Introduction to enzyme isolation and purification methods

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Introduction to enzyme isolation and purification methods. There are two types of enzymes produced by biological cells.: A type of enzyme that is produced within a cell and then secreted out of the cell to act is called an extracellular enzyme. Most of these enzymes are hydrolases. For example, the two amylase used in enzymatic production of glucose are secreted by Bacillus subtilis and root enzymes during fermentation. This type of enzyme is generally in high content and easy to obtain ； Another type of enzyme is not secreted outside the cell after being produced in the cell, but plays a catalytic role within the cell. It is called intracellular enzyme. For example, a series of chemical reactions in the fermentation production of citric acid, inosinic acid, and monosodium glutamate are carried out in the cell under the catalysis of a variety of enzymes. In the cell, enzymes are often combined with the cell structure and have a certain distribution area. The catalyzed reactions have a certain order, so that many reactions can be carried out in an orderly manner. The source of enzymes is mostly biological cells. Although the total amount of enzymes produced in biological cells is very high, the content of each enzyme is very low. For example, there are many types of hydrolytic enzymes in the mid-stage digestion of the pancreas, but the content of each enzyme varies greatly. Therefore, when extracting a certain enzyme, you should first select the material containing the most abundant enzyme according to your needs. For example, pancreas is a good material for extracting trypsin, chymotrypsin, amylase and lipase. Since the extraction of enzyme preparations from animal offal or plant fruits is limited by raw materials, if they cannot be comprehensively utilized, the cost will be very high. At present, most industries use the method of cultivating microorganisms to obtain a large number of enzyme preparations. There are many advantages to producing enzyme preparations from microorganisms. It is not limited by climate and geographical conditions, and most of the enzymes in animals and plants can be found in microorganisms. Microorganisms reproduce quickly and produce abundant enzymes. The yield can also be increased by breeding strains, and mass production can be achieved with cheap raw materials. Since in biological tissues, in addition to the enzyme we need, there are often many other enzymes and general proteins as well as other impurities. Therefore, when preparing an enzyme preparation, it must go through separation and purification procedures. Enzymes are proteins with catalytic activity. Proteins are easily denatured. Therefore, during the purification process of enzymes, strong acids and alkalis should be avoided and the operation should be maintained at a lower temperature. During the purification process, it is easier to track the whereabouts of the enzyme during the separation and purification process by measuring the catalytic activity of the enzyme. The catalytic activity of an enzyme can also be used as an indicator for selecting separation and purification methods and operating conditions. In each step of the entire enzyme separation and purification process, the total activity and specific activity of the enzyme must always be measured. Only in this way can we know how much enzyme is recovered after a certain step and how much the purity is improved, which determines the choice of a step. Enzyme isolation and purification generally involves three basic steps:: That is, extraction, purification, crystallization or preparation. First, the required enzyme is introduced into the solution from the raw material, which inevitably carries some impurities. Then the enzyme is selectively separated from the solution, or the impurities are selectively removed from the solution, and then a purified enzyme preparation is made. The following is a comprehensive introduction to common methods for enzyme separation and purification.: 1. Pretreatment and solid-liquid separation technology 1. Cell disruption (cell disruption) high-pressure homogenizer method: This method can be used to disrupt yeast, coliforms, pseudomonas, bacilli and even Aspergillus niger. The cell suspension is passed into a discharge hole with an adjustable pore size under high pressure. If the bacterial cells change from a high-pressure environment to a low-pressure environment, the cells will be easily broken. The cell disruption rate of the bacterial suspension once passed through the homogenizer is 12%-67%. The cell disruption rate is related to the type of cells. To achieve a cell disruption rate of more than 90%, the bacterial suspension must be passed through the homogenizer at least twice. It is best to increase the operating pressure and reduce the number of operations. However, it has been reported that when the operating pressure reaches 175Mpa, the crushing rate can reach 100%. When the pressure exceeds 70Mpa, the cell disruption rate increases slowly. The valve of the high-pressure homogenizer is an important factor affecting the cell disruption rate. Filamentous bacteria can clog the homogenizer valve, especially at high concentrations. Coliforms grown on rich media are more difficult to disrupt than those grown on synthetic media. mycozyme treatment: Egg white is rich in lysozyme, which is cheap and often used to lyse cells. The specific method is: micrococcus lysodeikticus) 43kg, put it in 0.5% sodium chloride solution to make the cell concentration 5% (dry weight), treat it with 0.68kg (dry weight) egg white at 35°C for 20 minutes, treat the obtained cell fragments with the same volume of ethanol, use a centrifuge to remove the cell fragments and intracellular proteins, and then increase the ethanol concentration to 75% (volume fraction) to obtain 1500g of catalase with a purity of 5%. 2. The centrifugal separation process can be divided into three types: centrifugal filtration, centrifugal sedimentation, and centrifugal separation. The equipment used includes filter centrifuges, sedimentation centrifuges, and centrifuges. The filter centrifuge has small holes on the drum wall and filter media on the wall. It can generally be used to handle situations where suspended solid particles are larger and the solid content is higher. Decanter centrifuge is used to separate solid-liquid separation with low solid concentration, such as bacteria in fermentation broth, proteins treated with salting-out method or organic solvents, etc. The separator is used to separate two mutually immiscible emulsions with slightly different densities or emulsions containing trace amounts of solid particles. The centrifuge system used in the biological field should not only meet the general requirements of a centrifuge, but also meet the technical requirements of biological production, including sterilization, cooling, and sealing to ensure that the product is not contaminated and does not pollute the environment. The modern centrifuge device includes the following three steps and is controlled by a program: Sterilization and cleaning-in-place of centrifugation and centrifugation systems. For example, the Alfa-Laval centrifuge product device has a double axial seal. The seal is composed of a silicon carbide moving ring and a fixed ring installed above and below the main shaft of the drum. The seal is continuously cooled and lubricated by water, which can prevent product contamination and prevent waste discharged during the production process from polluting the environment. The centrifuge is also a sealed pressure vessel that can be steam sterilized at a temperature of 121°C. The centrifuge equipment is equipped with a cooling jacket surrounding the centrifuge drum, which can fully cool the suspension and concentrated solids and effectively control the temperature, which is very important for biological products. For example, the BTPX205 centrifuge can be used for cell collection, purification of culture fluid, separation of cell debris, and extraction of vaccines, enzyme preparations, etc. The machine's other auxiliary systems and control systems are also relatively complete, such as pressure indicators, force gauges, temperature sensors and liquid level sensors. 3. Membrane separation technology. The main membrane separation technology used in the protein purification process is mostly ultrafiltration. When the static pressure decreases, the solution passes through the filter membrane with very small pore size, allowing the solutes with smaller molecular weight in the solution to pass through the membrane, while the large molecules are trapped on the surface of the membrane. Most ultrafiltration membranes are composed of a very thin functional membrane combined with a thicker support membrane. The functional membrane determines the pore size of the membrane, while the support membrane provides mechanical strength to resist static pressure. The advantages of ultrafiltration concentration are: The operating conditions are mild, there is no phase change, and there is no damage to biologically active substances. The ultrafiltration system is mainly composed of a feed liquid storage tank, a pump, an ultrafilter, and a permeate collection tank. The feed liquid is pumped into the ultrafilter through a pump, and water and low molecular weight substances are discharged out of the ultrafilter. The concentrated feed liquid circulates in the feed liquid storage tank, pump, and ultrafilter. When the feed liquid is concentrated to a certain multiple, it can be used as a concentrated feed liquid for further processing. The following issues should be paid attention to when ultrafiltration is used in the concentration and desalination process of protein substances.: First, during the ultrafiltration cycle, due to the frictional heat generation between the pump and impeller and the feed liquid, the temperature of the feed liquid will gradually increase, resulting in the loss of protein molecules. Therefore, the liquid storage tank should be equipped with a cooling system and an automatic temperature measurement and control system should be installed. Second, the loss of cofactors for some enzymes is a problem: Some enzymes contain cofactors with small molecular weights that are easily eliminated from the permeate during ultrafiltration. Therefore, a certain concentration of cofactors must be added before or after ultrafiltration. Ultrafiltration can also be combined with affinity chromatography to improve separation purity. Its working principle is: When the protein to be separated in the solution passes through the pores of the ultrafiltration membrane unhindered, if an affinity ligand is bound to one side of the membrane, the protein will bind to the ligand and aggregate on this side of the membrane. Other substances that are not bound to the ligand will pass through the pore and be carried away. The protein is then eluted with a suitable eluent, and the eluent is used for further separation and purification. 4. Principle of foam separation: Gas is passed into a solution containing multiple components. Due to the difference in surface activity of these components, some components will form foam on the surface of the solution. The stability of the foam depends on the operating conditions and the biological characteristics of the solution. Foam contains more surface-active ingredients, so the types and contents of foam components are different from those in the solution. In this way, the components in the solution are separated. Proteins are easier to adsorb to the gas-liquid interface, which is beneficial to the stability of their structure. The foam separation process is: Protein diffuses from the bulk solution to the air-liquid interface, a process that may be reversible or irreversible ； When molecules rearrange, it is generally believed that two types of films will be formed at the air-water interface, one is a dilute film and the other is a concentrated film, and the phenomenon of multiple molecules gathering together may occur. The protein film formed at the air-liquid interface can be a single layer or multiple layers. The type of membrane depends on the properties, structure and concentration of proteins in the bulk solution and at the air-liquid interface. The purpose of foam separation is, on the one hand, to increase the enrichment rate of enzyme protein (concentration of protein in foam/concentration of protein in initial solution), on the other hand to increase the extraction rate of enzyme protein (extraction rate of protein in foam/initial protein quality), or to maximize the distribution coefficient of a certain component in a multi-component mixture. 2. Extraction and precipitation 1. The commonly used salting out agent for salting out is anhydrous sulfate, which has high solubility and low price. Anhydrous sulfate has a strong ability to precipitate proteins, and its saturated solution can precipitate most proteins. Has no damaging effect on enzymes. pH control: The solubility and stability of the enzyme should be considered. At the isoelectric point of the enzyme, its solubility is minimum and it is easy to precipitate. However, some enzymes have poor stability at the isoelectric point, so the optimal pH value must be selected. Generally, it is required to consider the most suitable pH value for enzyme precipitation based on the most stable pH value of the enzyme. Once the optimal pH value is determined during operation, adjust the pH value of the enzyme solution with formic acid or alkali before adding an sulfate. Try to avoid fluctuations in the pH value of the solution to avoid damaging the stability of the enzyme. When adding anhydrous sulfate, pay attention to stirring and the adding speed of anhydrous sulfate. Generally, add slowly from less to more. Grind the anhydrous sulfate into fine powder as much as possible. temperature control: Some enzymes have better stability at higher temperatures and can be salted out at room temperature. However, for most enzymes, they should be operated at low temperatures as much as possible. Enzyme solution cleaning: After adding anhydrous sulfate, the enzyme solution should be allowed to stand for a period of time to allow the enzyme protein to completely precipitate. Do not stir the enzyme after it has been left to stand. 2． Organic solvent precipitation organic solvent selection: Organic solvents that can be used for enzyme protein precipitation include alcohols, such as methanol, ethanol, and isopropyl alcohol. Ethanol has good hydrophilic properties, which can prevent protein denaturation, and the solubility of enzyme proteins in it is also low. Organic solvent precipitation operation: Organic solvents generally denature proteins. When the temperature is high, the denatured protein molecules will become permanently inactive. Therefore, it is best to perform treatment with organic solvents below 0°C. Do not leave the enzyme protein precipitated with organic solvents for too long and add water to dissolve it as soon as possible. 3. Precipitation of polymer flocculants. Polymer flocculants, such as dextran and polyethylene glycol, compete with enzyme molecules for water molecules and have a dehydrating effect to precipitate the enzyme. The advantage of polyethylene glycol as a precipitating agent is that in aqueous solution, its concentration can reach 50%, and most proteins with a concentration of 6%-12% can be precipitated. This reagent does not require low-temperature operation and has a certain protective effect on protein stability. Polyethylene glycol will not be adsorbed, so it does not need to be removed before ion exchange adsorption. 4． Precipitating enzymes and other proteins with metal ions and complexes will form metal salts, which are less soluble. The disadvantage of using metal ions to precipitate is that the reversible changes after the enzyme interacts with the metal ions are poor, especially when using thiol derivatives, which bind] Metal ions will catalyze enzyme denaturation and inactivation. 5. Special reagent precipitation method using streptomycin can selectively remove nucleic acids, thereby precipitating intracellular enzymes. Streptomycin salt (concentration 0.5-1.0 mg/mg protein) is more effective than manganese ions in selectively precipitating nucleic acids, and the enzyme is not easily inactivated. 6． Affinity precipitation organically combines the high selectivity and low throughput characteristics of the affinity reaction with the large throughput and selectivity of the precipitation operation to form affinity precipitation technology. The ligand is coupled with a soluble carrier to form a carrier-ligand complex, which can precipitate under certain conditions after binding to biomolecules. The ligand-carrier complex can selectively bind to proteins. The pH value, ionic strength, protein concentration and other conditions in the solution have little influence on affinity binding. Only competitive ligands will reduce the affinity binding between the product and the original ligand, or even reverse the affinity binding. Methods to induce precipitation include: Ionic cross-linking ； Adding oppositely charged polymers ； Adding oppositely charged hydrophobic groups ； Change the pH value to induce hydrophobic precipitation ； Temperature induces precipitation. affinity binding: Add the affinity ligand to the solution containing the target protein, and adjust the conditions related to precipitation to make it conducive to affinity binding. washing: Non-specific binding may occur due to affinity precipitation in the treated crude solution, especially when using charged polymers. The effect of ion exchange will cause other proteins to co-precipitate, so the precipitate must be washed before isolating the target compound. The approach is: Add appropriate cleaning agent to redissolve the precipitate and then precipitate again ； Or, wash the pellet thoroughly before specific elution. During the above process, the target protein and ligand should always be kept in an affinity binding state. Separation of ligand-carrier complex and target protein: After the separation, it is necessary to ensure that the target protein and ligand-carrier complex are recovered. The target protein must reach a certain purity and the recovery rate must be high.