酶的分离工程-1.ppt
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1、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 Tim
2、e,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
3、 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 harvestin
4、g 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 : Pep
5、tidoglycan 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 differ
6、ence ( 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
7、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
8、 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,Analyti
9、cal 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
10、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 prepa
11、re 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) Sedim
12、entation 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
13、,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
14、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
15、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
16、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
17、 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 inter
18、fere 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
19、 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 c
20、oncentrations, 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 solubi
21、lity 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 precipita
22、tion 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(
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