生物分离工程课件

Bioseparation EngineeringChapter 1 Introduction1.1 Downstream processing in biotechnologyThe producing process of a biotechnological product is termed bioprocess,that can be divided into two processes as followsUpstream processing：strain development and bioreaction（enzyme catalyze reaction,microbial fermentation,plant/mammalian cell culture,etc）Downstream processing：the isolation and purification of biotechnological productsThe complexity of downstream processing is determined by the required purity of the product,in turn determined by its application.The downstream processing scheme normally can be divided into the following stagesBrothSolid-liquid separation Extracellular productIntracellular productCell disruptionRemoval of cell debrisPrimary isolationpurificationformulationFinal product1.2 Demands on the downstream processingStrictly monitor the DSP steps for keeping the activity of the product;Rapidly remove those impurities can affecting the stability of the final product;Generally it is necessary to apply special efficient methods to the purification of the product;Since many products are applied to food,pharmaceutical,and cosmetics,those substances harmful to mankind health must be removed;Since the product concentration is low in the culture broth,it is necessary to concentrate the broth for removing large amounts of water.Because those of unit operations are costly,the cost of DSP is increased observably.1.3 Separation mechanism and unit operation According to different separation principles the unit operations can be sorted into two groups：Mechanical separationObject：unhomogeneous phase systemMechanism：separation based on the difference of substances size and densityUnit operation：filtration,settling,centrifugation,etc.Mass transfer separationObject：homogeneous phase systemTransport/velocity separationMechanism：separation based on the migration velocity difference of solutes drove by bearing a applied forceUnit operation：ultrafiltration,reverse osmosis,electrophoresis,etc.Diffusion/equilibrium separation Mechanism：separation based on the difference of distribution of substances between the two phasesUnit operation：extraction,crystallization,adsorption,ion exchange,etc.1.4 Estimation for separation efficiency There are three criteria on assessing the efficiency of a downstream processing,i.e.concentration degree,isolation-purification degree,and recovery rateConcentration degreeGenerally can be represented as concentrated factorIsolation-purification degreeCan be represented by separation factor/coefficient Recovery rateChapter 2 Solid-liquid separation and cell disruption 2.1 Cell separation2.1.1 SettlingStokes settling velocity of global particleWhere d is particle diameter,s and L are the density of particle and liquid,separately,is resistance coefficient,Re is Reynolds number,and L is the viscosity of liquid.The volume of the cells is so small that its settling velocity is very slow.For accelerating settling process agglomeration of individual cells into large flocs is done by the addition of flocculating agents such as polycations,or inorganic salts.2.1.2 Centrifugation Centrifugation velocityWhere r is centrifugal radius,is rotary angular speed,N is revolution of centrifuge,and S is sedimentation coefficient.CentrifugationDifferential centrifugationIt is an unit operation commonly used in the biochemical industry.According to the characteristics of the broth,the aim of isolation,and the extent of separation requested,different components can be separated from the broth separately by selecting proper operational parameters in practice.Zonal centrifugationRate-zonal density gradient sedimentationIsopycnic density gradient sedimentationBesides sucrose,CsCl and NaBr can be used for achieving the density gradient,and applied to the separation of nucleic acid and lipoprotein,respectively.CentrifugesThe tubular bowl rotor centrifuge is commonly applied on a laboratory scale,and the types of tubular bowl and disc stack are commonly used on an industrial scale.The processing capacity of the tubular bowl centrifuge is described byWhere L is the length of the tube,r2 is the inside radius of the tube,and is usually called the area of centrifugal sedimentation,a function of the structure of the centrifuge and the operating conditions.2.1.3 FiltrationDefinition：a technology,apply filter media to retain the particle while allowing the passage of the liquid through the filter,is used to achieve solid-liquid separation.The flow through the filterWhere Q is the volume of the filtrate,A is the area of the filter,p is the pressure difference,L is the viscosity of the filtrate,Rm and Rc are the resistance of the filter medium and the cake,is the average specific resistance of the cake,and W is the weight of the dried cake.Before filtration pretreatment of the broth by addition of flocculating agents,their function have been described in Section 2.1.1,and precoating the filter medium with filter aids(diatomite,perlite,etc.)are usually required to improve the filtration velocity.Filtration equipmentFilter press and rotary drum vacuum filter are frequently used for clarification of the broths.2.2 Cell disruptionMany biotechnological products cant be excreted outside of the cells during microorganism grow.For collecting those products the first step must be rupturing of the microbial cells to release the intracellular compounds into the liquid phase.2.2.1 Cell structures The cell structures are quite different among a considerable variety of cells.The sequence of different cells being broken from difficult to easy can be listed as follows:plant cells,yeast cell,G+cells,G-cells,and animal cells.The goal of cell disruption is making the cell wall and/or cytoplasmic membrane damaged more or less to liberate the intracellular products.2.2.2 The principles of cell disruptionMechanical disruptionThe Cells structure is broken due to the cells being sheared and pressed by mechanical forces.As a general rule,the more small the size of the cells is,the more hard to be ruptured it is.Chemical/enzymatic means Treatment with chemicals/enzymes can damage the cell membranes/walls and render cells more permeable,that is available for release of intracellular products.2.2.3 The means of cell disruptionMechanical disruptionHigh-pressure homogenisation Principle:the cell suspension is forced at high pressure through an orifice of narrow internal diameter to emerge at atmospheric pressure.The sudden release of pressure creates a liquid shear capable of disrupting the cells.Where S is the disruption scale,p is operational pressure,N is cyclic times,k is the disruption velocity constant,correlation with the kind of the cells and operational temperature.Characteristic：It is feasible for disruption of yeast cells and the majority of bacteria cells,but not suitable for disruption of filamentous fungus.The influencing factors：pressure,cyclic times,temperature,etc.Bead millingPrinciple：agitation with glass in bead mills ruptures the cells by a combination of high shear and impact with the cells.The influencing factors：agitation speed,the concentration of cells,the operating time,the beads diameter,density,and loading density.Where S is disruption ratio,k is disruption velocity constant,correlation with the beads diameter,density,loading density,the concentration of cells,agitation speed,and the shape of the puddler,t is the operating time of batch operation,or can be written as t=V/Q(V is the effective volume of the chamber of the bead mill,and Q is the feed flux)at continue operation.Characteristic:The method can be widely applied to a variety of cells,but it is very poor on the available energy,the ability of the heat change must be considered in the cooling system design.And because many operating parameters can influence the disruption ratio,optimizing design of the processing is very complex.UltrasonicationPrinciple：cavitation.The influencing factors：the kind and concentration of the cells,and the energy of the ultrasonication.Characteristic:it is commonly used at laboratory scale;removal of the heat generated is difficult on a larger scale.Chemical methodsTreatment with chemicalsPrinciple：see 2.2.2Available chemicals:acid,alkali,organic solvents,detergent,chelates,chaotropic agents,etc.Enzymatic lysisPrinciple：see 2.2.2Available enzymes：Because there are different chemical components of cell wall among a variety of organisms,proper enzyme must be selected,e.g.lysozyme is suitable for treatment of bacteria;Zymolyase,-1,6-dextranase,or mannanase is used for yeast;and damaging plant cells need to apply cellulase.A combination of enzymatic/chemical lysis with mechanical disintegration has been suggested in enhancing the efficiencies of the respective methods,with savings in time and energy and the facilitation of subsequent processing.Physical meansOsmotic shock Principle：put cells into a solution of lower osmotic pressure suddenly from that of higher osmotic pressure,that result in a lot of water swarming into cells and bursting the periplasmic membrane.Characteristic：it is the most mild method of cell disruption,but only effective for animal cells that lack a cell wall.FreezethawPrinciple：because of water crystallizing quantities of crystal nucleus are formed in the cells during the cells are frozen rapidly,that can damage the structure of the cells.Generally freeze and thaw must be carried out again and again until the expectation for the ratio of cell disruption is met.Characteristic：it is only suitable for those cells whose wall is thinner,and difficult to be used on a larger scale.Summary:Since the structure among many species of cell and the property of products are much different,choice of the disruption methods has to be made empirically,at the same time taking into consideration the subsequent processing steps.Chapter 3 PrecipitationDefinition：a phenomena of solid aggregates formed in a solution,that is based on a decrease in solubility induced by external factors.Characteristic:precipitation is a elementary isolation technique.The purity of sediment is much lower than that of crystal.But high-purity products can be gotten by multistep operation.Application:it is widely applied to recovery of biotechnological products e.g.proteins.Commonly used methods3.1 Salting-out precipitation 3.1.1 Theory Definition：in a solution of increasing ionic strength the precipitation of proteins will happen,that is relative to a decrease solubility.Cohn empirical formula：Where S is the solubility of the protein,is a constant,Ks is salting-out constant,I is the ionic strength,ciand Zi are is molar concentration and number of charge,respectively.Mechanism:the addition of neutral salt can increase hydrophobic interactions between neutral protein molecules,that is widely accepted.3.1.2 The influencing factorsThe molecular weight and three-dimensional structure of different proteinsfor given proteinthe kind of inorganic salts（to Ks)Criteria of selecting a neutral salt:higher solubility；solubility is almost influenced by temperature；the density of the solution in which the neutral salt is dissolved is not much higher,that will facilitate the separation of the sediments by centrifugation.Most used neutral salt is(NH4)2SO4,besides Na2SO4 and NaCl can also be used.temperature and pH（to）under a higher ionic strength the solubility of proteins will decrease accompanied with the increase of temperature.when pH is close to pI，solubility of the protein is lowest.3.1.3 Unit operation of salting-outThe experimental steps for deciding the saturation used to precipitate given protein：take a small portion of material liquor,and equally divide into several part.And refrigerate to 0；separately calculate the additive amounts,that can make the solution reach the saturation from 20 to 100,and add according to the calculated results.At the same time keep the temperature at 0；After centrifugation dissolve the sediment and determine the concentration of total protein and that of given protein,respectively,at the same time determine corresponding concentration in the mother liquor.Compare the results and assure that the mensuration is reliable；Where W is the additive amount（g/L),S is the saturation,505 is the saturated concentration of(NH4)2SO4 at 0(g/L),and 0.285 is the saturated concentration of(NH4)2SO4 at 0(L/L).plot a figure to describe the correlation between the concentration of total protein and that of given protein and saturation of(NH4)2SO4 in the mother liquor，by that decide the additive amount based on the request for recovery of products.3.1.4 ApplicationBecause of many salts being remained in the sediment removal of salts must be carried out after salting-out precipitation.3.2 Isoelectric precipitationDefinition：isoelectric precipitation can be used for recovery of proteins,that is based on the principle of a decrease in solubility when pH of the solution is adjusted to pI.Operating condition：lower ionic strengthpHpIAs a rule it is applied to precipitation of hydrophobic proteins e.g.casein,and not suitable for hydrophilic proteins e.g.glutin.So applying fields is not wider than salting-out precipitaion.Characteristic：advantage：it is facilitated to subsequent operation；disadvantage：sometimes extremes of pH denature the products.3.3 Organic solvent precipitationPrinciple：reduced dielectric constant enhances electrostatic interactions between protein molecules.Operating condition：low ionic strengthpH is near pICharacteristic：advantage：the lower density of the organic solvent is convenient for separating sediment,and removal of salts isnt needed.disadvantage：proteins denaturing maybe happen,so low temperature required for operation.3.4 Another methods3.4.1 Thermal precipitationPrinciple：under higher temperature make heat sensitive proteins precipitate and achieve the separation of heat stable proteins.Operating condition：adjust pH of the solutionadd organic solvents.Characteristic：it is a separation method of making proteins denaturation,so there should be a difference of heat stability between the given protein and the impurity proteins,that must be known very clearly.3.4.2 Special agentsNon-ionic polymerMechanism：reduction in the effective quantity of water available for protein solvation.Agent in common use：PEGCharged polymerMechanism：complex formation between oppositely charged molecules leads to charge neutralization and precipitation.Available agents：acidic polysaccharides,CMC,etc.Polyvalent metal ionsMechanism：bond with some functional groups in the surface of protein molecule e.g.Ca2+and Mg2+can combine with carboxyl,Mn2+and Zn2+with nitrogenous compound and heterocyclic compound.Advantage：although lower protein concentration in the solution precipitation can be achieved too.after precipitation removal of metal ions is easy by using ion-exchange resin or chelating reagent.Chapter 7 Affinity purificationBioaffinityTo carry out life functions,biological systems undergo physical and chemical interactions that rely on variations in molecular selectively and binding strength.The particular set of physical and chemical interactions in which structure can play a major role in shaping is referred to as bioaffinity.Affinity purificationBioaffinity interactions have been employed in bioseparation processes,that lead to the appearance of affinity purification techniques such as affinity chromatography,affinity membrane,affinity precipitation,etc.7.1 Basic principle7.1.1 Molecular recognition processesFrom a molecular perspective,the binding process between a receptor and its ligand can be viewed as four continuous stepsElectrostatic interactionSolvent displacementSteric selecionCharge and conformation rearrangement7.1.2 Receptor-ligand interactionsIn the affinity,receptor-ligand interactions involve noncovalent interactions such asIonic bondsHydrogen bondsHydrophobic interactionsVan der waals forcesSpecific interactions result during the formation of different receptor-ligand complexes,and some of those can be applied to bioseparation for exampleAntibody-antigen interactionEnzyme-substrate interactionLectin-carbohydrate interaction7.2 Affinity chromatography7.2.1 Principle and operationPrincipleMolecular recognition forms the basis of adsorption and separation by affinity chromatography.One of the reactants in an affinity pair,the ligand,is immobilized on a solid matrix and is used to fish out the aim product(receptor).OperationProcess including four steps,that is adsorption,wash,elution,and reuse.Elution methodsSpecific elutionA free ligand molecule added into elution liquid.Nonspecific elutionIncreasing the ionic strengthChanging the pH of the buffer7.2.2 ligandExamples of ligands used for affinity chromatography of proteinLigand typeProtein typeBiospecific ligandsReceptorHormone Antibody Antigen Substrate/substrate analogue,inhibitor,cofactor enzymeLectins Glycoproteins Pseudobiospecific ligandsTriazine dyesDehydrogenases,kinases,and other proteinsMetal ionsMetal ion binding proteinsHydrophobic groupsVarious proteinsThe chemical coupling procedure for immobilization of a ligand is chosen so as to provideSatisfactory yields;Strong linkage to minimize ligand leakage during chromatographic operation;Minimal nonspecific interactions with biomolecules.7.2.3 Application and advantageA trend in downstream processing has been to exploit the specificity of affinity interactions earlier in the separation train so as to reduce the number of purification steps.Potentially,affinity chromatography possess very high resolving power.7.3 Affinity membrane7.3.1 Principle and operationMicro/macroporous membrane matrices with affinity ligands have been developed for binding proteins from the clarified feed pumped over them.Desorption of the protein is later performed using solutions as in affinity chromatography.7.3.2 AdvantageCan give similar high resolution separation as chromatographic methodsIncrease the speed of separation tremendously due to in membranes,liquid transport is by convection as opposed to the diffusional flow in gels.7.4 Affinity precipitation7.4.1 Principle Selectivity in precipitation has been introduced by use of affinity interactions.Creation of large complexes as a result of affinity interactions,as between antigen and antibody,is one mode of affinity precipitation,which is used in immunoprecipitation.7.4.2 ApplicationSelective precipitation of multimeric proteins(have more than one binding site for a ligand).The homobifunctional ligands(synthesized by coupling two ligand molecules by a spacer)are able to bridge different protein molecules thereby forming aggregates.With heterobifunctional ligands,where one functionality is responsible for the affinity binding and the other is exploited for the precipitation,it becomes possible to operate affinity precipitation in a more general mode.Chapter 4 ExtractionPrinciple：the substances can be purified or concentrated since partition coefficients of them are different between the two phases.The progress of extraction technique traditional organic solvent extraction liquid membrane and reverse micelle extraction aqueous two-phase and supercritical fluid extraction4.1 Basic concepts 4.1.1 Extractiondefinition：a unit operation employing liquid or SCF as solvents to extracting the product in the material.Classification：4.1.2 Back-extractionDefinition：an operation of target products being transferred from organic phase into a new aqueous phase under conditions different from the first extraction.Purpose：for farther purifying products or facilitating consequent separation.4.1.3 Physical extraction and chemical extractionPhysical extractionprinciple：the compound distributes itself between the two phases according to its physical preference.application：penicillin and other antibiotics.Chemical extraction(reactive extraction)principle：fat-soluble extractants can form fat-soluble complexes with the compound by chemical reaction,that car