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1.
M. Yakup Arica Hande Soydogan Gulay Bayramoglu 《Bioprocess and biosystems engineering》2010,33(2):227-236
Poly(2-hydroxyethyl methacrylate/ethylenglycol dimethacrylate) beads were grafted with poly(glycidylmethacrylate) via surface
initiated atom transfer radical polymerization. Epoxy groups of the grafted polymer were modified in to sulfone groups. Sulfonated
beads were characterized by swelling studies, FT-IR, SEM and elemental analysis, and were used for reversible immobilization
of lipase. Under given experimental conditions, the beads had an adsorption capacity of 44.7 mg protein/g beads. The adsorbed
lipase on beads retained up to 67.4% of its initial activity. The immobilized lipase exhibited improved thermal and storage
stabilities over those of the free enzyme. The immobilized lipase could desorb 1.0 M NaCl solution at pH 8.0, and the sulfonated
beads can be repeatedly charged with fresh enzyme after inactivation upon use. 相似文献
2.
Jianzhi Wang Guanghui Zhao Yanfeng Li Xiao Liu Pingping Hou 《Applied microbiology and biotechnology》2013,97(2):681-692
A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of Fe3O4/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic Fe3O4/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization. 相似文献
3.
G��lay Bayramo?lu Beg��m Alt?nta? M. Yakup Ar?ca 《Bioprocess and biosystems engineering》2011,34(2):127-134
Polyacrylonitrile film (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence
of potassium dichromate as an oxidizing agent. The conductive films were used for immobilization of uricase. The surface resistance
of the conductive film in this work was found to be 0.97 kΩ/cm. The maximum amount of immobilized enzyme on conductive film
containing 2.4% PANI was about 216 μg/cm2. The optimum pH for free and immobilized enzymes was observed at 7.0 and 7.5, respectively. The K
m values for free and immobilized uricase were found to be 94 and 138 μM, respectively. V
max values were calculated as 1.87 and 1.63 U/mg protein for the free and immobilized enzymes, respectively. Immobilized uricase
exhibited ~68% of its original activity even after 2 months of storage at 4 °C while the free enzyme lost its initial activity
within 4 weeks. 相似文献
4.
Silica gel bead coated with macroporous chitosan layer (CTS-SiO2) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu2+, Zn2+, Ni2+ ions, respectively on CTS-SiO2, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu2+-CTS-SiO2 with the value of 4980 ± 125 IU g−1 of the adsorbent, while the maximal adsorption capacity for trypsin on Zn2+ and Ni2+ loaded adsorbent was 3762 ± 68 IU g−1 and 2636 ± 53 IU g−1, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO2 beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO2 could effectively digest BSA revealed by HPLC peptide mapping. 相似文献
5.
The present study describes the preparation of poly(HEMA-co-GMA) reactive membranes that were grafted with polyethylenimine (PEI) following UV photo-polymerization. The immobilization of tyrosinase was carried out via multi-point ionic interactions based on ---NH2 groups of PEI and Cu(II) ions. Tyrosinase is a copper-dependent enzyme, which should show a binding affinity for the chelated Cu(II) ions on the membrane surfaces. The tyrosinase immobilization was positively correlated with the input enzyme amount in the immobilization medium. The maximum tyrosinase immobilization capacities of the poly(HEMA-co-GMA)–PEI and poly(HEMA-co-GMA)–PEI–Cu(II) membranes were 19.3 and 24.6 mg/m2, respectively. The enzyme activity when assessed at various pH and temperatures gave broader range for immobilized preparations when compared to free enzyme. The poly(HEMA-co-GMA)–PEI–Cu(II) tyrosinase membranes retained 82% of their initial activity at the end of 120 h of continuous reaction. Moreover, upon storage for 3 months the activity of the immobilized membranes retained 46% of their initial levels. After deactivation of the enzyme, the poly(HEMA-co-GMA)–PEI membrane was easily regenerated, re-chelated with the Cu(II) ions and reloaded with the enzyme for repeated use. The mild immobilization conditions, easy and rapid membrane preparation, one-step enzyme adsorption at substantially higher levels and membrane reusability are the beneficial properties of such systems and offers promising potential in several biochemical processes. 相似文献
6.
Sinan Akgl Yasemin Kaar Serpil
zkara Handan Yavuz Adil Denizli M. Yakup Arica 《Journal of Molecular Catalysis .B, Enzymatic》2001,15(4-6)
Poly(2-hydroxyethylmethacrylate) (pHEMA) based flat sheet membrane was prepared by UV-initiated photopolymerization technique. The membrane was then grafted with
-histidine. Catalase immobilization onto the membrane from aqueous solutions containing different amounts of catalase at different pH was investigated in a batch system. The maximum catalase immobilization capacity of the pHEMA–histidine membrane was 86 μg cm−2. The activity yield was decreased with the increase of the enzyme loading. It was observed that there was a significant change between Vmax value of the free catalase and Vmax value of the adsorbed catalase on the pHEMA–histidine membrane. The Km value of the immobilized enzyme was higher 1.5 times than that of the free enzyme. Optimum operational temperature was 5°C higher than that of the free enzyme and was significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed without loss of adsorption capacity or enzyme activity. 相似文献
7.
Catalase was immobilized on the chitosan film that is a natural polymer. Studies were done on free catalase and immobilized catalase on chitosan film concerning the determination of optimum temperature, optimum pH, thermal stability, storage stability, operational stability, and kinetic parameters. It was determined that optimum temperature for free catalase and immobilized catalase on chitosan film is 25 degrees C, and optimum pH is 7.0. It was found as K(m) = 25.16 mM, V(max) = 24042 μmole/min mg protein for free catalase, K(m) = 27.67 mM, V(max) = 1022 μmole/min mg protein for immobilized catalase on chitosan. It was observed that there was a big difference between V(max) value of the free catalase and V(max) value of immobilized catalase on chitosan film whereas there were minor changes in the value of K(m) for free catalase and immobilized catalase. It was found that storage stability at 5 degrees C for immobilized catalase stored wet is greater than free catalase and immobilized catalase stored dry, and immobilized catalase showed a operational stability. 相似文献
8.
Douglas S. Clark James E. Bailey Richard Yen Alan Rembaum 《Enzyme and microbial technology》1984,6(7):317-320
Radiation-mediated grafting of polyacrolein onto poly(methyl methacrylate) microspheres has been shown to activate the particles for chymotrypsin (EC 3.4.21.1) immobilization. Treatment of porous polystyrene/magnetite particles with polyacrolein produced very small enzyme loading enhancement and significantly increased substrate diffusional resistance. 相似文献
9.
Soybean beta-amylase (EC 3.2.1.2) wap immobilized on phenylboronate-agarose by strong interactive binding. The insoluble derivative was active and more stable to temperature changes than the free enzyme. The absence of enzyme leakage even in the presence of substrate was demonstrated. Changes in pH over a wide range (4.0-8.0) did not affect the stability of the complex. The support could be recovered by sorbitol elution, which demonstrated the reversibility of the binding. Since the enzyme was not retained on phenylagarose under similar conditions, we rejected hydrophobic interactions as a cause of the strong binding of the enzyme to phenylboronate-agarose. We suggest that the bonding of the enzyme to the phenylboronate ligand occurs by a charge transfer mechanism between the trigonal boronate and the side chain nitrogenated groups. It was concluded that phenylboronate-agarose has good properties as a support, which recommends its use for the preparation of immobilized enzymes. 相似文献
10.
In this article, chemical modification with catecholic derivative in solution and subsequent immobilization of catalase (CAT) on titania submicrospheres (450–500 nm) were described. Catalase was first reacted with 3-(3,4-dihydroxyphenyl) propionic acid activated via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry. The above chemically modified CAT bearing catechol groups was then covalently bound to the surface of titania through the facile chelation reaction between the catechol groups and titania. The immobilized CAT retained 60% catalytic activity with a high loading capacity of 500 mg/g titania. Meanwhile, the immobilized CAT displayed enhanced operational stability, thermal stability and storage stability compared with native, modified CAT counterparts. In repeated batches of decomposition of hydrogen peroxide, after 10 and 19 cycles, the immobilized CAT maintained about 90% and 75% of its initial activity, respectively. 相似文献
11.
12.
In this study, titanate nanotubes (TNTs) with desirable biocompatibility and hydrophilicity have been synthesized by a facile and cost-effective alkaline hydrothermal method, and used to immobilize the enzyme. The characterization results reveal that the prepared TNTs have a regular tubular morphology with a length about 100–180 nm and an outer diameter about 10 nm, and a BET specific surface area of 305.4 m2 g−1. Catalase (CAT), as the model enzyme, was pre-modified by 3-(3,4-dihydroxyphenyl) propionic acid (3,4-diHPP) via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry, and then covalently immobilized on the TNTs surface by the chelation of catechol groups with Ti4+ ions. It is found that TNTs exhibits excellent performances as the immobilized supporter of enzyme: the enzyme loading is as high as 820 mg g of support−1; the relative activity of immobilized enzyme is about 60% of that of free enzyme; the immobilized CAT demonstrates enhanced storage and recycling stability. 相似文献
13.
D I Metelitsa E I Pliugacheva I N Ermolenko I P Liubliner F N Kaputski? V E Pratsenko 《Prikladnaia biokhimiia i mikrobiologiia》1992,28(4):531-538
We optimized the conditions of the covalent binding of bovine liver catalase to phosphate-cellulose matrices (gauze, granules, and paper with various surface density) and to acetate-cellulose porous membranes of different productivity. The capacity of the catalase binding to the molded cellulose carriers depends on the physico-chemical characteristics of the latter. The maximum concentration of the bound catalase after periodate oxidation of the carriers at room temperature was determined. The catalytic activity of the immobilized enzyme was quantified; under optimal conditions it is 24% for acetate-cellulose membranes and 24.3% for phosphate-cellulose paper and reduces with the increase of the total protein binding capacity of both the carriers. 相似文献
14.
A streptavidin-biotin system was utilized to prepare an antibody-polyadenylic acid conjugate which was subsequently attached to commercially available magnetic beads, Dynabeads oligo(dT)25. Biotinylated polyadenylic acid was combined with streptavidin and the resulting polyadenylic acid-streptavidin was conjugated with an antibody-biotin derivative. The immobilized antibody-polyadenylic acid conjugate was separated from the reaction mixture by hybridization with complementary oligonucleotide immobilized on the surface of Dynabeads oligo(dT)25. The immobilized antibody-polyadenylic acid can be released from the carrier, utilizing low-ionic-strength buffers. The system is intended to be utilized in cell sorting, using immobilized antibodies against cell surface antigens. Dissociation of antibody-containing conjugate from magnetic beads is essential for the isolation of viable cells via positive cell sorting. 相似文献
15.
Wang XH Li DP Wang WJ Feng QL Cui FZ Xu YX Song XH 《International journal of biological macromolecules》2003,33(1-3):95-100
Chitosan and heparin were covalently immobilized onto a poly(lactic acid-co-glycolic acid) (PLGA) surface using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) in a 2-morpholinoethane sulfonic acid (MES) buffer system. The properties of the modified PLGA surface and the control were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). The water contact angle of the modified film was greatly decreased and the element content on the surface of the films changed correspondingly. Platelet adhesion assay showed that blood compatibility of the chitosan/heparin modified film was improved while hepatocyte culture indicated that the cell compatibility of the modified film was enhanced. 相似文献
16.
Teresita Díaz Ulf St»hl Francisco Batista-Viera Jan Carlsson 《Biotechnology Techniques》1995,9(7):533-538
Summary Pullulanase was provided with up to nine de novo thiol groups through a two-step procedure without substantially affecting its enzymatic activity. The chemically modified enzyme was immobilized via disulfide bond formation on two kinds of thiolreactive gels (pyridyldisulfide- and thiolsulfonate-substituted agarose). Thiolation of pullulanase improved both the immobilization yield and the apparent specific activity of the derivatives. 相似文献
17.
Three kinds of ampholites, i.e., 3-aminopropionic acid (NH2C2H4COOH), (2-aminoethyl)phosphonic acid (NH2C2H4PO3H2), and 2-aminoethane-1-sulfonic acid (NH2C2H4SO3H), were introduced into an epoxy group-containing polymer brush grafted onto a porous hollow-fiber membrane with a porosity of 70% and pore size of 0.36 microm. The amphoteric group density of the hollow-fiber ranged from 0.50 to 0.72 mmol/g. Three kinds of proteins, i.e., lactoferrin (Lf), cytochrome c (Cyt c), and lysozyme (Ly), were captured by the amphoteric polymer brush during the permeation of the protein solution across the ampholite-immobilized porous hollow-fiber membrane. Multilayer binding of the protein to the amphoteric polymer brush, with a degree of multilayer binding of 3.3, 8.6, and 15 for Lf, Cyt c, and Ly, respectively, with the (2-aminoethyl)phosphonic acid-immobilized porous hollow-fiber membrane, was demonstrated with a negligible diffusional mass-transfer resistance of the protein to the ampholite immobilized. The 2-aminoethane-1-sulfonic acid-immobilized porous hollow-fiber membrane exhibited the lowest initial flux of the protein solution, 0.41 m/h at a transmembrane pressure of 0.1 MPa and 298 K, and the highest equilibrium binding capacity of the protein, e.g., 130 mg/g for lysozyme. Extension and shrinkage of the amphoteric polymer brushes were observed during the binding and elution of the proteins. 相似文献
18.
The immobilization of penicillin G acylase on chitosan 总被引:3,自引:0,他引:3
19.
Ligands and proteins were covalently but reversibly immobilized on polyacrylamide gels using novel acrylic monomers whose syntheses are reported here. These reagents have an acrylyl group at one end for copolymerization into gels, an N-succinimidyl ester at the other allowing rapid immobilization of molecules having an available primary amino group, and a cleavable disulfide bond in the middle. Two immobilization methods were developed using these reagents. In the first method, a ligand with a primary amino group was treated with the immobilization reagent in anhydrous ethanol and the resulting amide derivative was purified and copolymerized with acrylamide and bisacrylamide resulting in the desired reversible immobilization. In the second method, the immobilization reagents (at densities up to 50 mumol/ml) were directly copolymerized with acrylamide and bisacrylamide to form activated gels of the desired shape and porosity. Proteins or other ligands in aqueous buffers were then added to the activated gels resulting in their covalent immobilization. Ligands or proteins immobilized using the methods reported here remained stably bound even when gels were subjected to boiling in detergents or high-ionic-strength buffers. Immobilized ligands were readily released (greater than 97%) from gels by treatment with quantitative amounts of aqueous dithiothreitol (DTT) under mild conditions. Immobilized proteins were also released (up to 87%) from the gels by DTT treatment. Small ligands (e.g., aminohexyl glycosides), active enzymes, and glycoproteins were immobilized, and then recovered, using these reagents. 相似文献
20.
Adhesion and viability of two enterococcal strains on covalently grafted chitosan and chitosan/kappa-carrageenan multilayers 总被引:1,自引:0,他引:1
Bratskaya S Marinin D Simon F Synytska A Zschoche S Busscher HJ Jager D van der Mei HC 《Biomacromolecules》2007,8(9):2960-2968
Chitosans are natural aminopolysaccharides, whose low cytotoxicity suggests their potential use for nonadhesive, antibacterial coatings on biomaterials implant surfaces. Here, the antiadhesive behavior and ability to kill bacteria upon adhesion ("contact killing") of chitosan coatings were evaluated for two strains of Enterococcus faecalis, isolated from clogged biliary stents. Chitosan coatings covalently grafted or applied as chitosan/kappa-carrageenan multilayers were characterized by ellipsometry, scanning force microscopy (SFM), X-ray photoelectron spectroscopy (XPS), and electrokinetic measurements. Decreases in initial bacterial deposition rates and the number of bacteria adhering in a more advanced state of the adhesion process were observed on both types of modified surfaces, with more pronounced effects on highly hydrated multilayers. Adhesion of negatively charged enterococci was slightly enhanced on chitosan-terminated multilayers, but antibacterial effect was absent on kappa-carrageenan-terminated multilayers. Thus, the efficacy of multilayers remains an interesting interplay between the promoting effect of cationically charged groups on adhesion of negatively charged bacteria and, on the other hand, their antibacterial effects. 相似文献