Glutaraldehyde-activated Chitosan Research Articles

Immobilisation of catalase purified from mushroom (Hydnum repandum) onto glutaraldehyde-activated chitosan and characterisation: Its application for the removal of hydrogen peroxide from artificial wastewater

Abstract Hydrogen peroxide (H2O2) is a chemical that is widely used in many industrial processes, and, except at certain concentrations, it is toxic in biological systems such as water and air. Among enzymes, catalases are important industrial enzymes because of their role in the conversion of hydrogen peroxide to water and molecular oxygen. Herein, catalase (CAT) from Hydnum repandum was purified 3.02-fold with a yield of 68.10% by three-phase partitioning (TPP) for the first time. The purified catalase was immobilised on glutaraldehyde-activated chitosan (Glu-Cts), and its applicability for the removal of hydrogen peroxide released from industrial processes was investigated. The results of the present study showed that the optimum pH and temperature were found to be 7.0 and 30°C for both free and immobilised catalase (CAT-Glu-Cts). The catalytic efficiency (V max/K m) of the immobilised enzyme increased 8-fold compared to the free enzyme. CAT-Glu-Cts was shown to have better pH, thermal stability, and storage stability than free CAT. In this study, >96% of 6 mM, 15 ve 24 mM H2O2 was removed from artificial wastewater after 2 h using immobilised catalase. We expect that CAT-Glu-Cts, obtained by purifying a plant-derived catalase and immobilising it into an environmentally friendly and biocompatible material, is a promising candidate that can be safely used for H2O2 removal in various branches of industry.

The Immobilization and Stabilization of Trypsin from the Porcine Pancreas on Chitosan and Its Catalytic Performance in Protein Hydrolysis

In this study, trypsin from the porcine pancreas was immobilized on a heterofunctional support prepared by activating chitosan (Chit) hydrogel with glutaraldehyde (GA), then functionalizing it with glycine (Chit–GA–Gly). The catalytic performance of the immobilized trypsin in the hydrolysis reactions was compared with the catalytic performance of the immobilized enzyme on glutaraldehyde-activated chitosan (Chit–GA) and chitosan hydrogel (Chit). The maximum concentration of immobilized protein on Chit–GA–Gly was approximately 16 mg·g−1 at pH 9.0 (5 mmol·L−1 buffer sodium carbonate) at 25 °C from an offered protein loading of 20 mg·g−1. This biocatalyst exhibited maximum specific activity (SA) of 33.1 ± 0.2 nmol·min−1·mg−1 for benzoyl-DL-arginine-p-nitroanilide (BAPNA) hydrolysis, twice as high as the enzyme immobilized on the classic Chit–GA support (SA values ranging between 6.7 ± 0.1 nmol·min−1·mg−1 and 8.1 ± 0.1 nmol·min−1·mg−1). The Elovich kinetic model was used to describe the adsorption process using low (3 mg·g−1) and high (20 mg·g−1) initial protein loadings. The optimum temperature for BAPNA hydrolysis catalyzed by the immobilized trypsin (60 °C) was 10 °C higher than that of its soluble form. Additionally, the immobilized enzyme was 16 to 20 times more stable than its soluble form at 50–55 °C. Thermodynamic studies were conducted to elucidate the kinetics of the thermal inactivation process of soluble and immobilized forms. Complete hydrolysis of bovine serum albumin (BSA) at 37 °C was achieved after 2 h using a soluble enzyme, while for its immobilized form, the hydrolysis yield was 47%. Reuse tests revealed that this biocatalyst retained 37% of its original activity after 10 successive hydrolysis batches. Based on these results, this support could be used as an interesting alternative for producing heterogeneous biocatalysts with high catalytic activity and thermal stability when producing protein hydrolysates.

Covalent Immobilisation of an Aspergillus niger Derived Endo-1,4-β-Mannanase, Man26A, on Glutaraldehyde-Activated Chitosan Nanoparticles for the Effective Production of Prebiotic MOS from Soybean Meal

An Aspergillus niger endo-1,4-β-mannanase, Man26A, was confirmed by FTIR and XRD to be immobilised on glutaraldehyde-activated chitosan nanoparticles via covalent bonding. The immobilisation (%) and activity yields (%) were 82.25% and 20.75%, respectively. The biochemical properties (pH, temperature optima, and stability) were then comparatively evaluated for both the free and immobilised Man26A. The optimal activity of Man26A shifted to a lower pH after immobilisation (pH 2.0–3.0, from pH 5 for the free enzyme), with the optimum temperature remaining unchanged (60 °C). The two enzymes exhibited identical thermal stability, maintaining 100% activity for the first 6 h at 55 °C. Substrate-specific kinetic analysis showed that the two enzymes had similar affinities towards locust bean gum (LBG) with varied Vmax values. In contrast, they showed various affinities towards soybean meal (SBM) and similar Vmax values. The immobilised enzyme was then employed in the enhancement of the functional feed/prebiotic properties of SBM from poultry feed, increasing mannooligosaccharides (MOS) quantities. The SBM main hydrolysis products were mannobiose (M2) and mannose (M1). The SBM-produced sugars could be utilised as a carbon source by probiotic bacteria; Streptococcus thermophilus, Bacillus subtilis, and Lactobacillus bulgaricus. The results indicate that the immobilised enzyme has the potential for use in the sustainable and cost-effective production of prebiotic MOS from agricultural biomass.

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.

Covalent Immobilization of Chondrostereum purpureum Endopolygalacturonase on Ferromagnetic Nanoparticles: Catalytic Properties and Biotechnological Application.

Pectinases are widely used in a variety of industrial processes. However, their application is limited by low catalytic processivity, reduced stability, high cost, and poor re-use compatibility. These drawbacks may be overcome by enzyme immobilization with ferromagnetic nanoparticles, which are easily recovered by a magnetic field. In this work, an endopolygalacturonase from Chondrostereum purpureum (EndoPGCp) expressed in Pichia pastoris was immobilized on glutaraldehyde-activated chitosan ferromagnetic nanoparticles (EndoPGCp-MNP) and used to supplement a commercial enzyme cocktail. No significant differences in biochemical and kinetic properties were observed between EndoPGCp-MNP and EndoPGCp, although the EndoPGCp-MNP showed slightly increased thermostability. Cocktail supplementation with EndoPGCp-MNP increased reducing sugar release from orange wastes by 1.8-fold and showed a synergistic effect as compared to the free enzyme. Furthermore, EndoPGCp-MNP retained 65% of the initial activity after 7 cycles of re-use. These properties suggest that EndoPGCp-MNP may find applications in the processing of pectin-rich agroindustrial residues.

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.

Immobilization of Laccase from Trametes versicolor on Chitosan Macrobeads for Anthracene Degradation

ABSTRACTAnthracene bioconversion was achieved by immobilized enzyme technology. An oxidation yield of 0.7 mg/L of polycyclic aromatic hydrocarbons reached 60% following 24 h of incubation with laccase from Trametes versicolor covalently immobilized on glutaraldehyde-activated chitosan at the optimal pH of 5 in the presence of diammonium 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the radical mediator. High-performance liquid chromatography indicated that the main product of anthracene oxidation was 9,10-anthraquinone which is less toxic than its precursor. Highly porous 3 mm diameter chitosan macrobeads were synthesized by precipitation in alkaline solution. Support activation with glutaraldehyde was confirmed by elemental analysis, thermogravimetry, and infrared spectroscopy. The bioreactor system was characterized for kinetic parameters obtaining a Michaelis–Menten constant of 0.13 mM and a maximum rate of 0.0011 µmol/min/mg, thermal stability, and reuse. The protein and glutaraldehyde concentrations were optimized to enhance the efficiency of the bioreactor.

Chemical improvement of chitosan-modified beads for the immobilization of Enterococcus faecium DBFIQ E36 L-arabinose isomerase through multipoint covalent attachment approach.

D-tagatose is produced from D-galactose by the enzyme L-arabinose isomerase (L-AI) in a commercially viable bioprocess. An active and stable biocatalyst was obtained by modifying chitosan gel structure through reaction with TNBS, D-fructose or DMF, among others. This led to a significant improvement in L-AI immobilization via multipoint covalent attachment approach. Synthetized derivatives were compared with commercial supports such as Eupergit(®) C250L and glyoxal-agarose. The best chitosan derivative for L-AI immobilization was achieved by reacting 4 % (w/v) D-fructose with 3 % (w/v) chitosan at 50 °C for 4 h. When compared to the free enzyme, the glutaraldehyde-activated chitosan biocatalyst showed an apparent activity of 88.4 U g (gel) (-1) with a 211-fold stabilization factor while the glyoxal-agarose biocatalyst gave an apparent activity of 161.8 U g (gel) (-1) with an 85-fold stabilization factor. Hence, chitosan derivatives were comparable to commercial resins, thus becoming a viable low-cost strategy to obtain high active L-AI insolubilized derivatives.

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.

Fructooligosaccharides synthesis by highly stable immobilized β-fructofuranosidase from Aspergillus aculeatus

The enzymatic synthesis of fructooligosaccharides (FOS) was carried out using a partially purified β-fructofuranosidase from the commercial enzyme preparation Viscozyme L. Partial purification of β-fructofuranosidase from Viscozyme L was done by batch adsorption using ion-exchange resin DEAE-Sepharose, showing a 6-fold increase in specific activity. The biocatalyst was then covalently immobilized on glutaraldehyde-activated chitosan particles. Thermal stability of the biocatalyst was evaluated at 50°C and 60°C, being around 100 times higher at 60°C when compared to the free enzyme. The immobilized biocatalyst was reused 50 times for FOS production (100min per batch at 50°C and pH 5.5) without significant loss of activity. The average yield (grams of FOS per grams of initial sucrose) was 55%. The immobilization process combined with partial purification method resulted in a derivative with activity of 1230Ut/g, which is among the best for FOS production.

Continuous production of β-cyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan

Cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on glutaraldehyde-activated chitosan spheres and used in a packed bed reactor to investigate the continuous production of β-cyclodextrin (β-CD). The optimum temperatures were 75°C and 85°C at pH 6.0, respectively for free and immobilized CGTase, and the optimum pH (5.0) was the same for both at 60°C. In the reactor, the effects of flow rate and substrate concentration in the β-CD production were evaluated. The optimum substrate concentration was 4% (w/v), maximizing the β-CD production (1.32g/L) in a flow rate of 3mL/min. In addition, the biocatalyst had good operational stability at 60°C, maintaining 61% of its initial activity after 100 cycles of batch and 100% after 100h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.

High stability of immobilized β-d-galactosidase for lactose hydrolysis and galactooligosaccharides synthesis

β-d-Galactosidase from Kluyveromyces lactis was immobilized on glutaraldehyde-activated chitosan and used in a packed-bed reactor for the continuous hydrolysis of lactose and the synthesis of galactooligosaccharides (GOS). The biocatalyst was tested for its optima pH and temperature, thermal stability in the presence of substrate and products, and operational stability. Immobilization increased the range of operational pH and temperature, and the enzyme thermal stability was sharply increased in the presence of lactose. Almost complete lactose hydrolysis was achieved for both milk whey and lactose solution at 37°C at flow rates up to 2.6mLmin−1. Maximal GOS concentration of 26gL−1 was obtained at a flow rate of 3.1mLmin−1, with a productivity of 186gL−1h−1. Steady-state operation for 15 days showed the reactor stability concerning lactose hydrolysis.

Immobilization of Keratinolytic Metalloprotease from Chryseobacterium sp. Strain kr6 on Glutaraldehyde-Activated Chitosan

Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support (qm) and dissociation constant (Kd) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at 65 degrees C. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.

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.

Stabilization of penicillin G acylase by immobilization on glutaraldehyde-activated chitosan

The objective of this work was to study enzyme immobilization on chitosan activated with glutaraldehyde, aiming to produce a cheap biocatalyst. Two different immobilization strategies were studied: one-point and multipoint covalent attachment to the solid matrix. The multipoint covalent attachment derivative had an 82% immobilization yield. It was 4.9-fold more stable than the free enzyme at 50°C and 4.5-fold more stable than soluble enzyme at pH 10.0. The one-point derivative had an 85% immobilization yield. It was 2.7-fold more stable than the free enzyme at 50°C and 3.8-fold more stable than soluble PGA at pH 10.0. Results indicated that chitosan can be loaded with PGA above 330 IU/g. Intraparticle diffusive effects, however, limited hydrolysis of penicillin G catalyzed by those derivatives at 37°C and 25°C. Operational stability assays were performed and the multipoint derivative exhibited a half-life of 40 hours.

Preparation and Characterization of Cellulase-Containing Liposomesand Their Immobilization Suitable for Enzymatic Hydrolysis of Cellulose

A commercial cellulase was entrapped in liposomes composed of phosphatidylcholine (65 mol%), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (5 mol%) and cholesterol (30 mol%) to prepare the cellulase-containing liposomes (CL) with the enzyme incorporated as much as possible into the membrane. The CL was then immobilized with chitosan gel beads to develop an efficient biocatalyst for enzymatic hydrolysis of cellulose. The activity of the CL prepared was determined with the membrane impermeable substrates such as insoluble filter paper (FP) and soluble carboxymethyl cellulose (CMC). The activity for FP was only several percents of that for CMC. The activity efficiency of the CL for either substrate was found to be about 5%, which suggests that around 5% of the whole cellulase contained in the CL were incorporated in the outer membrane of the liposomes. The CL was coupled with the glutaraldehyde-activated chitosan gel beads to obtain the immobilized cellulase-containing liposomes (ICL). The ICL was repeatedly used in the hydrolysis of both CMC and FP to examine its operational stability. The stability of the ICL was also compared to that of a conventionally immobilized cellulase (IC). As a result, although the activity of the ICL for FP or CMC decreased to 30% or 85%, respectively of the initial activity in its first use, the ICL almost maintained the respective activity for its subsequent uses. The activity of the IC for either substrate decreased steadily from around 70% to 50% of the initial one with the repeated use.