BIOTECHNOLOGY - V. H. Gerasymenko - 2006

Part II. Special Biotechnologies

Chapter 20. BIOTECHNOLOGY OF ENZYME PRODUCTION

20.3. PRODUCTION OF COMMERCIAL FORMS OF ENZYME PREPARATIONS

20.3.2. Purification of Enzyme Preparations

In some cases, enzyme preparations are used in an unpurified form: in the leather and alcohol industries, the degree of purification does not affect the quality of the finished product, while in animal husbandry, the Introduction of producer biomass and impurities into feed even increases its nutritional value. Conversely, in the food and microbiological industries, as well as in medicine, only sufficiently purified or even highly purified Enzymes can be used. The purification process results in an increased specific activity of the preparation. The dependence of $\alpha$-amylase activity on the degree of preparation purification is demonstrated using Asp. oryzae as an example (Table 20.3).

Class="center">Table 20.3

Purification sequence of Asp. oryzae $\alpha$-amylase

Among the Methods that allow obtaining enzyme preparations of the required degree of purity, Separation Methods based on differences in Protein solubility are widely used.

Separation is most often carried out by precipitating enzyme Proteins without losing their catalytic activity. Soluble enzymes can be precipitated using physical or chemical agents. Physical methods include heating, cooling, dilution, or solution concentration. Chemical agents used for precipitation include Salts of inorganic acids, most commonly ammonium sulfate, as well as organic Solvents (ethanol, acetone, dioxane, polyethylene glycol, dextran).

A high degree of enzyme purity is achieved through chromatography (adsorption, hydrophobic, covalent, ion-exchange, partition, and Gel chromatography), performed on gels of agarose, polyacrylamide, calcium phosphate, as well as on $Al$ oxide, silica gel, starch, Cellulose, etc.

Other Isolation and Purification methods include:

Gel filtration — fractionation of enzymes according to their molecular weight using Sephadexes, Bio-Gels, and Ultrogels;

rate-zonal centrifugation in a density gradient (of sucrose, glycerol, dextran, Glycogen, proteins, etc.);

Electrophoresis — where the enzyme solution is placed in a strong electric field that drives the movement of its ionized components;

isoelectric focusing — the enzyme is focused in a zone where the pH value equals its isoelectric point.

Of the listed enzyme separation and purification methods, only a few (ion-exchange and Affinity Chromatography) have been implemented in large-scale production. A promising approach for Enzyme Purification is The Use of aqueous two-phase systems containing polymers that stabilize the enzyme, thereby eliminating The Need for low temperatures.