Effect of the solvent on recognition properties of molecularly imprinted polymer specific for ochratoxin A

A molecularly imprinted polymer specific for the mycotoxin ochratoxin A has been synthesised using a non-covalent approach. The polymer has shown an excellent affinity and specificity for the target template in aqueous solutions. The binding experiments, NMR study and molecular modelling have proven that the template recognition by polymer originates from the shape complementarity of binding sites. The binding mechanism is critically depended on factors that affect the polymer conformation. Thus the variation in buffer concentration, pH and presence of organic solvent, which affect the polymer swelling or shrinking, had a profound effect on the polymer recognition properties.     

Indirect bioremediation: biodegradation of hydrocarbons on a commercial sorbent

Urea-formaldehyde polymer is currently used as asorbent for containment and clean up of hydrocarbons. The aerobic biodegradability of this polymer andhydrocarbons sorbed to the polymer were tested. Soilmicroorganisms readily grew on the polymer, and twoorganisms, a bacterium and a fungus, capable of growthon the polymer were isolated. However, biodegradationof the polymer was very slow and possibly incomplete. Biodegradation of the polymer was evident as a changein appearance of the polymer, but disappearance of thepolymer was not detectable in liquid culturesincubated for six months or soil cultures incubatedfor one month. Destruction of the polymer by soilmicroorganisms at ambient temperature does not appearto be practical. Degradation of ¹⁴C-labeledhexadecane and phenanthrene mixed with crude oil inliquid cultures inoculated with soil microorganismswas used as an estimate of general hydrocarbondegradation. When nitrogen was not limiting, therates of hexadecane and phenanthrene degradation werethe same, whether those hydrocarbons were sorbed tothe polymer or not sorbed. When nitrogen waslimiting, the polymer stimulated the rate ofhexadecane degradation but not the rate ofphenanthrene degradation. The polymer may stimulatehexadecane degradation by serving as a source ofnitrogen. However, optimal degradation of sorbedhydrocarbons requires nitrogen addition. The resultssuggest that it may be feasible to decontaminate spentpolymer by biodegradation of sorbed hydrocarbons.     

Mechanisms of Capsid Assembly around a Polymer

Capsids of many viruses assemble around nucleic acids or other polymers. Understanding how the properties of the packaged polymer affect the assembly process could promote biomedical efforts to prevent viral assembly or nanomaterials applications that exploit assembly. To this end, we simulate on a lattice the dynamical assembly of closed, hollow shells composed of several hundred to 1000 subunits, around a flexible polymer. We find that assembly is most efficient at an optimum polymer length that scales with the surface area of the capsid; polymers that are significantly longer than optimal often lead to partial-capsids with unpackaged polymer “tails” or a competition between multiple partial-capsids attached to a single polymer. These predictions can be tested with bulk experiments in which capsid proteins assemble around homopolymeric RNA or synthetic polyelectrolytes. We also find that the polymer can increase the net rate of subunit accretion to a growing capsid both by stabilizing the addition of new subunits and by enhancing the incoming flux of subunits; the effects of these processes may be distinguishable with experiments that monitor the assembly of individual capsids.     

Zeta potential as a measure of polyelectrolyte flocculation and the effect of polymer dosing conditions on cell removal from fermentation broth

Characterization of flocculation for cell removal from fermentation broth via polyelectrolyte addition is commonly based on qualitative methods such as physical appearance of the floc. The use of zeta potential as a quantitative measure of floc character was evaluated as an indicator of optimal polymer addition. Zeta potential was found to increase with increasing cationic polyelectrolyte dosage, but never reached zero regardless of the total amount of polymer added, indicating flocculation occurs at least partially through a bridging type mechanism. Experiments were conducted using various polymer concentrations (25-75 g/L) and dosing methods (batch, incremental and continuous addition) that resulted in variable overall polymer requirements to achieve optimum flocculation. Zeta potential was found to be constant at optimal floc character regardless of the total amount of polymer added, polymer concentration, or method of polymer addition. Experiments with two additional types of fermentation broth also showed characteristic zeta potentials at optimal flocculation. Polymer requirements to achieve a particular floc character can vary greatly, depending on polymer dosing conditions and fermentation batch. The effect of polymer dosing conditions on the polymer requirement to obtain optimal floc character was evaluated. Polymer dosing method and calcium concentration were both found to have a significant effect (P < 0.0001) with continuous polymer addition and high calcium concentration requiring less polymer than did batch polymer addition and low calcium concentration, respectively. Polymer dosing concentration did not significantly affect polymer requirement for optimal flocculation.     

Molecularly imprinted polymers from nicotinamide and its positional isomers

Imprinted polymers were prepared for nicotinamide and its positional isomers. The influence of porogenic solvent and functional monomer on recognition properties of the polymer was compared. The results indicated that two functional groups, the heterocyclic nitrogen and the amide group, in the nicotinamide or isonicotinamide molecule have a synergistic effect in binding to the polymer. The polymers prepared with nicotinamide and isonicotinamide can be used as HPLC stationary phase for the separation of positional isomers of nicotinamide or isonicotinamide, while the polymer prepared with picolinamide showed no specificity toward the template. The mechanisms for the differences in recognition are discussed. In addition to the retention of polymers to their templates the polymers also displayed excellent retention to nicotinic acid and isonicotinic acid, compounds structurally similar to the template. This dual recognition property of the polymer may be useful in circumstances where the preparation of a polymer for a specific template may be problematic because of poor stability or solubility.     

Alteration of poly(adenosine diphosphoribose) metabolism by ethanol: mechanism of action

We present evidence that ethanol alters intracellular poly(adenosine diphosphoribose) metabolism and we further describe the mechanism by which ethanol exerts its effect on polymer synthesis. One percent ethanol stimulates polymer accumulation as much as 2.5-fold but does not alter polymer degradation in intact cells following DNA damage. Ethanol directly stimulates polymer synthesis following low doses of DNA damage induce by deoxyribonuclease I in a nucleotide-permeable cell system that does not possess a functional polymer turnover system. Ethanol has no measurable effect on polymer synthesis in undamaged nucleotide-permeable cells or in permeable cells treated with high doses of deoxyribonuclease I. Ethanol concentrations that stimulate poly(adenosine diphosphoribose) polymerase activity in vitro specifically lower KDNA without affecting KNAD or Vmax. The results clearly show that ethanol alters the binding of this enzyme to the DNA component of chromatin and that this altered binding is responsible for the activation of the enzyme. Altered affinity of poly(adenosine diphosphoribose) polymerase and perhaps other regulatory proteins for chromatin may play an important role in the pathology of alcohol.     

Occurrence of glucose polymer in undifferentiated PC12 cells

A large glucose polymer was found, following pronase digestion, in PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was excluded from a Bio-Gel A-0.5 m column and adsorbed by immobilized concanavalin A-Sepharose from which it was eluted with 10 mM alpha-methylmannoside. Glucose was found to be the sole component monosaccharide. Except for those capable of degrading glycogen, no exo- or endo-glycosidases cleaved the polymer. This is the first report on the occurrence of a glucose polymer in undifferentiated PC12 cells.     

The development and application of a new affinity partitioning system for enzyme isolation and purification.

A reactive water-soluble polymer was synthesized by copolymerizing N-isopropylacrylamide and glycidyl acrylate. The reactive polymer could react with the amino groups of enzymes/proteins or other ligands to form an affinity polymer. As a model, the reactive polymer was allowed to react with paraaminobenzamidine, a strong trypsin inhibitor. The affinity polymer could easily form an aqueous two-phase system with either dextran or pullulan, and the phase diagram was compared favorably to that of the well-known polyethylene glycol-dextran system. Once trypsin was attracted to the affinity polymer dominant phase, the enzyme could be dissociated from the polymer at low pH. Owing to the N-isopropylacrylamide units, the affinity polymer could be isolated from the solution by precipitation at a low level of ammonium sulfate. The enzyme recovery was always greater than 50%, and the affinity polymer could be reused in several cycles of affinity partitioning and recovery.     

Attractive Protein-Polymer Interactions Markedly Alter the Effect of Macromolecular Crowding on Protein Association Equilibria

The dependence of the fluorescence of catalase upon the concentration of added superoxide dismutase (SOD) indicates that SOD binds to saturable sites on catalase. The affinity of SOD for these sites varies with temperature, and with the concentration of each of three nominally inert polymeric additives—dextran 70, Ficoll 70, and polyethylene glycol 2000. At room temperature (25.0°C) and higher, the addition of high concentrations of polymer is found to significantly enhance the affinity of SOD for catalase, but with decreasing temperature the enhancing effect of polymer addition diminishes, and at 8.0°C, addition of polymer has little or no effect on the affinity of SOD for catalase. The results presented here provide the first experimental evidence for the existence of competition between a repulsive excluded volume interaction between protein and polymer, which tends to enhance association of dilute protein, and an attractive interaction between protein and polymer, which tends to inhibit protein association. The net effect of high concentrations of polymer upon protein associations depends upon the relative strength of these two types of interactions at the temperature of measurement, and may vary significantly between different proteins and/or polymers.     

Hydrophobic salt-modified Nafion for enzyme immobilization and stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells. In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes. In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion. Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells. This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.