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 assay for the measurement of hydroperoxides

The enzyme glutathione (GSH) peroxidase can be used to measure hydroperoxides quantitatively, easily, and specifically. A timed reaction of GSH peroxidase, coupled with the oxidation of NADPH by GSH reductase, allows a direct spectrophotometric measurement of hydroperoxide. Addition of catalase prior to the addition of GSH peroxidase permits the distinction between hydrogen peroxide and organic hydroperoxides. The solvents that can be used with the assay include methanol, ethanol, water, and aqueous solutions of detergents such as Brij 35, Triton X-100, and cetyl trimethyl ammonium bromide. The utility of the method is demonstrated by the measurement of hydrogen peroxide and organic hydroperoxides formed upon ozonolysis of an unsaturated fatty acid.     

Characterisation and inhibition effect of cetrimide on collagenase produced byAspergillus flavus, isolated from mycotic ulcers

Collagenase was found to be the most important enzyme, produced by mycotic keratitis fungi. Therefore,Aspergillus flavus collagenase enzyme has been purified by ammonium sulphate precipitation, Sephadex G25 and DEAE-cellulose chromatography. Electrophoretic analysis for the purified enzyme indicated one subunit of molecular weight of 70–90 KDa when examining on SDS-PAGE. Cetrimide (cetyl trimethyl ammonium bromide) has been tested against the purified collagenase enzyme and indicated reversible competitive inhibitor (kis=0.15 mg/ml) with high promising activity. Cetrimide might be used to inhibit mycotic keratitis fungi.     

Bioaffinity-based an inexpensive and high yield procedure for the immobilization of turnip (Brassica rapa) peroxidase

This study demonstrates the immobilization of carbohydrate containing turnip peroxidase on an inexpensive bioaffinity adsorbent, Concanavalin A-cellulose support. The bioaffinity support was prepared simply by incubating cellulose powder with jack bean extract at 4 degrees C. Cellulose powder adsorbed 30 mg concanavalin A/g of the matrix. Concanavalin A adsorbed cellulose has been employed for the simultaneous purification and immobilization of glycoenzymes directly from ammonium sulphate fractionated proteins of turnip. The obtained bioaffinity support was quite effective in high yield immobilization of peroxidase from turnip and it retained 672 U/g. Turnip peroxidase immobilized on concanavalin A-cellulose support retained 80% of the initial activity. Immobilized turnip peroxidase preparation was quite resistant against the denaturation mediated by pH, heat, urea, guanidinium-HCl, Surf Excel, cetyltrimethylammonium bromide and water-miscible organic solvents; dimethyl formamide, dioxane and n-propanol. Low concentration of detergents like Surf Excel and cetyltrimethylammonium bromide enhanced the activity of soluble and immobilized turnip peroxidase.     

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.     

Highly efficient "tight fit" immobilization of alpha-chymotrypsin in mesoporous MCM-41: a novel approach using precursor immobilization and activation

The zymogen alpha-chymotrypsinogen A is bound to mesoporous silica MCM-41 with a protein loading of 170 mg/g solid (MCM-Z) by a simple stirring in aqueous tris-HCl buffer (pH 7.2). The bound zymogen is then activated with trypsin to obtain alpha-chymotrypsin immobilized on MCM-41 (MCM-E.I) that displays an effective enzyme activity corresponding to 65 mg protein/g of solid support (3250 BTEE units/g). A direct immobilization of commercially available alpha-chymotrypsin (MCM-E.II) gives lower loading (1250 BTEE units/g). Protein content of the solid support after immobilization is confirmed by thermogravimetric analysis (TGA). The enzyme is tightly bound to the support and can be used over 100 recycles over 1 week in aqueous as well as reverse micellar media. The immobilized enzyme (MCM-E.I) has been used for resolution of N-acetyl-dl-amino acid esters and racemic trans-4-methoxy-3-phenylglycidic acid (PGA) methyl ester.     

Characterization of dewatering process of activated sludge assisted by cationic surfactants

In this work, the dewatering of activated sludge assisted by cationic surfactants was investigated. Dose of dodecyl trimethyl ammonium bromide (DTAB) and cetyl trimethyl ammonium bromide (CTAB) resulted in the release of extracellular polymeric substances (EPS) from sludge and decrease in sludge negative charge. The surfactants significantly promoted sludge dewaterability, as reflected by decreased specific resistance of filtration (SRF) and water content in sludge cakes. The treated sludge were analyzed by combined use of differential scanning calorimetry (DSC), thermo gravimetric analysis (TGA) and Brunauer–Emmett–Teller (BET) analysis. CTAB was found more effective in releasing bound water from sludge than DTAB, due to its superior surface activity and strong adsorption/bridge capacities with sludge. The specific surface area and pore size of sludge cakes declined after surfactant treatment, indicating an enhanced compressibility. With these results, the surfactant-assisted dewatering mechanism could be attributed to the integrated effects of electrostatic neutralization, enhanced compaction and release of EPS and bound water. Our study clearly characterizes the accelerated dewatering process assisted by cationic surfactants, and demonstrates that cationic surfactants could be used as a potential agent for sludge dewatering.     

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.     

Improvement in tannase recovery using enzymatic disruption of mycelium in combination with reverse micellar enzyme extraction

Summary A high activity tannase (tannin acyl hydrolase EC 3.1.1.20) is synthetized in high yield by Aspergillus niger LCF 8. At the production optimum, the tannase is strongly bound to the mycelium and detachment of the enzyme by classical physical and chemical means, largely failed. Enzymatic hydrolysis of cell walls using a chitinase from Streptomyces griseus followed by reverse micellar tannase extraction resulted in a recovery of 43% active enzyme, i.e. an improvement in yield of 2.5 from a previous process. Best conditions were enzymatic hydrolysis of mycelium with chitinase at pH 6.0 and 25°C for 2.5 h followed by tannase extraction at pH 7.5 with isooctane containing 80 mM cetyl trimethyl ammonium bromide and stripping at pH 4.0 in the presence of 0.35 M NaCl.     

Covalent immobilization of cyclodextrin glucosyltransferase (CGTase) in activated silica and Sepharose

Cyclodextrin glucanotransferase is a non-Leloir glycosyltransferase that directly employs the free energy of cleavage of starch to produce cyclodextrins. In presence of appropriate acceptors, this enzyme synthesizes oligosaccharides containing alpha(1-->4) bonds. We have investigated the covalent immobilization of CGTase onto different activated supports. Silica was aminated and further activated with glutaraldehyde. The maximum amount of bound protein was about 4 mg CGTase per gram of support; however, the catalytic efficiency of the immobilized enzyme was lower than 6%. Sepharose 4B activated with cyanogen bromide (CNBr-activated Sepharose) and Sepharose 4B with a spacer arm of 1,6-diaminohexane (EAH Sepharose) were also assayed. These gels react with the amino and carboxylic groups of CGTase, respectively. With CNBr-activated Sepharose, a low percentage of enzyme was bound to the support but with a significant catalytic efficiency (29%). A higher recovery of protein was obtained with EAH Sepharose (62%), but only 2.4% of the initial activity was present in the immobilized biocatalyst. The results were discussed in terms of CGTase structure and mechanism. In addition, the solvent accessibility of amino or carboxylic groups, calculated using the NACCESS software, was considered.     

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.     

Glucose isomerase activity of streptomyces kanamyceticus

Summary Streptomyces kanamyceticus produces a significant level of intracellular glucose isomerase when grown in submerged culture. The optimum temperature for enzyme activity is 90°C, but the optimum pH is changed by the kinds of buffer solution used. The activity is higher at pH 7.0–9.5. Treatment of cells with cetyl trimethyl ammonium bromide extracts almost the same amount of the enzyme as ultrasonic treatment. The selection of the method of treatment for enzyme extraction depends, however, on the nature of cell growth in synthetic or complex medium.     

Redox-mediated oxidation and removal of aromatic amines from polluted water by partially purified bitter gourd (Momordica charantia) peroxidase

Here, the role of bitter gourd peroxidase has been investigated for the treatment of water contaminated with aromatic amines. Most of the aromatic amines were recalcitrant to the action of bitter gourd peroxidase. However, these aromatic amines were oxidized by bitter gourd peroxidase in the presence of a redox mediator, o-dianisidine HCl. The maximum oxidation of aniline was found to be in the buffer of pH 5.0 at 40 °C in the presence of 0.5 mM H₂O₂ and 0.15 mM o-dianisidine HCl. Aromatic amines oxidized and removed from wastewater were 65% aniline, 50% m-toluidine, 86% m-chloroaniline, 54% p-aminobenzoic acid, 61% diphenylamine and 95% N,N-dimethylaniline. Benzidine and p-nitroaniline were recalcitrant to the action of this enzyme even in the presence of o-dianisidine HCl. Complex mixtures of aromatic amines were treated by bitter gourd peroxidase. These mixtures were removed to varying extent, mixtures A, B and C were oxidized to 59%, 56% and 62%, respectively. Mixtures D, E and F were marginally oxidized to 30%, 14% and 16%, respectively.     

Comparative study of horseradish peroxidase immobilization on modification of a gold surface using a surface plasmon resonance method

Horseradish peroxidase is immobilized by a periodate method on the gold surfaces previously modified with 16-mercapto-hexadecanoic acid or with hydrogen disulfide and soybean trypsin inhibitor. The effect of gold surface modification conditions on the immobilization of the enzyme as well as on the properties of the immobilized glycoprotein are studied using surface plasmon resonance technique. Restoration of the ability to bind specific antibodies is demonstrated for the immobilized enzyme. The low level of non-specific antibody binding to the immobilized glycoprotein is also shown.     

Comparison of soluble and immobilized trypsin kinetics: Implications for peptide synthesis

The protease trypsin was immobilized to porous glass in both the presence and absence of acetylated soybean trypsin inhibitor (STI) to determine whether immobilization could alter enzyme activity in favor of aminolysis over hydrolysis. Actiive-site titration with 4-methylumbelliferylguanidinobenzoate (MUGB) showed that only about 10% of immobilized trypsin had catalytic activity. Immobilization in the presence of STI produced a higher yield of active enzyme accessible to the inhibitor but did not increase the total yield of MUGB-active immobilized enzyme. Thus, enzyme inactivation upon immobilization could not be attributed to an inaccessible enzyme orientation, nor did STI prevent inactivation by stabilizing the active-site conformation. Kinetic parameters were determined for soluble and immobilized trypsin for two esters, N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE), and two amides, N-benzoyl-L-arginine p-nitroanilide (BAPNA) and N-t-boc-leucylglycylarginine p-nitroanilide (LGRNA). In all cases, immobilization caused a greater decrease in k(cat) for amidase activity than for esterase activity. The ratio [k(cat)/ K(m) (ester)]/[k(cat)/K(m) (amide)] increased slightly or stayed the same (for I.GRNA) or decreased sharply (for BAPNA). Including STI during immobilization had little effect on the active enzyme's intrinsic kinetics. A direct comparison of energy diagrams and free energies of activation for BAEE and BAPNA indicates that immobilization raises the free energy barriers for both amide and ester hydrolysis and lowers the energy barrier for aminolysis. In practice, these effects should lower the amidase activity and increase the aminolysis-hydrolysis ratio, rendering the immobilized enzyme a more efficient catalyst for peptide synthesis. (c) 1993 John Wiley & Sons, Inc.     

The effect of beta-tubulin-specific antisense oligonucleotide encapsulated in different cationic liposomes on the suppression [correction of supression] of intracellular L. donovani parasites in vitro

An antisense oligonucleotide (20 mer) targeted to the parasite beta-tubulin gene and encapsulated in cationic liposomes, was used to test its antileishmanial activity in vitro. Cationic liposomes containing dioleyl trimethyl ammonium propane (DOTAP) were found to have higher antileishmanial activity (88% at 4 microM oligonucleotide) compared to two other liposomes with stearyl amine (SA) and cetyl trimethyl ammonium bromide (CTAB) as cations. Dot-blot experiments were performed to analyse the expression of beta-tubulin mRNA using beta-tubulin-specific radiolabelled DNA as a probe. When compared with their respective controls, beta-tubulin-specific gene expression was found to be diminished by treatment with a specific antisense oligonucleotide encapsulated in cationic liposomes (CTAB:DOPE) in a concentration-dependent manner. These experiments show that antisense oligonucleotides targeted to the beta-tubulin gene of Leishmania donovani inhibit beta-tubulin synthesis leading to the arrest of multiplication of intracellular parasites.     

Immobilization of trypsin on sand: Mode of binding

Acid-washed and heat-treated river sand was separated into different fractions by geochemical methods and immobilization of trypsin was carried out on the separated fractions using 3-aminopropyltriethoxysilane and glutaraldehyde. Scanning Electron Micrographs of the purified fraction (Sp. gr >2.5 and <2.8) of magnetically non-susceptible sand and quartz showed that the enzyme could be fixed on the supports. Malonic acid (16.3 nmol and 16.7nmol per g) appeared to be bound to alkylamine purified fraction of magnetically non-susceptible sand and alkylamine quartz, respectively. Studies on the effect of 6 M guanidine.HCl on immobilized trypsin demonstrated that immobilized trypsin had considerable stability against denaturation. The results obtained indicated that magnetically non-susceptible sand was found to be nearly as good as quartz for trypsin immobilization and that trypsin was covalently coupled to sand via 3-aminopropyltriethoxysilane and glutaraldehyde.     

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.     

Immobilized trypsin on hydrophobic cellulose decorated nanoparticles shows good stability and reusability for protein digestion

The preparation of biocatalysts based on immobilized trypsin is of great importance for both proteomic research and industrial applications. Here, we have developed a facile method to immobilize trypsin on hydrophobic cellulose-coated silica nanoparticles by surface adsorption. The immobilization conditions for the trypsin enzyme were optimized. The as-prepared biocatalyst was characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and elemental analysis. In comparison with free enzyme, the immobilized trypsin exhibited greater resistances against thermal inactivation and denaturants. In addition, the immobilized trypsin showed good durability for multiple recycling. The general applicability of the immobilized trypsin for proteomic studies was confirmed by enzymatic digestion of two widely used protein substrates: bovine serum albumin (BSA) and cytochrome c. The surface adsorption protocols for trypsin immobilization may provide a promising strategy for enzyme immobilization in general, with great potential for a range of applications in proteomic studies.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.