Preparation of immobilized proteins covalently coupled through silane coupling agents to inorganic supports

Enzymes were first immobilized on inorganic supports through silane coupling agents over 25 yr ago. Since that initial report, literally hundreds of laboratories have utilized this methodology for the immobilization of enzymes, antigens, antibodies, receptors, and other high and low mol wt compounds. Today silane coupling is one of the commonly used techniques in the arsenal of the biochemist for the binding of material of all sorts to inorganic surfaces. Inorganic materials come in a variety of shapes, sizes, and characteristics. Today silane coupling is one of the most used coupling methods for the preparation of biosensing devices. Sol-gel entrapped enzymes are also produced by the application of silane technology by the polymerization of the silane to form glass-like materials with entrapped protein. This review will discuss the general preparation and characterization of silane coupled proteins with special emphasis on enzymes and describe in detail the actual methods for the silanization and specific chemical coupling of proteins to the silanized carrier.     

Platinum nanocatalysts immobilized on latex supports

Several latex dispersions of different hydrophobicity were investigated with respect to their ability to adsorb platinum nanoparticles that had been reduced in their presence. Two reduction methods were tested, specifically the slower method of refluxing the alcoholic solutions and the more rapid method of reaction with KBH₄. The immobilization of the metal particles and their nanosize dimensions were demonstrated by transmission electron microscopy, and their catalytic activity was tested by the hydrogenation of cyclohexene as a model reaction. Some additional immobilized platinum nanoparticles were prepared in the presence of various protective polymers. This can lead to various advantages with respect to, for instance, the stability and the catalytic properties of these materials. Even in the presence of such additional protective polymers, the platinum nanoparticles remained immobilized for some of the hydrophobic latexes both before and after catalytic hydrogenations. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35 : 1207–1216, 1997     

Biocatalytic reactors based on ribonuclease A immobilized on macroporous monolithic supports

Immobilized enzyme reactors (IMERs) produced by the covalent attachment of ribonuclease A to macroporous methacrylate-based monolithic supports using different experimental approaches are discussed and compared. Enzyme immobilization was carried out by direct covalent binding, as well as through attachment via a polymer spacer. The kinetic properties of an IMER operating in either recirculation mode or zonal elution mode were studied. Additionally, the effect of flow rate on the bioconversion efficiency of each IMER sample was examined.

Horseradish peroxidase immobilized through its carboxylic groups onto a polyacrylonitrile membrane: comparison of enzyme performances with inorganic beaded supports

A hydrophilic polyacrylonitrile (PAN) flat sheet membrane was aminated (8.5 μmol of NH₂/mg of dry support) for covalent binding of horseradish peroxidase (HRP), mediated by the soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Silica microbeads derivatized by silanization, to yield an aminated support, and commercial aminated glass microbeads were also coupled to HRP with EDC or activated with glutaraldehyde. The immobilized enzyme activities were determined in a batch enzyme reactor with an external loop, the highest specific immobilized HRP activity being obtained on the glass support (55.8U/mg of protein). Continuous operational stability studies showed that hydrophilic PAN membrane led to the highest retention of HRP activity after an overall period of 35 h, with a normalized productivity of 59.5 μmol of H₂O₂ reduced/(h·U_{immob HRP}).     

Alkylation of anions on solid inorganic supports : an interpretation of the function of the supports

In the anionic alkylation (CH₃ CO₂K + n C₈ H₁₇ Br) on solid inorganic supports, silica impregnated with cationic surfactant appears to be as effective as alumina, while silica bearing ammonium groups covalently bonded to the support (SPHEROSIL QMA) exhibits increased effectiveness. An interpretation based upon the superficial charge of the solid surfaces is proposed.     

Chlorotitanium (IV) tetradentate Schiff‐base complex immobilized on inorganic supports: Support type and other factors having effect on ethylene polymerization activity

A titanium complex with [O,N,N,O]‐type tetradentate Schiff base (LTiCl₂), never used before in polymerization of olefins, was immobilized on silica‐ and magnesium‐type carriers, and it was used in ethylene polymerization. The conducted research revealed that the catalytic properties of the complex LTiCl₂ supported on those carriers were different for both the catalytic systems studied, and simultaneously they turned out different from those of the unsupported system. The supported catalysts require the use of Me₃Al, Et₃Al, or MAO as the activator to be able to offer high catalytic activities, whereas Et₂AlCl is needed for the nonsupported catalyst. This finding, together with considerable changes in polymerization yields and in properties of polymers versus composition of the catalytic system, suggest that there are different types of active sites in the studied catalysts. The catalyst anchored on the carrier produced in the reaction of MgCl₂·3.4EtOH with Et₂AlCl is definitely the most active one within the support systems tested. Its activity remarkably increases with the increasing reaction temperature. Moreover, that catalyst does not undergo deactivation over the studied period of time, irrespective of the type of the activator used and of the process temperature. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 4811–4821, 2009     

Affinity of trypsin for amidine derivatives immobilized on dextran-coated silica supports.

The pancreas contains two very analogous enzymes: trypsin and chymotrypsin. These two enzymes are very similar in their physicochemical characteristics and are therefore quite difficult to separate by classical purification procedures. They constitute a good model for affinity chromatography. It was previously demonstrated that amidine derivatives are able to interact strongly and specifically with these serine proteases and are often used as ligand in affinity chromatography. To understand the trypsin interaction mechanism, we synthesized different amidines and immobilised them with or without spacer arm on silica beads previously coated by dextran substituted with a calculated amount of positively charged diethylaminoethyl functions, in order to minimize the non-specific interactions of silanol groups of the silica material. First the affinity constant and the adsorption capacity of these supports for trypsin were determined in batch procedures, then they were used in affinity chromatography. The effects of ionic strength, pH and competitive inhibitors on proteins desorption were also studied. Last, to demonstrate the importance of passivation, the chromatographic performances of dextran-coated silica phases and a commercial support grafted with the same amidine were compared.     

Catalysts with mixed ligands on immobilized supports. Electronic and steric advantages

[reaction: see text] Immobilized dirhodium(II) catalysts having mixed chiral ligands enhance reactivity (AH = azetidinone) and influence stereoselectivity in cyclopropanation and carbon-hydrogen insertion reactions.     

Preparation and properties of enzymes immobilized on supports activated by metal ions

Summary 1. Preparations of trypsin, -chymotrypsin, and glucose oxidase immobilized on synthetic and polysaccharide supports charged with transition-metal ions contain 3–64 mg of protein per 1 g of support.2. The activity of immobilized enzymes amounts to 100–10,000 U/g. The activity yield is 2.2–90X%.3. The pH dependence of the enzymes is shifted in the alkaline direction by 0.26–1.19 units. The Michaelis constants and inhibitor constants have decreased by factors of 1.5–21.4. The mechanism of the fixation of enzymes is determined by the formation of coordination bonds and by inclusion in inorganic gels. The properties of the enzymes are due to the surface charge of the activated supports.Institute of Biochemistry, Academy of Sciences of the Lithuanian SSR, Vilnius. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 629–637, September–October, 1978.     

Palladium nanoclusters immobilized on defective nanodiamond-graphene core-shell supports for semihydrogenation of phenylacetylene

We report a nanocarbon material with nanodiamond(ND) core and graphene shell(ND@G) as a support for Pd nanocatalysts. The designed catalyst performed good selectivity of styrene(85.2%) at full conversion of phenylacetylene and superior stability under mild conditions. Supported Pd catalysts are characterized by means of high resolution transmission electron microscopy(HRTEM), Raman, X-ray diffraction(XRD), X-ray photoelectron spectroscopy(XPS) and H_2 temperature-programmed reduction(H_2-TPR).The results clearly show that formation of the strong metal-support interaction(SMSI) between Pd nanoclusters and the defective graphene shell helpfully modifies the selectivity and stability of the Pd-based catalysts.     

Platinum carbonyl derived catalysts on inorganic and organic supports: a comparative study

Fumed silica, silica gel, silica-alumina and cross-linked (5.5%) polystyrene have been functionalized with quaternary ammonium groups and the Chini cluster [Pt₁₂(CO)₂₄]²⁻ has been anchored onto these functionalized materials by ion pairing. A catalyst has also been prepared by the adsorption of Na₂[Pt₁₂(CO)₂₄] on unfunctionalized fumed silica. The catalytic activities of the resultant materials, and that of commercially purchased 5% platinum on alumina have been studied for the hydrogenation of a variety of unsaturated compounds. The substrates studied are: α-acetamidocinnamic acid, cyclohexanone, acetophenone, methyl pyruvate, ethyl acetoacetate, nitrobenzene and benzonitrile. Compared to the polystyrene supported catalyst, the inorganic oxide supported catalysts have higher surface areas and for most of the substrates have notably higher activities. The functionalized fumed silica-based catalyst gives higher conversions than functionalized silica gel and silica-alumina-based catalysts. In the hydrogenation of acetophenone and ethyl acetoacetate, the functionalized fumed silica-based catalyst show superior activity compared to the commercial platinum catalyst, and the catalyst made by conventional adsorption method. In benzonitrile hydrogenation with all the cluster-derived catalysts a hydrazine derivative is selectively formed, but when the commercial platinum catalyst is used benzyl amine is the main product.     

Microwave-assisted solvent-free synthesis of a quinoline-3,4-dicarboximide library on inorganic solid supports

Inorganic solid supports are useful media for the rapid and efficient synthesis of a library of quinoline-3,4-dicarboximides. In particular, wet clay K10 was shown to be the best medium for the condensation reaction between 2-methylquinoline-3,4-dicarboxylic anhydride and several primary amines. Microwave irradiation is essential for a rapid and complete conversion.     

Flow-injection determination of inorganic pyrophosphate with use of an enzyme thermistor containing immobilized inorganic pyrophosphate

Inorganic pyrophosphate immobilized on controlled-pore glass is used in a simple flow enzyme thermistor system. The heat produced in hydrolysis of pyrophosphate is enhanced. by using Tris-HCl buffer, pH 7.2, containing 1 mM magnesium chloride, as carrier stream. The calibration graph is linear for 0.1–20 mM pyrophosphate; 500 assays are possible without loss of enzyme activity. For 0.5-ml injections of 10 mM pyrophosphate, the relative standard deviation was 2.0% (n=30). A single determination takes 6 min. Calcuim and strontium interfere.     

Linkers and catalysts immobilized on oxide supports: New insights by solid-state NMR spectroscopy

This review article describes classical and modern solid-state NMR methods that allow to gain insight into catalyst systems where one or two metal complexes are bound to oxide supports via bifunctional phosphine linkers, such as (EtO)₃Si(CH₂)₃PPh₂. Many aspects of the immobilized molecular catalysts can be elucidated with the corresponding NMR technique. The bulk of the support can be studied, as well as the interface of the support with the ethoxysilane. With respect to the linkers, their structural integrity and mobility are as easy to investigate by classical CP/MAS and high-resolution magic angle spinning (HRMAS) NMR techniques, as their adsorption behavior. Even electrostatic bonding to the support via phosphonium groups can be proven by solid-state NMR. For the immobilized catalysts, leaching, and even “horizontal” translational mobility effects, as probed by HRMAS NMR under “realistic conditions” in the presence of solvents, are described.     

Ultrathin silica films immobilized on gold supports: fabrication, characterization, and modification

A novel method for covalent attachment of ultrathin silica films (thickness <10 nm) to gold substrates is reported. Silica layers were prepared using spin-coating of sol-gel precursor solutions onto gold substrates that were cleaned and oxidized using UV photo-oxidation in an ozone atmosphere. The gold oxide layer resulting from this process acts as a wetting control and adhesive agent for the ultrathin silica layer. Control of silica layer thickness between approximately 6 and 60 nm through modification of precursor solution composition or by repetitive deposition is demonstrated. Films were characterized using infrared spectroscopy, ellipsometry, atomic force microscopy, and cyclic voltammetry. For the standard deposition parameters developed here, films were determined to be 5.5 +/- 0.75 nm thick, and were stable in aqueous solutions ranging in pH from 2 to 10 for at least 30 min. Films contained nanoscopic defects with radii of 相似文献   

Effect of surface hydrophobicity/hydrophilicity of mesoporous supports on the activity of immobilized lipase

Taking advantage of the virtue of hydrophilic surface, lipase was firstly immobilized on SBA-15 as a support. Then the surface of the SBA-15 with enzyme entrapped inside the channels was modified by grafting with organic moieties. It has been found that the silylation with n-decyltrimethoxysilane (DE) and 3-(trimethoxysilyl)propyl methacrylate (MA) following the lipase immobilization increases the surface hydrophobicity. But the surface modified by MA shows more hydrophilicity than that modified by DE. The activity assay indicates that the hydrolytic activity for the hydrolysis of insoluble or partly soluble substrates increases with enhanced surface hydrophobicity.     

Effect of the nature of carbonaceous supports on the bioelectrocatalytic activity of peroxidase, immobilized on the supports, in the hydrogen peroxide reduction reaction

Electrochemical properties of such disperse carbonaceous materials as acetylene black AD-100, finely divided colloidal graphite (FCG), ultradisperse diamond (UDD), and carbon nanotubes (CNT) are examined. Effect of the nature of disperse carbonaceous supports on bioelectrocatalytic activity of adsorbed peroxidase (POD) in the hydrogen peroxide reduction reaction is investigated. It is shown that the hydrogen peroxide reduction on the biocatalysts studied proceeds in conditions of direct bioelectrocatalysis independently of the disperse-support type. It is also demonstrated that the biocatalysts’ activity depends on the structure and properties of the surface of the supports defining the magnitude of the POD adsorption in an orientation favorable for direct bioelectrocatalysis. Maximum activity is inherent in the catalysts manufactured on the basis of materials with moderate hydrophobic and hydrophilic properties. By the magnitude of the activity in the hydrogen peroxide reduction reaction, depending on the nature of the carbonaceous support, the fabricated catalysts (carbonaceous material with adsorbed POD) form the series AD-100, CNT > FCG > UDD.     

Synthesis and structure of metal complexes with organic polymers or inorganic supports

Macromolecular ligands have been widely used in the past two decades with the objective of preparing structurally defined heterogeneous catalysts from soluble organometallic complexes. This activity has been largely reconsidered and focused on few specific systems. In this connection the present paper reviews recent data concerning the preparation of macromolecular metal complexes derived from transition metals which can produce active catalytic complexes for olefin polymerization and oligomerization and comparison is made about the suitability of both organic resins (crosslinked polystyrene) and inorganic materials (silica, alumina and zeolites).     

Catalyzed Knoevenagel reactions on inorganic solid supports: Application to the synthesis of coumarine compounds

Knoevenagel reactions involving carbonyl compounds and active methylene derivatives have been studied in the presence of alumina, KSF and K10 montmorillonites. With addition of water the ester group undergoes hydrolysis. A route is disclosed for the synthesis of coumarine compounds catalyzed by solid supports.