Glutaraldehyde Activated Chitosan Beads Research Articles

α-Acetolactate decarboxylase immobilized in chitosan: A highly stable biocatalyst to prevent off-flavor in beer.

In this work, we studied the covalent immobilization of α-acetolactate decarboxylase (ALDC) on glutaraldehyde activated chitosan beads for application in beers to prevent off-flavor. The immobilized preparation was characterized regarding yield, efficiency, activity recovery, thermal and pH stabilities, and operational stability in beer. The concentration of activating agent was evaluated and at the best condition an immobilization yield of 84% and activity recovery of 9.1% were verified. Optimal pH was 6.0 for free and immobilized enzymes, however, the immobilized enzyme showed higher relative activity at the tested pH, with emphasis on the pH range from 5.0 to 6.5. Besides, immobilized enzyme showed stabilization factor of almost sevenfold at 60°C compared to free form. The immobilized biocatalyst was tested in repeated batches in beer for α-acetolactate conversion to acetoin, and after 12 cycles the enzyme presented 80% of its initial activity. To date, this is the most stable immobilized preparation of ALDC reported.

Immobilization of Candida Rugosa Lipase on the Glutaraldehyde-Activated Chitosan Beads

An immobilized enzyme is a biocatalyst that speeds up the conversion of a chemical reaction. The application of enzymes for chemical synthesis is an effort toward a responsible production initiative to ensure the sustainability of chemical synthesis. Therefore, in the present work, Candida rugosa lipase was immobilized onto glutaraldehyde-activated chitosan beads through covalent bond linkages. Chitosan is biodegradable and contains amine groups, which serve as bases for lipase binding via cross-linking with bifunctional cross-linkers like glutaraldehyde. The immobilization of lipase on the chitosan beads was confirmed by determining lipase activity through the hydrolysis of a standard substrate. The effect of lipase and glutaraldehyde concentrations on the immobilization and activity yield was investigated. In general, lipase and glutaraldehyde concentrations have a significant effect on immobilization and activity yield. The interaction between the investigated parameters is significant toward the activity yield rather than the immobilization yield. The optimized immobilization procedures give lipase activity up to 46 IU by using 0.013 g/mL lipase and 2 %/v/v glutaraldehyde. It was found that the immobilized enzyme was rather stable and could be recycled 7 times. Therefore, immobilization of lipase onto glutaraldehyde-activated chitosan support is feasible.

Process conditions optimization and study of pH and thermal stability of free and immobilized gutaminase-free recombinant L-asparaginase II of Pactobacterium carotovorum MTCC 1428 from Escherichia coli

L-asparaginase (EC 3.5.1.1) is used for the treatment of acute lymphocytic leukemia and food processing of starch rich foods for reducing the acrylamide formation. In our current efforts, we have immobilized the purified glutaminase-free recombinant Lasparaginase II of Pectobacterium carotovorum MTCC 1428 from Escherichia coli BL21 (DE3) on glutaraldehyde activated chitosan beads. The purified recombinant L-asparaginase II has no partial glutaminase activity which is a pre-requisite to reduce the possibility of side effects during the course of anticancer therapy. In order to study the best conditions for the performance of free enzymes and immobilized enzymes, response surface methodology was used to optimize the pH and temperature of the process conditions. It was found that the most favorable pH and temperature for the free enzyme were pH 7.83 and 47.64°C while for the immobilized enzyme, the optimum pH and temperature levels were found to be 7.88 and 48.07 °C. Furthermore, the thermal and pH studies of free and immobilized enzymes were studied under various combinations of pH and temperature and finally the thermodynamic parameters of free and immobilized glutaminase-free recombinant asparaginase were evaluated.

Batch synthesis of galactooligosaccharides from co-products of milk processing using immobilized β-galactosidase from Bacillus circulans

Milk whey (MW) and milk whey permeate (MWP) constitute the main part of co-products derived from milk processing. The use of the transgalactosylation activity of β-galactosidases is an excellent alternative to add value to these co-products since this is the route for the synthesis of galactooligosaccharides (GOS), a valuable prebiotic sugar. Hereby, we immobilized a β-galactosidase from Bacillus circulans onto glutaraldehyde-activated chitosan beads. The immobilized enzyme was characterized and the parameters affecting GOS synthesis (pH, temperature, and concentration of MW and MWP) were evaluated. The optimum pH and temperature of activity for the immobilized β-galactosidase were 50 °C and pH 6.0, respectively. The immobilization improves enzyme thermal stability by 2.9 times. In the batch process, the immobilized enzyme exhibited excellent operational stability at 40 °C, being able to hydrolyze 91% lactose after 17 cycles of reuse. Regarding GOS synthesis, the immobilized enzyme could be reused for 10 consecutive cycles retaining 74% of the initial activity at 50 °C. The maximum concentration of GOS achieved, using MW and MWP, was 159.4 g L−1 and 168.8 g L−1, respectively. Therefore, these results proved that it was possible to obtain, in high concentration, a useful and valuable prebiotic sugar from co-products of milk processing.

Immobilization of a peroxidase from Moringa oleifera Lam. roots (MoPOX) on chitosan beads enhanced the decolorization of textile dyes

The textile industry is essential, but it is also responsible for causing environmental problems, particularly the discharge of dyes. In this context, this study aimed to immobilize a previously purified peroxidase, called MoPOX, on glutaraldehyde-activated chitosan beads in order to improve the potential for textile dye decolorization. The chitosan beads were activated with 8% glutaraldehyde for 1 h, and the immobilization was performed at 30 °C, pH 5.2 for 4 h. Scanning Electron Microscopy (SEM) analyses were used to observe the differences in the chitosan beads after immobilization. The optimum temperature dropped from 70 °C to 30 °C after immobilization, but immobilized MoPOX demonstrated excellent heat stability. The optimum pH remained 5.2, while the apparent kinetic constant value (Km) of immobilized MoPOX (14.67 mM) was lower in comparison to free MoPOX (46.8 mM). The immobilized enzyme showed improved activity after long storage times, and it could retain 40 % of its original activity even after 5 cycles. The potential for decolorization of different textile dyes was considerably enhanced after immobilization, reaching more than 80 %. Also, MoPOX showed no toxicity towards Artemia salina. Overall, the findings point to the promising potential of using immobilized MoPOX as a biocatalyst in a variety of biotechnological applications.

Mutagenesis and Immobilization of ChitB-Protease for Induced De-staining and Goat Skin Dehairing Potentialities

Proteases with alkaline nature and requisite catalytic characteristics have been the supreme interest of numerous industrial sectors. The present study was focused on justifying the following aims, i.e., (1) protease producing strain improvement via physical and chemical mutagenesis, (2) protease optimization, purification, and immobilization on chitosan beads, and (3) de-staining and goat skin dehairing potentialities of chitosan beads immobilized protease (ChitB-protease). Briefly, a protease produced by a UV mutant Bacillus sp. MS1 was purified and characterized to present its scope for detergent and leather sectors. Two-step purification resulted up to 4.1-fold purification and 41% yield of Bacillus sp. MS1 protease. The purified fraction was identified as a monomeric with an apparent molecular weight of 28 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The protease was entrapped in glutaraldehyde-activated chitosan beads to improve its stability and reusability. The ChitB-protease represented the highest activity and immobilization efficiency of 10.31 U/mL and 86.43 ± 1.15%, respectively. ChitB-protease exhibited better catalytic activity in a broader pH range and higher temperature than free enzyme. Moreover, a significant improvement in the thermal resistance was also recorded after immobilization. ChitB-protease retained 70.53% of its initial activity after seven washings. SEM morphological analysis further confirmed the recycling efficiency of ChitB-protease. Furthermore, a complete de-staining and dehairing were observed within the short incubation period. These results suggest that this protease is potentially useful in many industrial applications.

Optimization of Enzyme Co-Immobilization with Sodium Alginate and Glutaraldehyde-Activated Chitosan Beads.

In this study, two different materials-alginate and glutaraldehyde-activated chitosan beads-were used for the co-immobilization of α-amylase, protease, and pectinase. Firstly, optimization of multienzyme immobilization with Na alginate beads was carried out. Optimum Na alginate and CaCl2 concentration were found to be 2.5% and 0.1M, respectively, and optimal enzyme loading ratio was determined as 2:1:0.02 for pectinase, protease, and α-amylase, respectively. Next, the immobilization of multiple enzymes on glutaraldehyde-activated chitosan beads was optimized (3% chitosan concentration, 0.25% glutaraldehyde with 3h of activation and 3h of coupling time). While co-immobilization was successfully performed with both materials, the specific activities of enzymes were found to be higher for the enzymes co-immobilized with glutaraldehyde-activated chitosan beads. In this process, glutaraldehyde was acting as a spacer arm. SEM and FTIR were used for the characterization of activated chitosan beads. Moreover, pectinase and α-amylase enzymes immobilized with chitosan beads were also found to have higher activity than their free forms. Three different enzymes were co-immobilized with these two materials for the first time in this study.

Chitosan beads immobilized manganese peroxidase catalytic potential for detoxification and decolorization of textile effluent

Textile industry has led to severe environmental pollution and is posing a serious threat to the ecosystems. Immobilized biocatalysts have gained importance as potential bio-remediating agent. Manganese peroxidase (MnP) was immobilized onto glutaraldehyde activated chitosan beads by crosslinking and employed for the degradation and detoxification of dyes in textile effluents. The efficiency of chitosan-immobilized MnP (CI-MnP) was evaluated on the basis of decolorization, water quality improvement and toxicity reduction. Maximum color removal of 97.31% was recorded and up to 82.40%, 78.30% and 91.7% reductions in COD, TOC, and BOD were achieved, respectively. The cytotoxicity of bio-treated effluents reduced significantly and 38.46%, 43.47% and 41.83% Allium cepa root length, root count and mitotic index were increased, respectively, whereas brine shrimp nauplii death reduced up to 63.64%. Mutagenicity (Ames test) reduced up to 73.44% and 75.43% for TA98 and TA100 strains, respectively. The CI-MnP retained 60% activity after 10 repeated decolorization batches. The CI-MnP showed excellent efficiency for the bioremediation of textile effluents and can be used for the remediation of toxic agents in wastewater. The monitoring of processed wastewater using bioassays is suggested to evaluate bio-efficiency of treatment method for safe disposal of effluents into water bodies.

Studies on Immobilization of Cutinases from Thermobifida fusca on Glutaraldehyde Activated Chitosan Beads

Aims: To evaluate and optimize the activity and stability performance of two recombinant cutinases of Thermobifida fusca, Cut1 and Cut2 on glutaraldehyde activated chitosan beads. Place and Duration of Study: Biochemical Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Assam India. Experiment conducted as a partial fulfillment to PhD degree from December, 2010 to January, 2014. Methodology: Purified cutinase were immobilized on chitosan beads by covalently coupling with glutaraldehyde. The biophysical properties of immobilized cutinase was Original Research Article British Biotechnology Journal, 4(10): 1049-1063, 2014 1050 analysed by FTIR, FESEM and the operational stability and activity of the immobilized cutinase was studied at different pH and temperature. Results: The optimal immobilization was achieved with 3% (v/v) glutaraldehyde activation and coupling pH of 8.5. Under this condition, 74% and 71% of immobilization was achieved for Cut1 and Cut2, respectively. Immobilized cutinase showed optimal activity at pH 8 with optimal functional range of pH 7.5 to 9 and 55oC with operational stability in the range of 45oC to 70oC. The reusability and storage stability was found to be 80% after 10 reuse cycles and 50% after 13 days, respectively as compared to its initial activity. There was no loss in activity even after repeated freeze drying. Conclusion: the cutinase immobilized on glutaraldehyde activated chitosan beads demonstrated better operational stability in comparison to free cutinase, showing chitosan as a potential support in the enzyme immobilization technology for industrial applications of T. fusca cutinases.

Immobilization and characterization of laccase and its use in the biodegradation of paper mill effluent.

Laccase from Aspergillus sp was immobilized on glutaraldehyde-activated chitosan beads. A comparative study between free and immobilized laccase was conducted and the potential of the resulting immobilized derivative in the biodegradation of pulp and paper mill effluent was evaluated. The immobilized laccase is more resistant to various denaturing conditions, which allows for the reduction of 65% of the phenols (total and low molecular weight) and loss of 60% of total color in the effluent. These results show the potential of the immobilized laccase in the biodegradation of phenols, the chemical agents responsible for the high toxicity of the effluent generated in cellulose pulp industries.

Immobilization of acid phosphatase from Vigna aconitifolia seeds on chitosan beads and its characterization

Acid phosphatase isolated from Vigna aconitifolia seeds was immobilized onto glutaraldehyde activated chitosan beads by crosslinking method. Chitosan beads activated with 2% of glutaraldehyde have demonstrated maximum immobilization yield (∼83%). The immobilized enzyme showed optimum activity at pH 7.0, while soluble form was maximally active in acidic range (pH 5.0). With respect to free form, immobilized acid phosphatase showed better activity in alkaline range. On the other side, immobilization does not affect the optimum temperature range i.e., both, soluble and immobilized acid phosphatase exhibited maximum activity at 60°C. The Km and Vmax values for the immobilized enzyme were calculated to be 0.37mM and 13.5U/mg. The immobilization on chitosan beads enhanced the shelf life of acid phosphatase. The immobilized enzyme retained its more than 50% hydrolytic activity for approximately two months. The immobilized acid phosphatase was reusable for more than 40 cycles of reaction.

Glutaraldehyde-Activated Chitosan Matrix for Immobilization of a Novel Cysteine Protease, Procerain B

Proteases have several applications in the food industry. We report the immobilization of procerain B, a novel cysteine protease, on glutaraldehyde-activated chitosan beads through covalent attachment. Glutaraldehyde not only serves as a cross-linking agent but also links the procerain B on the surface of bead through primary amine group (either lysine side chain or N-terminal) by Schiff base linkage. Immobilized procerain B was characterized for optimum functional range and stability with respect to pH and temperature. The chitosan-immobilized procerain B has broad pH and thermal optima. The effects of substrate concentration and reusability of immobilized beads were also studied. It showed nearly 50% activity until the 10th use.

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (∼99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent Km value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.

Preparation and adsorption property of chitosan beads bearing β-cyclodextrin cross-linked by 1,6-hexamethylene diisocyanate

Glutaraldehyde activated chitosan beads bearing β-cyclodextrin cross-linked by 1,6-hexamethylene diisocyanate was prepared, and characterized by IR, XRD and TG-DTA. Its adsorption property of hydroquinone was investigated by spectrophotometry. The adsorption capacity increases with increasing temperature and pH value. The adsorption process can be better simulated by Freundlich isotherm equation ( R 2 > 0.996 ) and the thermodynamic parameters such as free energy change, enthalpy change and entropy change were calculated. The adsorption of hydroquinone on chitosan beads bearing β-cyclodextrin cross-linked by 1,6-hexamethylene diisocyanate is a spontaneous, endothermic and random process.

Purification of Trypsin Inhibitor from Sweet Potato by Applying Immobilized Trypsin on Glutaraldehyde Activated Chitosan Beads

Immobilized trypsin on the glutaraldehyde activated chitosan beads was employed to purify trypsin inhibitor from the tuberous roots of sweet potato by means of affinity chromatography. The optimum conditions for the preparation of immobilized trypsin were investigated, and a maximum trypsin activity of 10 unit/g-beads was achieved in this study. Fifty-six percent of the activity of the trypsin inhibitor in the crude extract of sweet potato could be recovered through affinity chromatography. Seven visible bands by SDS-PAGE were suspected to be trypsin inhibitors, and all of the molecular weights were more than 20kDa.

Pigeonpea (Cajanus cajan L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications.

Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was observed at 4 degrees C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05 M Tris-acetate buffer, pH 7.3, at 4 degrees C had a t(1/2) of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77 degrees C, compared with 47 degrees C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations.

Production of isomaltooligosaccharides by a-glucosidase immobilized in chitosan beads and by polyethyleneimine-glutaraldehyde treated mycelia of Aspergillus carbonarious

Partially purified a-glucosidase from Aspergillus carbonarious, immobilized on glutaraldehyde-activated chitosan beads in a packed bed reactor, produced isomaltooligosaccharides at a yield of 60% (w/w) using 30% (w/v) maltose solution. Using intact mycelia attached with polyethyleneimine-glutaraldehyde, produced isomaltooligosaccharides at a yield of 46% (w/w) using 30% (w/v) maltose solution. Batchwise reaction stabilities were improved for chitosan beads immobilized enzyme and polyethyleneimine-glutaraldehyde treated mycelia as compared to mycelia without any treatment.