Immobilization of yeasts on titanium activated inorganic supports

Summary A study of the immobilization of yeast cells with invertase activity by the metal link method was performed. Baker's yeast cells were immobilized on titanium activated porous silica support and on its alkylamine and aldehyde derivatives, their initial activities being 19.6, 39.9 and 10.6 U/ml of reactor respectively. When crosslinking of the immobilized cells was performed, an initial activity of 48.2 U/ml was achieved on the titanium activated support. Batch long-term stability tests were car ried out for 400 hours and the crosslinked preparations showed an unsta ble behaviour compared with the very stable preparations obtained with the simple metal-link method.A higher activity (56.2 U/ml) was obtained when a titanium activated macroporous support, pumice stone, was used as cell carrier, which compared favourably with calcium alginate entrapped cells (17.7 – 31.3 U/ml)     

Immobilization of proteins on liposome surface

Different methods for the immobilization of proteins (enzymes and immunoglobulins) on liposomes are reviewed. These methods include adsorption, incorporation, covalent binding and binding of preliminarily modified proteins with the liposomal surface. Literature data are compared, optimal immobilization conditions are discussed and requirements for the immobilization processes are formulated. The possibility of using liposome-protein conjugates for drug targeting is especially discussed.     

Experimental estimation of the effect of antibiotics, immobilized on the surface of titanium implant, on the development of the inflammatory process

Titanium implants with immobilized lincomycin and ampicillin were studied on rats with respect to the effect on development of inflammatory reactions. The data on the concentrations of sialic acids in the blood during the postoperative period and the histological examinations showed that lower concentrations of sialic acids, early fibroblastic reaction, productive nature of inflammation in the main and rapid formation of the fibrous connective tissue capsule around the implants were the characteristic features and advantages of the use of the implants with the immobilized antibiotics as compared to the use of the routine titanium implants.     

Immobilization of immunoglobulin-G-binding domain of Protein A on a gold surface modified with biotin ligase

Protein A from Staphylococcus aureus specifically binds to the Fc region of immunoglobulin G (IgG) and is widely used as a scaffold for the immobilization of IgG antibodies on solid supports. It is known that the oriented immobilization of Protein A on solid supports enhances its antibody-binding capability in comparison with immobilization in a random manner. In the current work, we developed a novel method for the oriented immobilization of the IgG-binding domain of Protein A based on the biotinylation reaction from archaeon Sulfolobus tokodaii. Biotinylation from S. tokodaii has a unique property in that the enzyme, biotin protein ligase (BPL), forms a stable complex with its biotinylated substrate protein, biotin carboxyl carrier protein (BCCP). Here, BCCP was fused to the IgG-binding domain of Protein A, and the resulting fusion protein was immobilized on the BPL-modified gold surface of the sensor chip for quartz crystal microbalance through complexation between BCCP and BPL. The layer of the IgG-binding domain prepared in this way successfully captured the antibody, and the captured antibody retained high antigen-binding capability.     

Determination of the electron temperature in microplasma discharges excited on a titanium surface

Results are presented from experimental studies of the emission spectra of microplasma discharges excited on a titanium surface by a pulsed plasma flow. The excited discharges are maintained by current pulses with an amplitude of 200 A and a duration of 20 ms. Analysis of more than 100 spectral lines of titanium atoms and ions in the wavelength range of 350–800 nm shows that the electron temperature of a microplasma discharge is in the range of 0.2–1.3 eV.     

Immobilization of immunoglobulin on a quartz surface by BrCN without loss of antigen binding capability.

BrCN-activated quartz was used for the immobilization of monoclonal immunoglobulins. BrCN-activated quartz keeps its ability to quantitatively bind immunoglobulins for at least 8 h. The surface density of the immobilized immunoglobulins was 100 ng cm-2 and they were found to retain their ability to bind antigen in a molar ratio of 1:2 for not less than three months.     

Effect of surface roughness of ground titanium on initial cell adhesion

The effect of surface roughness of ground Ti on the initial adhesion of osteoblast-like U-2 OS cells was investigated in this study. Different numbers (#120, #600, and #1500) of SiC sandpaper and two Al2O3 polishing powder (0.3 and 1 microm) were used to prepare the metal specimens with varying degrees of surface roughness. Surface roughness (Ra) was measured by profilometry. Surface topography was observed using an atomic force microscope. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay was used to measure the optical density (OD) of specimens after 2 h of cell incubation. The OD value was analyzed by one-way ANOVA for analyzing the factor of surface roughness. Crystal violet staining technique was used to characterize the cell spreading. Results showed that the specimen of #1500 Ti (Ra: 0.15 microm) had the highest OD value. The specimens polished with 0.3 and 1 microm Al2O3 powder (Ra: 0.05 and 0.07 microm) exhibited the worst cell adhesion behavior. Contact guidance of cells could be observed on the rougher #600 and #120 specimens (Ra: 0.33 and 1.20 microm). This study concludes that the surface roughness (Ra: 0.05-1.20 microm) of ground Ti has a highly significant influence on the initial adhesion of osteoblast-like U-2 OS cells. The ground Ti with an Ra of 0.15 microm shows the optimal cell adhesion behavior with respect to either the rougher or smoother specimens.     

Immobilization of yeast cells by adhesion to glass surface using polyethylenimine

Summary A method has been described for immobilization of yeast cells by adsorption to glass surface using polyethylenimine for coating cells or glass or both. The immobilized yeast cells were found to be viable and adhered strongly as a monolayer, which could not be desorbed by washing with running tap water or under extreme conditions of pH and ionic strength or during repeated uses in sucrose solutions.     

Proteome analysis of the plasma protein layer adsorbed to a rough titanium surface

In this study a label-free proteomic approach was used to investigate the composition of the layer of protein adsorbed to rough titanium (Ti) after exposure to human blood plasma. The influence of the protein layer on the surface free energy (SFE) of the Ti was evaluated by contact angle measurements. Ti discs were incubated with blood plasma for 180?min at 37?°C, and the proteins recovered were subjected to liquid chromatography coupled to tandem mass spectrometry analysis. A total of 129 different peptides were identified and assigned to 25 distinct plasma proteins. The most abundant proteins were fibronectin, serum albumin, apolipoprotein A-I, and fibrinogen, comprising 74.54% of the total spectral counts. Moreover, the protein layer increased the SFE of the Ti (p?<?0.05). The layer adsorbed to the rough Ti surface was composed mainly of proteins related to cell adhesion, molecule transportation, and coagulation processes, creating a polar and hydrophilic interface for subsequent interactions with host cells.     

Immobilization of enzymes on crosslinked gelatin particles activated with various forms and complexes of titanium(IV) species

A number of methods of activating the surface of glutaraldehyde crosslinked gelatin beads with titanium(IV) compounds, for subsequent enzyme coupling, have been investigated. Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.1.3) was so immobilized using titanium(IV)-urea, -acrylamide, -citric acid and -lactose complexes; however, immobilized enzyme preparations with low activities were obtained (0.36–1.28 U g^?1). Activation with uncomplexed titanium(IV) chloride, however, of both moist and freeze-dried crosslinked gelatin particles resulted in highly active immobilized glucoamylase preparations (1.74–26.6 U g^?1). Dual immobilized enzyme conjugates of glucoamylase and invertase (β-d-fructofuranosidase, EC 3.2.1.26) were also prepared using this method. Invertase was served on the entrapped enzyme while glucoamylase was coupled on the surface of titanium(IV)-activated gelatin pre-entrapped invertase particles. A dual gelatin coupled glucoamylase/gelatin entrapped glucoamylase was prepared (3.8 U g^?1) and ～72.5% of the total combined activity was due to the surface bound enzyme.     

Immobilization of alkaline polygalacturonate lyase from Bacillus subtilis on the surface of bacterial polyhydroxyalkanoate nano-granules

Applied Microbiology and Biotechnology - Alkaline polygalacturonate lyase (PGL), one of the pectinolytic enzymes, has been widely used for the bioscouring of cotton fibers, biodegumming, and...     

Immobilization of oligonucleotides on glass surface using an efficient heterobifunctional reagent through maleimide-thiol combination chemistry

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl) cyclohexanamide (TPMC), was developed for the immobilization of thiol-modified oligonucleotides on an unmodified glass surface. The heterobifunctionality of the reagent was used for the construction of a DNA microarray in which the triethoxysilyl functionality has specificity toward a glass surface, whereas the maleimide functionality has thiol-modified oligonucleotides via a stable thioether linkage. Immobilization of DNA was achieved by two alternative approaches. In the first approach, the reagent TPMC was treated with oligonucleotides to get triethoxysilyl-oligonucleotide conjugate, which was then covalently attached via specific triethoxysilyl functionality to an unmodified glass surface. In the second approach, the reagent was first covalently linked with an unmodified glass surface to get maleimide functionality on a glass surface, which was then used for the immobilization of oligonucleotides via a stable thioether linkage. The applicability of the reagent was explored by hybridization studies with the fluorescein-labeled complementary DNA strand and in mismatch discrimination.     

Immobilization of immunoglobulins using lectins with an immune sensor method based on surface plasmon resonance

The effect of different lectins upon the response of immune sensor based on surface plasmon resonance (SPR) was investigated. Lectins affinity to carbohydrates of the IgG can be used to increase the density and orientation of IgG molecules at their immobilisation on the sensor surface. Conditions were elaborated for enhancement of immune sensor response in comparison with that one for bare or preliminary treated with dodecanthiol thin gold sensor surface. It was shown that human IgG revealed maximal affinity to wheat germ lectin and mouse monoclonal antibodies and rabbit IgG--to helix pomatia lectin. Pig antibodies, similar to human IgG, showed the greatest affinity to wheat germ lectin.     

Immobilization of Penicillium funiculosum cellulase on a soluble polymer

The three cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] components of Penicillium funiculosum have been immobilized on a soluble, high molecular weight polymer, poly(vinyl alcohol), using carbodiimide. The immobilized enzyme retained over 90% of cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], and exo-β-d-glucanase [1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21] activities. The bound enzyme catalysed the hydrolysis of alkali-treated bagasse with a greater efficiency than the free cellulase. The potential for reuse of the immobilized system was studied using membrane filters and the system was found to be active for three cycles.     

Effect of caries preventive products on the growth of bacterial biofilm on titanium surface

Fluorides may affect the oxide layer on titanium surface. Caries preventive mouthwashes or gels contain fluorides and are applied at low pH. The aim of the present work was to study whether various concentrations of fluoride at acidic pH cause changes in the surface structure on the polished region of Ti implants, and alter the adherence and colonization of bacteria. Commercially pure Ti grade 4 discs with a polished surface were treated with a mouthwash containing 0.025% fluoride, a gel containing 1.25% fluoride or a 1% aqueous solution of NaF (pH 4.5). The change of surface roughness of the samples and the colonization of Porphyromonas gingivalis strains were studied by scanning electron microscopy after 5 days of anaerobic incubation. The quantity of the bacterial protein was determined by protein assay analysis. Agents with high fluoride concentration at acidic pH increased the roughness of the Ti surface. A slight increase in the amount of bacteria was found on the surfaces treated with 1% NaF and gel in comparison with the control surface. This study suggested that a high fluoride concentration at acidic pH may hinder the development of a healthy transgingival epithelial junction on Ti implants, due to bacterial colonization.     

Immobilization of lipase by ultrafiltration and cross-linking onto the polysulfone membrane surface

In this study, a biphasic enzymatic membrane reactor was made by immobilizing Candida Rugosa lipase onto the dense surface of polysulfone ultrafiltration membrane by filtration and then cross-linking with glutaraldehyde solution. The reactor was further applied for the hydrolysis of olive oil, the performance of which was evaluated in respect of apparent reaction rate based on the amount of fatty acids extracted into the aqueous phase per minute and per membrane surface. It was found that the ultrafiltration and cross-linking process greatly improved the reaction rate per unit membrane area and the enzyme lifetime. The highest reaction rate reached 0.089 micromol FFA/min cm2 when the enzyme loading density was 0.098 mg/cm2. The results also indicated that the performance of lipase immobilized on the membrane surface was superior to that immobilized in the pores, and the apparent reaction rate and stability of immobilized lipases were improved greatly after cross-linking. It suggested that immobilization of enzymes by filtration and then cross-linking the enzymes onto the membrane surface is a simple and convenient way to prepare a high-activity immobilized enzyme membrane.     

Immobilization of carbohydrate epitopes for surface plasmon resonance using the Staudinger ligation

The detection of carbohydrate-protein interactions is often performed using techniques that require surface immobilization of the lectin or the glycan. A commonly used assay for lectin binding is surface plasmon resonance (SPR). We describe an implementation of the Staudinger ligation as a method to immobilize carbohydrate epitopes to a biosensor surface. This was accomplished by first introducing an azide functionality to a carboxymethyldextran surface, followed by reaction with a phosphane-modified carbohydrate ligand. The chemistry employed is extremely mild and was easily adapted to a commercial biosensor system. Using this approach, we investigated the binding of jacalin and wheat germ agglutinin (WGA) to galactose, lactose, and N-acetyl-lactosamine. We observed that WGA binding shows evidence of multivalent interaction with the surface. Additionally, we found that jacalin binding was influenced by the presence of a flexible and hydrophobic galactosyl aglycone.