Composition and structure of whey protein/gum arabic coacervates

Complex coacervation in whey protein/gum arabic (WP/GA) mixtures was studied as a function of three main key parameters: pH, initial protein to polysaccharide mixing ratio (Pr:Ps)(ini), and ionic strength. Previous studies had already revealed under which conditions a coacervate phase was obtained. This study is aimed at understanding how these parameters influence the phase separation kinetics, the coacervate composition, and the internal coacervate structure. At a defined (Pr:Ps)(ini), an optimum pH of complex coacervation was found (pH(opt)), at which the strength of electrostatic interaction was maximum. For (Pr:Ps)(ini) = 2:1, the phase separation occurred the fastest and the final coacervate volume was the largest at pH(opt) = 4.0. The composition of the coacervate phase was determined after 48 h of phase separation and revealed that, at pH(opt), the coacervate phase was the most concentrated. Varying the (Pr:Ps)(ini) shifted the pH(opt) to higher values when (Pr:Ps)(ini) was increased and to lower values when (Pr:Ps)(ini) was decreased. This phenomenon was due to the level of charge compensation of the WP/GA complexes. Finally, the structure of the coacervate phase was studied with small-angle X-ray scattering (SAXS). SAXS data confirmed that at pH(opt) the coacervate phase was dense and structured. Model calculations revealed that the structure factor of WP induced a peak at Q = 0.7 nm(-1), illustrating that the coacervate phase was more structured, inducing the stronger correlation length of WP molecules. When the pH was changed to more acidic values, the correlation peak faded away, due to a more open structure of the coacervate. A shoulder in the scattering pattern of the coacervates was visible at small Q. This peak was attributed to the presence of residual charges on the GA. The peak intensity was reduced when the strength of interaction was increased, highlighting a greater charge compensation of the polyelectrolyte. Finally, increasing the ionic strength led to a less concentrated, a more heterogeneous, and a less structured coacervate phase, induced by the screening of the electrostatic interactions.     

Self-healing fish gelatin/sodium montmorillonite biohybrid coacervates: structural and rheological characterization

Complex coacervation driven by associative electrostatic interactions was studied in mixtures of exfoliated sodium-montmorillonite (Na(+)-MMT) nanoplatelets and fish gelatin, at a specific mixing ratio and room temperature. Structural and viscoelastic properties of the coacervate phase were investigated as a function of pH by means of different complementary techniques. Independent of the technique used, the results consistently showed that there is an optimum pH value at which the coacervate phase shows the tightest structure with highest elasticity. The solid-like coacervates showed an obvious shear-thinning behavior and network fracture but immediately recovered back into their original elastic character upon removal of the shear strain. The nonlinear mechanical response characterized by single step stress relaxation experiments revealed the same trend for the yield stress and isochronal shear modulus of the coacervates as a function of pH with a maximum at pH 3.0 and lower values at 2.5 and 3.5 pHs, followed by a very sharp drop at pH 4.0. Finally, small-angle X-ray scattering (SAXS) data confirmed that at pHs lower than 4.0 the coacervate phases were dense and structured with a characteristic length scale (ξ(SAXS)) of ~7-9 nm. Comparing the ξ(SAXS) with rheological characteristic length (ξ(rheol)) estimated from low-frequency linear viscoelastic data and network theory, it was concluded that both the strength of the electrostatic interactions and the conformation of the gelatin chains before and during of the coacervation process are responsible for the structure and rigidity of the coacervates.     

Systematic of alcohol-induced simple coacervation in aqueous gelatin solutions

Turbidity measurements performed at 450 nm were used to follow the process of simple coacervation when 1% (w/v) aqueous alkali processed gelatin (type-B) solutions were titrated with methanol, ethanol, propanol, and tert-butyl alcohol at various pHs of the solution ranging from pH = 5 to 8 and ionic strengths varying from I = 0.01 to 0.1 M NaCl. The titration profiles clearly established the transition points in terms of the percentage of volume of alcohol added relative to that of solvent corresponding to the first occurrence of turbidity (Vt) and a point of turbidity maximum (Vp). Addition of more alcohol drove the system toward precipitation. The values of Vt and Vp characterized the initiation of intramolecular folding and intermolecular aggregate formation of the charge neutralized gelatin molecules and the subsequent micro coacervate droplet formation. The state of intermolecular aggregates and that of folded gelatin molecules could be characterized by dynamic laser light scattering experiments, which implied spontaneous segregation of particle sizes preceding coacervation. The aggregates constitute the coacervate phase while the folded gelatin molecules mostly stay dispersed in the supernatant. The data taken together reveal the role played by solution entropy in addition to that of electrostatic and solute-solvent interactions, which had been overlooked hitherto.     

Physicochemical and structural characterization of a polyionic matrix of interest in biotechnology, in the pharmaceutical and biomedical fields

This paper reports on the swelling degree and the rheological and structural characteristics of a hydrogel composed by chitosan and xanthan. The latter is a polyionic hydrogel obtained by complexation between the both polysaccharides. The swelling degree has been found to be influenced by the time of coacervation, the pH of the solution of chitosan used to form the hydrogel and the pH of the swelling solution. The molecular weight and the degree of acetylation of the chitosan also influence the swelling degree of this matrix. The connectivity between chitosan and xanthan affects the swelling degree of this matrix. A rheological study has been carried out in order to understand the formation of the coacervate and of the subsequent hydrogel. The evolution of the storage modulus with time during the coacervation has allowed to optimize the time of coacervation required for a subsequently hydrogel, with desirable swelling degree. The kinetics has shown that (a) the coacervate is formed in two distinct steps and (b) the storage modulus of the hydrogel reaches a stable plateau. The values of the storage modulus have been correlated with the swelling degree. The microscopic characterization has shown the presence of a porous network with a fibrillar structure. To complete the characterization studies fine powder of this hydrogel has been used to determine the surface, perimeter, Feret diameter and sphericity factor distribution of dry and hydrated (swollen) particles.     

Thermodynamic and hydrodynamic properties of human tropoelastin. Analytical ultracentrifuge and pulsed field-gradient spin-echo NMR studies

Tropoelastin is the soluble precursor of elastin that bestows tissue elasticity in vertebrates. Tropoelastin is soluble at 20 degrees C in phosphate-buffered saline, pH 7.4, but at 37 degrees C equilibrium is established between soluble protein and insoluble coacervate. Sedimentation equilibrium studies performed before (20 degrees C) and after (37 degrees C) coacervation showed that the soluble component was strictly monomeric. Sedimentation velocity experiments revealed that at both temperatures soluble tropoelastin exists as two independently sedimenting monomeric species present in approximately equal concentrations. Species 1 had a frictional ratio at both temperatures of approximately 2.2, suggesting a very highly expanded or asymmetric protein. Species 2 displayed a frictional ratio at 20 degrees C of 1.4 that increased to 1.7 at 37 degrees C, indicating a compact and symmetrical conformation that expanded or became asymmetric at the higher temperature. The slow interconversion of the two monomeric species contrasts with the rapid and reversible process of coacervation suggesting both efficiently incorporate into the coacervate. A hydrated protein of equivalent molecular weight modeled as a sphere and a flexible chain was predicted to have a frictional ratio of 1.2 and 1.6, respectively. Tropoelastin appeared as a single species when studied by pulsed field-gradient spin-echo NMR, but computer modeling showed that the method was insensitive to the presence of two species of equal concentration having similar diffusion coefficients. Scintillation proximity assays using radiolabeled tropoelastin and sedimentation analysis showed that the coacervation at 37 degrees C was a highly cooperative monomer-n-mer self-association. A critical concentration of 3.4 g/liter was obtained when the coacervate was modeled as a helical polymer formed from the monomers via oligomeric intermediates.     

聚皂凝胶对蛋白分子的吸附与释放

以牛血清白蛋白为对象,研究了丙烯酸－丙烯酰胺型９ＰＢｕＡＭＡ）和疏水改性的四乙烯五胺－环（ＨＰＴＥ）聚合物凝胶对蛋白质的及附与释放过程。在吸附过程中,当溶液ＰＨ值在牛血清白蛋白电点（４．７）附近时,凝胶蛋白抽的吸附值最大;而在释放过程中,ＰＨ＝７的释放率远大于其它ＰＨ值时的释放率。实验表明凝胶结构对蛋白质吸附与释放有较大影响。未疏水改性的凝胶与疏水改性的凝胶相比,前 的释放速率约为后者的２倍。结果     

Suitability of high-molecular-weight tissue-derived elastin polypeptides and their particles as cosmetic biomaterials

We aimed to determine the coacervation properties of high-molecular-weight (HMW) tissue-derived elastin (TDE) and to examine the potential use of TDE particles as a cosmetic biomaterial. TDE solutions were filtered and divided into three fractions (1–3) according to the molecular weight of the elastin. The turbidity of fraction 2, which contained a large portion (58%) of HMW elastin polypeptides (>100 kDa), was measured under several pH values (3.0–11.0) and NaCl concentrations (0–1000 mM) to examine its coacervation ability. HMW TDE exhibited coacervation under the physiological conditions (temperature, pH, and NaCl concentration) of the skin surface. We performed inclusion and release experiments using three model chemicals with different molecular weights and measured the size and zeta potential of the fraction 3 particles to investigate the suitability of HMW elastin polypeptides. Fraction 3, which contained a larger portion (64%) of HMW elastin polypeptides, displayed a strong coacervation property at a phase transition temperature of 19.8 ± 0.1°C. The inclusion ratio of the model chemical Biebrich Scarlet (BS) with a molecular weight of <600 was approximately 92.1 ± 0.7%. The release profiles of BS from the particles linearly increased and reached a plateau after 15 days. Moreover, the average size of the particles with BS was 474.2 ± 24.6 nm. The low-molecular-weight (LMW) elastin peptides have moisturizing and whitening functions for the skin. We concluded that TDE, as a mixture of HMW polypeptides and LMW peptides, can potentially serve as a multifunctional and effective cosmetic biomaterial.     

Analysis of the formation mechanism for thermoresponsive-type coacervate with functional copolymers consisting of N-isopropylacrylamide and 2-hydroxyisopropylacrylamide

We now report the formation mechanism of the thermoresponsive-type coacervate with the novel functional temperature-sensitive polymer, poly(N-isopropylacrylamide-co-2-hydroxyisopropylacrylamide) (poly(NIPAAm-co-HIPAAm)), synthesized in our laboratory. The effects of introducing the hydrophilic comonomer (HIPAAm) into the copolymer chains and adding salts on the behaviors of the coacervate droplets induced in the poly(NIPAAm-co-HIPAAm) aqueous solutions were investigated. Not only the particle sizes of the coacervate droplets but also the cloud points of the copolymer solutions could be modulated by the HIPAAm content incorporated in the copolymers. Moreover, the particle sizes of the coacervate droplets were also changed by adding salts. Namely, the particle sizes increased with the decreasing HIPAAm composition and increasing NaCl concentration. In addition, the 1H NMR and differential scanning calorimetric measurements suggested that as the HIPAAm content decreased or NaCl concentration increased, dehydration of the copolymers induced in the phase transition and/or separation became much easier. Therefore, on the basis of the findings obtained from these measurements, we determined that the particle sizes of the coacervate droplets induced in the temperature-sensitive polymers increased as the number of the water molecules, which are dissociated from the polymeric chains during the phase transition and/or separation, increased. Besides, to examine the separation of the model solutes, the aqueous two-phase separation with the coacervate droplets of poly(NIPAAm-co-HIPAAm) was carried out. The partitions of Methyl Orange as a model solute under both acidic (pH 2) and basic (pH 12) conditions were performed. The amount of Methyl Orange partitioned into the coacervate droplets at pH 12 is much greater than that at pH 2, which indicated that the coacervate droplets could recognize a slight difference in the polarity or structure between the model solutes.     

Protein purification by polyelectrolyte coacervation: influence of protein charge anisotropy on selectivity

The effect of polyelectrolyte binding affinity on selective coacervation of proteins with the cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDADMAC), was investigated for bovine serum albumin/β-lactoglobulin (BSA/BLG) and for the isoforms BLG-A/BLG-B. High-sensitivity turbidimetric titrations were used to define conditions of complex formation and coacervation (pH(c) and pH(?), respectively) as a function of ionic strength. The resultant phase boundaries, essential for the choice of conditions for selective coacervation for the chosen protein pairs, are nonmonotonic with respect to ionic strength, for both pH(c) and pH(?). These results are explained in the context of short-range attraction/long-range repulsion governing initial protein binding "on the wrong side of pI" and also subsequent phase separation due to charge neutralization. The stronger binding of BLG despite its higher isoelectric point, inferred from lower pH(c), is shown to result from the negative "charge patch" on BLG, absent for BSA, as visualized via computer modeling (DelPhi). The higher affinity of BLG versus BSA was also confirmed by isothermal titration calorimetry (ITC). The relative values of pH(?) for the two proteins show complex salt dependence so that the choice of ionic strength determines the order of coacervation, whereas the choice of pH controls the yield of the target protein. Coacervation at I = 100 mM, pH 7, of BLG from a 1:1 (w/w) mixture with BSA was shown by SEC to provide 90% purity of BLG with a 20-fold increase in concentration. Ultrafiltration was shown to remove effectively the polymer from the target protein. The relationship between protein charge anisotropy and binding affinity and between binding affinity and selective coacervation, inferred from the results for BLG/BSA, was tested using the isoforms of BLG. Substitution of glycine in BLG-B by aspartate in BLG-A lowers pH(c) by 0.2, as anticipated on the basis of DelPhi modeling. The stronger binding of BLG-A, confirmed by ITC, led to a difference in pH(?) that was sufficient to provide enrichment by a factor of 2 for BLG-A in the coacervate formed from "native BLG".     

Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymer

Genetic engineering has been used to construct hydrophobically modified fusion proteins of cutinase from Fusarium solani pisi and tryptophan-containing peptides. The aim was to enhance the partitioning of the tagged protein in a novel aqueous two-phase system formed by only one water-soluble polymer. The system was based on a hydrophobically modified random copolymer of ethylene oxide (EO) and propylene oxide (PO) units, HM-EOPO, with myristyl groups (C(14)H(29)) at both ends. The HM-EOPO polymer is strongly self-associating and has a lower critical solution temperature (cloud point) at 12 degrees C in water. At temperatures above the cloud point a two-phase system is formed with a water top phase and a polymer-enriched bottom phase. By adding a few percent of hydroxypropyl starch polymer, Reppal PES 200, to the system, it is possible to change the densities of the phases so the HM-EOPO-enriched phase becomes the top phase and Reppal-enriched phase is the bottom phase. Tryptophan-based peptides strongly preferred the HM-EOPO rich phase. The partitioning was increased with increasing length of the peptides. Full effect of the tag as calculated from peptide partitioning data was not found in the protein partitioning. When a short spacer was introduced between the protein and the tag the partitioning was increased, indicating a better exposure to the hydrophobic core of the polymer micelle. By adding a hydrophilic spacer between the protein and trp-tag, it was possible to increase the partitioning of cutinase 10 times compared to wild-type cutinase partitioning. By lowering the pH of the system and addition of NaCl, the partitioning of tagged protein was further increased towards the HM-EOPO phase. After isolating the HM-EOPO phase, the temperature was increased and the protein was back-extracted from the HM-EOPO phase to a fresh water phase.     

Hydrophobic modification of sodium alginate and its application in drug controlled release

Sodium alginate was hydrophobically modified by coupling of polybutyl methacrylate onto the alginate. The polybutyl methacrylate was previously prepared through polymerization of butyl methacrylate in the presence of 2-amino-ethanethiol as a chain transfer agent. The structure of the product was characterized by Fourier-transformed infrared spectrometry, nuclear magnetic resonance (¹HNMR) and thermogravimetry. The result of fluorescence analysis showed that the hydrophobicity of the modified alginate was obviously increased. The modified alginate conjugate was used for immobilization of bovine serum albumin in the presence of calcium chloride. In addition, the release behavior of the drug-loaded alginate in deionized water and Tris–HCl buffer solution (pH 7.2) was investigated. It was found that the modified sodium alginate possessed prolonged release behavior compared to unmodified sodium alginate, and it had potential application in controlled release as a drug carrier.     

Effect of dense gas CO2 on the coacervation of elastin

In this study for the first time the effect of high-pressure CO2 on the coacervation of alpha-elastin was investigated using analytical techniques including light spectroscopy and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging and circular dichroism (CD) spectroscopy. The coacervation behavior of alpha-elastin, a protein biopolymer, was determined at temperatures below 40 degrees C and pressures lower than 180 bar. At these conditions elevated pressures did not disrupt the ability of alpha-elastin to coacervate. It was feasible to monitor the presence of amide I, II, and III bands for alpha-elastin at high-pressure CO2 using ATR-FTIR imaging. At a constant temperature the peak absorption was substantially enhanced by increasing the pressure of the system. CD analysis demonstrated the preservation of secondary structure attributes of alpha-elastin exposed to dense gas CO2 at the pressure range investigated in this study. The lower critical solution temperature of alpha-elastin was dramatically decreased from 37 to 16 degrees C when the CO2 pressure increased from 1 to 50 bar, without a significant change after that. Carbon dioxide at high pressures also impeded the reversible coacervation of alpha-elastin solution. These effects were predominantly associated with the lowered pH of the aqueous solution and maybe the interaction between CO2 and hydrophobic domains of alpha-elastin.     

Intermolecular complexation and phase separation in aqueous solutions of oppositely charged biopolymers

Turbidity measurements performed at 450nm were used to follow the process of complex formation, and phase separation in gelatin-agar aqueous solutions. Acid (Type-A) and alkali (Type-B) processed gelatin (polyampholyte) and agar (anionic polyelectrolyte) solutions, both having concentration of 0.1% (w/v) were mixed in various proportions, and the mixture was titrated (with 0.01 M HCl or NaOH) to initiate associative complexation that led to coacervation. The titration profiles clearly established observable transitions in terms of the solution pH corresponding to the first occurrence of turbidity (pH(C), formation of soluble complexes), and a point of turbidity maximum (pH(phi), formation of insoluble complexes). Decreasing the pH beyond pH(phi) drove the system towards precipitation. The values of pH(C) and pH(phi) characterized the initiation of the formation of intermolecular charge neutralized soluble aggregates, and the subsequent formation of microscopic coacervate droplets. These aggregates were characterized by dynamic light scattering. It was found that Type-A and -B gelatin samples formed soluble intermolecular complexes (and coacervates) with agar molecules through electrostatic and patch-binding interactions, respectively.     

Coacervate-like microspheres from lysine-rich proteinoid

Microspheres form isothermally from lysine-rich proteinoid when the ionic strength of the solution is increased with NaCl or other salts. Studies with different monovalent anions and with polymers of different amino acid composition indicate that charge neutralization and hydrophobic bonding contribute to microsphere formation. The particles also form in sea water, especially if heated or made slightly alkaline. The microspheres differ from those made from acidic proteinoid but resemble coacervate dtoplets in some ways (isothermal formation, limited stability, stabilization by quinone, uptake of dyes). Because the constituent lysine-rich proteinoid is of simulated prebiotic origin, the study is interpreted to add emphasis to and suggest an evolutionary continuity for coacervation phenomena.     

Gum arabic-chitosan complex coacervation

The formation of electrostatic complexes of gum Arabic (GA) with chitosan (Ch), two oppositely charged polysaccharides, as a function of the biopolymers ratio (RGA/Ch), total biopolymers concentration (TBconc), pH, and ionic strength, was investigated. The conditions under which inter-biopolymer complexes form were determined by using turbidimetric and electrophoretic mobility measurements in the equilibrium phase and by quantifying mass in the precipitated phase. Results indicated that optimum coacervate yield was achieved at RGA/Ch = 5, independently of TBconc at the resulting pH of solutions under mixing conditions. High coacervate yields occurred in a pH range from 3.5 to 5.0 for RGA/Ch = 5. Coacervate yield was drastically diminished at pH values below 3.5 due to a low degree of ionization of GA molecules, and at pH values above 5 due to a low solubility of chitosan. Increasing ionic strength decreased coacervate yield due to shielding of ionized groups.     

Rheological interfacial properties of plant protein-arabic gum coacervates at the oil-water interface

This study concerns the interfacial properties of the plant proteins-arabic gum coacervates, which are involved in encapsulation processes based on complex coacervation. The results make it possible to deduce the prerequisite characteristics of the protein, which are involved in the coacervate interfacial properties. The influence of pH and concentration on protein interfacial properties was also studied so as to enable us to predict the best conditions to achieve encapsulation. It has been established that, to obtain a good encapsulation yield, the coacervate must show high surface-active properties and its adsorption on the oil droplets must be favored compared to the free protein adsorption. On the other hand, mechanical properties of the interfacial film made of the coacervate, appear to be a key parameter, as reflected by the dilational viscoelasticity measurements. When compared to the properties of the proteins films, an increase of the rigidity of the interfacial film was shown with the coacervates. It was also observed that viscoelastic properties of the coacervate film were strongly reduced, as well as the associated relaxation times. In acidic conditions, the coacervates containing alpha-gliadin are characterized by an interfacial viscoelastic behavior. This behavior reflects the softness of the interfacial film. This viscoelasticity allows also the formation of a continuous layer around the oil droplets to be encapsulated. Drop tensiometry is shown to be a method that could allow the most adapted protein to be selected and the conditions of the coacervation process to be optimized with regard to concentration and pH.     

Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies

The temperature dependence of the composition of coacervate and equilibrium phases is examined for the polypentapeptide of elastin (L -Val¹-L -Pro²-Gly³-L -Val⁴-Gly⁵)_n in water. This provides for the development of a phase diagram. CD data is presented that provides information on associated polypeptide structure changes that, when added to previous CD, nmr, and dielectric relaxation data at lower water composition, allow construction of a phase-structure diagram of the polypentapeptide–water system. The molecular-weight dependence of phase change (coacervation) is included. The volume–composition studies as a function of temperature also provide temperature coefficients of expansion and of composition important in analyzing the mechanism of elasticity.     

Low molecular weight chitosan-g-l-phenylalanine: Preparation,characterization, and complex formation with DNA

The grafting of l-phenylalanine onto low molecular weight chitosan is accomplished by using carbodiimide as a coupling agent. As increase in the amount of phenylalanine in feed, the grafting chain length increases, while a number of grafting chains hardly change. The obtained product, LMWCts-g-Phe, performs sphere with an average size of ～80 nm when the % grafting is less than 123. The complexes of the LMWCts-g-Phe and DNA (LMWCts-g-Phe/DNA) prepared by a complex coacervation method possess various shapes with an average size of ～50–150 nm and a negatively charged surface. The LMWCts-g-Phe and its complex show very reduced toxicity to fibroblast cells. The release of DNA from the complex is very fast in high pH media (tris buffer, pH 8.0 and carbonate buffer, pH 9.5), and relatively slow or more sustainable in neutral and low pH ones (PBS, pH 7.4 and citric acid/trisodium citrate buffer, pH 3.0). The results suggest that the LMWCts-g-Phe be an alternative promising carrier for negatively charged active molecules.     

Design and <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation of Capsaicin Transdermal Controlled Release Cubic Phase Gels

The purpose of this study was to design and investigate the transdermal controlled release cubic phase gels containing capsaicin using glycerol monooleate (MO), propylene glycol (1,2-propanediol, PG), and water. Three types of cubic phase gels were designed based on the ternary phase diagram of the MO–PG–water system, and their internal structures were confirmed by polarizing light microscopy (PLM) and small-angle X-ray scattering (SAXS). Release results showed the cubic phase gels could provide a sustained system for capsaicin, while the initial water content in the gels was the major factor affecting the release rate. Release kinetics was determined to fit Higuchi’s square-root equation indicating that the release was under diffusion control. The calculated diffusion exponent showed the release from cubic phase gels was anomalous transport. The unique structure of the cubic phases, capsaicin distributed in the lipid bilayers, and cubic phase gel swelling contributed to the release mechanism. The cubic phase gel may be an interesting application for transdermal delivery system of capsaicin in alleviating the post-incision pain.     

β-Cyclodextrin grafted polyethyleneimine hydrogel immobilizing hydrophobically modified glucose oxidase

Hydrogels which release their contents in response to glucose concentration were prepared by immobilizing glucose oxidase (GOD) into β-cyclodextrin grafted polyethyleneimine hydrogels (PEI-βCD hydrogel). For the tight immobilization, hydrophobically modified GOD (HmGOD) was prepared by reacting GOD with palmitic acid-N-hydroxysuccinimide ester (PA-NHS) in the molar ratio of 1:40. According to trinitrobenzene sulfonic acid (TNBS) assay, five palmitic acids were covalently attached to one GOD molecule. The activity of HmGOD was about 76% of native enzyme. The swelling ratios of HmGOD loaded hydrogels increased from about 960% to 1190% in 24h, when glucose concentration was varied from 0 to 100mg/dl. The % release in 48 h of fluorescein isothiocyanate dextran increased from about 53% to 89%, when glucose concentration was varied in the same range. Gluconic acid, produced by the enzymatic reaction, would protonate and swell the PEI-βCD hydrogel, leading to a higher release.