Tributyl phosphate degradation by immobilized cells of aCitrobacter sp.

Tributyl phosphate (TBP), a plasticizer and solvent, is used in nuclear fuel reprocessing, generating TBP wastes laden with residual uranium. ACitrobacter sp. accumulated heavy metals via a phosphohydrolase(s) that precipitated metals with inorganic phosphate liberated from an organic phosphate “donor” molecule (TBP). Mutant analysis suggested that TBP hydrolysis was not attributable to a previously documented acid phosphatase (monoesterase). Purified monoesterase had little activity against phospho di- and triesters, had no requirement for Mg²⁺ or Mn²⁺, and was EDTA-resistant. Conversely, TBP cleavage by immobilized cells was enhanced by Mg²⁺, and ininhibited by Mn²⁺ and EDTA. A separate phosphotri/diesterase was implicated.     

Synthesis of maltooligosyl fructofuranosides catalyzed by immobilized cyclodextrin glucosyltransferase using starch as donor

Cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on Eupergit C and used for the synthesis of maltooligosyl fructofuranosides employing soluble starch as donor and sucrose as acceptor. Using a weight ratio starch-sucrose of 1:2, the conversion of starch into acceptor products catalyzed by soluble and immobilized CGTases was higher than 80% in 48 h. Under these conditions, the reaction was selective for the formation of maltosyl fructofuranoside.     

Surface modification of proteins by covalent binding of acrylic polymers

Surface modification of enzymes for a potential use in therapy was obtained with a new type of tailor-made copolymers ofNacryloylmorpholine andN-acryloxysuccinimide. The first monomer was designed to confer solubility on the polymer, whereas the second was used to give it reactivity toward protein amino groups. The reactivity of polymers of different composition towards amino acid derivatives and model proteins, such as catalase and ribonuclease-A, is described. Water soluble and catalytically active enzyme derivatives were obained using copolymers prepared with a mixture of N-acryloxysuccinimide andn-acryloylmorpholine in a 1:99 molar ratio. At increasing molar ratio (3:97, 10:90) extensive crosslinking between polymer and enzymes takes place, yielding insoluble adducts.     

Effect of sodium dodecyl sulfate on lipase ofCandida lipolytica

The effects of sodium dodecyl sulfate on extracellular lipase produced byCandida lipolytica have been studied. The microorganism was grown in culture medium containing different sodium dodecyl sulfate concentrations added to the culture at different intervals of growth. The extracellular lipase activity was not detected when the treated culture supernatants were directly tested in Yeast Mold Agar-Triolein-Rhodamine plates, regardless of surfactant addition time and concentrations. However, after ammonium sulfate precipitation and dialysis, the extracellular lipase activity could be recovered. Therefore, the surfactant, under the experimental conditions used here, does not seem to be able to inhibit lipase production, but it does inhibit the enzyme activity because of its presence in the mixture of the reaction.     

Highly conformationally constrained halogenated 6-spiroepoxypenicillins as probes for the bioactive side-chain conformation of benzylpenicillin

Summary The halogenated 6-spiroepoxypenicillins are a series of novel semisynthetic-lactam compounds with highly conformationally restricted side chains incorporating an epoxide. Their biological activity profiles depend crucially on the configuration at position C-3 of that epoxide. In derivatives with aromatic-containing side chains, e.g., anilide, the 3R-compounds possess notable Gram-positive antibacterial activity and potent-lactamase inhibitory properties. The comparable 3S-compounds are antibacterially inactive, but retain-lactamase inhibitory activity.Using the molecular simulation programs COSMIC and ASTRAL, we attempted to map a putative, lipophilic accessory binding site on the PBPs that must interact with the side-chain aromatic residue. Comparative computer-assisted modelling of the 3R, and 3S-anilides, along with benzylpenicillin, indicated that the available conformational space at room temperature for the side chains of the 3R and the 3S-anilides was mutually exclusive. The conformational space for the more flexible benzylpenicillin could accommodate the side chains ofboth the constrained penicillin derivatives. By a combination of van der Waals surface calculations and a pharmacophoric distance approach, closely coincident conformers of the 3R-anilide and benzylpenicillin were identified. These conformers must be related to the antibacterial, bioactive conformer for the classical-lactam antibiotics. From these proposed bioactive conformations, a model for the binding of benzylpenicillin to the PBPs relating the three-dimensional arrangement of a putative lipophilic S₂-subsite, specific for the side-chain aromatic moiety, and the 3-carboxylate functionality is presented.This work has been reported in preliminary form at the 4th Royal Society of Chemistry International Symposium on Recent Advances in the Chemistry of-lactam Antibiotics, Churchill College, Cambridge, U.K., 3–6 July 1988.     

Cassava flour wastewater as a substrate for biosurfactant production

Five cassava flour wastewater (manipueira) preparations were tested as culture media for biosurfactant production by a wild-type Bacillus sp. isolate. No-solids (F), no-solids diluted (F/2), natural (I), natural diluted (I/2), and decanted (IPS) were the tested manipueira media. The microorganism was able to grow and to produce biosurfactant on all manipueira preparations. The media whose solids were removed (F and F/2) showed better results than preparations with the presence of solids (I, I/2, and IPS). No-solids medium (F) showed a surface tension of 26,59 mN/m and reciprocal of critical micelle concentration of over 100 and was selected as a potential substrate for biosurfactant production.     

Whey proteins eluted reversed phase gradients

The behavior of bovine whey proteins in Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) systems has been investigated. The Linear Solvent Strength (LSS) model has been applied to the separation of these proteins studying how their retention time and band broadening change when different gradient parameters are modified. From our results it is deduced that the LSS model describes the behaviour of the whey proteins in RP-HPLC. Also, it seems that t_s (the retention time for non-retained solutes) depends on the size of these proteins. The good fit observed between experimental data and the equations deduced from the LSS model allows the prediction of a gradient shape that permits a rapid analysis of the above mentioned proteins.     

Hydrophilic core-shell microspheres: a suitable support for controlled attachment of proteins and biomedical diagnostics

Functional hydrophilic microspheres (latex particles) have found various applications in life sciences and in medicine - particularly in latex diagnostic tests. This paper presents a comprehensive review of studies on latex particles with a hydrophilic interfacial layer composed of various hydrophilic polymers with reactive groups at the ends of macromolecules or at each monomeric unit along the chain. Typical examples of these hydrophilic polymers are poly(2-hydroxyethyl methyl methacrylate), poly(acrylic acid), poly(N,N-dimethylacrylamide), polysaccharides, poly(ethylene oxide) and polyglycidol. Hydrophilic microspheres with different morphologies (uniform or core-shell, see Figure) have been synthesized by emulsion and dispersion polymerizations. The chemical structure of polymers which constitute the interfacial layer of microspheres has been investigated using a variety of instrumental techniques (such as XPS, SSIMS and NMR) and analytical methods based on specific chemical reactions suitable for the determination of particular functional groups. Microspheres are exposed to contact with proteins in the majority of medical applications. This paper presents examples of studies on the attachment of these biomacromolecules to microspheres. The relation between the structure of the interfacial layer of microspheres and the ability of these particles for the covalent binding of proteins is discussed. Several examples of diagnostic tests, in which hydrophilic microspheres with adsorbed or covalently immobilized proteins were used as reagents, are presented. The paper also contains a short review of the application of magnetic hydrophilic particles for protein separation. Examples of hydrophilic latex particles used for hemoperfusion or heavy metal ion separation are presented. Hydrophilic microspheres with uniform or core-shell morphologies.     

Principle of a new immunoassay based on electrophoretic mobility of poly(styrene/alpha-tert-butoxy-omega-vinylbenzyl-polyglycidol) microspheres: application for the determination of helicobacter pylori IgG in blood serum

The principle of a novel latex diagnostic test for the determination of antibodies against Helicobacter pylori in blood sera is described. The test is based on the measurement of the electrophoretic mobility of the microspheres with immobilized H. pylori antigens. The electrophoretic mobility of these microspheres depends on the concentration of the antibodies against H. pylori in suspending medium. Particles with hydrophilic polyglycidol in the surface layer were used for the test. The microspheres were obtained by an emulsifier-free emulsion copolymerization of styrene and alpha-tert-butoxy-omega-vinylbenzyl-polyglycidol macromonomer (D(n) = 220 nm, diameter polydispersity factor D(w)//D(n) = 1.02). Activation of polyglycidol hydroxyl groups with cyanuric chloride allowed for covalent immobilization of H. pylori antigens. The fraction of H. pylori not specifically adsorbed onto the microspheres was negligible. Changes of the electrophoretic mobility of the microspheres with the surface concentration of the covalently immobilized H. pylori antigens Gamma = (1.6 +/- 0.3) . 10(-3) g m(-2) were suitable for the detection of the antibodies in the sera of patients with titer in the range (determined by the indirect ELISA test) from 1:500 to 1:32 000.     

Self-assembled monolayers of dendritic polyglycerol derivatives on gold that resist the adsorption of proteins

Highly protein-resistant, self-assembled monolayers (SAMs) of dendritic polyglycerols (PGs) on gold can easily be obtained by simple chemical modification of these readily available polymers with a surface-active disulfide linker group. Several disulfide-functionalized PGs were synthesized by N,N'-dicyclohexylcarbodiimide-mediated ester coupling of thioctic acid. Monolayers of the disulfide-functionalized PG derivatives spontaneously form on a semitransparent gold surface and effectively prevent the adsorption of proteins, as demonstrated by surface plasmon resonance (SPR) kinetic measurements. A structure-activity relationship relating the polymer architecture to its ability to effectuate protein resistance has been derived from results of different surface characterization techniques (SPR, attenuated total reflectance infrared (ATR-IR), and contact-angle measurements). Dendritic PGs combine the characteristic structural features of several highly protein-resistant surfaces: a highly flexible aliphatic polyether, hydrophilic surface groups, and a highly branched architecture. PG monolayers are as protein resistant as poly(ethylene glycol) (PEG) SAMs and are significantly better than dextran-coated surfaces, which are currently used as the background for SPR spectroscopy. Due to the higher thermal and oxidative stability of the bulk PG as compared to the PEG and the easy accessibility of these materials, dendritic polyglycerols are novel and promising candidates as surface coatings for biomedical applications.