Preparation of PEGylated uricase attached magnetic nanowires and application for uric acid oxidation.

The present study aims to immobilize the uricase enzyme on magnetic nanowires and to examine its potential for use in the treatment of gout. For this, Au/Ni/Au nanowires were synthesized using a polycarbonate membrane template by the sequential electrodeposition of Au, Ni, and Au, respectively. The uricase enzyme was covalently attached to these nanowires and was also coated with PEG. Optimum enzymatic conditions, kinetic parameters, thermal, storage, and operational stability were determined by performing enzymatic activity tests of free and immobilized uricase. Additionally, the efficacy of both enzyme preparations in artificial human serum and the presence of protease was also investigated. Experimental results showed that immobilized uricase showed higher stability than free uricase in all studied conditions. The potential of immobilized uricase to oxidize uric acid in artificial serum was also investigated and it was found that immobilized preparation demonstrated approximately 6 times higher activity than that of the free enzyme. The results of this study showed that uricase-attached nanowires oxidized uric acid effectively and are promising in the treatment of gout.

Inulinase Immobilized Lectin Affinity Magnetic Nanoparticles for Inulin Hydrolysis.

In this presented paper, concanavalin A-modified cysteine-functionalized Fe3O4/Ag core/shell magnetic nanoparticles were synthesized and used as a support material for inulinase enzyme, which has been intensively used for the preparation of high-fructose syrup by hydrolyzing inulin. Inulinase adsorption capacity of Con A-functionalized Ag-coated magnetic nanoparticles was optimized by changing medium pH, temperature, and initial inulinase concentration, and maximum inulinase adsorption capacity was found to be 655.32 mg/g nanoparticle by using 1.00 mg/mL of inulinase solution in pH 3.0 buffer system at 25 °C. Finally, efficient inulin degradation capacity of the inulinase immobilized magnetic nanoparticles was demonstrated by TLC studies and released fructose amount was determined as 0.533 mg/mL only within the 5 min of hydrolysis. This newly developed hydrolysis strategy holds considerable promise to produce high-fructose syrup in many industries.

Bacteria killer enzyme attached magnetic nanoparticles.

The objective of the present work was to develop immobilized lysozyme systems through adsorption on magnetic nanoparticles for potential usage in bacteria killing studies. For this, magnetic poly(HEMA-GMA) nanoparticles were prepared by surfactant free emulsion polymerization technique and functionalized with dye ligand Reactive Green 5. Synthesized magnetic nanoparticles were then characterized by FTIR, SEM, EDX and ESR studies. Particle size range of the polymers was found to be as 90-120 nm. Magnetic behavior was also demonstrated by ESR with the g value of 2.48. Maximum lysozyme loading was found to be as 1045.1 mg/g nanopolymer. Repeated usability of the magnetic nanoparticles was also studied. Immobilized form of lysozyme protected 85.85% of its initial activity at the end of the immobilization process. Bacteria killing capacity of the lysozyme immobilized magnetic nanoparticles were investigated by using Micrococcus lysodeikticus bacteria and it was demonstrated that all bacteria were successfully destroyed by the lysozyme immobilized magnetic nanoparticles within 5 min.

Enzymatic Activity of Urokinase Immobilized onto Cu2+-Chelated Cibacron Blue F3GA-Derived Poly (HEMA) Magnetic Nanoparticles.

In this presented work, magnetic poly(2-hydroxyethyl methacrylate) (p (HEMA)) nanoparticles were synthesized by surfactant-free emulsion polymerization technique. Cibacron Blue F3GA was covalently attached to the magnetic p (HEMA) nanoparticles and Cu2+ ions were then chelated with dye molecules. Synthesized magnetic nanoparticles were spherical with the diameter of 80 nm and exhibited magnetic character. Incorporation rate of Cibacron Blue for magnetic nanoparticles was found to be 28.125-µmol/g polymer. Loaded amount of Cu2+ ions was calculated as 10.229-µmol/g polymer. These Cu2+-Cibacron Blue F3GA-derived magnetic p (HEMA) nanoparticles were used for urokinase adsorption under different conditions (i.e., pH, enzyme initial concentration, ionic strength, temperature). Maximum adsorption capacity was found to be 630.43-mg/g polymer, and it was observed that Langmuir adsorption isotherm was applicable in this adsorption process. The adsorbed urokinase was desorbed from the Cu2+-Cibacron Blue F3GA-derived magnetic p (HEMA) nanoparticles by using 1.0 M of NaCl with the desorption rate of 96%. It was also demonstrated that adsorption capacity did not change significantly after five adsorption/desorption cycles.

Ultrasound-propelled nanowire motors enhance asparaginase enzymatic activity against cancer cells.

Ultrasound-(US) propelled nanowires consisting of Au/Ni/Au/PEDOT-PPy-COOH segments are modified with asparaginase enzyme and applied as an effective anti-cancer agent. After immobilization of asparaginase onto the surface of the nanowire motors, the enzyme displays enhanced thermal and pH stabilities, improved resistance towards protease, and higher affinity for the substrate. The fast motion of the motor-carrying asparaginase leads to greatly accelerated biocatalytic depletion of asparagine and hence to a significantly enhanced inhibition efficacy against El4 lymphoma cancer cells (92%) as compared to free enzyme counterpart (17%) and other control groups. Such enhanced enzymatic activity against cancer cells is attributed to the fast motion of the motors which facilitates the interaction between the enzyme and the cancer cells. While asparaginase and EL4 tumor cells are used as a model system in the present study for cancer cell inhibition, the same mechanism can be expanded to other types of enzymes and biomolecules for the corresponding biofunctions.

Immobilization of alcohol dehydrogenase onto metal-chelated cryogels.

In this presented work, poly(HEMA-GMA) cryogel was synthesized and used for the immobilization of alcohol dehydrogenase. For this, synthesized cryogels were functionalized with iminodiacetic acid and chelated with Zn(2+). This metal-chelated cryogels were used for the alcohol dehydrogenase immobilization and their kinetic parameters were compared with free enzyme. Optimum pH was found to be 7.0 for both immobilized and free enzyme preparations, while temperature optima for free and immobilized alcohol dehydrogenase was 25 °C. Kinetic constants such as K(m), V(max), and k(cat) for free and immobilized form of alcohol dehydrogenase were also investigated. k(cat) value of free enzyme was found to be 3743.9 min(-1), while k(cat) for immobilized enzyme was 3165.7 min(-1). Thermal stability of the free and immobilized alcohol dehydrogenase was studied and stability of the immobilized enzyme was found to be higher than free form. Also, operational stability and reusability profile of the immobilized alcohol dehydrogenase were investigated. Finally, storage stability of the free and immobilized alcohol dehydrogenase was studied, and at the end of the 60 days storage, it was demonstrated that, immobilized alcohol dehydrogenase was exhibited high stability than that of free enzyme.

Dye functionalized cryogel columns for reversible lysozyme adsorption.

In this study, poly (methyl methacrylate-glycidyl methacrylate) [poly(MMA-GMA)] cryogels were prepared by radical cryocopolymerization of MMA with GMA as a functional comonomer. Reactive Green 19 dye was then attached to the cryogel by nucleophilic substitution reaction, and this dye-attached cryogel column was used for lysozyme adsorption. Characterization of the cryogel was performed by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, Brunauer-Emmett-Teller, and energy dispersive X-ray analysis. Pore size of the cryogels was 15-30 µm and pores were interconnected structure. Attached amount of Reactive Green 19 to cryogel support was calculated as 106.25 µmol/g cryogel. Lysozyme adsorption studies were carried out by using a continuous system. It was found that the maximum amount of lysozyme adsorption (32 mg/g cryogel) obtained from experimental results was found to be approximately same with the calculated Langmuir adsorption capacity (33 mg/g cryogel). Desorption of adsorbed lysozyme was carried out by using 1.5 M NaCl in pH 4.5 acetate buffer, and desorption yield was found to be 97.4%. Cryogels were very stable, and it was found that there was no remarkable reduction in the adsorption capacity at the end of ten adsorption-desorption cycles. As a result, Reactive Green 19-attached cryogels have great advantages such as easy preparation, rapid adsorption, and desorption, being economic and allowing the direct separation of proteins.

A novel affinity disks for bovine serum albumin purification.

The adsorption characteristics of bovine serum albumin (BSA) onto the supermacroporous poly(hydroxyethylmethacrylate)-Reactive Green 19 [p(HEMA)-RG] cryogel disks have been investigated in this paper. p(HEMA) cryogel disks were prepared by radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Reactive Green (RG) 19 was covalently attached to the p(HEMA) cryogel disks. These disks were used in BSA adsorption studies to interrogate the effects of pH, initial protein concentration, ionic strength, and temperature. BSA adsorption capacity of the p(HEMA)-RG cryogel disk was significantly improved after the incorporation of RG. Adsorption capacity reached a plateau value at about 0.8 mg/mL at pH 4.0. The amount of adsorbed BSA decreased from 37.7 to 13.9 mg/g with increasing NaCl concentration. The enthalpy of BSA adsorption onto the p(HEMA)-RG cryogel disk was calculated as -58.4 kJ/mol. The adsorption equilibrium isotherm was fitted well by the Freundlich model. BSA was desorbed from cryogel disks (over 90 %) using 0.5 M NaSCN, and the purity of desorbed BSA was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The experimental results showed that the p(HEMA)-RG cryogel disks have potential for the quick protein separation and purification process.

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels.

In this study, poly(2-hydroxyethyl methacrylate-glycidylmethacrylate) [poly(HEMA-GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N'-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA-GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30-50 µm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA-GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA-GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0M NaCI at pH8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS-PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase.

Reversible immobilization of urease by using bacterial cellulose nanofibers.

In this work, bacterial cellulose nanofibers were produced by using the Gluconacetobacter hansenii HE1 strain. These nanofibers were derivatized with dye affinity ligand Reactive Green 5, and these newly synthesized dye-attached nanofibers were used for affinity adsorption of urease. Reactive Green 5-attached nanofibers were characterized by Fourier transform infrared spectroscopy, SEM, and energy-dispersive x-ray spectroscopy analysis. Some adsorption conditions which significantly affect the adsorption efficiency were investigated. The maximum urease adsorption capacity was found to be 240 mg/g nanofiber in pH 6.0 and at room temperature. Dye-free plain nanofibers also used for studying nonspecific urease adsorption onto plain nanofibers and nonspecific adsorption were found to be negligible (3.5 mg/g nanofiber). Prepared dye-attached nanofibers can be used in five successive adsorption/desorption steps without any decrease in their urease adsorption capacity. The desorption rate of the adsorbed urease was found to be 98.9 %. The activity of the urease was also investigated, and it was found that free and desorbed urease from the dye-attached nanofibers showed similar specific activity.

Immobilization of inulinase on concanavalin A-attached super macroporous cryogel for production of high-fructose syrup.

In this study, concanavalin A (Con A)-attached poly(ethylene glycol dimethacrylate) [poly(EGDMA)] cryogels were used for immobilization of Aspergillus niger inulinase. For this purposes, the monolithic cryogel column was prepared by radical cryocopolymerization of EGDMA as a monomer and N,N'-methylene bisacrylamide as a crosslinker. Then, Con A was attached by covalent binding onto amino-activated poly(EGDMA) cryogel via glutaraldehyde activation. Characterization of cryogels was performed by FTIR, EDX, and SEM studies. Poly(EGDMA) cryogels were highly porous and pore size was found to be approximately 50-100 µm. Con A-attached poly(EGDMA) cryogels was used in the adsorption of inulinase from aqueous solutions. Adsorption of inulinase on the Con A-attached poly(EGDMA) cryogel was performed in continuous system and the effects of pH, inulinase concentration, and flow rate on adsorption were investigated. The maximum amount of inulinase adsorption was calculated to be 27.85 mg/g cryogel at 1.0 mg/mL inulinase concentration and in acetate buffer at pH 4.0. Immobilized inulinase was effectively used in continuous preparation of high-fructose syrup. Inulin was converted to fructose in a continuous system and released fructose concentration was found to be 0.23 mg/mL at the end of 5 min of hydrolysis. High-fructose content of the syrup was demonstrated by thin layer chromatography.

Synthesis and characterization of amino acid containing Cu(II) chelated nanoparticles for lysozyme adsorption.

Immobilized metal ion affinity chromatography (IMAC) is a useful method for adsorption of proteins that have an affinity for transition metal ions. In this study, poly(hydroxyethyl methacrylate-methacryloyl-L-tryptophan) (PHEMATrp) nanoparticles were prepared by surfactant free emulsion polymerization. Then, Cu(II) ions were chelated on the PHEMATrp nanoparticles to be used in lysozyme adsorption studies in batch system. The maximum lysozyme adsorption capacity of the PHEMATrp nanoparticles was found to be 326.9 mg/g polymer at pH 7.0. The nonspecific lysozyme adsorption onto the PHEMA nanoparticles was negligible. In terms of protein desorption, it was observed that adsorbed lysozyme was readily desorbed in medium containing 1.0 M NaCl. The results showed that the metal-chelated PHEMATrp nanoparticles can be considered as a good adsorbent for lysozyme purification.

Metal-chelating nanopolymers for antibody purification from human plasma.

The purification of immunoglobulin G (IgG) from human plasma was performed by using a novel metal-chelated adsorbent with nano size. The non-porous nanoparticles were produced by surfactant free emulsion polymerization of ethylene glycol dimethacrylate (EDMA) and 2-methacryloylamidohistidine (MAH). Then, Cu(II) ions were chelated on the nanoparticles. The nano-poly(EDMA-MAH) nanoparticles were characterized by Fourier transform infrared, scanning electron microscope, atomic force microscope and elemental analysis. The non-porous nanoparticles were spherical form and have 100-250 nm size distribution. The maximum IgG adsorption capacity of the Cu(II) chelated nanoparticles was found to be 463 mg/g polymer at pH 7.0 in HEPES buffer. Desorption of IgG was performed by 1.0 M NaCl and desorption rate was found to be 97 %. IgG was obtained from human plasma with purity of 94 % (up to 578 mg/g polymer). The non-porous nanoparticles allowed one-step purification of IgG from human plasma.

Purification of papain using reactive green 5 attached supermacroporous monolithic cryogel.

Supermacroporous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] monolithic cryogel was prepared by radical cryocopolymerization of HEMA with N,N'-methylene bisacrylamide as crosslinker. Reactive Green 5 dye was immobilized to the cryogel with nucleophilic substitution reaction, and this dye attached cryogel column was used for affinity purification of papain from Carica papaya latex. Reactive Green 5-immobilized poly(HEMA) cryogel was characterized by swelling studies, Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersive X-ray analysis. Maximum papain adsorption capacity was found to be 68.5 mg/g polymer while nonspecific papain adsorption onto plain cryogel was negligible (3.07 mg/g polymer). Papain from C. papaya was purified 42-fold in single step with dye attached cryogel, and purity of papain was shown by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Concanavalin A immobilized poly(ethylene glycol dimethacrylate) based affinity cryogel matrix and usability of invertase immobilization.

Concanavalin A (Con A) immobilized supermacroporous poly(ethylene glycol dimethacrylate) [poly(EGDMA)] monolithic cryogel column was prepared by radical cryocopolymerization of EGDMA as a monomer and N,N'-methylene-bisacrylamide as a crosslinker. Bioligand Con A was then immobilized by covalent binding onto poly(EGDMA) cryogel via glutaraldehyde activation [Con A-poly(EGDMA)]. Con A-poly(EGDMA) cryogel was characterized by swelling studies and scanning electron microscopy. The monolithic cryogel contained a continuous polymeric matrix having interconnected pores of 10-50 µm size. The equilibrium swelling degree of the cryogel was 15.01 g H2O/g dry cryogel. Con A-poly(EGDMA) cryogel was used in the adsorption/desorption of invertase from aqueous solutions. The maximum amount of invertase adsorption from aqueous solution in acetate buffer was 55.45 mg/g polymer at pH 5.0. Con A-poly(EGDMA) cryogels were used for repetitive adsorption/desorption of invertase without noticeable loss in invertase adsorption capacity after 10 cycles.

A novel support for antibody purification: fatty acid attached chitosan beads.

Linoleic acid attached chitosan beads [poly(LA-Ch)] (1.25 microm in diameter) are obtained by the formation of amide linkages between linoleic acid and chitosan. Poly(LA-Ch) beads are characterized by FTIR, TEM, and swelling studies. Poly(LA-Ch) beads are used for the purification of immunoglobulin-G (IgG) from human plasma in a batch system. The maximum IgG adsorption is observed at pH 7.0 for HEPES buffer. IgG adsorption onto the plain chitosan beads is found to be negligible. Adsorption values up to 136.7 mg/g from aqueous solutions are obtained by poly(LA-Ch) beads. IgG adsorption saw an increase as a result of increasing temperature. Higher amounts of IgG are adsorbed from human plasma (up to 390 mg/g) with a purity of 92%. The adsorption phenomena appeared to follow a typical Langmuir isotherm. It is observed that IgG could be repeatedly adsorbed and desorbed without significant loss when we take into account the adsorption amount. It is concluded that the poly(LA-Ch) beads allowed one-step purification of IgG from human plasma.

Antioxidant activity and proline content of leaf extracts from Dorystoechas hastata.

Dorystoechas hastata (D. hastata) is a monotypic plant endemic to Antalya province of Turkey. D. hastata leaves are used to make a tea locally called "çalba tea". Diethyl ether (E), ethanol (A), and water (W) were used for the sequential preparation of extracts from dried D. hastata leaves. A hot water extract (S) was also prepared by directly boiling the powdered plant in water. The antioxidant activities of the extracts were tested by ferric thiocyanate (FTC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging methods. E extract exhibited the greatest antioxidant activity with FTC method, whereas S extract exhibited the lowest IC50 value (6.17±0.53µg/ml) for DPPH radical scavenging activity. Total phenolic contents of the extracts were estimated by Folin-Ciocalteu method and S extract was found to contain the highest amount (554.17±20.83mg GAE/g extract) of phenolics. Extract A contained highest flavonoid content and there was a inverse linear correlation (R(2)=0.926) between IC50 values for DPPH radical scavenging activity and flavonoid contents of all extracts. Reducing power of extracts increased in a concentration-dependent manner. S extract was found to possess higher reducing power than equivalent amount of ascorbic acid at 20 and 25µg/ml concentrations. Linear correlation between reducing power and concentration of E, A, and W extracts (R(2)>0.95) was observed. A, W, and S extracts contained relatively high levels of proline. The results presented suggest that D. hastata may provide a natural source of antioxidants and proline.