Ca-alginate Beads Research Articles

Prosopis alba exudate gum as novel excipient for fish oil encapsulation in polyelectrolyte bead system

In this work, a bottom-up approach based on the study of polyelectrolyte interactions was performed in order to evaluate the effect of Prosopis alba exudate gum as novel excipient for fish oil encapsulation in composed calcium-alginate-chitosan beads. Emulsion and beads properties such as oil distribution, encapsulation efficiency, yield, microstructure and thermo-oxidative protection were evaluated. Alginate and gum exert a synergistic effect on emulsion stability properties, which conducted to better oil distribution in the beads and higher encapsulation efficiencies (98%) and yield (89%). The positive effect of including the gum as wall material was observed in terms of a higher oil retention capacity of the alginate beads, improved oxidative thermal stability and better microstructural features. Present results are promising and allowed considering P. alba gum as a novel non-conventional polyelectrolyte for improving Ca-alginate beads microstructure and stability with the added benefit of taking advantage of an available resource currently untapped.

Free and Ca-Alginate Beads Immobilized Horseradish Peroxidase for the Removal of Reactive Dyes: an Experimental and Modeling Study.

The aim of this work was to remove the dyes Reactive Blue 221 (RB 221) and Reactive Blue 198 (RB 198) of synthetic effluent using the immobilized enzyme horseradish peroxidase (HRP) in Ca-alginate beads. Experimental parameters affecting the dye removal process such as the effect of pH, temperature, hydrogen peroxide concentration, mass capsules, and reuse were evaluated, and a numerical model of mass transfer was developed. A maximum removal of 93 and 75%, respectively, for the dyes RB 221 and RB 198, at pH 5.5 and temperature of 30°C, concentration of hydrogen peroxide of 43.75μM for dye RB 221 and 37.5μM for the dye of RB 198 was obtained. A removal reaction of 180min for RB 221 and 240min for RB 198 was observed. Three reuse cycles of use of immobilized enzyme were achieved for both dyes. The numerical model proposed led to a good fit compared to experimental data. The HRP enzyme immobilized in Ca-alginate capsules showed a great potential for biotechnological applications, especially for the removal of reactive dyes.

Calcium gluconate as cross-linker improves survival and shelf life of encapsulated and dried Metarhizium brunneum and Saccharomyces cerevisiae for the application as biological control agents

Calcium chloride (CC) is the most common cross-linker for the encapsulation of biocontrol microorganisms in alginate beads. The aim of this study was to evaluate if calcium gluconate (CG) can replace CC as cross-linker and at the same time improve viability after drying and rehydration, hygroscopic properties, shelf life and nutrient supply. Hence, the biocontrol fungi Metarhizium brunneum and Saccharomyces cerevisiae were encapsulated in Ca-alginate beads supplemented with starch. Beads were dried and maximum survival was found in beads cross-linked with CG. Beads prepared with CG showed lower hygroscopic properties, but a higher shelf life for encapsulated fungi. Moreover, we demonstrated that gluconate has a nutritive effect on encapsulated fungi, leading to increased mycelium growth of M. brunneum and to enhanced CO2 release from beads containing Saccharomyces cerevisiae. The application of CG as cross-linker will pave the way towards increasing drying survival and shelf life of various, especially drying-sensitive microbes.

Continuous generation of fructose from Taraxacum officinale tap root extract and inulin by immobilized inulinase in a packed-bed reactor

Abstract Production of exo-inulinase from newly isolated Aspergillus niger was statistically optimized using dandelion tap root extract (% fresh w/v) resulting in a ~2.5 fold (2337 nkat/ml) enhancement. Exo-inulinase (1166 nkat/mg) immobilized in Ca-alginate beads was used for continuous generation of fructose from crude inulin extracted from Taraxacum officinale (dandelion) and chicory juice. Immobilized enzyme was optimally active at 50 °C and pH 5.0 and the kinetic parameters evaluated were 3.74 mM ( K m ) and 1.62 µm/min ( V max ). Continuous generation of fructose syrup from low-cost dandelion root extract and chicory inulin using a packed bed bioreactor prepared with exo-inulinase immobilized in alginate beads was demonstrated. Successive degradation of dandelion oligosaccharides and fructans to pure fructose was observed over 30 days period. The bioreactor had a half-life of 15 days and more than 90% (w/w) yield of fructose was observed. The study demonstrated use of a fructan-rich common weed for continuous production of fructose.

Immobilization of a microbial consortium on Ca-alginate enhances degradation of cationic surfactants in flasks and bioreactor

Free cells and Ca-alginate encapsulated cells of Aeromonas hydrophila MFB03 removed in shake flasks up to 65% of tetradecyltrimethylammonium bromide (TTAB) 30 mg l−1. While planktonic cells were unable to utilize 50 mg l−1 benzalkonium chloride (BAC) after 48 h of incubation, the immobilized cells of A. hydrophila MFB03 removed up to 90% of BAC (25–210 mg l−1) in the same period. A microbial immobilized consortium formed by A. hydrophila MFB03 and Pseudomonas putida A (ATCC 12633) was able to degrade, after 24–30 h, 65% of TTAB and 100% of BAC, both added at 50 mg l−1. BAC was completely removed by the consortium encapsulated in a stirred tank bioreactor at 30 °C, 100 rpm and 0.024 g beads ml−1 of medium, after 12 h of incubation; reaching a degradation rate five times greater than the one obtained in shake flasks (0.415 vs. 0.089 mg−1 h−1, respectively). The system completely removed, with the same efficiency, 50 mg l−1 of BAC for 3 consecutive cycles. These results indicate that the use of Ca-alginate beads containing cells of the consortium can be considered a proper method to achieve degradation of cationic surfactants.

Matrix resistance stress: A key parameter for immobilized cell growth regulation

Abstract Microenvironmentally restricted yeast cell growth within Ca-alginate beads with and without entrapped gas bubbles was considered based on experimental data. Cell growth dynamics was described by (1) the dimensionless cell number density as a function of the cell growth time and (2) the cell distribution per bead cross sections. One of the key control parameters for bioprocess optimization is the matrix resistance stress generated during immobilized cell expansion. The dynamics of the increase in matrix stress was described theoretically based on a multi-scale mathematical model. In order to estimate and reduce the accumulation of matrix stress we considered repeated stress relaxation cycles in separate rheological experiments without immobilized cells. The results revealed that the increase in resistance stress within the Ca-alginate matrix was significant (∼7 kPa) after 10 repeated cycles, even under a low compression strain of 2% per cycle. The stress could be reduced by using the Ca-alginate matrix with entrapped gas bubbles. The final cell concentration within the beads with entrapped bubbles was 3.3 times higher in comparison with the beads without bubbles. The bubbles could locally amortize the compression effects within the surrounding cell clusters.

Calcium-alginate microparticles for sustained release of catechin prepared via an emulsion gelation technique

Catechin-loaded Ca-alginate beads and microparticles were prepared by an emulsion gelation method using sunflower oil for efficient sustained release of catechin. The emulsion was prepared by sequential mixing of alginate, oil, and oleic acid ester as an emulsifier. Encapsulation efficiency (EE) and inhibition of catechin release of the beads were significantly increased approximately to 453.83 and 148.71% by the emulsion gelation technique, respectively (p<0.05). For the microparticles, the highest inhibition of catechin release after 1 h of incubation (78.82%) was observed at the microparticles prepared by 5% (w/w) oil, 3% (w/w) alginate, 4% (w/v) CaCl2, and 200 mg catechin with the most hydrophilic emulsifier, decaglycerol mono-ester. Moreover, the catechin release was sustained at acidic conditions and increased with increase in pH of release medium. These results suggest that catechin encapsulation within Ca-alginate particles by emulsion gelation method can be an effective delivery system for catechin.

Characterization and Screening of a Novel Multiparticulate Pulsatile Delivery of Aceclofenac

In the present investigation, sodium alginate based multiparticulate system overcoated with time and pH dependent polymer was studied in the form of oral pulsatile system to achieve pulsatile with sustained release of aceclofenac for chronotherapy of rheumatoid arthritis seven batches of micro beads with varying concentration of sodium alginate (2-5 %) were prepared by ionotropic-gelation method using CaCl2 as cross-linking agent. The prepared Ca-alginate beads were coated with 5% Eudragit L100 and filled into pulsatile capsule with varying proportion of plugging materials. Drug loaded microbeads were investigated for physicochemical properties and drug release characteristics. The mean particle sizes of drug-loaded microbeads were found to be in the range 596±1.1 to 860 ± 1.2 micron and %DEE in the range of 65-85%. FT-IR and DSC studies revealed the absence of drug polymer interactions. The release of aceclofenac from formulations F1 to F7 in buffer media (pH 6.8) at the end of 5h was 65.6, 60.7, 55.7, 41.2, 39.2, 27 and 25% respectively. Pulsatile system filled with eudragit coated Ca-alginate microbeads (F2) showed better drug content, particle size, surface topography, in-vitro drug release in a controlled manner. Different plugging materials like Sterculia gum, HPMC K4M and Carbopol were used in the design of pulsatile capsule. The pulsatile system remained intact in buffer pH 1.2 for 2 hours due to enteric coat of the system with HPMCP. The enteric coat dissolved when the pH of medium was changed to 7.4. The pulsatile system developed with Sterculia gum as plugging material showed satisfactory lag period when compared to HPMC and Carbopol.

Developing an antibacterial biomaterial

Development and production of antibacterial materials were gained momentum because of the increasing infectional diseases in recent years. Antibacterial products are used in many industries, such as, textile, dye and package industries and medical technology. Short duration antibacterial affect is the main problem for these products. Therefore, the development of materials that exhibit long duration antimicrobial activity, is needed for industry. In this study, the free carboxylic acid groups of alginate were activated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and amoxicillin (AMX) was covalently immobilized to alginate. AMX, D-α-amino-p-hydroxybenzyl penicillin trihydrate, is one of the most frequently used β-lactam antibiotics that has been employed to treat and reduce the spread of bacterial infections in human, with the benefits of maintaining animal welfare. At the end of the study, an antibacterial biomaterial was developed. The optimum reaction medium, that immobilization of AMX to Ca-alginate beads, was determined as; 1mg of EDC, pH 5, 50mM of phosphate buffer, 5 pieces of Ca-alginate beads, incubation temperature was 4°C, mixing speed was 150rpm, initial amount of AMX (IAA) was 4mg and incubation time 1.5h. Ca-alginate beads and AMX immobilized Ca-alginate beads were tested for antibacterial affects against Escherichia coli (ATCC 8739, gram negative) and Staphylococcus aureus (ATCC 6538, gram positive) bacterial strain. According to the SEM images and ATR-FTIR spectrums of AMX immobilized Ca-alginate and Ca-alginate beads were analyzed, the immobilization process was proved successfully. Developed biomaterial was antibacterial according to antibacterial tests.

Effect of air-blast drying and the presence of protectants on the viability of yeast entrapped in calcium alginate beads with an aim to improve the survival rate.

Five yeast strains, Saccharomyces cerevisiae D8, M12, and S13; Hanseniaspora uvarum S6; and Issatchenkia orientalis KMBL5774, isolated from Korean grapes, were entrapped in Ca-alginate beads, which are non-toxic, simple to use, and economical. Ca-alginate beads containing yeast cells were soaked in protective solutions, such as skim milk, saccharides, polyols, and nitrogen compounds, before air-blast drying to improve the yeast survival rate and storage ability. The results showed that both entrapment in Ca-alginate beads and soaking in protective agents favorably affected the survival of all strains. The microenvironment formed by the beads and protective agents can protect the yeast cells from harsh environmental conditions, such as low water (below 10%). All the yeast strains entrapped in Ca-alginate beads showed greater than 80% survival and less than 11% water content after air-blast drying at 37°C for 5h. In addition, air-blast dried cells of S. cerevisiae D8, M12, S13; H. uvarum S6; and I. orientalis KMBL5774 entrapped in 2% Ca-alginate beads and soaked in protective agents (10% skim milk containing 10% sucrose, 10% raffinose, 10% trehalose, 10% trehalose, and 10% glucose, respectively) after air-blast drying at 37°C for 5h showed 90, 87, 92, 90, and 87% viability, respectively. All dried entrapped yeast cells showed survival rates of at least 51% after storage at 4°C for 3months.

The influence of solution viscosities and surface tension on calcium-alginate microbead formation using dripping technique

Currently, there is a growing interest for encapsulation of bioactive ingredients using calcium alginate microspheres or beads. Diffusion of the Ca in the alginate droplets provokes an ionic gelation and their conversion to hydrogel beads. The main objective of this study is to investigate the influence of physical properties of alginate and CaCl2 solutions on alginate droplet formation and penetration into gelling bath, in size and shape of Ca-alginate beads. The droplet formation and penetration in the calcium bath was investigated using a high-speed video recording. Viscosity of alginate solution was modified by changing the alginate concentration (from 10 to 30 g/L) and the viscosity of the bath was modified by using different water/glycerol mixes (0–90% of glycerol) to prepare the CaCl2 solution. Surface tension of CaCl2 solution is reduced by adding different concentrations of surfactants (Tween 20) in a range of 0.01–1 g/L. Droplets detach from the tip with a tear shape. In all conditions tested, the droplets reach a spherical shape in less than 15 ms after detachment and less than 25 mm from the tip. Spherical beads are obtained when the kinetic energy is high enough to break the surface resistance of the calcium bath and droplet viscosity high enough to avoid deformations. Penetration depth of alginate droplets were mainly affected by viscosity and surface tension of CaCl2 solution. When viscosity and surface tension of CaCl2 solution increases, sphericity decrease and shape deformations are observed. The surfactant addition enhanced penetration and prevented shape deformations of Ca-alginate beads.

Immobilization of Lycinibacillus fusiformis B26 cells in different matrices for use in turquoise blue HFG decolourization

Abstract The decolourization of Turquoise Blue HFG by immobilized cells of Lysinibacillus fusiformis B26 was investigated. Cells of L. fusiformis B26 were immobilized by entrapment in agar and calcium alginate matrices and attached in pumice particles. The effects of operational conditions (e.g., agar concentrations, cell concentrations, temperature, and inoculum amount) on microbial decolourization by immobilized cells were investigated. The results revealed that alginate was proven to be the best as exhibiting maximum decolourization (69.62%), followed by agar (55.55%) at 40°C. Pumice particles were the poorest. Optimum conditions for agar matrix were found: concentration was 3%, cell amount was 0.5 g and temperature was 40°C (55.55%). Ca-alginate beads were loaded with 0.5, 1.0 and 2.0 g of wet cell pellets and the highest colour removal activity was observed with 2.0 g of cell pellet at 40°C for alginate beads. Also, 0.5 and 1.0 g of pumice particles that were loaded with 0.25 and 0.5 g of cell pellets respectively were used and the results were found very similar to each other.

Development of a CO2 -releasing coformulation based on starch, Saccharomyces cerevisiae and Beauveria bassiana attractive towards western corn rootworm larvae.

CO2 is known as an attractant for many soil-dwelling pests. To implement an attract-and-kill strategy for soil pest control, CO2 -emitting formulations need to be developed. The aim of the present work was to develop a slow-release bead system in order to bridge the gap between application and hatching of western corn rootworm larvae. We compared different Ca-alginate beads containing Saccharomyces cerevisiae for their potential to release CO2 over a period of several weeks. The addition of starch improved CO2 release, resulting in significantly higher CO2 concentrations in soil for at least 4 weeks. The missing amylase activity was compensated for either by microorganisms present in the soil or by coencapsulation of Beauveria bassiana. Formulations containing S. cerevisiae, starch and B. bassiana were attractive for western corn rootworm larvae within the first 4 h following exposure; however, when considering the whole testing period, the maize root systems remained more attractive for the larvae. Coencapsulation of S. cerevisiae, starch and B. bassiana is a promising approach for the development of attractive formulations for soil applications. For biological control strategies, the attractiveness needs to be increased by phagostimuli to extend contact between larvae and the entomopathogenic fungus growing out of these formulations. © 2016 Society of Chemical Industry.

Mutagenicity and cytotoxicity assessment of biodegraded textile effluent by Ca-alginate encapsulated manganese peroxidase

Present study was aimed to appraise the potential of free and immobilized manganese peroxidase (MnP) for the decolorization and detoxification of textile effluent. MnP was immobilized in Ca-alginate beads at optimized conditions of sodium alginate, calcium chloride and enzyme concentrations. Maximum effluent decolorization of 87.4% was achieved in the presence of H2O2 (1mmoll−1), 1-hydroxybenzotriazole (1mmoll−1), pH (5.0) and temperature (40°C) for 5h of incubation time. The effluent treated at optimized conditions was subjected to toxicity evaluation. The cytotoxicity was evaluated using Allium cepa, brine shrimp and heamolytic bioassays; whereas mutagenicity was tested using Ames test of both treated and untreated effluent. The cytotoxicity of treated sample reduced significantly and A. cepa showed increase in root length, root count and mitotic index up to 38.46%, 43.47% and 41.83%, respectively, whereas red blood cells (RBCs) lysis reduced 69.84% and brine shrimp nauplii death reduced up to 63.64% in immobilized MnP treated effluent. The mutagenicity reduced up to 73.44% and 75.43% for TA98 and TA100 strains, respectively. The Ca-alginate beads encapsulated MnP revealed promising bio-catalytic efficiency and could be used for the decolorization and detoxification of textile effluents.

A Formulation, Optimization and Evaluation of Controlled Released Alginate Beads Loaded-Flurbiprofen

While flurbiprofen (FLB) may cause local gastrointestinal problems such as sensation of warming or burning in mouth and stomach, it is recommended to be limited its therapeutic uses or use it as a modified-release preparation for only once daily-use using Calcium-alginate (Ca-alginate) beads. Ca-alginate beads were prepared by dropping sodiumalginate solution containing FLB into calcium chloride solution. The prepared Ca-alginate beads loaded-FLB were characterized by optical-microscope at different positions. The weight and swelling percent of the beads were measured for freshly and dried prepared beads. The release of the formed beads was studied in different releasing medias. Finally, after optimizing all conditions the formed beads were used to reduce the paw edema as an anti-inflammatory dosage form. The release of FLB from the formed beads in a phosphate buffer (0.2 M, pH 7.4) was slow and complete due to the FLB solubility at this pH. Kinetic analysis of the release-profile of FLB from the formed beads was obeyed the diffusion mechanism. The post carrageenan injection showed reduction of swelling in groups treated with Ca-alginate beads loaded-FLB with longer duration compared with the free FLB. The results showed that a simple and evaluated method for preparation of a modified-release Ca-alginate beads loaded-FLB for release for controlling of FLB with therapeutic purposes.

Adsorption and desorption of Zn(II) and Cu(II) on Ca- alginate immobilized activated rice bran

Ca-alginate immobilized activated rice bran has been used for adsorption of Zn(II) and Cu(II) from aqueous solution. The effect of the pH, kinetics model, adsorption isotherm and desorption on the adsorption performance was investigated. Activated rice bran was immobilized by the entrapment in alginate beads. The adsorption strength of Ca-alginate immobilized activated rice bran was compared to Ca-alginate and non-immobilized activated rice bran. The concentrations of adsorbed ions were analyzed using Atomic Absorption Spectrophotometer (AAS). The result showed that pH of 4.0 and the contact time of 120 min are the optimum condition for adsorption of Zn(II) and Cu(II). The adsorption kinetic of Zn(II) and Cu(II) followed the pseudo-second-order model with adsorption rate constant 4.9 x 10-2 and 3.14 g.mg-1.min-1, respectively. The both adsorption processes obeyed Langmuir isotherm with adsorption capacity of 2.03 and 2.42 mg.g-1 of adsorbent, respectively. The strength of Zn adsorption on Ca-alginate immobilized activated rice bran (86.63%) was more effective compared to Ca-alginate beads (60.96%) and activated rice bran (43.85%). The strength of Cu adsorption was 80.00%, 61.50% and 22.10%, respectively. The desorption of Zn(II) and Cu(II) showed that recovery percentage of the adsorption was 76.56% and 57.80% with the condition of using HCl 0.1 M as desorption agent for 1 hour.

The Influence of Dopants on the Effectiveness of Alginate Beads in Immobilized Cell Reactors.

Zymomonas mobilis immobilized in doped calcium alginate (Ca-alginate) was successfully employed for the production of ethanol in an immobilized cell reactor. Polyethylene oxide and F127 dimethacrylate were evaluated as potential dopants for Ca-alginate beads to decrease lag time and increase initial ethanol yield. The influence of the type and concentration of the dopant on the effectiveness of the microbe immobilized in Ca-alginate beads to produce ethanol was studied, and results were compared to the widely used 2% Ca-alginate with no dopants, which acted as control. Immobilized cell reactors that were operated using beadsdoped with 0.25% polyethylene oxide (PEO) reached an ethanol yield of ∼70% in 24h, which was significantly higher than an ethanol yield of 25% obtained for the control reactor operated using undoped Ca-alginate beads. This study shows that the use of water-soluble dopants can potentially reduce the lag phase and thus improve the initial production yield of immobilized cell reactors, likely due to an increase in porosity and diffusion rate of the doped beads.

Dye decolorization and detoxification potential of Ca-alginate beads immobilized manganese peroxidase.

BackgroundIn view of compliance with increasingly stringent environmental legislation, an eco-friendly treatment technology of industrial dyes and effluents is a major environmental challenge in the color industry. In present study, a promising and eco‐friendly entrapment approach was adopted to immobilize purified manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum IBL-05 on Ca-alginate beads. The immobilized MnP was subsequently used for enhanced decolorization and detoxification of textile reactive dyes).ResultsMnP isolated from solid-state culture of G. lucidum IBL-05, presented highest immobilization yield (83.9 %) using alginate beads prepared at optimized conditions of 4 % (w/v) sodium alginate, 2 % (w/v) Calcium chloride (CaCl2) and 0.5 mg/ml enzyme concentration. Immobilization of MnP enhanced optimum temperature but caused acidic shift in optimum pH of the enzyme. The immobilized MnP showed optimum activity at pH 4.0 and 60 °C as compared to pH 5.0 and 35 °C for free enzyme. The kinetic parameters Km and Vmax of MnP were significantly improved by immobilization. The enhanced catalytic potential of immobilized MnP led to 87.5 %, 82.1 %, 89.4 %, 95.7 % and 83 % decolorization of Sandal-fix Red C4BLN, Sandal-fix Turq Blue GWF, Sandal-fix Foron Blue E2BLN, Sandal-fix Black CKF and Sandal-fix Golden Yellow CRL dyes, respectively. The insolubilized MnP was reusable for 7 repeated cycles in dye color removal. Furthermore, immobilized MnP also caused a significant reduction in biochemical oxygen demand (BOD) (94.61-95.47 %), chemical oxygen demand (COD) (91.18-94.85 %), and total organic carbon (TOC) (89.58-95 %) of aqueous dye solutions.ConclusionsG. lucidum MnP was immobilized in Ca-alginate beads by entrapment method to improve its practical effectiveness. Ca-alginate bound MnP was catalytically more vigorous, thermo-stable, reusable and worked over wider ranges of pH and temperature as compared to its free counterpart. Results of cytotoxicity like hemolytic and brine shrimp lethality tests suggested that Ca-alginate immobilized MnP may effectively be used for detoxification of dyes and industrial effluents.

Protective effect of soy powder and microencapsulation on freeze-dried Lactobacillus brevis WK12 and Lactococcus lactis WK11 during storage

Food-grade cryoprotective agents, i.e., skim milk, soy powder, yeast extract, and trehalose were tested for viability of lactic acid bacteria (LAB, Lactobacillus brevis WK12 and Lactococcus lactis WK11) during freeze drying. Ten percentage of soy powder showed a strong protective effect upon viability of both LAB, showing 1.85×1011 CFU/mL for L. brevis WK12 and 1.89×1011 CFU/mL for L. lactis WK11. Microencapsulation in Ca-alginate beads increased viability of LAB, compared to nonencapsulated cells. Moreover, there was a synergetic effect on the stability of LAB when encapsulated LAB in Ca-alginate beads were soaked in 10% of soy powder prior to freeze-drying. When temperature increased, however, survival of LAB decreased. Four weeks after storage the highest viability was observed at -18°C for L. brevis WK12 and L. lactis WK11, showing 1.80×1011 CFU/mL and 1.78×1011 CFU/mL, respectively. These results suggest that soy powder may be useful for enhancing viability of LAB with microencapsulation.

Performance Improvement of Ca-Alginate Bead Cross- Linked Laccase from Trametes versicolor IBL-04

Extracellular laccase was produced by Trametes versicolor IBL-04 using corn cobs as a substrate under pre-optimized culture conditions. A 64-kDa laccase enzyme was purified and immobilized on calcium alginate beads using glutaraldehyde as a cross-linking reagent. Maximum enzyme immobilization efficiency (89%) was observed with 2-mm calcium-alginate beads that were developed using 4% (w/v) sodium alginate in 2% (w/v) calcium chloride solution. Immobilization of laccase enhanced the optimum temperature but caused an acidic shift in the optimum pH of the enzyme. The immobilized enzyme showed optimum activity at pH 3.0 and 60 °C as compared to pH 4.5 and 45 °C for free laccase. The kinetic constants Km and Vmax of laccase were significantly altered by immobilization. The affinity of enzyme toward its substrate increased (Km decreased), leading to enhanced catalytic efficiency (Vmax increased). Scanning electron microscopy (SEM) was performed to characterize the free and enzyme-bound immobilization matrix. Free and immobilized enzymes also were used for decolorization of the Reactive T Blue dye (030905 GWF) for three days. The free and immobilized laccases decolorized the dye by 65% and 92%, respectively, in 72 h. The immobilized enzyme retained 68% of its original activity after three cycles of repeated reuse for dye decolorization, indicating the usefulness of immobilized laccase in repeated industrial batch operations.