High-yield method for immobilization of enzymes

Two types of polyethylenimine-coated glass microbeads (13–44 μm) were synthesized and used for the immobilization of glucose oxidase from Aspergillus niger and catalase from A. niger and beef liver. The two types of beads were distinguishable by differences in their surface topography. Immobilizations were performed by adsorption followed by treatment with glutaraldehyde. The immobilized-enzyme activities per unit support of all of the enzymes tested were compared with and found to be superior to the immobilized activities attainable on aminopropyl-activated glass microbeads. When enzyme was present in less than saturating amounts, the coated beads were able to remove 100% of the glucose oxidase activity initially present in the immobilization solution, with 78–87% of that activity expressed on the support surface. Bound glucose oxidase was more stable to thermal inactivation than native enzyme.     

Co-immobilization of manganese peroxidase from Phlebia radiata and glucose oxidase from Aspergillus niger on porous silica beads

Manganese peroxidase (MnP) from Phlebia radiata and glucose oxidase from Aspergillus niger were co-immobilized on porous silica beads. Immobilization of both enzymes on the same carrier provided an integrated system in which H₂O₂ required by MnP was produced by glucose oxidase. The immobilization process resulted in a decrease of both enzymatic activities and substrate affinities. However, immobilization improved the stability of MnP against H₂O₂ or high pH, as well as the storage stability of this enzyme.     

Novel polymeric microspheres: Synthesis,enzyme immobilization,antimutagenic activity,and antimicrobial evaluation against pathogenic microorganisms

New polymeric microspheres containing azomethine ( 1a ‐ 1c and 2a ‐ 2c ) were synthesized by condensation to compare the enzymatic properties of the enzyme glucose oxidase (GOx) and to investigate antimutagenic and antimicrobial activities. The polymeric microspheres were characterized by elemental analysis, infrared spectra (FT‐IR), proton nuclear magnetic resonance spectra, thermal gravimetric analysis, and scanning electron microscopy analysis. The catalytic activity of the glucose oxidase enzyme follows Michaelis‐Menten kinetics. Influence of temperature, reusability, and storage capacity of the free and immobilized glucose oxidase enzyme were investigated. It is determined that immobilized enzymes exhibit good storage stability and reusability. After immobilization of GOx in polymeric supports, the thermal stability of the enzyme increased and the maximum reaction rate (V_max) decreased. The activity of the immobilized enzymes was preserved even after 5 months. The antibacterial and antifungal activity of the polymeric microspheres were evaluated by well‐diffusion method against some selected pathogenic microorganisms. The antimutagenic properties of all compounds were also examined against sodium azide in human lymphocyte cells by micronuclei and sister chromatid exchange tests.     

Simultaneous co-immobilization of glucose oxidase and catalase in their substrates

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (Florisil®) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide, which is the substrate of CAT, was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in the presence of both their substrates: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in the presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in the presence of their substrates highly improved the activity and reusability of both enzymes.     

Application of Exchangeable Biochemical Reactors with Oxidase‐Catalase‐Co‐immobilizates and Immobilized Microorganisms in a Microfluidic Chip‐Calorimeter

Several methods for the quantitative detection of different compounds, e.g., L‐amino acids, sugars or alcohols in liquid media were developed by application of an automatic measuring unit including a fluid chip‐calorimeter FCC‐21. For this purpose, enzymes were immobilized covalently on the inner and outer surface of CPG (controlled porous glass)‐spherules with an outer diameter of 100 μm and filled into a micro flow‐through reaction chamber (V_R = 20 μL). The design of the measuring cell allows for easy insertion into the calorimeter device of a stored series of comfortably pre‐fabricated measuring cells. These cells can be filled with different enzyme immobilizates. Different oxidases were used and co‐immobilized with catalase for the improvement of the detection sensitivity. A signal amplification could be achieved up to a factor of 3.5 with this configuration. β‐D‐glucose, ethanol and L‐lysine could be detected in a range of 0.25–1.75 mM using glucose oxidase, alcohol oxidase and lysine oxidase. The group of oxidases in combination with the enzymatic catalysis of the intermediate H₂O₂ allows the quantitative detection of a large number of analytes. A good measurement and storage stability could be achieved for several weeks by this immobilization method. In addition to enzyme‐based detection reactions, it was shown that living microorganisms can be immobilized in the reaction chamber. Thus, the system can be used as a whole‐cell biosensor. The quantitative detection of phenol in the range of 10–100 μM could be performed using the actinomycete Rhodococcus sp. immobilized on glass beads by means of embedding into polymers.     

Glucose‐6‐phosphate dehydrogenase encapsulated in silica‐based hydrogels for operation in a microreactor

The future of hydrogen as fuel strongly depends on the possibility to produce it in an economic and clean way. Hydrogen can be produced from carbohydrates and water under mild conditions by means of a multistep synthetic pathway (13 enzymes) with very high yield. Crossover inhibitions and different optimal conditions of involved enzymes hinder the use of one‐pot approach. Immobilization of enzymes in coupled individual reactors may avoid this problem. This work deals with the immobilization in silica‐based hydrogels of one key enzyme of this pathway: glucose 6‐phosphate dehydrogenase from Leuconostoc mesenteroides. The carriers were prepared with an ethylene glycol‐modified silane, two polymers (polyethylene oxide and Pluronic®) and amino groups created by 3‐aminopropyltriethoxysilane. These parameters influenced the enzymatic activity after immobilization. Gels prepared by addition of polyethylene oxide gave the best results and were used as monoliths in microreactors with two different geometries. The systems showed a high operational stability but a low effective enzyme activity. Enzyme leaching and a nonideal flow pattern may account for the low activity observed. This work is possibly the first one dealing with the immobilization of glucose 6‐phosphate dehydrogenase in silica‐based gels for its application in flow‐through microreactors.     

Covalent immobilization of enzymes within micro-aqueous organic media

Traditional covalent immobilization of enzymes was mostly operated within water phase. However, most of enzymes are flexible when they are in water environment, and the covalent reactions generally lead to complete or partial activity losing due to the protein conformational changes.This paper examined enzyme covalent immobilization operated in micro-aqueous organic media, to display the differences between two environments of immobilization within water and micro-aqueous organic solvent by activity and stability determination of the resulting immobilized enzymes. Catalase, trypsin, horseradish peroxidase, laccase and glucose oxidase have been employed as model enzymes. Results showed the thermal, pH and reusable stabilities of the micro-aqueous organic covalently immobilized enzymes were improved when compared with the immobilized enzymes within water. Micro-aqueous covalent immobilization showed a remarkable advantage in remaining the enzymes catalytic activity for all the five enzymes compared with the traditional water phase immobilization. And the optimum pH values for both immobilization within water and micro-aqueous organic media shifted slightly.     

Immunoaffinity layering of enzymes

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae. Received: 16 November 1998 / Received revision: 22 March 1999 / Accepted: 26 March 1999     

Quantitating intraparticle O2 gradients in solid supported enzyme immobilizates: Experimental determination of their role in limiting the catalytic effectiveness of immobilized glucose oxidase

Enzymatic O₂‐dependent oxidations are receiving increased attention for use in fine chemicals synthesis. Solid supported oxidation catalysts often show poor efficiency due to pronounced O₂ diffusion restriction. Internal O₂ supply therefore constitutes a key parameter for optimizing the enzyme immobilization. We herein describe an optical sensing method for quantitation of space‐averaged intraparticle O₂ concentrations in porous Sepabeads carriers. The method applies phosphorescence lifetime measurements on Sepabeads labeled with an O₂ sensitive indicator dye. Using glucose oxidase immobilized at different loadings (0.005–12 mg/g) on labeled Sepabeads, we analyzed in real time during the enzymatic reaction the formation of O₂ concentration differences between bulk liquid and the intraparticle environment. We show that the O₂ gradient at apparent steady state increased with increasing enzyme loading, so that O₂ eventually became totally depleted from inside the highly loaded carriers. We also show that the residual intraparticle O₂ concentration was correlated with the catalytic effectiveness factor (η) of the enzyme immobilizate used, thus providing a direct measure of the magnitude of O₂ diffusion limitation. Once corrected for diffusional effect, η was no longer dependent on enzyme loading and its constant value now described the intrinsic activity of immobilized glucose oxidase. Three common procedures of enzyme immobilization, involving adsorption, cross‐linking, and covalent attachment, are shown to differ widely concerning the obtained intrinsic activity. Therefore, intraparticle O₂ concentration data enable distinction between diffusional restriction and activity loss as the two principal factors limiting the effectiveness of immobilized O₂ dependent enzymes, and thus they inform rational design of an optimally active oxidation biocatalyst on solid support. Biotechnol. Bioeng. 2013; 110: 2086–2095. © 2013 Wiley Periodicals, Inc.     

Chemical thiolation strategy: A determining factor in the properties of thiol-bound biocatalysts

Immobilization of enzymes on thiolsulphinate-agarose, a thiol-reactive support, is a unique method which allows reversible covalent immobilization under mild conditions, so excellent immobilization and activity yields are obtained. It allows both the formation of stable bonds as well as enzyme desorption and matrix regeneration. The impact of the source of the enzyme's thiol group involved in the immobilization (native, reduced disulphide or chemically introduced) on the properties of the resulting biocatalysts was studied using three β-galactosidases from Escherichia coli, Kluyveromices lactis and Aspergillus oryzae as a model. Chemical thiolation, which generates changes at surface exposed lysines, produced derivatives similar to their soluble counterparts. However, the reduction of native disulphide bonds prior to immobilization lead to very variable activity and stability of the derivatives depending on the accessibility and location of the disulphide bonds in the enzyme structure.     

Covalent immobilization of glucose oxidase onto poly(styrene-co-glycidyl methacrylate) monodisperse fluorescent microspheres synthesized by dispersion polymerization

In this study, micron-sized poly(styrene-co-glycidyl methacrylate) (PSt-GMA) fluorescent microspheres of 5.1microm in diameter were synthesized via dispersion polymerization of styrene and glycidyl methacrylate in the presence of 1,4-bis(5-phenyloxazol-2-yl) benzene (POPOP), which provided surface functional groups for covalent immobilization of enzymes. In an effort to study the biocompatibility of the microspheres' surface, glucose oxidase and beta-d-(+)-glucose were selected as a catalytic system for enzymatic assays. A colorimetric method was adopted in evaluating enzymatic activity by introducing horseradish peroxidase (HRP). Both the immobilization amount and the apparent activity of immobilized glucose oxidase from Aspergillus niger (GOD) were determined at different conditions. The results show that the immobilized enzymes retained approximately 28 to 34% activity, as compared with free enzymes, without pronounced alteration of the optimum pH and temperature. Kinetics studies show that the corresponding values of K(m) and V(max) are 23.2944 mM and 21.6450M/min.mg GOD for free enzymes and 35.1780 mM and 15.4799M/min.mg GOD for immobilized enzymes. The operational stability studies show that immobilized GOD could retain nearly 50% initial activity after being washed 20 times. The results suggest that the resultant PSt-GMA fluorescent microspheres provide a suitable surface for covalent immobilizing biomolecules; therefore, they have the potential of being used in fluorescence-based immunoassays in high-throughput screening or biosensors.     

Conventional and microwave‐assisted SPPS approach: a comparative synthesis of PTHrP(1–34)NH2

Attracted by the possibility to optimize time and yield of the synthesis of difficult peptide sequences by MW irradiation, we compared Fmoc/tBu MW‐assisted SPPS of 1–34 N‐terminal fragment of parathyroid hormone‐related peptide (PTHrP) with its conventional SPPS carried out at RT. MWs were applied in both coupling and deprotection steps of SPPS protocol. During the stepwise elongation of the resin‐bound peptide, monitoring was conducted by performing MW‐assisted mini‐cleavages and analyzing them by UPLC‐ESI‐MS. Identification of some deletion sequences was helpful to recognize critical couplings and as such helped to guide the introduction of MW irradiations to these stages. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Immobilization and stabilization of α-galactosidase on Sepabeads EC-EA and EC-HA

α-Galactosidase from tomato has been immobilized on Sepabead EC-EA and Sepabead EC-HA, which were activated with ethylendiamino and hexamethylenediamino groups, respectively. Two strategy was used for the covalent immobilization of α-galactosidase on the aminated Sepabeads: covalent immobilization of enzyme on glutaraldehyde activated support and cross-linking of the adsorbed enzymes on to the support with glutaraldehyde. By using these two methods, all the immobilized enzymes retained very high activity and the stability of the enzyme was also improved. The obtained results showed that, the most stable immobilized α-galactosidase was obtained with the second strategy. The immobilized enzymes were characterized with respect to free counterpart. Some parameters effecting to the enzyme activity and stability were also analyzed. The optimum temperature and pH were found as 60 °C and pH 5.5 for all immobilized enzymes, respectively. All the immobilized α-galactosidases were more thermostable than the free enzyme at 50 °C. The stabilities of the Sepabead EC-EA and EC-HA adsorbed enzymes treated with glutaraldehyde compared to the stability of the free enzyme were a factor of 6 for Sepabead EC-EA and 5.3 for Sepabead EC-HA. Both the free and immobilized enzymes were very stable between pH 3.0 and 6.0 and more than 85% of the initial activities were recovered. Under the identical storage conditions the free enzyme lost its initial activity more quickly than the immobilized enzymes at the same period of time. The immobilized α-galactosidase seems to fulfill the requirements for different industrial applications.     

Enzyme stabilization by glutaraldehyde crosslinking of adsorbed proteins on aminated supports

The stabilization achieved by different immobilization protocols have been compared using three different enzymes (glutaryl acylase (GAC), D-aminoacid oxidase (DAAO), and glucose oxidase (GOX)): adsorption on aminated supports, treatment of this adsorbed enzymes with glutaraldehyde, and immobilization on glutaraldehyde pre-activated supports. In all cases, the treatment of adsorbed enzymes on amino-supports with glutaraldehyde yielded the higher stabilizations: in the case of GOX, a stabilization over 400-fold was achieved. After this treatment, the enzymes could no longer be desorbed from the supports using high ionic strength (suggesting the support-protein reaction). Modification of the enzymes immobilized on supports that did not offer the possibility of react with glutaraldehyde showed the same stability that the non modified preparations demonstrating that the mere chemical modification did not have effect on the enzyme stability. This simple strategy seems to permit very good results in terms of immobilization rate and stability, offering some advantages when compared to the immobilization on glutaraldehyde pre-activated supports.     

Microwave‐assisted TFA cleavage of peptides from Merrifield resin

Microwave‐assisted (MW) reactions are of special interest to the chemical community due to faster reaction times, cleaner reactions and higher product yields. The adaptation of MW to solid phase peptide synthesis resulted in spectacular syntheses of difficult peptides. In the case of Merrifield support, used frequently in synthesis of special peptides, the conditions used in product cleavage are not compatible with off‐resin monitoring of the reaction progress. The application of MW irradiation in product removal from Merrifield resin using trifluoroacetic acid (TFA) was investigated using model tetrapeptides and the effects were compared with standard trifluoromethanesulphonic acid (TFMSA) cleavage using elemental analysis as well as chromatographic (HPLC) and spectroscopic (IR) methods. The deprotection of benzyloxycarbonyl and benzyl groups in synthetic bioactive peptides was analyzed using LC‐MS and MS/MS experiments. In a 5 min microwave‐assisted TFA reaction at low temperature, the majority of product is released from the resin, making the analytical scale MW‐assisted procedure a method of choice in monitoring the reactions carried out on Merrifield resin due to the short reaction time and compatibility with HPLC and ESI‐MS conditions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β‐galactosidase

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead‐Glu) or carboxyl groups through acid solution (Immobead‐Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β‐galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead‐Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10–500 mg g^?1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g^?1 support. Gal immobilized on Immobead‐Glu and Immobead‐Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half‐lifes than the soluble enzyme, where the half‐lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:934–943, 2018     

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm². The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.     

Simultaneous co-immobilization of glucose oxidase and catalase in their substrates

Glucose oxidase (GOD) and catalase (CAT) were simultaneously co-immobilized onto magnesium silicate (florisil) by covalent coupling. Glucose was added in immobilization mixture and hydrogen peroxide which is the substrate of CAT was produced in coupling mixture during immobilization time. Therefore, co-immobilization of GOD and CAT was carried out in presence of both their substrate: glucose and hydrogen peroxide, respectively. The effect of glucose concentration in immobilization mixture on activities of GOD and CAT of co-immobilized samples were investigated. Maximum GOD and CAT activities were determined for samples co-immobilized in presence of 15 and 20 mM glucose, respectively. Co-immobilization of GOD and CAT in presence of their substrates highly improved the activity and reusability of both enzymes.     

Characterization of immobilized enzymes in polyurethane foams in a dynamic bed reactor

-d-Galactosidase (E 3.2.1.23) from Aspergillus oryzae was immobilized with polyurethane foam (PUF). Among several immobilization methods attempted in this work, the immobilized enzyme preparation by in-situ co-polymerization between enzyme and prepolymer HYPOL 3000 showed the highest activity. The intrinsic kinetics of PUF-immobilized enzyme was determined in a dynamic bed reactor, used to increase transport rates. The immobilization mechanism in PUF was studied by measurements of immobilized enzyme kinetics and by using scanning electron microscopy combined with immuno-gold labeling techniques. The results showed that immobilization was predominantly by covalent bonding between primary amino groups of -d-galactosidase and isocyanate groups of the prepolymers. Entrapment in the PUF micropores assisted the immobilization of enzymes, and adsorption on the surface of macropores was not important for immobilization. The bicinchoninic acid method was applied for the determination of PUF loading capacity and specific enzyme activity and used to determine enzyme deactivation during immobilization.     

Immobilization of glucose oxidase on chitosan-based porous composite membranes and their potential use in biosensors

The glucose oxidase (GO_x) enzyme was immobilized on chitosan-based porous composite membranes using a covalent bond between GO_x and the chitosan membrane. The chitosan-based porous membranes were prepared by the combination of the evaporation- and non-solvent-induced phase separation methods. To increase the membrane conductivity, carbon nanotubes (CNTs) were added to the chitosan solution. The resulting membranes were characterized in terms of water permeability, surface morphology and surface chemistry. Enzyme immobilization was performed on the chitosan membranes with and without activation using glutaraldehyde (GA). Three different configurations of working electrodes were evaluated to investigate the potential use of the modified membranes in biosensors. The results show that enzyme immobilization capacity was greater for membranes that had been activated than for membranes that had not been activated. In addition, activation increased the stability of the enzyme immobilization. The immobilization of GO_x on chitosan-based membranes was influenced by both pH and the concentration of the enzyme solution. The presence of CNTs significantly increased the electrical conductivity of the chitosan membranes. The evaluation of three different configurations of working electrodes suggested that the third configuration, which was composed of an electrode-mediator-(chitosan and carbon nanotube) structure and enzyme, is the best candidate for biosensor applications.