课件：酶的提取和分离纯化.ppt

It should be remembered that most industrial enzymes contain relatively little active enzyme ( 10% w/w, including isoenzymes and associated enzyme activities), the rest being due to inactive protein, stabilisers, preservatives, salts and the diluent which allows standardisation between production batches of different specific activities. Approaches to maintain enzyme activity The key to maintaining enzyme activity is maintenance of conformation, so preventing unfolding, aggregation and changes in the covalent structure. Three approaches are possible: use of additives, the controlled use of covalent modification, and enzyme immobilisation In general, proteins are stabilised by increasing their concentration and the ionic strength of their environment. Neutral salts compete with proteins for water and bind to charged groups or dipoles. This may result in the interactions between an enzymes hydrophobic areas being strengthened causing the enzyme molecules to compress and making them more resistant to thermal unfolding reactions. Not all salts are equally effective in stabilising hydrophobic interactions, some are much more effective at their destabilisation by binding to them and disrupting the localised structure of water. Ammonium sulphate and potassium hydrogen phosphate are a powerful enzyme stabilisers whereas sodium thiosulphate and calcium chloride destabilise enzymes. Many enzymes are specifically stabilised by low concentrations of cations which may or may not form part of the active site, for example Ca2+ stabilises a-amylases and Co2+ stabilises glucose isomerases. At high concentrations (e.g. 20% NaCl), salt discourages microbial growth due to its osmotic effect. In addition ions can offer some protection against oxidation to groups such as thiols by salting-out the dissolved oxygen from solution. Effect of ions on enzyme stabilisation ? increased chaotropic effect Cations Al3+, Ca2+, Mg2+, Li+, Na+, K+, NH4+, (CH3)4N+ Anio