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.     

Application of immobilized horseradish peroxidase onto modified acrylonitrile copolymer membrane in removing of phenol from water

A non-modified and modified with NaOH and ethylenediamine ultrafiltration membranes prepared from AN copolymer have been used as carriers for the immobilization of horseradish peroxidase (HRP) enzyme. The amount of bound protein onto the membranes and the activity of the immobilized enzyme have been investigated as well as the pH and thermal optimum, and the thermal stability of the free and immobilized HRP. The experiments have proved that the modified membrane is a better support for the immobilization of HRP enzyme. The latter has shown a greater thermal stability than the free enzyme.A possible application has been studied for reducing phenol concentration in water solutions through oxidation of phenol by hydrogen peroxide, in the presence of free and immobilized HRP enzyme on modified AN copolymer membranes. A higher degree of the phenol oxidation has been observed in the presence of the immobilized enzyme. A total removal of phenol has been achieved in the presence of immobilized HRP at concentration of the hydrogen peroxide 0.5 mmol L^?1 and concentration of the phenol in the model solutions within the interval 5–40 mg L^?1. A high degree of phenol oxidation (95.4%) has been achieved in phenol solution with 100 mg L^?1 concentration in the presence of hydrogen peroxide and immobilized HRP, which demonstrates the promising opportunity of using the enzyme for bioremediation of waste waters, containing phenol.The immobilized HRP has shown good operational stability. Deactivation of the immobilized enzyme to 50% of the initial activity has been observed after the 20th day of the enzyme operation.     

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 gelatin — poly (HEMA) copolymer preparation and characterization

Urease was immobilized onto gelatin-poly (HEMA) copolymer by covalent linkage. Maximum amount of urease was immobilized onto the support at a pH of 8.5. The optimal pH of the immobilized urease was similar to that of free urease; the optimal temperature showed an increase of 10 °C over the free enzyme. The stability of the immobilized urease for a range of pH, temperature and shelf life was greater than the corresponding values for the free enzyme. The same result was obtained for k _m also.Grateful acknowledgement is made to CSIR, Govt. of India for the research associateship conferred on Dr. M. Chellapandian which helped the progress of this piece of research investigation.     

Immobilization of urease on vermiculite

Urease (EC 3.5.1.5) of high activity was obtained when the enzyme was immobilized on vermiculite crosslinked with 2.5% glutaraldehyde in chilled EDTA-phosphate buffer (pH 5.5). The highest activity of the immobilized enzyme was at 65°C and pH 6.5 while the optimum temperature for free urease was found to be 25°C. The thermal stability of immobilized urease was observed to be much better than that of the free urease. When stored at 4°C, urease immobilized on vermiculite retained 69 to 81% of its activity after 60 days and 61 to 75% of its original activity was retained after 4 repeated uses.     

Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes

The urease was immobilized onto nanoporous alumina membranes prepared by the two-step anodization method, and a novel piezoelectric urea sensing system with separated porous alumina/urease electrode has been developed through measuring the conductivity change of immobilized urease/urea reaction. The process of urease immobilization was optimized and the performance of the developed urea biosensor was evaluated. The obtained urea biosensor presented high-selectivity monitoring of urea, better reproducibility (S.D. = 0.02, n = 6), shorter response time (30 s), wider linear range (0.5 μM to 3 mM), lower detection limit (0.2 μM) and good long-term storage stability (with about 76% of the enzymatic activity retained after 30 days). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples.     

Biosorption of hexavalent chromium onto raw and chemically modified Sargassum sp

Hexavalent chromium biosorption by raw algae is always accompanied with significantly high organic leaching. In this study, hydrochloric acid, sodium hydroxide, calcium chloride, formaldehyde, and glutaraldehyde were used for modification of raw Sargassum sp. seaweed (RSW), in order that the modified seaweed (MSW) has a lower organic leaching while the metal biosorption capacity is comparable to the RSW. The result shows that the chemical modification by 0.2% formaldehyde achieves such goals. The biosorption of both RSW and MSW is highly pH dependent. At the optimal pH of 2.0, the maximum biosorption capacities of MSW and RSW are 1.123 and 0.601 mmol g(-1), respectively. The surface treatment improves the reduction capacity of the biosorbents. The instrumental analysis demonstrates that the Cr(VI) biosorption is controlled by redox, ion exchange and coordination reactions, of which alcohol, carboxyl, amino and sulphonic groups play important roles. The complete uptake of hexavalent chromium is achieved in 20 h. The chemical reduction for Cr(VI) to Cr(III) is pH dependent and controls the overall chromium removal kinetics.     

Immobilization of urease using Amberlite MB-1

Amberlite MB-1 was used to immobilize urease (EC.3.5.1.5). The thermal stability of the immobilized urease was better than that of the free urease. Its highest activity was obtained at 75?°C and at pH 6.5 while the optimum temperature for the free urease was found to be 25?°C. Urease immobilized on Amberlite MB-1 retained 65% of the original activity after 5 repeated uses and 62% of the activity after 60 days when stored at 4?°C.     

Improved antifouling properties of PVDF membranes modified with oppositely charged copolymer

Biofouling resulting from the attachment of microorganisms communities to the membrane surface is the major obstacle for the widespread application of membrane technology. This work develops a feasible approach to prepare an anti-biofouling poly(vinylidene fluoride) (PVDF) membrane. A copolymer that possessed oppositely charged groups was first synthesized via radical copolymerization with methyl methacrylate, 2-methacryloxy ethyltrimethyl ammonium chloride and 2-acrylamide-2-methyl propane sulphonic acid as monomers. The copolymer was blended with the PVDF powder to prepare the antifouling membrane via the immersed phase inversion method. The antifouling properties of the modified PVDF membrane were studied by X-ray photoelectron spectroscopy, field emission scanning electron microscopy, water contact angle measurement, zeta-potential measurement, protein adsorption, microbial adhesion and filtration experiments. The modified PVDF membrane showed limited adsorption and adhesion of protein bovine serum albumin and microbes (Escherichia coli and Saccharomyces cerevisiae) with increasing copolymer concentration in the casting solution. The modified PVDF membrane exhibited excellent antibiofouling properties.     

Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes

Purified porin OmpF from Escherichia coli outer membrane was chemically modified by acetylation and succinylation of amino groups and by amidation of the carboxyl groups. Native and chemically modified porins were incorporated into lipid bilayer membranes and the permeability properties of the pores were studied. Acetylation and succinylation of the porin trimers had almost no influence on the single channel conductance in the presence of small cations and anions and the cation selectivity remained essentially unchanged as compared with the native porin. Amidation had also only little influence on the single channel conductance and changed the pore conductance at maximum by less than 50%, whereas the cation selectivity of the porin is completely lost after amidation. The results suggest that the structure of the porin pore remains essentially unchanged after chemical modification of the pores and that their cation selectivity is caused by an excess of negatively charged groups inside the pore and/or on the surface of the protein. Furthermore, it seems very unlikely that the pore contains any positively charged group at neutral pH.     

Injectable and sustained delivery of human growth hormone using chemically modified Pluronic copolymer hydrogels

A new injectable biodegradable hydrogel system with thermosensitive sol-gel transition behavior was developed. A series of A-B-A triblock copolymers consisting of Pluronic copolymer end-capped with D- or L-lactic acid oligomers (PL-LA(n)) with various chain lengths (n = 5,12) was synthesized. It was assumed that a pair of two triblock copolymers with enantiomeric oligolactide chains, when blended in an equimolar mixture, would form more stable, self-assembled, and stereocomplexed (ST) hydrogels. A series of blend hydrogels encapsulating human growth hormone (hGH) was prepared by varying blend ratios between PL and stereocomplexed PL copolymers. They showed sustained release of hGH via an erosion-dependent mechanism. The hydrogel with a 5% blending ratio exhibited the most delayed mass erosion as well as sustained protein release patterns in vitro possibly due to the formation of a fish-net like 3-D mesh structure. The effect of incubation condition on hGH release and degradation behaviors was also assessed.     

Immobilization of yeast cells onto hydroxyalkyl methacrylate gels modified by spacers of different lengths

Summary The potential of hydroxyalkyl methacrylate gel as a support for yeast cell immobilization by covalent attachment was investigated with respect to the length of spacer used and the manner of its activation. Saccharomyces paradoxus cells were immobilized successfully onto glutaraldehyde activated glycyl-, -alanyl-and -aminocaproyl-derivatives of the above mentioned gel modified with hexamethylenediamine. Results suggest that covalent attachment via spacer is strongly influenced by cell surface characteristics.     

Immobilization of horseradish peroxidase onto kaolin

Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer–Emmett–Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of H₂O₂) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi–Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes.     

Immobilization of antibodies onto glass wool

The immobilization of antibodies onto solid phases in an efficient and activity-retaining form is an important goal for both research and industry. Methods have been developed for the site-directed attachment of antibodies to agarose by oxidation of the carbohydrate moieties in their Fc region. Similar attachment to silianized supports have not been as successful. Here we describe a novel combination protocol for the site-directed attachment of periodate oxidized, goat polyclonal antibodies to glass wool fibers activated with 3-aminopropyltriethoxysilane. The study demonstrates that this procedure results in effective immobilization of polyclonal antibodies that retain their antigen-binding capacity. This protocol should prove useful in the development of more efficient and effective glass-based immunosupports.     

Immobilization of a soluble chemically thermostabilized enzyme

Cellobiase was coupled to a dialdehyde dextran by reductive alkylation in the presence of sodium cyanoborohydride. The resulting conjugate, obtained without loss of enzymic activity, presents properties of thermoresistance largely superior to those of native enzyme: the rate of inactivation is reduced compared to that of native enzyme and its optimal temperature of activity is 70-75 degrees C instead of 65 degrees C. Finally the conjugate presents increased longevity when subjected to experiments of operational stability; its hydrolytic activity is maintained at 60 degrees C in a 10% (w/v) cellobiose solution for more than 100 h whereas the native enzyme is inactivated after 45 h. The cellobiase-dextran conjugate was immobilized by covalent coupling on aminated silica by reductive alkylation in the presence of NaBH(3)CN. The characteristics of thermoresistance of this stabilized and immobilized conjugate were studied and compared to those of a preparation of native cellobiase immobilized on a silica support activated with glutaraldehyde. Analysis of the thermoresistance of these two cellobiase preparations clearly shows that immobilization has maintained and even enhanced their properties. In particular, the operational stability, measured at 68 degrees C on 10% (w/v) cellobiose shows an increased longevity of the stabilized and immobilized enzyme for 120 h compared to 60 h for the native immobilized enzyme. Two successive incubations of these cellobiase derivatives show that it is possible to obtain 2.5 times more glucose with the stabilized-immobilized enzyme than with the immobilized preparation. The procedure described above enables us to prepare a thermostabilized immobilized cellobiase.     

Immobilization of urease within a thin channel ultrafiltration cell

Urease [urea amidohydrolase, EC 3.5.1.5] has been immobilized within a thin channel ultrafiltration cell. Loss of enzymic activity as a result of concentration polarization and other causes was minimized. The flow characteristics of the reactor were fully characterized by analysis of the distribution of residence times (using F diagrams) and kinetic data were also obtained for the immobilized enzyme. These data show that under certain conditions the thin channel ultrafiltration reactor can be considered to be an ideally mixed vessel. After almost 8 days of continuous operation it was found that 15% of the original enzyme activity remained.