Covalent Binding Method Research Articles

IMMOBILIZTION OF ORGANOPHOSPHORUS HYDROLASE ENZYME ON FERRIC MAGNETIC NANOPARTICLES AND INVESTIGATION OF IMMOBILIZED ENZYME STABILITY

In the present study, organophosphorus hydrolase enzyme on Functionalized ferric magnetic nanoparticles was immobilized by the covalent binding method. The Optimized amount for parameters of mg EDAC/mg nanoparticles, enzyme units (U)/mg nanoparticles, reaction time, and pH were determined to be 6.125, 0.1341, 3 h and 6.15 respectively. The amount of immobilization yield according to the enzyme activity was obtained to be 70% and also the amount of immobilized enzyme on nanoparticles was 0.25 U/mg nanoparticles. Stability studies showed significant increase in immobilized enzyme stability at 4, 25 and 45°C. The stability of Immobilized enzyme showed a 6.3-fold increase in comparison to free enzyme at 4°C. The results demonstrated that the pH stability of the immobilized enzyme significantly increased in comparison with free enzyme. The immobilized enzyme was usable and recoverable for seven cycles. The results depicted that 80% of enzyme activity was retained after fifth cycle. FTIR test showed the covalent binding of enzyme to magnetic nanoparticles’ surface and the modified enzyme magnetic nanoparticles property was superparamagnetic by vibrating sample magnetometer test.

Immobilization of native type I collagen on polypropylene fabrics as a substrate for HepG2 cell culture.

Background/aims The critical part of a bio-artificial liver device is establishment of a bioreactor filled with liver cells. However, it is still unclear how to maintain benign cell function while achieving the sufficient cell quantity. In the current study, we aim to establish a novel carrier for the culture of HepG2 cells, a liver cell line, by modifying polypropylene nonwoven fabrics with native type I collagen. Methods "Piranha" solution, KH-550 and glutaraldehyde subsequently were used to bridge native type I collagen and polypropylene nonwoven fabrics. The type I collagen-coupled polypropylene nonwoven fabric was characterized by XPS, SEM, ATR-FTIR and water contact angle measurement. Furthermore, the biocompatibility between HepG2 cells and fiber film is evaluated by the ability of cell proliferation, albumin secretion, as well as urea synthesis. Results The coating of collagen onto polypropylene fabrics was more efficient using the chemical covalent binding method than direct immersion, which was validated by the presence of collagen-related elements and chemical bond. The adding of collagen in polypropylene fabrics promoted hydrophilicity and HepG2 cell adherence. Additionally, enhanced cell proliferation, increased albumin secretion and urea synthesis were observed in HepG2 cells growing on collagen-coated polypropylene fabrics. Conclusions The collagen coated polypropylene nonwoven fabrics, acting as a feasible substrate for HepG2 cell culture, may be used as a promising liver cell carrier for artificial liver reactor.

A Portable Evanescent Wave Optic Fiber Immunosensor for Sensitive and Rapid Detection of 2,4-D in Water Samples

A portable evanescent wave optic ?ber immunosensor was developed for detection of 2,4-dichlorophenoxyacetic acid(2,4-D) using a indirect competitive immunoassay. In this paper, hapten conjugates 2,4-D-OVA were immobilized with covalent binding methods. After pre-reacting, 2,4-D-antibody-Cy5.5 in sample specifically recognized the 2,4-D antigens binding sites on surface of the optical fiber probe. Under optimum conditions, 2,4-D could be detected in less than 18 min for each assay cycle. The regeneration of the optic ?ber surface allowed more than 200 times without losing performance. The limits of detection of 0.039 ug/L and the quantitative detection range of 0.47 - 81.02 ug/L were obtained when the concentration of 2,4-D was 1 mg/L. This immunosensor shows great potential in rapid simultaneous detection of 2,4-D in waters samples.

Recent advances and development of supported ionic liquids

Ionic liquids (ILs), as a new class of green medium, have unique properties, such as low melting point, low vapor pressure, wide electrochemical window, adjustable structures. Supported ILs, combined of ILs with different kinds of carriers, retain original fluidity or structural characteristics of ILs. They are highly reactive, highly selective, easy to recycle and low-energy consuming, which break the traditional restriction of ILs in industrial applications and provide new routes for large-scale application of ILs. This review summarized the recent progress of supported ILs and introduced their classification and preparation methods including impregnation, covalent binding and sol-gel method. Then the screening-predicting model and recent advances of calculation are elaborated. Finally, the application progress of supported ILs in catalysis and separation and its future development are discussed.

Covalent immobilization of cellulase in application of biotransformation of ginsenoside Rb1

Cellulase was immobilized on the surface of carrageenan beads activated by using polyethyleneimine and glutaraldehyde, then immobilized cellulase was used to transform ginsenoside Rb1. In this work, we studied the functionalization of carrageenan with amine terminal functional group for the covalent immobilization of cellulase. The process of immobilization was performed by amination process on the surface of the carrageenan gel beads with polyethylenimine followed by activation with glutaraldehyde to finally immobilize the enzyme. Different factors affecting the amination and activation processes were studied and their effects on the catalytic activity of the immobilized cellulase were followed, then Fourier transform infrared spectroscopy and scanning electron microscopy were applied to verify the process. Furthermore, the optimum conditions of transformation of ginsenoside Rb1 were also studied and showed better stability for the immobilized enzyme. The best results were obtained using carrageenan as support and covalent binding method, which consisted of amination process (0.5% PEI (pH 8))) for 3h and 3% GA in activation process for 3h. These experiments gave optimum reaction condition of temperature at 60°C and pH 5.0. Enzyme after immobilization exhibited high activity with broader pH and temperature range. Full conversion and kinetic parameters Km and Vmax for them were also studied. The Km of immobilized enzyme, 3.64mM, is lower than free one, 4.82mM; and the Vmax of immobilized enzyme, 0.04mM, is lower than free one, 0.06mM. Results detected also show after using for 5 consecutive times, the immobilized enzyme can retain 60% of its initial activity.

The immobilisation of proteases produced by SSF onto functionalized magnetic nanoparticles: Application in the hydrolysis of different protein sources

Alkaline proteases produced from protein-rich waste (hair waste and soya residues) by solid state fermentation (SSF) were immobilised onto functionalized magnetic iron oxide nanoparticles (MNPs) using glutaraldehyde as a crosslinking agent. The covalent binding method had a better immobilisation yield compared to simple adsorption, retaining 93%–96% (459±106U/mg nanoparticles, 319±34U/mg nanoparticles) of hair waste and soya residues proteases, respectively after crosslinking with 5% glutaraldehyde for 6h. However, the adsorption immobilisation yield was 47%–54% after 8h for both proteases. MNPs and immobilised proteases were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and electron diffraction. Our results indicated successful crosslinking between the proteases and amino-functionalized MNPs. The operational stability (pH and temperature) and storage stability of free and immobilised enzyme were also analysed. Despite the fact that the optimum pH of free and immobilised proteases was identical in the alkaline region, the immobilised proteases reached their optimum condition at higher temperatures (40°C–60°C). After 2 months of storage at 4°C, the immobilised proteases showed good stability, retaining more than 85% of their initial activity. The high magnetic response of MNPs render an ease of separation and reusability, which contributes to the residual activity of both immobilised proteases on MNPs retaining more than 60% of their initial values after seven hydrolytic cycles. These results showed the enhancement of the stability of the crosslinking interactions between the proteases and nanoparticles. The immobilised proteases were capable of hydrolysing selected proteins (casein, oat bran protein isolate, and egg white albumin). However, differences in the degree of hydrolysis were observed, depending on the combination of the protease and type of substrate used.

Screening of thrombin inhibitors from phenolic acids using enzyme-immobilized magnetic beads through direct covalent binding by ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry

Thrombin was immobilized on dynabeads®M-270 epoxy by direct covalent binding method for the first time. The enzyme coated magnetic beads were combined with ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry technique to establish a simple, rapid and reliable approach for screening thrombin inhibitors from Danshen preparation. The conjugation of thrombin to the magnetic beads was characterized using scanning electron microscope, transmission electron microscope and infrared spectroscopy, and the enzyme activity was determined by the analysis of enzyme-bead ratio and peak areas of target compounds. Several factors including amount of magnetic beads, type of elution solvent, incubation temperature and time were optimized. Additionally, two thrombin-bound compounds (protocatechuic aldehyde and salvianolic acid C) in Danshen injection were validated by conventional inhibitory assay and the IC50 values were 286.11 and 66.09μg/mL, respectively. Our findings suggested that the proposed method was efficient in screening active compounds from medicinal plants.

Phosvitin phosphopeptide preparation using immobilised trypsin and enhancing calcium absorption in growing rats

We demonstrate a new, efficient method for the preparation of phosvitin phosphopeptides using immobilised-trypsin enzymolysis technology. Immobilised trypsin was prepared using a covalent binding method, and then was added to degrade egg yolk phosvitin for the production of phosphopeptides. In our results, the prepared immobilised trypsin demonstrated a higher hydrolysing activity toward phosvitin than free trypsin, and the hydrolysing activity was retained well even after trypsin was repeatedly used up to five times. Interestingly, the phosvitin phosphopeptides prepared with immobilised trypsin demonstrated a lower N/P ratio and a higher calcium-binding efficiency than those prepared with free trypsin. Furthermore, phosphopeptides significantly increased the rate of calcium absorption and serum calcium content in vivo. Based on these results, we conclude that trypsin immobilised onto chitosan has a greater phosvitin hydrolysing activity than free trypsin, and the prepared phosphopeptides can be used as a new calcium supplement to significantly increase calcium absorption in growing rats.

Immobilisation of organophosphate hydrolase on mesoporous and Stöber particles

Hexahistidine-tagged organophosphate hydrolase (His6-OPH), an organophosphate degrading enzyme, immobilised by physical adsorption and covalent binding methods on mesoporous silica (MPS) particles, was examined as a biocatalyst for organophosphate hydrolysis within an aqueous medium and for providing an efficient tool in the detection/detoxification of paraoxon. His6-OPH was immobilised on mesoporous silica particles of different pore size distributions (10–40 and 6 nm) and sizes (5 μm and 500 nm), through covalent attachment and physical adsorption. The biocatalysts showed good activities and enhanced stabilities with respect to the free enzymes, depending on the material and used immobilisation technique. Biomaterial based on hexahistidine-tagged organophosphorus hydrolase (His6-OPH) was developed by enzyme immobilisation into/onto two types of silica particles with different porosity. While adsorbed enzymes showed higher activities, covalent attached enzymes showed minimal or no losses in activity as well as reusability.

Design and Construction of Magnetic Nanoparticles Incorporated with a Chitosan and Poly (vinyl) Alcohol Cryogel and its Application for Immobilization of Horseradish Peroxidase

Magnetic nanoparticles were synthesized by simple co-precipitation method in aqueous medium. The structural characteristics of the powders were studied by XRD and the sizes of nanoparticles were measured with dynamic light scattering. The prepared magnetic nanoparticles were about 43nm in diameter and were then subsequently incorporated into a chitosan and poly (vinyl alcohol) cryogel and applied to immobilize the horseradish peroxidase through covalent binding method (MNP-CS-PVA-HRP cryogel microbar). The MNP-CS-PVA- HRP cryogel microbar was applied for the detection of hydrogen peroxide. The horseradish peroxidase can catalytically oxidize the substrate of hydrogen peroxide and o-dianisidine, generating blown colour that are proportional to the concentrations of hydrogen peroxide. The results showed that the absorption values at 430nm increased with the hydrogen peroxide concentrations ranging from 10-1,000mM. The immobilized horseradish peroxidase on magnetic nanoparticles retained enzyme activity up to the 10th reusable. The proposed approach confirmed that the magnetic nanoparticles not only possessed enzyme activity but also showed potential application in varieties of simple, robust, and cost-effective analytical methods in the future.

Effect of spacer modification on enzymatic synthetic and hydrolytic activities of immobilized trypsin

A thorough comparison of spacer-mediated covalent and non-covalent immobilization of trypsin on micro-magnetic particles was accomplished in the present study. Trypsin was coupled via diaminoalkanes, aminoalkanoic acids, bovine serum albumin (BSA), and biotin-derivate spacers onto magnetic particles. A comparison of resulting synthetic and hydrolytic activities after immobilization was performed. Whereas hydrolytic trypsin activity was measured employing N-α-benzoyl-dl-arginine 4-nitroanilide (BAPNA) assay, synthetic trypsin activity was measured employing a dipeptide synthesis assay. Within spacer-mediated trypsin immobilization, diaminoalkanes, aminoalkanoic acids and biotin spacers showed up to 40% increased synthetic specific activity of trypsin compared to the spacer-free method. Within the hydrolytic reaction type, coupling of trypsin via diaminoalkanes and biotin spacers resulted in a specific activity increase of up to 30%. BSA-bound trypsin displayed only minor increasing effects on both activities of trypsin. Furthermore, protein loading-dependent specific synthetic and hydrolytic activities were evaluated for 8-aminooctanoic acid, 12-aminododecanoic acid and 1,12-diamonododecane as spacers and compared to the direct covalent binding method. The protein binding capacity of spacer-modified particles was lower compared to the direct binding method. Synthetic activity of 8-aminooctanoic acid-bound trypsin was higher than in the case of the spacer-free method over a broad protein loading range with an up to 10-fold increase. The hydrolytic activity was increased by 64% using 8-aminooctanoic acid as spacer within a lower protein loading range. Spacer-bound trypsin showed a slightly lowered reusability over ten sequential cycles compared to spacer-free covalent binding method.

Preparation of Immobilized Recombinant Tubulin Beta(TuBb) on Chitosan Nanoparticles by Covalent Binding Method

Objective In order to screen TuBb inhibitors, this paper describes the preparation of immobilized TuBb on chitosan nanoparticles. Methods TuBb was immobilized onto chitosan nanoparticles by covalent binding method. Results The results of the univariate test indicated that the highest immobilized yield can be obtained when the optimal immobilization condition was 1 mg of TuBb, 0.5 mol/L of buffer solution with pH 6.5, immobilization 30 min and immobilization at 0-4 °C. Conclusions The authors conclude that the immobilized TuBb maintain the catalysis properties and can be used as the screening of TuBb inhibitors.

Quantitative Analysis of a Promising Cancer Biomarker, Calretinin, by a Biosensing System Based on Simple and Effective Immobilization Process

AbstractCalretinin (CAL) is calcium binding protein, and its levels in blood and cerebrospinal fluids are increased, since its expression is increased various cancer types. A novel biosensor system fabricated by immobilization of a specific antibody to CAL, anti‐Calretinin (anti‐CAL), onto a gold electrode surface via an effective covalent binding method using mercaptohexanol, epichlorohydrin, and ethanolamine was reported for the sensitive, selective, and accurate analysis of CAL. The proposed biosensor showed a linear calibration range between 1 ng/mL and 5 ng/mL. LOD and LOQ values were determined as 0.11 ng/mL and 0.38 ng/mL, respectively. The standard deviation related to the reproducibility of the new biosensor system was calculated as 3.95 %. Lastly, in order to state the applicability of the biosensor to early diagnosis of CAL in practice, artificial serum samples spiked with CAL have been analyzed by the proposed biosensor.

Immobilization of Aspergillus niger lipase on chitosan-coated magnetic nanoparticles using two covalent-binding methods

Aspergillus niger lipase immobilization by covalent binding on chitosan-coated magnetic nanoparticles (CMNP), obtained by one-step co-precipitation, was studied. Hydroxyl and amino groups of support were activated using glycidol and glutaraldehyde, respectively. Fourier transform infrared spectrometry, high-resolution transmission electron microscopy and thermogravimetric analysis confirmed reaction of these coupling agents with the enzyme and achievement of a successful immobilization. The derivatives showed activities of 309.5 ± 2.0 and 266.2 ± 2.8 U (g support)(-1) for the CMNP treated with glutaraldehyde and with glycidol, respectively. Immobilization enhanced the enzyme stability against changes of pH and temperature, compared to free lipase. Furthermore, the kinetic parameters K m and V max were determined for the free and immobilized enzyme. K m value quantified for enzyme immobilized by means of glutaraldehyde was 1.7 times lowers than for free lipase. High storage stability during 50 days was observed in the immobilized derivatives. Finally, immobilized derivatives retained above 80% of their initial activity after 15 hydrolytic cycles. The immobilized enzyme can be applied in various biotechnological processes involving magnetic separation.

An in-line photonic biosensor for monitoring of glucose concentrations.

This paper presents two PDMS photonic biosensor designs that can be used for continuous monitoring of glucose concentrations. The first design, the internally immobilized sensor, consists of a reactor chamber, micro-lenses and self-alignment structures for fiber optics positioning. This sensor design allows optical detection of glucose concentrations under continuous glucose flow conditions of 33 μL/h based on internal co-immobilization of glucose oxidase (GOX) and horseradish peroxidase (HRP) on the internal PDMS surface of the reactor chamber. For this design, two co-immobilization methods, the simple adsorption and the covalent binding (PEG) methods were tested. Experiments showed successful results when using the covalent binding (PEG) method, where glucose concentrations up to 5 mM with a coefficient of determination (R2) of 0.99 and a limit of detection of 0.26 mM are detectable. The second design is a modified version of the internally immobilized sensor, where a microbead chamber and a beads filling channel are integrated into the sensor. This modification enabled external co-immobilization of enzymes covalently onto functionalized silica microbeads and allows binding a huge amount of HRP and GOX enzymes on the microbeads surfaces which increases the interaction area between immobilized enzymes and the analyte. This has a positive effect on the amount and rate of chemical reactions taking place inside the chamber. The sensor was tested under continuous glucose flow conditions and was found to be able to detect glucose concentrations up to 10 mM with R2 of 0.98 and a limit of detection of 0.7 mM. Such results are very promising for the application in photonic LOC systems used for online analysis.

Direct Immobilization of Glucose Oxidase on the Hydrophilic Copolymer Support Containing Oxirane

Glucose oxidase (GOD) (EC 1.1.3.4) is an oxido-reductase that catalyses the oxidation action of glucose to hydrogen peroxide and D-glucono-δ-lactone [. GOD is widely used in the determination of free glucose in body fluids, in vegetal raw material and in the food industry [. It also has many applications in biotechnologies, typically enzyme assays for biochemistry including biosensors in nanotechnologies. Free GOD will be denatured and inactivated rapidly due to its structural instability under extreme pH or temperature [. Four immobilizing methods, including physical adsorption, covalent binding, crosslinking and embedding methods, were used to improve their economic feasibility. Covalent binding combined active functional group monomers of carriers onto enzymes [4-. Researches showed that oxirane groups can react with-NH2 and-HS of enzymes under mild conditions so that the enzyme molecules were immobilized on the copolymer support containing oxirane [.

Preparation of clay–poly(glycidyl methacrylate) composite support for immobilization of cellulase

A composite material was synthesized by grafting of glycidyl methacrylate onto clay using surface initiation atom transfer radical polymerization (SI-ATRP) technique. Epoxy group of the grafted p(GMA) chains was reacted with hexamethylenediamine (HMDA). The composite material was characterized using scanning electron microscopy (SEM) and FTIR. Cellulase from Trichoderma reesei was immobilized on the aminated composite particles via adsorption and covalent binding methods. The amounts of adsorbed enzyme on the aminated composite particles were 43.4mg/g. The recovered activities of the adsorbed and covalently immobilized cellulase were found to be 87.7% and 73.2% for the substrate, carboxymethyl cellulose (CMC, 1.0g/L). The pH stabilities and thermo-stabilities, repeated use and storage stabilities of both immobilized cellulase preparations were evaluated. The immobilized cellulase preparations have better stabilities and higher retained activities with respect to pH, temperature and storage than those of the free enzyme. Operational stability of the covalently immobilized cellulase was tested in a continuous flow system, and the activity loss was about 4% at the end of 48h operation period.

Immobilization on magnetic nanoparticles of the recombinant trehalose synthase from Deinococcus geothermalis

In our study the gene encoding trehalose synthase from Deinococcus geothermalis was cloned and overexpressed in Escherichia coli Rosetta (DE3)pLysS. Wild-type trehalose synthase has been purified from host protein after cell disruption and precipitation at 20% ammonium sulphate saturation. Recombinant trehalose synthase was immobilized onto glutaraldehyde activated silanized magnetic ferrous-ferric oxide by using covalent binding method. The morphology and surface of the obtained particles were characterized using SEM. These images show that all samples have a particle size below 30nm. The obtained immobilized preparation has specific activity of 0.134U/g support when measured at 40°C using maltose as substrate. Immobilization process was almost fully completed after 30min of the reaction at 30°C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10mM. No significant differences in optimal pH and temperature were observed upon immobilization. The immobilized trehalose synthase exhibited mass transfer limitation, which is reflected by higher KM and activation energy values. In addition, immobilized trehalose synthase was easily separated from the reaction medium by an external magnetic field and retained 82% of its initial activity after successive twelve repeated batches reaction.

Mesoporous silicas synthesis and application for lignin peroxidase immobilization by covalent binding method

Immobilization of enzymes on mesoporous silicas (MS) allows for good reusability. MS with two-dimensional hexagonal pores in diameter up to 14.13 nm were synthesized using Pluronic P123 as template and 1,3,5-triisopropylbenzene as a swelling agent in acetate buffer. The surface of MS was modified by the silanization reagents 3-aminopropyltriethoxysilane. Lignin peroxidase (LiP) was successfully immobilized on the modified MS through covalent binding method by four agents: glutaraldehyde, 1,4-phenylene diisothiocyanate, cyanotic chloride and water-soluble carbodiimide. Results showed that cyanotic chloride provided the best performance for LiP immobilization. The loaded protein concentration was 12.15 mg/g and the immobilized LiP activity was 812.9 U/L. Immobilized LiP had better pH stability. Acid Orange II was used to examine the reusability of immobilized LiP, showing more than 50% of the dye was decolorized at the fifth cycle.

Immobilization of Candida rugosa lipase on hexagonal mesoporous silicas and selective esterification in nonaqueous medium

Candida rugosa lipases (CRLs) immobilized by physical adsorption, cross-linking and covalent binding methods on a MSU-H type mesoporous silica previously modified organically by different strategies, respectively, were examined as biocatalysts for esterification of conjugated linoleic acid (CLA) and ethanol in nonaqueous medium. MSU-H silica was modified by nonionic surfactant of triblock copolymer Pluronic P123, amino-functionalization and glutaraldehyde-grafting and confirmed by FT-IR analysis. Interaction mechanisms of CRLs and supports involve covalent and non-covalent interactions including electrostatic repulsion and hydrophobic interaction at pH 7. The immobilized CRLs containing surfactant were prepared by cross-linking via entrapping CRL aggregates inside the pores of silicas. The surfactant located inside the silicas could interfacially activate the immobilized CRLs and favored catalytic esterification. The biocatalyst containing 38wt.% of surfactant afforded 1111.1U/mg of specific activity about eight times higher than soluble CRL, and maximal 56.7% of total CLA esterification with 96.5% of 9c, 11t-CLA isomer esterification degree. The immobilized-CRL with 64.5mg/g of loading amount of protein exhibited maximal hydrolytic activity of 2945.3U/g-support for grafting glutaraldehyde. This derivative showed a high level of esterification activity and operational stability and remained 43.2–46.9% of total esterification for 32h consecutive four runs.