Preparation of enzymes for sale

Once the enzyme has been purified to the desired extent and concentrated, the manufacturer's main objective is to retain the activity. Enzymes for industrial use are sold on the basis of overall activity. Often a freshly supplied enzyme sample will have a higher activity than that stated by the manufacturer. This is done to ensure that the enzyme preparation has the guaranteed storage life. The manufacturer will usually recommend storage conditions and quote the expected rate of loss of activity under those conditions. It is of primary importance to the enzyme producer and customer that the enzymes retain their activity during storage and use. Some enzymes retain their activity under operational conditions for weeks or even months. Most do not.

To achieve stability, the manufacturer uses all the subtleties at their disposal. Formulation is an art and often the precise details of the methods used to stabilise enzyme preparations are kept secret or revealed to customers only under the cover of a confidentiality agreement. Sometimes it is only the formulation of an enzyme that gives a manufacturer the competitive edge over rival companies. 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.

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 (discussed further in Chapter 3).

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 enzyme's 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 (the chaotropic effect, Table 2.4). From this it can be seen why 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.

Table 2.4. 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 (e.g., 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 ice-crystal formation at sub-zero temperatures. Some hydrophilic polymers (e.g., polyvinyl alcohol, polyvinylpyrrolidone and hydroxypropylcelluloses) stabilise enzymes by a process of compartmentalisation whereby the enzyme-enzyme and enzyme-water interactions are somewhat replaced by less potentially denaturing enzyme-polymer interactions. They may also act by stabilising the hydrophobic effect within the enzymes. Many specific chemical modifications of amino acid side chains are possible which may (or, more commonly, may not) result in stabilisation. A useful example of this is the derivatisation of lysine side chains in proteases with N-carboxyamino acid anhydrides. These form polyaminoacylated enzymes with various degrees of substitution and length of amide-linked side chains. This derivatisation is sufficient to disguise the proteinaceous nature of the protease and prevent autolysis.

Important lessons about the molecular basis of thermostability have been learned by comparison of enzymes from mesophilic and thermophilic organisms. A frequently found difference is the increase in the proportion of arginine residues at the expense of lysine and histidine residues. This may be possibly explained by noting that arginine is bidentate and has a higher pK_a than lysine or histidine (see Table 1.1). Consequently, it forms stronger salt links with bidentate aspartate and glutamate side chains, resulting in more rigid structures. This observation, among others, has given hope that site-specific mutagenesis may lead to enzymes with significantly improved stability (see Chapter 8). In the meantime it remains possible to convert lysine residues to arginine-like groups by reaction with activated ureas. It should be noted that enzymes stabilised by making them more rigid usually show lower activity (i.e., V_max) than the 'natural' enzyme.

Enzymes are very much more stable in the dry state than in solution. Solid enzyme preparations sometimes consist of freeze-dried protein. More usually they are bulked out with inert materials such as starch, lactose, carboxymethylcellulose and other poly-electrolytes which protect the enzyme during a cheaper spray-drying 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 compatible with the final use of the enzyme'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 company. 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).