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 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⁺).     

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.     

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.     

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.     

植物乳杆菌亚油酸异构酶的分离纯化及其性质研究

经硫酸铵分级沉淀、阴离子交换层析和凝胶过滤,由植物乳杆菌(LactobacillusplantarumL 2 9)分离纯化得到亚油酸异构酶,分子量为4 3ku。对其酶学性质进行研究,结果表明,温度37℃、pH 6 0时酶活性较高;Co2 + 、Fe2 + 可提高酶的活性,Cu2 + 、Zn2 + 则对酶活力有抑制作用;该酶作用于亚油酸的Km=2 5 3×10 -5mol/L ,Vmax=2 5 7×10 -8mol/ (min·mg)。     

Purification and characterization of phosphofructokinase from the yeast Kluyveromyces lactis

Phosphofructokinase from Kluyveromyces lactis was purified by 180-fold enrichment, elaborating the following steps: cell disruption, polyethylene glycol precipitation, affinity chromatography, size exclusion chromatography on Sepharose 6B and on Bio-Sil SEC 400 and ion exchange chromatography. The homogeneous enzyme exhibits a molecular mass of 845±20 kDa as determined by sedimentation equilibrium measurements and a specific activity of 100 units/mg protein. The apparent sedimentation coefficient was found to be s_20,C=20·7±0·6 S and no significant dependence on the protein concentration was observed in a range from 0·2 to 8 mg protein/ml. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed two bands corresponding to molecular masses of 119±5 kDa and 102±5 kDa, respectively. Thus, the enzyme assembles as octamer composed of two types of subunits. From Western blot analysis applying subunit-specific monoclonal antibodies raised against Saccharomyces cerevisiae phosphofructokinase and from the determination of the N-terminal amino acid sequence, the conclusion was drawn that the 102 kDa-subunit corresponds to the β-subunit of the S. cerevisiae enzyme. In contrast to bakers' yeast phosphofructokinase, the K. lactis enzyme exhibits no cooperativity with respect to the substrate fructose 6-phosphate. Both activators AMP and fructose 2,6-bisphosphate decrease the Michaelis constant with respect to this substrate. The enzyme from K. lactis is also inhibited by ATP. Fructose 2,6-bisphosphate or AMP diminish the ATP-inhibition. In contrast to the phosphofructokinase from S. cerevisiae, where fructose 2,6-bisphosphate turned out to be more efficient than AMP, both activators exert similar effects on the K. lactis enzyme. © 1997 John Wiley & Sons, Ltd.     

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.     

Copper–zinc superoxide dismutase from the marine yeast Debaryomyces hansenii

We have isolated the cytosolic form of Cu–Zn superoxide dismutase (SOD) from the marine yeast Debaryomyces hansenii. This enzyme has a subunit mass of 18 kDa. The preparation was found to be heterogeneous by IF electrophoresis with two pI ranges: 5·14–4·0 and 1·6–1·8. The enzyme preparation had a remarkably strong stability at pH 6·0–7·0, surviving boiling for 10 min without losing more than 60% of activity. On Western blots, this enzyme was recognized by antibodies raised in rabbits against D. hansenii extracts, while only a weak cross‐reaction could be detected using antibodies generated against either Saccharomyces cerevisiae or bovine erythrocyte Cu–Zn SODs. In sequencing analysis, a peptide obtained by trypsin digestion was found to have 85% identity to the S. cerevisiae Cu–Zn SOD. Copyright © 1999 John Wiley & Sons, Ltd.     

AN APPROACH TO THE IDENTIFICATION OF A ß-GLUCAN SOLUBILASE FROM BARLEY1

A ß-glucan solubilase activity was demonstrated in barley extract. This enzyme catalyzed the dissolution of barley ß-glucan by releasing a product having a narrow molecular weight distribution and a molecular weight of about 20,000. The enzyme was partially purified by ion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Bio-Gel P-100. Although carboxypeptidase activity was present in the crude extract of barley flour the partially purified ß-glucan solubilase did not hydrolyse N-CBZ-Phe-ala. Examination of extracts from different barley tissues indicated that the ß-glucan solubilase activity was associated with the husks only; a large portion of the activity was extractable from whole barley kernels. About 85% of the enzyme activity in crude extracts from barley flour was retained after 40 min at 62°C. However, the enzyme was much more heat-labile in extracts of whole barley kernels. The pH of maximal activity was found to be about pH 5.7 and results from column chromatography suggested that the enzyme had a low pl value and a MW between 5 × 10⁴ and 6 × 10⁴.     

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.     

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).     

Multiple molecular forms of finger millet subtilisin and amylase inhibitors

Nine varieties of finger millet (Eleusine coracana Gaertn) including a wild form were screened for proteinase and α-amylase inhibitory activities. Subtilisin inhibitory activity was present in all the varieties examined and was highest in the wild form, while the cultivated varieties had more trypsin inhibitory activity. Isoelectric focusing of a 60% ammonium sulphate fraction of the wild form, before and after complexing with subtilisin, showed multiple forms of subtilisin inhibitors one of which was also active against trypsin. Isoforms of finger millet subtilisin inhibitors were isolated from the ammonium sulphate fraction by ion exchange chromatography on DEAE-Sephadex followed by SP-Sephadex chromatography. Copurification of trypsin and α-amylase inhibitory activities with subtilisin inhibitory activity was observed during cation exchange chromatography. PAGE analysis revealed them to be charge isomers with pI in the range 5·5–6·0. SDS–PAGE gave an estimate of 12 000 mol wt for each of them. Finger millet subtilisin inhibitors were more active on bacterial proteinases than on bovine trypsin and chymotrypsin.     

Purification of a cyanide-sensitive superoxide dismutase from soya beans: A food-compatible enzyme preparation

A method of purifying superoxide dismutase (SOD; EC 1.15.1.1), free from lipoxygenase (EC 1.13.11.12) and catalase (EC 1.11.1.6), from ethanol-treated soya beans is described, Preparations with the highest activity were obtained from new season's beans. Although not homogeneous by electrophoretic analysis, the preparation was 300-fold purified. The enzyme has a molecular weight of 36 300 and a subunit molecular weight of 18 000. Its sensitivity to cyanide suggests that it is a typical cupro-zinc SOD. Isoelectric focusing showed the presence of three separate charged species with isoelectric points of pH 4·73 (the major species), 4·55 (the minor species), and 4·37. The enzyme withstands 45 min at 65°C without loss of activity, and loses only half its activity in 30 min at 75°C at pH values between 5 and 8. Severe losses in activity at pH 4 and below are observed at 75°C. The enzyme can be stabilised for storage at 4°C in 60% (v/v) glycerol.     

Purification and Some Properties of Glutaminase fromActinomucor taiwanensis,Starter of Sufu

Glutaminase of Actinomucor taiwanensis was purified approximately 96-fold with a yield of 18%, by sequential fractionation with ammonium sul-phate, anion exchange with DEAE-Sepharose CL-6B and gel filtration with Sephacryl S-200. The pH and temperature optima of purified glutaminase were 8·0 and 45°C, respectively. Glutaminase was stable at a temperature up to 35°C and at pH values of 6·0–8·0. The molecular weight was 80000 as determined from SDS-PAGE. The enzyme activity was markedly inhibited by HgCl₂. In the presence of 100 g litre⁻¹ NaCl, the enzyme activity was inhibited 50%.     

Purification and Characterization of a Pullulan-Hydrolyzing Glucoamylase from Sclerotium rolfsii

The pullulan-hydrolyzing enzyme from the culture filtrates of Sclerotium rolfsii grown on soluble starch as a carbon source has been purified by ultrafiltration (Amicon, PM-10), ion-exchange chromatography (DEAE-Cellulose DE-52) and gel filtration chromatography (Bio-Gel P-150). The enzyme moved as a single band in non-denaturing polyacrylamide gel electrophoresis carried out at pH 2.9 and 7.5. The relative molecular mass of the enzyme was estimated to be 64.000 D by SDS-PAGE and 66.070 D by gel filtration on Bio-Gel P150. The enzyme hydrolyzed pullulan optimally at 50°C between pH 4.0–4.5, whereas, soluble starch was optimally hydrolyzed at a pH of between 4.0–4.5 and at 65°C. The Michaelis constant (Km) for pullulan was 5.13mg·ml⁻¹ (V_max 1.0U · mg⁻¹) and for soluble starch, it was 0.6mg · ml⁻¹ (V_max 8.33 U · mg⁻¹). The enzyme was observed to be a glycoprotein (12–13% carbohydrate by weight) and had a strong affinity for Concanavalin A. The enzyme hydrolyzed α-D-glucans in an exo-manner, which resulted in the release of glucose as the sole product of hydrolysis. Acarbose, a maltotetraose analog, was found to be a potent inhibitor of both pullulan and starch hydrolysis (100% inhibition at 0.06 μM). The enzyme has been characterized as a glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.     

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.     

Chicken pancreatic enzymes for clinical use: 2. Preparation and storage stability of lipase-rich pancreatin

A lipase-rich pancreatin was prepared from chicken pancreas by extracting the pancreatic pulp with phosphate-buffered saline (pH8·0), precipitating the soluble enzyme by cold isopropanol, and drying under vacuum. Loss of lipase activity was 8% on a laboratory scale (20 g) and 43% on a pilot scale (200 g). Precipitation of the enzyme by acetone resulted in lipase activity losses of 35 and 70%, respectively. Protease and lipase activity was compared with that of several commercial preparations of porcine origin. The chicken pancreatin exhibited good stability when stored over a period of 4 months at 45°C. However, storage in an atmosphere of high water activity (a_w = 0·8) resulted in the activation of trypsinogen and chymotryp-sinogen and consequent inactivation of amylase and lipase.     

Isolation and characterisation of four trypsin-chymotrypsin inhibitors from lentil seeds

Twenty-three proteinase inhibitors were isolated from Syrian local small lentils (Lens culinaris) by ammonium sulphate fractionation of the acidic extract followed by affinity chromatography on anhydrotrypsin-Sepharose. They all inhibited human and bovine trypsin and chymotrypsin. Three inhibitors (LCI-11·7, -3·3 and -4·6) were separated and purified to homogeneity by anion exchange chromatography and preparative isoelectric focusing (IEF) with immobilised pH gradients; a fourth (LCI-2·2) required additional reversed-phase high-pressure liquid chromatography. The four inhibitors were similar in their amino acid composition, with high cystine and aspartic acid/asparagine content, and lack of free sulphydryl groups, methionine and tryptophan. The calculated minimum number of amino acid residues per molecule, the calculated molecular masses confirmed by gel liquid chromatography, gel-permeation high-pressure liquid chromatography and sodium-dodecylsulphate polyacrylamide gel electrophoresis, and the isoelectric points determined by IEF (immobilised pH gradients and carrier ampholytes) were 84, 77, 68 and 60 residues per molecule, 9200, 8500, 7200 and 6750, and 5·26, 5·88, 6·80 and 7·80 for LCI-1·7, -2·2, -3·3 and -4·6, respectively. All four inhibitors inhibited human trypsin less than bovine trypsin, and human chymotrypsin more than the bovine enzyme. All these properties are in accordance with the classification of the four lentil inhibitors as members of the Bowman-Birk proteinase inhibitor family. © 1998 Society of Chemical Industry.     

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.