Simultaneous covalent immobilization of glucose oxidase and catalase onto chemically modified acrylonitrile copolymer membranes

Ultrafiltration membranes from acrylonitrile copolymer were chemically modified with different concentrations of hydrogen peroxide (from 5 to 30% H₂O₂). The amount of the amide groups in the modified membranes was determined. The water flow and permeability coefficients of the initial and modified membranes were also researched. The modified membranes were used as carriers for covalent immobilization of the dual enzyme system of glucose oxidase and catalase (GOD+CAT). It was found that the best matrices for immobilization of the dual system were membranes modified with 20% H₂O₂ and the optimal activity ratio was GOD : CAT = 1 : 5. The glucose conversion efficiency with the dual enzyme system was twice as high as that of bound GOD alone. Some of the basic characteristics (optimum pH, optimum temperature, pH, temperature stability, and storage stability) of the dual enzyme system were determined and compared with characteristics of free and bound enzymes. The catalytic parameters of the enzyme reaction (K_m and V_max) were determined with GOD immobilized alone and with the dual system GOD+CAT. The higher rate observed with the dual enzyme system clearly showed the advantage and the efficiency of the immobilized system. Glucose oxidase without catalase was deactivated by H₂O₂ more rapidly than the immobilized dual GOD+CAT system. These experimental evidences can be explained by the protecting effect of catalase on glucose oxidase from inhibition by H₂O₂. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91: 4057–4063, 2004     

Comparative study of poly(vinyl alcohol)‐based support materials for the immobilization of glucose oxidase

BACKGROUND: The performances of four types of glucose oxidase (GOD) immobilization materials based on poly(vinyl alcohol) (PVA) were compared. The matrices of interest were chemically‐linked PVA, freeze‐thawed PVA cryogel, tetramethoxysilane (TMOS) sol‐gel‐PVA hybrid material, and alumina sol‐gel‐PVA hybrid material. RESULTS: Overall, the membranes showed good sensitivity except for the chemically cross‐linked PVA. However, the main differences with the enzyme immobilization methods were enzyme leakage and values of K_m^app. CONCLUSION: Freeze‐thawed PVA‐GOD membranes and TMOS‐PVA, which showed satisfactory sensitivity and adequate value of K_m^app, were quite promising as support materials for immobilizing GOD. Copyright © 2007 Society of Chemical Industry     

Covalent immobilization of β‐galactosidase onto electrospun nanofibers of poly (AN‐co‐MMA) copolymer

Immobilization of β‐galactosidase in poly (acrylonitrile‐co‐methyl methacrylate) poly (AN‐co‐MMA) Nanofibers was studied by electrospinning, and a spacer‐arm i.e., (Polyethyleneimine (PEI)) was covalently attached by the reaction of carbonyl groups of Poly (AN‐co‐MMA) nanofibers. β‐galactosidase was then covalently immobilized through the spacer‐arm of the Poly (AN‐co‐MMA) nanofibers by using glutaraldehyde (GA) as a coupling agent. Nanofibers mode of interaction was proven by FTIR and thermal gravimetric analysis and supported by morphological changes recognized through SEM examination. Factors affecting the modification process such as PEI concentration, reaction time, and reaction temperature have been studied. Its influence on the amount of coupled PEI was consequently correlated to the changes of the catalytic activity and the retained activity of immobilized enzyme, the main parameters judging the success of the immobilization process. Evidences of Poly (AN‐co‐MMA) nanofibers modification were extracted from morphological changes recognized through SEM examination. The maximum activity (V_max) and michaelis constant (K_m) of immobilized enzyme were found to be 8.8 μmole/min mg protein and 236.7 mM, respectively. Stabilities of the immobilized β‐galactosidase were obviously improved. The optimum temperature for β‐galactosidase immobilized on the spacer‐arm attached nanofiber was 5°C higher than that of the free enzyme and was also significantly broader. The immobilized β‐galactosidase had better resistance to temperature inactivation than did the free form. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Covalent immobilization of penicillin G acylase onto amine-functionalized PVC membranes for 6-APA production from penicillin hydrolysis process. II. Enzyme immobilization and characterization

This article describes the covalent immobilization of penicillin G acylase (PGA) onto glutaraldehyde-activated NH₂-PVC membranes. The immobilized enzyme was used for 6-aminopenicillanic acid production from penicillin hydrolysis. Parameters affecting the immobilization process, which affecting the catalytic activity of the immobilized enzyme, such as enzyme concentration, immobilization's time and temperature were investigated. Enzyme concentration and immobilization's time were found of determine effect. Higher activity was obtained through performing enzyme immobilization at room temperature. Both optimum temperature (35°C) and pH (8.0) of immobilized enzyme have not been altered upon immobilization. However, immobilized enzyme acquires stability against changes in the substrate's pH and temperature values especially in the higher temperature region and lower pH region. The residual relative activities after incubation at 60°C were more than 75% compared to 45% for free enzyme and above 50% compared to 20% for free enzyme after incubation at pH 4.5. The apparent kinetic parameters K_M and V_M were determined. K_M of the immobilized PGA (125.8 mM) was higher than that of the free enzyme (5.4 mM), indicating a lower substrate affinity of the immobilized PGA. Operational stability for immobilized PGA was monitored over 21 repeated cycles. The catalytic membranes were retained up to 40% of its initial activity after 10.5 working h. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Synthesis and characterization of binary copolymers of methyl methacrylate with glycidyl methacrylate and 2‐hydroxy ethyl methacrylate as carriers for cellulase

Cellulase was immobilized directly on methyl methacrylate‐glycidyl methacrylate copolymer (MMA‐co‐GMA) and methyl methacrylate‐2‐hydroxy ethyl methacrylate copolymer (MMA‐co‐HEMA) by covalent attachment and crosslinking methods. The properties of the immobilized cellulase were investigated and compared with those of the free one. For the assays carried out through crosslinking method at 25°C and pH 7, the retained activities were found to be 91.92% and 74.63%, respectively, for MMA‐co‐GMA and MMA‐co‐HEMA crosslinked with 0.1% of 1‐cyclohexyl‐3‐(2‐morpholino‐ethyl) carbodiimide metho‐p‐toluenesulfonate (CMCT), respectively. The immobilized cellulase had better stability and higher retained activities with respect to pH, temperature, and storage stability than the free one. In the repeated use experiments, the immobilized cellulase using (MMA‐co‐GMA)‐CMCT (0.1%) and (MMA‐co‐HEMA)‐CMCT (0.1%) did not change after 10 and eight times of repeated use and maintained 67% and 62% from their original activities after 25 times, respectively. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Preparation of glucose oxidase immobilized in different carriers using radiation polymerization

Glucose oxidase (EC 1.1.3.4) was immobilized on different polymeric materials using different immobilization techniques (entrapping by γ‐irradiation, and covalent binding using epichlorohydrin). Studies were carried out to increase the thermal stability of glucose oxidase (GOD) for different applications. The activity and stability of the resulting biopolymers have been compared with those of free GOD. The effect of different polyvinyl alcohol/polyacrylamide (PVA/PAAm) compositions of the copolymer carrier on the enzymatic activity of the immobilized GOD was studied. The maximum enzymatic activity was obtained with the composition ratio of PVA/PAAm of 60:40. The behaviour of the free and immobilized enzyme was analysed as a function of pH. A broadening in the pH profile (5.5–8) was observed for immobilized preparations. The activity and stability of the resulting biopolymers produced by immobilization of GOD onto different carriers have been compared, in both aqueous and organic media, with those of the free GOD. The enzyme's tolerance toward both heat and organic solvent was enhanced by immobilization onto polymers. The addition of different concentrations of organic solvents (10–50%, v/v) to the enzyme at higher temperature (60 °C) was found to stabilize the enzyme molecule. The strongest stabilizing effect on the enzymatic activity was achieved at a concentration of 10%. Copyright © 2005 Society of Chemical Industry     

Immobilization of glucose oxidase on nonwoven fabrics with bombyx mori silk fibroin gel

The enzyme glucose oxidase (GOD) was immobilized on the nonwoven fabrics, which have excellent properties in diffusivity of substrates, mechanical strength, and handling, with Bombyx mori silk fibroin gel. The nonwoven fabrics of silk fibroin, viscose rayon, poly-ethyleneterephthalate, 6-nylon, and polypropylene with activated surface by fluoline treatment were used. The stabilities of GOD to heat or pH changes were much improved by the immobilization as well as the case of the GOD immobilized in the silk fibroin membrane. Among nonwoven fabrics, silk fibroin was the most excellent support material for the immobilization of GOD although all nonwoven fabrics used here are able to be used as the support materials. The increase of the sensitivity was observed when the glucose sensor was made with the GOD immobilized on nonwoven silk fabrics as four times compared with the case of the GOD immobilized in the silk fibroin membrane.     

Construction of a choline biosensor through enzyme immobilization on a poly(2‐hydroxyethyl methacrylate)‐grafted Teflon film

An amperometric choline biosensor was constructed by immobilizing choline oxidase (ChO) on poly(2‐hydroxyethyl methacrylate) (PHEMA)‐grafted Teflon (polytetrafluoroethylene, PTFE) film. Grafting was achieved by γ irradiation. PHEMA‐grafted Teflon films were activated with epichlorohydrin or glutaraldehyde to achieve covalent immobilization of enzyme onto the film. To decrease the diffusional barrier caused by the enzyme‐immobilized film, the film was stretched directly on the electrode. The PHEMA‐grafted Teflon film, therefore, had to have appropriate mechanical properties. Glucose oxidase (GOD) was used in the determination of optimum immobilization conditions, then these were applied to ChO. With GOD, the effect of activation type and film position in electrode on enzyme activity was studied and the highest catalytic activity was obtained when the enzyme was immobilized using glutaraldehyde and the film was stretched over the electrode surface. Further studies revealed that the films activated with glutaraldehyde, immobilized in 2 mg/mL ChO concentration, and stretched directly on the electrode were suitable (specific activity, 0.427 ± 0.068 U mg^?1) for use in the choline biosensor. The linear working range of this biosensor was found to be 52–348 μM, with a 40 ± 5 μM minimum detection limit. The response of the sensor, however, decreased linearly upon repeated use. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

Covalent immobilization of trypsin onto thermo‐sensitive poly(N‐isopropylacrylamide‐co‐acrylic acid) microspheres with high activity and stability

Poly(N‐isopropylacrylamide‐co‐acrylic acid) (P(NIPAM‐co‐AA)) microspheres with a high copolymerized AA content were fabricated using rapid membrane emulsification technique. The uniform size, good hydrophilicity, and thermo sensitivity of the microspheres were favorable for trypsin immobilization. Trypsin molecules were immobilized onto the microspheres surfaces by covalent attachment. The effects of various parameters such as immobilization pH value, enzyme concentration, concentration of buffer solution, and immobilization time on protein loading amount and enzyme activity were systematically investigated. Under the optimum conditions, the protein loading was 493 ± 20 mg g^?1 and the activity yield of immobilized trypsin was 155% ± 3%. The maximum activity (V_max) and Michaelis constant (K_m) of immobilized enzyme were found to be 0.74 μM s^?1 and 0.54 mM, respectively. The immobilized trypsin showed better thermal and storage stability than the free trypsin. The enzyme‐immobilized microspheres with high protein loading amount still can show a thermo reversible phase transition behavior. The research could provide a strategy to immobilize enzyme for application in proteomics. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43343.     

Pore morphology control and hydrophilicity of polyacrylonitrile ultrafiltration membranes

The effects of different solvents (dimethyl formamide: DMF and dimethylsulfoxide: DMSO) on the solubility of polyacrylonitrile (PAN) were investigated by the phase diagrams of H₂O/DMF/PAN and H₂O/DMSO/PAN ternary systems through cloud‐point titration method at low polymer concentration. The influences of polymer concentrations and temperatures on the morphologies of PAN ultrafiltration membranes were elucidated. The morphologies of fabricated UF membranes were characterized by scanning electron microscopy (SEM) and atomic force microscope (AFM), and the basic performance of ultrafiltration including pure water flux and rejection of BSA were explored. At 25°C, the pure water flux of ultrafiltration membranes at the lower PAN content (16 wt % PAN in 84 wt % DMSO) reached 213.8 L/m/bar and the rejection of BSA was 100%. Interestingly, the water flux of UF membranes dramatically decreased to 20.6 L/m/bar (20 wt %) and 2.9 L/m/bar (24 wt %) when increasing PAN concentrations in DMSO. On the other hand, the hydrophilicity of membranes can be enhanced by increasing coagulation temperatures and polymer concentrations which were characterized by static contact angle, fitting well with the variation tendency of roughness. Although there are many works concerning on the effects of phase inversion conditions on the performance of PAN UF membranes, to our best knowledge, there is seldom works focusing on investigating the membrane hydrophilicity trend by adjusting phase inversion conditions. To disclose the reason of the enhanced hydrophilicity, the water and glycol contact angles of various membranes were measured and the surface tensions were presented. The results illustrated that the enhanced hydrophilicity of PAN UF membranes fabricated at higher temperatures or higher polymer concentrations was due to the higher polarity on membrane surface. Since the vast majority of ultrafiltration membranes in labs and in industrial scale have been fabricated by immersion phase inversion method, this work can provide a guidance to obtain hydrophilic PAN UF membranes by adjusting the process of phase inversion. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41991.     

Chitosan‐grafted poly(hydroxyethyl methacrylate‐co‐glycidyl methacrylate) membranes for reversible enzyme immobilization

Epoxy group‐containing poly(hydroxyethyl methacrylate/glycidyl methacrylate), p(HEMA/GMA), membrane was prepared by UV initiated photopolymerization. The membrane was grafted with chitosan (CH) and some of them were chelated with Fe(III) ions. The CH grafted, p(HEMA/GMA), and Fe(III) ions incorporated p(HEMA/GMA)‐CH‐Fe(III) membranes were used for glucose oxidase (GOD) immobilization via adsorption. The maximum enzyme immobilization capacity of the p(HEMA/GMA)‐CH and p(HEMA/GMA)‐CH‐Fe(III) membranes were 0.89 and 1.36 mg/mL, respectively. The optimal pH value for the immobilized GOD preparations is found to have shifted 0.5 units to more acidic pH 5.0. Optimum temperature for both immobilized preparations was 10°C higher than that of the free enzyme and was significantly broader at higher temperatures. The apparent K_m values were found to be 6.9 and 5.8 mM for the adsorbed GOD on p(HEMA/GMA)‐CH and p(HEMA/GMA)‐CH‐Fe(III) membranes, respectively. In addition, all the membranes surfaces were characterized by contact angle measurements. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 3084–3093, 2007     

Immobilization of glucose oxidase and acetylcholinesterase onto modified polyamide sorbent

Two types of polyamide (PA) sorbents with high specific area were prepared. The effects of solvent type, concentrations of formic acid, and polymer on the porosity characteristics were studied. The sorbent with the highest specific area was obtained by using C₂H₅OH—HCOOH solvent (60% HCOOH) and the rest of the experiments were carried out with this type of sorbent. The possibility of applying the PA sorbent as carrier for immobilization of glucose oxidase (GOD) and acetylcholinesterase (AChE) was investigated. In order to increase the active groups content (necessary for enzyme immobilization), the sorbent was modified with dimethylaminoethylmethacrylate (DMAEM) and 2-acrylamido-2-methylpropensulfonic acid. The amount of the active groups introduced during the modification and the degree of hydrophilicity were determined. The quantity of bound protein and relative activity of GOD and AChE immobilized onto unmodified and modified sorbents were studied. Optimum pH and temperature of the immobilized GOD and AChE were also determined. The influence of three phosphoroorganic compounds on the activity of the immobilized AChE was investigated. Tetrachlorvinvos was found to be the strongest inhibitor, while AChE immobilized onto PA sorbent modified with DMAEM showed the highest stability. The possibility of using immobilized GOD and AChE in a flow-injection system for determination of the concentrations of glucose and phosphoroorganic compounds was studied. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 68:323–329, 1998     

Synthesis and characterization of PMMA composites activated with starch for immobilization of L‐asparaginase

Poly (methyl methacrylate) (PMMA)–starch composites were prepared by emulsion polymerization technique for L‐asparaginase (L‐ASNase) immobilization as highly activated support. The hydroxide groups on the prepared composites offer a very simple, mild and firm combination for enzyme immobilization. The pure PMMA and PMMA‐starch composites were characterized as structural, thermal and morphological. PMMA‐starch composites were found to have better thermal stability and more hydrophilic character than pure PMMA. L‐ASNase was immobilized onto PMMA‐starch composites contained the different ratio of starch (1, 3, 5, and 10 wt %). Immobilized L‐ASNase showed better performance as compared to the native enzyme in terms of thermal stability and pH. K_m value of immobilized enzyme decreased approximately eightfold compared with the native enzyme. In addition to, immobilized L‐ASNase was found to retain 60% of activity after 1‐month storage period at 4 °C. Therefore, PMMA‐starch composites can be provided more advantageous in terms of enzymatic affinity, thermal, pH and storage stability as L‐ASNase immobilization matrix. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43421.     

Immobilization of invertase onto dimer acid‐co‐alkyl polyamine

Invertase was immobilized onto the dimer acid‐co‐alkyl polyamine after activation with 1,2‐diamine ethane and 1,3‐diamine propane. The effects of pH, temperature, substrate concentration, and storage stability on free and immobilized invertase were investigated. Kinetic parameters were calculated as 18.2 mM for K_m and 6.43 × 10^?5 mol dm^?3 min^?1 for V_max of free enzyme and in the range of 23.8–35.3 mM for K_m and 7.97–11.71 × 10^?5 mol dm^?3 min^?1 for V_max of immobilized enzyme. After storage at 4°C for 1 month, the enzyme activities were 21.0 and 60.0–70.0% of the initial activity for free and immobilized enzyme, respectively. The optimum pH values for free and immobilized enzymes were determined as 4.5. The optimum temperatures for free and immobilized enzymes were 45 and 50°C, respectively. After using immobilized enzyme in 3 days for 43 times, it showed 76–80% of its original activity. As a result of immobilization, thermal and storage stabilities were increased. The aim of this study was to increase the storage stability and reuse number of the immobilized enzyme and also to compare this immobilization method with others with respect to storage stability and reuse number. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1526–1530, 2004     

Synthesis and characterization of poly(HEMA‐co‐MMA)‐g‐POSS nanocomposites by combination of reversible addition fragmentation chain transfer polymerization and click chemistry

New hybrid poly(hydroxyethyl methacrylate‐co‐methyl methacrylate)‐g‐polyhedral oligosilsesquioxane [poly(HEMA‐co‐MMA)‐g‐POSS] nanocomposites were synthesized by the combination of reversible addition fragmentation chain transfer (RAFT) polymerization and click chemistry using a grafting to protocol. Initially, the random copolymer poly(HEMA‐co‐MMA) was prepared by RAFT polymerization of HEMA and MMA. Alkynyl side groups were introduced onto the polymeric backbones by esterification reaction between 4‐pentynoic acid and the hydroxyl groups on poly(HEMA‐co‐MMA). Azide‐substituted POSS (POSS? N₃) was prepared by the reaction of chloropropyl‐heptaisobutyl‐substituted POSS with NaN₃. The click reaction of poly(HEMA‐co‐MMA)‐alkyne and POSS? N₃ using CuBr/PMDEATA as a catalyst afforded poly(HEMA‐co‐MMA)‐g‐POSS. The structure of the organic/inorganic hybrid material was investigated by Fourier transformed infrared, ¹H‐NMR, and ²⁹Si‐NMR. The elemental mapping analysis of the hybrid using X‐ray photoelectron spectroscopy and EDX also suggest the formation of poly(HEMA‐co‐MMA)‐anchored POSS nanocomposites. The XRD spectrum of the nanocomposites gives evidence that the incorporation of POSS moiety leads to a hybrid physical structure. The morphological feature of the hybrid nanocomposites as captured by field emission scanning electron microscopy and transmission electron microscopic analyses indicate that a thick layer of polymer brushes was immobilized on the POSS cubic nanostructures. The gel permeation chromatography analysis of poly(HEMA‐co‐MMA) and poly(HEMA‐co‐MMA)‐g‐POSS further suggests the preparation of nanocomposites by the combination of RAFT and click chemistry. The thermogravimetric analysis revealed that the thermal property of the poly(HEMA‐co‐MMA) copolymer was significantly improved by the inclusion of POSS in the copolymer matrix. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Immobilization of β-galactosidase onto magnetic poly(GMA–MMA) beads for hydrolysis of lactose in bed reactor

In the present study, novel magnetic beads were prepared from glycidylmethacrylate and methylmethacrylate via suspension polymerization in the presence of a cross-linker (i.e. ethylenedimethylmethacrylate). The magnetic poly(GMA–MMA) beads were characterized with scanning electron microscope, FT-IR and ESR spectrophotometers. The reactive character of the epoxy groups allowed the attachment of the amino groups. The aminated magnetic beads were used for the covalent immobilization of β-galactosidase via glutaric dialdehyde activation. The maximum amount of immobilized β-galactosidase on the magnetic poly(GMA–MMA) beads was 9.87 mg/g support. The values of Michaelis constants K_m for immobilized β-galactosidase was significant larger, indicating decreased affinity by the enzyme for its substrate, whereas V_max values were smaller for the immobilized β-galactosidase. However, the β-galactosidase immobilized on the magnetic poly(GMA–MMA) beads resulted in an increase in enzyme stability with time. Optimum operational temperature for immobilized enzyme was 5 °C higher than that of the free enzyme and was significantly broader. Finally, a bed reactor with β-galactosidase immobilized was used for hydrolysis of lactose. The enzyme reactor operated continuously at 35 °C for 60 h and the immobilized enzyme lost about 12% of its initial activity after this period.     

Immobilization of invertase onto crosslinked poly(p‐chloromethylstyrene) beads

Invertase was immobilized onto poly(p‐chloromethylstyrene) (PCMS) beads that were produced by a suspension polymerization with an average size of 186 μm. The beads had a nonporous but reasonably rough surface. Because of this, a reasonably large external surface area (i.e., 14.1 m²/g) could be achieved with the proposed carrier. A two‐step functionalization protocol was followed for the covalent attachment of invertase onto the bead surface. For this purpose, a polymeric ligand that carried amine groups, polyethylenimine (PEI), was covalently attached onto the bead surface by a direct chemical reaction. Next, the free amine groups of PEI were activated by glutaraldehyde. Invertase was covalently attached onto the bead surface via the direct chemical reaction between aldehyde and amine groups. The appropriate enzyme binding conditions and the batch‐reactor performance of the immobilized enzyme system were investigated. Under optimum immobilization conditions, 19 mg of invertase was immobilized onto each gram of beads with 80% retained activity after immobilization. The effects of pH and temperature on the immobilized invertase activity were determined and compared with the free enzyme. The kinetic parameters K_M and V_M were determined with the Michealis–Menten model. K_M of immobilized invertase was 1.75 folds higher than that of the free invertase. The immobilization caused a significant improvement in the thermal stability of invertase, especially in the range of 55–65°C. No significant internal diffusion limitation was detected in the immobilized enzyme system, probably due to the surface morphology of the selected carrier. This result was confirmed by the determination of the activation energies of both free and immobilized invertases. The activity half‐life of the immobilized invertase was approximately 5 times longer than that of the free enzyme. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 1268–1279, 2002     

Immobilization of enzymes onto modified polyacrylonitrile membranes: Application of the acyl azide method

Chemical reactions toward acyl azide activated polyacrylonitrile (PAN) and conditions for membrane surface modifications are described. Ultrafiltration (UF) membranes were prepared from PAN homopolymer and copolymer with methyl acrylate. Besides hydrazide formation and nitrosation, a new method to introduce acyl azide groups into carboxyl modified PAN, using azido transfer with diphenyl phosphoryl azide, was developed. Chemical conversions were characterized, especially with Fourier transform infrared spectroscopy. The heterogeneous modifications are not chemically selective due to side reactions and/or incomplete conversion. The pore structure is altered predominately via modified polymer swelling causing changed UF fluxes and selectivities. However, for the modification via PAN reaction with hydroxyl amine, acid hydrolysis, and azido transfer, the initial membrane separations performance is qualitatively preserved. Using the acyl azide ḿethod, amylo-glucosidase (AG) (EC 3.2.1.3) was immobilized onto the modified PAN UF membranes, enabling hydrolysis of starch or maltose to glucose. Enzyme activity was assayed depending on previous chemical modification (azide content) and immobilization (pH) conditions as well as hydrolysis parameters (substrate, conversion during diffusion or UF). The best results (up to 600 mU/cm² at 40°C and pH 5.0) were obtained after modification of PAN membranes via carboxyl creation and azido transfer. AG convalently bound to PAN is not influenced much in its catalytic properties (K_m = 3.48 and 3.1 mmol/L for free and bound AG, respectively, with maltose at 40°C and pH 5.0). Under UF conditions, AG effective activity can be improved by the convective flow through the membrane. UF selectivity for the polymer starch determines effective substrate concentrations in the membrane, thus affecting observed activities and product purities in the filtrate. © 1996 John Wiley & Sons, Inc.     

Glucose oxidase immobilization onto a plasma-induced graft copolymerized polymeric membrane modified by poly(ethylene oxide) as a spacer

Plasma-induced graft copolymerization of acrylic acid, which was incorporated onto polyethylene (PE) film, was prepared. A bisamino poly(ethylene oxide) (PEO) was immobilized onto the poly(acrylic acid) (PAAc)-grafted PE membrane to modify the surface properties. The samples were characterized by ESCA. A respective chemical shift of Ar plasma-treated and control polymeric film was revealed by ESCA. The presence of the grafted PAAc and PEO was also verified. Glucose oxidase (GOD) was immobilized onto this novel grafted polymeric film with and without PEO being used as a spacer. The Michaelis constant, K_m, and the maximum reaction velocity, V_max, were estimated for the free and the immobilized GOD. GOD immobilized onto the polymeric films with and without a spacer obeyed Michaelis kinetics. The Michaelis constant, K_m, was larger for the immobilized GOD than for the free one whereas V_max was smaller for the immobilized GOD. The bioactivity of PEO-modified PAAc-grafted PE membrane (PAAc–PEO–GOD) was higher than that of PAAc-grafted PE membrane (PAAc–GOD). The pH and thermal stabilities of the immobilized GOD without a spacer (PAAc–GOD) were higher than those of the immobilized GOD with a spacer (PAAc–PEO–GOD) and the free form. © 1993 John Wiley & Sons, Inc.     

Epoxy‐derived pHEMA membrane for use bioactive macromolecules immobilization: Covalently bound urease in a continuous model system

Poly(2‐hydroxyethylmethacrylate) (pHEMA) membranes were prepared by UV‐initiated photopolymerization of HEMA in the presence of an initiator (α‐α′‐azobisisobutyronitrile, AIBN). The epoxy group, i.e., epichlorohydrin, was incorporated covalently, and the urease was immobilized onto pHEMA membranes by covalent bonding through the epoxy group. The retained activity of the immobilized enzyme was found to be 27%. The K_m values were 18 and 34 mM for the free and the immobilized enzymes, respectively, and the V_max values were found to be 59.7 and 16.2 U mg⁻¹ for the free and the immobilized enzyme. The optimum pHs was 7.2 for both forms, and the optimum temperature for the free and the immobilized enzymes were determined to be 45 and 50°C, respectively. The immobilized urease was characterized in a continuous system and during urea degradation the operational stability rate constant for the immobilized enzyme was found to be 5.83 × 10⁻⁵ min⁻¹. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2000–2008, 2000