Immobilization of organophosphorus hydrolase enzyme by covalent attachment on modified cellulose microfibers using different chemical activation strategies: Characterization and stability studies

The plant cellulose powder was activated by two different methods using 1,4-butanediol diglycidyl ether(BTDE)and 1,1′-Carbonyldiimidazole(CDI) as the chemical coupling agents.Organophosphorus hydrolase(OPH) from Flavobacterium ATCC 27551 was immobilized on any of activated support through covalent bonding.The optimal conditions of affecting parameters on enzyme immobilization in both methods were found, and it was demonstrated that the highest activity yields of immobilized OPH onto epoxy and CDI treated cellulose were 68.32%and 73.51%, respectively.The surface treatment of cellulose via covalent coupling with BTDE and CDI agents was proved by FTIR analysis.The kinetic constants of the free and immobilized enzymes were determined, and it was showed that both immobilization techniques moderately increased the Kmvalue of the free OPH.The improvements in storage and thermal stability were investigated and depicted that the half-life of immobilized OPH over the surface of epoxy modified cellulose had a better growth compared to the free and immobilized enzymes onto CDI treated support.Also, the pH stability of the immobilized preparations was enhanced relative to the free counterpart and revealed that all enzyme samples would have the same optimum pH value for stability at 9.0.Additionally, the immobilized OPH onto epoxy and CDI activated cellulose retained about 59% and 68% of their initial activity after ten turns of batch operation, respectively.The results demonstrated the high performance of OPH enzyme in immobilized state onto an inexpensive support with the potential of industrial applications.     

The immobilization of enzymes onto hydrolyzed polyethylene-g-co-2-HEMA

A study of the immobilization of trypsin and other enzymes onto hydrolyzed poly(2-hydroxyethyl methacrylate)-g-co-polyethylene using hydroxyl and carboxyl activating agents has been undertaken. Some emphasis was placed on the immobilized trypsin system which involved examination of the variation of (i) the extent of hydrolysis of the graft copolymer, (ii) the concentration of activating agent, and (iii) the temperature of coupling. With the trypsin system, an increase in carbodi-imide concentration gave an increase in the amount of protein immobilized but a marked decrease in the retention of enzymic activity. Comparison of the behavior of the free with the immobilized enzyme showed that satisfactory yields were obtained and the immobilized system has an extended pH profile and good stability and thus would have broad applicability. The kinetic factors were examined further, and the role of the graft copolymer chains in the immobilized system is discussed.     

Enzyme immobilization on polyglycidylmethacrylate graft-copolymer of different polysaccharides

Comparative results obtained in preparing and characterizing samples of enzymes immobilized by reaction with polyglycidylmethacrylate (PGMA) copolymers with different polysaccharide matrices are reported. Sepharose copolymers having between 25 and 50% synthetic polymer were used to find the best immobilization conditions of horseradish peroxidase (HRP) and glucose-oxidase (GOD) (pH, time, temperature, enzyme cncentration). Activity, enzyme loading and coupling efficiency of immobilized HRP and GOD are greatly dependent on the type of matrix while the polymer content is less important. Coupling efficiencies between 0.8 and 1.5% have been obtained for HRP samples, whereas for GOD samples coupling efficiencies three times greater were obtained. HRP and GOD immobilized samples show K_m′ values greater than those of corresponding free enzymes and this indicates diffusion limitation phenomena. Storage, thermal and operational stability were also studied. In general the storage stability could be considered satisfactory (50% residual activity after 360 days). Sepharose and starch HRP-copolymers had an improved thermal stability compared with that of free enzyme. Residual activity found in continuous operation tests carried out on HRP-immobilized samples turned out to be dependent on support. HRP-PGMA-Cellulose sample gave the best results (50% residual activity after 16 days). PGMA-graft-copolymers have also been used to immobilize other enzymes such as α-amylase, α-chymotrypsin and cellulase.     

动态膜分离式固定化酶反应过程分析

测定了实验系统下固定化酶的本征动力常数,从动力学角度分析研究了所开发研制的动态膜分离式固定化酶反应器（DMIR）的反应过程,给出了无外扩散限制条件下的DMIR反应器的数学模型,并验证了实验方法的可靠性。     

Optimization of covalently coupling enzymes to polymeric membranes: EPR studies of papain

In an effort to better understand bioreactor systems, papain (EC 3.4.22.2) was covalently immobilized onto vinyl alcohol/vinyl butyral copolymer (PMB) membrane by means of glutaraldehyde (GA), 1,1'-carbonyldiimidazole (CDI), or 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP). Various kinetic and performance characteristics of the immobilized papain were evaluated. It was found that the characteristics of the membranebound papain depended on the immobilization methods. The CDI- and FMP-immobilized papain bioreactors showed better storage and thermal stability than did the GA-immobilized papain bioreactor, although the apparent Michaelis constant, K_m, of the GA-immobilized papain was closer to the free enzyme than to the corresponding CDI- and FMP-immobilized enzymes. In separate experiments, a 6-carbon spacer was inserted between the membrane surface and the covalently bound enzyme. It was found that the insertion of a spacer reduced the disturbance of the enzyme systems, resulting in K_m values intermediate between the free and directly bound enzymes for all three immobilization methods. Electron paramagnetic resonance spectroscopy was also used to investigate the conformational change and the active site structure of papain. It was found that the active site SH group of papain immobilized with a 6-carbon spacer had faster motion than that of directly bound enzyme, but slower motion than that of the free enzyme. With both direct-coupling and with a spacer, the SH group motion at the active site of papain by CDI and FMP immobilizations was similar, but slower than the corresponding GA immobilization. The conformational changes of the active site of papain upon immobilization with and without a spacer were in agreement with the functional properties of the enzyme. There was a good correlation between the motion of spin-labeled cysteine in the active site of papain and kinetic properties of this protease: As motion slowed, K_m increased and V_max decreased. Of the immobilization procedures used, GA immobilization with a spacer yielded kinetic and structural characteristics most similar to the free enzyme while providing increased stability and reusability relative to the latter. © 1993 John Wiley & Sons, Inc.     

Study on the activity and stability of urease immobilized onto nanoporous alumina membranes

Nanoporous alumina membranes were employed as substrate materials for urease immobilization. Anodic porous alumina was prepared by the two-step anodization of high purity aluminum. By controlling anodization conditions, the nanoporous structure with desired dimension was obtained. Urease immobilization onto nanoporous alumina membranes was performed by four different protocols. Effect of pore diameter, pore length and immobilization methods on the activity and stability of immobilized enzyme was discussed in detail. The results show that the enzymes immobilized onto porous alumina with big pore diameter possess high activity and poor stability as compared to small pore diameter. The effect of pore length is complicated, the activity of enzyme increases with the increasing pore length for big pore size; while for correspondingly small pore size, enzymatic activity slightly depends on pore length. The immobilization methods have a slight effect on enzymatic activity, whereas enzyme immobilization by chitosan coating and reticulation with glutaraldehyde exhibits a good long-term stability as compared to that only via physical adsorption.     

Immobilization of DNA on poly(glycidyl methacrylate-co-ethylene dimethacrylate), bead cellulose and sepharose

Summary The copolymer of glycidylmethacrylate with ethylene-dimethacrylate, its derivatives, bead cellulose and Sepharose were used for immobilization of DNA via new coupling methods: reaction with oxirane, acylhydrazide hydrazide, and diazonium salt. The susceptibility of immobilized DNA for DNAse was tested. The most efficient immobilization was achieved on carriers with acylhydrazide groups. The binding capacity of the carrier and the susceptibility of immobilized DNA for the DNase depends on the length of the spacer. The immobilized DNA is stable in solution for several weeks, and in the lyophilized state for several months.     

Pullulanase immobilization on natural and synthetic polymers

This work describes the immobilization of pullulanase onto two different polymers; agarose activated with epichlorohydrin and trichlorotriazine and casein activated with epichlorohydrin, in addition to a synthetic copolymer, butylacrylate–acrylic acid (BuA/AAc). Immobilization by covalent binding yields stable enzyme activity. The operational stability of the free and immobilized enzymes showed that the enzyme immobilized by a crosslinking technique using glutaric dialdehyde (GA) showed poor durability and the relative activity decreased sharply due to leakage after repeated washing, while the enzymes immobilized by covalent bonds resulted in a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4–6 months showed that the free enzyme lost most of its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzymes to doses of γ‐radiation (0.5–10 Mrad) resulted in complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to γ‐radiation up to a dose of 5 Mrad. Nuclear Magnetic Resonance (¹H NMR) and FTIR measurements were carried out to confirm the structure of the polymer as well as the immobilization process of the enzyme onto the polymeric carrier. The unique biochemical characteristics, mode of action and utility of the environmentally compatible pullulanase in starch conversion are well known. Using pullulanase with β‐amylase in starch saccharification processes can increase maltose yield by 20–25%. © 2001 Society of Chemical Industry     

Covalent immobilization of porcine pancreatic lipase on amorphous AlPO4 and other inorganic supports

Porcine pancreatic lipase (PPL) was covalently immobilized through the formation of an aromatic Schiff's-base obtained by reaction of the ε-amino group of lysine residues in the enzyme and the aromatic aldehyde function of activated supports. The enzymatic activities of different immobilized PPL systems on amorphous AlPO₄ and several inorganic supports at different pH values and temperatures were determined by hydrolysis of ethyl acetate. The nature of the support material critically affects the efficiency of enzyme immobilization as well as enzyme stability. All the supports examined, with the exceptions of cellulose and natural sepiolite, exhibited good properties for enzyme attachment. AlPO₄ was the best support for enzyme immobilization, giving the highest percentage binding of enzyme (90%) and the highest retention of enzyme activity after immobilization (92·8%). © 1998 SCI     

Behavior of immobilized glucose oxidase on membranes from polyacrylonitrile and copolymer of methylmethacrylate‐dichlorophenylmaleimide

Two new ultrafiltration membranes were obtained from a polymer mixture, containing 60% polyacrylonitrile (PAN) and 40% copolymer of methylmethacrylate‐dichlorophenylmaleimide (MMA‐DCPMI). Membrane 1 (MB1) contains 40% DCPMI of the copolymer, and membrane 2 (MB2) contains 15% of the copolymer. The pore size, the specific surface, the water content, the water flux, and the selectivity were determined for the two membranes. The presence of dichlorophenylmaleimide in the copolymer ensures the preparation of membranes suitable for direct covalent enzyme immobilization without further modifications. These membranes were used for immobilization of glucose oxidase (GOD). High amount of bound protein was found on each of the membranes. High relative activities of the immobilized GOD were achieved, 72% for MB1 and 68% for MB2. The properties of the immobilized enzyme (GOD) were determined: optimum pH and temperature and pH, thermal, and storage stability, and then compared with the properties of the native enzyme. The kinetic parameters of the enzyme reaction, Michaelis constant (K_m) and maximum reaction rate (V_max), were also investigated. The results obtained showed that the ultrafiltration membranes prepared from the mixture of PAN and the copolymer MMA‐DCPMI were suitable for use as carriers for the immobilization of GOD. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 4334–4340, 2006     

Laccase functionalized cellulose acetate for the removal of toxic combustion products

The ability of Trametes villosa laccase immobilized on cellulose acetate to reduce/eliminate combustions toxicants was investigated using a model enzyme filter design. In the initial stages, various strategies of grafting laccase onto cellulose acetate polymers including partial deacetylated cellulose acetate followed by generation of reactive groups using either periodate or 2,2,6,6-tetramethylpiperidinyl-1-oxy radical (TEMPO) and the use of different spacer arms [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC); 1,4-butanediol diglycidyl ether (BDGE)] and 3-aminopropyltriethoxysilane was investigated. The best process for effective immobilization of laccase onto both cellulose acetate powders and tows were those involving partial deacetylation, TEMPO activation to generate carboxylic groups, treatment with EDAC as a spacer arm followed by adding the enzyme. This procedure resulted in 45 units/mg laccase activity (28% increase in activity of immobilized enzyme) measured using ABTS as substrate as compared to the other strategies used to immobilize laccase. Further, the immobilized enzyme was able to oxidize > 60% of toxicants resorcinol, hydroquinone and methylcatechol passing through the model enzyme filter. This study therefore demonstrates the great possibility of immobilizing laccase onto modified cellulose acetate and the great potential application of immobilized laccase to remove toxicants during combustion.     

Bioactive polymers

Summary The immobilization of invertase on carboxymethyl cellulose acid chloride and the factors influencing the reaction (enzyme/support ratio, duration and pH) are studied. The activity of both coupled and free enzymes varies with temperature and pH of the incubation reaction. The values of the Michaelis-Menten constant are higher for the immobilized enzyme.     

Immobilization of glucose oxidase and electron-mediating groups on the film of 3-methylthiophene/thiophene-3-acetic acid copolymer and its application to reagentless sensing of glucose

Enzyme electrodes were prepared by covalent immobilization of glucose oxidase (GOx) on the films of conducting copolymer obtained by electrochemical copolymerization of 3-methylthiophene and thiophene-3-acetic acid. Onto the enzyme electrodes, 2,5-dihydroxyphenyl (DHP) groups were introduced as redox mediators by the reaction of 2,5-dihydroxybenzaldehyde using alkylenediamines as linker molecules. The mediator-carrying enzyme electrodes were applied to reagentless glucose sensing system. It was found that response current to glucose was increased by introducing DHP groups, and the longer the alkylene chain of used alkylenediamine was, the higher the response current became. For a comparison, poly-l-lysine was linked onto the films of the conducting copolymer in advance, and immobilization of GOx and then introduction of DHP groups were carried out. The mediator-carrying electrode prepared thus gave higher amperometric response than those prepared with alkylenediamines, suggesting that amino groups of poly-l-lysine functioned effectively as the sites for binding GOx and DHP groups.     

Immobilization of some biomolecules onto radiation‐grafted polyethylene beads for possible use in immunoassay applications

Functionalization of polyethylene (PE) beads was accomplished via radiation induced graft copolymerization of acrylic acid/acrylamide (AAc/AAm) binary comonomer of different compositions onto such beads. Factors affecting the grafting yield were optimized and the occurrence of the grafted chains was confirmed by following the FTIR spectra of the grafted beads. SEM analyses were used to follow the variation of the morphology of the grafting and immobilization onto PE beads. Some bio‐active molecules such as Follicle‐stimulating hormone (FSH), Luteinizing hormone (LH), Thyroid‐stimulating hormone (TSH), and Prolactin were immobilized to the radiation functionalized PE beads. The parameters may affect the immobilization process such as degree of grafting, temperature, and pH of the coupling buffer and the coupling period were investigated. The obtained results show that the grafting of AAc offers a better immobilization environment than those of AAm and their copolymer. It is found that the highest immobilization degree would be achieved at pH 7 and 37°C for 24 h. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

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     

Systematic determination of intrinsic reaction parameters in enzyme immobilizates

For the rational design of processes using immobilized enzymes a mechanistic kinetic model is required, which accounts for all kinetic and thermodynamic phenomena, including the enzyme reaction, the mass transfer of the reactants between both phases, and their diffusion inside the immobilizate. For the example of enzymes immobilized in hydrogel beads suspended in an organic solvent, such a mechanistic kinetic model was obtained by a model-based experimental analysis approach. It was proven that the usually applied concentration measurements in the bulk phase are not sufficient to draw mechanistic conclusions. The most suitable measurement technique was found to be the quantification of the concentration along the radius of the hydrogel bead. These line scans, achieved by two-photon laser scanning microscopy, for the first time allowed to estimate intrinsic reaction and mass transfer parameters simultaneously. Thus, the obtained intrinsic parameter estimates for the biphasic hydrogel system could be directly compared with those obtained in individual systems. This comparison revealed for the first time that the enzyme reaction was not significantly affected by the mild hydrogel encapsulation. However, a significant impact on the transport parameters was found that underlines the need for analyzing the real reaction system using mechanistic models.     

Grafting of acrylate monomers onto chlorinated rubber

Grafting of chlorinated rubber with ethylacrylate and butyl methacrylate in xylene solution has been studied using benzoyl peroxide as the initiator. It is observed that the grafting reaction follows conventional kinetic behavior under the present experimental conditions. The energies of activation obtained for both monomers were 10.8 and 10.95 K.cals, respectively. The grafting efficiency and percent grafting were calculated. Additional proof for grafting onto chlorinated rubber was also obtained by characterization of the graft copolymer by gel permeation chromatography.     

Grafting Study and Antifungal Activity of a Carboxymethyl Cellulose Derivative

Graft copolymerization of appropriate monomers onto cellulose and its derivatives can enhance their characteristics and consequently expand their potential applications. Carboxymethyl cellulose (CMC) was prepared and characterized by FTIR spectroscopy and XRD. Graft copolymerization of acrylic acid sodium salt (AAs) onto CMC using ammonium persulfate (APS) as a free radical initiator was carried out under nitrogen atmosphere in aqueous solution. Occurrence of grafting was confirmed by comparison of FTIR spectra of CMC and the graft copolymers as well as the XRD patterns and thermal analysis. The effects of concentration of AA, temperature, concentration of APS and reaction time on the grafting yield were investigated by determining the grafting percentage and grafting efficiency. With other conditions kept constant, the obtained optimum grafting conditions were: CMC = 0.2 g, [AAs] = 2 mM, [APS] = 7.5 mM, temperature = 70°C and reaction time = 2 h. A preliminary study was then carried out to evaluate the antifungal activity of the prepared graft copolymer. This preliminary investigation of the prepared graft copolymers showed that they may be tailored and exploited to expand the utilization of these systems in medical applications.     

Protein Corona Hinders N-CQDs Oxidative Potential and Favors Their Application as Nanobiocatalytic System

The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.     

葡萄糖氧化酶固定条件的优化

以醋酸纤维素(CA)为原料,采用溶液浇铸法成膜。用物理包埋法将荧光指示剂固定CA膜中。以戊二醛为交联剂,将葡萄糖氧化酶固定于膜表面。研究了高碘酸钠、乙二胺、戊二醛、给酶量、pH值等固定化条件对酶活的影响,确定了优化的固定条件:在室温下与0.5 mol/L的高碘酸钠反应30 min;与0.04 mol/L的乙二胺反应150 min时;与溶度为1.5%(v/v)的戊二醛交联120 min;在4℃条件下,在pH6.5、给酶量35 mg/mL的酶液中交联21 h。利用SEM分析表明,荧光指示剂均匀分布膜中,成膜质量较好。通过优化固定条件,把荧光指示剂和葡萄糖氧化物酶(GOD)同时固定醋酸纤维素膜上,可以得到同时具有光敏感性和酶催化能力的复合敏感膜。