ABSTRACT
Serine proteinase subtilisin 72 was covalently attached to the beads of poly(vinyl alcohol)-cryogel, a macroporous hydrogel prepared by the freeze-thaw technique. The immobilized enzyme was examined as a catalyst in the synthesis of protected peptides Z-Ala-Ala-Xaa-Phe-pNA (Xaa = Leu, Glu, Lys) in acetonitrile/dimethylformamide mixtures. Immobilized subtilisin catalyzed with high yield the formation of peptide bonds between Phe-pNA and acyl donors including those with free carboxylic group and non-protected C-terminal basic and acidic amino acid residues.
Subject(s)
Acetonitriles/pharmacology , Dimethylformamide/pharmacology , Peptides/chemical synthesis , Subtilisins/drug effects , Subtilisins/metabolism , Acetonitriles/chemistry , Bacillus subtilis/enzymology , Catalysis , Dimethylformamide/chemistry , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/drug effects , Enzymes, Immobilized/metabolism , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Peptide Biosynthesis , Peptides/chemistry , Peptides/metabolism , Polyvinyl Alcohol/chemistry , Serine Endopeptidases/drug effects , Serine Endopeptidases/metabolism , Time FactorsABSTRACT
This is a review discussing the production and properties of cryogels (from the Greek kappa rho iota sigma (kryos) meaning frost or ice), immobilization of ligands in cryogels and the application of affinity cryogels in bioseparation. Cryotropic gel formation proceeds in a non-frozen liquid microphase existing in the macroscopically frozen sample. Due to the cryoconcentration of gel precursors in the non-frozen liquid microphase, cryogelation is characterised by a decrease in the critical concentration of gelation and an increase in gelation rates compared with traditional gelation at temperatures above freezing point. Cryogels can be obtained through the formation of both physically and covalently cross-linked heterogeneous polymer networks. Interconnected systems of macropores and sponge-like morphology are typical for cryogels, allowing unhindered diffusion of solutes of practically any size. Most of the water present in spongy cryogels is capillary bound and can be removed mechanically by squeezing. The properties of cryogels can be regulated by the temperature of cryogelation, the time the sample is kept in a frozen state and freezing/thawing rates, by the nature of the solvent and by the use of soluble and insoluble additives. The unique macroporous morphology of cryogels, in combination with osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of large entities such as protein aggregates, membrane fragments, viruses, cell organells and even whole cells. Special attention is given to immunosorption of viruses on cryogel-based sorbents. As chromatographic materials, cryogels can be used both in bead form and as spongy cylindrical blocks (monoliths) synthesized inside the chromatographic column. The macroporous nature of cryogels is also advantageous for their application as matrices in the immobilization of biocatalysts operating in both aqueous and organic solvents. New potential applications of cryogels are discussed.
