Extraction, isolation, and characterization of globulin proteins from Lupinus albus

Lupin has recently been added to the list of allergens requiring mandatory advisory labeling on foodstuffs sold in the European Union, and since December 2008, all products containing even trace amounts of lupin must be labeled correctly. Lupin globulins consist of two major globulins called α-conglutin (11S and "legumin-like") and β-conglutin (7S and "vicilin-like") and another additional two globulins, γ-conglutin and δ-conglutin, which are present in lower amounts. We report on a methodology to facilitate the extraction of each of these proteins using centrifugation and isolation by anion-exchange chromatography followed by size-exclusion chromatography. The isolated subunits were characterized using reducing and non-reducing polyacrylamide gel electrophoresis, western blotting, and peptide mass fingerprinting, all of which revealed that the individual protein subunits are highly pure and can be used as immunogens for the production of antibodies specific for each of the conglutin fractions, as well as standards, and the extraction protocol can be used for the selective extraction of each of the subunits from foodstuffs, thus facilitating a highly accurate determination of the lupin concentration. Furthermore, the subunits can be used to elucidate information regarding the toxicity of each of the subunits, by looking at their interaction with the IgE antibodies found in the serum of individuals allergic to lupin, providing critical information for the definition of the requirements of analytical assays for the detection of lupin in foodstuffs.     

Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba Var. Alameda)

A phytate-degrading enzyme was purified approximately 2190-fold from germinated 4-day-old faba bean seedlings to apparent homogeneity with a recovery of 6% referred to the phytase activity in the crude extract. It behaves as a monomeric protein of a molecular mass of approximately 65 kDa. The phytate-degrading enzyme belongs to the acidic phytases. It exhibits a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 148 micromol L(-1) and k(cat) = 704 s(-1) at 35 degrees C and pH 5.0. The faba bean phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyzes phytate in a stepwise manner. The first hydrolysis product was identified as D/L-myo-inositol(1,2,3,4,5)pentakisphosphate.     

Purification and partial characterization of three turnip (Brassicanapus L. var. esculenta D.C.) peroxidases

Three turnip peroxidases (fractions C1, C2, and C3) were partially purified and characterized, to permit study of their feasibility for use in clinical and enzyme immunoassays. These fractions represented 20% of the initial activity, and fractions C1 and C2 were purified to homogeneity. The optimum pH was between 5.0 and 5.5, while optimum temperature ranged from 40 to 55 degrees C. The ABTS K(m) values for the two acidic fractions (C2 and C3) were 0.70 and 0.42 mM, respectively; about 5 times lower than that reported for the acidic commercial horseradish peroxidase (HRP). Fraction C3 had 4 times higher K(m) value than commercial cationic HRP. The molecular weights determined by SDS-PAGE ranged from 39.2 to 42.5 kDa. Activation energies for inactivation were 113 (C1), 130 (C2), and 172 kJ/mol (C3) which are higher or comparable to other peroxidase isoenzymes reported. Fractions C1 and C3 represent an alternative source of peroxidase because of their higher purification yield and specific activity, when compared to fraction C2.     

One- and two-dimensional electrophoretic identification of IgE-binding polypeptides of Lupinus albus and other legume seeds

The prevalence of food allergies in the world population requires integrated approaches to identify new potential allergens, especially those of plant origin. The aim of this work was the allergen in vitro analysis of Lupinus albus seed proteome, a promising food protein source, and the assessment of IgE cross-reactivities with other more diffused legume species. A combination of one- and two-dimensional gel electrophoresis and immunoblotting analyses with specific IgGs for band identification and lupin-sensitized patients' circulating IgEs for allergenicity studies has been used. Two lupin proteins, namely, conglutin gamma and 11S globulin basic subunits, strongly reacted with all patients' sera. Also, cross-reactivities with the homologous polypeptides of other legume species were observed. Otherwise, no reaction at all was detected with a 2S-type lupin protein. This global electrophoretic approach has allowed the identification of a new potential lupin allergen and confirmed the cross-reactivity among the legume 11S globulin basic subunits.     

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 a trypsin inhibitor from Plathymenia foliolosa seeds

A novel trypsin inhibitor (PFTI) was isolated from Plathymenia foliolosa (Benth.) seeds by gel filtration chromatography on a Sephadex G-100, DEAE-Sepharose, and trypsin-Sepharose columns. By SDSPAGE, PFTI yielded a single band with a M(r) of 19 kDa. PFTI inhibited bovine trypsin and bovine chymotrypsin with equilibrium dissociation constants (K(i)) of 4 x 10(-8) and 1.4 x 10(-6) M, respectively. PFTI retained more than 50% of activity at up to 50 degrees C for 30 min, but there were 80 and 100% losses of activity at 60 and 70 degrees C, respectively. DTT affected the activity or stability of PFTI. The N-terminal amino acid sequence of PFTI showed a high degree of homology with various members of the Kunitz family of inhibitors. Anagasta kuehniella is found worldwide; this insect attacks stored grains and products of rice, oat, rye, corn, and wheat. The velvet bean caterpillar (Anticarsia gemmatalis) is considered the main defoliator pest of soybean in Brazil. Diatraea saccharalis, the sugar cane borer, is the major pest of sugar cane crops, and its caterpillar-feeding behavior, inside the stems, hampers control. PFTI showed significant inhibitory activity against trypsin-like proteases present in the larval midguts on A. kuehniella and D. saccharalis and could suppress the growth of larvae.     

Purification and characterization of a beta-glucosidase from Citrus sinensis var. Valencia fruit tissue

A preliminary survey demonstrated activity for alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-arabinosidase, beta-D-glucosidase, beta-D-xylosidase, and beta-D-galactosidase in orange fruit flavedo and albedo tissue. alpha-L-Rhamnosidase was not detected. Subsequently, a beta-glucosidase was purified from mature fruit rag tissue (composed of intersegmental septa, squeezed juice sacs, and fruit core tissue) of Citrus sinensis var. Valencia. The beta-glucosidase exhibited low levels of activity against p-nitrophenyl-beta-D-fucopyranoside (13.5%) and p-nitrophenyl-alpha-D-glucopyranoside (7.0%), compared to its activity against p-nitrophenyl-beta-D-glucopyranoside (pNPG, 100%). The enzyme was purified by a combination of ion exchange (anion and cation) and gel filtration (Superdex and Toyopearl HW-55S) chromatography. It has an apparent molecular mass of 64 kDa by denaturing electrophoresis or 55 kDa by gel filtration chromatography (BioGel P-100). Hydrolysis of pNPG demonstrated a pH optimum between 4.5 and 5.5. At pH 5.0 the temperature optimum was 40 degrees C. At pH 5.0 and 40 degrees C the K(m) for pNPG was 0.1146 mM and it had a V(max) of 5.2792 nkatal x mg(-1) protein (katal = 0.06 International Units = the amount of enzyme that produces, under standard conditions, one micromol of product per min). Of the substrates tested, the enzyme was most active against the disaccharide cellobiose (1-->4), but was not active against p-nitrophenyl-beta-D-cellobioside. High levels of activity also were observed with the disaccharides laminaribiose (1-->3), gentiobiose (1-->6), and sophorose (1-->2). Activity greater than that observed with pNPG was obtained with the flavonoids hesperetin-7-glucoside and prunin (naringenin-7-glucoside), salicin, mandelonitrile-beta-D-glucoside (a cyanogenic substrate), and sinigrin (a glucosinolate). The enzyme was not active against amygdalin, coniferin, or limonin glucoside.     

Quantitative sandwich ELISA for the determination of lupine (Lupinus spp.) in foods

The use of lupine in foods has increased considerably during the past decade, reflected by a corresponding increase in reported lupine-induced allergic incidents. Lupine allergy may arise either by primary sensitization or by clinical cross-reactivity in peanut-allergic persons. Detection of lupine proteins in food has previously been based on the use of patient serum. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of lupine in processed foods was developed, using a polyclonal rabbit antilupine capture antibody and a biotinylated conjugate of the same antibody for detection. The antibody was highly specific for lupine, apart from minor cross-reactivities to other legumes. The assay had a detection limit of 1 mug/g and was successfully used to quantify lupine protein in various food matrixes. Recoveries ranged from 60 to 116%, while the intra-and interassay coefficients of variation were <6% and <21%, respectively.     

The acquisition of cadmium by Lupinus albus L., Lupinus angustifolius L., and Lolium multiflorum Lam.

Das Cadmiumaneignungsvermögen von Lupinus albus L., Lupinus angustifolius L. und Lolium multiflorum Lam. Mehrere Pflanzenarten mobilisieren Bodenphosphate (P) und Kationen wie Fe und Al durch die Exsudation organischer Anionen und Protonen. Deshalb untersuchten wir das Cd‐Aneignungsver‐mögen von P‐, Fe‐, Al‐mobilisierenden Arten (Lupinus albus L., Lupinus angustifolius L.) im Vergleich zu einer nicht mobili‐sierenden Pflanzenart (Lolium multiflorum Lam.). Die Pflanzen wuchsen in zwei stark unterschiedlichen Böden (saurer Humuspodsol, karbonathaltiger Lössunterboden). Die Cd‐Aufnahme in die Sprosse war bei Weidelgras 5 bis 10 mal höher als bei Blauen bzw. Weißen Lupinen. Dieses Ergebnis hat mehrere Ursachen: 1. Das Wurzellängen/Sprossmasseverhältnis des Weidelgrases ist 2—3 mal größer als das der Lupinenpflanzen. 2. Bei Weidelgras wird ein größerer Teil des aufgenommenen Cd in die Sprosse verlagert. 3. Die Cd‐Aufnahme bei Lupinen ist im sauren Boden (Podsol) und bei P‐Mangel auch im Kalkboden niedriger als bei Weidelgras. Während im Podsol die Cd‐Konzentration der Bodenlösung unter Lupine geringer war als in der Kontrolle (Gefäße ohne Pflanzen), war sie im Kalkboden höher. Bei den Lupinen war der Efflux organischer Säureanionen, vor allem Citrat und Malat, um den Faktor 10—100 höher als bei Weidelgras. Diese Exsudation kann zu einer hohen Cd‐Komplexierung, insbesondere durch Citrat, in der Rhizosphärenbodenlösung führen (˜ 85%). Diese Ergebenisse deuten darauf hin, dass das komplexierte Cd von den Wurzeln schlechter aufgenommen wird als das freie Cd.     

Biochemical characterization of cystine lyase from broccoli (Brassica oleracea var. italica)

Cystine lyase is the enzyme responsible for off-aroma deterioration in fresh unblanched broccoli. In this research, cystine lyase purification from broccoli has been optimized. Only one protein peak with cystine lyase activity was found during purification. Broccoli cystine lyase was purified 100-fold to homogeneity. L-Cystine, L-cysteine-S-sulfate, L-djenkolic acid, and some S-alkyl-L-cysteines and their sulfoxides are substrates, but the enzyme had negligible activity with L-cystathionine. A K(m) value of 81.2 microM was found for L-cystine. Inhibition and K(i) determinations indicated that L-cysteine is a linear noncompetitive inhibitor with a K(i) of 5 mM and DL-homocysteine is a competitive inhibitor with a K(i) of 1.5 mM. The molecular weight of cystine lyase was determined to be 100 kDa by three methods, with two subunits of 48 kDa each and a carbohydrate content of 3%. Further characterization included cofactor quantification, the effects of temperature and pH on activity and stability, and amino acid composition.     

Biological activity of alpha-galactoside preparations from Lupinus angustifolius L. and Pisum sativum L. seeds.

Biological activity tests were performed on alpha-galactoside preparations obtained from Lupinus angustifolius L. cv. Mirela (alkaloid-rich) and Pisum sativum L. cv. Opal seeds. The studies included the following tests: acute toxicity, cytotoxic test, delayed type hypersensitivity (DTH), plaque-forming cell number (IgM-PFC), and influence on the growth of bifidobacteria and coliform presence in rat colon. Results of these studies showed that alpha-galactosides from lupin and pea seeds were essentially nontoxic. Their acute toxicity (LD(50)) in mice was >4000 mg kg(-1) of body weight. alpha-galactoside preparations were not cytotoxic for mouse thymocytes in vitro. The in vitro test shows that oligosaccharides from lupin and pea are utilized by selected beneficial colon bacterium strains. The in vivo experiment demonstrated that alpha-galactosides from legume significantly influenced the growth of bifidobacteria in rats colon. Simultaneously, the decrease of the coliform presence was observed. The chemical composition of the tested preparations had no significant effect on their biological activity.     

Influence of lupin (Lupinus luteus L. cv. 4492 and Lupinus angustifolius L. var. zapaton) and fenugreek (Trigonella foenum-graecum L.) germination on microbial population and biogenic amines

Microbial population and bioactive amine profile and levels of two lupin species (Lupinus luteus L. cv. 4492 and Lupinus angustifolius L. var. zapaton) and fenugreek (Trigonella foenum-graecum L.) seeds as affected by germination were investigated. Microbial population increased considerably mainly in the first stage of germination (2 days), then small changes in bacterial numbers were observed up to 5 days to levels between 7.8 and 8.9 log colony-forming units/g. Microorganisms belonging to the Enterobacteriaceae family were dominant for the legumes tested. Ungerminated legume seeds contained putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. Bioactive amine levels found in fenugreek seeds were between 3- and 4-fold higher than those found in lupin seeds. The highest total amine levels were found in fenugreek seeds [162 mg/kg of dry weight (dw)], followed by L. angustifolius var. zapaton seeds (84 mg/kg of dw) and, finally, L. luteus cv. 4492 (46 mg/kg of dw) seeds. The concentration of individual amines showed a gradual rising trend during the germination period in all tested sprouts, reaching levels >3 times higher than those found in ungerminated seeds. After 5 days of germination, the fenugreek sprouts contained the highest amount of total bioactive amines. Tyramine was the predominant amine in both lupin varieties, whereas cadaverine was the main bioactive amine detected in fenugreek. The results of this work thus indicated that microbial population and biogenic amine levels in the studied lupin and fenugreek sprouts are not a risk for healthy consumers or for individuals with restricted activity of detoxification enzymes.     

Bacterial removal of quinolizidine alkaloids and other carbon sources from a Lupinus albus aqueous extract

Two Gram-negative bacterial strains capable of using lupanine, the predominant quinolizidine alkaloid in Lupinus albus, as a sole carbon source were isolated from soil in which L. albus and L. luteus had been grown [Santana, F. M. et al. J. Ind. Microbiol. 1996, 17, 110-115]. In the present study, we present results suggesting that these isolates are of potential interest for removing lupanine and other quinolizidine alkaloids (QA) from the effluent resulting from the wet processing of Lupinus seeds, at temperatures within the range 20-34 degrees C. Growth in L. albus aqueous extract was diauxic, with a first period of rapid growth leading to the simultaneous consumption of a significant part of the initial concentration of QA (3 g L(-1), being 2 g L(-1) lupanine) and amino acids (1.5 g L(-1)). This period was followed by a second period of slower growth corresponding to the subsequent partial utilization (25%) of the carbohydrates (initial concentration of 20 g L(-1)) together with further removal of QA and amino acids. Despite the differences detected in the susceptibility of the two strains to lupanine toxicity, in particular at supraoptimal temperatures, and in the efficiency of lupanine catabolism, their performance on L. albus extract did not vary significantly.     

Isolation and characterization of antioxidant protein fractions from melinjo (Gnetum gnemon) seeds

The protein from the seeds of melinjo ( Gnetum gnemon ) was purified using a precipitation method and ion exchange chromatographic techniques to identify the potent antioxidant and free radical scavenging activities. Two antioxidant protein fractions were isolated from G. gnemon seed with molecular weights of approximately 30 kDa (Gg-AOPI) and 12 kDa (Gg-AOPII) by SDS-PAGE. The N-terminal amino acid sequence of Gg-AOPII is Gly-Asn-Gly-Lys-Ala-Thr-Val-Ala-Ile-Leu-Val-Lys-Glu-Lys-Val-Glu-Tyr-Gly-Glu-Glu, and the result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis showed that they were distinct from each other; no protein in database matching was found to both Gg-AOPI and Gg-AOPII. The antioxidant or free radical scavenging activities of Gg-AOPs were investigated by employing in vitro assay systems including the inhibition of linoleic acid autoxidation, scavenging effect on α,α-diphenyl-β-picrylhydrazyl free radical (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), reducing power, chelating abilities of metal ions Cu(2+) and Fe(2+), and protections against hydroxyl radical-mediated DNA damages. The result showed that two protein fractions exhibited significant (p < 0.05) antioxidant activities against free radicals such as DPPH, ABTS, and superoxide anion and showed activities similar to those of glutathione (G-SH) and BHT in a linoleic acid emulsion assay system. Moreover, Gg-AOPI and Gg-AOPII also exhibited notable reducing power and strong chelating effect on Fe(2+) and protected hydroxyl radical induced oxidative DNA damage. The data obtained by the in vitro systems obviously established the antioxidant potency of Gg-AOPs.     

Analysis of Lupinus albus storage proteins by two-dimensional electrophoresis and mass spectrometry

A laboratory-prepared total protein extract (TPE) and a lupin protein isolate (LPI-E) produced in a pilot plant were submitted to a detailed two-dimensional (2DE) proteomic investigation. Recent findings have indicated that in an established rodent model of hyperlipidemia, moderate daily intakes of LPI-Es lead to a reduction of total and low-density lipoprotein cholesterol levels, and the knowledge of the actual composition of the protein sample used in that study is at the basis of further structure/action investigations. The experimental results indicate that the semi-industrial procedure used for the production of LPI-E damages only marginally the proteins. It does, however, cleave some disulfide bridges and induce mild proteolysis, as confirmed by the higher number of resolved protein spots in the low Mr and acidic pI region of the 2DE map. Out of 72 spots submitted to mass spectrometry and compared with available protein databases, 42 correspond to fragments of beta-conglutin, the 7S globulin of lupin, spanning between positions 37 and 495 of the protein sequence. Using the bioinformatic tool BlastP, these peptides were compared to the alpha'-subunit of beta-conglycinin, the 7S globulin of soybean, this being the most active hypocholesterolemic component of soybean protein, as shown by in vitro and in vivo experiments. At least 18 peptides derived from beta-conglutin, having a percentage identity higher than 50% and a similarity percentage higher than 70% vs the alpha'-subunit of beta-conglycinin, are likely candidates to be the biologically active components of lupin protein.     

Purification and characterization of calpastatin from grass prawn muscle (Penaeus monodon)

Calpastatin, a specific calpain inhibitor was purified to electrophoretical homogeneity from grass prawn (Penaeus monodon) muscle by 100 degrees C heat-treatment, DEAE-Sephacel, and Q-Sepharose chromatographs. No significant change in the inhibitory activity of crude calpastatin was observed even after 20 min incubation at 100 degrees C, pH 7.0. The purified prawn calpastatin had a molecular weight (M(r)) of 80 and 88.7 kDa determined by SDS-PAGE and Sephacryl S-200 HR gel filtration, respectively. According to the active site titration, the purified calpastatin revealed four beef mu-calpain and two beef m-calpain binding domains, respectively. It was stable during 1 h of incubation at 30 degrees C under pH 4.5-10.0 and shown to be a highly specific inhibitor for calpain.     

Purification and characterization of trimethylamine-N-oxide demethylase from jumbo squid (Dosidicus gigas)

Trimethylamine-N-oxide demethylase (TMAOase) was purified from Jumbo squid (Dosidicus gigas) and characterized in detail herein. The TMAOase was extracted from squid with 20 mM Tris-acetate buffer (pH 7.0) containing 1.0 M NaCl, followed by acid treatment and heat treatment. Then it was purified by deithylaminoethyl-cellulose and Sephacryl S-300 chromatography, subsequently resulting in an 839-fold purification. The molecular mass of the TMAOase was defined to be 17.5 kDa. The optimum pH of the purified TMAOase was 7.0, and its optimum temperature was confirmed to be 55 degrees C. The TMAOase was stable to heat treatment up to 50 degrees C and stable at pH 7.0-9.0. Reducing agents such as DTT, Na2SO3, and NADH were effective at activating TMAOase, and ethylenediaminetetraacetic acid, as well as Mg2+ and Ca2+, could also enhance the activity of TMAOase remarkably, whereas the TMAOase could be significantly inhibited by tea polyphenol, phytic acid and acetic acid. In addition, the TMAOase converted TMAO to dimethylamine and formaldehyde stoichiometrically with a K(m) of 26.2 mM.     

Purification and characterization of amylases from small abalone (Sulculus diversicolor aquatilis)

Amylases II-1 and II-2 with molecular weights of 55.7 and 65 kDa, respectively, were purified to electrophoretical homogeneity from small abalone (Sulculus diversicolor aquatilis) by ammonium sulfate fractionation, Sepharose CL-6B, CM-Sepharose CL-6B, and Sephacryl S-100 chromatographs. They had optimal temperatures of 45 and 50 degrees C and an optimal pH of 6.0. The purified amylases were stable at pH 5.0-8.0 and 6.0-8.0, respectively. They were completely or partially inhibited by Hg(2+), Cu(2+), Cd(2+), Zn(2+), iodoacetamide, phenylmethanesulfonyl fluoride, and N-ethylmaleimide, suggesting the existence of cysteine at their active sites. Digestion tests against various polysaccharides suggested that the purified amylases II-1 and II-2 are neoamylases which can hydrolyze both alpha-1,4 and alpha-1,6 glucosidic bonds. Amylase II-2 might be an exo- and II-1 an endo-/exo-amylase.     

Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus)

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.