Before enzymes can be used in industrial applications, they must be extracted from their biological sources. Each extraction or purification step increases production costs, thereby reducing the enzyme yield. Specific enzyme activity can be enhanced through additional purification steps, but these also require extra costs.
From the biological source, the protein content of the enzyme must be high to facilitate the extraction process. Another important factor is maintaining enzyme activity. The maximum activity must be preserved during enzyme preparation. Factors such as heat, proteolysis, pH changes, oxidation, and loss of cofactors can reduce enzymatic activity.
Extracellular enzymes are sometimes used without isolation or purification. On the other hand, intracellular enzymes require cell disruption to release the enzymes from the cells into the solution before purification. Isolating intracellular enzymes often involves separating complex biological mixtures. Extracellular enzymes are typically released into the medium with only a few other components. Native conformation is achieved under specific pH, temperature, and ionic strength conditions.
Solid-liquid separation is necessary to remove cellular debris after cell disruption. Intracellular enzymes may contain nucleic acids, which can interfere with purification procedures by increasing viscosity. Nucleic acids must be removed by adding nucleases or through precipitation.
1. Biological Material Preparation
Animal organs should be transported and stored at low temperatures to maintain enzymatic activity. The organs need to be cleaned of fat and connective tissue before freezing. Frozen organs can be chopped using machines typically used in the meat industry, and enzymes can be extracted with a buffer solution. In addition to mechanical grinding, enzymatic digestion can also be used. Fat attached to organs interferes with subsequent purification steps and can be removed with organic solvents.
Plant material can be ground with various grinders, and the desired enzymes can be extracted with buffer solutions. Cells can also be disrupted through pretreatment with lytic enzymes. Microorganisms are an important source of enzymes. Genes can be transferred into microorganisms to enable them to produce proteins they do not naturally produce. Alternatively, modifying a microorganism's genome can alter the properties of proteins, making them easier to isolate and purify.
1.1. Cell Disruption by Mechanical Methods
High-pressure homogenization is the most common method of cell disruption. Wet grinding of cells in a high-speed ball mill is another effective method. Ultrasonics are generally used in laboratories. Other methods include high-pressure nitrogen, French press, and freeze-thaw cycles.
1.2. Cell Disruption by Chemical Methods
Cells can often be disrupted by chemical lysis. Osmotic shock and modification of ionic forces through chemical agents are standard procedures in which the cell wall structure is altered to allow the subsequent extraction of cellular contents.
1.3. Cell Disruption by Enzymatic Methods
Enzyme-based methods (e.g., lysozymes) or autolysis have been described in the literature as techniques for cell disruption.
2. Solid Matter Separation
After cell disruption, the next step is the separation of extracellular or intracellular enzymes from the cells or cell fragments, respectively. This operation is challenging due to the small size of bacterial cells and the slight difference in density between the cells and the fermentation medium.
2.1 Filtration
Continuous filtration is used in the industry. Large cells, such as yeast cells, can be removed by sedimentation. The filtration rate depends on filter surface area, pressure, viscosity, and the resistance of the filter cake and medium. Vacuum filtration is typically the method of choice as biological materials are easily compressible. The future of this method depends on the development of suitable membranes.
2.2 Centrifugation
Effective centrifuges have been developed to separate cells and cell fragments in a continuous process. Plant and organ residues can be separated using centrifuges or simpler filters.
2.3 Extraction
An elegant method for isolating intracellular enzymes is liquid-liquid extraction in a two-phase aqueous system. The first extraction step separates the cell fragments.
2.4 Flocculation and Floatation
Flocculation is the process by which destabilized particles aggregate, come into contact, and form larger aggregates. Flocculating agents are additives that increase the degree of flocculation in a suspension.
3. Concentration
The enzyme concentration in the starting material is often very low. The volume of material to be processed is generally large, and significant amounts of waste must be eliminated. Therefore, to achieve economical purification, the starting material volume must be reduced by concentration. Only mild concentration procedures that do not inactivate the enzymes can be used. These include thermal methods, precipitation, and increasingly, membrane filtration.
3.1 Thermal Methods
Only brief thermal treatments can be used for concentration, as enzymes are thermolabile. Rotary evaporators can be employed for this purpose.
3.2 Precipitation
Enzymes are complex protein molecules with ionizable and hydrophobic groups that interact with the solvent. Proteins can be aggregated and eventually precipitated by modifying their environment. Precipitation is a simple procedure for concentrating enzymes. High salt concentrations affect the water molecules surrounding the protein and modify the electrostatic forces responsible for solubility. Ammonium sulfate is commonly used for precipitation, making it an effective agent for enzyme concentration.
Organic solvents affect enzyme solubility by reducing the dielectric constant of the medium. This modifies the solvation effect of the water molecules surrounding the enzyme, increases protein-protein interactions, and leads to aggregation and precipitation. Polymers such as polyethyleneimines and poly(ethylene glycols) of different molecular weights are also used to concentrate enzymes.
Protein solubility is strongly influenced by pH and is minimal at the isoelectric point, where the net charge is zero. Since most proteins have isoelectric points in the acidic range, this process is also referred to as acid precipitation.
3.3 Ultrafiltration
A semi-permeable membrane allows the separation of solvent molecules from larger enzyme molecules, as only smaller molecules can pass through the membrane when osmotic pressure is exceeded. This principle is used in all membrane separation processes, including ultrafiltration. Membranes for ultrafiltration are available and exclude molecules between 1000 and 300,000 Daltons.
4. Purification
For many industrial applications, partially purified enzyme preparations will suffice; however, enzymes for analytical and medical use must be highly purified. Special procedures for enzyme purification include crystallization, electrophoresis, and chromatography.
4.1 Crystallization
Enzyme crystallization is the formation of solid enzyme particles with defined shape and size. An enzyme can be crystallized or form protein-protein interactions by creating solvent conditions that lead to enzyme supersaturation. Enzymes that have been crystallized for commercial production include cellulase, glucose isomerase, subtilisin, and alcohol oxidase.
4.2 Electrophoresis
Electrophoresis is used to isolate pure enzymes at the laboratory scale. Depending on the conditions, the following procedures may be used: zone electrophoresis, isotachophoresis, or porosity gradients. Heat generated during electrophoresis and convection interferences are problems when scaling up this method. An interesting development in industrial electrophoresis is a continuous process where the electric field is stabilized by rotation.
4.3 Chromatography
Chromatography is fundamental to enzyme purification. Molecules are separated based on their physical properties (size, shape, charge, hydrophobic interactions), chemical properties (covalent binding), or biological properties (biospecific affinity).
