Hydrophobic adsorption and covalent immobilization of Candida antarctica lipase B on mixed-function-grafted silica gel supports for continuous-flow biotransformations

Adsorption onto solid supports has proven to be an easy and effective way to improve the mechanical and catalytic properties of lipases. Covalent binding of lipases onto the support surface enhances the active lifetime of the immobilized biocatalysts. Our study indicates that mesoporous silica gels grafted with various functions are ideal supports for both adsorptive and covalent binding for lipase B from Candida antarctica (CaLB). Adsorption of CaLB on phenyl-functionalized silica gels improved in particular its specific activity, whereas adsorption on aminoalkyl-modified silica gels enabling covalent binding with the proper reagents resulted in only moderate specific activity. In addition, adsorption on silica gels modified by mixtures of phenyl- and aminoalkyl silanes significantly increased the productivity of CaLB. Furthermore, CaLB adsorbed onto a phenyl/aminoalkyl-modified surface and then treated with glutardialdehyde (GDA) as cross-linking agent provided a biocatalyst of enhanced durability. Adsorbed and cross-linked CaLB was resistant to detergent washing that would otherwise physically deactivate adsorbed CaLB preparations. The catalytic properties of our best immobilized CaLB variants, including temperature-dependent behavior were compared between 0 and 70 °C with those of two commercial CaLB biocatalysts in the continuous-flow kinetic resolutions of racemic 1-phenylethanol rac-1a and 1-phenylethanamine rac-1b.     

Investigation of polymer and nanoparticle properties with nicotinic acid and p-aminobenzoic acid grafted on poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) via click chemistry

In this study, the grafting of nicotinic acid and p-aminobenzoic acid (PABA) onto poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) was performed by Huisgen's 1,3-dipolar cycloaddition, also known as click chemistry. Concentrations used for grafting were 0.10, 0.20, and 0.30 molar ratios with respect to caproyl units. The grafted copolymers were successfully obtained at all ratios as confirmed by NMR, GPC, and FT-IR. According to the DSC results, the polymorphisms of these grafted copolymers were mostly changed from semicrystalline to amorphous depending on the type and the amount of grafting compounds. TGA thermograms showed different thermal stabilities of the grafted copolymers compared to the original copolymers. Cytotoxicity results from HUVEC models suggested that the toxicity of grafted nanoparticles increased with the molar ratios of grafting units. Due to differences in molecular structure between nicotinic acid and PABA, physicochemical properties (particle size and surface charge) of grafted copolymer nanoparticles were substantially different. With increasing molar ratio of the grafting units, the particle size of blank nanoparticles tended to increase, resulting from an increase in the hydrophobic fragments of the grafted copolymer. Ibuprofen was chosen as a model drug to evaluate the interaction between grafted copolymers and loaded drug. After ibuprofen loading, the particle size of the loaded nanoparticles of both grafted copolymers increased compared to that of the blank nanoparticles. Significant differences in loading capacity between nicotinic acid and PABA grafted copolymer nanoparticles were clearly shown. This is most likely a result of different compatibility between each grafting compound and ibuprofen, including hydrogen bond interaction, π-π stacking interaction, and steric hindrance.     

Effects of Chromium Propionate on Egg Production,Egg Quality,Plasma Biochemical Parameters,and Egg Chromium Deposition in Late-Phase Laying Hens

This study was conducted to investigate the effects of chromium propionate on egg production, egg quality, plasma biochemical parameters and egg chromium deposition in late-phase laying hens. Four hundred thirty-two 60-weeks old laying hens were divided into four groups of 108 birds per group according to egg production. The dietary treatments consisted of the basal diet adding with 0, 200, 400, and 600 μg/kg chromium as chromium propionate. All laying hens were given feed and water ad libitum for 8 weeks. The addition of 400 μg/kg Cr as chromium propionate increased egg production (P?<?0.01) during the later 4 weeks, but decreased albumen height, yolk color score, and Haugh unit of eggs. Six hundred micrograms per kilogram Cr as chromium propionate supplementation improved shell thickness (P?<?0.05). 200 μg/kg Cr as chromium propionate supplementation decreased the uric acid concentration by 31 % (P?<?0.05). However, supplemental Cr did not affect the egg chromium deposition of hens (P?>?0.05). These data indicated that feeding of late-phase laying hens with chromium propionate could improve egg production, increase eggshell thickness, but do not result in abnormal levels of chromium deposition in eggs.     

Insulin adsorption on coated silica based supports grafted with N-acetylglucosamine by liquid affinity chromatography

Silica beads are coated with dextran carrying a calculated amount of positively charged diethylassminoethyl groups (DEAE) in order to neutralize negative charged silanol groups at the silica surface and in this way to minimize non specific interactions between silica surface and proteins in solution. Dextran-coated silica supports are potentially excellent stationary phases for high-performance liquid chromatography of proteins. These supports combine the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. These supports (silica-dextran-DEAE = SID) are easily functionalized by grafting N-acetylglucosamine (GlcNAc) using conventional coupling methods. The performances of the support bearing GlcNAc are studied by high-performance liquid affinity chromatography (HPLAC) of insulin, the hypoglycemic peptide hormone of the human organism. The study shows that these supports exhibit a reversible and specific affinity towards insulin and allow separations with high purification yields. Moreover, the influence of different physico-chemical parameters (pH, NaCl and insulin concentration) on insulin retention on the support was analysed. This allowed us to optimize the conditions of adsorption and to better understand the interaction mechanisms between insulin and GlcNAc as biospecific ligand.     

Well-defined lactose-containing polymer grafted onto silica particles

Reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-O-meth-acryloyloxyethoxyl-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-beta-D-glucopyranoside (MAEL) was performed directly in CHCl3 solutions using cumyl dithiobenzoate (CDB) as the chain transfer agent to give well-defined glycopolymers. The chemical composition and structure of the glycopolymer were characterized by 1HNMR, FTIR, and SEC. The living glycopolymer chains were subsequently grafted onto gamma-methacryloxypropyl-trimethoxy (MPTMS) modified silica particles. The acetyl groups of the poly(MAEL) grafted onto the silica gel particles were converted to the hydroxyl groups with CH3ONa/CH3OH, thus obtaining silica gel particles modified with well-defined lactose-carrying polymer.     

Efficacy of Dietary Chromium (III) Supplementation on Tissue Chromium Deposition in Finishing Pigs

The study was conducted to evaluate the efficacy of different forms of trivalent chromium (Cr) supplementation on tissue chromium deposition in finishing pigs. A total of 96 pigs with an initial average body mass 65.57±1.05 kg were blocked by body mass and randomly assigned to four treatments with three replicates. Pigs were offered one of four diets including a control diet or the control diet supplemented with 200 μg/kg chromium from either chromium chloride (CrCl(3)), chromium picolinate (CrPic) or chromium nanocomposite (CrNano) for 40 days. During the trial, all pigs were given free access to feed and water. After feeding trial, eight pigs from each treatment were slaughtered for samples collection. The results showed that supplemental CrNano increased Cr content in blood, longissimus muscle, heart, liver, kidney, jejunum, and ileum (P<0.05). Supplemental Cr from three sources increased Cr excretion from all feces (P<0.05). Urinary Cr excretion was increased by CrNano or CrPic supplementation significantly. These results suggested that chromium nanocomposite exhibited more effective on tissue Cr deposition in pigs, which indicated higher absorption compared with CrCl(3) and CrPic.     

In situ characterization of antibody grafting on porous monolithic supports

The efficient immobilization of antibodies on monolithic support is one of the most critical steps when preparing immunoaffinity supports. In this work, the ADECA (amino density estimation by colorimetric assay) method was adapted to tridimensional supports (in a dynamic mode) and proved to be efficient to characterize the antibodies grafting efficiency on 15.3±0.9mg porous glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA) monolithic columns. The amount of grafted antibodies measured in situ on the monolith by ADECA (8.2±0.2μg of antibodies per milligram of monolith) was consistent with values obtained by bicinchoninic acid assay (BCA) after crushing the monolith. ADECA was shown to be less time-consuming and more versatile than BCA. The ADECA method was further implemented to thoroughly study and optimize the antibody grafting conditions (influence of pH and kinetics of the grafting step) on GMA-based monoliths and to check the covalent nature of the antibody/surface linking and its stability. Using the total amount of grafted antibodies and the amount of recognized antigen, we found that 65±6% of antibodies were able to capture their antigen. Finally, the grafting of Fab and F(ab')(2) fragments demonstrated that no significant improvement of the global binding capacity of the monolith was obtained.     

New phase supports for liquid-liquid partition chromatography of biopolymers in aqueous poly(ethyleneglycol)-dextran systems. Synthesis and application for the fractionation of DNA restriction fragments

New phase supports for liquid-liquid partition chromatography, using aqueous poly(ethyleneglycol)-dextran systems have been developed by grafting linear polyacrylamide chains on to premanufactured chromatographic supports carrying primary or secondary aliphatic hydroxyl functions on their surface. Columns prepared from such supports have a higher binding capacity for the dextran-rich stationary phase and much higher performances than columns prepared from cellulose, the previously used phase support for this system. Test separations of DNA restriction fragments, ranging from 11 base pairs to 3829 base pairs, document a high resolution for DNA fragments larger than 200 base pairs.     

Aminopropyl silica gel as a solid support for preparation of glycolipid immunoadsorbent and purification of antibodies.

Aminopropyl silica gel was prepared from porous silica gel and was used as a solid support for immunoadsorbent in the purification of anti-glycolipid antibodies. For neutral glycosphingolipids, a carboxyl function was generated by oxidation of the olefinic double bond of the sphingosine moiety, whereas for gangliosides the carboxyl group of sialic acid was used to couple with aminopropyl silica gel in the presence of a carbodiimide. These compounds were used for purifying anti-glycolipid antibodies from serum of immunized rabbits. The antibodies bound to the su-strate were released by 2 M potassium thiocyanate and their immunological properties were studied. Aminopropyl silica gel may be preferred over conventional organic solid supports for the following reasons: 1) faster flow rate; 2) higher capacity; 3) easier handling; 4) more economical; and 5) lower susceptibility to microbial attack.     

Cytotoxicity of mesoporous silica nanomaterials

We here measure the toxicity of MCM-41, a mesoporous silica nanomaterial, two of its functionalized analogs, AP-T, which has grafted aminopropyl groups and MP-T, which has grafted mercaptopropyl groups, and spherical silica nanoparticles (SiO₂), toward human neuroblastoma (SK-N-SH) cells. Since the particles studied are not soluble in aqueous media, the metric used to report the cytotoxicity of these materials is a new quantity, Q₅₀, which is the number of particles required to inhibit normal cell growth by 50%. Determining the number of particles per gram of material applied to the cells required both the calculated and experimentally determined surface areas of these nanomaterials. This study shows that Q₅₀ increases in the order, MCM-41 < MP-T < AP-T ≈ SiO₂, showing that on a per particle basis, MCM-41 is the most cytotoxic material studied. For the three mesoporous silica materials in this study, cytotoxicity appears related to the adsorptive surface area of the particle, although the nature of the functional group cannot be ruled out. Silica nanospheres have the lowest surface area of the particles studied but since they exhibit a Q₅₀ value similar to that of AP-T, shape may also be important in the cytotoxicity of these materials.     

The effects of dietary chromium supplementation on some blood parameters in sheep

The effects of dietary inorganic chromium on some biochemical parameters were determined in lambs fed either a control diet or a 200-ppb or 400-ppb chromium-supplemented diet. The live weight of the animals were measured and jugular blood samples were collected prior to supplementation (d 0) and on d 20, 40, and 55. On d 55, three animals from each group were slaughtered to measure subcutaneous fat. Sera were analyzed for glucose, triglyceride, total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol, total protein, albumin, ALT, AST, and GGT levels. Chromium supplementation had no significant effect on live weight, but subcutaneous fat was reduced significantly in both chromium groups. There was a slight decrease in glucose concentrations in the 200-ppb chromium group, although only the differences on d 55 were significant. Triglyceride levels in both chromium groups were lower than the control group with marked differences in the 400-ppb chromium group. HDL cholesterol levels increased in both treatment groups compare to control, although the differences in the 400-ppb chromium group on d 40 were significant. Serum Cr concentrations slightly but not significantly increased in both chromium groups. No significant differences were found in total and LDL cholesterol, total protein, albumin, ALT, AST, and GGT levels. In conclusion, chromium supplementation may affect carbohydrate and lipid metabolisms and lipid deposition in lambs.     

Size control of silica nanoparticles and their surface treatment for fabrication of dental nanocomposites

Nearly monodispersed silica nanoparticles having a controlled size from 5 to 450 nm were synthesized via a sol-gel process, and then the optimum conditions for the surface treatment of the synthesized silica nanoparticles with a silane coupling agent (i.e., 3-methacryloxypropyltrimethoxysilane (gamma-MPS)) were explored to produce dental composites exhibiting enhanced adhesion and dispersion of silica nanoparticles in the resin matrix. The particle size was increased by increasing amounts of the catalyst (NH4OH) and silica precursor (tetraethylorthosilicate, TEOS) and by decreasing the amount of water in the reaction mixtures regardless of solvents used for the synthesis. The particle size prepared by using ethanol as a solvent was significantly larger than that prepared by using methanol as a solvent when the composition of the reaction mixture was fixed. The nanosized particles in the 5-25 nm range were aggregated. The amount of grafted gamma-MPS on the surface of the synthesized silica nanoparticles was dependent on the composition of the reaction mixture when an excess amount of gamma-MPS was used. When surface treatment was performed at optimum conditions found here, the amount of the grafted gamma-MPS per unit surface area of the silica nanoparticles was nearly the same regardless of the particle size. Dispersion of the silica particles in the resin matrix and interfacial adhesion between silica particles and resin matrix were enhanced when surface treated silica nanoparticles were used for preparing dental nanocomposites.     

Immobilization of enzymes on porous silica supports

Four silica supports differing in pore dimensions were activated by treatment with SiCl₄ and then with ethylenediamine to obtain alkylamine groups on the silica surface. Three enzymes, peroxidase from cabbage, glucoamylase from Aspergillus niger C and urease from soybean were immobilized on these supports using glutaraldehyde as coupling agent. It was found that the protein content, the retained enzymatic activity and the storage stability of the silica supported enzymes were considerably affected by support pore size and enzyme molecular weight, the factors which are supposed to alter protein distribution inside the support pores. The highest activity was found for peroxidase and glucoamylase attached to the silica with the widest pores, but their loss in activity during storage was considerable. The urease retained less activity after immobilization, but its storage stability was excellent.     

Stabilization of prostaglandin synthetase by immobilization of goat seminal microsomes on silica gel-G

Prostaglandin synthetase was immobilized by adsorption of goat vesicular microsomes on silica gel containing CaSO4 (silica gel G). Repeated cycles of enzymatic conversion of arachidonic acid to prostaglandin by the immobilized microsomes increased the product yield by 1.5 fold, in comparison to the same by free microsomal particles. The presence of Ca2+ in silica gel is responsible for this improved yield of prostaglandin as the divalent metal ion stabilized prostaglandin synthetase activity in a remarkable way. Microsomal particles immobilized on solid supports like alumina G and controlled pore glass were not very effective.     

Preparation of molecularly imprinted polymers for artemisinin based on the surfaces of silica gel

Artemisinin is an effective antimalarial drug isolated from the herbal medicine Artemisia annua L. Molecular imprinting is a technique of preparing molecularly imprinted polymers (MIPs) which can specifically recognize the imprinted template molecules. In this work, silica gel were used as supporting matrix, and vinyltriethoxysilane (VTES) was grafted onto its surface. The preparation of MIPs for artemisinin was performed on the surfaces of the modified silica gel using artemisinin as the template, acrylamide (AM) and methacrylic acid (MAA) as the functional monomers, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and 2,2'-azo-bis-isobutyronitrile (AIBN) as the initiator. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and pore size analysis were used to characterize the prepared MIPs. The adsorption kinetic curve, adsorption isotherm and selective adsorption were measured by static method. The adsorption reached equilibrium at about 10 h, while fast adsorption took place during the first 2-3 h. The maximum adsorption capacity has been found to be 37.13 mg/g according to calculation with Langmuir-Freundlich isotherm. The electivity coefficients of MIPs for artemisinin with respect to artemether and arteether were 2.88 and 3.38, respectively. The results showed that the MIPs possessed good specific adsorption capacity and selectivity for artemisinin.     

Review: Selective ligand-exchange adsorbents prepared by template polymerization

Highly selective ligand-exchange absorbents have been prepared by template polymerization, a process in which the target molecule serves as a template for assembly of specific recognition sites. In an effort to develop materials suitable for chromatographic separations, thin coatings of the selctive templated polymers have been grafted to two reactive macroporous supports, poly(trimethylolpropane trimethacrylate) (TRIM), and propylmethacrylate-derivatized silica beads. The precursor polymer prepared from the trifunctioal TRIM monomer is macroporous and highly crosslinked, providing a stable structure for surface grafting. The TRIM precursor polymer and various surface-grafted copolymers have been characterized by scanning electron microscopy (SEM) and IR, (13)C NMR, and XPS spectroscopic techniques. Composite adsorbents have also been prepared using propylmethacrylate-modified silica particles. While equilibrium rebinding selectivites for both types of surface-templated materials are similar to those reported previously for bulk-polymerized template polymers, the composite materials are far better suited to chromatographic separatios. Highly similar bis-imidazole substrates can be separated by ligand-exchange chromatography on these new templated adsorbents. (c) 1995 John Wiley & Sons, Inc.     

Grafting of material-binding function into antibodies Functionalization by peptide grafting

Quite recently, a few antibodies against bulk material surface have been selected from a human repertoire antibody library, and they are attracting immense interest in the bottom-up integration of nanomaterials. Here, we constructed antibody fragments with binding affinity and specificity for nonbiological inorganic material surfaces by grafting material-binding peptides into loops of the complementarity determining region (CDR) of antibodies. Loops were replaced by peptides with affinity for zinc oxide and silver material surfaces. Selection of CDR loop for replacement was critical to the functionalization of the grafted fragments; the grafting of material-binding peptide into the CDR2 loop functionalized the antibody fragments with the same affinity and selectivity as the peptides used. Structural insight on the scaffold fragment used implies that material-binding peptide should be grafted onto the most exposed CDR loop on scaffold fragment. We show that the CDR-grafting technique leads to a build-up creation of the antibody with affinity for nonbiological materials.     

HPLC enantioseparation on cellulose tris(3,5-dimethylphenylcarbamate) as a chiral stationary phase: Influences of pore size of silica gel,coating amount,coating solvent,and column temperature on chiral discrimination

Cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) was coated on large-pore silica gels and used as a chiral stationary phase (CSP) for high-performance liquid chromatographic separation of enantiomers. The influences of pore size of silica gel, coating amount of CDMPC, coating solvent, and column temperature on chiral discrimination were investigated. CSPs prepared with a large-pore silica gel having a small surface area showed higher chiral recognition. The amount of CDMPC adsorbed on the silica gel influenced the chiral recognition of some racemates. Loading capacity of racemates increased with an increase of the amount of CDMPC supported on the silica gel, and a CSP coated with 45% CDMPC by weight can be used for both analytical and semi-preparative scale separations. The CDMPC, coated using acetone as the coating solvent, exhibited, in many cases, higher enantioselectivity than that obtained with tetrahydrofuran F as the coating solvent. © 1996 Wiley-Liss, Inc.     

Take percentage and conditions of cultured epithelium were improved when basement membrane of the graft was maintained and anchoring mesh was added

To achieve a higher take rate for epithelial grafts, this study investigated grafting techniques. Seventy-seven nude mice received flap grafting in which cultured human epithelium was grafted inside the flap, and 55 nude rats received transplantation of epithelium to a full-thickness skin defect. In each group, four models were studied, including model 1, in which epithelium was cultured with the conventional method; model 2, in which epithelium was cultured with fibrin gel to avoid sheet damage, then absorptive mesh was incorporated into the epithelium for anchoring to the graft bed; model 3, in which epithelium was cultured with fibrin gel and combined with absorptive mesh and artificial dermis containing fibroblasts; and model 4, in which the model 2 epithelium was grafted after artificial dermis was transplanted. The take for these models was evaluated grossly and histologically. The results show that the take percentage of models 2 and 3 was significantly higher than that of model 1 (conventional epithelium) and that there was no significant difference between model 3 (simultaneous grafting) and model 4 (two-step grafting). The difference in the take percentages of the grafts to the flap and to the full-thickness skin defect was also insignificant. In immunohistochemistry, human keratin appeared in all epidermis layers and diversification of the layer was observed in models 2, 3, and 4. In these three models, type IV collagen appeared in the basal layer and the formation of basal membrane was confirmed. These findings suggest that epithelia cultured on fibrin gel and combined with absorptive mesh could be used in a new technique for better, more stable take.     

Effect of polymer grafting density on silica nanoparticle toxicity

Nanoparticles are commonly engineered with a layer of polymers on the surface used to increase their stability and biocompatibility, as well as providing multifunctional properties. Formulating the nanoparticle size and surface properties with polymers directly affects the way these nanoparticles interact with a biological system. Many previous studies have emphasized the importance of nanoparticle size and surface charge in affecting their toxicity in cells. However, the potential weakness in many of these studies is that the polymer grafting densities on nanoparticles have been disregarded during toxicity evaluation. In the current study, we hypothesized that the density of polymers on nanoparticles will affect their toxicity to cells, especially for nanoparticle cores that are toxic themselves. To address this issue, we synthesized a range of RAFT (reversible addition fragmentation chain transfer) polymers bearing different surface charges and coated them onto silica nanoparticles (SiNPs) with different grafting densities. The in vitro cytotoxicity of these SiNPs was evaluated using the MTT (thiazolyl blue tetrazolium bromide) assay with Caco-2 cells. We found that neutral (biocompatible) polymers with a high grafting density on SiNPs were effective at protecting the cells from the toxicity of the silica core. High cellular toxicity was only observed for cationic polymer-SiNPs, while all other neutral and anionic polymer-SiNPs induced limited cellular toxicity. In contrast, the toxic effects induced by low density polymer-coated SiNPs were mostly attributed to the silica core, while the polymer coatings had a limited contribution. These findings are important indicators for the future evaluation of the toxicological profile of polymer-coated nanoparticles.