Disaccharides, enzyme stabilization

Summarizing the results of many investigations, monosaccharides and such derivatives as D-mannitol and D-glucitol are rather weak acceptors. Disaccharides, including such acceptor products as isomaltose, are much better acceptors, except for certain molecules, for instance leucrose, which is not an acceptor.29,46,47 The decrease of enzyme activity with time has been described in terms of a first-order reaction. The inactivation parameters have been calculated for the immobilized enzyme. The inactivation constants kd were 0.0135 (1/d) when maltose was the acceptor (stabilizing), and 0.029 (1/d) when fructose was the acceptor.38... 

Whereas the use of disaccharides and some polysaccharides gave excellent results when drying the free enzyme, we experienced very poor results when we attempted to use the same stabilizers with immobilized enzymes or enzymes adsorbed onto solid surfaces. 

The biological properties of HA in aqueous solution is controlled by reversible tertiary structures, as defined by NMR spectroscopy. Evidence suggests a ft-pleated sheet-like array stabilized by H- and hydrophobic bonds. Easy transitions between secondary and tertiary structures occur that are convenient mechanisms for switching between functions. The 20 kDa or 50-disaccharide unit is around the size at which such stable tertiary structures are expected to form.199,200 Polymers greater than 20 kDa provide the preferred substrate for Hyal-2. The enzyme cleaves at a much slower rate once the HA substrate loses tertiary structure. The hyaladherins may also provide additional substrate specificity.201 The array of hyaladherins that bind to tertiary HA structures may differ from those that bind to HA chains with exclusively secondary structure. The substrate specificity of Hyal-2 may depend on a combination of differences in bound hyaladherins and on secondary versus tertiary structure. 

Procollagen(I) is an example of a protein that undergoes extensive posttransla-tional modifications. Hydroxylation reactions produce hydroxyproline residues from proline residues and hydroxylysine from lysine residues. These reactions occur after the protein has been synthesized (Fig. 49.3) and require vitamin C (ascorbic acid) as a cofactor of the enzymes, for example, prolyl hydroxylases and lysyl hydroxylase. Hydroxyproline residues are involved in hydrogen bond formation that helps to stabilize the triple helix, whereas hydroxylysine residues are the sites of attachment of disaccharide moieties (galactose-glucose). 

Enzyme spraying onto a heated fluidized bed of inert particles US 4,617,272 Disaccharide-stabilized enzyme preparation EP 0,501,375 A1... 

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