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.     

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.     

Interference of 5-hydroxytryptamine in the assay of glucose by glucose oxidase:peroxidase:chromogen based methods

The interference of 5-hydroxytryptamine with the assay of glucose by glucose oxidase:peroxidase:chromogen methods is demonstrated. Evidence is presented that this interference results from competition by 5HT and other indoles, which can act as hydrogen donors to the peroxidase: H₂O₂ system, with the chromogenic hydrogen donors guaiacol, o-dianisidine and ABTS 2,2′-azino-di[3-ethylbenzthiazoline-6-sulphonic acid] ammonium salt commonly used in glucose assay systems.     

Upsurge of particulate peroxidase in ripening banana fruit

Peroxidase was isolated from the pulp of ripening banana fruit and assayed with o-dianisidine as hydrogen-donor. Cell macerates contained soluble and particle-bound peroxidase. Soluble peroxidase levels did not appreciably differ in pre-climacteric, climacteric and post-climacteric fruit. Particulate peroxidase levels increased 3-fold with the initiation of the respiration climacteric and gradually declined with the onset of senescence. Bound peroxidase was released from cell wall and membrane fractions with washing in 0–8 M CaCl₂.     

Hymenolepis diminuta: Biochemical properties of peroxidase activity in mitochondria

The biochemical properties of a peroxidase previously localized cytochemically in the mitochondria of Hymenolepis diminuta were determined. The method chosen was the o-dianisidine procedure in which the decomposition of hydrogen peroxide has been followed spectrophotometrically. Peroxidase activity was initially demonstrated in the mitochondrial pellet. Subsequently, mitochondrial pellets were sonicated and the membrane and supernatant fractions were tested for peroxidase activity. Enzyme activity was demonstrated in the membrane fraction. The enzyme displayed a pH optimum of 5.0, was ascorbate sensitive, and was inhibited by excess H₂O₂. Neither peroxidase nor catalase were observed in any other fraction of the tapeworm tissue, confirming previous cytochemical investigations.     

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.     

Substrate specificity of peroxidase isoenzymes for hydrogen donors

The investigation of the substrate specificity of the anionic peroxidase isoenzymes, isolated from the zone of differentiation of the primary roots ofZea mays, for some representatives of phenolic compounds and aromatic amines, as hydrogen donors, is reported. The investigation was carried out electrophoretically with peroxidase isoenzymes partially purified by a combination of gel filtration by Sephadex G-25 and Sephadex G-100. A difference in the substrate specificity of the individual isoenzymes is observed. It was established that the anionic peroxidase isoenzymes showed a similarity in total number and relative activity on staining with bivalent phenols and difference on staining with trivalent phenols, as hydrogen donors. A greater number of isoenzymes was stained with benzidine ando-dianisidine and a lesser number witho- andp-phenylendiamine. The substrate specificity of the peroxidase isoenzymes was compared for guaiacol and benzidine. The substrate specificity of peroxidase soenzymes was discussed as regards their diverse role in the plant metabolism.     

Calcium alginate–starch hybrid support for both surface immobilization and entrapment of bitter gourd (Momordica charantia) peroxidase

Calcium alginate–starch hybrid gel was employed as an enzyme carrier both for surface immobilization and entrapment of bitter gourd peroxidase. Entrapped crosslinked concanavalin A–bitter gourd peroxidase retained 52% of the initial activity while surface immobilized and glutaraldehyde crosslinked enzyme showed 63% activity. A comparative stability of both forms of immobilized bitter gourd peroxidase was investigated against pH, temperature and chaotropic agent; like urea, heavy metals, water-miscible organic solvents, detergent and inhibitors. Entrapped peroxidase was significantly more stable as compared to surface immobilized form of enzyme. The pH and temperature-optima for both immobilized preparations were the same as for soluble bitter gourd peroxidase. Entrapped crosslinked concanavalin A–bitter gourd peroxidase showed 75% of the initial activity while the surface immobilized and crosslinked bitter gourd peroxidase retained 69% of the original activity after its seventh repeated use.     

A new sensitive colorimetric assay for peroxidase using 3,3'-diaminobenzidine as hydrogen donor

A new simple colorimetric assay for measurement of peroxidase activity using 3,3′-diaminobenzidine tetrahydrochloride as hydrogen donor is described. The DAB is stable under the usual assay conditions, and its rate of auto-oxidation is negligible. Under optimal conditions, a linear relationship is found between peroxidase concentration and the rate of oxidation of 3,3′-diaminobenzidine tetrahydrochloride (ΔA_405nm/min). Using horseradish peroxidase, the DAB method appears more sensitive than the o-dianisidine and the guaiacol assays for peroxidase. This method can also be used for measurement of peroxidase activity in tissue fractions.     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

Kinetics of fenugreek peroxidase isoenzymes

Peroxidase from fenugreek seedlings was separated into 6 isoenzymes; 4 on CM-cellulose and 2 on DEAE-cellulose. The kinetics of these peroxidase isoenzymes with regard to o-dianisidine and catechol are described.     

Transformations of Aromatic Compounds by Nitrosomonas europaea

Benzene and a variety of substituted benzenes inhibited ammonia oxidation by intact cells of Nitrosomonas europaea. In most cases, the inhibition was accompanied by transformation of the aromatic compound to a more oxidized product or products. All products detected were aromatic, and substituents were often oxidized but were not separated from the benzene ring. Most transformations were enhanced by (NH₄)₂SO₄ (12.5 mM) and were prevented by C₂H₂, a mechanism-based inactivator of ammonia monooxygenase (AMO). AMO catalyzed alkyl substituent hydroxylations, styrene epoxidation, ethylbenzene desaturation to styrene, and aniline oxidation to nitrobenzene (and unidentified products). Alkyl substituents were preferred oxidation sites, but the ring was also oxidized to produce phenolic compounds from benzene, ethylbenzene, halobenzenes, phenol, and nitrobenzene. No carboxylic acids were identified. Ethylbenzene was oxidized via styrene to two products common also to oxidation of styrene; production of styrene is suggestive of an electron transfer mechanism for AMO. Iodobenzene and 1,2-dichlorobenzene were oxidized slowly to halophenols; 1,4-dichlorobenzene was not transformed. No 2-halophenols were detected as products. Several hydroxymethyl (-CH₂OH)-substituted aromatics and p-cresol were oxidized by C₂H₂-treated cells to the corresponding aldehydes, benzaldehyde was reduced to benzyl alcohol, and o-cresol and 2,5-dimethylphenol were not depleted.     

Sensitive method for determination of peroxidase activity in tissue by means of coupled oxidation reaction

A simple colorimetric one-step method for determination of peroxidase activity in tissue is described. The method utilizes enzymically released H₂O₂ from glucose oxidation and o-dianisidine as hydrogen donor. The sensitivity of the method is at least ten times greater than existing methods. The influence of H₂O₂ concentration, buffer composition, and catalase interference is studied and discussed. An alternative fluorometric modification is briefly described.     

Partial characterization of peroxidase isoenzymes from rust-affected wheat leaves

Four anodic peroxidase isoenzymes from wheat leaves were purified by column chromatography and their kinetic behavior with common substrates were examined. One isoenzyme is more active in wheat resistant to stem rust fungi and differed from the others in carbohydrate content and also by a specific activity 2–4-fold higher with non-physiological electron donors. As a substrate, eugenol exhibited kinetic behavior different from p-phenylenediamine, guaiacol or o-dianisidine with all isoenzymes. All four isoenzymes showed similar pH and temperature optima and kinetic behavior and apparent K_m values for both H₂O₂ and non-physiological electron donors.     

Kinetics of chemically modified lignin peroxidase and enzymatic oxidation of aromatic nitrogen-containing compounds

Lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium was chemically modified by reductive alkylation with benzyl, naphthyl and anthracyl moieties, thereby increasing its superficial hydrophobicity. The three chemical modifications altered the kinetic behaviour of the enzyme in 10% acetonitrile with four different substrates: carbazole, pinacyanol, pyrene and veratryl alcohol. Benzyl modification of lignin peroxidase increased the catalytic efficiency (k _cat/K _m,app) 2.7 times for carbazole oxidation. Thirteen N-containing compounds, including pyrroles, pyridines, and aromatic amines, were tested to determine whether they could be oxidized by lignin peroxidase in 10% acetonitrile. All the pyrrole analogues and all the amines tested were oxidized, but none of the pyridine analogous reacted. Some products were isolated and analyzed by high-resolution mass spectrometry. Most were dimers or polymers and, in some cases, these contained oxygen atoms. The possibility of bitumen and petroleum modifications using this enzyme is discussed.     

Peroxidases from marine microalgae

Peroxidase activity was detected in cell-free extractsof strains of three species of the marine microalgae,Porphyridium purpureum, Phaeodactylumtricornutum and Dunaliella tertiolecta. However, no bromo- or chloroperoxidase activity wasdetected in any, using the standard 2-chlorodimedoneassay. Only the extract from P. purpureumoxidized iodide and this peroxidase was partiallypurified via anion-exchange chromatography. KI ando-dianisidine assay of the fractions indicatedthat only one peroxidase was present. Characterization of the thermally labile enzymesuggested that it is a heme-containing peroxidase,with a molecular weight of approximately 36,000.     

A spectrophotometric procedure for determining the activity of various rat tissue oxidases.

A continuous spectrophotometric method suitable for the determination of the activities of several peroxisomal oxidases in rat tissue homogenates is described. The assay involves the continuous spectrophotometric measurement of the reaction product, H₂O₂, by coupling it to the reduction of a chromogen, o-dianisidine, with horseradish peroxidase. Catalase interference was overcome using azide to inhibit its activity and a H₂O₂ standard curve used to quantitate oxidase activity in terms of microkatals per milliliter of enzyme.     

Mechanisms of peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate

Peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1–100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.     

Inhibition of horseradish peroxidase byN-ethylamide ofo-sulfobenzoylacetic acid

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence ofN-ethylamide ofo-sulfobenzoylacetic acid (EASBA) at pH 5.0–7.5. In the oxidation ofo-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases bothK _m andV _m in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.     

Anaerobic Metabolism of 1-Amino-2-Naphthol-Based Azo Dyes (Sudan Dyes) by Human Intestinal Microflora

The rates of metabolism of Sudan I and II and Para Red by human intestinal microflora were high compared to those of Sudan III and IV under anaerobic conditions. Metabolites of the dyes were identified as aniline, 2,4-dimethylaniline, o-toluidine, and 4-nitroaniline through high-performance liquid chromatography and liquid chromatography electrospray ionization tandem mass spectrometry analyses. These data indicate that human intestinal bacteria are able to reduce Sudan dyes to form potentially carcinogenic aromatic amines.