1.Cc phng php lng ta proteinPhng php ta c s dng tng i rng ri thu
nhn cc phn t sinh hc m nht l protein. ta, ngi ta c th dng nhiu cch
khc nhau: ta bng mui, ta bng cc dung mi hu c hoc thay i pH ca dung
dch c cha protein. 1. Ta bng mui y l cch ph bin ta protein. Kh nng
ha tan ca protein ty thuc vo nhiu yu t: c tnh l ha t nhin ca
protein, pH, nhit , nng ca mui nng mui thp, tnh tan ca protein tng
nh (salting in). Tuy nhin, nng mui cao, tnh tan ca protein gim mnh
(salting out). u tin, gi thuyt salting in nng mui thp c gii thch .
Trong dung dch, protein c bao bc xung quanh bi cc ion mui mang in
tch tri du. Chnh c tnh ny gia tng hot tnh ca cc dung mi, lm gim cc
phn t protein khng mang in tch, do lm tng tnh tan ca protein trong
dung mi. Gi thuyt ca Debye Huckel cho rng: tnh tan ca protein c biu
din bng mt hm logarit, gi tr ca hm t l vi cn bc hai ca cng ion
trong dung dch. Thut ng salting out trong mi trng c nng mui cao c
gii thch bi Kirkwood. S gia tng lng ion mui trong dung dch lm gim
qu trnh Solvate ha, gim tnh ha tan ca protein dn n s ta. nng mui
cao, ha tan tun theo cng thc sau ca Cohn: log S = B - KI Trong S:
ha tan ca protein B: hng s (ty thuc vo chc nng ca protein, pH v
nhit ) K: hng s salting out (tu thuc vo pH, hn hp v lng mui c trong
dung dch) I: cng ion ca mui. th hm log biu din ha tan ca protein
bin thin theo nng mui: Nhng ng biu din khc nhau ty theo tng protein
khc nhau. V th, khi mun tch 1 protein t mt hn hp gm nhiu protein
khc nhau, ta c th la chn nng mui thch hp sao cho ph hp nht vi
protein mc tiu. dc ca ng salting out m t trn ph thuc vo chc nng ca
protein v nng mui, khng ph thuc vo nhit v pH. Ngoi ra,
nu trng lng phn t ca protein tng th lng mui cn cho phng php ta
gim xung. Hiu qu ta protein ca cc anion mui khc nhau th khc nhau, c
th xp theo th t gim dn nh sau: citrate > phosphate > sulphate
> acetate/chloride > nitrate > thiocyanate. 2. Ta protein
bng cc dung mi hu c Khi thm dung mi hu c vo mi trng, hng s in mi ca
mi trng gim xung. Cng thc biu din mi quan h gia ha tan v hng s in
mi: ln (S/Sw) = (A/RT) (1/Dw - 1/D) Trong S: ha tan ca protein
trong dung mi hu c Sw: ha tan ca protein trong nc D: hng s in mi ca
mi trng sau khi b sung dung mi hu c vo Dw: hng s in mi ca nc R: hng
s kh T: nhit tuyt i A: hng s kh Cng thc trn cho thy: khi thm dung
mi hu c vo mi trng, hng s in mi tng ln, kh nng ha tan ca protein
gim, v th to s kt ta. Tuy nhin, cc dung mi hu c li c i lc vi cc b
mt k nc ca phn t protein. Kt qu l chng lm bin tnh protein trong sut
qu trnh ta. Do , khi ta, ch nn s dng cc dung mi hu c nng thp (mc d
mt s dung mi nh: 2 methyl 2,4 pentanediol (MPD), dimethyl Sulfoxide
(DMSO) v Ethanol c th c s dng nng cao. 3. Ta protein bng phng php
im ng in Khi thay i pH ca mi trng, mc ta ca protein cng thay i. pH
thp, protein tch in dng v nhm amide b proton ha (thu nhn proton).
gi tr pH cao, protein tch in m v cc nhm carbocyl trong phn t
protein b mt i proton (mt H+). Ti gi tr pI (Isoelectrics point im
ng in), protein khng tch in. iu ny lm gim tnh tan ca protein v
protein khng cn kh nng tng tc vi mi trng, khi , cc phn t protein s
tch ra khi mi trng. Hin tng ny c gii thch bng phng trnh Cohn. Phng
php ny thng dng cho cc protein u nnh (c im ng in khong 4.6)
4. Ta protein bng cc non ionic polymer Phng php ny c thc hin bng
cch cho thm non ionic vo trong dung dch protein. Khi thm vo, s lng
cc phn t nc c th tng tc c vi protein gim xung. Ngi ta thng s dng
dextrans v polyethylen glycols. i = i0 + RT (ln mi + fii mi + fij +
mj) Trong i0: in th ha hc chun ca phn t i i: in th ha hc ca phn t i
R: hng s kh T: nhit tuyt i mi, mj: nng molar ca phn t i v j fii: h
s tng tc ca phn t i fij: h s tng tc gia phn t i v j Khi protein tn
ti nng cao, hay pH mi trng vt ra ngoi im ng in mi xy ra qu trnh tng
tc gia cc cu t trong protein. Mum thm loi non ionic polymer no vo
trong dung dch protein ty thuc vo trng lng phn t ca protein . Cng
tng t nh ta protein bng phng php im ng in. Ti 1 gi tr pH no (sau
khi thm non ionic polymer) th kh nng ha tan ca protein cng gim
xung. Cc ion non ionic polymer thng s dng l cc dung mi hu c nh
Ethanol hay Acetone. Cc dung mi khc t s dng v kh thao tc v c th gy
bin tnh protein. Ch : - Nhit thp lm tng hiu qu ta v gim s bin tnh
protein. - Nng ion: 0.05 0.2. - i vi cht tan c trng lng phn t cng
cao th lng dung mi cn cho qu trnh ta cng t. Theo Scopes (1982), khi
bt u ta protein bng Acetone, nn tun theo cng thc sau: [(v/v)%] =
1.8 - 0.12 ln [MW] Trong (v/v)% l th tch dung mi cn cho qu trnh ta
tnh theo % MW: trng lng phn t ca cht tan Nu trong hn hp c s hn din
ca 2 protein, tnh tan ca protein ny s gim v s hin din ca protein
kia. Thun li ca phng php ta bng cch s dng non ionic polymers l
protein sn phm bn, v c th s dng nhit phng. 5. Ta bng ion kim
loi
Trong phng php ta ny, ion kim loi s gn vi mt phn ca phn t
protein. Thun li ca phng php ny l c th ta c protein trong mt dung
dch rt long. Cc ion kim loi c th chia lm ba nhm. Cc ion nh Mn2+,
Fe2+, Co2+, Ni2+, Zn2+ v Cd2+ s gn cht vo cc acid carboxylic v cc
hp cht c cha nitrogen. Cc ion nh Ca2+, Ba2+, Mg2+ v Pb2+ s gn vi
nhm chc acid carboxylic. Cc ion nh Ag2+, Hg2+, v Pb2+ s gn cht vi
cc nhm c cha lu hunh.
2. Cc phng php sc k2.1.Khi nim chung v sc k.Cc Phuong php sc k u
da trn vic p dng hin tng hp ph.mc nh ca pp ny l: _gn cc cht trao i
mun thu nhn trong dung dnh ln men vi nng rt thp v ko tinh khit, ln
mt cht hp ph rn thch hp, sao dung dung mi thch hp tch cc sn phm
trao ddoooir cht ra khi cht hp ph. _nh vy phng php ny cng mt lc lm
nhiu nhim v nh: tch, tinh ch, c c sn phm. _sc k l phng php c x dng
rng ri trong cng ngh sau ln men tinh ch cc sn phm trao i cht. _c
nhiu loi sc k khc nhau v mi loi ph hp cho vic tinh ch tng sn phm
nht nh.
2.2. Cc phng php sc k: 2.2.1 sc k hp ph_Da trn c im cc sn phm
trao i cht khc nhaucos i lc khc nhau vi cc cht hp ph (nh oxit nhm,
than hot tnh, tinh bt, xelluloce). Sn phm trao i cht sau khi c hp
ph c tch ra khi cht hp ph bang dung mi thch hp.
2.2.2. sc k trao i ion._Da vo s khc nhau ca cc hng s phn ly ca
tng loi cation, anion, cc phn t c gn vo cc cht trao i cation hoc
anion, mc khc nhau, sau tch sn phm trao i cht c hp ph bng cc dung
dnh c ion lc mnh hn, nh dung dnh mui.
2.2.3. Sc k gel:_ S dng cc gel c cc l khc. Cc phn t c kch thc b
hn cc l s chui vo bn trong cc l. trong khi cc phn t c kch thc ln hn
s ko b tri . i bng cch cho cc dung dnh ln men i qua cc ct gel nh vy
th c th tch cc phn t ca cc sn phm trao i khc ra theo kch thc ca
chng. V vy phng php ny gi l phng php lc phn t.
2.2.4. Sc k i lc:_ Trong phng php ny cc cht mang cc nhm ha hc c
i lc c bit vi cc sn phm trao i, v vy sn phm c gn chn lc vo cht
mang. Tt c cc thnh phn khc ca dung dnh ln men s i qua, sau khi hp
ph hon tt, th sn phm c vt ra bang cc cht ha hc thch hp. _Thnh phn
ca cht hp ph i lc: D h thng sc k i lc hot ng cn cc iu kin sau y:
Tay ni phi c gn vi cc cht mang sao cho phn t protein gn c mt cch d
dng. Spacer nm gia cc cht mang v tay ni phi to iu kin cho phn t
protein tip cn d dng vi tay ni. Tng tc ko c hiu phi yu sao cho ngoi
tr phn t protein cn tch, cc protein cn li ko c gn vi cht hp ph, Lin
kt ca tay ni v cht mang phi bn trong diu kin tin hnh sc k cng nh qu
trnh ti sinh hp ph.
