Immobilization of acetylcholinesterase on new modified acrylonitrile copolymer membranes

The three new dual-layer matrices (polyacrylonitrile (PAN) membranes coated with physically bound chitosan (CHI)—PANCHI-A and chemically bound chitosan—PANCHI-B and PANCHI-C) for immobilization of acetylcholinesterase (AChE) were obtained. The chemical-modified PAN membrane (PAN-NaOH + ethylenediamine (EDA)) was used as a base for the prepared dual-layer membranes. For chemical chitosan bound membrane, chitosan was tethered onto the membrane surface to form a dual-layer biomimetic membrane in the presence of glutaraldehyde (GA). The basic characteristics (amount of amino groups, hydrophilicity and transport characteristics) of the chitosan-modified membranes were investigated. The SEM analyses were shown essential morphology change in the different chitosan membranes.The relative activities and V_max of the covalently immobilized enzyme on PANCHI-B and PANCHI-C membranes were higher than that on PANCHI-A membrane and chemical-modified membrane with NaOH + EDA. K_m values for the different modified membranes are lower for the chitosan-treated membranes. The pH and temperature optimum of immobilized enzyme were determined. The bound enzymes on PANCHI-B and PANCHI-C have higher thermal and storage stability in comparison with AChE on PANCHI-A membrane and free enzyme.     

Immobilization of acetylcholinesterase on new modified acrylonitrile copolymer membranes

The three new dual-layer matrices (polyacrylonitrile (PAN) membranes coated with physically bound chitosan (CHI)—PANCHI-A and chemically bound chitosan—PANCHI-B and PANCHI-C) for immobilization of acetylcholinesterase (AChE) were obtained. The chemical-modified PAN membrane (PAN-NaOH + ethylenediamine (EDA)) was used as a base for the prepared dual-layer membranes. For chemical chitosan bound membrane, chitosan was tethered onto the membrane surface to form a dual-layer biomimetic membrane in the presence of glutaraldehyde (GA). The basic characteristics (amount of amino groups, hydrophilicity and transport characteristics) of the chitosan-modified membranes were investigated. The SEM analyses were shown essential morphology change in the different chitosan membranes.The relative activities and V_max of the covalently immobilized enzyme on PANCHI-B and PANCHI-C membranes were higher than that on PANCHI-A membrane and chemical-modified membrane with NaOH + EDA. K_m values for the different modified membranes are lower for the chitosan-treated membranes. The pH and temperature optimum of immobilized enzyme were determined. The bound enzymes on PANCHI-B and PANCHI-C have higher thermal and storage stability in comparison with AChE on PANCHI-A membrane and free enzyme.     

Poly(acrylonitrile)chitosan composite membranes for urease immobilization

(Poly)acrylonitrile/chitosan (PANCHI) composite membranes were prepared. The chitosan layer was deposited on the surface as well as on the pore walls of the base membrane. This resulted in the reduction of the pore size of the membrane and in an increase of their hydrophilicity. The pore structure of PAN and PANCHI membranes were determined by TEM and SEM analyses. It was found that the average size of the pore under a selective layer base PAN membrane is 7 microm, while the membrane coated with 0.25% chitosan shows a reduced pore size--small or equal to 5 microm and with 0.35% chitosan--about 4 microm. The amounts of the functional groups, the degree of hydrophilicity and transport characteristics of PAN/Chitosan composite membranes were determined. Urease was covalently immobilized onto all kinds of PAN/chitosan composite membranes using glutaraldehyde. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity (94%) was measured for urease bound to PANCHI2 membranes (0.25% chitosan). The basic characteristics (pH(opt), pH(stability), T(opt), T(stability), heat inactivation and storage stability) of immobilized urease were determined. The obtained results show that the poly(acrylonitrile)chitosan composite membranes are suitable for enzyme immobilization.     

Immobilization of urease on nanostructured polymer membrane and preparation of urea amperometric biosensor

A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%.     

Molecular weight and pH effects of aminoethyl modified chitosan on antibacterial activity in vitro

Aminoethyl modified chitosan derivatives (AEMCSs) with different molecular weight (Mw) were synthesized by grafting aminoethyl group on different molecular weight chitosans and chitooligosaccharide. FTIR, (1)H NMR, (13)C NMR, elemental analysis and potentiometric titration results showed that branched polyethylimine chitosan was synthesized. Clinical Laboratory Standard Institute (CLSI) protocols were used to determine MIC for Gram-negative strain of Escherichia coli under different pH. The antibacterial activity of the derivatives was significantly improved compared with original chitosans, with MIC values against E. coli varying from 4 to 64 μg/mL depending on different Mw and pH. High molecular weight seems to be in favor of stronger antibacterial activity. At pH 7.4, derivatives with Mw above 27 kDa exhibited equivalent antibacterial activity (16 μg/mL), while oligosaccharide chitosan derivative with lower Mw (~1.4 kDa) showed decreased MIC of 64 μg/mL. The effect of pH on antibacterial activity is more complicated. An optimal pH for HAEMCS was found around 6.5 to give MIC as low as 4 μg/mL, while higher or lower pH compromised the activity. Cell integrity assay and SEM images showed evident cell disruption, indicating membrane disruption may be one possible mechanism for antibacterial activity.     

Immobilization of a protease on modified chitosan beads for the depolymerization of chitosan

Neutral protease was immobilized on chitosan (CS), carboxymethyl chitosan (CMCS), and N-succinyl chitosan (NSCS) hydrogel beads. And the biocatalysts obtained were used to prepare low molecular weight chitosan (LMWC) and chitooligomers. Weight-average molecular weight of LMWC produced by neutral protease immobilized on CS, CMCS and NSCS hydrogel beads were 3.4 kDa, 3.2 kDa and 1.9 kDa, respectively. The effects of immobilization support and substrate on enzymatic reaction were analyzed by measuring classical Michaelis-Menten kinetic parameters. The FT-IR, XRD and potentiometric determination results indicated decrease of molecular weight led to transformation of crystal structure, but the degree of N-deacetylation and chemical structures of residues were not changed compared to initial chitosan. The degree of polymerization of chitooligomers was mainly from 2 to 7. We observed a strong dependence of the immobilized enzyme properties on the chemical nature of the supports, which leads to different microenvironment of neutral protease and changes the hydrolyzing process.     

Immobilization of β-galactosidase on modified polypropilene membranes

A new immobilized system: β-galactosidase-modified polypropylene membrane was created. It was obtained 13 different carriers by chemical modification of polypropylene membranes by two stages. The first stage is treatment with K(2)Cr(2)O(7) to receive carboxylic groups on membrane surface. The second stage is treatment with different modified agents ethylendiamine, hexamethylenediamine, hydrazine dihydrochloride, hydroxylamine, o-phenylenediamine, p-phenylenediamine, N,N'-dibenzyl ethylenediamine diacetate to receive amino groups. The quantity of the amino groups, carboxylic groups and the degree of hydrophilicity of unmodified and modified polypropilene membranes were determined. β-Galactosidase was chemically immobilized on the obtained carries by glutaraldehyde. The highest relative activity of immobilized enzyme was recorded at membrane modified with 10% hexamethylenediamine (Membrane 5) - 92.77%. The properties of immobilized β-galactosidase on different modified membranes - pH optimum, temperature optimum, pH stability and thermal stability were investigated and compared with those of free enzyme. The storage stability of all immobilized systems was studied. It was found that the most stable system is immobilized enzyme on Membrane 5. The system has kept 90% of its initial activity at 300th day (pH=6.8; 4°C). The stability of the free and immobilized β-galactosidase on the modified membrane 5 with 10% HMDA in aqueous solutions of alcohols - mono-, diol and triol was studied. The kinetics of enzymatic reaction of free and immobilized β-galactosidase on the modified membrane 5 at 20°C and 40°C and at the optimal pH for both forms of the enzyme were investigated. It was concluded that the modified agent - hexamethylenediamine, with long aliphatic chain ensures the best immobilized β-galactosidase system.     

Catalytic activity and the stability of horseradish peroxidase increase as a result of its incorporation into a polyelectrolyte complex with chitosan

The incorporation of horseradish peroxidase into polyelectrolyte complexes with chitosans of different molecular weights (MW 5–150 kDa) yielded highly active and stable enzyme preparations. As a result of the selection of optimal conditions for the formation of peroxidase-chitosan complexes, it was found that 0.1% chitosan with a MW of 10 kDa had the strongest activatory effect on peroxidase (activation degree, >70%) in the reaction of o-dianisidine oxidation by hydrogen peroxide. The complex formed by 0.001% chitosan with a molecular weight of 150 kDa was most stable: when immobilized on foamed polyurethane, it retained at least 50% of the initial activity for 550 days. The highest catalytic activity was exhibited in a 0.05 M phthalate buffer (pH 5.9–6.2) by the complex containing 0.006–0.009% chitosan in the indicator reaction. The activatory effect of the polysaccharide on the enzyme was determined by its influence on the binding and conversion of the reducting substrate peroxidase.     

Effect of the molecular weight of chitosan on its antiviral activity in plants

The effect of the molecular weight of chitosan on its ability to suppress systemic infection of bean mild mosaic virus in bean (Phasoleus vulgaris L.) plants was studied. The enzymatic hydrolysate of low-molecular-weight chitosan was successively fractionated by ultrafiltration through membranes with decreasing pore size. In total, four chitosan fractions with a weight-average molecular weight varying from 1.2 to 40.4 kDa were obtained. It was shown that the treatments of bean plants with these fractions (chitosan concentration, 10 or 100 microg/ml) inhibited virus accumulation and systemic propagation. The degree of chitosan-induced antiviral resistance increased as the molecular weight of chitosan decreased. The monomers comprising the chitosan molecule-glucosamine and N-acetylglucosamine--exhibited no antiviral activity.     

Poly(ethylene oxide)-grafted thermoplastic membranes for use as cellular hybrid bio-artificial organs in the central nervous system

Poly(acrylonitrile-co-vinyl chloride) (PAN/VC) anisotropic membranes were chemically modified with poly(ethylene oxide) (PEO) (5000 and 20,000 g/mol) by one of two aqueous reactions: (a) acid hydrolysis of the nitrile group to a carboxylic acid with which amine-terminated PEO (PEO-NH(2)) reacted or (b) base reduction of the nitrile group to an amine with which PEO-succinimide (PEO-SC) reacted. Approximately 1.3% of the bulk material was modified with PEO-NH(2) whereas 1.8 to 3.5% was modified with PEO-SC as determined by proton nuclear magnetic resonance ((1)H NMR) and attenuated total reflectance Fourier transform infrared (ATR FTIR) spectra. Approximately 50 to 75% less bovine serum albumin (BSA) adsorbed to PEO-grafted single skin fibers than to unmodified PAN/VC. Transport properties of modified and unmodified fibers were compared by passive diffusion, convective nominal molecular weight cutoff, and hydraulic permeability. Neither hydraulic permeability nor nominal molecular weight cutoff of BSA changed appreciably after surface modification with PEO indicating that pore structure was not adversely affected by the chemistry involved in grafting poly(ethylene oxide). However, in the absence of any membrane conditioning, the apparent diffusion of alpha-chymotrypsinogen (24,000 g/mol) was enhanced in PEO-grafted PAN/VC fibers possibly as a result of reduced sorption of the permeating protein. In vivo biocompatibility in the brain tissue of rats was judged by histological assessment of the host's cellular response to fibers implanted for 30 days; biocompatibility of both PAN/VC and PAN/VC-g-PEO was satisfactory but improved slightly with PEO grafting. (c) 1994 John Wiley & Sons, Inc.     

Surface functionalization of porous polypropylene membranes with polyaniline for protein immobilization

Commercial porous polypropylene membranes were chemically modified with polyaniline (PANI) using ammonium persulfate as the oxidizer. The influence of polymerization conditions on the membrane properties was studied by adsorption analysis and membrane permeability. The PANI-coated polypropylene (PANI/PP) membranes possessed high affinity toward the proteins, which can be immobilized onto the membrane surface through physical adsorption or covalent immobilization. The quantity of immobilized horseradish peroxidase (HRP) and its activity depended on the quantity and quality (oxidation level) of PANI. The storage conditions for PANI/PP membranes containing immobilized HRP were studied. HRP immobilized on the PANI/PP membrane was shown to retain 70% of its activity after 3-month storage at +5 degrees C, suggesting that this material can be used for practical application, such as in bioreactors as enzyme membranes.     

Biochemical characterization of the PH-20 protein on the plasma membrane and inner acrosomal membrane of cynomolgus macaque spermatozoa

Preparations of sperm membranes (plasma membranes and outer acrosomal membranes) and denuded sperm heads were isolated from macaque sperm, and the PH-20 proteins present were characterized by Western blotting, hyaluronic acid substrate gel analysis, and a microplate assay for hyaluronidase activity. Because we have shown previously that PH-20 is located on the plasma membrane and not on the outer acrosomal membrane, the PH-20 in the membrane preparations was presumed to be plasma membrane PH-20 (PM-PH-20). PM-PH-20 had an apparent molecular weight of 64 kDa and the optimum pH for its hyaluronidase activity was 6.5. The PH-20 associated with denuded sperm heads was localized by immunogold label to the persistent inner acrosomal membrane (IAM) and was presumed to be IAM-PH-20, which included a major 64 kDa form and a minor 53 kDa form. The 53 kDa form was not detected in extracts of denuded sperm heads from acrosome intact sperm that were boiled in nonreducing sample buffer, but was present in extracts of sperm heads from acrosome reacted sperm and in the soluble material released during the acrosome reaction, whether or not the samples were boiled. Substrate gel analysis showed that the hyaluronidase activity of the 53 kDa form of PH-20 was greatest at acid pH, and this activity was probably responsible for the broader and lower optimum pH of IAM hyaluronidase activity. When hypotonic treatment was used to disrupt the sperm acrosome and release the acrosomal contents, less than 0.05% of the total hyaluronidase activity was released. The PH-20 protein released by hypotonic treatment was the 64 kDa form and not the 53 kDa form, suggesting that its source might be the disrupted plasma membranes. Our experiments suggest that the soluble form of hyaluronidase, which is released at the time of the acrosome reaction, is derived from the IAM. This soluble hyaluronidase is composed of both the 64 kDa form and 53 kDa form of PH-20. The 53 kDa form appears to be processed from the 64 kDa form at the time of the acrosome reaction. Mol. Reprod. Dev. 48:356–366, 1997. © 1997 Wiley-Liss, Inc.     

Development of Silica Gel-Supported Modified Macroporous Chitosan Membranes for Enzyme Immobilization and Their Characterization Analyses

The present work was aimed at developing stability enhanced silica gel-supported macroporous chitosan membrane for immobilization of enzymes. The membrane was surface modified using various cross-linking agents for covalent immobilization of enzyme Bovine serum albumin. The results of FT-IR, UV–vis, and SEM analyses revealed the effect of cross-linking agents and confirmed the formation of modified membranes. The presence of silica gel as a support could provide a large surface area, and therefore, the enzyme could be immobilized only on the surface, and thus minimized the diffusion limitation problem. The resultant enzyme immobilized membranes were also characterized based on their activity retention, immobilization efficiency, and stability aspects. The immobilization process increased the activity of immobilized enzyme even higher than that of total (actual) activity of native enzyme. Thus, the obtained macroporous chitosan membranes in this study could act as a versatile host for various guest molecules.     

Neurotoxin of the black widow spider and its interaction with receptors from the rat brain

A presynaptic neurotoxin isolated from the venom of the Central Asia spider karakurt (Black Widow Spider, Latrodectus mactans tredecimguttatus) is shown to consist of two identical subunits of mol. weight about 118 kDa. The iodinated neurotoxin binds to the rat brain synaptosomal plasma membranes with Kd 0.1 nM (Bmax 0.1 pmol/mg of protein) at 37 degrees C, and with Kd 0.35 nM (Bmax 0.2 pmol/mg of protein) at 5 degrees C. At intermediate temperatures both types of receptors are detectable. It is supposed that the dimeric form of the toxin interacts with a single class of receptors possessing lateral mobility in the membrane. By the use of different bifunctional reagents it is revealed that the neurotoxin interacts with a presynaptic membrane protein of mol. weight 95 kDa. A protein of the same size accompanied by a 71 kDa protein was isolated by the affinity chromatography of solubilized synaptosomal membranes on the absorbent, containing immobilized neurotoxin.     

Effect of the molecular weight of chitosan on its antiviral activity in plants

The effect of the molecular weight of chitosan on its ability to suppress systemic infection of bean mild mosaic virus in bean (Phaseolus vulgaris L.) plants was studied. The enzymatic hydrolysate of low-molecular-weight chitosan was successively fractionated by ultrafiltration through membranes with decreasing pore size. In total, four chitosan fractions with a weight-average molecular weight varying from 1.2 to 40.4 kDa were obtained. It was shown that the treatments of bean plants with these fractions (chitosan concentration, 10 or 100 μg/ml) inhibited virus accumulation and systemic propagation. The degree of chitosan-induced antiviral resistance increased as the molecular weight of chitosan decreased. The monomers comprising the chitosan molecule—glucosamine and N-acetylglucosamine—exhibited no antiviral activity.     

Probing the modes of antibacterial activity of chitosan. Effects of pH and molecular weight on chitosan interactions with membrane lipids in Langmuir films

Chitosan, a cationic biopolymer derived from chitin, has been described as having antibacterial activity. The modes of this activity, however, have not been established. One mode proposed is that chitosan perturbs bacterial cell membranes. To validate this proposal, in this study we investigated chitosan interactions with lipids in Langmuir monolayers as model membranes. The interactions were assessed by monitoring differences in the shape of the compression isotherms measured in the absence and presence of chitosan in the subphase (acetate buffer). To appraise the contribution of electrostatic interactions versus hydrogen bonding and hydrophobic interactions, three membrane lipids differing in charge were studied-anionic dipalmitoylphosphatidylglycerol (DPPG), zwitterionic dipalmitoylphosphatidylcholine (DPPC), and neutral cholesterol-and the pH of the subphase was varied between 3.5 and 6.0. In addition, the impact of the molecular weight of chitosan on the interactions was assessed at pH 3.5. It was found that while chitosan had a negligible effect on DPPC monolayers over the pH range studied, it distinctly affected DPPG and cholesterol monolayers. The effect on DPPG was found to decrease with increasing pH, that at pH 3.5 being ascribed to the charge-mediating action of chitosan on the local ionic environment and that at higher pHs to the intercalation of chitosan to the monolayers. Practically independent of pH, the effect of chitosan on cholesterol was accounted for by the formation of cholesterol-chitosan hydrogen bonds. Chitosan of lower molecular weight facilitated the interactions with all the three lipids studied. The results obtained may be helpful in identifying the mode of antibacterial activity of chitosan versus other modes that involve the disturbance of cell life cycles.     

Immobilization of glucose oxidase on chitosan-based porous composite membranes and their potential use in biosensors

The glucose oxidase (GO_x) enzyme was immobilized on chitosan-based porous composite membranes using a covalent bond between GO_x and the chitosan membrane. The chitosan-based porous membranes were prepared by the combination of the evaporation- and non-solvent-induced phase separation methods. To increase the membrane conductivity, carbon nanotubes (CNTs) were added to the chitosan solution. The resulting membranes were characterized in terms of water permeability, surface morphology and surface chemistry. Enzyme immobilization was performed on the chitosan membranes with and without activation using glutaraldehyde (GA). Three different configurations of working electrodes were evaluated to investigate the potential use of the modified membranes in biosensors. The results show that enzyme immobilization capacity was greater for membranes that had been activated than for membranes that had not been activated. In addition, activation increased the stability of the enzyme immobilization. The immobilization of GO_x on chitosan-based membranes was influenced by both pH and the concentration of the enzyme solution. The presence of CNTs significantly increased the electrical conductivity of the chitosan membranes. The evaluation of three different configurations of working electrodes suggested that the third configuration, which was composed of an electrode-mediator-(chitosan and carbon nanotube) structure and enzyme, is the best candidate for biosensor applications.     

Covalent immobilization of acetylcholinesterase on a novel polyacrylic acid‐based nanofiber membrane

In this study, polyacrylic acid‐based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from Electrophorus electricus was covalently immobilized onto polyacrylic acid‐based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid‐based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1 mM Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Mg²⁺, Ca²⁺ ions on the stability of the immobilized AChE was also investigated. According to the Michaelis–Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis–Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733 mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE‐based biosensors.     

Effect of Molecular Weight and Degree of Acetylation on Adjuvantive Properties of Chitosan Derivatives

The hemostatic and immunostimulating activity and cytotoxicity were determined for a number of chitosans differing in molecular weight (from 3 to 510 kDa) and degree of acetylation (from 1 to 25 mol%) that were used as adjuvants in inactivated poliomyelitic, influenza, and live influenza vaccines. It has been shown that the hemostatic activity of chitosan increased sharply with an increase in its molecular weight. In oligochitosan with a molecular weight of <16 kDa, it was smaller by a factor of 15–100 than in chitosan with a molecular weight of 20–510 kDa. The level of increase in the immunogenicity of vaccines containing oligochitosan as adjuvants was not lower than that for the vaccine including high-molecular chitosan. However, the immunostimulatory activity of oligochitosan depended on the degree of acetylation, reaching a maximum value at 6 mol%. It was shown that all oligochitosans and chitosans with a molecular mass below ~50 kDa showed almost no cytotoxicity at a concentration of ≤2.5 mg/mL, which enable their use as adjuvants for inactivated and live vaccines at the optimal ratio of molecular weight to the degree of acetylation.     

DNA protection from extracapsular nucleases, within chitosan- or poly-L-lysine-coated alginate beads

DNA was immobilized within alginate matrix using an external or an internal calcium source, and then membrane coated with chitosan or poly-L-lysine. Membrane thickness increased with decreasing polymer molecular weight and increasing degree of deacetylation (chitosan). Beads were exposed to a 31,000 molecular weight nuclease to determine the levels of DNA protection offered by different membrane and matrix combinations. Almost total hydrolysis of DNA was observed in alginate beads following nuclease exposure. Less than 1% of total double-stranded DNA remained unhydrolyzed within chitosan- or poly-L-lysine-coated beads, corresponding with an increase in DNA residuals (i.e. double- and single-stranded DNA, polynucleotides, bases). Chitosan membranes did not offer sufficient DNA protection from DNase diffusion since all of the double-stranded DNA was hydrolyzed after 40 min of exposure. Both chitosan and poly-L-lysine membranes reduced the permeability of alginate beads, shown by enhanced retention of DNA residuals after DNase exposure. The highest level of DNA protection within freshly prepared beads was obtained with high molecular weight (197,100) poly-L-lysine membranes coated on beads formed using an external calcium source, where over 80% of the double-stranded DNA remained after 40 min of DNase exposure. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DS-DNA recoveries of 60% for chitosan-coated, 90% for poly-L-lysine-coated, and 95% for uncoated alginate beads.