Hydrophilic Support Research Articles

Immobilization of catalase on a novel polymer support, crosslinked polystyrene ethylene glycol acrylate resin: Role of the macromolecular matrix on enzyme activity

AbstractCrosslinked polystyrene ethylene glycol acrylate resin (CLPSER) was developed for the immobilization of the enzyme catalase by the introduction of a crosslinker, O,O′‐bis(2‐acrylamidopropyl) poly(ethylene glycol)1900, to styrene. The crosslinker was prepared by the treatment of acryloyl chloride with O,O′‐bis(2‐aminopropyl) poly(ethylene glycol)1900 in the presence of diisopropylethylamine. The resin was characterized with IR and 13C‐NMR spectroscopy. The catalytic activity of the catalase‐immobilized system of CLPSER was compared with divinylbenzene‐crosslinked polystyrene, ethylene glycol dimethacrylate crosslinked polystyrene, and 1,4‐butanediol dimethacrylate crosslinked polystyrene systems. Crosslink levels of 2, 8, and 20 mol % were evaluated. Among these crosslinked systems, the 2 mol % system was found to be most suitable to support catalytic activity. When a long flexible hydrophilic poly(ethylene glycol) crosslink, introduced between the polystyrene (PS) backbone and functional groups was used for immobilization, the extent of coupling and enzyme activity increased. Depending on the nature of the support, the catalytic activity of the system varied. The hydrophilic CLPSER support was most efficient for immobilization compared to the other PS‐based supports. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 8–19, 2005

Design of well balanced hydrophilic–lipophilic catalytic surfaces for the direct and selective monoesterification of various polyols

The transesterification process is a well known reaction of organic chemistry. However, the monoesterification of unprotected polyols such as glycerol or sucrose is much more complex and the design of selective catalysts is becoming a huge challenge in order to avoid many protection and deprotection steps, harmful for the cost and the environmental impact of the resulting process. In this study, we showed that the control of the hydrophilic–lipophilic balance of heterogeneous catalysts is a crucial key in order to tune both the catalyst activity and the monoester selectivity. Indeed, whereas homogeneous guanidine led to low selectivity toward monoesters, its anchorage on a hydrophilic solid support such as silica allowed us to prepare two basic hybrid organic–inorganic materials able to selectively afford monoesters in high yield and in an environmentally-friendly process, at low temperature and starting from an equimolecular mixture of unprotected polyols and various fatty methyl esters.

A thin carboxymethyl cellulose culture substrate for the cellulase-induced harvesting of an endothelial cell sheet

Engineered tissues constructed with two-dimensionally organized cells provide promising parts for reconstructing damaged tissues. Here we propose a new method for fabricating a 2D sheet made of an endothelial cell monolayer. First a culture substrate was prepared by treating the glass surface with an amine-terminated organosilicon derivative, followed by the covalent attachment of a thin carboxymethyl cellulose (CMC) layer. Fibronectin was immobilized onto the CMC-coated surface to promote cell adhesion. These surfaces were characterized step by step by means of contact angle measurement and X-ray photoelectron spectroscopy. Porcine aortic endothelial cells adhered to the culture substrate and consequently formed a confluent monolayer. When the substrate–cell composite was immersed in a cellulase solution, a cell sheet was spontaneously detached from the substrate due to enzymatic digestion of the CMC layer. The cell–cell connections were well preserved in the cell sheet, even after detachment from the substrate, most likely due to the fact that cellulase is harmless to mammalian cells. The cell sheet could be transferred to other culture dish with the aid of a hydrophilic membrane support, retaining the proliferation activity of the cells. The results obtained in this study demonstrate that cellulase treatment of the CMC layer is a rational and efficient method for obtaining a 2D cell sheet.

Chemical and thermal denaturation of crystalline bacterial S‐layer proteins: An atomic force microscopy study

Crystalline monomolecular cell surface layers, S-layers, are one of the most common outermost cell envelope components of the prokaryotic organisms (bacteria and archaeda) that protects them from competitive habitats. Since isolated S-protein subunits are able to re-assemble into crystalline arrays on lipid films and solid supports making biomimetic surfaces, S-layer technology is currently used in nanobiotechnology. An important aspect of the biomimetic surfaces built with S-layers is their stability under extreme solvent conditions or temperature. Chemical (pH, alcohol) and physical (thermal) denaturant conditions were employed to test the stability of S-layers. Recrystallized bacterial surface layers from Bacillus sphaericus (SbpA) on hydrophilic silicon wafers loses the crystalline structure at 80% ethanol/water mixtures, the change in structure being reversible after treating the surface with buffer solution. SbpA on silicon supports denatures at pH 3 and at 70 degrees C, and the process is irreversible. Cross-linking of SbpA enhances the stability for high ethanol and acidic conditions, but it does not improve thermal stability. Recrystallized SbpA on secondary cell wall polymer (SCWP), a natural environment for the protein layer, is more resistant to ethanol and pH exposure than recrystallized SbpA on hydrophilic silicon supports. Atomic force microscopy (AFM) was used to monitor the loss of stability and the changes in protein layer conformation.

Novel Methodology toward Deep Desulfurization of Diesel Feed Based on the Selective Elimination of Nitrogen Compounds

π-Acceptor molecules covalently attached on hydrophilic support were used to selectively remove neutral nitrogen heterocyclic compounds from diesel feed by a charge transfer mechanism. Functionalized hydrophilic polymers can effectively adsorb nitrogen heterocyclic compounds with a high selectivity toward sulfur heterocyclic compounds from model and real feed. The results showed that charge transfer processes coupled with an ion-exchange process to selectively remove basic nitrogen compounds are efficient enough to produce denitrogenated feed in high yield. This study was conducted to determine whether trends in hydrodesulfurization (HDS) activity with lower sulfur content were mainly the result of lower reactivity of hindered sulfur compounds or due to nitrogen species inhibition. The inhibition effects of nitrogen compounds on HDS at conditions commonly used in the hydrotreatment of gas oil feedstocks has been experimentally determined. This study suggests that the selective removal of nitrogen compounds from gas oil strongly enhanced the deep desulfurization.

The influence of cell and substratum surface hydrophobicities on microbial attachment

This study investigated the role of hydrophobic/hydrophilic interaction between bacterial and support surfaces in microbial adhesion, and a model that correlates microbial adhesion and relative cell-hydrophobicity defined as the ratio of cell-support surface hydrophobicity over cell-support hydrophilicity was derived. This model quantitatively describes how cell hydrophobic and hydrophilic interactions affect microbial adhesion, and offers deep insights into the thermodynamic mechanisms of microbial adhesion. The proposed model was verified by literature data. It appears that a high cell-hydrophobicity strongly facilitates microbial adhesion on both hydrophobic and hydrophilic support surfaces.

Catalytic Activity of Myoglobin Immobilized on Zirconium Phosphonates

The adsorption and catalytic activity of myoglobin (Mb) on zirconium phosphonates (a-zirconium benzenephosphonate (alpha-ZrBP), a-zirconium carboxyethanephosphonate (alpha-ZrCEP), and a novel layered zirconium fluoride aminooctyl-N,N-bis(methylphosphonate) (ZrC8)) were investigated. The maximum adsorption was reached after 16 h of contact and was greater on hydrophobic supports such as alpha-ZrBP and ZrC8 compared to hydrophilic supports such as alpha-ZrCEP. The equilibrium adsorption isotherms fitted the Langmuir equation, suggesting the presence of a monolayer of protein molecules on the support surfaces. The catalytic activities of free Mb and of the obtained biocomposites were studied in terms of the oxidation of two aromatic substrates, o-phenylenediamine and 2-methoxyphenol (guaiacol), by hydrogen peroxide. The oxidation catalyzed by immobilized myoglobin followed the Michaelis-Menten kinetics, similar to oxidation by free Mb. The kinetic parameters, kcat and KM, were significantly affected by the adsorption process. Mb/alpha-ZrCEP was the most efficient biocatalyst obtained, probably because of the hydrophilic nature of the support. The effect of immobilization on the stability of Mb toward inactivation by hydrogen peroxide was also investigated, and an increased resistance was found. The biocomposites obtained can be stored at 4 degrees C for months without a significant loss of catalytic activity.

The influence of cell and substratum surface hydrophobicities on microbial attachment.

This study investigated the role of hydrophobic/hydrophilic interaction between bacterial and support surfaces in microbial adhesion, and a model that correlates microbial adhesion and relative cell-hydrophobicity defined as the ratio of cell-support surface hydrophobicity over cell-support hydrophilicity was derived. This model quantitatively describes how cell hydrophobic and hydrophilic interactions affect microbial adhesion, and offers deep insights into the thermodynamic mechanisms of microbial adhesion. The proposed model was verified by literature data. It appears that a high cell-hydrophobicity strongly facilitates microbial adhesion on both hydrophobic and hydrophilic support surfaces.

An artificial receptor for glycoproteins.

We describe the rational design, synthesis and development of a sterilizable biomimetic ligand for the affinity purification of glycoproteins. Based on mimicking the principles of natural carbohydrate recognition, a putative library of 196 glycoprotein-binding synthetic ligands was designed and synthesized on a polymeric support. Ligand 11/11, based on a triazine scaffold and immobilized on a hydrophilic support, was identified as the "lead" ligand. The carbohydrate recognizing the potential of the "lead" ligand was revealed by reduced binding of a periodate oxidized model glycoprotein, and by "sharp" elution profiles achieved with borate buffer eluents. Specific elution and competitive binding experiments determined the monosaccharide specificity of 11/11 in the order mannoside > glucoside > galactoside. The diastereo-selective performance of ligand 11/11 was quantified and reaffirmed by analytical affinity chromatography and (1)H-NMR, in the order galactoside < glucoside < mannoside with binding affinities (K(a), M(-1)) in the 63-214 and 20-83 M(-1) range, respectively. Partition coefficient analysis revealed binding constants towards glycoproteins in the 10(4) M(-1) range, that compared favourably with the affinities of carbohydrate binding lectins for glycoproteins, such as concanavalin A. Molecular modelling studies of ligand 11/11 revealed the formation of a pre-organized apolar "tweezer-like" cavity, containing complementary nitrogenous hydrogen bond donor and acceptor groups that formed selective interactions with the equatorial 3- and 4-hydroxyl groups of saccharides.

Azlactone-reactive polymer supports for immobilizing synthetically useful enzymes: Part I. Pig liver esterase on dispersion polymer supports

Covalent attachment of pig liver esterase (E.C.3.1.1.1) to cross-linked dispersion polymer supports was effectively accomplished using azlactone [5(4H)-oxazolone] reactive groups. The binding process required about one hour at room temperature, and it was imperative that a relatively high concentration of a salt co-solute be present along with the enzyme. Under these conditions the enzyme rapidly bound onto the polymeric supports via hydrophobic interaction and then covalent attachment proceeded at effective rates. Up to 10 wt.% of the enzyme could be quantitatively bound to supports with retention of high levels of catalytic function, e.g., 68% specific activity at 4 wt.%. The non-reactive content of the polymeric support and especially the hydrophilic–hydrophobic balance were shown to be very important, with the hydrophilic supports providing the more favorable environment for hydrolytic esterase activity.

A novel hydrophilic support, CoFoam, for enzyme immobilization.

Lipases from different sources (porcine pancreas, Mucor miehei and Candida antarctica B) were covalently immobilized on a hydrophilic polyurethane composite (CoFoam). Their hydrolytic activities assayed with tributyrin were 0.55, 2.1 and 447 U g(-1), respectively. The activity of the C. antarctica B composite in the synthesis of methyl oleate in hexane was 8.8 U g(-1) compared to 60.6 U g(-1) for commercial Novozyme 435. The advantage of the CoFoam composite lies in the low pressure drop in a packed-bed reactor at fairly large flow rates. For example, at flow rates of 10-12 l min(-1), the pressure drop over 15 cm is typically 3 kPa.

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir–Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: First, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 μF/cm 2 for DPhyPC bilayers and 0.75 and 0.77 μF/cm 2 for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 MΩ cm 2 were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.

Bacterial Biofilm in Seawater: Cell Surface Properties of Early-attached Marine Bacteria

The development of antifouling strategies in seawater requires knowledge of the physico-chemical properties of the cell surfaces of early adherent bacteria. The hydrophilic, electrostatic and the Lewis acid-base cell surface properties of eleven marine bacteria were characterized. Although these bacteria adhered to a hydrophilic support immersed for 3 and 6 h, they presented various physico-chemical properties. Eleven strains possessed a hydrophilic surface and five a hydrophobic surface. Although the majority of the bacteria presented an electron-donating character, some could not generate Lewis acid-base interactions with the support. On the other hand, all strains possessed an isoelectric point ranging from 2.2 to 3.4 and were negatively charged at the pH of seawater. Hydrophilicity was a preponderant property among these bacteria, but other properties should not be ignored. The development of new antifouling paints must take account all the possible interaction levels used by the bacteria to adhere to an immersed surface.

Investigation of particle-based and monolithic columns for cation exchange protein displacement chromatography using poly(diallyl-dimethylammonium chloride) as displacer

The overall topic of the investigation was the separation of basic proteins by cation exchange displacement chromatography. For this purpose two principal column morphologies were compared for the separation of ribonuclease A and α-chymotrypsinogen, two proteins found in the bovine pancreas. These were a column packed with porous particles (Macro-Prep S, 10 μm, 1000 Å) and a monolithic column (UNO™ S1). Both columns are strong cation exchangers, carrying SO 3 −-groups linked to a hydrophilic polymer support. Poly(diallyl-dimethylammonium chloride) (PDADMAC), a linear cationic polyelectrolyte composed of 100–200 quaternary pyrrolidinium rings, was used as displacer. The steric mass action (SMA) model and, in particular, the operating regime and dynamic affinity plots were used to aid method development. To date the SMA model has been applied primarily to simulate non-linear displacement chromatography of proteins using low molar mass displacers. Here, the model is applied to polyelectrolytes with a molar mass below 20 000 g mol −1, which corresponds to a degree of polymerization below 125 and an average contour length of less than 60 nm. The columns were characterized in terms of the adsorption isotherms (affinity, capacity) of the investigated proteins and the displacer.

Solid-phase handling of hydrophobins: immobilized hydrophobins as a new tool to study lipases.

Hydrophobins are fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach. This attribute can be used to introduce hydrophobic foci on the surface of hydrophilic supports where hydrophobins are attached by covalent binding. In this paper, we report the binding of Pleurotus ostreatus hydrophobins to a hydrophilic matrix (agarose) to construct a support for noncovalent immobilization and activation of lipases from Candida antarctica, Humicola lanuginosa, and Pseudomonas flourescens. Lipase immobilization on agarose-bound hydrophobins proceeded at very low ionic strength and resulted in increased lipase activity and stability. The enzyme could be desorbed from the support using moderate concentrations of Triton X-100, and its enantioselectivity was similar to that of lipases interfacially immobilized on conventional hydrophobic supports. These results suggest that lipase adsorption on hydrophobins follows an "interfacial activation" mechanism; immobilization on hydrophobins offers new possibilities for lipase study and modulation and reveals a new application for fungal hydrophobins.

Nucleotide isolation by boronic acid functionalized hydrophilic supports

Abstract Aminophenyboronic acid (APBA) carrying-gel beads were proposed as alternative sorbents for nucleotide isolation. For the preparation of two different types of gel beads with different surface characteristics, two hydrophilic monomers, 2-hydroxypropylmethacrylate (HPMA) and polyethyleneglycol methacrylate (PEGMA) were selected as the base materials. The spherical gel beads were obtained by the suspension copolymerization of HPMA or PEGMA with epoxypropyl methacrylate (EPMA) by using ethyleneglycol dimethacrylate (EGDMA) as the crosslinker. A boronic acid carrying ligand, m -aminophenylboronic acid (APBA) was covalently attached onto the both types of gel beads via the direct chemical reaction between epoxypropyl and amino groups. The equilibrium APBA binding capacities of HPMA and PEGMA gel beads were 47 and 34 mg g −1 , respectively. The reversible adsorption-desorption behavior of β-nicotinamide adenine dinucleotide (β-NAD) was investigated comparatively by using APBA carrying-HPMA and PEGMA based gel beads as sorbents in batch-fashion. While no-significant non-specific β-NAD adsorption was observed onto the plain (i.e. non-derivatized) gel beads, the equilibrium β-NAD adsorption capacities of APBA carrying-HPMA and PEGMA based gels were determined as 11 and 4 mg g −1 , respectively. The desorption yields higher than 90% could be achieved with both types of gels. The results indicated that the proposed materials could be efficiently used as pseudospecific-sorbent for nucleotide isolation.

A solid-Supported phosphine-Free ruthenium alkylidene for olefin metathesis in methanol and water

The synthesis and olefin metathesis activity in protic solvents of 7, a phosphine-free ruthenium alkylidene bound to a hydrophilic solid support are reported. This heterogeneous catalyst promotes relatively efficient ring closing- and cross-metathesis reactions in both methanol and water. The potential utility of homogeneous catalyst 2 for olefin metathesis in methanol is also demonstrated.

Synthesis of Hydrophilic Polar Supports Based on Poly(dimethylacrylamide) via Copper-Mediated Radical Polymerization from a Cross-Linked Polystyrene Surface: Potential Resins for Oligopeptide Solid-Phase Synthesis

A series of cross-linked polystyrene-based resins, Wang, Merrifield, and primary amino functional beads, have been transformed into initiators for transition-metal-mediated radical polymerization. Wang and amino functional resins have been condensed with 2-bromoisobutyryl bromide to give approximately quantitative conversion to initiator groups as monitored by FTIR and gel-phase 13C NMR spectrometry. In each case efficient polymerization of N,N-dimethylacrylamide (DMAc) has been demonstrated. Polymerization occurs with the RuCl2(PPh3)3 system in conjunction with Al(OiPr)3 with the reaction proceeding most efficiently with a toluene/ethanol solvent mixture which gives the best swelling of the composite particle. Surprisingly, copper(I) bromide with the pyridine imine type catalysts also led to efficient polymerization. Polymerization proceeds effectively directly from chloromethyl functional Merrifield resins given a 75% increase in weight at 130 °C after 55 h. Yields are significantly greater for derivati...

Normal and Lateral Forces between Lipid Covered Solids in Solution: Correlation with Layer Packing and Structure

We report on the normal and lateral forces between controlled-density mono- and bilayers of phospholipid co-adsorbed onto hydrophobic and hydrophilic solid supports, respectively. Interactions between 1,2-dioleoyl- sn-glycero-3-phosphocholine layers were measured using an atomic force microscope. Notable features of the normal force curves (barrier heights and widths) were found to correlate with the thickness and density of the supported lipid layers. The friction and normal force curves were also found interrelated. Thus, very low friction values were measured as long as the supported layer(s) resisted the normal pressure of the tip. However, as the applied load exceeded the critical value needed for puncturing the layers, the friction jumped to values close to those recorded between bare surfaces. The lipid layers were self-healing between measurements, but a significant hysteresis was observed in the force curves measured on approach and retraction, respectively. The study shows the potential of using atomic force microscopy for lipid layer characterization both with respect to structure and interactions. It further shows the strong lubricating effect of adsorbed lipid layers and how this varies with surface density of lipids. The findings may have important implications for the issue of joint lubrication.

Electrically communicating three-dimensional cardiac tissue mimic fabricated by layered cultured cardiomyocyte sheets.

Recent progress in stem cell biology is likely to provide implantable sources of human cardiomyocytes in the near future. This possibility has encouraged cardiac tissue engineering. To construct heart-like tissue, we have exploited the capabilities of novel cell culture surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), to produce intact viable monolayer cell sheets simply by reducing culture temperature. Cultured chick embryonic cardiomyocyte sheets detached from PIPAAm-grafted surfaces were layered into tissue-like laminate stacks using hydrophilic support and transfer membranes. The layered cell sheets rapidly adhered to each other, establishing cell-to-cell connections characteristic of heart tissue, including desmosomes and intercalated disks. Bilayer cell sheets pulsed spontaneously and synchronously, altering their characteristic pulsing frequency with applied electric stimulation transmitted across the sheets. These results demonstrate that electrically communicative three-dimensional cardiac constructs can be achieved by stacking monolayer cardiomyocyte sheets. Cardiac tissue engineering based on this technology will facilitate new in vitro heart models and may prove useful for in vivo cardiovascular tissue repair.