synthase in preference for the lipid substrates, inhibition by nucleoside monophosphates and stabilization by Mn²⁺ and phospholipids.     

Isoelectric fractionation and some properties of a protease from soyabean seeds

A protease, capable of hydrolysing benzoyl DL -arginine p-nitroanilide(BAPA), and L-amino acid β-naphthylamide derivatives, was purified, by isoelectric focusing in the region pH 3–6, from dormant and 6-day germinated soyabean seeds. The enzyme was focused at pH 4·80. The K_m value using BAPA as substrate was found to be 5·03 × 10⁻⁴M . Maximum activity of the enzyme towards BAPA was obtained in the pH 8·2–8–5 region. Slight activation was observed in the presence of 0·05 M concentration of Ca²⁺ and Mg²⁺ ions. The protease lacked caseinolytic activity, and was not inhibited by Kunitz soyabean trypsin inhibitor.     

Purification,crystallisation and characterisation of carboxypeptidase from wheat bran

A carboxypeptidase was purified and crystallised from wheat bran. Disc gel electrophoresis at pH 4·0 and ultracentrifugal analysis revealed that the enzyme was essentially homogeneous. The sedimentation constant and isoelectric point were determined to be 6·3 S and 6·0, respectively. The molecular weight of the enzyme was estimated to be 118,000 by a gel filtration method. The enzyme liberated carboxyl terminal amino acid residues from a wide range of N-substituted dipeptides and tripeptides which contain l-proline. It had a pH optimum at pH 4·0 for Z-Glu-Tyr (Z-benzyloxycarbonyl). The K_m and k_cat values for Z-Glu-Tyr at pH 4·0 and 30°C were 0·19 mm and 20 s^?1, respectively. The enzyme hydrolysed Z-Gly-Pro-Leu-Gly-Pro and bradykinin sequentially at pH 4·0 from their carboxyl terminal amino acid residues. The enzyme activity was completely inhibited by DFP.     

Winged bean lipoxygenase—Part 2: Physicochemical properties

Winged bean lipoxygenase (linoleate: oxygen oxidoreductase EC 1.13.11.12) isoenzymes FI and FII were isolated and purified according to the method of Truong et al. (1980).FI and FII were both highly specific for linoleic acid. They exhibited optimal activity at pH 6·0 and 5·8, respectively at 30°C. An activation energy of 4·5 kcal mol^?1 was calculated for this lipoxygenase within the temperature range of 30–50°C.At 0·075% Tween 20, FI and FII had K_m values for linoleic acid of 0·44 and 0·37 × 10^?3M, respectively, compared to 0·4 × 10^?3M for the crude enzyme. Maximal activity was obtained at 1·6 × 10^?3M. Higher levels of Tween 20 inhibited the lipoxygenase activity.Both isoenzymes had identical average molecular weight of 80 000 daltons by gel filtration and SDS gel electrophoresis.FI and FII isoenzymes were strongly inhibited by Hg⁺⁺, Mn⁺⁺, Mg⁺⁺ and Fe⁺⁺⁺ and activated by Zn⁺⁺, Co⁺⁺ and Fe⁺⁺. A difference in the degree of inhibition or activation was observed between FI and FII response. Ca⁺⁺ inhibited both FI and FII but the former was more sensitive to Ca⁺⁺. KCN also inhibited the two isoenzymes.Among the antioxidants tested, butylated hydroxytoluene and butylated hydroxyanisole most effectively inhibited both FI and FII at only 10^?6M. Sulphydryl reagents such as iodoacetamide and dithiothreitol have little effect on the lipoxygenase isoenzyme activity.The lipoxygenase isoenzymes were more stable at neutral pH. The enzyme in the crude extract and especially in situ was more stable to heat treatment.     

Purification and characterization of highly active and stable polyphosphatase from Saccharomyces cerevisiae cell envelope

Saccharomyces cerevisiae cell envelope polyphosphatase was isolated in highly active and stable form by extraction from cells with zwittergent TM-314 followed by chromatography of the extract on phosphocellulose and QAE-Sephadex in the presence of 5 mM -MgCl₂, 0·5 mM -EDTA and 0·1% Triton X-100. The enzyme possessed a specific activity of 220 U/mg and after 30 days retained 87% of its activity at ?20°C. Polyphosphatase molecular mass was determined to be about 40 kDa by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme hydrolysed polyphosphates with various chain lengths (n = 3–208), had low activity for GTP and did not split pyrophosphate, ATP and p-nitrophenylphosphate. On polyphosphates with chain lengths n = 3, 9 and 208, K_m values were 1·7 × 10^?4, 1·5 × 10^?5 and 8·8 × 10^?7 M respectively. Polyphosphatase was most active and stable at pH 6·0–8·0. The enzyme showed maximal activity at 50°C. The time of half inactivation of polyphosphatase at 40, 45 and 50°C was 45, 10 and 3 min, respectively. In the absence of divalent cations and also with Ca²⁺ or Cu²⁺, the enzyme showed practically no activity. The ability of divalent cations to activate polyphosphatase was reduced in the following order: Co²⁺ > Mg²⁺ > Mn²⁺ > Fe²⁺ > Zn²⁺. Polyphosphatase was completely inhibited by 1 mM -ammonium molybdate and 50 μM -Zn²⁺ or Cu²⁺ (in the presence of Mg²⁺).     

The enzymic release of fatty acids from phosphatidylcholine in green peas (Pisum sativum)

‘Phospholipid acyl-hydrolase’ (PLAH), an enzymic activity releasing fatty acid from phosphatidylcholine (PC), has been identified and characterised in green peas. The K_m value for PC dipalmitoyl ester was 0·167 mm. The enzymic activity possessed a pH optimum of 5·6 and was stable for 20 min only at that pH value. The optimum temperature was 45°C and thermal sensitivity was indicated by a 94% decrease in activity upon exposure of the enzyme to 55°C for 3 min, and by an exponential decrease in activity upon storage at 4°C for 1 week. The enzyme was optimally activated by 2·0 mm calcium chloride at pH 5·6, and the optimal concentration of sodium dodecyl sulphate was 0·75 mg ml^?1. Pea PLAH was non-competitively inhibited by sodium cyanide, EDTA and p-chloromercuribenzoate, with no activity in the presence of mercuric chloride. The results from this study are related to those of other workers on lipid-degrading enzymes in peas, and a pathway is proposed for the enzymic degradation of endogenous lipids in fresh or unblanched frozen peas during post-harvest storage.     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Effects of high pressure on papain activity and structure

Papain was progressively inactivated by increasing pressures in both phosphate (pH 5·0 and 6·8) and Tris (pH 6·8) buffer, but in all systems the effect at pressures up to 600 MPa was minimal and independent of the temperature. However, at 800 MPa, significant losses were found. These losses at 800 MPa were more marked when pressure treatment was at 60°C than when at 20°C. Even though inactivation occurred at 800 MPa, electrophoretic and calorimetric analysis indicated there was little change in size or conformation of the enzyme. The loss in activity, though, was very dependent on the oxygen concentration during pressure treatment, and it is suggested that oxidation of the thiolate ion at the active site, to SO₂⁻ or SO₃⁻, is the major reason for the pressure inactivation of papain. © 1997 SCI.     

ISOLATION AND CHARACTERISATION OF THE AMYLOTIC SYSTEM OF SCHWANNIOMYCES CASTELLII

The amylolytic system of Schwanniomyces castellii has been isolated and purified by means of ultrafiltration followed by polyacrylamide gel electrophoresis. Both α-amylase and glucoamylase were purified. α-Amylase activity was stable from pH 5·5 to 6·5 and glucoamylase activity was stable at a more acidic range of pH 4·2 to 5·5. The optimal temperature of α-amylase activity was between 30 and 40°C with rapid deactivation at 70°C. The optimal temperature of glucoamylase was 40 to 50°C with rapid decline of activity at 60°C. The K_m of α-amylase with soluble starch as the substrate was 1·15 mg/ml and the K_m of glucoamylase with the same substrate was 10·31 mg/ml. Glucoamylase was able to hydrolyze α-1, 4 and α-1,6 glucosidic linkages, as demonstrated by its ability to hydrolyse maltose and isomaltose respectively, whereas α-amylase could hydrolyse α-1,4 glucosidic linkages only. α-Amylase was shown to be a glycoprotein, whereas no carbohydrates were associated with glucoamylase.     

ADENOSINE TRIPHOSPHATASE OF SACCHAROMYCES CARLSBERGENSIS

The ATPase activity of whole cells of anaerobically grown Saccharomyces carlsbergensis was low, but EDTA or glucose revealed ATPase activity. The ATPase was activated by Mg⁺⁺ and Ca⁺⁺; excess Ca⁺⁺ was inhibitory. The optimum pH was 8·0 and optimum temperature about 40° C. The enzyme liberated less than two moles of phosphate from one mole of ATP and other nucleoside triphosphates and released phosphate slowly from ADP; nucleoside monophosphates were not hydrolysed. The enzyme was inhibited by atebrin, sodium azide and ADP, but was unaffected by ouabain, sodium fluoride or 2,4-dinitrophenol.     

Kinetics of degradation of sorbic acid in aqueous glycerol solutions

Degradation of sorbic acid in aqueous glycerol solutions at pH 4·0 over the a_w range 0·71–1·00 and the temperature range 40°–60°C was found to follow first-order reaction kinetics and to conform to the Arrhenius equation. Activation energy values obtained were 5·8 kcal mol^?1 and 7·8 kcal mol^?1 for systems at 0·80 a_w with and without added Co⁺⁺, respectively. The rate of sorbic acid degradation was observed to increase with decreasing a_w (i.e. increasing glycerol concentration). The presence of added Co⁺⁺ decreased the rate of sorbic acid breakdown at any particular a_w or temperature. Browning of sorbate solutions during storage was markedly inhibited by Co⁺⁺.     

Purification and Characterization of a Halotolerant Alkaline Serine Protease from Penicillium citrinum YL-1 Isolated from Traditional Chinese Fish Sauce

A halotolerant alkaline serine protease from Penicillium citrinum YL-1 which was isolated from traditional Chinese fish sauce was purified by ammonium sulfate precipitation, dialysis, and DEAE 52-Cellulose column, thereby resulting in a 4.66-fold increase in specific activity (110.68 U/mg). The molecular weight (MW) was estimated to be 32.27 kDa using SDS-PAGE analysis. The protease exhibited optimal activity toward the substrate casein at pH 8.0 at 40°C and was stable at pH 6.0–8.0 and 4–30°C. Activity was inhibited by NaCl and retained at 28.3, 21.4 and 18.1% of the initial activity after incubation for 6 h at 20, 25 and 30% NaCl concentrations, respectively. The enzyme was stimulated by Mn²⁺ and inhibited by K⁺, Ca²⁺, Zn²⁺, Mg²⁺, Fe²⁺, and Fe³⁺. K_m and V_max of the protease for casein were 1.93 mg/ml and 56.81 μg/(min·ml), respectively. Protease activity was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), which confirmed the serine protease nature of the enzyme. The protease can hydrolyze tilapia protein in the absence or presence of NaCl (5–30%), thus suggesting that this protease is more halotolerant than the protease from other bacteria with high salinity resistance based on the current literature. These properties make the halotolerant alkaline serine protease a suitable candidate enzyme for fish protein hydrolysis during fish sauce fermentation.     

Purification and characterisation of basic ascorbate oxidase from satsuma mandarin (Citrus unshiu Marc)

Basic ascorbate oxidase of the multiple enzyme forms existing in young fruit of satsuma mandarin (Citrus unshiu Marc) has been separated and subsequently purified to electrophoretic homogeneity through (NH₄)₂SO₄ fractionation and chromatographies on DEAE-Toyopearl 650M, CM-Sephadex C-50 and Sephadex G-100. The native molecular weight was estimated to be 141 kDa by gel filtration and composed of two non-identical subunits with an apparent mass of 74 kDa and 62 kDa. The optimum pH was found to be 5.5 with reasonable stability between pH 5 and 8. The enzyme had an optimum temperature at 45°C and was stable up to 50°C upon heat treatment for 5 min. The presence of sodium diethyldithiocarbamate, metabisulphite and potassium cyanide completely inhibited the enzyme activity. Fluoride also inhibited the activity substantially at higher concentrations. Other tnonovalent and divalent metal ions did not have inhibitory effects.     

Injury,Inhibition and Inactivation ofEscherichia coli O157:H7 by Potassium Sorbate and Sodium Nitrite as Affected by pH and Temperature

The effect of potassium sorbate (0–2 g litre⁻¹) and sodium nitrite (0–1 g litre⁻¹) on the growth of four strains of Escherichia coli O157: H7 in tryptic soya broth at various pH levels (pH 4·0–7·0 for sorbate, pH 5·0–8·0 for nitrite) were determined at 37°C and 4°C. Among the pH levels tested, sorbate and nitrite exhibited the highest antimicrobial activity at pH 4·0 and 5·0, respectively. At pH 5·0 and 37°C, the presence of 500 mg litre⁻¹ sorbate or 200 mg litre⁻¹ nitrite completely inhibited the growth of E coli O157: H7. While at higher pH levels, 2 g litre⁻¹ sorbate or 1 g litre⁻¹, nitrite, the highest concentration tested, did not show significant antimicrobial action against the test organisms. At 4°C and pH 5·0, the inoculated test organisms did not showed any significant growth in preservative-free control media. Different degree of inactivation and injury was observed when E coli O157: H7 strain 933 was stored in TSB (pH 5·0) containing 1 g litre⁻¹ sorbate or nitrite at 37°C. At 4°C, inactivation and injury of E coli O157: H7 cells was not observed in the medium containing sorbate or nitrite throughout the 24 h experimental period.     

Purification and Properties of Beta-galactosidase from Streptococcus thermophilus

A β-galactosidase from Streptococcus thermophilus was purified to homogeneity by ammonium sulfate and acetone fractionation, gel filtration on Sephadex G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme preparation exhibited an optimum pH at 6.6–7.0 and an optimum temperature of 57°C. The enzyme was stable at pH 6.8–7.0. Km and V_max for the enzyme, using ortho-nitrophenyl β-D-galactopyranoside as the substrate, were 0.25 mM and 83 μmoles/mg protein/min, respectively. It was strongly inhibited by Hg⁺⁺, Ag⁺, and Cu⁺⁺ as well as pchloro-mercuri benzoate. The enzyme had a molecular weight of about 6 × 10⁵ and was highly specific for β-galactoside bonds.     

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.