Bioresponsive systems based on crosslinked polysaccharide hydrogels

Detection of bacterial and fungal contamination is of extreme importance in the fields of medical products or food packaging. Here a diagnostic tool based on pectinase and cellulase triggered release of a dye from a crosslinked polysaccharide matrix was developed. The hydrogel-based bioresponsive matrix consisted of carboxymethylcellulose (CMC) as a substrate for cellulases and polygalacturonate (PGA) as substrate for pectinases. To improve the stability of the hydrogels, methacrylic groups were inserted as crosslinking molecules. For polymerisation, two different methods were used, namely UV and thermal crosslinking. Controlled release triggered by extracellular enzymes of potentially pathogenic/contaminating microorganisms was investigated by the incorporation of Alizarin into the hydrogels. UV polymerised hydrogels turned out to be more suitable than thermal crosslinked polymers. Integration of such polymer based bioresponsive systems in medical surfaces or package systems could therefore act as an in situ monitoring system for detecting the presence of bacteria or fungi like e.g. Aspergillus species.     

Solute adsorption and enzyme immobilization on chitosan beads prepared from shrimp shell wastes

The equilibrium and kinetics of adsorption of reactive dye RR222 and Cu²⁺, and the activity of immobilization of acid phosphatase, on highly swollen chitosan beads were examined at 30°C. The chitosan was prepared from shrimp shell wastes and was cross-linked with different dosages of glutaraldehyde or glyoxal (100–80,000 mg/l). It was shown that the amounts of solute adsorption and the immobilization capacity of acid phosphatase on cross-linked chitosan beads were substantially affected by their degree of cross-linking. The cross-linking rate of chitosan with glutaraldehyde could be described by a pseudo-second-order equation and the cross-linking equilibrium by the Freundlich equation. This provided an experimental method to control the degree of cross-linking of chitosan beads. Finally, the activity and lifetime of the immobilized enzyme were measured to evaluate the application potential.     

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%.     

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)).     

Dye decolorisation by laccase entrapped in copper alginate

A novel immobilisation system was developed for dye decolorisation using laccase produced by Ganoderma sp. KU-Alk4. The enzyme showed high efficiency in dye decolorisation when entrapped in Cu–Al and Cu-alginate beads. The former gave the highest activity but the enzyme activity survived longer in the latter. An experimental design of two 3 × 3 Latin Square experiments was applied to evaluate the effects of three different alginate compositions (low, intermediate and high mannuronate), concentration of alginate, (1.5, 3.0 and 4.5% w/v) and concentration of cross-linking agent, CuSO₄ (0.075, 0.15 and 0.225 M) on the decolorisation of indigo carmine dye and residual laccase activity in beads. The most significant factor for residual activity was the concentration of the cross-linking agent (P < 0.05) followed by alginate composition (P < 0.1). Increasing the alginate concentration resulted in only small increase in the dye decolorisation. However, higher laccase activity remained in 3.0% w/v alginate beads. Maximal dye decolorisation was achieved when 3.6% w/v low mannuronate alginate and 0.15 M CuSO₄ was used. Optimal conditions were confirmed in an extended experimental run. Results are presented from 9 successive batch runs over 12 days, reaching 96% removal of the dye (216 mg/l).     

Biocatalytic modification of polyethylene terephthalate fibres by esterases from actinomycete isolates

The modification of polyethylene terephthalate (PET) fibres by extracellular enzymes produced by actinomycetes was investigated. Cultivation of isolates in media containing PET yarn and suberin, a plant polyester composed of aliphatic and aromatic moieties, induced the production of p-nitrophenyl butyrate hydrolyzing enzymes. Incubation of enzyme preparations from the isolates M5, M9 and Thermomonospora fusca KW3b with PET yarn resulted in an increase in the absorbance of the reaction mixtures at 240 nm indicating the release of terephthalic acid or its esters catalyzed by the enzymes. The results of dyeing of enzyme-treated PET fabrics with a reactive dye (CI Reactive Red 2) indicated an increase in hydroxyl groups at the fibre surfaces as a result of the enzyme treatment.     

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.     

Biocatalytic modification of polyethylene terephthalate fibres by esterases from actinomycete isolates

The modification of polyethylene terephthalate (PET) fibres by extracellular enzymes produced by actinomycetes was investigated. Cultivation of isolates in media containing PET yarn and suberin, a plant polyester composed of aliphatic and aromatic moieties, induced the production of p-nitrophenyl butyrate hydrolyzing enzymes. Incubation of enzyme preparations from the isolates M5, M9 and Thermomonospora fusca KW3b with PET yarn resulted in an increase in the absorbance of the reaction mixtures at 240 nm indicating the release of terephthalic acid or its esters catalyzed by the enzymes. The results of dyeing of enzyme-treated PET fabrics with a reactive dye (CI Reactive Red 2) indicated an increase in hydroxyl groups at the fibre surfaces as a result of the enzyme treatment.     

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.     

Design and development of hydrogel beads for targeted drug delivery to the colon

The purpose of this research was to develop and evaluate a multiparticulate system of chitosan hydrogel beads exploiting pH-sensitive property and specific biodegradability for colon-targeted delivery of satranidazole. Chitosan hydrogel beads were prepared by the cross-linking method followed by enteric coating with Eudragit S100. All formulations were evaluated for particle size, encapsulation efficiency, swellability, and in vitro drug release. The size of the beads was found to range from 1.04±0.82 mm to 1.95±0.05 mm. The amount of the drug released after 24 hours from the formulation was found to be 97.67%±1.25% in the presence of extracellular enzymes as compared with 64.71%±1.91% and 96.52%±1.81% release of drug after 3 and 6 days of enzyme induction, respectively, in the presence of 4% cecal content. Degradation of the chitosan hydrogel beads in the presence of extracellular enzymes as compared with rat cecal and colonic enzymes indicates the potential of this multiparticulate system to serve as a carrier to deliver macromolecules specifically to the colon and can be offered as a substitute in vitro system for performing degradation studies. Studies demonstrated that orally administered chitosan hydrogel beads can be used effectively for the delivery of drug to the colon. Published: July 13, 2007     

Development and characterization of simulant pancreatic islets

Insulin is stored in pancreatic islets as a zinc-insulin complex, and stimulating the islets results in the release of insulin and zinc. Simulant pancreatic islet beads have been developed using agarose beads (50-250 micro m diameter) derivatized with iminodiacetic acid that have been loaded with zinc. A qualitative comparison of the simulant beads with pancreatic islets has been made by staining with dithizone and a zinc-binding fluorescent dye, TSQ. The binding capacity of simulant beads was determined to be 34 micro mol Zn(2+)/g of dried beads using anodic stripping voltammetry. Hydrochloric acid was used to release zinc from beads to mimic the secretion of insulin from pancreatic islets and a release profile was established. The simulant beads can be used to optimize the islet isolation process and reduce the use of real islets in method development.     

An exo-poly-alpha-D-galacturonosidase implicated in the regulation of extracellular pectate lyase production in Erwinia chrysanthemi.

Pectic enzymes in the supernatants of Erwinia chrysanthemi cultures in late-logarithmic-phase growth on D-galacturonan were resolved into three components: two pectate lyase isozymes and an exo-poly-alpha-D-galacturonosidase previously unreported in this organism. The hydrolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, preparative electrofocusing in Ultrodex gel, and gel filtration through Ultrogel AcA54. The enzyme had a specific activity of 591 mumol/min per mg of protein, a pI of 8.3, a molecular weight of 67,000, a pH optimum of 6.0, and a Km of 0.05 mM for D-galacturonan. Analyses of reaction mixtures by paper chromatography revealed that the enzyme released only digalacturonic acid from D-galacturonan. The action of the hydrolytic enzyme on D-galacturonan labeled at the nonreducing end by partial digestion with pectate lyase revealed that it rapidly released 4,5-unsaturated digalacturonic acid from 4,5-unsaturated pectic polymers. The production of extracellular exo-poly-alpha-D-galacturonosidase was coordinately regulated with pectate lyase production. The action patterns of the two enzymes appeared complementary in the degradation of pectic polymers to disaccharides that stimulated pectic enzyme production and supported bacterial growth.     

Detoxification of azo dyes mediated by cell-free supernatant culture with manganese-dependent peroxidase activity: effect of Mn2+ concentration and H2O2 dose

White-rot fungi (WRF) are capable of degrading complex organic compounds such as lignin, and the enzymes that enable these processes can be used for the detoxification of recalcitrant organopollutants. The aim of this study is to evaluate a system based on the use of an in vitro ligninolytic enzyme for the detoxification of recalcitrant dye pollutants. The dyes selected for investigation were the anionic and cationic commercial azo dyes, basic blue 41 (BB41), acid black 1 (AB1), and reactive black 5 (RB5). A supernatant, cell-free culture of WRF with manganese peroxidase activity was used to investigate its degradative capacity under various conditions, and concentrations of cofactors, H(2)O(2) and Mn(2+). The assays were carried out using a 2(2) experimental designs whose variables were concentration of Mn(2+) (33 and 1,000 μM) and semicontinuous dosage of the H(2)O(2) (0.02 and 0.10 μmol) added at a frequency of 0.2 min(-1). The response variables analyzed were the efficiency and the initial rate of the decolorization process. The dye concentrations considered ranged from 10 to 200 mg L(-1). AB1 and RB5 were decolorized over the entire interval of concentrations studied; reaching efficiencies between 15 and 95%. Decolorization of up to 100 mg L(-1), BB41 had less than 30% efficiency. The decay of the concentration of AB1 was interpreted by two-stage kinetics model, with the exception of the condition of 33 μM Mn(2+)-0.02 μmol of H(2)O(2) in which only one stage was observed. For all assays performed with 33 μM Mn(2+), the initial rate of the decolorization process was found to be dependent on the dosage of H(2)O(2). The results of this study can be applied to the development bioreactors for the degradation of recalcitrant pollutants from the textile industry and may be used as a model for expanding the use of extracellular enzyme supernatants in bioremediation.     

Linoleic acid-induced endothelial cell injury: Role of membrane-bound enzyme activities and lipid oxidation

High plasma levels of linoleic acid (18:2) may injure endothelial cells, resulting in decreased barrier function of the vascular endothelium. The effects of linoleic acid on endothelial barrier function (transendothelial movement of albumin), membrane-bound enzyme activities, and possible autooxidation of linoleic acid under experimental conditions were studied. The exposure of endothelial monolayers to 18:2 for 24 hr at 60, 90, and 120 μM. fatty acid concentrations caused a significant increase in transendothelial movement of albumin, with maximum albumin transfer at 90 μM. Fatty acid treatment resulted in the increased appearance of cytosolic lipid droplets. Activities of the membrane-bound enzymes, angiotensin-converting enzyme (ACE), and Ca²⁺-ATPase increased steadily with increasing time of cell exposure to 90 μM 18:2, reaching significance at 24 hr. Treatment of endothelial cultures with up to 120 μM 18:2 did not cause cytotoxicity, as evidenced by a nonsignificant change in cellular release of [₃H]-adenine. Incubation of 18:2-supplemented serum-containing culture media with 1000 μM 18:2 at 37°C for up to 48 hr did not result in formation of autooxidation products. These results suggest that 18:2 itself, and not its oxidation products, plays a major role in disrupting endothelial barrier function.     

Inhibition of Ca2+/calmodulin-dependent phosphatase activity by regucalcin in rat liver cytosol: Involvement of calmodulin binding

The regulatory effect of regucalcin on Ca²⁺/calmodulin-dependent phosphatase activity and the binding of regucalcin to calmodulin was investigated. Phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine in rat liver cytosol was significantly increased by the addition of Ca²⁺ (100 μM) and calmodulin (0.30 μM). Thess increases were clearly inhibited by the addition of regucalcin (0.50–1.0 μM) into the enzyme reaction mixture. The cytosolic phosphoamino acid phosphatase activity was significantly elevated by the presence of anti-regucalcin monoclonal antibody (0.2 μg/ml), suggesting that endogenous regucalcin in the cytosol has an inhibitory effect on the enzyme activity. This elevation was prevented by the addition of regucalcin (0.50 μM). Purified calcineurin phosphatase activity was significantly increased by the addition of calmodulin (0.12 μM) in the presence of Ca²⁺ (1 and 10 μM). This increase was completely inhibited by the presence of regucalcin (0.12 μM). The inhibitory effect of regucalcin was reversed by the addition of calmodulin with the higher concentration (0.36 μM). Regucalcin has been demonstrated to bind on calmodulin-agarose beads by analysis with sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The present study demonstrates that regucalcin inhibits Ca²⁺/calmodulin-dependent protein phosphatase activity in rat liver cytosol, and that regucalcin can bind to calmodulin. J. Cell. Biochem. 71:140–148, 1998. © 1998 Wiley-Liss, Inc.     

Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16.

The four pectate lyases (EC 4.2.2.2) secreted by Erwinia chrysanthemi EC16 have been individually produced as recombinant enzymes in Escherichia coli. Oligogalacturonates formed from polygalacturonic acid during reactions catalyzed by each enzyme have been determined by high-performance liquid chromatography analysis. PLa catalyzes the formation of a series of oligomers ranging from dimer to dodecamer through a random endolytic depolarization mechanism. PLb and PLc are trimer- and tetramer-generating enzymes with an identical combination of endolytic and exolytic mechanisms. PLe catalyzes a nonrandom endolytic depolymerization with the formation of dimer as the predominant product. The pectate lyases secreted by E. chrysanthemi EC16 represent a battery of enzymes with three distinct approaches to the depolymerization of plant cell walls.     

Cloning and characterization of an esophageal-gland-specific pectate lyase from the root-knot nematode Meloidogyne javanica

Root-knot nematodes (Meloidogynejavanica) are obligate sedentary endoparasites that must penetrate the host root to initiate their life cycle. Many enzymes are secreted by the nematode to facilitate host penetration; required enzymes may include pectate lyases and cellulases. Using differential screening, a class III pectate lyase, Mj-pel-1 (M. javanica pectate lyase 1), was cloned from a library enriched for esophageal gland genes. DNA gel blotting confirmed that the Mj-pel-1 gene was of nematode origin and a member of a small multigene family. In situ hybridization localized the expression of Mj-pel-1 to the basal cells of the esophageal glands, while immunolocalization detected the protein in the esophageal glands as well as on the exterior of the nematode, confirming that the protein is secreted. When MJ-PEL-1 was expressed in Pichia pastoris, the resulting protein was active. The pH optimum of MJ-PEL-1 was 10.0, and the enzyme was five times more active on pectate than on pectin. Like other class III pectate lyases, MJ-PEL-1 also displayed an absolute requirement for Ca2+. The root-knot nematode migrates through the middle lamella of the plant root; therefore, MJ-PEL-1 may be an important enzyme early in the infection process.     

Effect of chloroquine on cultured fibroblasts: release of lysosomal hydrolases and inhibition of their uptake.

Incubation of normal human fibroblasts with 1–5 μM chloroquine at physiological pH for 8 hr produces granular cytoplasmic inclusions, release of lysosomal enzymes into the medium and decrease of intracellular lysosomal enzyme activities. The effects are dose dependent and reversible. The uptake of arylsulfatase A into fibroblasts genetically deficient in arylsulfatase A (grown from skin biopsies of patients with metachromatic leukodystrophy) is completely inhibited by pretreating the cells with 5 μM chloroquine. Arylsulfatase A, which has been taken up as exogenous enzyme from the medium into the cells, is partially released into the culture medium upon incubation with chloroquine. The data suggest that chloroquine competes with the binding of lysosomal enzymes to the cell membrane and to the membranes of pinocytotic vacuoles and causes release of previously internalized exogenous enzyme.