There are two types of enzymes produced by biological cells: one is an enzyme that is produced by intracellular secretion and then secreted outside the cell, called an extracellular enzyme. Most of these enzymes are hydrolases, such as the two amylases used in the enzymatic production of glucose, which are secreted by the fermentation of Bacillus subtilis and root enzymes. These enzymes are generally high in content and easy to obtain; another type of enzyme is not secreted outside the cell after it is produced in the cell, but acts as a catalyst in the cell, called intracellular enzymes such as citric acid, inosinic acid, and MSG. The series of chemical reactions carried out in the fermentation production are carried out in a cell under the catalysis of various enzymes. In the cell, the enzyme often binds to the cell structure, and there is a certain distribution area, and the catalytic reaction has a certain order. Many reactions of the disc separator can be carried out in an orderly manner.
The source of the enzyme is mostly biological cells. Although the total amount of enzyme produced in biological cells is very high, the content of each enzyme is very low. For example, there are many types of hydrolyzing enzymes in the mid-stage digestion of pancreas, but the contents of various enzymes vary greatly.
Therefore, when extracting an enzyme, the material most abundant in this enzyme should be selected first, and the pancreas is a good material for extracting trypsin, chymotrypsin, amylase and lipase. Since the extraction of the enzyme preparation from the animal's internal organs or plant fruits is limited by the raw materials, if it cannot be comprehensively utilized, the cost is large. At present, most methods for culturing microorganisms are used in the industry to obtain a large amount of enzyme preparations. There are many advantages in the production of enzyme preparations from microorganisms, which are not limited by climatic and geographical conditions, and the enzymes in animals and plants can be found in microorganisms. Microorganisms are fast-growing, and the amount of enzymes is abundant. It is also possible to select strains. Increase production and mass production with cheap raw materials.
In biological tissues, in addition to one of the enzymes we need, there are often many other enzymes and general proteins and other impurities. Therefore, in order to prepare an enzyme preparation, it is necessary to undergo a purification process.
Enzymes are catalytically active proteins, and proteins are easily denatured. Therefore, strong acid and alkali should be avoided during the purification of the enzyme and kept at a lower temperature. It is relatively easy to track the fate of the enzyme during the separation and purification process by measuring the catalytic activity of the enzyme during the purification process. The catalytic activity of the enzyme can be used as an indicator for selecting the separation and purification method and the operating conditions. At each step of the separation and purification process of the whole enzyme, the total activity and specific activity of the enzyme are always determined, so that the recovery can be known through a certain step. How many enzymes, the purity is increased, which determines the one-step trade-off.
The separation and purification of the enzyme generally involves three basic steps: extraction, purification, crystallization or formulation. First, the desired enzyme is introduced into the solution from the raw material, at which time some impurities are inevitably entrapped, and then the enzyme is selectively separated from the solution, or the impurities are selectively removed from the solution, and then purified. Enzyme preparation. The following is a comprehensive introduction to the common methods of separation and purification of enzymes:
1. Pretreatment and solid-liquid separation technology
Cell disruption
High-pressure homogenizer method: This method can be used to break yeast, coliform, pseudomonas, bacilli and even Aspergillus. The cell suspension is passed under high pressure into an orifice with adjustable pore size. The cells are transferred from a high pressure environment to a low pressure environment, and the cells are easily broken. The cell breakage rate of the bacterial suspension once through the homogenizer is between 12% and 67%. The rate of cell disruption is related to the type of cell. To achieve a cell breakage rate of more than 90%, at least pass the bacterial suspension through the homogenizer 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 175 MPa, the breaking rate can reach 100%. When the pressure exceeds 70 MPa, the cell breakage rate rises more slowly. The valve of the high pressure homogenizer is an important factor affecting the cell breakage rate. Filamentous bacteria can block the valves of the homogenizer, especially when the concentration of bacteria is high. It is more difficult to break on rich media than coliforms grown on synthetic media.
Capsule enzyme treatment: Egg white is rich in lysozyme, which is cheap and is commonly used to lyse cells. The specific method is: 43kg of micrococcus lysodeikticus, placed in 0.5% sodium chloride solution, the cell concentration is 5% (dry weight), treated with 0.68kg (dry weight) of egg white at 35 ° C for 20min The obtained cell debris was treated with the same volume of ethanol, and the cell debris and intracellular protein were removed by a centrifuge, and the ethanol concentration was increased to 75% (volume fraction) to obtain 1500 g of a hydrogen peroxide having a purity of 5%.
2. Centrifugation The centrifugal separation process can be divided into three types: centrifugal filtration, centrifugal sedimentation, and centrifugal separation. The equipment used is a filter centrifuge, a decanter centrifuge, and a centrifuge. The filter centrifuge has a small hole in the drum wall and a filter medium on the wall, which can generally be used for the treatment of large suspended solid particles and high solid content. The decanter centrifuge is used to separate solid-liquid separation with low solid concentration, such as bacteria in the fermentation broth, protein treated by salting out or organic solvent, and the like. The separator is used to separate two immiscible, slightly different density emulsions or emulsions containing traces of solid particles.
The centrifuge system used in the biological field, in addition to the general requirements of the centrifuge, should also meet the technical requirements of biological production, including sterilization, cooling, sealing, to ensure that the product is free from pollution and does not pollute the environment. Modern centrifuge equipment consists of the following three steps and is programmed to control: centrifugation, centrifugation, and in-situ cleaning. For example, the Alfa-Laval centrifuge product has a double axial seal. The seal consists of a silicon carbide moving ring and a retaining ring mounted on the top and bottom of the drum. The seal is continuously cooled and lubricated by water to prevent product contamination. It can also prevent environmental pollution caused by waste discharged during the production process. The centrifuge, in turn, is a closed pressure vessel that can be steam sterilized at 121 ° C. The centrifuge is equipped with a cooling jacket surrounding the centrifuge drum for adequate cooling of the suspension and concentrated solids. And can effectively control the temperature, which is very important for biological products. For example, BTPX205 centrifuge can be used for cell collection, purification of culture solution and separation of cell debris, and can be used for extraction of vaccines, enzyme preparations and the like. The machine's other auxiliary systems and control systems are also perfect, such as pressure indicators, power meters, temperature sensors and liquid level sensors.
3. Membrane separation technology The membrane separation technology mainly used in protein purification process is mostly ultrafiltration. The hydrostatic lowering solution passes through a filter membrane having a very small pore size, so that the solute having a smaller molecular weight in the solution is transmitted through the membrane, and the macromolecule is trapped on the surface of the membrane. Most ultrafiltration membranes consist 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: mild operating conditions, no phase change, no damage to biologically active substances.
The ultrafiltration system mainly consists of a liquid storage tank, a pump, an ultrafilter, and a permeate collection tank. The feed liquid is pumped into the ultrafilter, and the water and low molecular weight substances are discharged from the ultrafilter, and the concentrated feed liquid is Circulating in the liquid storage tank, pump, and ultrafilter. When the feed liquid is concentrated to a certain multiple, it can be used as a concentrated liquid for further treatment.
When applying ultrafiltration to the concentration and desalination of proteinaceous materials, the following problems should be noted: First, during the ultrafiltration cycle, the temperature of the feed liquid will gradually increase due to the frictional heat release from the pump and the impeller and the feed liquid. Will cause loss of protein molecules. Therefore, the liquid storage tank should be equipped with a cooling system and an automatic temperature measurement and control system. Second, the loss of cofactors of some enzymes is a problem: some enzymes contain cofactors, which have a small molecular weight and are easily removed from the permeate during ultrafiltration. Therefore, a certain concentration is added before or after ultrafiltration. Cofactor.
Ultrafiltration can also be combined with affinity chromatography to increase separation purity. The working principle is: when the protein to be separated in the solution passes through the pores of the ultrafiltration membrane unimpeded, if an affinity ligand is bound to one side of the membrane, the protein will bind to the ligand and thus aggregate. This side of the membrane. Other substances that do not bind to the ligand will be carried through the hole. The protein is then eluted with a suitable eluent for further separation and purification.
4. Foam separation principle: the gas is introduced into the solution containing various components. Since the surface activity of these components is different, some components will form a foam on the surface of the solution, and the stability of the foam depends on the operation. Conditions and biological characteristics of the solution. The foam contains more surface active ingredients, so the types of foam components and their contents are different from those in the solution. In this way, the components in the solution are separated.
Protein is easier to adsorb and the gas-liquid interface, which is conducive to the stability of its structure. The foam separation process is: the diffusion of protein from the main solution to the gas-liquid interface, the process may be reversible or irreversible; the molecular rearrangement is generally considered to form two types of membranes at the air-water interface, one It is a thin film, and the other is a thick film, which may occur by a plurality of molecules. The protein film formed at the gas-liquid interface may be a single layer or a multilayer. The type of membrane depends on the nature, structure and concentration of the protein in the bulk solution and the gas-liquid interface.
The purpose of foam separation is to increase the enrichment rate of the enzyme protein (concentration of protein in the foam / protein concentration in the initial solution), and on the other hand to increase the extraction rate of the enzyme protein (protein extraction rate / initial protein quality in the foam) Or to maximize the partition coefficient of a component in a multi-component mixture.
Second, extraction, precipitation
1. Salting out The commonly used salting-out agent is ammonium sulfate, which has high solubility and low price. Ammonium sulfate has a strong ability to precipitate proteins, and its saturated solution can precipitate most proteins. No damage to the enzyme.
pH control: It should be considered from the solubility and stability of the enzyme. At the isoelectric point of the enzyme, the solubility is minimal and easy to precipitate, but some enzymes have poor stability at the isoelectric point, so choose the optimal pH. It is required to consider the pH of the most suitable enzyme precipitation under the premise of the most stable pH of the enzyme. Once the optimum pH value is determined during the operation, the formic acid or alkali adjusts the pH of the enzyme solution before the addition of ammonium sulfate, and the pH fluctuation of the solution should be avoided as much as possible to avoid destroying the stability of the enzyme. Attention should be paid to the addition of ammonium sulfate, and attention should be paid to the rate of addition of ammonium sulfate, generally from less to more, slowly added, and the ammonium sulfate is ground to a fine powder as much as possible.
Temperature control: Some enzymes have good stability at higher temperatures, and can be subjected to salting out at normal temperature, while most enzymes are operated at low temperatures as much as possible.
Net solution of enzyme solution: After adding ammonium sulfate, the enzyme solution should be allowed to stand for a period of time, so that the enzyme protein is completely precipitated. After the enzyme is allowed to stand, do not stir it.
2. Organic solvent precipitation Organic solvent selection: Organic solvents which can be used for the precipitation of enzyme proteins include alcohols and the like, such as methanol, ethanol, and isopropanol. Ethanol has a good hydrophilic property and prevents denaturation of proteins, and the solubility of the enzyme protein therein is also low.
Organic solvent precipitation operation: Organic solvents generally denature proteins, and when the temperature is high, the denatured protein molecules become permanently inactivated. Therefore, it is preferred to carry out the treatment with an organic solvent at 0 ° C or lower. Do not leave the enzyme protein precipitated with an organic solvent for too long. Add water to dissolve as soon as possible.
3. Polymer flocculant precipitation Polymer flocculants, such as dextran and polyethylene glycol, compete with enzyme molecules for water molecules and have dehydration to precipitate the enzyme. The advantage of polyethylene glycol as a precipitant is that in aqueous solution, the concentration can reach 50%, and the protein 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 is not adsorbed and therefore does not have to be removed prior to ion exchange adsorption.
4. The precipitation of enzymes and other proteins with metal ions and complexes forms metal salts with low solubility. The disadvantage of precipitation with metal ions is that the reversible change of the enzyme after interaction with the metal ions is poor, especially with the sulfhydryl derivative, which binds to the metal ion which catalyzes the denaturation of the enzyme and is inactivated.
5. Precipitation with special reagents Streptomycin can be used to selectively remove nucleic acids, thereby allowing intracellular enzymes to precipitate. The streptomycin salt (concentration of 0.5-1.0 mg/mg protein) is better for the selective precipitation of nucleic acids than manganese ions, and the enzyme is not easily deactivated.
6. Affinity precipitation The affinity-precipitation technique is formed by organically combining the high selectivity of the affinity reaction with the low throughput characteristics of the precipitation operation and the large amount of the treatment. The ligand is coupled with a soluble carrier to form a carrier-ligand complex, which can be precipitated under certain conditions after binding to the biomolecule.
The ligand-carrier complex can selectively bind to the protein, and the pH, ionic strength and protein concentration in the solution have little influence on the affinity binding, and only the competitive ligand will lower the product and the original ligand. The affinity of the combination, even the reversal of affinity binding.
Methods for directing precipitation are: ion crosslinking; addition of oppositely charged polymers; addition of oppositely charged hydrophobic groups; changes in pH to induce hydrophobic precipitation; temperature induced precipitation.
Affinity binding: The affinity ligand is added to the solution containing the protein of the target, and the conditions related to the precipitation are adjusted to facilitate the affinity binding.
Washing: Non-specific binding may occur in the presence of affinity precipitation in the treated crude liquid, especially with charged polymers. The effect of ion exchange will cause other proteins to co-precipitate, so the precipitate is washed before separating the target. . The practice is to re-dissolve the precipitate by adding a suitable cleaning agent and re-precipitate; or wash the precipitate thoroughly before specifically eluting. In the above process, the target protein and the ligand are always in an affinity binding state.
Separation of the ligand-carrier complex from the protein of interest: After the separation, it is ensured that the protein of interest and the ligand-carrier complex are recovered, and the target protein is required to have a certain purity, and the recovery rate is high.
