Stability and reactivity of acid phosphatase immobilized on composite beads of chitosan and ZrO2 powders

Equal weights of chitosan and ZrO2 powders were mixed in acetic acid solution to prepare the composite beads. They were then cross-linked with glutaraldehyde and stored with and without freeze-drying before use. The physicochemical properties of acid phosphatase immobilized on four types of the supports (wet/dried pure chitosan beads, wet/dried chitosan-ZrO2 composite beads) were compared. Various parameters including glutaraldehyde concentration, cross-linking time, enzyme concentration, temperature, and pH on enzyme activity were studied. It was shown that the activity yield of enzyme immobilized on the dried chitosan-ZrO2 beads was the highest, and the relative activity remained above 83.2% within pH 2.9-5.8. Regardless of wet or dried beads, the Michaelis constant KM and maximum rate of reaction Vmax of acid phosphatase immobilized on chitosan-ZrO2 composite beads were 1.8 times larger than those on pure chitosan beads. Of the four immobilized enzymes, the use of wet chitosan-ZrO2 bead as the support showed the lowest thermal deactivation energy (78 kJ mol(-1)).     

Stability and catalytic kinetics of acid phosphatase immobilized on composite beads of chitosan and activated clay

The stability of acid phosphatase immobilized on composite beads was studied. The beads were prepared from equal weights of cuttlebone chitosan and activated clay and were cross-linked with glutaraldehyde. The immobilized enzyme maintained 90% of its original activity after 50 times of reuse. The immobilized acid phosphatase revealed acceptable thermal and pH stabilities over a broad experimental range. Thermal deactivation of immobilized enzyme was also examined by first-order kinetics and the deactivation energy was determined. The kinetics of a model reaction catalyzed by the immobilized acid phosphatase was finally investigated by the Michaelis–Menten equation.     

The influence of the degree of cross-linking on the adsorption properties of chitosan beads

The influence of the degree of cross-linking (DCL) on chitosan beads was studied. Chitosan was prepared from the exoskeleton of Cape rock-lobsters, collected from the surroundings of Cape Town, South Africa. The chitosan beads were characterized; the beads water contents and pKa varied in the range of 90-96% and 4.3-6.0, respectively, and were found to decrease with increasing DCL (0.0-34.0%). A pH-model, which described the reversibility of the metal adsorbed onto the beads, was used to predict the equilibrium properties of copper adsorption onto the cross-linked beads. The model accounts for the effect of pH and the important model parameters, the equilibrium adsorption constant (Kads) and to a lesser extent the adsorbent adsorption capacity (qmax) showed to decrease with the DCL. The adsorbent capacity and the adsorption constant were determined as 3.8-5.0mmol/g chitosan and (9-90)x10(-4), respectively. The adsorption kinetics could be described using a shrinking core model and the effective diffusion coefficient (Deff) was determined as (8.0-25.8)x10(-11)m2/s. It was found that Deff decreases with the DCL mainly due to the decreased in water content of the beads at high DCL.     

Adsorption behaviour of Fe(II) and Fe(III) ions in aqueous solution on chitosan and cross-linked chitosan beads

A batch adsorption system was applied to study the adsorption of Fe(II) and Fe(III) ions from aqueous solution by chitosan and cross-linked chitosan beads. The adsorption capacities and rates of Fe(II) and Fe(III) ions onto chitosan and cross-linked chitosan beads were evaluated. Chitosan beads were cross-linked with glutaraldehyde (GLA), epichlorohydrin (ECH) and ethylene glycol diglycidyl ether (EGDE) in order to enhance the chemical resistance and mechanical strength of chitosan beads. Experiments were carried out as function of pH, agitation period, agitation rate and concentration of Fe(II) and Fe(III) ions. Langmuir and Freundlich adsorption models were applied to describe the isotherms and isotherm constants. Equilibrium data agreed very well with the Langmuir model. The kinetic experimental data correlated well with the second-order kinetic model, indicating that the chemical sorption was the rate-limiting step. Results also showed that chitosan and cross-linked chitosan beads were favourable adsorbers.     

Immobilization of a keratinolytic protease from Purpureocillium lilacinum on genipin activated-chitosan beads

Keratinase from Purpureocillium lilacinum LPSC # 876 was immobilized on chitosan beads using two different cross-linking agents: glutaraldehyde and genipin. For its immobilization certain parameters were optimized such as cross-linker concentration, activation time and activation temperature. Under optimum conditions, enzyme immobilization resulted to be 96 and 92.8% for glutaraldehyde and genipin, respectively, with an activity recovery reaching up to 81% when genipin was used. The immobilized keratinase showed better thermal and pH stabilities compared to the soluble form, retaining more than 85% of its activity at pH 11 and 74% at 50 °C after 1 h of incubation. The residual activity of immobilized keratinase remained more than 60% of its initial value after five hydrolytic cycles. The results in this study support that glutaraldehyde could be replaced by genipin as an alternative cross-linking eco-friendly agent for enzyme immobilization.     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

壳聚糖球固定化酵母细胞的研究

以戊二醛为交联剂,将壳聚糖球交联引入醛基,然后将交联的壳聚糖球浸泡在酵母细胞悬浮液中,制备了固定化酵母细胞壳聚糖球。以苯乙酮酸为底物,催化合成了D-扁桃酸。最优固定化条件是戊二醛的质量分数w(GA)=1%,酵母细胞与交联壳聚糖球的质量比m(Y):m(CB)0=0.5,交联时间为6h,固定化时间为18h,底物浓度为10mmol/L,在此条件下反应最大转化率和产物光学纯度分别高达67.86%和98.05?。固定化酵母壳聚糖球具有良好的重复使用性和贮存稳定性。     

Preparation and evaluation of chitosan/carrageenan beads for controlled release of sodium diclofenac

The polyelectrolyte complex (PEC) hydrogel beads based on chitosan (CS) and carrageenan (CR) have been studied as a controlled release device to deliver sodium diclofenac (DFNa) in the simulated gastrointestinal condition. Various factors potentially influencing the drug release (ie, CS/CR proportion, DFNa content, types and amount of cross-linking agents) were also investigated. The optimal formulation was obtained with CS/CR proportion of 2/1 and 5% (wt/vol) DFNa. The controlled release of the drug from this formulation was superior to other formulations and was able to maintain the release for approximately 8 hours. Upon cross-linking with glutaric acid and glutaraldehyde, the resulting beads were found to be more efficient for prolonged drug release than their non-cross-linking counterparts. The bead cross-linked with glutaraldehyde was able to control the release of the drug over 24 hours. The difference in the drug release behavior can be attributed to the differences in ionic interaction between the oppositely charged ions and to the concentrations of the drug within the beads, which depends on the compositions of the formulation and the pH of the dissolution medium. The release of drug was controlled by the mechanism of the dissolution of DFNa in the dissolution medium and the diffusion of DFNa through the hydrogel beads.     

Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.     

Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.     

Study of molybdate ion sorption on chitosan gel beads by different spectrometric analyses

Molybdate ion uptake both by raw chitosan and by glutaraldehyde cross-linked chitosan beads was investigated. This study focused on the identification of sorption mechanisms by means of several analytical procedures such as infra-red and reflectance spectrophotometries and CP-MAS 13C NMR analyses. Although the amine functions of glucosamine residues remain the major sites of interaction with the metal species, other functional groups can also be involved. It is certainly the case with carbonyl functions provided by the glutaraldehyde structure in cross-linked sorbents. Due to the large size of the polynuclear hydrolysed molybdate species, the sorption may involve several monomer units, resulting in additional linkages between the polymer chains. This behaviour can be confirmed by the chemical shifts of the carbon atoms observed by CP-MAS 13C NMR on raw chitosan beads, showing that the carbon atoms supporting the amino sites are not the only atoms affected by molybdate ion sorption. Moreover, cross-linking promotes a partial reduction of molybdenum species in the presence of some unreacted aldehyde groups.     

Glutaraldehyde and glyoxal cross-linked chitosan microspheres for controlled delivery of centchroman

Glutaraldehyde and glyoxal cross-linked microspheres were prepared using chitosan with different molecular weights (MWs) and degrees of deacetylation (DDAs) for sustained release of centchroman under physiological conditions. The DDA in chitosan was determined by different methods, and the samples were categorized as chitosan with low (48%), medium (62%), and high (75%) DDA. The size and shape of the microspheres were determined by scanning electron microscopy (SEM), and hydrophobicity was determined by adsorption of Rose Bengal dye on microspheres cross-linked with glutaraldehyde or glyoxal. The effect of MW, DDA, and degree of cross-linking in microspheres was studied on the degree of swelling, as well as by the loading and release of centchroman. The glyoxal cross-linked microspheres were more compact and hydrophobic and showed better sustained release in companion to chitosan microspheres and glutaraldehyde cross-linked microspheres. The linear fractional release of centchroman with the square root of time indicated a Fickian behavior of centchroman, and the microspheres also showed zero-order release kinetics for centchroman.     

Bacterial cellulose-chitosan composite hydrogel beads for enzyme immobilization

In this work, we report the preparation of bacterial cellulose (BC)-chitosan composite hydrogel beads by co-dissolution of BC and chitosan in 1-ethyl-3-methylimidazolium acetate and subsequent reconstitution with distilled water. The BC-chitosan hydrogel beads were used as enzyme supports for immobilizing Candida rugosa lipase by physical adsorption and covalent cross-linking. BC-chitosan hydrogel beads immobilized lipase more efficiently than microcrystalline cellulose (MCC)-chitosan hydrogel beads. The amount of protein adsorbed onto BCchitosan beads was 3.9 times higher than that adsorbed onto MCC-chitosan beads, and the catalytic activity of lipase was 1.9 times higher on the BC-chitosan beads. The lipase showed the highest thermal and operational stability when covalently cross-linked on BC-chitosan hydrogel beads. The half-life time of the lipase cross-linked on BC-chitosan bead at 60°C was 22.7 times higher than that of free lipase. Owing to their inherent biocompatibility and biodegradability, the BC-chitosan composite hydrogel beads described here could be used to immobilize proteins for various biomedical, environmental, and biocatalytic applications.     

Catalytic properties of lipases immobilized onto ultrasound-treated chitosan supports

Ultrasound sonication has been utilized to produce fragmentation of chitosan polymer and hence increase the chitosan surface area, making it more accessible to interactions with proteins. In this context, we have investigated the catalytic properties of lipases from different sources immobilized onto ultrasound-treated chitosan (ChiS) pre-activated with glutaraldehyde (ChiS-G). Atomic force microscopy indicated that ChiS-G displays a more cohesive frame without the presence of sheared/fragmented structures when compared with ChiS, which might be attributed to the cross-linking of the polysaccharide chains. The immobilization efficiency onto ChiS-G and ChiS were remarkably higher than using conventional beads. In comparison with the free enzymes, lipases immobilized onto ChiS show a slight increase of apparent K_m and decrease of apparent V_max. On the other hand, immobilization onto ChiS-G resulted in an increase of V_max, even though a slight increase of K_m was also observed. These data suggest that the activation of chitosan with glutaraldehyde has beneficial effects on the activity of the immobilized lipases. In addition, the immobilization of the lipases onto ChiS-G displayed the best reusability results: enzymes retained more than 50% of its initial activity after four reuses, which might be attributed to the covalent attachment of enzyme to activated chitosan. Overall, our findings demonstrate that the immobilization of lipases onto ultrasound-treated chitosan supports is an effective and low-cost procedure for the generation of active immobilized lipase systems, being an interesting alternative to conventional chitosan beads.     

Thermal inactivation and reactivity of beta-glucosidase immobilized on chitosan-clay composite

The dried and wet chitosan-clay composite beads were prepared by mixing equal weights of cuttlebone chitosan and activated clay and then spraying drop-wise through a syringe, with and without freeze-drying, respectively. These beads were then immersed in 5 g/L of glutaraldehyde solution at a dosage of 0.5 g/L and were cross-linked, which were finally used as supports for beta-glucosidase immobilization. The properties of the enzyme immobilized on wet- and dried-composite beads were compared. Kinetic modeling of thermal inactivation of free and immobilized enzymes was also investigated. For a given enzymatic reaction, the rate constant related to the decomposition of the enzyme-substrate complex to final product and the uncomplexed enzyme using dried-composite immobilized enzyme was larger than those using both free and wet-composite immobilized enzymes.     

Preparation of cross-linked tyrosinase aggregates

Tyrosinase from mushroom was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and cross-linking with glutaraldehyde. The effects of precipitation and cross-linking on CLEA activity were investigated and the immobilized tyrosinase was characterized. Sixty percent ammonium sulfate saturation and 2% glutaraldehyde were used; a 3-h cross-linking reaction at room temperature, at pH 7.0 was performed; particle sizes of the aggregates were reduced; consequently, 100% activity recovery was achieved in CLEAs with enhanced thermal and storage stabilities. Slight changes in optimum pH and temperature values of the enzyme were recorded after immobilization. Although immobilization did not affect V_max, substrate affinity of the enzyme increased. Highly stable CLEAs were also prepared from crude mushroom tyrosinase with 100% activity recovery.     

Adsorption of Cr(VI) and As(V) ions by modified magnetic chitosan chelating resin

Cross-linked magnetic chitosan anthranilic acid glutaraldehyde Schiff's base (CAGS) was prepared for adsorption of both As(V) and Cr(VI) ions and their determination by ICP-OES. Prepared cross-linked magnetic CAGS was investigated by means of SEM, FTIR, wide angle X-ray diffraction (WAXRD) and TGA analysis. The adsorption properties of cross-linked magnetic CAGS resin toward both As(V) and Cr(VI) were evaluated. Various factors affecting the uptake behavior such as pH, temperature, contact time, initial concentration of metal ions, effect of other ions and desorption were studied. The equilibrium was achieved after about 110 min and 120 min for As(V) and Cr(VI), respectively at pH = 2. The adsorption kinetics followed the mechanism of the pseudo-second order equation for all systems studied, evidencing chemical sorption as the rate-limiting step of adsorption mechanism and not involving a mass transfer in solution. The equilibrium data were analyzed using the Langmuir, Freundlich, and Tempkin isotherm models. The best interpretation for the equilibrium data was given by Langmuir isotherm, and the maximum adsorption capacities were 58.48 and 62.42 mg/g for both Cr(VI) and As(V), respectively. Cross-linked magnetic CAGS displayed higher adsorption capacity for Cr(VI). The adsorption capacity of the metal ions increased with increasing temperature under optimum conditions in case of Cr(VI), but decreased in case of As(V). The metal ion-loaded cross-linked magnetic CAGS were regenerated with an efficiency of greater than 88% using 0.2 M sodium hydroxide (NaOH).     

Biological ferrous sulfate oxidation by A. ferrooxidans immobilized on chitosan beads

The immobilization of Acidithiobacillus ferrooxidans cells on chitosan and cross-linked chitosan beads and the biooxidation of ferrous iron to ferric iron in a packed-bed bioreactor were studied. The biofilm formation was carried out by using a glass column reactor loaded with chitosan or cross-linked chitosan beads and 9 K medium previously inoculated with A. ferrooxidans cells. The immobilization cycles on the carrier matrix with the bioreactor operating in batch mode were compared. Then, the reactor was operated using a continuous flow of 9 K medium at different dilution rates. The results indicate that the packed-bed reactor allowed increasing the flow rate of medium approximately two fold (chitosan) and eight fold (chitosan cross-linked) without cells washout, compared to a free cell suspension reactor used as control, and to reach ferric iron productivities as high as 1100 and 1500 mg l(-1) h(-1) respectively. Scanning electron microscopy micrographs of the beads, infrared spectroscopy and the X-ray diffraction patterns of precipitates on the chitosan beads were also investigated.     

Preparation and characterization of sodium hexameta phosphate cross-linked chitosan microspheres for controlled and sustained delivery of centchroman

The cross-linked microspheres using chitosan with different molecular weights and degree of deacetylation have been prepared in presence of sodium hexameta polyphosphate (SHMP) as physical cross-linker. The degree of cross-linking through electrostatic interactions in chitosan microspheres has been evaluated by varying the charge density on chitosan and varying degree of dissociation of sodium hexameta polyphosphate by solution pH. The degree of deacetylation and molecular weight of chitosan has controlled electrostatic interactions between hexameta polyphosphate anions and chitosan, which played significant role in swelling, loading and release characteristics of chitosan microspheres for centchroman. The microspheres prepared by hexameta polyphosphate anions cross-linker were compact and more hydrophobic than covalently cross-linked microspheres, which has been attributed to the participation of all amino groups of chitosan in physical cross-linking with added hexameta polyphosphate anions. The microspheres prepared under different experimental conditions have shown an initial step of burst release, which was followed by a step of controlled release for centchroman. The extent of drug release in these steps has shown dependence on properties of chitosan and degree of cross-linking between chitosan and added polyanions. The degree of swelling and release characteristics of microspheres was also studied in presence of organic and inorganic salts, which shown significant effect on controlled characteristics of microspheres due to variations in ionic strength of the medium. The initial step of drug release has followed first order kinetics and become zero order after attaining an equilibrium degree of swelling in these microspheres. The microspheres prepared using chitosan with 62% (w/w) degree of deacetylation and molecular weight of 1134 kg mol⁻¹ have shown a sustained release for centchroman for 50 h at 4% (w/w) degree of cross-linking with SHMP.     

Amino acid modified chitosan beads: Improved polymer supports for immobilization of lipase from Candida rugosa

Amino acid modified chitosan beads (CBs) for immobilization of lipases from Candida rugosa were prepared by activation of a chitosan backbone with epichlorohydrin followed by amino acid coupling. The beads were analyzed by elemental analysis and solid state NMR with coupling yields of the amino acids ranging from 15 to 60%. The immobilized lipase on unmodified chitosan beads showed the highest immobilization yield (92.7%), but its activity was relatively low (10.4%). However, in spite of low immobilization yields (15–50%), the immobilized lipases on the amino acid modified chitosan beads showed activities higher than that of the unmodified chitosan beads, especially on Ala or Leu modified chitosan beads (Ala-CB or Leu-CB) with 49% activity for Ala-CB and 51% for Leu-CB. The immobilized lipases on Ala-CB improved thermal stability at 55 °C, compared to free and immobilized lipases on unmodified chitosan beads and the immobilized lipase on Ala-CB retained 93% of the initial activity when stored at 4 °C for 4 weeks. In addition, the activity of the immobilized lipase on Ala-CB retained 77% of its high initial activity after 10 times of reuse. The kinetic data (k_cat/K_m) supports that the immobilized lipase on Ala-CB can give better substrate specificity than the unmodified chitosan beads.