生物分离工程英文课件电子教案

1、生物分离工程英文课件1.1 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 cel

2、l 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 follow

3、ing stagesBrothSolid-liquid separation Extracellular productIntracellular productCell disruptionRemoval of cell debrisPrimary isolationpurificationformulationFinal product1.2 Demands on the downstream processing Strictly monitor the DSP steps for keeping the activity of the product; Rapidly remove t

4、hose 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

5、 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 pr

6、inciples the unit operations can be sorted into two groups： Mechanical separation Object：unhomogeneous phase system Mechanism：separation based on the difference of substances size and density Unit operation：filtration, settling, centrifugation, etc. Mass transfer separation Object：homogeneous phase

7、system Transport/velocity separation Mechanism：separation based on the migration velocity difference of solutes drove by bearing a applied force Unit operation：ultrafiltration, reverse osmosis, electrophoresis, etc. Diffusion/equilibrium separation Mechanism：separation based on the difference of dis

8、tribution of substances between the two phases Unit 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,

9、 and recovery rate Concentration degree Generally can be represented as concentrated factorXCXPXTCTPTccmccm Isolation-purification degree Can be represented by separation factor/coefficient Recovery rateCPXWXPTWTPXTXCXPTCTPAAccccmmcccc%100%100TCCTPPTCCTPPcVcVRcFcFRChapter 2 Solid-liquid separation a

10、nd cell disruption 2.1 Cell separation2.1.1 Settling Stokes settling velocity of global particleLLgeeegLsLbsgdvRRRdvfgdfgdf1102442616142233Where 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 viscosit

11、y 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 v

12、elocitysvrN224222292SrrNdvLLssgrNZrNrFc2222244ZgWhere r is centrifugal radius, is rotary angular speed, N is revolution of centrifuge, and S is sedimentation coefficient. Centrifugation Differential centrifugation It is an unit operation commonly used in the biochemical industry. According to the ch

13、aracteristics 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 centrifugation Rate-zonal density gradient sedimentation Isopycnic density gradient

14、sedimentation Besides sucrose, CsCl and NaBr can be used for achieving the density gradient, and applied to the separation of nucleic acid and lipoprotein, respectively. Centrifuges The tubular bowl rotor centrifuge is commonly applied on a laboratory scale, and the types of tubular bowl and disc st

15、ack are commonly used on an industrial scale. The processing capacity of the tubular bowl centrifuge is described byggvgLrvQ2222Where 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 cent

16、rifuge and the operating conditions.2.1.3 Filtration Definition：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 filtercmLRRpAdtdQAWRcWhere Q is the volume of the filt

17、rate, 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 a

18、ddition 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 equipment Filter press and rotary drum vacuum filter are frequently us

19、ed for clarification of the broths.2.2 Cell disruption Many 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.

20、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 ce

21、ll wall and/or cytoplasmic membrane damaged more or less to liberate the intracellular products . 2.2.2 The principles of cell disruption Mechanical disruption The 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

22、 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 disruptionH ig h -p re s s u reh o m o g

23、e n is a tio nB e a d m illin gC ra s hU ltra s o n ic a tio nM e c h a n ic a lT re a tm e n t w ithc h e m ic a lsE n z y m a tic ly s isC h e m ic a l/e n z y m a t icO s m o tic s h o c k F re e z e -th a wP h y s ic a lM e t h o d s f o r d is r u p t io n o f c e lls Mechanical disruption High

24、-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.baNkpS11lnWhere S is the disruption scale, p is o

25、perational 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. Th

26、e influencing factors：pressure, cyclic times, temperature, etc. Bead milling Principle：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 di

27、ameter, density, and loading density.ktS11lnWhere 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 w

28、ritten 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 coo

29、ling system design. And because many operating parameters can influence the disruption ratio, optimizing design of the processing is very complex. Ultrasonication Principle：cavitation. The influencing factors：the kind and concentration of the cells, and the energy of the ultrasonication. Characteris

30、tic: it is commonly used at laboratory scale; removal of the heat generated is difficult on a larger scale. Chemical methods Treatment with chemicals Principle：see 2.2.2 Available chemicals : acid, alkali, organic solvents, detergent, chelates, chaotropic agents, etc. Enzymatic lysis Principle：see 2

31、.2.2 Available 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 c

32、ellulase. 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 means Osmotic shock Principle：put cells into a soluti

33、on 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. Freezethaw Pri

34、nciple：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. Ch

35、aracteristic：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 t

36、aking into consideration the subsequent processing steps.Chapter 3 Precipitation Definition：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 se

37、diment 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. ionsmetalpolyvalentpolymeredargchpolymerionicnonagentsSpecialionprecipitatThermalOthersionprecipitatsolv

38、entOrganicionprecipitatcIsoelectriionprecipitatoutSaltingnecipitatioPr 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：IK

39、Sslog221iiZcIWhere 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 mole

40、cules, that is widely accepted. 3.1.2 The influencing factors The molecular weight and three-dimensional structure of different proteins for given protein the kind of inorganic salts （to Ks) Criteria of selecting a neutral salt: higher solubility； solubility is almost influenced by temperature； the

41、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 solubil

42、ity 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-out The experimental steps for deciding the saturation used to precipitate given protein： take a small portion of material liquor, an

43、d 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；212285. 01505SSSW After centrifugation dissolv

44、e 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 saturati

45、on, 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 de

46、cide the additive amount based on the request for recovery of products.3.1.4 Application Because of many salts being remained in the sediment removal of salts must be carried out after salting-out precipitation. Practical examples of applying precipitation to recovery of proteinsSaturation of (NH4)2

47、SO4productSourceOne stepTwo stepRecoveryratePurificationmultipleHumaninterferonCell cultureliquid30（mother liquor）80（sediment）991.7Interleukin-2Cell cultureliquid35（mother liquor）85（sediment）73.57.0monoclonalantibodyCell cultureliquid50（sediment）10083.2 Isoelectric precipitation Definition：isoelectr

48、ic 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 strength pHpI As a rule it is applied to precipitation of hydrophobic proteins e.g. casein, and not suitable f

49、or 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 precipitation Principle：reduced dielectric constant

50、 enhances electrostatic interactions between protein molecules. Operating condition： low ionic strength pH is near pI Characteristic： advantage：the lower density of the organic solvent is convenient for separating sediment, and removal of salts isnt needed. disadvantage：proteins denaturing maybe hap

51、pen, so low temperature required for operation.3.4 Another methods3.4.1 Thermal precipitation Principle：under higher temperature make heat sensitive proteins precipitate and achieve the separation of heat stable proteins. Operating condition： adjust pH of the solution add organic solvents. Character

52、istic： 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 agents Non-ionic polymer Mechanism：reduction in the effective quantity of water avai

53、lable for protein solvation. Agent in common use：PEG Charged polymer Mechanism：complex formation between oppositely charged molecules leads to charge neutralization and precipitation. Available agents：acidic polysaccharides, CMC, etc. Polyvalent metal ions Mechanism：bond with some functional groups

54、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

55、using ion-exchange resin or chelating reagent.Chapter 7 Affinity purification Bioaffinity To 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 inte

56、ractions in which structure can play a major role in shaping is referred to as bioaffinity. Affinity purification Bioaffinity interactions have been employed in bioseparation processes, that lead to the appearance of affinity purification techniques such as affinity chromatography, affinity membrane

57、, affinity precipitation, etc.7.1 Basic principle7.1.1 Molecular recognition processes From a molecular perspective, the binding process between a receptor and its ligand can be viewed as four continuous steps Electrostatic interaction Solvent displacement Steric selecion Charge and conformation rea

58、rrangement7.1.2 Receptor-ligand interactions In the affinity, receptor-ligand interactions involve noncovalent interactions such as Ionic bonds Hydrogen bonds Hydrophobic interactions Van der waals forces Specific interactions result during the formation of different receptor-ligand complexes, and s

59、ome of those can be applied to bioseparation for example Antibody-antigen interaction Enzyme-substrate interaction Lectin-carbohydrate interaction7.2 Affinity chromatography7.2.1 Principle and operation Principle Molecular recognition forms the basis of adsorption and separation by affinity chromato

60、graphy. 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). Operation Process including four steps, that is adsorption, wash, elution, and reuse. Elution methods Specific elution A free ligand molecule added into