【正文】 tive 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: 1. use of additives, 2. the controlled use of covalent modification, and 3. enzyme immobilisation ? In general, proteins are stabilised by increasing their concentration and the ionic strength of their environment. Neutral salts pete with proteins for water and bind to charged groups or dipoles. This may result in the interactions between an enzyme39。s hydrophobic areas being strengthened causing the enzyme molecules to press 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 aamylases and Co2+ stabilises glucose isomerases. ? At high concentrations (. 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 saltingout the dissolved oxygen from solution. Effect of ions on enzyme stabilisation ? increased chaotropic effect Cations Al3+, Ca2+, Mg2+, Li+, Na+, K+, NH4+, (CH3)4N+ Anions SCN, I, ClO4, Br, Cl, SO42, HPO42, citrate3 increased stabilisation ? Low molecular weight polyols (. glycerol, sorbitol and mannitol) are also useful for stabilising enzymes, by repressing microbial growth, due to the reduction in the water activity, and by the formation of protective shells which prevent unfolding processes. Glycerol may be used to protect enzymes against denaturation due to icecrystal formation at subzero temperatures. ? Some hydrophilic polymers (. polyvinyl alcohol, polyvinylpyrrolidone and hydroxypropylcelluloses) stabilise enzymes by a process of partmentalisation whereby the enzymeenzyme and enzymewater interactions are somewhat replaced by less potentially denaturing enzymepolymer interactions. They may also act by stabilising the hydrophobic effect within the enzymes. ? Important lessons about the molecular basis of thermostability have been learned by parison of enzymes from mesophilic and thermophilic anisms. A frequently found difference is the increase in the proportion of arginine residues at the expense of lysine and histidine residues. ? It should be noted that enzymes stabilised by making them more rigid usually show lower activity (. Vmax) than the 39。natural39。 enzyme . ? Enzymes are very much more stable in the dry state than in solution. Solid enzyme preparations sometimes consist of freezedried protein. ? More usually they are bulked out with inert materials such as starch, lactose, carboxymethylcellulose and other polyelectrolytes which protect the enzyme during a cheaper spraydrying stage. ? Other materials which are added to enzymes before sale may consist of substrates, thiols to create a reducing environment, antibiotics, benzoic acid esters as preservatives for liquid enzyme preparations, inhibitors of contaminating enzyme activities and chelating agents. Additives of these types must, of course, be patible with the final use of the enzyme39。s product. ? Enzymes released onto the market should conform to a number of quality procedures including regulatory requirements, which are legal and mandatory. This is provided by the quality assurance (QA) within the pany. Enzyme products must be consistent as appropriate to their intended use. This may be ensured by good manufacturing practice (GMP) and further checked by quality control (QC).