Highly concentrated water-in-oil emulsions as novel reaction media for protease-catalysed kinetically controlled peptide synthesis

Abstract

High-internal-phase-ratio-emulsions (HIPREs) or gel emulsions, formulated with a large amount of water (80.0-99.5% w/w), were investigated as reaction media for α-chymotrypsin-catalysed peptide synthesis under kinetic control using Ac--Phe-OEt and H--Leu-NH 2 as model substrates. Both the initial reaction rate and dipeptide yield were examined as a function of the structure of the non-ionic polyoxyethylene alkyl ether type surfactant, alkyl chain length of the oil component, temperature and aqueous buffer content. Dipeptide yields of 70% were achieved in gel emulsions formulated with 90% w/w aqueous buffer. In these systems, the reaction performance was found to be independent of the gel emulsion system (i.e. surfactant and oil) and therefore of the water-oil interfacial tension. Interestingly, α-chymotrypsin showed superactivity at surfactant concentrations ranging between 0.2 and 0.8% w/w, that is, at 99.5 and 98.0% w/w water content, respectively. Furthermore, high dipeptide yields (90-94%) were achieved in the gel emulsions studied at very high substrate concentrations and thus with undissolved reactants. Under these conditions, examples of α-chymotrypsin-catalysed dipeptide synthesis on an analytical and preparative scale were conducted.

Effect of Incorporation of Multiwalled Carbon Nanotubes on the Microstructure and Flow Behavior of Highly Concentrated Emulsions

“…HCEs are extensively used in the food industry, paints, pharmaceuticals, cosmetics, and petroleum-based industries. In addition, HCEs are widely used as reaction media in chemical reactions − and as templates for the preparation of low-density organic and inorganic substances. − The preparation of stable HCEs with volume fractions exceeding the critical value is only possible by using extremely polydisperse systems and/or by the deformation of spherical droplets into polyhedrons . HCEs maintain their original structure at such high values of ϕ because of the polydispersity and the “compression” of spherical droplets within the system …”

Preparation of Span 80/oil/water highly concentrated emulsions: Influence of composition and formation variables and scale-up

“…. ), cosmetics [10], skin protective films and topical drug delivery [11,12], membrane supports, filters [13], preparation of low-density organic and inorganic foams (macroporous and meso/macroporous materials) [14,15], as novel reaction media in chemical and enzyme-catalysed reactions [15][16][17], etc.…”

Proteases as Powerful Catalysts for Organic Synthesis

“…A biphasic reaction medium is another option for performing peptide synthesis since the partition of the peptide products from the aqueous phase that contains the enzyme to the organic phase drives the equilibrium towards synthesis, with the additional benefit that the product is no longer subjected to hydrolysis [213,214,[219][220][221]. An alternative to the classic biphasic system has been proposed by Clap es et al [225] as reaction medium for enzymatic peptide synthesis using water-in-oil emulsion with a high water content (95%) which differs from conventional reverse micelles where the water phase containing the enzyme represents a small percentage of the volume which corresponds mostly to the oil phase [226][227][228]. However, reaction rates in biphasic systems are low because of the limitation of substrate diffusion and because proteases tend to denature at the water/solvent interface [202,224].…”