Evgeny N. Vulfson

Biologically active peptides and enzymatic approaches to their production

This review briefly surveys various classes of biologically active and flavor peptides that have been isolated and characterized in recent years, and analyzes emerging trends and advances in biotechnological methods for their production.

Application of enzymes to the synthesis of surfactants

Recent progress in the application of isolated enzymes to the preparation of surface-active compounds demonstrates the feasibility of an alternative to organosynthetic methods. Processes such as the syntheses of monoglycerides, sugar fatty acid esters, (lyso)phospholipids, anomerically pure alkyl glycosides and amino acid-based surfactants are discussed, highlighting some of the advantages of enzymatic methods over conventional organic syntheses and whole-cell systems.

Directed nucleation of calcite at a crystal-imprinted polymer surface

The finely tuned properties of natural biominerals and composites reflect the remarkable level of control that is exercised over the size, shape and organization of the constituent crystals. Achieving this degree of control over synthetic materials might therefore lead to superior material properties. Organic small molecules, polymers or surfactant mesophases have been used to guide the growth and morphology of inorganic materials via steric constraints or structure-directing interactions. Here we show that synthetic polymers can be imprinted with motifs of crystal surfaces so as to template the growth of specific crystal phases. Our polymers, imprinted with calcite, are able to induce the nucleation of calcite under conditions favouring the growth of aragonite (another polymorph of calcium carbonate). The synthesis of the polymers, based on the principles of molecular imprinting, involves the adsorption of functional monomers to a calcite surface, followed by co-polymerization with a crosslinker to create an imprint of the crystal surface. Subsequent removal of the calcite template yields a polymer matrix with a surface functionality mirroring the crystal face and able to promote the nucleation of calcite. We expect that the molecular-imprinting approach to directed nucleation can be applied to crystals other than calcite.

Comparison of polymer coatings of capillaries for capillary electrophoresis with respect to their applicability to molecular imprinting and electrochromatography

Abstract In molecular imprinting (MI), interactive monomers and suitable cross-linking agents are polymerized in the presence of a template. Once the template has been removed, the remaining space acts as a highly specific binding site for the template or analogs thereof, due to the unique three-dimensional arrangement of interaction points. Several steps are involved in producing such a polymer coat inside a capillary electrophoresis capillary. First, the silanization of the inner surface of the capillary with a suitable silane is necessary, to link a monolayer of unsaturated groups suitable for polymerization to the capillary surface. These monomeric groups are then integrated into the three-dimensional polymer coat produced in the next step. MI-capillary coatings ideally are highly porous and of a thickness, δ, which is smaller than the inner radius, r, of the capillary in question. Porous polymer networks can be produced by dispersive polymerization using a suitable solvent (porogen). However, the exact conditions for producing a coating suitable for capillary zone electrophoresis had to be determined experimentally. Seven porogens, namely hexane, toluene, tetrahydrofuran, acetonitrile, CHCl3, dimethyl sulfoxide and dimethylformamide, and two cross-linkers, namely ethyleneglycoldimethacrylate and divinylbenzene, at concentrations of between 5 and 20% (v/v) were investigated. In about 20% of the combinations, a polymer coat of the desired qualities was obtained. The applicability of the MI capillaries to specific separations was demonstrated for the separation of a racemic mixture of S(+)- and R(−)-2-phenylpropionic acid. trans-3(3-Pyridyl)-acrylic acid was used as the interactive monomer in this case.

Molecularly imprinted nanoparticles prepared by core‐shell emulsion polymerization

Submicron core-shell polymer particles, with molecularly imprinted shells, were prepared by a two-stage polymerization process. Particles of this type, prepared with a cholesterol-imprinted ethyleneglycol dimethacrylate shell and in the absence of porogen, were found to be 76 nm in diameter with a surface area of 82 m2 g−1. Cholesterol uptake from a 1 mM solution in isohexane was measured at both 10 and 30 mg mL−1, with the imprinted polymer showing considerable binding (up to 57%). Imprinted but not hydrolyzed and hydrolyzed nonimprinted polymers showed very low uptakes (≤4.5%) and a phenol-imprinted polymer showed reduced binding (36%) under the same conditions. Imprinted shells were also prepared over superparamagnetic polymer cores and over magnetite ferrocolloid alone. The cholesterol binding to magnetic particles was very similar to that of equivalent nonmagnetic materials. Magnetic particles could be sedimented in as little as 30 s in a magnetic field.

The synthesis of sub-micron magnetic particles and their use for preparative purification of proteins

A novel one-step protocol for the preparation of sub-micron magnetic particles as small as 30 nm in diameter has been developed. The surface of the particles was functionalized with carboxyl groups ( approximately 1 COOH per 15A2) to facilitate the attachment of affinity ligands. The high surface area of the resulting beads, combined with the high density of functional groups on the surface, makes them ideally suited for the preparative purification of proteins as was demonstrated by the efficient isolation of trypsin (36 mg per gram of particles) from pancreatic extract.

Enzymic solvent-free synthesis of sugar acetal fatty acid esters

Abstract An enzymic solvent-free acylation of sugar acetals with a range of long-chain fatty acids (lauric, myristic, palmitic, stearic) and their methyl esters is described. The lipase-catalysed reaction was carried out at 1:1 and 2:1 molar ratios of fatty acid or methyl ester of fatty acid to sugar acetal to obtain mono- or diesters, respectively. A range of 6 monoesters of 1,2-O-isopropylidene- d -glucofuranose and 1,2:3,4-Di-O-isopropylidene- d -galactopyranose as well as 5-mono- and 3,5-diesters of 1,2-O-isopropylidene- d -xylofuranose were prepared. The synthesis was performed at 75°C in open vials or under vacuum, to provide an equilibrium shift towards the final products via evaporation of water or methanol produced in the course of the reaction. Typically yields of 50–90% were obtained under optimal conditions.

Enzymatic synthesis of food ingredients in low-water media

Abstract Current and potential applications of enzymes in low-water media to the synthesis of food ingredients/additives are described. Examples given include biotransformations of fats and oils, and the synthesis of emulsifiers, flavours, peptides and oligosaccharides. Advantages associated with biotechnological methods of production are briefly discussed.

Alkyl-β-glucoside synthesis in a water-organic two-phase system

SummaryAlmond β-glucosidase, adsorbed on XAD-4, has been shown to catalyse alkyl-β-glucoside synthesis in a water organic two-phase system; a concentrated glucose solution and water immiscible alcohols were used as the aqueous and the organic phase respectively. The reaction yield of approximately 10 g/l was achieved with a wide range of primary alcohol. The half-life of the biocatalyst was estimated to be 25 days in the described system.

Identification of inorganic crystal-specific sequences using phage display combinatorial library of short peptides: A feasibility study

The feasibility of using a combinatorial phage display library of decapeptides to identify ligands which can interact with the surface of a crystal was assessed using geological calcium carbonate as a model. Two relatively strong binding clones were identified by ELISA, sequenced and the encoded oligopeptides were prepared by solid phase synthesis and their properties compared with those of casein hydrolysate.