《酶的分離工程》ppt課件

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 PurityStableCostTime Pu

2、rification typically involves three steps1) 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 Prepa

3、ration of Crude Enzymes Endoenzyme: intracellularMost enzymes of the metabolic pathways.Exoenzyme: extracellularBreak down (hydrolyze) large food molecules or harmful chemicals. Example: cellulase, amylase, penicillinase.When harvesting broth cultures, how are cells separated from the broth?Clarifie

4、d liquidRotating BowlRotating scrollFrame FilterHow to improve filter velocity? 1Flocculation and Agglomeration 2 Decrease viscosity 3Filter aid Cell Disruption The main component of cell wall Bacteria : Peptidoglycan Yeast：Dextran，Mannose，Protein Mycelial fungus: Chitin, Dextran Gram Positive Bacte

5、rial Cell WallGram Negative Cell WallFungus Cell WallMechanical methodsGrinding ( in liquid nitrogen, ball mill Dry wayHomogenization mortar , homogenizer)- Wet wayPhysical methodstemperature difference freezing and thawing pressure difference( osmotic shock)ultrasonicationChemical treatmentorganic

6、solventsdetergents：Triton X-100，Tween used if enzyme is in lipid membrane Enzyme lysisautolysis extra enzymeWays to break cell Bead MillCascading beadsCells being disruptedRolling beadsSonicatorSonicatorSonicatorSonicator Disrupts tissue by creating vibrations which cause mechanical shearing of the

7、cell wall.After breaking the cell1) Keep temperature low2) Purify as soon as possible3) Avoid oxidation 4) Avoid contamination Cooling and protease inhibition are importantto recover the enzyme! Enzyme Extraction From plant and animal tissue. To achieve maximum solubility and activity of the enzyme.

8、 Extract methods Solvent or Solutionextract targetsalting liquid0.020.5mol/L NaCl solutionacid solutionpH26 aqueous solutionalkali solutionpH812aqueous solutionorganic solventwater-miscible organic solventMethods for Extraction of Enzymes Unit 2 Methods of Purification Centrifugation Preparative cen

9、trifugation Analytical centrifugation Preparative centrifugation Collect material cells precipitated macromolecules Subcellular fractionationAnalytical Centrifugation Sedimentation Coefficient (s) is the velocity per Fc, or s = v/2r unit is Svedberg ,where 1 S = 10-13 secRelative Centrifugal Force a

10、nd Rotation Per Minute expressed as x gravity RCF = Fc/Fg = 2r/980 = (rpm)/30 RCF = 1.119 10-5 (rpm)2r The unit is “gSpeed(rpm) Important noticelow speed centrifuge 6000 R.T.equilibriumhigh speed centrifuge6000 25000 freezing equilibrium exactlyultra speed centrifuge 25000freezingvacuum system equil

11、ibrium exactlyTypes of CentrifugesEquilibriumSet tempreatureSet rpmTiming Centrifugation Methods Centrifugation Methods Differential centrifugation Differential centrifugationHigh speed High speed Sedimentation velocity Sedimentation velocitySedimentation equilibriumSedimentation equilibriumSuper hi

12、gh speed 1 1Differential Centrifugation Differential Centrifugation (Gravity Centrifugation) (Gravity Centrifugation) Separate supernatant and pellet by mass Separate supernatant and pellet by mass and densityand density prepare cell lysate prepare cell lysate subject to centrifugation subject to ce

13、ntrifugation centrifugal force centrifugal force time (g min) time (g min) tube size and shape tube size and shape rotor angle rotor angle re-centrifuge supernatant re-centrifuge supernatant Problems contamination large particles contaminated with smaller particles resolution particles of similar si

14、zes not separated vibrations and convection currents2) Sedimentation VelocityRate Zonal p s(大大) separates primarily by masscommon media: sucrose3) Sedimentation equilibrium Isodensity s小小 p s p s時(shí)時(shí), V 0, V 0,樣品順離心力方向沉降樣品順離心力方向沉降 p s p s時(shí)時(shí), V 0, V 0.1M0.1M, the charged molecules are , the charged mol

15、ecules are quickly precipitated because the quickly precipitated because the excess ions (not bound to the protein) excess ions (not bound to the protein) compete with proteins for the compete with proteins for the solvent.solvent.Salting-out effect: ions take all water, expose the nonpolar surface;

16、 solubility decrease!+-+-OH-H+OH-H+OH-H+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 enh

17、ancing 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. Fin

18、ally, 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)2SO4which is cheap, effecti

19、ve, does not disturb structure and is very soluble.Salting out (Ammonium sulfate precipitation) Salt concentration is indicted in Percentage saturation飽和度飽和度 Volume of saturated (NH4)2SO4 Saturation ratio Total solution volume How to achieve desired percentage of ammonium sulfate？ Add -saturated sol

20、ution -dry powderHow 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

21、 be redissolved in buffer for further purification Which protein will ppt first? (hydrophobic or hydrophilic?) Fractional salting outDifferent proteins precipitate at different salt concentration. serumglobulinalbumin(NH4)2SO4(NH4)2SO450% saturationsaturatedprecipitateprecipitate Salting out curvepr

22、otein(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 minutes4) collect precipitated protein by centrifugati

23、on5) re-dissolved in buffer for further purification lImportant factors: l1) ionic strength l S = solubility of the proteinKs： salt-specific constant : idealized solubility I : the ionic strength of the solutionlog S = - Ks I log S = - Ks I l2) pH: pIl3) temperaturellow ionic strength ,T. protein so

24、lubilitylhigh ionic strength ,T. protein solubilityl4) protein concentration: moderate2. 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 an

25、d precipitates it.Common organic solvent: acetone ethanol methanol，2protein volumelImportant factorsl1)Temperature： low 0l Because.la. Some proteins might be denatured by heat produced.lb. Increase enzyme yieldT. , solubility l2) pH：pIl3) Protein concentration: moderateSalting-inSalting-outOrganic s

26、olventReagentsNaCl (monovalent)(NH4)2SO4 (divalent)Acetone, ethanol, methanolRemarksThe reverse process of salting-in is not salting-out, it is the dialysis process.1) Non-polar proteins will be precipitated earlier.2) Protein is very stable in(NH4)2SO4 .1) Some proteins might bedenatured by heat pr

27、oduced.2) Factors facilitate precipitation: larger protein, pH close to protein pI.3) Lipophilic protein might be dissolved more readily.Comparison of two methodsl 3. Isoelectric point precipitationl Change in pHl - Enzymes are least soluble at pI l - Different enzymes have different pI l Using meth

28、odIt seldom be used aloneoften 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 RemarksPEG will precipitates without denaturing .Its precipit

29、ation 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 (酪蛋白酪蛋白) ,denatured in boiling milk, will not be precipitated.Protein is not denatured

30、by salting out .Extraction A method to separate compounds based on their relative solubilities in two different immiscible liquids. Organic solventwater 1. Aqueous two-phase extractionATPE Special cased of liquid-liquid extraction Two types of aqueous two-phase systems:Polymer-polymer two-phase syst

31、eme.g.: dextran and PEGPolymer-salt two-phase systeme.g. PEG and KClPEGdextran 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. Biphasic systemMonophasic

32、 systemDextran % w/wTie linesPEG% 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 s

33、ubstance is defined as a supercritical fluid.2. Supercritical Fluid ExtractionSFElSF combines desirable properties of gases and liquidslSolubility of liquidslPenetration power of gasesSolvent (SF)SolutePhaseDensity(g/ml)Diffusion coefficient(cm2/s)Viscosity(g/cm/s)Gas10-310-110-4SCF0.30.910-310-410-

34、410-3Liquid110-510-2 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. lDensity of SF and solubility of a solute in it can be changed in a continuous manner by change of pressureSupercritical Fluid Extraction Proc

35、ess-flexibilitylLiquid-SF extractionlSimilar to liquid-liquid extractionlExamples:lRemoval of alcohol from beerlSolid-SF extraction lSimilar to solid-liquid extraction (leaching)lExamples:lRemoval of caffeine from coffee beans3. Reversed micelle extractionReversed Micelles: surface active agent orga

36、nic solvent Q: Whats the different between reversed micelle extraction and solvent extraction ? FiltrationProtein solution through a membrane which retains the protein of interest.This method is less likely to cause denaturation.membrane separationDiffusion membrane Microfiltration Ultrafiltration R

37、everse osmosispressure membraneElectro dialysis dialysisElectro 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 soluti

38、on.Concentrate protein sample.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. Pres

39、sure membranePressure difference driven processMicrofiltrationMF 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. UltrafiltrationUF Usually used to further

40、separate any contaminants able to pass through the microfiltration membrane using a pressure gradient .Small molecules are filtered out by pressureUsed for concentrating proteinsAlternatively, centrifugation with dialysis membrane超濾濃縮安裝超濾濃縮安裝 c. Reverse osmosis c. Reverse osmosisRORO Osmosis is the “movement of a solventthrough a semi-permeable membrane into a solution of