课件：酶的分离工程.ppt

Chapter 4 Purification of Enzymes Proteins are very diverse. They differ by size, shape, charge, hydrophobicity, and their affinity for other molecules. All these properties can be exploited to separate them from one another so that they can be studied individually.,Objectives Purity Stable Cost Time,Purification typically involves three steps 1) Preparation of a crude extract from harvested cells 2) Fractionation: Separation of a mixture of proteins into various fractions according to some property (e.g. size, charge, solubility) 3) Separation of protein from solvents and concentration,Unit 1 Preparation of Crude Enzymes Endoenzyme: intracellular Most enzymes of the metabolic pathways. Exoenzyme: extracellular Break down (hydrolyze) large food molecules or harmful chemicals. Example: cellulase, amylase, penicillinase.,Solid/Liquid Separation,-centrifugation and filtration,When harvesting broth cultures, how are cells separated from the broth?,Decanter Centrifuge,Clarified liquid,Rotating Bowl,Rotating scroll,Frame Filter,How to improve filter velocity? 1）Flocculation and Agglomeration 2） Decrease viscosity 3）Filter aid,Cell Disruption The main component of cell wall Bacteria : Peptidoglycan Yeast：Dextran，Mannose，Protein Mycelial fungus: Chitin, Dextran,Gram Positive Bacterial Cell Wall,Gram Negative Cell Wall,Fungus Cell Wall,Mechanical methods,Grinding ( in liquid nitrogen, ball mill ）Dry way Homogenization （mortar , homogenizer)- Wet way,Physical methods,temperature difference （ freezing and thawing ） pressure difference( osmotic shock) ultrasonication,Chemical treatment,organic solvents detergents：Triton X-100，Tween （used if enzyme is in lipid membrane ）,Enzyme lysis,autolysis extra enzyme,Ways to break cell,Bead Mill,Sonicator,Sonicator,Disrupts tissue by creating vibrations which cause mechanical shearing of the cell wall.,After breaking the cell 1) Keep temperature low 2) Purify as soon as possible 3) Avoid oxidation 4) Avoid contamination Cooling and protease inhibition are important to recover the enzyme!,Enzyme Extraction From plant and animal tissue. To achieve maximum solubility and activity of the enzyme.,Methods for Extraction of Enzymes,Unit 2 Methods of Purification Centrifugation Preparative centrifugation Analytical centrifugation,Preparative centrifugation Collect material cells precipitated macromolecules Subcellular fractionation,Analytical Centrifugation Sedimentation Coefficient (s) is the velocity per Fc, or s = v/2r unit is Svedberg ,where 1 S = 10-13 sec,Relative Centrifugal Force and Rotation Per Minute expressed as x gravity RCF = Fc/Fg = 2r/980 = (rpm)/30 RCF = 1.119 10-5 (rpm)2r The unit is “g”,Types of Centrifuges,How to use a centrifuge?,Equilibrium Set tempreature Set rpm Timing,Centrifugation Methods Differential centrifugationHigh speed Sedimentation velocity Sedimentation equilibrium,Super high speed,1）Differential Centrifugation (Gravity Centrifugation) Separate supernatant and pellet by mass and density prepare cell lysate subject to centrifugation centrifugal force time (g min) tube size and shape rotor angle re-centrifuge supernatant,Problems contamination large particles contaminated with smaller particles resolution particles of similar sizes not separated vibrations and convection currents,2) Sedimentation Velocity Rate Zonal p s(大) separates primarily by mass common media: sucrose 3) Sedimentation equilibrium Isodensity s（小） p s（大） equilibrium separates by density common media: CsCl,每种纯样品成份在梯度液中的沉降速度（cm/s） d2(p - s )2r V= 18 式中 d：样品颗粒的直径（cm） p ：样品颗粒的密度（g/cm3） s ：密度梯度液的密度（ g/cm3 ） 当 p s时, V 0,样品顺离心力方向沉降 p s时, V 0,样品逆离心力方向上浮 p = s时 V=0,样品停止沉降或上浮，“稳定“在这一位置,Common Features Centrifugation in a dense medium -increases stability -provides greater resolution,Comparison of centrifuges,Gravity Centrifugation (No density),Utilize gravity force to separate particles from the solution,Differential Cent.,Density Gradient Cent.,Zone Centrifugation (Precast) (step-wise),Isodensity equilibration (CsCl gradient forming),Sample: protein (similar density, but different in MW),Sample: nucleic acid (similar MW, but different in density),High speed,Ultracentrifugation,Two ultracentrifuge types,General Procedures Prepare gradient 2) Apply sample 3) Centrifuge 4) Collect and analyze fractions,Large-scale Purification of Viruses 1. Growth of the virus in large-scale equipment 2. Removal of virus-enriched culture fluid 3. Concentration of the virus particles by precipitation 4. Final purification by density-gradient centrifugation 5. Crystallization of virus,Precipitation Decrease solubility This unit operation serves to concentrate and fractionate the target product from various contaminants.,1. Salting out Change in ionic strength Salt effects protein solubility- Salting in : Addition of salt at low ionic strength can increase solubility of a protein. Salting out: Proteins tend to aggregate and precipitate from high ionic strength solution.,Salting in Protein has no net charge at its pI, that leads to the binding between proteins via ionic interactions, and precipitation. Salt can interfere these ionic interactions and separate bound protein molecules.,Salting-in effect: decrease proteinprotein electrostatic interaction; solubility increase!,Salting out (Can be used for fractionation) Beyond a certain ionic strength（0.1M）, the charged molecules are quickly precipitated because the excess ions (not bound to the protein) compete with proteins for the solvent.,Salting-out effect: ions take all water, expose the nonpolar surface; solubility decrease!,Precipitation,Protein molecules are dehydrated by strong salt solution. The charges of protein molecules are neutralized.,At low concentrations, the presence of salt stabilizes the various charged groups on a protein molecule, thus attracting protein into the solution and enhancing the solubility of protein. This is commonly known as salting-in. However, as the salt concentration is increased, a point of maximum protein solubility is usually reached. Further increase in the salt concentration implies that there is less and less water available to solubilize protein. Finally, protein starts to precipitate when there are not sufficient water molecules to interact with protein molecules. This phenomenon of protein precipitation in the presence of excess salt is known as salting-out.,Used to selectively precipitate proteins, often with (NH4)2SO4 which is cheap, effective, does not disturb structure and is very soluble.,Salting out (Ammonium sulfate precipitation),Salt concentration is indicted in Percentage saturation（饱和度） Volume of saturated (NH4)2SO4 Saturation ratio Total solution volume,How to achieve desired percentage of ammonium sulfate？ Add -saturated solution -dry powder,How to making X% solution from Xo% solution?,The effect of salt on different proteins may differ: Certain proteins precipitate from solution under conditions in which others remain quite soluble. Once the protein is precipitated (not denatured) can separate by centrifugation pellet can be redissolved in buffer for further purification Which protein will ppt first? (hydrophobic or hydrophilic?),Fractional salting outDifferent proteins precipitate at different salt concentration.,serum,globulin,albumin,(NH4)2SO4,50% saturation,saturated,precipitate,precipitate,Salting out curve protein(mg) or enzyme activity 10 20 30 40 50 60 70 80 90 100(NH4)2SO4 percentage of saturated,In brief, the procedure goes as follows: obtain protein solution of interest 2) add (NH4)2SO4 to a chilled, stirring solution 3) allow to stir for 15-30 minutes 4) collect precipitated protein by centrifugation 5) re-dissolved in buffer for further purification,Important factors: 1) ionic strength,S = solubility of the protein Ks： salt-specific constant : idealized solubility I : the ionic strength of the solution,log S = - Ks I,2) pH: pI 3) temperature low ionic strength ,T. protein solubility high ionic strength ,T. protein solubility 4) protein concentration: moderate,2. Precipitation with organic solvents Decrease in dielectric constant Organic solvent decreases the water activity and the dielectric constant of the solution, which then decreases the solubility of the protein and precipitates it.,Common organic solvent: acetone ethanol methanol，2protein volume,Important factors 1)Temperature： low （0） Because a. Some proteins might be denatured by heat produced. b. Increase enzyme yield（T. , solubility ） 2) pH：pI 3) Protein concentration: moderate,Comparison of two methods,3. Isoelectric point precipitation Change in pH - Enzymes are least soluble at pI - Different enzymes have different pI,Using method It seldom be used alone（often used to remove undesired protein）. Because： Many proteins have similar pI. Proteins have some solubility at pI.,4. Non-ionic hydrophilic polymers （Polyethylene glycol （PEG ）precipitation） Molecular weight: 600020000 Remarks PEG will precipitates without denaturing . Its precipitation effects is very high.,5. Selective denaturation A negative methodleaves the desired protein active in solution; heat; extreme pH; organic solvents ;,Relationship between denaturation and precipitation? Casein (酪蛋白) ,denatured in boiling milk, will not be precipitated. Protein is not denatured by salting out .,Extraction A method to separate compounds based on their relative solubilities in two different immiscible liquids.,1. Aqueous two-phase extraction（ATPE） Special cased of liquid-liquid extraction Two types of aqueous two-phase systems: Polymer-polymer two-phase system e.g.: dextran and PEG Polymer-salt two-phase system e.g. PEG and KCl,PEGdextran system The “upper phase“ is formed by the more hydrophobic polyethylene glycol (PEG), which is of lower density than the “lower phase,“ consisting of the more hydrophilic and denser dextran solution.,Aqueous two-phase extraction,Biphasic system Monophasic system,Dextran % w/w,Tie lines,PEG% w/w,For every substance, there is a critical temperature (Tc) and pressure (Pc) above which no applied pressure can force the substance into its liquid phase. If the temperature and pressure of a substance are both higher than the Tc and Pc for that substance, the substance is defined as a supercritical fluid.,What is a Supercritical Fluid?,2. Supercritical Fluid Extraction（SFE）,Supercritical Fluid Extraction,SF combines desirable properties of gases and liquids Solubility of liquids Penetration power of gases,Solvent (SF),Solute,The Choice of the SFE Solvent,Carbon dioxide is the most commonly used SCF, due primarily to its low critical parameters (31.1C, 73.8 bar), low cost and non-toxicity.,Density of SF and solubility of a solute in it can be changed in a continuous manner by change of pressure,Supercritical Fluid Extraction Process-flexibility,SFE: Types,Liquid-SF extraction Similar to liquid-liquid extraction Examples: Removal of alcohol from beer Solid-SF extraction Similar to solid-liquid extraction (leaching) Examples: Removal of caffeine from coffee beans,3. Reversed micelle extraction Reversed Micelles: surface active agent organic solvent,Q: Whats the different between reversed micelle extraction and solvent extraction ?,Filtration Protein solution through a membrane which retains the protein of interest. This method is less likely to cause denaturation.,membrane separation,Diffusion membrane,Microfiltration Ultrafiltration Reverse osmosis,pressure membrane,Electro dialysis,dialysis,Electro membrane,1. Diffusion membrane （ concentration difference driven process） dialysis Dialysis tubing has pores with a specific molecular weight cut-off that allows smaller molecules (salt) to pass. Purposes: Reduce ionic strength of the solution. Concentrate protein sample.,Dialysis,Typically, process involves several changes of buffer so that the salt concentration in the sample is reduced to acceptable level.,What happens during dialysis? Why is dialysis an important technique in protein purification? Q: Why is blood red ? How to testify?,2. Pressure membrane （Pressure difference driven process） Microfiltration（MF） Microfiltration is a filtration process which removes contaminants from a fluid (liquid & gas) by passage through a microporous membrane (0.1 to 10 m).,Some examples for microfiltration,b. Ultrafiltration（UF ） Usually used to further separate any contaminants able to pass through the microfiltration membrane using a pressure gradient .,Small molecules are filtered out by pressure Used for conce