Purification and characterization of a novel peroxidase from bitter gourd (Momordica charantia)

Peroxidase from bitter gourd was purified by three step purification scheme; ammonium sulphate fractionation, gel filtration and affinity chromatography. The enzyme was purified 42 fold with the retention of 67% of the initial activity. The enzyme exhibited its maximum activity at pH 5.6 and 40 degrees C. The enzyme retained half of its activity even after 1 h incubation at 60 degrees C. Molecular weight of the purified glycosylated bitter gourd peroxidase determined by Sephacryl S-100 and SDS-PAGE was 43 kDa. The stokes radius, diffusion coefficient and sedimentation coefficient of the purified peroxidase were 27.3 A, 8.17 x 10(-7) cm(2)/sec and 3.74 S, respectively. K(m) for o-dianisidine and ABTS were 1.3 and 4.9 mM, respectively. The activity of the enzyme was inhibited by sulfide, azide and L-cysteine. The carbohydrate content and sulfydryl groups of the enzyme were 25% (w/w) mass of the protein and 16 mmoles/mole of the protein, respectively.     

Partially purified bitter gourd (Momordica charantia) peroxidase catalyzed decolorization of textile and other industrially important dyes

The aim of this study was to evaluate the enzymatic action of partially purified bitter gourd peroxidase for the degradation/decolorization of complex aromatic structures. Twenty-one dyes, with a wide spectrum of chemical groups, currently being used by the textile and other important industries have been selected for the study. Here, for the first time we have shown peroxidases from Momordica charantia (300 EU/gm of vegetable) to be highly effective in decolorizing industrially important dyes. Dye solutions, containing 50-200 mg dye/l, were used for the treatment with bitter gourd peroxidase (specific activity of 99.0 EU/mg protein). M. charantia peroxidases were able to decolorize most of the textile dyes by forming insoluble precipitate. When the textile dyes were treated with increasing concentration of enzyme, it was observed that greater fraction of the color was removed but four out of eight reactive dyes were recalcitrant to decolorization by bitter gourd peroxidase. Step-wise addition of enzyme to the decolorizing reaction mixture at the interval of 1h further enhanced the dye decolorization. The rate of decolorization was enhanced when the dyes were incubated with fixed quantity of enzyme for increasing times. Decolorization of non-textile dyes resulted in the degradation and removal of dyes from the solution without any precipitate formation. Decolorization rate was drastically increased when the textile and other industrially important non-textile dyes were treated with bitter gourd peroxidase in presence of 1.0 mM 1-hydroxybenzotriazole. Complex mixtures of dyes were prepared by taking three to four reactive textile and non-textile dyes in equal proportions. Each mixture was decolorized by more than 80% when treated with the enzyme in presence of 1.0 mM 1-hydroxybenzotriazole. Our data suggest that the peroxidase/mediator system is an effective biocatalyst for the treatment of effluents containing recalcitrant dyes from textile, dye manufacturing, dyeing and printing industries.     

A role of glycosyl moieties in the stabilization of bitter gourd (Momordica charantia) peroxidase

The possible role of carbohydrate moieties in the stabilization of proteins has been investigated by using bitter gourd peroxidase as a model system. A comparative study of glycosylated and non-glycosylated isoenzymes of bitter gourd peroxidase was performed at various temperatures, pH, water-miscible organic solvents, detergents and chaotropic agent like urea. The pH-optima and temperature-optima of both glycosylated and non-glycosylated isoforms of bitter gourd peroxidase remained unchanged. The probes employed were changes in the enzyme activity and fluorescence. The glycosylated form of peroxidase retained greater fraction of enzyme activity against the exposure caused by various physical and chemical denaturants. The unfolding of both forms of enzyme in the presence of high urea concentrations, studied by fluorescence, indicated greater perturbations in the conformation of non-glycosylated preparation. The different properties examined thus indicated that glycosylation plays an important role in the stabilization of native conformation of proteins against the inactivation caused by various types of denaturants.     

Polyclonal antibodies mediated immobilization of a peroxidase from ammonium sulphate fractionated bitter gourd (Momordica charantia) proteins

Polyclonal antibody bound Sepharose 4B support has been exploited for the immobilization of bitter gourd peroxidase directly from ammonium sulphate precipitated proteins. Immunoaffinity immobilized bitter gourd peroxidase exhibited high yield of immobilization. IgG-Sepharose 4B bound bitter gourd peroxidase showed a higher stability against heat, chaotropic agents (urea and guanidinium chloride), detergents (cetyl trimethyl ammonium bromide and Surf Excel), proteolytic enzyme (trypsin) and water-miscible organic solvents (propanol, THF and dioxane). The activity of immobilized bitter gourd peroxidase was significantly enhanced in the presence of cetyl trimethyl ammonium bromide and after treatment with trypsin as compared to soluble enzyme.     

Use of bitter gourd (Momordica charantia) peroxidase together with redox mediators to decolorize disperse dyes

In this study, salt fractionated bitter gourd (Momordica charantia) peroxidase was used for the decolorization of water-insoluble disperse dyes; Disperse Red 17 and Disperse Brown 1. Effect of nine different redox mediators; bromophenol, 2,4-dichlorophenol, guaiacol, 1-hydroxybenzotriazole, m-cresol, quinol, syringaldehyde, violuric acid, and vanillin on decolorization of disperse dyes by bitter gourd peroxidase has been investigated. Among these redox mediators, 1-hydroxybenzotriazole was the most effective mediator for decolorization of both the dyes by peroxidase. Bitter gourd peroxidase (0.36 U/mL) could decolorize Disperse Red 17 maximally 90% in the presence of 0.1 mM 1-hydroxybenzotriazole while Disperse Brown 1 was decolorized 65% in the presence of 0.2 mM 1-hydroxybenzotriazole. Maximum decolorization of these dyes was obtained within 1 h of incubation at pH 3.0 and temperature 40°C. The application of such enzyme plus redox mediator systems may be extendable to other recalcitrant and water insoluble synthetic dyes using novel redox mediators and peroxidases from other new and cheaper sources.     

Extraction of peroxidase from bitter gourd (Momordica charantia) by three phase partitioning with dimethyl carbonate (DMC) as organic phase

Three phase partitioning (TPP) is most renowned technique used for extraction and purification of natural products. In previous studies of TPP, t-butanol is mainly used as an organic phase. This is the first report that explores ability of dimethyl carbonate (DMC) in the field of TPP as an alternate solvent for t-butanol. In the present study TPP process with t-butanol and DMC as organic phase along with different salts was applied to waste bitter gourd powder to obtained peroxidase enzyme. DMC was found to be compatible with most of salts such as ammonium sulphate and sodium citrate and explored as more efficient solvent than t-butanol. This TPP system provides 4.84 fold purity of peroxidase enzyme at optimum source concentration of 0.15 g/mL, with a system comprising DMC as organic phase, sodium citrate (20%) as salt, agitation speed 120 rpm, pH 7, temperature 30 °C and extraction time of 3 h. Present study has aimed for extraction and separation of peroxidase from bitter gourd waste with TPP technique and ensures the scope of carbonated solvents in extraction and purification of proteins.     

Induction of anti-inflammatory responses by dietary Momordica charantia L. (bitter gourd)

We assessed the immunomodulatory activity of Momordica charantia L. (bitter gourd), a vegetable that has been reported to possess various bioactivities. We examined the effect of bitter gourd on intestinal immunity by monitoring the TGF-beta and IL-7 secretion from Caco-2 cells and the IL-10 and IL-12 secretion from THP-1 cells that are used as in vitro models of the intestinal epithelium and monocyte/macrophages, respectively. We also determined the in vivo immunological responses of rats fed on bitter gourd for 3 weeks. We found that bitter gourd induced a decrease in the intestinal secretion of IL-7 and an increase in the secretions of TGF-beta and IL-10, these effects reflecting the bitter gourd-induced changes in systemic immunity, i.e., a decrease in the number of lymphocytes, increases in the populations of Th cells and NK cells, and increase in the Ig production of lymphocytes. Dietary bitter gourd may therefore induce both intestinal and also systemic anti-inflammatory responses.     

Inheritance of gynoecism in bitter gourd (Momordica charantia L.)

The inheritance of sex expression in cucumber (Cucumis sativus) and other cucurbits is well documented; however, the genetics of female sex (gynoecism) expression in bitter gourd (Momordica charantia) has not been described. Inheritance of gynoecism in bitter gourd was studied in a 100% gynoecious line (Gy263B). The F(2) and testcross segregation data revealed that gynoecism in Gy263B is under the control of a single, recessive gene. Following the gene nomenclature of cucurbits, it is proposed that the gene symbol, gy-1, be assigned for the expression of gynoecism in bitter gourd.     

Thermodynamic analysis of porphyrin binding to Momordica charantia (bitter gourd) lectin.

Owing to the use of porphyrins in photodynamic therapy for the treatment of malignant tumors, and the preferential interaction of lectins with tumor cells, studies on lectin-porphyrin interaction are of significant interest. In this study, the interaction of several free-base and metalloporphyrins with Momordica charantia (bitter gourd) lectin (MCL) was investigated by absorption spectroscopy. Difference absorption spectra revealed that significant changes occur in the Soret band region of the porphyrins on binding to MCL. These changes were monitored to obtain association constants (Ka) and stoichiometry of binding. The tetrameric MCL binds four porphyrin molecules, and the stoichiometry was unaffected by the presence of the specific sugar, lactose. In addition, the agglutination activity of MCL was unaffected by the presence of the porphyrins used in this study, clearly indicating that porphyrin and carbohydrate ligands bind at different sites. Both cationic and anionic porphyrins bind to the lectin with comparable affinity (Ka =10(3)-10(5) m(-1)). The thermodynamic parameters associated with the interaction of several porphyrins, obtained from the temperature dependence of the Ka values, were found to be in the range: DeltaH degrees = -98.1 to -54.4 kJ.mol(-1) and DeltaS degrees =-243.9 to -90.8 J.mol(-1).K(-1). These results indicate that porphyrin binding to MCL is governed by enthalpic forces and that the contribution from binding entropy is negative. Enthalpy-entropy compensation was observed in the interaction of different porphyrins with MCL, underscoring the role of water structure in the overall binding process. Analysis of CD spectra of MCL indicates that this protein contains about 13%alpha-helix, 36%beta-sheet, 21%beta-turn, and the rest unordered structures. Binding of porphyrins does not significantly alter the secondary and tertiary structures of MCL.     

New ribosome-inactivating proteins from seeds and fruits of the bitter gourd Momordica charantia

A new ribosome-inactivating protein (RIP) (δ-momorcharin) and a candidate RIP (ε-momorcharin) were isolated, respectively, from the seeds and fruits of the bitter gourd Momordica charantia, by affinity chromatography on Affi-gel blue gel and ion exchange chromatography on Mono S with a fast protein liquid chromatography (FPLC) system. Both δ- and ε-momorcharins were adsorbed on Affi-gel blue gel and were eluted from the cation exchange Mono S column earlier than α- and β-momorcharins, the two previously reported RIPs, δ- and ε-momorcharins possessed a molecular weight of 30 and 24 kDa respectively and inhibited cell-free translation in rabbit reticulocyte lysate with an IC₅₀ of 0.15 and 170 nM. The sequence of the first seven N-terminal amino acids in δ-momorcharin was DVNFGLA, which was different from the N-terminal sequences DVSFRLS and DVNFDLS for α- and β-momorcharins respectively.     

The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica charantia)

The complete amino acid sequence of ribonuclease (RNase MC) from the seeds of bitter gourd (Momordica charantia) has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with trypsin, lysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The protein contains 191 amino acid residues and has a calculated molecular mass of 21,259 Da. Comparison of this sequence with sequences of the fungal RNases, RNase T2, and RNase Rh, revealed that there are highly conserved residues at positions 32-38 (TXHGLWP) and 81-92 (FWXHEWXKHGTC). Furthermore, the sequence of RNase MC was found to be homologous to those of Nicotiana alata S-glycoproteins involved in self-incompatibility sharing 41% identical residues.     

Fluorimetric studies of the binding of Momordica charantia (bitter gourd) lectin with ligands.

The association constants for the binding of a series of ligands with a galactose-specific lectin from Momordica charantia (bitter gourd) has been determined through the ligand-induced quenching of protein fluorescence. Analysis of the iodide quenching suggested that there is a slight increase in the accessibility of tryptophan residues of the lectin on binding lactose.     

Purification and characterization of charantin, a napin-like ribosome-inactivating peptide from bitter gourd (Momordica charantia) seeds.

A peptide designated charantin, with a molecular mass of 9.7 kDa, was isolated from bitter gourd seeds. The procedure comprised affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono S and gel filtration on Superdex 75. The N-terminal sequence of charantin exhibited marked similarity to that of the 7.8-kDa napin-like peptide previously isolated from bitter gourd seeds. Charantin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 400 nm, a potency lower than that of the previously reported small ribosome-inactivating protein gamma-momorcharin (IC50 = 55 nm) which also exhibited an abundance of arginine and glutamate/glutamine residues. Charantin reacted positively in the N-glycosidase assay, yielding a band similar to that formed by the small ribosome-inactivating proteins gamma-momorcharin and luffin S.     

Horseradish peroxidase for the removal of carcinogenic aromatic amines from water

A new method has been developed for the removal of carcinogenic aromatic amines from industrial aqueous effluents. It includes the treatment of aqueous solutions containing the carcinogens with horseradish peroxidase and hydrogen peroxide. Such treatment results in a nearly complete precipitation of carcinogenic aromatic amines from water due to enzymatic crosslinking. This method was used to remove ten recognized human carcinogens from water: benzidine and its derivatives, naphthylamines, 4-aminobiphenyl, and p-phenylazoaniline. The dependence of the removal efficiency of the peroxidase treatment on the concentrations of the enzyme, H₂O₂ and a carcinogen and also on pH and the duration of the treatment was studied. The enzymatic removal of carcinogens from water was confirmed by both chemical and toxicological assays.     

Crystal structure of a ribonuclease from the seeds of bitter gourd (Momordica charantia) at 1.75 A resolution.

The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acid residues with four disulfide bridges and belongs to the RNase T(2) family, including fungal RNases typified by RNase Rh from Rhizopus niveus and RNase T(2) from Aspergillus oryzae. The crystal structure of RNase MC1 has been determined at 1.75 A resolution with an R-factor of 19.7% using the single isomorphous replacement method. RNase MC1 structurally belongs to the (alpha+beta) class of proteins, having ten helices (six alpha-helices and four 3(10)-helices) and eight beta-strands. When the structures of RNase MC1 and RNase Rh are superposed, the close agreement between the alpha-carbon positions for the total structure is obvious: the root mean square deviations calculated only for structurally related 151 alpha-carbon atoms of RNase MC1 and RNase Rh molecules was 1.76 A. Furthermore, the conformation of the catalytic residues His-46, Glu-105, and His-109 in RNase Rh can be easily superposed with that of the possible catalytic residues His-34, Glu-84, and His-88 in RNase MC1. This observation strongly indicates that RNase MC1 from a plant origin catalyzes RNA degradation in a similar manner as fungal RNases.     

Energetics of carbohydrate binding to Momordica charantia (bitter gourd) lectin: an isothermal titration calorimetric study

Physico-chemical and carbohydrate binding studies have been carried out on the Momordica charantia (bitter gourd) seed lectin (MCL). The lectin activity is maximal in the pH range 7.4-11.0, but decreases steeply below pH 7.0. The lectin activity is mostly unaffected in the temperature range 4-50 degrees C, but a sharp decrease is seen between 50 and 60 degrees C, which could be correlated to changes in the structure of the protein as seen by circular dichroism and fluorescence spectroscopy. Isothermal titration calorimetric studies show that the tetrameric MCL binds two sugar molecules and the binding constants (Kb), determined at 288.15 K, for various saccharides were found to vary between 7.3 x 10(3) and 1.52 x 10(4)M(-1). The binding reactions for all the saccharides investigated were essentially enthalpy driven, with the binding enthalpies (DeltaHb) at 288.15 K being in the range of -50.99 and -43.39 kJ mol(-1), whereas the contribution to the binding reaction from the entropy of binding was negative, with values of binding entropy (DeltaSb) ranging between -99.2 and -72.0 J mol(-1)K(-1) at 288.15 K. Changes in heat capacity (DeltaCp) for the binding of disaccharides, lactose and lactulose, were significantly larger in magnitude than those obtained for the monosaccharides, methyl-beta-D-galactopyranoside, and methyl-alpha-D-galactopyranoside, and could be correlated reasonably well with the surface areas of these ligands. Enthalpy-entropy compensation was observed for all the sugars studied, suggesting that water structure plays an important role in the overall binding reaction. CD spectroscopy indicates that carbohydrate binding does not lead to significant changes in the secondary and tertiary structures of MCL, suggesting that the carbohydrate binding sites on this lectin are mostly preformed.     

Amino acid sequences and disulfide bridges of serine proteinase inhibitors from bitter gourd (Momordica charantia LINN.) seeds

Three serine proteinase inhibitors, MCTI-I, MCTI-II, and MCEI-I, were isolated from bitter gourd (Momordica charantia LINN.) seeds. MCTI-I and MCTI-II were inhibitors for trypsin and MCEI-I was an elastase inhibitor. Their amino acid sequences and the positions of disulfide bridges of MCTI-II were determined to be as follows. (sequence; see text)     

苦瓜主要品质性状的遗传变异、相关和通径分析

对23份苦瓜材料的10个品质性状进行了研究。结果表明,苦瓜维生素C、还原糖、有机酸、果瘤、果色、果刺和苦味遗传变异系数大,遗传力高;水分含量遗传变异系数很小;风味遗传变异系数较小,遗传力低。苦瓜4个内在营养品质之间相关性小,而4个外观品质性状之间关系密切。两个味觉品质与4个内在营养品质之间相关性小,但与4个外观品质之间关系密切。内在营养品质与外观品质之间有一定关系。通径分析提出通过降低果实的苦味和有机酸含量来提高风味品质;通过水分含量和果瘤两性状的直接选择来实现对维生素C的间接选择。     

The oxidation of N-substituted aromatic amines by horseradish peroxidase

The mechanism of N-dealkylation by peroxidases of the Ca2+ indicator quin2 and analogs was investigated and compared with the mechanism of N-dealkylation of some N-methyl-substituted aromatic amines. Nitrogen-centered cation radicals were detected by ESR spectroscopy for all the compounds studied. Further oxidation of the nitrogen-centered cation radicals, however, was dependent upon the structure of the radical formed. In the case of quin2 and analogs, a carbon-centered radical could be detected using the spin trap 5,5-dimethyl-1-pyrroline N-oxide. By using the spin trap 2-methyl-2-nitrosopropane (tert-nitrosobutane), it was determined that the carbon-centered radical was formed due to loss of a carboxylic acid group. This indicated that bond breakage most likely occurred through a rearrangement reaction. Furthermore, extensive oxygen consumption was detected, which was in agreement with the formation of carbon-centered radicals, as they avidly react with molecular oxygen. Thus, reaction of the carbon-centered radical with oxygen most likely led to the formation of a peroxyl radical. The peroxyl radical decomposed into superoxide that was spin trapped by 5,5-dimethyl-1-pyrroline N-oxide and an unstable iminium cation. The iminium cation would subsequently hydrolyze to the monomethyl amine and formaldehyde. In the case of N-methyl-substituted aromatic amines, carbon-centered radicals were not detected during the peroxidase-catalyzed oxidation of these compounds. Thus, rearrangement of the nitrogen-centered radical did not occur. Furthermore, little or no oxygen consumption was detected, whereas formaldehyde was formed in all cases. These results indicated that the N-methyl-substituted amines were oxidized by a mechanism different from the mechanism found for quin2 and analogs.