1. Separasi Dan Purifikasi

7/23/2019 1. Separasi Dan Purifikasi

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Separasi dan

Purifkasi dalam Bioteknologi

Jumeri M. Wikarta,Ph.D



Fermentation broth

• Combination of insoluble, gelatinous biomass,the nutrient uid, and the soluble metabolitesresulting from the fermentation operation.

• Fluidity for stirring or aeration is ± 3-7% dryweight of biomass.

• Baterial fermentation for !C" will produe abroth of 3% #$ in whih the slurry is &%'by (olume) wet biomass and *&%

interpartile uid.



Typial Produt !onentrations"ea#ing Fermenters





• +f the desired produt is etraellular, itis only neessary to lter the biomassfrom the broth and isolate the produt

from the uid.• +f a produt is intraellular, a ell

disruption step must rst be employed.

• +f the produt is also water soluble, this

disruption should be performed while thebiomass is still in a slurry form.



/C0$/1 2+40"/54+0!

• Filtration and entrifugation are unitoperations in whih the suspended solids areseparated from the uid phase.

• 6rying is the remo(al of moisture or sol(entfrom solid partiles

• /(aporation is the remo(al of moisture orsol(ent from a solution.

• +n rystalliation, the onditions of a solutionare ad8usted to hange the solubility of oneof the dissol(ed ompounds so that it lea(esthe solution as a solid.



Separation and $eo#eryTehni%ues



9 5 : &,9nm

• ;iroltration, ultraltration, and re(erseosmosis are membrane proesses in whihseparation is based on di<erenes in abilityto ow through a thin barrier that separatestwo uids.

• ;iroltration is a hydraulially dri(enproess using a membrane with a pore siein the 9&& to 3&&& 5 range.

• 2ltraltration, the pore sie is 9&-9=> 5,while for re(erse osmosis, the pore sie is3-9& 5.



• 5dsorption, ion ehange, olumn hromatography,and a?nity hromatography grouped as reo(erytehni@ues in whih the remo(ed ompound or

solute establishes an e@uilibrium between sites ona solid phase material and the solution.

• +n adsorption, the remo(ed speies is bonded tothe solid phase material by polarity or weaA

hemial bonds.• +on ehange reo(ers material by the interhangeof ions between the li@uid and solid phases.

• Column hromatography may use adsorpti(e, ionehange or moleular sie(e materials to separate

solutes whih are rst loaded onto a olumn ofthe separation material and then eluted in suh amanner that the indi(idual solutes are olleted inseparate frations.

• +n a?nity hromatography, the remo(ed speies isbound with a high le(el of seleti(ity to ligands



• +n sol(ent etration, the remo(ed ompoundestablishes an e@uilibrium distribution betweenimmisible sol(ents, usually water and an organili@uid.

• /letrophoresis and eletrodialysis are separationtehni@ues that separate harged moleules orions using an eletri eld.

• /letrophoresis separates harged omponents byaentuating small di<erenes in ioni mobility inan eletri eld using a mo(ing arrier uid.

• /letrodialysis onentrates omponents on thebasis of eletromigration through ioni membranes.



Per&orated bo'l or basket typeentri&uge

•2sed with a lterbag of nylon,terylene, or otton.

• 4he feed is added

ontinually.•#hen the bowl islled with thebiomass aAe, freshwashing li@uid an

be added todisplae the residualbroth uid retained

in the aAe.



Tubular bo'l entri&uge

5 ontinuousfeed of materialpasses through themahine. 5s soon

as the bowl is fullof the biomassaAe, theentrifuge isstopped, strippeddown, the aAeremo(ed, themahine leanedand

the se@uene



Multi hamber solid bo'l entri&uge

•5llows three or moretime the apaity ofthe tubular bowl.

• 4he disassembly and

leaning of thisentrifuge is moredi?ult.



(o))le disk entri&uge

• 0perate ontinuouslysine the solids areautomatially remo(edfrom the bowl.

• 6isharges a ontinuousstream of onentratedsolid-slurry.



Desludger or intermittent dishargeentri&uge

as anintermittentdisharge ofsolids with lower

water ontentthan the noleentrifuge.



Sroll entri&uges

2ses a srew within a rotatingbowl to allow ontinuous remo(al

of the solid and the li@uidortions.



!harateristis o& Di*erent Types o&!entri&uges



$eo#ery andpurifation o&itri aid



+solationproedure &or

sodiumla#ulanate



Purifationproedure &ormirooalnulease



eo(ery proessesfor human insulin,humangrowth hormone andhuman leuAoyteinterferon synthesiedfrom /. oli



Why puri&y Proteins

• Charaterie – Funtion

– 5ti(ity

– !truture

• !tudy protein regulation and proteininterations

• 2se in assays

• "rodue 5ntibodies• "erform strutural analysis by -ay

and Crystallography



-o' to puri&y Proteins

Biohemists eploit the ways indi(idualproteins di<er from one another, suh assolubility, sie, or harge.

!pei

binding



!lassial Protein Purifation MethodsThe ldest/ 0mmonium Sul&ate

Frationation



Dialysis 1 2etting $id o& 3(-456S4 or

!hange Bu*ers



+solation of "roteins fromCells;any di<erent proteins eists within one ell

D ;any steps needed to etrat protein ofinterest, and separate from many

ontaminants

D Before puriation begins, protein mustbe released from ell by

homogeni)ation



Compartmentalization provides an opportunity

for a purification step

e

Protein profile for compartments of gram-negative prokaryotes



!ell Disruption

!hemial/ alAali, organi sol(ents,detergents

7n)ymati/ lysoyme, gluanases,hitinase

Physial/ osmoti shoA,freeeEthaw

Mehanial/ soniation,homogeniation, wetmilling, Frenh press



!hemial Disruption

Detergents suhas Trition 89:;; or(P4; anpermeabili)e ellsby solubili)ingmembranes.

Detergents an bee<pensi#e,denatureproteins, andmust be remo#ed



Press

!ells are plaedin a stainless

steel ontainer.0 tight fttingpiston is

inserted andhigh pressuresare applied to&ore ellsthrou h a small



-omogeni)ation

•!ells are plaed in alosed #essel 3usuallyglass5.

•0 tight ftting plunger isinserted and rotated'ith a do'n'ard &ore.

•!ells are disrupted asthey pass bet'een theplunger and #essel 'all.

•0lso, shaking 'ith glass

beads 'orks, B=T/ Frition > -eat



Soniation

A sonicator can be

immersed directly into

a cell suspension. Thesonicator is vibrated

and high frequency

sound waves disruptcells.



6i<erential entrifugation



Protein purifation

Protein mixture applied to column

Solvent (buffer) applied to top,

flowed through column

Different proteins interact with

matrix to different extents, flow

at different rates

Proteins collected separately indifferent fractions



Column Chromatography

9.!+/GHel ltration '!ieelusion )

=.C5H/G+on ehange

3.!"/C+F+C B+6+HG5?nity

Moleules an be separated on the basis o&/



ow #e Het "roteins 0ut ofCells



$eyling Preparati#e-P"!



$eyling Preparati#e-P"!



Hel Filtration

!eparates moleules aording to di<erenes in

sie as they pass through a gel ltration mediumpaA in a olumn

5 Aey position in the puriation of thousandsenymes, polyssaharides, nulei aids, protein,

and other biologial maromoleules.;oleules do not bind to the hromatographymedium so bu<er omposition does not diretlya<et resolution

4he li@uid inside the pores is sometimes re<eredto as the stationary phase and this li@uid is ine@uilibrium with the li@uid outside the partiles,re<ered to as the mobile phase



Hel Filtration

Can be performed in the presene of essential

ions or ofator, detergent, urea, at high or lowioni strength, at 37IC or in the old roomaording to the re@uirements of the eperiment

4he sample one mo(es down the bed as eluent

is added to the top 4he small moleules whih di<use into the gelbeads are delayed in their passage down theolumn ompare with the large moleules whih

anJt di<use into the gel and mo(e ontinuouslydown the olumn in the owing eluent

4he large moleules thus lea(e the olumn rstfollowed by the smaller moleules in the order intheir sie

l fltration hromatography



l fltration hromatography



!ie /lusionEHel-ltration



Si)e9e<lusion hromatography

!ie elusion



!ie-elusionhromatography

5bsorbane at =K& is used to identifyprotein-ontaining frations. 1ou analso perform an enyme spei assay.



!olumns !onneted toFration !olletors



/lution prole 'hromatogram) of gel ltration



$esolution in gel fltration

Fators in?uene /- !ample (olume- 4he ratio of sample(olumeto olumn (olume- Column dimension- "artile sie- "artile sie distribution- "aAing density- "ore sie of the partiles- Flow rate- $isosity of the sampleand bu<er

$esolution@ degree o&



$esolution@ degree o&separation

s L 9.> M two omponent are present ine@ual proportions

s an be inreased by N

- 2sing longer olumn

- Hel medium with smaller partiles andEor greaterseleti(ity

- !mall sample (olume

- Oow ow rate



Separation time

ange from a few minutes to manyhours

5ording to the di?ulty of the

separation, the gel medium, theolumn length, and the ow rate

+mpro(e the resolution also lead to

longer separation time

S l l



Sample #olume

esolution depend onthe ratio of sample(olume to olumn

(olume

Oarge sample toolumn (olume ratiogi(ing lower resolution

$eomended sample #olume as a



$eomended sample #olume as aper ent o& the total bed #olume

&or good resolution

Medium $eomended sample#olume 3A t5

!ephade

!epharose!epharose CO!epharyl !uperde prepgrade

!uperose prepgrade!uperde!uperose

= P >

= P >= P >9 P =9 P =9 P =

&.>&.>

Moleular 'eight



Moleular 'eightdetermination

5 gel should be hosen, so that thesampleJs epeted ;# falls on the linearpart of the seleti(ity ur(e and in the

middle of a suitable range of alibrationstandard

+f the ;# is unAnown, a gel with widefrationation range '!epharyl ) will be

most suitable

2el fltration



media seletionguide



2 l Filt ti M di



2el Filtration Media



+on9e<hange 30dsorption5 hromatography

!eparation depends upon the re(ersibleadsorption of harged solute moleules toimmobilied ion ehange groups ofopposite harge.

"robably the most fre@uently used for theseparation and puriation of proteins,polypeptides, nulei aids,polynuleotides, and other harged

biomoleules.#idespread appliability, its high resol(ingpower, its high apaity, and the simpliityand ontrollability of the method.

+on9e<hange



ghromatogra

phy



+on /hangeD +nteration based on o(erall

harge 'less spei thana?nity)

D Cation ehange

D 5nion ehange





+on-/hange



+on /hangehromatography

+f p mobile phase :7.=

4hen harge of the proteinsN '-) '-) 'Q)

'Q)

-- Q Q

Q

Q

Q

Q

Q

Q-

--

--

-Q

Q

Q

Q

Q

Q

Q

Q

Q

Q

Q

Q

5nion ehange olumn : Q harged

F ti l d i



Funtional groups used on ionehangers

Step'ise elution &or ion e<hange



Step'ise elution &or ion e<hange

0Cnity hromatography




!eparates proteins on the basis of a re(ersible

interation between a protein 'or group ofproteins) and a spei ligand oupled to ahromatography matri.

Can be used to separate ati(e biomoleules from

denatured or funtionally di<erent forms, toisolate pure substanes present at lowonentration in large (olumes of rude sampleand also to remo(e spei ontaminants

igh seleti(ity, hene high resolution, andusually high apaity for the protein's) of interest.

"uriation an be in the order of se(eralthousand-fold and reo(eries of ati(e material

are generally (ery high.

0Cnity



hromatography

5? it Ch t h



5?nity ChromatographyD2ses spei binding properties of moleulesEproteins

D!tationary phase has a polymer that an be o(alently linAedto a ompound alled a ligand that speially binds toprotein




0Cnity hromatographyseparation by biologial

binding interations

wash

porous

bead

glutathione

elute

GST apply sample

thrombin siteprotein of interest

/ampleN 2ST 92lutathione

H!4-tagged proteins bindto gluthatione on beads

on-speially orweaAly

bound proteins washedo<

H!4-tagged proteinseluted

with glutathione

Protein purifation by hromatography



Protein purifation by hromatography

What sort o& e%uipment do



% p'e need



-o' to pak the olumn



-o' to pak the olumn

5 puriation table for a



5 puriation table for ahypothetial enyme

Separating and #isuali)ing proteins



Separating and #isuali)ing proteinsSDS9P027/ Sodium dodeyl sul&ate polyarylamide gel eletrophoresis

9. eat sample with !6! and β -meraptoethanol

!6! : 6etergent 'anioni)- 6enatures proteins- Coats proteins- /ah protein has similar massEharge ratio

β-meraptoethanolE644- redues disulde bonds

=. !eparate on polyarylamide gel

- polymer of arylamineEbis-arylamide- 4/;/6, ammonium persulfate atalyst for polymeriation- "rotein migrates through gel matri in eletri eld.

7stimating the moleular



g'eight o& protein

Protein Properties



Protein Properties

• Proteins denatured by heating them in a sample buffer

containing SDS• The proteins no longer have any secondary, tertiary, or

quaternary structure

• esultant proteins taAe on a rod-liAe shape and auniform negati(e harge- to-mass ratio proportional to

their moleular weights



4he e@uipment of !6!



4he e@uipment of !6!-"5H/

"reparing gel



"reparing gel

Documents

1. Separasi Dan Purifikasi