《酶的分離工程》

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. -centrifugation and filtration When harvesting broth cultures, how ar

4、e cells separated from the broth? Clarified liquid Rotating BowlRotating 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 l Bacteria : Peptidoglycan l Yeast：Dextran，Mannose，Protein

5、l Mycelial fungus: Chitin, Dextran l 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 ） pressur

6、e 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 Cascading beads Cells being disrupted Rolling beads SonicatorSonicator SonicatorSonic

7、ator 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

8、 From plant and animal tissue. To achieve maximum solubility and activity of the enzyme. Extract methods Solvent or Solution extract target salting liquid0.020.5mol/L NaCl solution acid solutionpH26 aqueous solution alkali solutionpH812 aqueous solution organic solventwater-miscible organic solvent

9、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

10、velocity per Fc, or s = v/2r unit is Svedberg ,where 1 S = 10-13 sec Relative Centrifugal Force and 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” Speed(rpm) Important notice low speed centrifuge 6000 R.T. equilibrium high spee

11、d centrifuge 6000 25000 freezing equilibrium exactly ultra speed centrifuge 25000freezingvacuum system equilibrium exactly Types of Centrifuges Equilibrium Set tempreature Set rpm Timing Centrifugation Methods Differential centrifugationHigh speed Sedimentation velocity Sedimentation equilibrium Sup

12、er high speed 1 1）Differential Centrifugation Differential Centrifugation (Gravity 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 su

13、pernatant 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 大) separates primarily by mass common media: sucrose 3) Sedimentation equilibrium Iso

14、density s（?。?（?。?p s時(shí)時(shí), , V V 0,0,樣品順離心力方向沉降樣品順離心力方向沉降 p s時(shí)時(shí), , V V 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 decreas

15、e! + + + + + + + - - - - - - - + + + + + + + - - - - - - - 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 at

16、tracting 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

17、 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,

18、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（飽和度）（飽和度） Volume of saturated (NH4)2SO4 Saturation ratio Total solution volume How to achieve desired p

19、ercentage 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

20、 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. serumglobulinalbumin (NH(NH4 4) )2 2SOSO4 4 50% sa

21、turation saturated precipitateprecipitate Salting out curve lprotein(mg) or enzyme activity 10 20 30 40 50 60 70 80 90 100(NH(NH4 4) )2 2SOSO4 4 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) al

22、low to stir for 15-30 minutes 4) collect precipitated protein by centrifugation 5) re-dissolved in buffer for further purification lImportant factors: l1) ionic strength l S = solubility of the protein KsKs： salt-specific constant : : idealized solubility I : the ionic strength of the solution log S

23、 = - Klog S = - Ks s I I l2) pH: pIpI l3) temperature llow ionic strength , ,T. protein solubility lhigh ionic strength , ,T. protein solubility l4) protein concentration: moderate 2. Precipitation with organic solvents Decrease in dielectric constant l Organic solvent decreases the water activity a

24、nd 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 lImportant factors 1)1)Temperature： low （0 0） Because. a.a. Some proteins might be denatured by heat produced. b. Incre

25、ase enzyme yield（T. , solubility ） 2) pH2) pH：pIpI 3)3) Protein concentration: moderate Salting-inSalting-outOrganic solvent Reagents NaCl (monovalent) (NH4)2SO4 (divalent) Acetone, ethanol, methanol Remarks The reverse process of salting-in is not salting-out, it is the dialysis process. 1) Non-pol

26、ar proteins will be precipitated earlier. 2) Protein is very stable in (NH4)2SO4 . 1) Some proteins might be denatured by heat produced. 2) Factors facilitate precipitation: larger protein, pH close to protein pI. 3) Lipophilic protein might be dissolved more readily. Comparison of two methods 3. Is

27、oelectric point precipitation l Change in pH - Enzymes are least soluble at pI - Different enzymes have different pI l 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 p

28、olymers （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 ; Rela

29、tionship between denaturation and precipitation? Casein (酪蛋白酪蛋白) ,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. Organic solvent water 1

30、. Aqueous two-phase extraction（ATPE） Special cased of liquid-liquid extraction Two types of aqueous two-phase systems: Polymer-polymer two-phase system le.g.: dextran and PEG Polymer-salt two-phase system le.g. PEG and KCl PEGdextran system The upper phase is formed by the more hydrophobic polyethyl

31、ene glycol (PEG), which is of lower density than the lower phase, consisting of the more hydrophilic and denser dextran solution. Biphasic system Monophasic system Dextran % w/w Tie linesPEG% w/w For every substance, there is a critical temperature (Tc) and pressure (Pc) above which no applied press

32、ure 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. 2. Supercritical Fluid Extraction（SFE） lSF combines desirable properties of gases and liquids So

33、lubility of liquids Penetration power of gases Solvent (SF) Solute PhaseDensity (g/ml) Diffusion coefficient (cm2/s) Viscosity (g/cm/s) Gas10-310-110-4 SCF0.30.910-310-410-410-3 Liquid110-510-2 Carbon dioxide is the most commonly used SCF, due primarily to its low critical parameters (31.1C, 73.8 ba

34、r), 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 pressure Supercritical Fluid Extraction Process-flexibility lLiquid-SF extraction Similar to liquid-liquid extraction Examples: lRemoval of alcohol from beer lSolid-SF ex

35、traction Similar to solid-liquid extraction (leaching) Examples: lRemoval of caffeine from coffee beans 3. Reversed micelle extraction lReversed Micelles: surface active agent organic solvent Q: Whats the different between reversed micelle extraction and solvent extraction ? Filtration Protein solut

36、ion 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 di

37、fference 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. Typically, process involves several changes of buffer so that the salt conce

38、ntration 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 pro

39、cess 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 concentrating proteins Alternatively, centrifugation with dialysis membrane 超濾濃縮裝置超濾濃縮裝置 c. c. Reverse osmosis（RO） Osmosis is the “movement of a solvent through a semi-permeable membrane into a solution of higher solute co