Trng i Hc Bch Khoa Tp.

H Ch Minh Khoa K Thut Ha Hc B MN CNG NGH SINH HC

BK
TP HCM

TH NGHIM PROTEIN - ENZYME

SVTH: Nguyn Th Thanh Loan Nguyn Th Hng H Nguyn Xun Qunh C Kim Th GVHD: Hunh Nguyn Anh Khoa

60701307 60700701 60702005 60702415

TP HO CH MINH 2/2011

MC LC
I. Dng c th nghim ha cht ging 1. Dng c th nghim 2. Ha cht s dng 3. Ging S Thao tc tin hnh 1. Giai on chun b 2. Giai on nhn ging 3. Giai on nui cy thu nhn enzyme amylase 4. Giai on bn tinh khit Lit k cng tc th nghim Ti liu tham kho

II. III.

IV. V.

NI DUNG
I. DNG C TH NGHIM HA CHT GING 1. Dng c th nghim ng nghim Que cy vng a petri Pipette 1, 5, 10 ml Erlen 250 ml Pipette t ng Erlen 100 ml Eppendorf Bercher 500 ml ng li tm Bercher 250 ml My lc Bercher 1000 ml B n nhit n cn Bng khng thm Gi ng nghim Gc Bnh tia Giy nhm a khuy My o OD 2. Ha cht s dng Ha cht pha mi trng H tinh bt Dch chit nm men Peptone MgSO4 NaCl

CaCl2 Agar m phosphate 0,1M Tween 20, Tween 40, Tween 80, Natri lauryl sulphate

Ha cht kim tra hot tnh enzyme H tinh bt m phosphate 0,1M pH 6,8 HCl I/KI 3. Ging: ng ging cha chng Bacillus Subtilis CM3 II. S S qui trnh sn xut enzyme amylase t Bacillus Subtilis

Ging Bacillus Subitlis CM3 Nhit 60oC Tc lc 150 rpm Thi gian 38h

Mi trng nhn ging

Nhn ging

Gi ging

Kim tra bng sc k bn mng

Mi trng nui cy

Nui cy thu nhn enzyme

Nhit 60oC Tc lc 150 rpm Thi gian 38h pH 6,8

Xc nh hot tnh enzyme

Ammonium sulphate

Bn tinh sch

in di SDS PAGE (kim tra)

Enzyme amylase th

III. THAO TC TIN HNH 1. Giai on chun b a. Chun b ging S dng chng Bacillus Subtilis CM3 phn lp t phn b Ging c gi trong mi trng thch nghing 40C b. Pha mi trng Pha 800 ml mi trng Thnh phn mi trng H tinh bt 0,8 g Dch chit nm men 0,16g Peptone 0,4g MgSO4 0,04 NaCl 0,04g CaCl2 0,012g Agar 1,6g Nc ct n 800ml Chnh pH = 7 trc khi hp kh trng Hp kh trng 121oC trong 15 pht Ly 10ml mi trng thm 12g Agar, un nh to mi trng ng nht c. Chun b dng c - Hp kh trng 121oC trong 15 pht Mi trng pha 2 ng nghim c khong 3 ml mi trng(c thm 12g Agar to thch nghing), y nt bng, bao gi 20 erlen c y nt bng, bao gi - 4 a petri ra sch, bao gi ri em sy ? oC trong ? pht - Sau khi hp kh trng xong, tin hnh mi trng vo a petri v erlen - 2 ng nghim c t nghing sao cho lng mi trng chim chiu cao ng nghim. yn cho thch c li. B mt thch phng, khng g gh, nhn, lin tc. - Tt c cc a petri, ng nghim, erlen sau khi cho mi trng vo c a vo t c ch nhit n nh cho vi sinh vt pht trin kim tra v khun ca mi trng trong vng 1-2 ngy. Sau ly ra quan st, loi b nhng ng nghim, a petri, erlen c vi sinh vt pht trin. - Cc ng nghim, a petri, b nhim, cn loi b bng cch em hp bng my autoclave sau em ra sch, ro v sy kh dng c 2. Giai on nhn ging a. Chun b - Mi trng - 15 x erlen 100ml cha 20ml mi trng (A) lp ng chun v ng cong sinh trng. - 2 x erlen 500ml cha 200ml mi trng (B) nhn ging. - 2 thch nghing (C) tr ging trc v sau khi phn lp. - 4 thch a (D) phn lp.

b. Tin hnh: - Cy chuyn t ng ging ra 1 ng thch nghing C1, 600C trong 38h sau em tr trong t lnh gi ging. - Cy ria t ng ging ra 2 a petri D2, D3 nhit 600C. Lm tiu bn, quan st di knh hin vi kim tra thun ca ging. Nu b nhim, chn nhng khun lc c trng cy ln a D3, D4 v 1 thch nghing C2 tr li ging thun. 600C trong 38h. Sau em tr trong t lnh. - Chn khun lc c trng cy vo erlen B1,B2 to ging cp 1. B1, B2 nhit 600C trong 38h, 150 rpm. Sau 38h tr trong t lnh. - Canh gi cy 5ml ging t B1 vo cc erlen A, lc 150 rpm nhit 600C sao cho n gi th nghim thu c cc mu 0, 4, 8, 14, 16, 20, 24, 28, 32, 38, 40, 44, 48, 52 , 56h lp ng chun v ng cong sinh trng. Thi gian cy nh sau: + 2 ngy trc khi lp ng chun, 7h cy ging vo erlen A1, A2, A3. + 2 ngy trc khi lp ng chun, 17h cy ging vo erlen A4, A5, A6. + 1 ngy trc khi lp ng chun, 7h cy ging vo erlen A7, A8, A9. + 1 ngy trc khi lp ng chun, 17h cy ging vo erlen A10, A11, A12. + Vo ngy lp ng chun, 7h sng cy ging vo erlen A13, A14,A15 Thi gian ly mu lp ng chun: K hiu M0 M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 Thi gian 0h 4h 8h 14h 16h 20h 24h 28h 32h 38h 40h 44h 48h 52h 56h Gi ly 7h 11h 15h 7h 9h 13h 7h 11h 15h 7h 9h 13h 7h 11h 15h Ly t erlen A13, A14, A15 A13, A14, A15 A13, A14, A15 A10, A11, A12 A10, A11, A12 A10, A11, A12 A7, A8, A9 A7, A8, A9 A7, A8, A9 A4, A5, A6 A4, A5, A6 A4, A5, A6 A1, A2, A3 A1, A2, A3 A1, A2, A3 Lng ly

5 ml

Ly 5ml t cc erlen A pha long o OD =600nm (canh sao cho OD nm trong khong 0,15 n 0,45) v m s t bo bng bung m hng cu lp ng chun v ng cong sinh trng. Nhn ging chun b cho qu trnh ln men thu - amylase: s dng erlen B2 cy ging trn

nh tnh enzyme - amylase bng sc k bn mng Sn phm ca hot ng xc tc ca enzyme -amylase c xc nh bng cc to nhng chm trn bn mng silica gel hot ha 80oC trong 30 pht Sau nhng vo butanol: ethanol: nc (50:30:20), kh qua m 32oC. ng n s c quan st nh dung mi nitrate bc acetone (0,1 ml AgNO3 bo ha trong 20 ml acetone) 3. Giai on nui cy thu enzyme a. Chun b - 3 x erlen 100ml cha 25 ml mi trng, sau cy 1ml ging B.Subtilis CM3 t erlen B2 b. Tin hnh - Tin hnh nui cy lc, vn tc my lc 150 rpm. Lu lng khng kh c duy tr mc 1vvm (th tch kh/th tch mi trng/pht). - iu chnh cc thng s vt l ca mi trng trong sut qu trnh tin hnh nui cy: pH 6.8, nhit 600C,thi gian 38h B sung cc cht hot ng b mt (0.02%):Tween 20,Tween 40,Tween 80 v Sodium lauryl sulphate (SLS) lm tng s sn xut enzyme. - Thc hin trn 3 erlen ly trung bnh - Sau khi kt thc qu trnh (sau 38h) , cc enzyme ngoi bo s c thu v ly tm 8000 x g trong 15 pht 40C - Qa trnh sn xut enzyme v s pht trin ca vi sinh vt s c theo di trong my -lc nhit 600C di tc lc l 150 rpm. - S pht trin ca B.Subtilis: c xc nh bng phng php quang hc-o cng quang hc ca mi trng bc sng quang ph UV-Vis l 600nm Kho st hot tnh - Amylase: Da trn s thay i cng mu xanh ca phn ng thu phn tinh bt ca enzyme -amylase theo chiu hng gim dn Hn hp phn ng gm: 0,2ml enzyme (enzyme ngoi bo) 0,25ml h tinh bt 0,1% 0,5ml dd m phosphaste (0,1M; pH 6,8) Tt c c 500C trong 10 pht phn ng xy ra Thm 0,25ml HCl (0.1N) ngng phn ng v cho thm 0,25 ml I/KI (2%KI/0,2%I) thc hin phn ng mu. Sau ,o cng quang hc (OD) ca phn ng mu xanh bng quang ph UV-Vis bc sng 690nm 1 n v hot tnh enzyme c nh ngha l khi lng enzyme lm gim 0,01% mu xanh 50oC trong 1 pht mi 1 ml hn hp 4. Giai on bn tinh sch Phng php 1 Dch lc c li tm lc ly tm 8000x g trong 20 pht 4C loi b cc t bo. Phn dch ni c cho vo ammonia sulphate 50% 4C t trong chu nc ta protein. Protein kt ta c thu bng cch ly tm 8000x g

4C v ha tan vo mt th tch ti thiu ca cht m Phosphate (0,1M, pH=6). Dung dch enzyme c thm tch ngc 4C vi cng cht m trong 24h 4C c khuy lin tc v thc hin 3 ln, Sau dng phng php sc k trao i ion tch enzyme: Gi th dng l DEAE-cellulose. DEAE-cellulose dng bt kh c ha vo mt lng nc va to gel, cho gel va to vo ct (nhi ct gel) v c cn bng bng cht m (cng cht m ban u). Ta c c dung dch enzyme c thm tch (cht thm tch qua mng bn thm) bng my c quay chn khng (Rotary Evaporator) 50C v cho chy t t vo ct nhi DEAE-cellulose vi tc khong 0,6ml/pht , sau ta ra chit bng 200ml gradient NaCl (0-0,1M). Cc phn on 10ml c thu, trong gm protein khng hot ng v enzyme hot tnh. Cc phn on c tp trung v c c li bng my c quay chn khng 50C thu c enzyme bn tinh khit, enzyme thu c c in di SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide) xc nh khi lng phn t.

Phng php 2: Thu dch ln men ri em li tm 5000 vng/ pht trong 20 pht 4C Thu dch sau li tm l ch phm enzyme th Bo qun ch phm enzyme th 4oC trong t lnh Ly cn t t lnh t t vo dch lc enzyme lm lnh, khuy rt nh cn ho u vi dch enzyme. Sau , hn hp ny vo t lnh. Lng cn dng kt ta enzyme, dng 2 lt cn cho 1 lt enzyme (thng gp 2 - 2,5 ln lng dch enzyme). Sau 15 - 24 gi, hn hp s phn thnh 2 lp. em ly tm 5000 vng/ pht hoc lc thu protein - enzyme dng ta. Protein - enzyme dng ta ny cn cha nhiu nc. Nc tn ti trong s lm gim hot tnh enzyme. Do , cn phi loi nc bng cch sy kh nhit 30C40C. in di SDS - PAGE S dng gel polyacrylamide 12% Protein c nhum bng Coomassie Brilliant Blue 0,2% Khi lng phn t ca amylase bn tinh sch c c on da trn phn t protein nh du chun bit trng lng (14,3 97,4 kDa) IV. LIT K CNG TC TH NGHIM

Thi gian 21/2 8h 8h30 9h30 11h 11h30 24/2 25/2

Cng vic

Ngi thc hin

-Nhn dng c,kim tra -Pha mi trng,gi dng c -Hp,dn v sinh -Sau khi hp, thch nghing -Ct mi trng -Cy chuyn, cy ria phn lp -Quan st hnh thi. Nu nhim, cy tr ging thun. -Nhn ging cp 1 Cy ging vo A1, A2, A3 Cy ging vo A4, A5, A6 Cy ging vo A7, A8, A9 Cy ging vo A10, A11, A12 Cy ging vo A13, A14, A15 -Ly mu, pha long, o OD -m s t bo bng bung m hng cu -Dn dp v sinh -Ly mu, pha long, o OD -m s t bo bng bung m hng cu -Dn dp v sinh Nui cy ging -Chun b -Cy ging (dung pippet 2ml v trng) -iu chnh cc thng s vt l trongqu trnh nui cy -B sung cht ph tr -,lc theo iu kin -Dn v sinh -Chun b -Ly tm,thu nhn enzyme th theo iu kin -Tr enzyme trong t lnh Kho st hot tnh enzyme th -Chun b -Phn ng thu phn tinh bt ca enzyme -amylase th -Phn ng mu ca tinh bt vi dd I/KI -o OD -Dn dp v sinh -Chun b

26/2 7h 17h 27/2 7h 17h 28/2 7h 7h-11h30

13h-16h

5/3 16h30 17h-17h30

7/3 6h30 7h-9h

9h-9h30 9h30-11h

11h-11h30 14/3 8h

8h30 15/3 8h 8h30-11h

-Ta enzyme bng cn trong t lnh - enzyme cha cn trong t lnh -Chun b -Ly tm hoc lc -Sy kh -Bo qun -Dn dp v sinh

11h-11h30