Simultaneous purification and immobilization of bitter gourd (Momordica charantia) peroxidases on bioaffinity support

This paper demonstrates the construction of an inexpensive bioaffinity adsorbent by simply incubating Sephadex G 50 matrix with jack bean meal extract at room temperature. Sephadex G 50 adsorbed 17 mg Con A (concanavalin A) per g of the matrix. Con A‐adsorbed Sephadex was employed for the immobilization of glycoenzymes directly from ammonium sulfate‐fractionated proteins of bitter gourd. The obtained bioaffinity support was very efficient for high yield immobilization of peroxidases from bitter gourd and it bound nearly 425 enzyme units per g of the matrix. Bitter gourd peroxidase immobilized on lectin–Sephadex support showed a very high effectiveness factor, ‘η,’ of 1.25. Immobilized BGP preparation was quite stable against the denaturation induced by pH, heat, urea, Triton X 100, Tween 20, SDS, Surf Excel and water‐miscible organic solvents: dimethyl sulfoxide and dimethyl formamide. Low concentration of detergents like SDS, Tween 20, and Triton X 100 enhanced the activity of soluble and immobilized bitter gourd peroxidase. Peroxidase bound to the bioaffinity support exhibited very high resistance to proteolysis caused by the trypsin treatment. Con A–Sephadex‐bound bitter gourd peroxidase retained 85% of its initial activity after treatment with 2.5 mg trypsin per cm³ of incubation mixture for 1 h at 37 °C while the soluble enzyme lost nearly 40% of the initial activity under similar incubation conditions. Immobilized bitter gourd peroxidase preparation appeared to be more rigid to proteolysis mediated by trypsin compared with soluble bitter gourd peroxidase. Copyright © 2004 Society of Chemical Industry     

Matrix physicochemical properties affect activity of entrapped chlorophyllase

Chlorophyllase, a membrane glycoprotein, was entrapped in various matrices, including alginate, alginate–silicate mixed gel, TMOS‐based sol–gel, and their hydrophobically‐modified counterparts. Chlorophyllase activity was affected by the physicochemical properties of the matrix, demonstrating lower activity in organically‐modified matrices compared with the corresponding hydrophilic matrices. The advantage of adopting organically‐modified matrices to facilitate the transfer of hydrophobic substrate is likely compromised by detrimental interaction between chlorophyllase and the hydrophobic components. This hypothesis is at least partly substantiated by the negligible activity demonstrated by free chlorophyllase in hydrophobic micellar media. Even though activity yields exceeded 50% in alginate beads, the release profile reveals that alginate matrix is too porous to retain chlorophyllase. Although alginate–silicate mixed gel more effectively confined chlorophyllase in the matrix, only 16% of the activity was recovered. In contrast, inorganic sol–gel yielded a chlorophyllase preparation with mass yield above 90%, and activity yield above 50%. Doping additives did not improve activity yield, which could be explained by the lower specific surface area and pore volume, and hence possible restricted accessibility of chlorophyllase by its substrate. Water/silane ratio was found to affect the sol–gel‐entrapped chlorophyllase activity by influencing the gelation time and physical properties of the gel. Copyright © 2005 Society of Chemical Industry     

Antibacterial activity of chitosan–alginate sponges incorporating silver sulfadiazine: Effect of ladder‐loop transition of interpolyelectrolyte complex and ionic crosslinking on the antibiotic release

Hydrogel membranes prepared from polyelectrolyte complex (PEC) have been used for repair of wounds and controlled antibacterial release. A simple method, based on homogenizing interpolyelectrolyte complex, has been developed to prepare a chitosan–alginate sponge with high stability. The spongelike chitosan–alginate hydrogel can be used as a wound dressing for the sustained release of silver sulfadiazine (AgSD) in a controlled way. In this study, we evaluated the effect of electrolyteic properties of chitosan and alginate on the characteristics of the prepared chitosan–alginate PEC. All types of the spongelike chitosan–alginate hydrogels exhibited superabsorbent properties. However, only the chitosan–alginate hydrogel prepared by the interpolyelectrolyte complex of alginate with low pH of chitosan, and that prepared by the interpolyelectrolyte complex of chitosan with high pH of alginate, can keep their stability after swelling in PBS solution. FTIR analysis suggests that the protonated amino groups on chitosan and the ionized carboxylic groups on alginate should be responsible for the formation of a stable ladder‐type of chitosan–alginate PEC. Ionic crosslinking is helpful to increase the stability of the loop‐type of chitosan–alginate PEC. The release of AgSD from chitosan–alginate PEC sponges could be controlled by the variation of ladder‐loop structural transition of chitosan–alginate PEC and the ionic crosslinking of the chitosan–alginate complex. The antibacterial ability of AgSD‐incorporated PEC sponges was examined in agar plate against Pseudomonas aeruginosa and Staphylococcus aureus. The result suggests that the PEC sponges containing antimicrobial agents should effectively suppress bacterial proliferation to protect the wound from bacterial invasion. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 538–549, 2005     

Shear‐Induced Fabrication of Multiple Parallel Alginate Gel Filaments Using Alginate–Polyethylene Glycol Blend

A rigid assembly of alginates is formed in aqueous media primarily via hydrogen bonding between guluronic units. A flow of aqueous alginate solution in a co‐flow capillary can form alginate gel fibers by contact with Ca²⁺ ions in sheath flow. Mixing with polyols [e.g., polyethylene glycol (PEG)] facilitates the shaping of the alginate assembly because PEG disrupts the assembly of the extended alginate chains to instead form alginate–PEG complexes that exhibit shear‐thinning behavior. The shear‐induced fibrous domains of the globular alginate–PEG complexes can be partitioned by a PEG‐rich phase, resulting in multiple parallel alginate gel filaments when the strong ionic‐field‐induced PEG‐rich phase is adjusted and an alginate–PEG complex phase is used as the aqueous two‐phase separation system.     

Controlled delivery of diclofenac sodium from calcium alginate beads loaded with a drug–resin complex

This study focused on a detailed investigation of the release of the nonsteroidal anti‐inflammatory drug diclofenac sodium from strong anion resin particles, entrapped in ionotropically crosslinked alginate beads, in simulated gastric and intestinal fluids at 37°C. The percentage drug released from the beads in media of various pH values in 6 h was nearly 68.8 ± 2.6%, whereas, for the same duration, the drug–resin complex particles released 87.6 ± 3.2% drug. The amount of drug released from the beads depended on the composition of the beads, their degree of crosslinking, and the size of the crosslinker ions. Finally, the value of the release exponent was found to be 0.56, which thus indicated the diffusion‐controlled mechanism of drug release from the alginate beads © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Properties of cathepsin B immobilized in calcium alginate beads

Cathepsin B (EC 3.4.22.1), purified from goat brain, was immobilized in calcium alginate beads in the presence of bovine serum albumin. The immobilized enzyme retained ∼63% of the original activity and could be used for seven successive batch reactions with retention of 22–30% of the initial activity. Immobilized cathepsin B hydrolysed α-N-benzoyl-D ,L -arginine-β-naphthylamide (BANA) maximally at pH 5·5, exhibiting a shift of 0·5 pH unit from that of the soluble enzyme (pH optima 6·0). It showed enhanced stability in acidic as well as alkaline environments in comparison to the free enzyme. The optimal temperature and thermal stability were not altered significantly after immobilization. The K_m value for the immobilized enzyme was two-fold higher than for the soluble enzyme.     

Chitosan/calcium–alginate beads for oral delivery of insulin

A mild chitosan/calcium alginate microencapsulation process, as applied to encapsulation of biological macromolecules such as albumin and insulin, was investigated. The microcapsules were derived by adding dropwise a protein-containing sodium alginate mixture into a chitosan–CaCl₂ system. The beads containing a high concentration of entrapped bovine serum albumin (BSA) as more than 70% of the initial concentration were achieved via varying chitosan coat. It was observed that approximately 70% of the content is being released into Tris-HCl buffer, pH 7.4 within 24 h and no significant release of BSA was observed during treatment with 0.1M HCl pH 1.2 for 4 h. But the acid-treated beads had released almost all the entrapped protein into Tris-HCl pH 7.4 media within 24 h. Instead of BSA, the insulin preload was found to be very low in the chitosan/calcium alginate system; the release characteristics were similar to that of BSA. From scanning electron microscopic studies, it appears that the chitosan modifies the alginate microspheres and subsequently the protein loading. The results indicate the possibility of modifying the formulation in order to obtain the desired controlled release of bioactive peptides (insulin), for a convenient gastrointestinal tract delivery system. © 1996 John Wiley & Sons, Inc.     

Optimization of lactic acid production from whey by L casei NRRL B‐441 immobilized in chitosan stabilized Ca‐alginate beads

The production of lactic acid from whey by Lactobacillus casei NRRL B‐441 immobilized in chitosan‐stabilized Ca‐alginate beads was investigated. Higher lactic acid production and lower cell leakage were observed with alginate–chitosan beads compared with Ca‐alginate beads. The highest lactic acid concentration (131.2 g dm^?3) was obtained with cells entrapped in 1.3–1.7 mm alginate–chitosan beads prepared from 2% (w/v) Na‐alginate. The gel beads produced lactic acid for five consecutive batch fermentations without marked activity loss and deformation. Response surface methodology was used to investigate the effects of three fermentation parameters (initial sugar, yeast extract and calcium carbonate concentrations) on the concentration of lactic acid. Results of the statistical analysis showed that the fit of the model was good in all cases. Initial sugar, yeast extract and calcium carbonate concentrations had a strong linear effect on lactic acid production. The maximum lactic acid concentration of 136.3 g dm^?3 was obtained at the optimum concentrations of process variables (initial sugar 147.35 g dm^?3, yeast extract 28.81 g dm^?3, CaCO₃ 97.55 g dm^?3). These values were obtained by fitting of the experimental data to the model equation. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in lactic acid production using alginate–chitosan‐immobilized cells. Copyright © 2005 Society of Chemical Industry     

Chitosan/polyethylene glycol–alginate microcapsules for oral delivery of hirudin

A mild chitosan/calcium alginate microencapsulation process, as applied to encapsulation of biological macromolecules such as albumin and hirudin, was investigated. The polysaccharide chitosan was reacted with sodium alginate in the presence of calcium chloride to form microcapsules with a polyelectrolyte complex membrane. Hirudin-entrapped alginate beads were further surface coated with polyethylene glycol (PEG) via glutaraldehyde functionalities. It was observed that approximately 70% of the content is being released into Tris-HCl buffer, pH 7.4 within the initial 6 h and about 35% release of hirudin was also observed during treatment with 0.1 M HCl, pH 1.2 for 4 h. But acid-treated capsules had released almost all the entrapped hirudin into Tris-HCl, pH 7.4 media within 6 h. From scanning electron microscopic and swelling studies, it appears that the chitosan and PEG have modified the alginate microcapsules and subsequently the protein release. The microcapsules were also prepared by adding dropwise albumin-containing sodium alginate mixture into a PEG– CaCl₂ system. Increasing the PEG concentration resulted in a decrease rate of albumin release. The results indicate the possibility of modifying the formulation to obtain the desired controlled release of bioactive peptides (hirudin), for a convenient gastrointestinal tract delivery system. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 70: 2143–2153, 1998     

Evaluation of alginate–chitosan bioadhesive beads as a drug delivery system for the controlled release of theophylline

This study describes the preparation of mucoadhesive alginate–chitosan beads containing theophylline intended for colon‐specific delivery. The calcium alginate beads were coated with chitosan by the ionotropic hydrogelation method with a polyelectrolyte complex reaction between two oppositely charged polyions. The release profiles of theophylline from the beads were determined by ultraviolet–visible absorption measurement at 272 nm. Scanning electron microscopy was used for morphology observation. The in vitro mucoadhesive tests for particles were carried out with the freshly excised jejunum of Sprague‐Dawley rats. The bead particles, which ranged in size from 200 to 400 μm, exhibited excellent mucoadhesive properties. The results showed that the formulated coated beads succeeded in controlling the release of theophylline over a 24‐h period. In conclusion, the release of theophylline was found to be dependent on the composition of the beads, the component polymer and its possible interactions, and the bioadhesiveness. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Chemoselective hydrolysis of methyl 2‐acetoxybenzoate using free and entrapped esterase in K‐carrageenan beads

Porcine liver esterase was entrapped in natural polysaccharides K‐carrageenan and retention of its activity was determined using p‐nitrophenyl acetate as the substrate. The optimum pH for esterase activity of entrapped enzyme showed a little shift towards acidic side. Immobilized enzyme showed improved thermal and storage stability. The entrapped esterase retained 50% of its activity after eight repetitive cycles. Michaelis constant K_m for the free and entrapped enzymes was almost same indicting no conformational change during immobilization. Maximum velocity V_max was observed to decrease on immobilization. The free and entrapped esterase was used for selective hydrolysis of methyl 2‐acetoxybenzoate to methyl 2‐hydroxybenzoate in batch process as well as in a fixed bed reactor. The hydrolysis was observed to be 99% within 2 h for free as well as immobilized enzyme in batch process. The rate of hydrolysis was found to depend on pH. The turn over number of selective hydrolysis in batch and fixed bed reactor was 3.08 × 10⁶ and 1.19 × 10⁷, respectively. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Viscoelastic sol–gel state of the chitosan and alginate solution mixture

The rheological behavior of chitosan/alginate solutions was investigated in relation to gelation and polyelectrolyte complex (PEC) formation. Before mixing, the chitosan and the alginate solutions were both homogeneous fluids. However, heterogeneity developed after mixing, accompanied by a serious increase of viscosity. To determine the sol–gel state of the solutions, the viscoelastic variables, such as the dynamic storage modulus (G′) and loss modulus (G″), the loss tangent, and the viscoelastic exponents for G′ and G″, were obtained. Depending on the concentration, the chitosan/alginate solutions revealed unexpected rheological behavior. At a polymer concentration of 1.0 wt %, the chitosan/alginate solution was in a viscoelastic gel state, whereas, at higher concentrations, viscoelastic sol properties were dominant. A viscoelastic gel state for the chitosan/alginate solution was induced based on the weak formation of fiber‐shaped precipitates of a PEC at a low polymer concentration. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 1408–1414, 2007     

Bioartificial dextran and poly[ethylene‐co‐(vinyl alcohol)] hybrid sponges as support for proteins

A biological–synthetic hybrid material was prepared as a support for proteinic molecules. To control the conformational stability and ‘vitality’ of the entrapped compound, an enzyme, which catalyses a chemical reaction, was chosen. The physical immobilisation of α‐amylase on a novel biological–synthetic hybrid material was realised using a synthetic (poly[ethylene‐co‐(vinyl alcohol)]) and a biological (dextran) component. The supports were obtained in the form of porous sponges using the phase inversion process. The kinetic parameters of enzymatic hydrolysis of soluble starch were obtained by determining the maltose (reaction product) concentration in the test solution by a spectrophotometric method. A high amount of enzyme (10 and 15 mg per g of polymer matrix) was successfully entrapped in the polymer support and a rather good enzyme activity of entrapped α‐amylase and a constant activity for repeated use, compared with native α‐amylase, were observed. The results obtained clearly indicated that the bioartificial polymer sponges, showing an immobilisation of α‐amylase with maintenance of the catalytic function of the enzyme, can be used as suitable supports for proteins. Copyright © 2005 Society of Chemical Industry     

Optimized synthesis,characterization, and antibacterial activity of an alginate–cupric oxide bionanocomposite

In this research, the structural features and optimal conditions for the synthesis of an alginate–CuO nanocomposite with the highest antibacterial activity were investigated. CuO nanoparticles (NPs) and the alginate biopolymer were synthesized chemically and biologically, respectively. Nine nanocomposite compounds were produced on the basis of the Taguchi method with different levels of CuO NPs and the alginate biopolymer nanocomposite with different stirring times. Fourier transform infrared spectroscopy, high‐resolution field emission scanning electron microscopy, and energy‐dispersive X‐ray spectroscopy analysis confirmed the formation of the nanocomposites. The nanocomposite synthesized with 8 mg/mL copper oxide NPs and 2 mg/mL alginate biopolymer with 60 min of stirring time showed the highest antibacterial activity. The results of two colony forming units and disk‐diffusion methods indicated a stronger antibacterial activity of the alginate–CuO nanocomposite compared with those of its components. The alginate–CuO nanocomposite showed the potential ability to act as an antimicrobial agent against Gram‐negative and Gram‐positive bacteria. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 45682.     

Recyclable plant tannin‐chelated Rh(III) complex catalysts for aqueous–organic biphasic hydrogenation of quinoline

BACKGROUND: Synthetic ligands have conventionally been used for the preparation of homogenous Rh complex catalyst but biomass has rarely been utilized for this purpose. In the present investigation, plant tannins (natural polyphenols) were used as water‐soluble ligands for the preparation of homogenous Rh³⁺ complex catalysts. RESULTS: Based on X‐ray photoelectron spectroscopy (XPS) and proton nuclear magnetic resonance (HNMR) analyses, the preparation mechanism of these complex catalysts was proven to involve chelating interactions between Rh³⁺ and the adjacent phenolic hydroxyls of plant tannins. As a result, the use of plant tannin promoted aqueous‐organic biphasic interactions and the plant tannin‐chelated Rh³⁺ complex catalysts exhibited much higher catalytic activity than commercial Rh complex catalysts in the biphasic hydrogenation of quinoline. Furthermore, the plant tannin‐chelated Rh³⁺ complex can be reused three times without significant loss of catalytic activity CONCLUSION: Our experimental results suggested that black wattle tannin (BWT) can be used as water‐soluble ligands for the preparation of highly active and recyclable Rh³⁺ complex catalysts. Copyright © 2012 Society of Chemical Industry     

Polyurethane‐incorporated chitosan/alginate core–shell nano‐particles for controlled oral insulin delivery

Chitosan (CS) and polyurethane‐chitosan (PU‐CS) nano‐particles (NPs) were prepared for the core formation by complex coacervation method whereas alginate (ALG) and PU‐ALG were crosslinked by ionic gelation method to form the protective shell‐layer over the core. Effects of PU incorporation either within the core or shell or both were investigated by different in vitro and in vivo parameters. Fourier transform infrared (FTIR) spectroscopy of different compositions of nano‐particles showed distinct characteristic peaks for CS, PU, and ALG, indicating their presence in variable ratios. Significance of polyurethane‐incorporated systems towards insulin encapsulation efficiency, swelling parameters, insulin release, and in vivo pharmacological effect were also studied. Particle sizes, zeta potential, morphological analysis, mucoadhesion study, and in vivo acute toxicity studies of these core–shell nano‐particles were also performed. Bioavailability of insulin ranged from 9.04% to 11.6% for polyurethane‐incorporated chitosan‐alginate core–shell nano‐particle formulations which was significantly higher than the insulin bioavailability of basic CS/ALG core–shell nano‐particle system. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46365.     

Chitosan‐coated alginate–polyvinyl alcohol beads for encapsulation of silicone oil containing pyrene: a novel method for biodegradation of polycyclic aromatic hydrocarbons

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are potential hazards in the environment owing to their toxic, carcinogenic and recalcitrant nature. Biodegradation of these compounds, although effective compared with other treatment techniques, is problematic owing to its low aqueous solubility and negligible bioavailability. The present study reports a novel method for biodegradation of PAHs using an encapsulated form of the pollutant in chitosan‐coated alginate–polyvinyl alcohol (PVA) beads. RESULTS: A suitable combination of 3% (w/v) PVA, 100 g L^?1 non‐ionic surfactant Brij 30 and 0.3 silicone oil fraction in the formulation was found to be optimal in the preparation of stable emulsion. The emulsion obtained was admixed with alginate (3% w/v) to prepare suitably sized microspheres by an emulsion gelation technique, which were later coated with chitosan to yield a maximum pyrene encapsulation efficiency of 90.7%. Pyrene in silicone oil at concentration as high as 2 g L^?1, when delivered through the chitosan coated alginate–PVA beads, was completely degraded by Mycobacterium frederiksbergense within 10 days without any significant lag phase. CONCLUSION: Using chitosan‐coated alginate–PVA beads sustained release of pyrene and subsequent biodegradation by M. frederiksbergense were achieved. Using the present system, complete degradation of pyrene was attained even at its very high initial concentration and within a short time period. Further advantage offered by this system seems to be negligible toxic effect of pyrene and solvents on the degrading microorganisms since these were in an encapsulated form and were not in direct contact with the organism. Copyright © 2010 Society of Chemical Industry     

Investigation of Phenol Removal using Sol‐Gel/Alginate Immobilized Soybean Seed Hull Peroxidase

In this study, the removal of phenol using immobilized soybean seed hull peroxidase was investigated. The soybean peroxidase (SBP) enzymes were extracted from the raw soybean seed hulls and entrapped within hybrid (silica sol‐gel/alginate) particles. An aqueous sol‐gel route was preferred over an alkoxide route to prepare the silica sol‐gel suspension. A cluster of nozzles was designed for the bulk production of the biocatalysts. Upto 85% phenol was polymerized using the fabricated biocatalysts under the optimized conditions. Viability studies and repeated application/regeneration of entrapped enzymes were carried out. Surface conditions and the pore structure of the particles were studied using SEM before the reaction and after the reaction. The study indicates that these particles are promising biocatalysts for fluidized bed or packed bed reactors. This study represents the first investigation of enzymatic reactions that form a coating on the immobilized biocatalyst.     

Macroscopic N‐halamine biocidal polymeric beads

The particle size of N‐halamine biocidal polymers was methodically modified forming beads of different sizes by blending water‐insoluble N‐halamine polyurethane with sodium alginate as the matrix and loading heterocyclic rings onto modified silica gels. The biological activity of the prepared beads and halogenated modified silica derivatives was evaluated against examples of Gram‐positive (Staphylococcus aureus) and Gram‐negative (Escherichia coli) bacteria. The recycling possibilities and the optimum preparation conditions of the blended beads were investigated; blending prehalogenated polyurethane (5%, w/v) with sodium alginate (3%, w/v) followed by crosslinking with CaCl₂ (10%, w/v) at 40°C are the optimum preparation conditions for the alginate beads. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Preparation and characterization of alginate hydrogel membranes crosslinked using a water‐soluble carbodiimide

The preparation of alginate hydrogel membranes by the film immersion method was optimized for maximum crosslinking using swelling measurements as an indicator of the degree of crosslinking. The variables investigated were the concentration of the nonsolvent (ethanol) for sodium alginate, water‐soluble carbodiimide (WSC) concentration, and pH of the crosslinking medium. Optimum conditions resulted when the crosslinking medium contained 60 vol % ethanol and 100 mM WSC at pH 4. Membranes prepared using different ethanol concentrations (100 mM WSC, pH 4) and different WSC concentrations (60 vol % ethanol, pH 4) were investigated using infrared spectroscopy. The spectra showed the characteristic ester linkage (crosslinking) band at 1698 cm^?1 in cases where swelling measurements indicated that crosslinking had occurred. Differential scanning calorimetry of noncrosslinked and crosslinked membranes indicated that crosslinking increased the crystallinity of the membrane. Durability trials showed that membranes crosslinked using the optimum conditions determined in this work retained all weight when immersed in water for 32 days. Membranes prepared using these conditions possessed the characteristics required for use in the pervaporation separation of ethanol–water mixtures. These membranes also have potential as protective coatings for hydrophobic, microporous membranes in the membrane distillation and osmotic distillation concentration of feeds containing surface active components. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 747–753, 2003