H v tn Nguyn Cao Tr Nguyn Hong Minh Tu

MSSV 1015901 1015916 BO CO THC TP SINH HA C S

Bi 3: ENZYME BROMELAIN
I. NGUYN TC: - Bromelain l mt protease c nhiu trong tri thm. kho st hot tnh ca bromelain, ngi ta cho enzyme thy phn c cht protein l casein, sn phm to thnh l cc poptode ngn hay cc amino acid, trong c tyrosin. Sau dng phn ng mu vi thuc th Folin xc nh lng tyrosin, t xc nh hot tnh ca ezyme - Trong bi thc tp ny, chng ta s to mu dch chit nc thm cha enzyme, sau xc nh hot tnh bng cch tnh hot ca bromelain v so snh. II. TM TT QU TRNH: - u tin cn trch dch thm c cha enzyme bromelain bng cch vt ly nc thm v ly tm tc 6000 vng/pht trong 10 pht. - Sau chun b cc ng nghim, mi b mu 3 ng nghim v cho theo th t bng sau: ng Tyrosine Khng Dung dch casein 1,2% trong m phosphate (ml) 2,5 2,5 yn nhit phng trong 5 pht Dung dch trichloroacetid acid 5% (ml) 5 5 Dung dch HCl 0,2N (ml) 0,5 Dung dch tyrosine mu (ml) 0,5 Dch nc th mc cha enzyme bromelain (ml) Lc u, yn nhit phng trong 10 pht Dung dch tricloroacetid acid 5% (ml) Dch nc thm c cha enzyme bromelain (ml) 0,2 0,2 yn nhit phng khong 10 pht, sau em lc Dung dch NaOH 0,5N (ml) 5 5 Dung dch thuc th Folin (ml) 1 1 Lc k, yn trong 15 pht ri tin hnh o OD Mu 2,5

0,5 0,2 5

5 1

III. KT QU: - Tnh gi tr OD trung bnh ca mi l ng nghim bng cch ly gi tr OD trung bnh ca mi l ng nghim tr i gi tr OD trung bnh ca l ng nghim khng:

Kt qu: Tyrosin 2 0,505 0,492 0,232 Khng 2 0,250 0,260 0 Mu 2 0,369 0,379 0,119

ng nghim OD OD trung bnh OD trung bnh -

1 0,456

3 0,514

1 0,273

3 0,257

1 0,392

3 0,376

Tnh hot : Hot (UI/ml) enzyme bromelain c biu th l s micro gam tyrosin sinh ra do s thy phn casein ca enzyme c trong 1 ml dung dch (hay 1mg hn hp cha enzyme) trong 1 pht Hot c tnh theo cng thc:

T suy ra hot tnh ca enzyme:

Trong : - 450: s micro gam tyrosine c trong 1ml dung dch tyrosine - ODM: Gi tr OD trung bnh ca mu - ODT: Gi tr OD trung bnh ca mu tyrosine - x1: th tch dung dch tyrosine mu (0,5 ml). - x2: th tch dch cha enzyme bromelain (0,2 ml) - t: thi gian thy phn (10 pht) n v hot tnh UI ( /pht) = 1 tyrosine sinh ra trong 1 pht. IV. THO LUN V GII THCH: - Bromelain l nhm endoprotease, thnh phn c cha nhm sulfurhydryl, c kh nng phn ct cc lin kt peptid ni phn t protein thnh cc on nh l cc peptid v cc acid amin. Bromelain bao gm 3 hot tnh: peptidase, amidase v esterase. - C ch tc ng: + S tham gia ca nhm SH ca cysteine, nhm imidazole ca histidine v nhm disulfur trong hot ng thy phn ca bromelain. Nhm SH tham gia to acyl-thioester trung gian vi nhm carboxyl ca c cht ( v tr cc lin kt peptide b ct). Nhm imidazole lm cht trung gian nhn gc acid v chuyn cho nhm anion ca cht nhn khc. Cu ni S-S c vai tr duy tr cu trc khng gian ca bromelain. + u tin, bromelain kt hp vi protein v thy phn s b cho ra polypeptide v acid amin. Protein kt hp vi nhm SH ca enzyme khin n b ester ha ri nhm imidazole s kh ester gii phng enzyme, acid amin v peptide. + giai on u ion Zn2+ c vai tr quan trng ,chng kt hp vi nhm SH ca trung tm hot ng hnh thnh mercaptide phn ly yu, nh vy m nhm SH trong tm hot ng b ester ha bi c cht, cu trc khng gian c bo v n nh. - Trong th nghim, trichloroacetic acid (TCA) c dng kt ta hon ton lng casein v enzyme, xt th t cho TCA vo cc ng nghim ta thy ng Mu TCA c cho vo sau cng, mc ch l thi gian cho bromelain hot ng, ng Tyrosine v ng khng, ta kt ta casein ngay t u (TCA c cho vo sau khi cho casein) nn bc cui khi cho bromelain vo th enzyme cng khng th tc dng c vi c cht v lng enzyme ny cng b kt ta bi TCA - Dung dch HCl 5% c s dng nhm mc ch to mi trng thch hp cho bromelain hot ng, mi trng hi acid, pH khong 6-7. ng tyrosine khng thm HCl v ban u tyrosine c pha vi dung dch HCl - ng khng, theo l thuyt th tt c cc protein, enzyme u b kt ta bi TCA, nhng khi cho phn ng vi thuc th Folin th dung dch vn cho mu xanh, iu ny chng t trong ng vn c tyrosine. Nhng y tyrosine khng phi do qu trnh thy phn to ra m l do lng tyrosine t do (cc acid amin t do trong casein v dch nc thm) c trong dung dch, cc acid amin ny khng b kt ta bi TCA v chnh cc acid amin ny phn ng vi folin. V ta khng th kim sot c cc acid amin t do, nn ta lm ng khng khi tnh hot tnh, ta dng ng mu tr vi ng khng (ng khng c dng nh ng i chng), vic lm ny s lm gim sai s ca kt qu.

H v tn Nguyn Cao Tr Nguyn Hong Minh Tu

MSSV 1015901 1015916 BO CO THC TP SINH HA C S

Bi 4 : NH LNG VITAMIN C BNG IOD

I. NGUYN TC : Vitamin C (ascorbic acid) C6H8O6 cos 2 dng ng phn quang hc D v L. Dng D khng c hot tnh sinh hc. Bi ny kho st v L-ascorbic acid. Acid ny gp trong t nhin 2 dng oxi ha v kh. xc nh nhanh hm lng ca vitamin C trong nguyn liu (vin vitamin C) khi khng c cht mu, ngi ta s dng phng php chun iod. Tt c ascorbic acid b oxid ha bi iod. Phn iod cn tha s phn ng vi dung dch h tinh b cho mu xanh tm. Khi xut hin mu ny chng t phn ng kt thc.

II. TM TT QU TRNH : - Xc nh h s chun x : Chun 10 ml dung dch Na2S2O3 0,1N bng dung dch Iod 0,1N vi thuc th l h tinh bt 1% (dng 1ml). Lm li 2 ln v ly gi tr trung bnh. - Chun b mu : + Cn vin thuc vitamin C : Cn c vin, cn phn v vin thuc => khi lng mu (g) + Cho mu vo bnh nh mc 100ml ng sn 1 t dung dch HCl 2,5%, sau lc u tan ht mu, cho thm dung dch HCl 2,5% n vch - nh phn 20 ml dch chit c cha vitamin C bng dung dch Iod 0,1N vi thuc th l h tinh bt 1% (dng 1ml). Lm li thm 2 ln na v ly gi tr trung bnh. III. Kt qu : - S gam mu : 0,3919 0,0548 = 0,3371 g - H s iu chnh x : Ln 1 Th tch Iod 0,1N cn dng chun dch chit (ml) Gi tr trung bnh (ml) H s iu chnh - Th tch Iod (ml) cn chun : 10,2 10,25 1,025 Ln 2 10,3

Ln 1 Ln 2 Ln 3 Th tch Iod 0,1N cn dng chun 6,1 6,2 6,3 dch chit (ml) Gi tr trung bnh 6,2 Th tch dung dch Iod 0,1N cn thit 31,775 tc dng vi tng th tch dch chit S gam vitamin C c trong m gam mu 0,27962 S gam vitamin C c trong 100g mu 82,949 - Vi cc cng thc: + Th tch dung dich Iod 0,1N cn thit tc dng vi tng th tch dch chit vitamin C

+ S gam vitamin C c trong m gam mu vt:

+ S gam vitamin C c trong 100g mu vt:

Trong : IV. a: Th tch dung dch Iod 0,1N dng khi chun (ml) x: H s iu chnh ca dung dch Iod 0,1N V: Tng th tch ca dch chit vitamin C (100ml) v: Th tch dch chit Vitamin C ly chun (20ml) m: Khi lng mu vt (g) 8,8: 1ml dung dch Iod 0,1N tng ng vi 8,8mg ascorbic acid. THO LUN V GII THCH: Cn xc nh h s iu chnh v: Nng ca dung dch Iod d b thay i trong qu trnh bo qun, nn mi ln s dng dung dch ta cn phi xc nh li h s iu chnh xc nh c nng thc t ca dung dch, i vi bi thc tp ny dung dch chun l Na2S2O3. C th d on c th tch dung dch Iod cn dng l khong gn 10ml. C ch tc dng ca phn ng vitamin C v Iod: + Ta c Iod tng i t tan trong nc, nhng iu ny c th ci thin bng cch pha Iod v Iodua to thnh triiodua: I2 + I -> I3 + Sau triiodua oxid ha vitamin C to dehydroascorbic acid, khi vitamin C ht th iod d s phn ng vi tinh bt vo to phc xanh tm, vic chun kt thc
-

C6H8O6 + I3- + H2O -> C6H6O6 + 3I- + 2H+

(phn ng ascorbic acid b oxid ha bi Iod)