Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria

Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.     

A novel serine protease cryptolepain from Cryptolepis buchanani: purification and biochemical characterization

A novel protease is purified to homogeneity from the latex of a medicinally important plant Cryptolepis buchanani of family Apocynaceae (formerly Asclepiadaceae). The enzyme named cryptolepain has a molecular mass of 50.5 kDa. The isoelectric point and extinction coefficient (epsilon280nm1%) are 6.0 and 26.4, respectively. Cryptolepain contains 15 tryptophans, 41 tyrosines, and eight cysteine residues forming four disulfide bridges. The detectable carbohydrate moiety in the enzyme was found to be 6-7%. Cryptolepain hydrolyzes denatured natural substrates like casein, azocasein, and azoalbumin with high specific activity. The protease is exclusively inhibited by serine protease inhibitors phenylmethansulfonyl fluoride and diisopropyl fluorophosphate. Hydrolysis of azoalbumin by the cryptolepain is optimal in the pH range of 8-10 and temperatures of 65-75 degrees C. The enzyme shows high stability against pH (2.5-11.5), temperature (up to 80 degrees C), and chemical denaturants. The Km value of the enzyme was found to be 10 microM with azocasein as the substrate. The N-terminal sequence of cryptolepain is unique and shows only little homology to other known serine proteases, which makes this enzyme an ideal candidate for our ongoing biochemical and structure-function investigations of proteases. Easy availability of the latex and simple purification procedures make the enzyme a good system for exploring the biophysical chemistry of serine proteases as well as applications in the food industry.     

A high cysteine containing thiol proteinase from the latex of Ervatamia heyneana: purification and comparison with ervatamin B and C from Ervatamia coronaria

A cysteine protease, with a high cysteine content and a high degree of amino terminal sequence homology with ervatamins B and C, has been purified from the latex of Ervatamia heyneana (Family Apocynaceae). The enzyme designated as heynein (M(r) = 23 kDa) has a comparatively high cysteine content (11), high isoelectric point (10.8), and high stability against pH (2.5-11.5), temperature (63 degrees C, 15 min), strong denaturants, and organic solvents. The enzyme has high specific activities for natural substrates such as casein and azoalbumin. The pH and temperature optima are pH 8.0-8.5 and 52 +/- 2 degrees C, respectively. Hydrolysis of synthetic substrates and digestion of bovine serum albumin confirm a distinct specificity of heynein as compared to ervatamins and papain. Also, heynein has distinct immunogenicity as monitored by enzyme-linked immunosorbent assay and Ouchterlony's double immunodiffusion. Strong enzyme activation by reducing agents such as beta-mercaptoethanol, dithiothreitol, and strong enzyme inhibition by thiol proteinase inhibitors such as E-64 and iodoacetic acid have evidenced heynein to be a cysteine protease. High stability, specific activity, and easy purification may make heynein a potential protease for food and biotechnology applications.     

Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae)

A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibited high thermal stability (inactivation required heating for 60 min at 75 degrees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 and molecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value approximately 9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme.The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.     

Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory)

A new serine protease from the latex of Ipomoea carnea spp. fistulosa (Morning glory), belonging to the Convolvulaceae family, was purified to homogeneity by ammonium sulfate fractionation followed by cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24 kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. The pH and temperature optima for proteolytic activity were 6.5 and 65 degrees C, respectively. The extinction coefficient (epsilon2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35 tryptophan, 76 tyrosine, and seven cysteine residues. The effect of several inhibitors such as iodoacetic acid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsin inhibitor, HgCl2, 3S-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid, N-ethyl maleimide, ethylene glycol-bis(alpha-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and o-phenonthroline indicates that carnein belongs to the family of serine proteases. The enzyme is not prone to autolysis even at very low concentrations. The N-terminal sequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarity to those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilase family endopepetidases.     

Isolation and thermal characterization of an acidic isoperoxidase from turnip roots

An acidic peroxidase (pI approximately 2.5) was purified from turnip roots (TAP), and its thermal properties were evaluated. TAP is a monomeric protein having a molecular weight (MW) of 49 kDa and a carbohydrate content accounting for 18% of the MW. The yield of pure TAP was relatively high ( approximately 2 mg/kg of fresh roots), with a specific activity of 1810 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) units/mg at pH 6. The activity increased 4-fold at the optimum pH (4.0) to 7250 ABTS units/mg, higher than that of most peroxidases. TAP was heat stable; heat treatment of 25 min at 60 degrees C resulted in 90% initial activity retention, whereas an activity of 20% was retained after 25 min of heating at 80 degrees C. TAP regained 85% of its original activity within 90 min of incubation at 25 degrees C, following heat treatment at 70 degrees C for 25 min. Thermal inactivation caused noticeable changes in the heme environment as evaluated by circular dichroism and visible spectrophotometry. TAP was rapidly denatured by heating in the presence of 1.0 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but the Soret band and activity were fully recovered by adding an excess of Ca(2+). This is further evidence that Ca(2+) plays an important role in the stability of TAP. The high specific activity of TAP, together with its relatively high thermal stability, has high potential for applications in which a thermally stable enzyme is required.     

Purification, characterization, and solvent-induced thermal stabilization of ficin from Ficus carica

Ficin (EC 3.4.22.3), a cysteine proteinase isolated from the latex of a Ficus tree, is known to occur in multiple forms. Although crude ficin is of considerable commercial importance, ficin as such has not been fully characterized. A major ficin from the commercial crude proteinase mixture preparation of Ficus carica was purified and characterized. The purified enzyme was homogeneous in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography and is a single polypeptide chain protein with a molecular mass of 23 100 +/- 300 Da as determined by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF). The enzyme was active in the pH range of 6.5-8.5, and maximum activity was observed at pH 7.0. The N-terminal core sequence of ficin has homology with N-terminal sequences of plant cysteine proteinases. The enzyme contains three disulfide bonds and a single free cysteine residue at the active site. The effect of co-solvents, such as sorbitol, trehalose, sucrose, and xylitol, on the thermal stability of ficin was determined by activity measurements, fluorescence, and thermal denaturation studies. The apparent thermal denaturation temperature (T(m)) of ficin was significantly increased from the control value of 72 +/- 1 degrees C in the presence of all co-solvents. However, the maximum stabilization effect was observed in terms of thermal stabilization by the co-solvent trehalose.     

Muscadine Grape ( Vitis rotundifolia ) and Wine Phytochemicals Prevented Obesity-Associated Metabolic Complications in C57BL/6J Mice

The objective of this study was to determine the effects of muscadine grape or wine (cv. Noble) phytochemicals on obesity and associated metabolic complications. Muscadine grape or wine phytochemicals were extracted using Amberlite FPX66 resin. Male C57BL/6J mice were given a low-fat diet (LF, 10% kcal fat), high-fat diet (HF, 60% kcal fat), HF + 0.4% muscadine grape phytochemicals (HF+MGP), or HF + 0.4% muscadine wine phytochemicals (HF+MWP) for 15 weeks. At 7 weeks, mice fed HF+MGP had significantly decreased body weights by 12% compared to HF controls. Dietary MGP or MWP supplementation reduced plasma content of free fatty acids, triglycerides, and cholesterol in obese mice. Inflammation was alleviated, and activity of glutathione peroxidase was enhanced. Consumption of MGP or MWP improved insulin sensitivity and glucose control in mice. Thus, consumption of muscadine grape and wine phytochemicals in the diet may help to prevent obesity-related metabolic complications.     

Reactivation of broccoli peroxidases: structural changes of partially denatured isoenzymes

Structural changes involved in the reactivation of peroxidases (PODs) from broccoli and horseradish (HRP) following heat denaturation were investigated by using circular dichroism and absorption spectroscopy. Cooling heat-treated enzymes resulted in rapid refolding of the secondary structure into an inactive structural species, similar in conformation to the native enzyme. Reassociation of heme to the refolded peroxidase, as well as molecular rearrangement of the structure around the heme, occurs during incubation at approximately 25 degrees C and results in the return of biological activity. The secondary structure of neutral broccoli POD (N) is relatively heat labile, resulting in a rapid loss of activity, but the level of reactivation is high because the structure at the heme pocket is relatively stable. Acidic broccoli POD and HRP are more heat stable than N, but have a low degree of reactivation. Loss of activity is due primarily to alteration of the structure at the heme pocket. Effects of bovine serum albumin and pH on reactivation of PODs are also discussed. Keywords: Peroxidase; reactivation; horseradish; broccoli; circular dichroism; absorption spectroscopy.     

Characterization of different commercial soybean peroxidase preparations and use of the enzyme for N-demethylation of methyl N-methylanthranilate To produce the food flavor methylanthranilate

The potential of different peroxidase preparations for the N-demethylation of methyl N-methylanthranilate to produce the food flavor methylanthranilate (MA) was investigated. All tested peroxidase preparations were able to catalyze the N-dealkylation. The tested soybean preparations vary widely with respect to their heme content. Furthermore, the operational stability of purified soybean peroxidase (SP) is at least 25-fold lower than that of horseradish peroxidase and only 5-fold higher than that of microperoxidase 8. Thus, the presence of a large protein chain around a porphyrin cofactor in a peroxidase is, by itself, insufficient to explain the observed differences in operational stability. Despite its relatively low operational stability, SP proved to be the most efficient biocatalyst for the production of MA with high yield and purity, especially observed at the high temperature and low pH values at which SP appeared to be optimally active.     

Extractability and Subsequent Biodegradation of PAHs from Contaminated Soil

The biodegradation of polyaromatic hydrocarbons (PAHs) has been well documented; however, the biodegradation of PAHs in contaminated soil has proved to be problematic. Sorption of PAHs to soil over time can significantly decrease their availability for extraction much less than for biodegradation. In this study the ability of various organic solvents to extract PAHs from coal tar-contaminated soil obtained from former manufactured gas plant (MGP) sites was investigated. Solvents investigated included acetone/hexane, dichloromethane, ethanol, methanol, toluene, and water. The extraction of MGP soils with solvents was investigated using soxhlet extraction, multiple soxhlet extractions, sonication, and brief agitation at ambient temperature with a range of solvent concentrations. Of particular interest was the documentation of the recalcitrance of PAHs in weathered MGP soils to extraction and to bioremediation, as well as to demonstrate the ease with which PAHs extracted from these soils can be biodegraded. The efficiency of extraction of PAHs from MGP soils was found to be more dependent upon the choice of solvent. The environmentally-benign solvent ethanol, was shown to be equal to if not better than acetone/hexane (the EPA recommended solvent) for the extraction of PAHs from MGP soils, brief contact/agitation times (minutes) using small quantities of ethanol (2 volumes or less) can achieve nearly quantitative extraction of PAHs from MGP soils. Moreover aqueous slurries of an MGP soils experienced less than 10% biodegradation of PAHs in 14 days while in the same period about 95% biodegradation was acieved using PAHs extracted from this soil by ethanol and subsequently added to aqueous bacterial suspensions.     

Influence of the extent of hemoglobin hydrolysis on the digestive absorption of heme iron. An in vitro study

This study was designed to assess the interactions of heme with peptides produced by enzyme hydrolysis of hemoglobin, and their relationship with heme iron absorption. Bovine hemoglobin was hydrolyzed by pepsin or by subtilisin, which differ in their hydrolysis processes. The hydrolysis rate ranged from 0 (native hemoglobin) to 15%. Heme solubility and heme-peptides interactions were compared to iron absorption by the Ussing chamber model, at intestinal pH (7.5). Increasing hemoglobin hydrolysis enhanced iron absorption; the highest value was reached between 8 and 11% hydrolysis, whatever the enzyme used. Comparing the products of hydrolysis of the two enzymes showed that heme iron absorption depends not only on its solubility, but relies mainly on the balance between the strength of heme-peptides and the polymerization rate of heme.     

Purification and properties of a neutral peroxidase isozyme from turnip (Brassica napus L. Var. Purple Top White Globe) roots

A neutral peroxidase isozyme (pI 7.2) from turnip roots (TNP) was purified to homogeneity and partially characterized. TNP is a monomeric glycoprotein with 9.1% carbohydrate content and a molecular weight of 36 kDa. Optimum pH values for activity using 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and guaiacol as H donors were 4.5 and 5.5, whereas the K(m) values were 0.7 and 3.7 mM, respectively. The ABTS K(m) was approximately 7 times higher than that reported for basic commercial horseradish peroxidase (HRP-C). TNP retained approximately 70% activity after 11 min of heating at 65 degrees C, whereas the activation energy for inactivation (132 kJ/mol) was higher than or comparable to that of other peroxidases. The low ABTS K(m) and high specific activity (1930 units/mg) gave a high catalytic efficiency (500 M(-1) s(-1)). These properties make TNP an enzyme with a high potential as an alternative to HRP in various applications.     

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.     

Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp

Polyphenol oxidase (EC 1.10.3.1, PPO) in the pulp of banana (Musa sapientum L.) was purified to 636-fold with a recovery of 3.0%, using dopamine as substrate. The purified enzyme exhibited a clear single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight of the enzyme was estimated to be about 41000 and 42000 by gel filtration and SDS-PAGE, respectively. The enzyme quickly oxidized dopamine, and its K(m) value for dopamine was 2.8 mM. The optimum pH was at 6.5, and the enzyme activity was stable in the range of pH 5-11 at 5 degrees C for 48 h. The enzyme had an optimum temperature of 30 degrees C and was stable even after a heat treatment at 70 degrees C for 30 min. The enzyme activity was completely inhibited by L-ascorbic acid, cysteine, sodium diethyldithiocarbamate, and potassium cyanide. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

Cloning, heterologous expression and properties of a recombinant active turnip peroxidase

Turnip (Brassica napus) roots peroxidase isoforms have been used in diagnostic kits and can also efficiently polymerize phenolic compounds from wastewaters. Heterologous expression of a turnip acidic peroxidase (BnPA) was investigated to increase availability of this widely used enzyme. The mature BnPA was ligated into the pET28a(+) vector and used to transform Escherichia coli Rosetta 2. Recombinant BnPA peroxidase was overexpressed and accumulated in inclusion bodies from which it was purified to homogeneity by immobilized metal affinity chromatography under denaturing conditions. Peroxidase activity was observed after a refolding process under oxidative conditions. The yield of pure recombinant BnPA was 29 mg L(-1) of culture with a specific activity of 981 ± 20 ABTS units mg(-1) at optimal conditions (pH 6, 45 °C). Recombinant BnPA showed similar kinetic properties compared to native turnip peroxidase, and its secondary structure evaluated by circular dichroism comprised 20% α-helix, 32% β-sheet and 48% random structure. Recombinant BnPA showed high yield and good kinetic properties which are key steps for future structure-function studies and biotechnological applications.     

Effect of Bonding Forces on Corn Starch Isolation

Chemical treatments with a number of low‐toxicity or nontoxic reagents were applied to corn slurry to investigate the disruption or weakening of common bonding forces between corn starch and proteins, such as hydrogen bonds, disulfide bonds, electrostatic interactions, or combinations thereof, to improve the corn starch isolation process. Starch and proteins could be easily separated by disrupting disulfide bonds with 1% l ‐cysteine (w/v). The most effective reagents for hydrogen bonds and electrostatic interactions were 3M urea and pH 7.5 separately. The sequence treatment of hydrogen bonds, disulfide bonds, and electrostatic interactions (namely, sequence treatment of 1M urea, 1% l ‐cysteine, and pH 7.5) led to the highest amount of starch in corn slurry and facilitated the corn starch isolation.     

Purification and biochemical properties of dipeptidyl peptidase I from porcine skeletal muscle

Dipeptidyl peptidase I (DPP I; EC 3.4.14.1) was purified from porcine skeletal muscle after several steps such as heat treatment, ammonium sulfate fractionation, gel filtration chromatography, and HPLC anion exchange chromatography. The purified enzyme showed a native molecular mass of approximately 200 kDa on Sephacryl S-200 column chromatography. Two protein bands of 65 and 42 kDa were obtained by SDS-PAGE, indicating its oligomeric nature. Maximum activity was reached at pH 5.5 and 55 degrees C. DPP I shared some common substrate specificities, both on synthetic derivatives and on real peptides, with porcine muscle DPP III. The enzyme required reducing agents for full activation, although the halide requirement was not proved. DPP I was inhibited by the assayed cysteine peptidase inhibitors except p-CMB. The serine peptidase inhibitor 3, 4-DCI also inhibited the enzyme as did the divalent cations Co(2+), Mn(2+), and Zn(2+). On the basis of its properties, DPP I may contribute to the generation of dipeptides during the processing of meat and/or meat products, including cooked ham.     

Soybean peroxidase-catalyzed oxidation of luminol by hydrogen peroxide

Anionic soybean peroxidase Glycine max (SbP) is shown to efficiently catalyze luminol oxidation by hydrogen peroxide. Contrary to horseradish peroxidase, the presence of p-iodophenol in the reaction medium affects slightly the efficiency of SbP catalysis. A maximal intensity of chemiluminescence, produced through this enzymatic reaction, was detected at pH 8.4-8.6. Contrary to anionic palm tree peroxidase, in the presence of SbP, chemiluminescence intensity increases with the reaction buffer concentration. The detection limit of SbP in the reaction of luminol oxidation is 0.3 x 10(-12) M. Therefore, high sensitivity in combination with the long-term chemiluminescent signal is indicative of good prospects for application of this enzyme in enzyme immunoassay with chemiluminescent detection.     

Myoglobin species with enhanced prooxidative activity is formed during mild proteolysis by pepsin

Pepsin proteolysis at pH approximately 4 resulted in a lowering of the (pseudo)peroxidase activity of metmyoglobin both at physiological pH and at meat pH, as measured by a peroxidase assay with H(2)O(2) and ABTS as substrates. In contrast, the mildly proteolyzed myoglobin had a strongly enhanced prooxidative effect on lipid oxidation in an oil in water methyl linoleate emulsion compared to native metmyoglobin, as evidenced by rates of oxygen depletion. More severe proteolysis of metmyoglobin at lower pH values near the optimum for pepsin did not result in a similar enhancement of prooxidative activity. The mildly proteolyzed metmyoglobin had spectral characteristics in agreement with a relative stabilization of the iron(II) state. On the basis of the observed effects of metal chelators, of lipophilic and hydrophilic peroxides and of radical scavengers on oxygen depletion rates, it is suggested that the increased prooxidative effect is due to radicals formed by cleavage of lipid peroxides by iron(II)/iron(III) cycling of a heme pigment with affinity for the lipid/water interface.