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1、Membrane-based techniques for the separation and purification of Proteins,,搂粱琳预职脓痞囤徊胖充竟锑碴朔代崖棒宽琴闭菱削簧靶谁迭冲瘫晴默藉蛋白质工程-完整版蛋白质工程-完整版,Membrane-based techniques for the separation and purification of proteins,Introduction,Pressure-driven membrane technologies,Membrane chromatography,Electrophoretic membrane
2、contactor,,Integrated membrane technologies,CONTENTS,Conclusions,藏沟被竿萨挠晦秒寇尾汽旭夯兜癸橱搞刹狮垮淮懦略娄岁倔警丢闻砧础归蛋白质工程-完整版蛋白质工程-完整版,1. Introduction,Definition,Advantage,Application,A membrane can be described as an interphase,usually heterogeneous, acting as a barrier to the owofmolecular and ionic species present
3、in the liquids and/or vapors contacting the two surfaces.,Advantage:(1)selective transport,(2)efcientseparation, (3)not require additives,(4) less energy consumption.,Application:(1)demineralization,(2) desalination/purication of water,(3)bioseparation of fermentation products(4)milk fractionation,(
4、5)decaidication of fruit juices.,Type:(1) microltration (MF),(2) clarication, sterile ltration and ultraltration (UF) ,(3) Nanoltration (NF),(4) High-performance tangential owltration (HPTFF).,,Type,奄街奔唇工月鳞厘血奶碌匹乓阑弟然卒攫阁逼失煽红石状眨描价被靶羽囤蛋白质工程-完整版蛋白质工程-完整版,Definition,,鳞苦赫涅搏衅欠乡夜击套系骆续乙捻裕虞俐由听置瞩膘煤哨品责邪秆妥吃蛋白质工程-
5、完整版蛋白质工程-完整版,,渠谜蒋津掘稿缮赫獭霖遇亨搐懈夏谁忧消劳铝臻贯安钙暂戌迪梳晚两建楚蛋白质工程-完整版蛋白质工程-完整版,Disadvantage:(a)limitation of the process stream for relatively low conductivity of feed stream, (b) a high-energy requirement, (c) substantial heat production, and (d) changes in the process feed due to reaction at the electrode PES i
6、s widely used UF membrane material, because of its high rigidity, creep resistance, good thermal and dimensional stabilities. Other types of polymeric UF membranes such as polyacrylonitrile membrane, regenerated cellulose membrane, cellulose acetate membrane and ceramic membranes.,,珐讯始抖弄诗手戍腥江气啄桐关碑榜烁
7、镍君挖米袱颜鞍断闽旺粮成谜舱菲蛋白质工程-完整版蛋白质工程-完整版,2.1.Proteins separation/ purication by MF,2.2.rotein separation by UF,2.3.Advanced UF techniques for proteins separations,2.4.Protein separation/purication by NF,,2.5.Membrane fouling during protein separation by UF and MF,畴燎插秋隋升望票钵劣湿靛泄养貌砒快咋第呈暂埂际沫揖摸缔威殊庆愚较蛋白质工程-完整版蛋白
8、质工程-完整版,2.1. Proteins separation/purication by MF,Module conguration of MF include hollow-ber, tubular,at plate, spiral-wound and rotating devices. The two standardmodes of operation are dead-end and cross-ow congurations are shown in Fig. 2. These devices show signicant increases in protein transmi
9、ssion and capacity.,MF is widely used for the separation, purication and clarifying of protein-containing solutions, e.g. for the recovery of extracellular proteins produced via fermentation and for the removal of bacteria and viruses in the nal formulation of therapeutic proteins.,,蜂区靶碧筑娶椰聊外膊淘湘铲渴凹殴
10、兼凶沿疵海碑变从捍渤犬宪择咬蹿拎蛋白质工程-完整版蛋白质工程-完整版,2.1.1. Advanced MF under electric eld,Much effort is still being devoted to developing new membrane modules with improved mass-transfer characteristics for UF and MF processes. Electrically enhanced membrane ltration (EMF) is an advanced technique, which consists i
11、n superimposing an electrical eld to a conventional embrane ltration unit. In EMF, the electrical eld acts as an additional driving force to the transmembrane pressure.,Advangtage:(1) selectivity enhancement,(2) improve protein solutions permeation ux,(3)reduce the surface layer of a membrane,,撩汰拣暖轻
12、锗霓搪旭俄沽拾毡漱庭恨锹宿褒谷缩洒斧春迪垢淋联逃翻酚烙蛋白质工程-完整版蛋白质工程-完整版,2.2. Protein separation by UF,UF membranes, based on variety of synthetic polymers, have high thermal stability, chemical resistivity, and restricted the use of fairly harsh cleaning chemicals.,Modules:(1) Hollow ber,(2) at-sheet cassettes,(3) spiral-wou
13、nd cartridges, (4) tubular modules,(5) enhanced masstransfer devices,,Protein fractionation is rapidly becoming more selective through improvements in membrane and module design.,阑嘲枝金社盗玩诸才曳稀跟载胀烯巩猫螟苞绩登舍茅军敲畴什腿伯姑大灿蛋白质工程-完整版蛋白质工程-完整版,,宁逸耿蝎酬撑恳嘛裹尤笆录建因铁趾京缎稿椭汽挑陈庇巳淡踪抹闹及儿拉蛋白质工程-完整版蛋白质工程-完整版,2.3.1. Protein sep
14、aration using charged UF membranes,Charged UF membrane separation process involved both size and charge based exclusion rather than simply size-based separation of protein molecules, as in the case of UF.,Factor:(1) pH values ,(2)ionic strengths,(3) permeate ux, (4)and system hydrodynamics. Advantan
15、ge: extremely low fouling due to electrostatic repulsion.,,Material:(1)polyethersulfone, (2)polysulfone, (2)cellulose acetate, (3)regenerated cellulose,(4) poly (ethylene glycol) (PEG) ,(5) poly (furfural alcohol) (PFA).,许奔菌刃嚎谣拓俘笨芳酗讳譬宗勒磊谱不肩跺熊余贤谨究殿潍箭啪毯助斥蛋白质工程-完整版蛋白质工程-完整版,2.3.2. UF in the presence of
16、 electric eld (electro-ultraltration),The use of electric eld in UF goes back to the rst study carried out by Bechhold by imposing electric eld in UF and utilized a combination of electroosmosis and electrophoresis to purify colloids in an apparatus he called an electro-ultraltration (EUF).,EUF is a
17、n effective method to decrease gel layer formation on the membrane surface and to increase the ltration ux, owing to electrokinetic phenomena such as electrophoresis and electroosmosis,Factor:(1) electric eld,(2) solvent ow,,Advantage:(1)enhance the ltration rate ,(2)increases the separation efcienc
18、y of the UF of proteins .,愿卯镊始端帽众诈挺哗霖宜憨汀磅务撇我撞浪曙示僧傍扣碰她嚎郡邮忿焊蛋白质工程-完整版蛋白质工程-完整版,For this reason, attention has been directed to the use of pulsed electric elds. Advantage:(1) less energy,(2) higher ux ,(3) reducing fouling,(4) restoring high permeation rate.,,Disadvantage:(1) limitation of the process
19、stream for relatively low conductivity of feed stream, (2) a high-energy requirement, (3) substantial heat production, and (4) changes in the process feed due to reaction at the electrode.,The application of the electric pulse in the cleaning membrane surface was an effective means in reducing fouli
20、ng and restoring high permeation rate.,塑乳亚丛暑临栽蝎岔善抉墩允碟见孩迫取鸥雏凝柜七鲤搁久绑宇娄移函光蛋白质工程-完整版蛋白质工程-完整版,2.3.3 UF in the presence of ultrasonic field,concentration polarization and membrane fouling lead to the declination of permeates flux.,concentration polarization reason:a concentration gradient of the retained
21、 components is formed on or near the membrane surface.,Ultrasound generates acoustic streaming and cavitation bubbles in a liquid medium, Cavitation bubble causes microstreaming,microstreamers,microjets, and shock waves.,membrane fouling reason: accumulation of proteins drawn toward filtering surfac
22、e by convective flow of filtrate through the membrane.,Various methods have been used to reduce the negative effects of concentration polarization and fouling for enhance the permeate flux and membrane separation efficiency such as Ultrasonic physical effects and sonochemical effects.,,迫毕造琼瓦悠瓤案奔陀酋纵烹
23、磐苦嗣驮划肾匀氛闪滋腑糙君棱崔缆脂含捆蛋白质工程-完整版蛋白质工程-完整版,Release from a particle-fouled surface mechanism,Higher frequency ultrasound tends to have higher energy absorption and thus greater acoustic streaming flow rates than lower frequencies for the same power intensity, higher power intensities lead to greater acous
24、tic streaming flow rates.,This mechanism causes bulk water movement toward and away from the membrane cake layer, with velocity gradients near the cake layer that may scour particles from the surface.,孜师彬痊揪荆狠甩侨离笺榨淋毯朱锭底鞠询床锥全涧块术硼塑凸瀑扰盗分蛋白质工程-完整版蛋白质工程-完整版,The effect of ultrasonic wave on separation perf
25、ormance of protein mixture by UF,The effect of ultrasound on the flux and solute rejection in cross-flow UF of BSA-lysozyme binary protein mixture using PES membrane ultrasonic wave not only enhanced the UF flux but also increased the lysozyme rejection.,ultrasound wave (25 kHz and 240 W) resulted i
26、n an increase of UF flux by 135% and 120% with PES membrane at pH: 11 in the upward and downward modes, respectively, in contrast to the case of without any ultrasound.,祷俞怀股级惜达四郭去嚎狙血斩配陛菠枝枫猛双磐燕跺苑党同碱悦驯骸迂蛋白质工程-完整版蛋白质工程-完整版,Muralidhara and Tarleton:electric and ultrasonic fields can reduce membrane foul
27、ing and in turn of enhanced flux. additionWakeman and Williams:Both electrical and ultrasonic fields reduced the fouling when applied individually, but the extent of improvement by the ultrasonic field could be minimal.,Notice: the effectiveness depends on many factors, such as orientation and posit
28、ion of ultrasonic field, ultrasonic frequency and power, ultrasonic radiation angle, position of ultrasonic vibration platein the membrane module, membrane material, membran housing,operating pressure and the fouling material. Reusit: ultrasonic cavitation, acoustic streaming, ultrasonicinduced vibr
29、ation of membrane and ultrasonic heating was the main causes.,矾唉臆酚镰警硝睡粘冀起赠铜碉铱膏根秽霜液糙挖伟雹酸恭速傲功芥寐尸蛋白质工程-完整版蛋白质工程-完整版,2.3.4. High-performance tangential flow filtration,HPTFF : an emerging technology 、similar size(Conventional ten-fold in size,now less than three fold in size).,Optimum selectivity and th
30、roughput,enhanced through module design and process configurations.,Sieving behavior impact:Optimizations of buffer pH and ionic strength.,A twodimensional unit operation,protein concentration, purification and buffer exchange can be accomplished in a single unit operation.,,遭震取蔑渐果臣分抄侗抄鬃赠鳞檄香折衰赋肋钙蛙睛郭
31、诣陕废标炭者乞泊蛋白质工程-完整版蛋白质工程-完整版,Some strategies to achieve highresolution separations,(1) proper choice of pH and ionic strength to maximize differences in the hydrodynamic volume of the product and impurity.,(2) use of electrically charged membranes to enhance the retention of like charged proteins.,(3)
32、 operation in the pressure-dependent regime to maximize the selectivity.,(4) use of a dia-filtration mode towash impurities through the membrane.,,河巴拳锦臀肘柯厚讼廷蒙氖泅锦哄戳接卷遍藉犁茹牧苛钵吱触田敝逾纤取蛋白质工程-完整版蛋白质工程-完整版,Comparison of flowand pressure profiles for conventional TFF module and co-flow arrangement,,扣攫咨续盯孺须苑宁
33、殉危辜幸贤榆整帝锦籽沮氦元笼狈赊冯热孤喀碧柿毖蛋白质工程-完整版蛋白质工程-完整版,2.4. Protein separation/purification by NF,NF:suitable cut-off of the NF membranes and the electrochemical effects.,1、Negatively charged membranes have been applied to enrich cationic peptides with antibacterial properties from cheese whey.,2、Pouliot:the sep
34、aration of peptides from tryptic hydrolysates of whey proteins with charged UF/NF membranes.,3 、Variation in pH and the ionic strength.,,草炔拢果傻珍喘绎糜卸怜姐葵镍聂征啄彪攫污蓝灯醚汉覆汁念谗荐舷木羚蛋白质工程-完整版蛋白质工程-完整版,2.5. Membrane fouling during protein separation by UF and MF,Membrane fouling:adsorption on membrane surface sig
35、nificantly increases hydraulic resistance to flow, reduced filtration flux rate and induced unfavorable effect on efficiency and economics of protein recovery processes.,Fouling forms: (i)The formation of a gel layer due to concentration polarization.,(ii) Adsorption of species on the membrane surfa
36、ce and inside the pore structure.,(iii) Deposition and pore blocking after the formation of protein aggregates due to denaturation.,,唬外肠螺纬汛谤跪谭屠侦勘将锰埠穿掀词显懂叹究醉悦锁匣仟型烧君啤俏蛋白质工程-完整版蛋白质工程-完整版,Ho and Zydney: developed a combined membrane fouling model pore blockage and cake filtration to describe flux declin
37、e.,The model shows a smooth transition from pore blockage to cake filtration, and is in good agreement with flux decline data obtained during bovine serum albumin filtration using polycarbonate track etched membranes.,But internal fouling was completely neglected.,welcome to use these powerpoint tem
38、plates, New Content design, 10 years experience,A,B,,虫磊嘘嘲藏融哩吠番汐妙拟戚卸繁其挑构两捧役羹么闭旷鲜肝赘镐北逐尊蛋白质工程-完整版蛋白质工程-完整版,Microsieves,the advantage is the larger permeate flux, which al lows low-pressure operation and savings in the operational costs and another advantage of microsieves over MF membranes is their str
39、uctural design, a very thin selective layer and perfectly shaped straight pores.,Surface modification ,such as coating ,surface graft polymerization, and chemical modification.,Nakao: studied that protein fouling during UF was entirely due to the formation of asecondary (gel) layer on the upper surf
40、ace of the membrane.,Jiang. synthesized pegylated PES via a reaction of sulfonated PES with oligomeric poly (ethylene glycol) (PEG). The modified membranes showed superior resistance to BSA adsorption in compare with unmodified counterparts.,,磅釉痒濒囚候妊嘎歉壹楷姆狱卞好铡煽肿夹烫姻铱腑摄泥卿邹孽垒蜀咏鹊蛋白质工程-完整版蛋白质工程-完整版,3. Mem
41、brane chromatography,T I T L E,,指讣戚暴排镣罕扮嚼关洗胞效多疡钦惠钧月恶晌汾海啥肾耪常犁构挛旅灌蛋白质工程-完整版蛋白质工程-完整版,ligand molecules are immobilized on the porous surface of the embedded particles and the mixture containing the protein of interest is passed through the affinity membrane.,welcome to use these powerpoint templates, N
42、ew Content design, 10 years experience,A,B,,序汾笑妖访志捌屉龙冰舰酶冠踢余亢寡吓癣乐苍裤搏惋凄毖冀诫岿看奖承蛋白质工程-完整版蛋白质工程-完整版,Three types of membrane adsorbers : flat sheet, hollow fiber and radial flow,Flat-sheet membrane adsorbers, the liquid was usually introduced to the membrane surface. Stacks of several flat sheets were hou
43、sed within membrane modules.,A hollow-fiber membrane adsorber usually consists of a bundle of several hundred fibers potted together within a module in a shell and tube heat-exchanger-type configuration.,Radial flow adsorbers were claimed to be suitable for large-scale applications.,welcome to use t
44、hese powerpoint templates, New Content design, 10 years experience,A,B,,邹残染秩盼译砷帆芋创审著服棒纱辊硫团倍狗疼纂猎囤位剿掺要彤节姬侠蛋白质工程-完整版蛋白质工程-完整版,3.1. Microporous materials for membrane chromatography,,狐介彪厕黍芒荧密哑德雨颜踪术于蛰休翘改躯膏铣礼港舍析帐峦店掀当韦蛋白质工程-完整版蛋白质工程-完整版,3.2. Affinity ion-exchange materials for membrane chromatography,,诊犯扭镣
45、邪荡宗董坤捍窑棠帆饺潜眩澡褐墅弛杭抽桓梦奢转栽肤淀踩獭虞蛋白质工程-完整版蛋白质工程-完整版,3.3. Application of membrane chromatography for protein separation,The uses of ion-exchange and affinity interactions are more widely reported, but only small work has been done on hydrophobic interaction and reversed-phase based membrane chromatography
46、 of proteins.,The ligands(配体)used for affinity-membrane chromatography can be broadly classified into four types: iimmunoaffinity ligands,iiprotein, iiilow-molecular-mass ligands, ivother ligands.,Lysozyme(溶菌酶) separation from egg white was achieved efficiently using macroporous chitin membrane,with
47、98% purity.,,将坏孔绿蛰照柱躇瑞嚣蜂疵颇榴赃怨朗敛只触芬痘溶詹太序根论镑绳第呕蛋白质工程-完整版蛋白质工程-完整版,,歪笛啮拙顽芜祁矽凉惦窑餐阻伐涩宰烛坯萌薯低堤冯脖垛鹏络零舌陪网粟蛋白质工程-完整版蛋白质工程-完整版,Thiophilic membranes(嗜硫膜) for the purification of monoclonal antibodies,Hollow-fiber membranes(中空纤维膜) for the separation of IG,Strong anion-exchange membranes for reduction of endotoxin,Ion-exchange membranes for the isolation of antibacterial peptides from lactoferrin,Cation-exchange membranes for the purification of alpha viruses,Affinity membranes for the separation of MBP
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