Isolation and Purification of Enzymes .ppt
- 2. Enzymes are biological catalysts and are protein in nature with an exception of ribozyme, an RNA catalyzing the RNA splicing in eukaryotes. Almost, all the metabolic reactions, in the living systems, are catalyzed by the enzymes. Mostly enzymes are found within the cells, there are few enzymes especially in microbes which are secreted in the medium by the microbe and are called extra-cellular enzymes. To study an enzyme in vitro, it is essential to isolate it from the cell. It is preferable to purify too at least up to some extent before studying its characteristics.
- 3. • For isolation of the enzyme, cell has to be ruptured using a suitable isolation medium and suitable rupturing technique. However, it is also important to select tissue, organism for isolating the enzyme. • Selection of tissue/ organism: There is no hard and fast rule for selecting tissue and/ or organism for the isolation of an enzyme, it is always preferable to select a source enriched in that particular enzyme. • Selection of the isolating medium: distilled water as isolating (homogenizing) medium. However, generally a buffer of a suitable ionic concentration and pH is preferred in order to maintain the pH and ionic concentration in the medium.
- 4. • A certain ionic strength of the buffer is essential for maintaining the buffering capacity. However, much higher ionic strength is also avoided since some times, high ionic concentration may be inhibitory to the activity of the enzyme. • All the enzymes are pH sensitive. Every enzyme is stable in a particular pH range only. Every enzyme shows enzyme activity in a particular pH range only. However, mostly for isolation, pH of the medium is little different than the pH at which enzyme activity is measured. The pH of the buffer is maintained according to the nature of the enzyme. Selection of the pH should be such that isolated enzyme should be in fully active form.
- 5. Enzyme solubilisation techniques •Almost all the enzymes (with few exceptions) are heat labile and not much stable at room temperature, •The entire process of enzyme isolation, purification is carried out at 0-4o C using a cold room. However, enzyme work can also be done without a cold room if precautions of cold conditions are followed. •All the isolation medium components should be in chilled condition. •The component of the homogenization technique like pestle and mortar, bowl of the Waring blender should also be in chilled condition. While homogenizing in a pestle and mortar, it should be surrounded by the ice flakes. In case of Waring blender bowl, many people also wrap a cloth wet with chilled water.
- 6. Techniques used for enzyme isolation As mentioned above, generally enzymes are isolated in the cold condition (at 0 to 4o C). For the purpose, homogenizing medium as well as container should be in the chilled condition. It is preferable to homogenize the tissue in a cold room. The following are the commonly used techniques for enzyme homogenization: 1. Pestle and mortar: Pestle and mortar is a moderate technique for tissue homogenization. Mechanical breakdown occurs during the process. Sometimes, grinding is done in the presence of purified sand or glass beads for aberration. Pestle and mortar is considered to be a moderate grinding technique and rupturing of the cell organelles does not occur if isotonic grinding medium without detergent is used.
- 8. 2. Blenders: Waring blender (commonly called as mixie) is comparatively harsh technique of grinding the tissue compared to pestle and mortar and is mostly used for homogenizing the harder tissues (generally the plant tissues). Waring blender is first operated at low speed for few seconds and then at medium speed(s) for few seconds before bringing it at high speed. Time of grinding at various speeds is decided according to the nature of the tissue being ground. If homogenization has to be done for a little longer time, then it is generally done after few seconds interval after every minute of grinding at high speed to avoid heating during operation of the Waring blender. If the worker is interested in isolating intact cell organelles, then Waring blender is not a preferred technique.
- 10. 3. Ultra-Sonicator: This technique of rupturing the cells is generally used for microbial/ bacterial cells. Ultra-sonicator generates low as well as high wavelength ultrasonic waves. For the purpose, a suitable probe depending on the volume of the homogenizing medium is selected and connected with the ultra- sonicator. The container having cells and homogenizing (isolating) medium is put in chilled condition by covering the container with ice. There is much generation of heat during ultra-sonication, therefore, ultrasonic waves are thrown in the sample after few seconds interval, every 10 to 15 seconds ultrasonication.
- 12. 4. Vir-Tis homogenizer: This is considered to be a mild technique and generally used for homogenization of soft tissues such as animal tissues. Here a motorized pestle with teeth like aberrations is used. With Vir-Tis homogenizer, generally no rupturing of cell organelles occurs during grinding provided isotonic medium with no detergent is used.
- 14. 5. Potter Elvejm homogenizer: This is also a mild technique and is used for homogenization of soft animal tissues. Potter Elvejm Homogenizer is a simple equipment having a pestle like glass rod with teeth like aberrations on its tip. There are down aberrations in the tube too on which teeth of the rod are fitted during up and down process of the rod. Up and down process of the pestle is done manually by hand or by mechanical device.
- 16. 6. Razor blade: It is comparatively very mild technique. It is generally used only for isolation of intact cell organelles for the purpose of studying the intracellular localization of the enzyme proteins. In the technique, razor blade is used for chopping the tissue in the presence of isolating medium. Although the technique is good for the isolation of intact organelles, but it is unable to rupture all the cells. Therefore, there is low recovery of the enzyme due to left out of un- ruptured or partially ruptured cells. These un-ruptured or partially ruptured cells are removed as cell debris after centrifugation. 7. Extrusion method: This method relies on the principle that forcing a cell suspension at high pressure through a narrow orifice will provide a rapid pressure drop. This is a powerful mean of disrupting cells especially from bacteria.
- 18. 8. Lytic enzymes: Cell wall and cell membrane lytic enzymes like cellulase, pectinase, xylanase, pectin methyl esterase, lysozyme etc. can be used for rupturing the cells. Enzymes being costly are not commonly used for making cell free preparation for isolation of enzymes. In plant tissue culture, lytic enzymes are used to prepare protoplast. 9. Freeze - Thaw: With certain susceptible microbes and eukaryotic cells, repeated freezing and thawing results in extensive membrane lesions with release of periplasmic and intracellular proteins.
- 19. 10. Acetone powder: Drying with acetone is a good method for rupturing the cell membrane. Using acetone powder of the tissue may be prepared which may be stored in a Deep freezer for a long time. It forms a convenient starting material from which the enzyme may be extracted with the isolating medium, whenever required. However, one has to take much precautions of low temperature (generally –20oC ), otherwise, acetone may denature the enzyme protein.
- 20. Enzyme purification • The purification of a particular enzyme involves removal of other substances (proteins as well as non- proteins) present in the preparation. • Purification of an enzyme protein is generally a multi- step process exploiting a range of biophysical and biochemical characteristics such as its relative concentration in the source, solubility, charge, size (molecular weight), hydrophobicity/ hydrophilicity of the target protein. • Enzyme purification is a costly affair and also the time consuming. Therefore, it is necessary to have an idea of the degree of purity necessary for the intended use of the targeted enzyme.
- 21. • In general, design of the purification technique/ protocol should be focused on: (i) high recovery, (ii) highly purified enzyme protein, (iii) reproducibility of the methods, (iv) economical use of the chemicals (reagents) (v) shorter time for complete purification.
- 22. • Enzymes are more unstable in dilute solutions. Therefore, while designing the purification procedure, initially emphasis is given on concentrating the protein concentration in the sample rather than purification. • After concentration, emphasis is given to purification (removal of unwanted proteins) and lesser loss of enzyme activity of the targeted enzyme. • Commonly, the first step in enzyme purification is based on fractionation of proteins on the basis of solubility of proteins in aqueous solutions of salts or organic solvents.
- 23. Fractionation of the proteins on the basis of solubility Salt fractionation: It is one of the best and oldest method to remove unwanted proteins in enzyme purification. It is carried out by the stepwise addition of suitable salt to the crude enzyme preparation or supernatant. Ammonium sulphate is most commonly used due to its high water solubility, high degree of purity and its cheap rate. Further it has no deleterious effects on most enzyme-proteins. After each addition of salt care is taken to ensure complete dissolution and the formation of a homogeneous solution. The unwanted precipitated proteins are discarded by centrifugation.
- 24. Organic solvent fractionation: The experimental procedure for organic solvent fractionation is similar to that for salt fractionation method. It is based upon difference in the solubility of protein in aqueous solutions and organic solvents such as ethanol, acetone or butanol. Organic solvent fractionation is usually carried out at -10 to 20ºC. This technique always leads to loss of some enzyme activity. Organic polymer fractionation: The method of organic polymer fractionation is similar to organic solvent fractionation except that an organic polymer is used in low concentration to achive enzyme- protein purification.
- 25. Chromatographic separation of the enzyme proteins •Chromatography is a laboratory technique for the separation of a mixture. •The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. •The various constituents of the mixture travel at different speeds, causing them to separate. •The separation is based on differential partitioning between the mobile and stationary phases.
- 27. • For enzyme purification, commonly used chromatography techniques are: (i) Ion exchange chromatography; (ii) Adsorption chromatography; (iii) Gel filtration chromatography and (iv) Affinity chromatography.
- 28. • In general, the procedure of carrying the work is same in all types of chromatography. First, the enzyme protein sample to be purified is applied onto the pre-equilibrated column and thereafter, the sample from the column is eluted with buffer with a series of steps of different solute concentrations, with a gradient of solute or with a specific ligand for the desired enzyme protein. The effluent eluted out from the column is collected as a series of fractions using a fraction collector, tested for enzyme activity and protein.
- 29. Ion exchange chromatography The basic principle involved in ion exchange chromatography is binding of charged proteins on the ion exchanger by electrostatic attraction (ionic bonds) between charged groups on the proteins and opposite charges on the exchanger. Unbound proteins are removed from the column by washing with the same medium used for pre-equilibrium. Bound proteins are eluted by passing buffer of higher ionic strength (using salts like sodium or potassium chloride) or by using buffer of different pH. Fractions of the effluent are collected and analyzed for the desired enzyme activity.
- 30. In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.
- 31. Adsorption chromatography • Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phases accounts for the separation of different solutes. • Afterwards, proteins are eluted from the column matrix by using a suitable elution buffer either having change in ionic concentration or pH.
- 32. The commonly used matrices in adsorption chromatography are: (i) calcium phosphate gel; (ii) alumina gel and (iii) hydroxylapatite gel.
- 33. Gel filtration (Molecular sieve) chromatography • Gel filtration also known as gel permeation chromatography lacks an attractive interaction between the stationary phase and solute. • The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. • The pores are normally small and exclude the larger solute molecules, but allow smaller molecules to enter the gel, causing them to flow through a larger volume. • This causes the larger molecules to pass through the column at a faster rate than the smaller ones. • The commonly used gel filtration gels are of dextran, agarose, polyacrylamide.
- 35. Affinity chromatography • This is the most selective type of chromatography employed. • It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. • For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.
- 37. Electrophoretic techniques Electrophoretic techniques are more commonly used for analytical purpose for checking the purity of the enzyme protein. The characteristic mobility of the enzyme proteins in electric field is the basis for the electrophoretic purification techniques for proteins. Most commonly used electrophoretic technique is Polyacrylamide Gel Electrophoresis (PAGE). In PAGE, proteins are separated on the basis of charge as well as size. Polyacrylamide gel has the advantage that pore size can be controlled by varying the concentration of acryl amide and N, N/ - methylene bis acryl amide.
- 38. Ultrafiltration Although ultrafiltration is used to concentrate the sample, ultrafiltration is also capable of removing low molecular weight substances. In ultra-filtration, protein sample is fed into a cell fitted with a membrane, which retains high molecular weight proteins while solvent and low molecular weight molecules are filtered through the membrane. In ultra-filtration, positive or negative pressure using nitrogen gas is put into the solution or solvent collecting chamber, respectively to maintain flow across the membrane. For retaining and concentrating low molecular weight proteins, smaller pore size ultra-filtration membranes are commercially available. Use of successive large to small pore size membranes may result in enzyme purification as well as concentration of the sample.
- 39. Dialysis The separation of particles in a liquid on the basis of differences in their ability to pass through a membrane. Dialysis is used for removal of low molecular weight contaminants, salt etc. Animal membranes, collodion, colloids deposited in porous pots, cellophane tubing etc are the commonly used membranes for dialysis. The dialysis membranes used are thin films of highly polymerized substances which swell in aqueous solvent resulting in a type of molecular sieve. Through the pores in the molecular sieve, low molecular weight substances will pass while high molecular weight proteins will be retarded.
- 40. Crystallization •After considerable degree of purification, enzyme protein may be tried for crystallization. However, crystallization is considered to be a difficult task. Generally ammonium sulfate or other salts such as sodium sulfate is added to the concentrated enzyme protein sample till slight turbidity. Thereafter, the preparation is kept to stand in cold condition. After storage for 24 to 48 hours, crystals of the enzyme protein may appear. It is pertinent to mention that crystallization is not an evidence of purity.
- 41. • The first crystals may contain other proteins too. On re-crystallization (after dissolving the crystals in a suitable medium and repeating the process the same way), specific activity of the crystals may increase.