2.2 THU NHN ENZYMECC PHNG PHP CHIT TCH ENZYME T T BOCC PHNG PHP TINH SCH ENZYMEKIM TRA TINH SCH

ENZYME NI BOENZYME NGOI BO

CC PHNG PHP CHIT TCH Phng php c hc: ng ha bng p sut cao (55MPa): ch lm lnh Nghin bi t vi tc cao bng cc ht thy tinh. Cc ht thy tinh thng c kch thc 0.2-1mm Lnh ng: Huyn ph t bo di dng bt nho c lm lnh ng -200C, ri nn di p sut cao qua cc l hp ca my nn.Tuy nhin thit b ny ch c th s dng gin on tng m nh (10 kg/h).

Phng php khng s dng c hc: Dung gii mng t bo vi khun bng: phng php sc thm thu, cht ty ra (tritol X-100, tween 20), hoc enzyme (lysozyme). Sng siu m (Ultrasounds) X l kim pH gia 11,5 v 12,5 s lm thy phn mng t bo v do s gii phng enzyme. Ch p dng cho cc enzyme bn trong mi trng kim t nht trong khong 20 30 pht. Ngi ta thng dng phng php ny trch ly asparaginase t Erwinia chrysathemi.

i vi t bo ng vt: D tch chit do thiu thnh t bo Cht bo v cc m lin kt phi c loi b trc khi tin hnh tch chit. Vi khun v nm Thnh t bo phi c x l trc bng enzyme (c th s dng lysozyme hoc chitinase/3-glucanase) T bo thc vt Ph v khng bo c th lm hng enzyme Enzyme mng S dng cht ty ra (detergent c th l anionic, cationic, neutral) Ch loi chn cht ty ra khng nh hng n giai on tinh sch sau, c bit l tinh sch bng sc k.

CC PHNG PHP TINH SCH1. DA VO KHI LNG V KCH THC Siu ly tm (Ultracentrifugation): Khi lng phn t l nhn t chnh phn tch. Khng phi l phng php hiu qu thu enzyme t dch chit hay dch nui cy, dng loi b cc cht bn. Lc gel (Gel filtration: Mr ~ hundreds of thousands) Sephadex, Bio-Gel P, Sephacryl, and Sepharose: thng t tin v tn thi gian. Thng c s dng cc bc sau ca qu trnh tinh sch.

Thm tch (Dialysis: Mr ~ tens of thousands): Thng c s dng loi b mui, cc cht ha tan hu c . Da vo s chnh lch nng Mng bn thm (semi-permeable membrane)

Siu lc (Ultra-filtration): Cc phn t nh c lc ra ngoi bng p sut S dng mng lc c kch thc nh c s dng cho vic c c protein Phng php thay th: Ly tm bng mng thm tch. Lu : mng khng c tng tc vi enzyme

2. DA VO PHN CC (POLARITY) Sc k trao i ion (ion-exchange chromatography): Da vo c tnh tnh in ca enzyme Dung dch cha enzyme cn tinh sch phi c nng mui thp v c iu chnh n pH thch hp. Enzyme c tch in tri du vi cc ht trong pha tnh ca ct sc k.

C 2 loi: sc k trao i ion m v sc k trao i ion dng.

C 2 loi: sc k trao i ion m v sc k trao i ion dng.

C 2 loi: sc k trao i ion m v sc k trao i ion dng.

Enzyme c gii hp ph bng dung dch nng mui cao Gradient nng mui Ti mt nng mui xc nh tinh sch c th tng ln gp 10 ln

Question???

Sc k k nc (hydrophobic interaction chromatography):Ph thuc vo cc amino acid khng phn cc trn b mt ca enzyme Cc ht ca pha tnh c gn vi cc nhm alkyl (nh Octyl, phenyl ...) Dung dch cha enzyme cn tch c nng mui cao. Dung dch gii hp ph c nng mui gim dn (hoc thm cc dung mi hu c nh ru)

in di (Electrophoresis): in di theo im ng im (isoelectric focusing)

3. DA VO KH NNG HA TAN Thay i pH: Enzyme thng t ha tan ti im ng in pI, do khng cn lc y gia cc enzymeKt ta phn on bng mui: Cc i phn t c tch in nh enzyme thng rt t ha tan trong nc, vic thm cc ion (cc loi mui c in ly mnh) s lm tng kh nng ha tan ca enzyme. Tuy nhin, khi vt qua mt ngng nht nh, vic thm cc loi ion ny s lm cho cc i phn t b kt ta nhanh chng. Mi loi enzyme c ha tan ti cc nng mui khc nhau. Thng s dng ammonium sulfate.

Gim hng s in mi Bng cch thm vo dung dch hn hp ca nc v dung mi hu c (acetone hoc ethanol) Nhng im khng thun li: Lm vic ti nhit thp ( t hiu qu cao thng -20oC) C th lm bt hat mt vi enzyme. Tiu tn thi gian Hiu sut thu protein thp. C th s dng PEG (poly ethylene glycol) vi trng lng phn t t 4000-6000.

4. DA CC V TR LIN KT C HiU Sc k i lc: C cht hoc cht c ch c gn ln pha tnh. Gii hp ph bng lc ko ca c cht, hoc s thay i pH, hoc bng mnh ca ion.

Sc k i lc.

Sc k i lc: Kh khn: Vic gn c cht hoc cht c ch ln pha tnh c th rt kh Lin kt c cht vo pha tnh c th ngn cn s lin kt ca c cht vi enzyme. Kh khn trong vic gii hp ph. Gii quyt: s dng protein lai (fusion protein- protein c gn thm cc ui) Glutathione-S-transferase : glutathione Maltose binding protein : maltose Hexahisitidine: Ni 2+ (gii hp ph bng imidazole hoc s dng thrombine ct v tr c hiu c thm vo cng ui His).

Sc k hp ph min dch: C nh khng th i vi Sepharose c tin x l vi CNBr. t c tinh sch cao hn. Sc k Dye ligand Cibacron Blue F3G-A c th lin kt vi mt s lng ln cc enzyme dehydrogenases and kinases Procion Red HE-3B lin kt tt vi cc enzyme dehydrogenase ph thuc NADP+ Sc k cng ha tr (Covalent chromatography) Phn tch cc enzyme giu cysteine c th s dng ht pha tnh c gn nhm thiol.

5. LA CHN PHNG PHP???Khng i hi tinh sch caoHm lng enzyme lnTiu tn thi gianKt ta bng cn, acetone, kt ta phn on vi amonisulfatei hi tinh sch caoHm lng enzyme nhTiu tn thi gianSc k cti hi tinh sch caoHm lng enzyme nhNhanhNhng t tinSc k FPLC, HPLC

NH GI TINH SCH Kim tra hat enzyme: nh gi hat enzyme Ch cc cht c ch v cofactor.

QUESTION????Hy vit quy trnh tinh sch Adenylate kinase t dch chit bp tht ln, bit rng: Enzyme c i lc cao vi adenosine monophosphate (AMP) Cc loi enzyme kinase c kch thc khc nhau ng k. C th kt tinh ti nng (NH4)2SO4 bo ha?

2.3 BO QUN ENZYME Nn bo qun lnh Trnh nh nng mt tri chiu trc tip. Nn bo qun ti pH trung tnh Nu bo qun enzyme nhit ng c cn b sung glycerol 50%, enzyme ch c th gi hot tnh 3 chu k ng v tan bng

Dng dung dch Ch phm enzyme c c c hoc pha long n nng thch hp B sung cc cht bo qun v cht n nh: 2-mercaptoethanol or DTT(Dithiothreitol), Protease inhibitor PMSF (Phenylmethylsulfonyl flouride), NaN3 (cht khng khun). Kh trng bng siu lc. Bo qun nhit thp (4oC), thi gian bo qun ngn.

2-mercaptoethanol or DTT(Dithiothreitol)*

Dng bt Sy thng hoa (freeze drying) L mt dng sy m sn phm dng ng c s c cho vo thit b c chn khng cao. Ti y nc s c lm bc hi khng qua trng thi lng, v v vy sn phm c sy kh. Trc khi sy thng hoa cn b sung cc cht n nh v d nh carbohydrate. c s dng rng ri cho cc sn phm nhy cm vi nhit cao nh enzyme, vi sinh vt, thuc Tuy nhin, mt vi enzyme b bin tnh ti nhit qu thp.

Dng bt Sy phun (spray drying) S dng nhit cao vi thi gian tip xc ngn vi tng git dung dch nn s lm bc hi nc nhanh. Enzyme t dch nui cy c c c bng siu lc. R tin Tuy nhin s dng nhit cao c th lm ph hng hat tnh enzyme Gii php: s dng lp bo v (cc mui khng ha tan nh tribasic calcium phosphate v tinh bt hoc cellulose)

*Cc ht lc gel thng c s dng*http://www.mikeblaber.org/oldwine/bch5425/lect30/lect30.htm*http://www.mikeblaber.org/oldwine/bch5425/lect30/lect30.htm*http://www.mikeblaber.org/oldwine/bch5425/lect30/lect30.htm*http://www.mikeblaber.org/oldwine/bch5425/lect30/lect30.htmhttp://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-JP/products/hydrophobic-interaction-chromatography-hic/*http://www.encorbio.com/protocols/AM-SO4.htm : tnh ton nng mui cn dng.Ammonium Sulfate Precipitation Ammonium sulfate precipitation was one of the earliest forms of protein purification. By increasing the concentration of ammonium sulfate in steps, differential precipitation of the protein mixture would occur. Centrifugation of the precipitate resulted in cuts of protein populations, significantly enriching the purity over the starting material. The precipitate would normally resolubilize once the salt was removed, either by filtration or by dialysis.

*Gim hng s in mi s lm cho cc phn t khng cn y nhau nas dng PEG (poly ethylene glycol) bt li trong vic loi b PEG, c th loi b bng siu lc hoc bng vic thm ethanol (PEG ha tan trong ethanol nhng protein th khng).**http://www.mikeblaber.org/oldwine/bch5425/lect30/lect30.htmImidazole c i lc vi Ni2+* CNBr treated Sepharose- Cyanogen bromide

*http://www.patentstorm.us/patents/4180917/description.html chun b mu cho sy thng hoahttp://www.jmcs.org.mx/PDFS/V55/3/10.-%20Martinez.pdf*http://www.google.com/patents/US4233405*