Influence of the microenvironment on the activity of enzymes immobilized on Teflon membranes grafted by γ-radiation

The effect of the microenvironment and immobilization method on the activity of immobilized β-galactosidase was investigated. Immobilization was done on Teflon membranes grafted with different acrylic monomers by γ-radiation and activated by two different coupling agents through the functional groups of the grafted monomers. 2-Hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAA) were grafted on the membrane, and 1,6-hexamethylenediamine (HMDA) was used as a spacer. Glutaraldehyde or cyanuric chloride were used as coupling agents to bind the enzyme to the membrane. Four different catalytic membranes were obtained using the same solid support. Direct comparison between the isothermal behaviour of the biocatalyst in its free and immobilized form was carried out. In particular the dependence of the isothermal activity on the temperature and pH was studied and the kinetic parameters determined. The influence of the microenvironment on the observed activity of the four membranes was evidenced and discussed. The way of improving the yield of these catalytic membranes is discussed also.     

Enzymatic activity of immobilized enzyme determined by isothermal titration calorimetry

The activity of adsorbed β-glucosidase onto spherical polyelectrolyte brushes (SPBs) is investigated by UV-Vis spectroscopy and isothermal titration calorimetry (ITC). By comparing the results of these two methods, we demonstrate that ITC is a precise method for the study of the activity of immobilized enzymes. The carrier particles used for immobilization here consist of a polystyrene core onto which poly(acrylic acid) chains are grafted. High amounts of enzyme can be immobilized in the brush layer at low ionic strength by the polyelectrolyte-mediated protein adsorption (PMPA). Analysis of the activity of β-glucosidase was done in terms of Michaelis-Menten kinetics. Moreover, the enzymatic activity of immobilized enzyme is studied by ITC using cellobiose as substrate. All data show that ITC is a general method for the study of the activity of immobilized enzymes.     

Adhesive cell cultivation on polymer particle having grafted epoxy polymer chain

In this study, we synthesized a new cell immobilization support having poly(glycidyl methacrylate) as a graft polymer chain and used this support for cell cultivation. Base polymer particle was synthesized by suspension polymerization and epoxy polymer chain was extended from particle surface on graft polymerization. Produced polymer particles had broad particle size distribution ranging from 20 to 1000 μm and the degree of polymerization of grafted polymer chain was ranged from 500 to 1000. The effects of various factors, such as grafted polymer chain length and its surface density, composition of base polymer network and graft polymer chain, on the cell growth of murine fibroblast cell line (MS-5 cell) on polymer particle were studied. This polymer particle could cultivate not only fibroblast cell line but also epidermal cell line (HeLa cell), osteoblast cell line (MC3T3E1 cell), and chondrocyte cell line (ch-8 cell) on its surface. Growth rate is almost the same as that of cells using poly(styrene) tissue culture dish. To apply this cell cultivation system for examination of cell co-culture, HeLa cell immobilized on 100 μm of polymer particle was successfully co-cultured with MS-5 cell immobilized on 300 μm of polymer particle for four weeks.     

Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene–divinylbenzene) (P(S–DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The K _m value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene–divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.     

Immobilization of glucose oxidase in polypyrrole/polytetrahydrofuran graft copolymers

Glucose oxidase (GOD) was immobilized in four different conducting polymer matrices, namely: polypyrrole, (PPy), poly(pyrrole-graft-polytetrahydrofuran), (1) and (3); and poly(pyrrole-graft-polystyrene/polytetrahydrofuran), (2). The kinetic parameters V(max) and K(m), and the optimum temperature were determined for both immobilized and native enzymes. The effect of electrolysis time and several supporting electrolytes, p-toluenesulfonic acid, p-toluene sulfonic acid (PTSA), sodium p-toluene sulfonate, sodium p-toluene sulfonate (NaPTS), and sodium dodecyl sulfate, sodium dodecyl sulfate (SDS), on enzyme immobilization were investigated. The high K(m) value (59.9 mM) of enzyme immobilized in PPy was decreased via immobilization in graft copolymer matrices of pyrrole. V(max), which was 2.25 mM/min for pure PPy, was found as 4.71 mM/min for compound (3).     

Sequential immobilization of urease to glycidyl methacrylate grafted sodium alginate

Objective of this study is to realize appropriate enzyme immobilization onto a suitable support material and to develop a model which enables reactions catalyzed with different enzymes arranged in order. Thence, this model was potential for developing a multi-enzyme system. The reactions need more than one enzyme can be realized using immobilized form of them and the enzymes will be in one support at wanted activities. In this study, sodium alginate was used as immobilization material and glycidyl methacrylate was grafted onto sodium alginate. Thus reactive epoxy groups were added to sodium alginate which also has carboxyl groups. Average molecular weight of sodium alginate was determined using Ubbelohde viscosimetri. The molecular mass of sodium alginate was calculated as 15,900 Da. Graft polymerization was made in two steps. Firstly, sodium alginate was activated with benzophenone using UV-light at 254 nm. Secondly, glycidyl methacrylate was grafted under UV-light at 365 nm onto activated sodium alginate. Grafted glycidyl methacrylate was determined gravimetric and titrimetric. Additional groups after grafting were showed with FT-IR spectrum. 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide was used for immobilization urease from carboxyl groups at pH 5.0. Suitable 1-ethyl-3-(3-dimetylaminopropyl)-carbodiimide/–COOH ratio was found 1/10 and immobilized product activity was 197 U/g support. Reaction medium pH was 8.0 for immobilization from epoxy group. Optimum immobilization reaction time was found as 2 h and immobilized product activity was 285 U/g support. Sequential immobilization of urease to glycidyl methacrylate grafted sodium alginate was made from –COOH and epoxy groups, respectively.     

Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc.     

Preparation and properties of immobilized amyloglucosidase on nonporous PS/PNaSS microspheres

Nonporous polystyrene/poly(sodium styrene sulfonate) (PS/PNaSS) microspheres were used for immobilization of amyloglucosidase and the properties of immobilized enzyme was studied and compared with those of free enzyme. Sulfonated groups on the PS/PNaSS microspheres present a very simple, mild, and time-saving process for enzyme immobilization. Nonporous microspheres provide their surface for immobilization of enzyme and prevent the diffusion limitation problem in the pore. Despite the high concentration of bound enzyme the influence of immobilization on kinematic parameters, K(m) and V(max), is relatively low compare to other porous supports. Simple and time-saving immobilization procedure as well as the effects of pH and temperature on immobilized enzyme also showed that the PS/PNaSS microspheres could be good support.     

Polyaniline as a support for urease immobilization

Polyaniline synthesized by chemical oxidative polymerization was used as an immobilization support for jack bean urease. Such immobilized enzyme has a good catalytic activity, storage stability, and reusability. Properties of free and immobilized urease were compared. Blends of polystyrene, cellulose acetate and poly(methyl methacrylate) with polyaniline were used for urease immobilization as well.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes

The present study describes the preparation of poly(HEMA-co-GMA) reactive membranes that were grafted with polyethylenimine (PEI) following UV photo-polymerization. The immobilization of tyrosinase was carried out via multi-point ionic interactions based on ---NH₂ groups of PEI and Cu(II) ions. Tyrosinase is a copper-dependent enzyme, which should show a binding affinity for the chelated Cu(II) ions on the membrane surfaces. The tyrosinase immobilization was positively correlated with the input enzyme amount in the immobilization medium. The maximum tyrosinase immobilization capacities of the poly(HEMA-co-GMA)–PEI and poly(HEMA-co-GMA)–PEI–Cu(II) membranes were 19.3 and 24.6 mg/m², respectively. The enzyme activity when assessed at various pH and temperatures gave broader range for immobilized preparations when compared to free enzyme. The poly(HEMA-co-GMA)–PEI–Cu(II) tyrosinase membranes retained 82% of their initial activity at the end of 120 h of continuous reaction. Moreover, upon storage for 3 months the activity of the immobilized membranes retained 46% of their initial levels. After deactivation of the enzyme, the poly(HEMA-co-GMA)–PEI membrane was easily regenerated, re-chelated with the Cu(II) ions and reloaded with the enzyme for repeated use. The mild immobilization conditions, easy and rapid membrane preparation, one-step enzyme adsorption at substantially higher levels and membrane reusability are the beneficial properties of such systems and offers promising potential in several biochemical processes.     

Modification and characterization of cellulosic cotton fibers for efficient immobilization of urease

Cotton fibers were first grafted by polyacrylonitril in the presence of KMnO(4) and oxalic acid as a combined redox initiator. Moreover, modification of the grafted cotton fibers was done by changing the nitrile group (-CN) into hydrazidine group through the reaction with hydrazine hydrate, then the fibers were activated by glutaraldehyde to introduce free aldehyde groups which were able to react with amino groups of urease to form Schiff's base, and result in cotton fibers immobilized urease. The efficiency of the immobilization was evaluated by examining the relative enzymatic activity of enzyme before and after the immobilization of urease. The results showed that the optimum temperature of immobilized urease was 35°C, which was higher than that of the free enzyme (30°C), and the immobilized urease exhibited a higher relative activity than that of free urease over 35°C. The optimal pH for immobilized urease was 6.5, which was lower than that of the free urease (pH 7.0), and the immobilization resulted in stabilization of enzyme over a wider pH range. The kinetic constant value (K(m)) of immobilized urease was higher than that of the free urease. However, the thermal and operational stabilities of immobilized urease have been improved greatly.     

Immobilization of xylan-degrading enzymes from<Emphasis Type="Italic"> Melanocarpus albomyces</Emphasis> IIS 68 on the smart polymer Eudragit L-100

Xylanase of Melanocarpus albomyces IIS 68 was immobilized on Eudragit L-100. The latter is a copolymer of methacrylic acid and methyl methacrylate and is a pH-sensitive smart polymer. The immobilization was carried out by gentle adsorption and an immobilization efficiency of 0.82 was obtained. The enzyme did not leach off the polymer even in the presence of 1 M NaCl and 50% ethylene glycol. The K(m) of the enzyme changed from 5.9 mg ml(-1) to 9.1 mg ml(-1) upon immobilization. The V(max) of the immobilized enzyme showed an increase from 90.9 micro mol ml(-1) min(-1) (for the free enzyme) to 111.1 micro mol ml(-1) min(-1). The immobilized enzyme could be reused up to ten times without impairment of the xylanolytic activity. The immobilized enzyme was also evaluated for its application in pre-bleaching of eucalyptus kraft pulp.     

Immobilization of Lipase from Mucor Miehei Onto Poly (Ethylene) Based Graft Copolymers

Lipase from Mucor miehei was immobilized covalently onto hydrolyzed poly(ethylene)-g.co-hydroxyethyl methacrylate (PE-HEMA). This hydrolysis of the copolymer was achieved using 0.1 M NaOH over different periods of time, under controlled conditions. The graft copolymers and their hydrolyzed equivalents were characterized by scanning electron microscopy (SEM) and by differential scanning calorimetry analysis (DSC). Water sorption studies were undertaken to provide a measure of relative hydrophobicity of the samples.

The lipase immobilization reaction was studied in order to assess the effects of controlling various important parameters. These include the nature of the buffering medium, the time over which the immobilization was allowed to occur, the concentration of the activating and coupling agent used (CMC) and the concentration of enzyme employed during attempts at effective immobilization. The immobilized lipase was used in the hydrolysis of triolein (glycerol trioleate). From this study, the apparent K_M, the optimum pH for hydrolysis and the optimum temperature for hydrolysis were revealed.

The suitability of hydrolyzed poly(ethylene)-g.co-HEMA as a support in the immobilization of lipase was assessed by determination of the amount of lipase coupled to the support and by assessment of the retention of activity of the immobilized lipase after its exposure to the immobilization reagents, procedure and conditions.     

Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K_m) and the maximum rate of reaction (V_max) were calculated from the Lineweaver–Burk plot. The K_m and V_max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.     

Immobilization of alpha-chymotrypsin on poly(urethane-graft-acrylic acid)

A study was made of the immobilization of alpha-chymotrypsin (alpha-CT) onto a previously well characterized synthetic polyurethane grafted with acrylic acid P(U-g-AA). The P(U-g-AA) had previously been prepared using 2,2'-azo-bis-isobutyronitrile (AIBN) as a radical initiator and acrylic acid as monomer in the presence of an unsaturated polyurethane in solution at 60 degrees C. Some kinetic parameters of both the native enzyme and the enzyme immobilized on the P(U-g-AA) were evaluated. Using a Lineweaver-Burk plot (double reciprocal), it was found that the Michaelis-Menten constant (Km(for the immobilized enzyme was (4.0 +/- 0.9) x 10(-3) M and that of the free enzyme was (3.0 +/- 0.2) x 10(-3) M. The enzyme alpha-chymotrypsin was immobilized on the grafted polyurethane micelles/aggregates with about 45% retention of activity. Also the immobilized alpha-CT retained this activity for at least 6 weeks. The immobilized enzyme was found to have a maximum stability at 43 degrees C compared with 36 degrees C in the case of free enzyme, and the pH optimum was shifted from pH 6.6 to pH 8.2. The long-term operational stability of the enzyme was investigated and this is of interest since the enzyme is probably trapped physically in a micellar environment. The assay of the enzyme was carried out in 0.01 M phosphate buffer, pH 7.5, using p-nitrophenyl acetate as a substrate. No inhibition of alpha-CT in the presence of the synthetic ungrafted and grafted polyurethane was observed.     

Preparation and application of poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan hydrogels for immobilization of lipase

In the present of this study, two novel polymeric matrixes that are poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan was synthesized and applied for immobilization of lipase. For the immobilization of enzyme, two different immobilization procedures have been carried out via covalently binding and entrapment methods. On the free and immobilized enzymes activities, optimum pH, temperature, storage and thermal stability was investigated. The optimum temperature for free, covalently immobilized and entrapped enzymes was found to be 30, 35 and 30 degrees C, respectively. Optimum pH for both free and immobilized enzymes was also observed at pH 8. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for free and immobilized lipases. Furthermore, the reuse numbers of immobilized enzymes also studied. It was observed that after 40th use in 5 days, the retained activities for covalently immobilized and entrapped lipases were found as 39% and 22%, respectively. Storage and thermal stability of enzyme was also increased by as a result of immobilization procedures.     

Synthesis of tethered-polymer brush by atom transfer radical polymerization from a plasma-polymerized-film-coated quartz crystal microbalance and its application for immunosensors

This study synthesizes a tethered surface-grafted poly(acrylic acid) with quartz crystal microbalance (QCM) surfaces and provides detailed analysis of their properties and application. A tethered polyelectrolyte brush of poly(acrylic acid) is generated by first covering the substrate with a plasma-polymerized allyl alcohol (pp-AA) film, changing the polymerization initiators (bromination), and then grafting through atom transfer radical polymerization (ATRP) of tert-butyl acrylate (t-BA); these initiators are immobilized on a surface and exposed to a monomer. Finally, we convert the poly(t-BA) brush into poly(acrylic acid) through hydrolysis. We use the QCM technique to measure configuration change of the tethered poly(acrylic acid) grafted chains with two different degrees of polymerization (DP=50,200) in aqueous solutions at three different pH values (4.0, 4.8, and 5.4). The tethered poly(acrylic acid) grafted QCM shows that repeatable frequency responses are induced by pH change of solution. These frequency responses of large DP for pH are 20 times larger than responses of lower DP for pH. The frequency response of antibody immobilization on tethered poly(acrylic acid) grafted QCM (DP=200) and its frequency response of immunoreaction are 10 times larger than conventional immobilization methods by cysteamine with glutalaldehyde coupling of the antibody. The tethered poly(acrylic acid) grafted QCM can increase the frequency response for pH, the immobilization amount of antibody, and immunosensor response.     

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters.

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.     

Use of chemically modified PMMA microspheres for enzyme immobilization

Modified poly(methyl methacrylate) (PMMA) microspheres, about 7microm in diameter, carrying aldehyde groups on their surfaces were synthesized and used as the support for enzyme immobilization. The immobilizing behavior as well as the properties of immobilized enzyme was studied. The amount of bound enzyme can be extended to 76.8mg g(-1) support, which is relatively much higher than other supports. The kinetic investigation derived from three typical models shows that the practical process is more complicated than the ideal condition, with one or more interactions being involved in the immobilization process. The K(m) value is actually larger and V(max) is smaller in the immobilized form than those in the free form. The increased resistance of the immobilized enzyme against the changes of temperature indicates that immobilizing enzyme onto the modified microspheres is useful for enzyme immobilization.