GVHD:Ts V NGC BI NHM 3

A. ENZYME PROTEASE: I. TNG QUAN V ENZYME PROTEASE II. NGUN THU NHN ENZYME PROTEASE III. CC YU T NH HNG N HOT ENZYME IV. NG DNG CHUNG CA ENZYME PROTEASE B. NG DNG C TH (BROMELIN)

I.TNG QUAN V ENZYME PROTEASE:

- Nhm enzyme protease xc tc qu trnh thy phn lin kt peptide (-CO-NH)n trong phn t protein, polypeptide n sn phm thy phn cui cng l cc acid amin. Ngoi ra, nhiu protease cng c kh nng thy phn lin kt este v vn chuyn amine

M HNH ENZYME PROTEASE THY PHN PHN T PROTEIN

Phn loi protease

Cn c vo cc tiu ch: Da vo c ch phn ng ca enzyme tham gia. Da vo pH ti thch cho hot ng ca enzyme nh: protease acid, protease kim, protease trung tnh. Tnh c hiu c cht ca enzyme : endopeptidase v exopeptidase Ngun thu nhn enzyme:enzyme t ng vt,enzyme t thc vt,enzyme t vi sinh vt.

Phn loi protease:

Peptidase (Protease) (E.C.3.4)

Exopeptidase (E.C. 3.4.11-17)

Endopeptidase (E.C. 3.4.21-99)

Serine proteinase
Aminopeptidase Cystein proteinase Aspartic proteinase Metallo proteinase

Carboxypeptidase

 Da vo vi tr tc ng trn mch polypeptide, exopeptidase c phn chia thnh 2 loi: Aminopeptidase: xc tc thy phn lin kt peptide u N t do ca chui polypeptide gii phng ra mt amino acid, cc polypeptide.

Carboxypeptidase: xc tc thy phn lin kt peptide u C ca chui polypeptide gii phng ra mt amino acid hoc cc polypeptide.

 Da vo ng hoc ca c ch xc tc, endopeptidase c chia thnh 4 nhm:

Serin proteinase: l nhng proteinase cha nhm OH ca gc Serine trong trung tm hot ng (TTH) v c vai tro c bit quan trong i vi hot ng xc tc ca enzyme. Gm 2 nhm nh l chymotrypsin v subtilicin .

Cystein proteinase: cc proteinase cha nhm SH trong TTH bao gm: protease thc vt (papain, bromelin,ficin), mt vi protease ng vt. Cc enzyme ny thng hot ng vng pH trung tnh.

Aspartic protease: l 1 loi protease c cha carboxyl trong TTH, thng hot ng pH acid. Metallo protease: l nhm proteinase c tm thy vi khun, cc vi sinh vt bc cao, hot ng vng pH trung tnh v hot gim mnh di tc dng ca EDTA Ngoi ra, protease c phn loi mt cch n gin thnh 3 nhm: - Protease acid: pH 2-4 - Protease trung tnh: pH 7-8 - Protease kim: pH 9-11

II.NGUN THU NHN ENZYME PROTEASE:

- Ngun ng vt Ty tng (ngun lu i): Tripsin
D dy b: Renin bin i casein

thnh paracasein c kh nng kt ta trong mi trng sa c nng Ca2+ -Ngun thc vt: + Bromelain: thu t qu, chi da, v da (Pineapple plant) +Papain: thu t nha ca l, thn, qu u ( Carica papaya) +Ficin: thu t nha thn cy co ( Ficus carica )

-Ngun vi sinh vt : +vi khun: lng protease c sn xut 59% lng enzyme s dng Bacillus subtilis, B.mesentericus, B.thermorpoteoliticus v mt s ging thuc chi Clostridium +nm: Aspergillus oryzase, A.terricola, A.fumigatus, A.saitoi +x khun: Streptomyces grieus, S.fradiae, S. Trerimosus

III.CC YU T NH HNG N HOT ENZYME:

nh hng ca nng enzyme nh hng ca nng c cht nh hng ca cc cht km hm nh hng ca cc cht hot ha nh hng ca nhit nh hng ca nng mui Ngoi cc yu t trn hot enzyme cn ph thuc vo nhiu yu t khc nh: nh sng (c bit l tia t ngoi), sng siu m, tia bc x

IV.NG DNG CHUNG CA PROTEASE:

Trong cng nghip thc phm: Trong cng nghip ch bin thit: protease thy phn mt phn protein trong thit, lm cho thit c mt mm thch hp v tng hng vi cho thit. - Cc enzyme thng dng + Protease thc vt: papain, bromelain, ficin + Protease vi sinh vt: t nm mc, x khun Sn xut dich m: t Streptomyces fradiae tch c ch phm keratineza thy phn c keratin rt c gi tri sn xut dich m t da, lng v

Trong cng nghip sa: protease c dng sn xut pho mat nh hot tnh lm ng t sa ca chng. - VD: renine, pepsine, mt s protease vi sinh vt: A.candidus, P.roquerti, P.mesentericus Trong cng nghip sn xut bnh: ch dng prorease giai on cc ngn amino acid v peptide t do phn ng vi mt s thnh phn ca bt lm gim thi gian o trn, tng do v lm nhuyn bt, to xp v n tt hn. Trong cng nghip sn xut bia: dng protease thy phn c gii hn protein ca bia to thnh cc peptid c trong lng phn t trung bnh to bot cho bia v sau khi thanh trng khng to kt ta. - VD: Protease ca A.oryzae dng thy phn protein trong ht ng cc to iu kin x l bia tt hn

Trong cng nghip da: protease c s dng lm mm da nh s thu phn s b lng, tch cc m lin kt pha ngoi ra, ch con lp collagen to lp mng v mm da. - VD: + Protease c tch t vi khun: P.mesentevicus, P.suptilis. + T nm mc: A.oryzae, A.flavus + T x khun: S.fradiae, S.griseus, S.rimosus Trong cng nghip dt: y l protease c s dng x l bn ngoi cc si t, lm nhim v kt dnh cc si t. Protease c tc dng thy phn lp protein serisin lm dnh bt cc si t t nhin, lm bong v tch ri cc loi t tm do lm gim lng ha cht ty trng

Trong ch bin thy sn: tng lng nc mm nh thy phn protein thnh cc acid amin lm tng hiu sut thu hi m ca nc mm.

Mt s ng dng khc: iu ch dich m thy phn lm cht dinh dng, cht tng vi trong thc phm v sn xut mt s thc n king. Protease ca nm mc v vi khun phi hp vi amilase to thnh hn hp enzyme dng lm sc n gia sc, c ngha ln trong chn nui gia sc v gia cm. Sn xut keo ng vt, cht git tng hp git ty cc cht bn protein, sn xut m phm S dng nhiu enzyme protease sn xut thuc h tr tiu ha, nu cao ng vt, cha bnh nghn mch mu, tiu vim vt thng.

I. Tng quan v enzyme bromelain

1. c im ca enzyme bromelain:
Bromelain l tn goi chung cho nhm enzyme thc vt c cha nhm SH, c kh nng phn gii protein c thu nhn t ho Bromeliaceae , c bit l cy da (thn, chi, tri, v). Bromelain chim 50% protein trong qu da. C kh nng thy phn mnh v hot ng tt pH = 6-8 Trong lng phn t khong 33000 Da Trong dich chit c cha mt t peroxydase, photphatase acid v cht cn protease

2. Cu to ha hoc:
Bromelain l mt glycoprotein, mi phn t c glycan gm 3 manose, 2 glucosamine, 1 xylose v 1 fructose. Si hydrate carbon ny lin kt hon vi vi si polypeptide Bromelain c thnh phn amino acid thay i trong khong 321-144 amino acid (thn), 283-161 amino acid (qu). Bromelain thn l mt si polypeptide c amino acid u amin l valine v u carbonhydrate l glycine. Bromelain qu c amino acid u amin la alanine

3. Cu trc khng gian:

Cu trc bc 1: Ser-Val-Lys-Asn-Gln-Asn-Pro-Cys-Gly-Ala-Cys-Tryp-Gly-Cys-Lys Trong trung tm hot ng c cha cysteine v 2 si polypeptide lin kt vi nhau bng cu ni -S-S-. Phn t dng hnh cu. Trong phn t c cha nhm sulfhydryl , 5 cu ni S-S- v cc ion Zn2+ c vai tro trong duy tr cu trc khng gian ca enzyme.

4. Tnh cht vt l:*: Tnh bng phng php sa lng khuch tn

**:Tnh t hng s sa lng v nht bn trong ***:Tnh bng phng php Archibald Hng s sa lng
Hng s khuch tn Th tch ring phn nht bn trong Ch s ma st im ng in S hp thu S D V [I] f/fo pI A1% cm 280nm 2,73S 7.77x10-7cm2/s 0,743ml/g 0,038dl/g 1,26 9,55 20,1

Trong lng phn t

33.200* 32.100** 35.500***

II. Phng php thu nhn bromelin:

Quy trnh cng ngh
Chi, thn, l da

Xay nhuyn

Loc

Ly tm 1

Kt ta

Hp ph

Siu loc

Ly tm 2

Dich

Sy

bromelin

1.Thu nhn Bromelin t da:

a. Ph v t bo: Qu thn da c xay nhuyn ph v t bo m da Ta c th dng phng php ng ha va ph v t bo va gim nht ca dich chit

b. Thu dich enzyme th:

Qu da (chi ) c xay nhuyn, vt k, loc b b, thu dich loc ly tm dich loc vi tc 6000 vng/pht trong 10 pht loi b cht x, thu c dich chit c cha bromelain.

c. Tch enzyme:
Cc phng php: - Phng php kt ta: Ta bng acetone: thm 1 th tch acetone lnh hoc 20% acetone lnh vo mt th tch dd nc da sau ly tm, trong 1 gi to 0-4oC. Ly tm hn hp 6000 vng /pht, trong 5 pht, loi b ta. Thm 2 th tch acetone lnh, 1h 0-4oC, ly tm thu ta v ra ta bng acetone lnh.

Ta bng ethanol: lm lnh dd nc da sau ly tm, cho ethanol 96o theo t l 4 da : 1 cn, trn u to 0oC t 3-4h. Ly tm hn hp 6000 vong/pht , trong 5 pht. Ra ta bng acetone v thu nhn ta. Ta bng ammonium sulphate: chun bi 1l dd nc da sau ly tm, cho t t 532g amonium sulphate vo khuy u. yn nhit phng 10-15, ly tm 6000 vong/pht trong 5 pht thu nhn ta

- Phng php hp th : C th dng kaolin/betonite hp ph. Cu to kaolin: cu to thnh tng lp, phn tn cao nn tnh hp ph tt, c kh nng to hnh kt dnh, kch thc < 1m, trn b mt c cha nhiu ion OHv O2Tin hnh: kaolin kh ( hoc ngm cho trng n) + dd nc da, t k 25mg kaolin/ ml dd nc da. Khuy u bng my khuy t, ri ly tm thu ta ( l bromelin-kaolin)

2.Tinh sch bromelain bng sc k loc gel:

a. Nguyn tc: - K thut sc k loc gel dng tch nhng phn t c kch thc , trong lng phn t khc nhau bng cch cho chng i qua ct gel. - Nhng phn t c kch thc nh lot vo bn trong l gel s bi tr hon v di chuyn chm qua ct.

 Trong khi nhng phn t ln hn di chuyn bn ngoi cc ht gel nn s di chuyn nhanh v c thot khi ct sm hn cc phn t nh

b. Chun bi ho cht v dng c:

Dng c v thit bi: - H thng sc k ct p sut thp, hng Bio-Rad, M - Phu gel - Bnh ht chn khng - Flow Adaptor 1,5 cm - Ct sc k Bio-Rad c kch thc 30x1,5cm - Bnh ng dd m - ng nghim 50 ci - Voi ht chn khng kh bot kh cho cc dd

 Ha cht
- Gel Bio-gel P-100, hng Bio-Rad c c tnh: dng ht min, kch thc ht 45-90m; kh nng ngm nc: 12ml/1g gel kh; phm vi phn tch: 5000-100000 Daltons - m phosphate 0,1M vi pH=7 kh bot trc khi dng

c. Tin hnh
Chun bi gel: - Cn 5g gel Bio-gel P-100 kh, cho bt gel t t vo dd m phosphate 0,1M ng trong cc. - i vi cc gel t Bio-gel P-30 n Bio-gel P-100 cn 12h 20oC hydrate ha hoc 4h nu bt u 100oC. Sau khi th huyn ph ng nht ca cc ht gel hnh thnh khng cn phi khuy n inh trong sut qu trnh hydrate ha

- Sau khi hydrate ha hon ton, gn lp ni trn b mt. Chuyn dd vo bnh ht chn khng kh kh ca dd t 10-15, thnh thong lc nh bnh, khng dng a khuy v c th lm h gel - Thm dd m kh kh vi th tchs gp 2 ln th tch lp nn, v lc nh. n inh cho n khi 90-95% s ht n inh. Gn hoc loi lp ni trn b mt bng cch ht loi cc ht min. Lp li 4 ln loi hn 90% ht min cn tr qu trnh loc gel

- Gn phu, gel vo ct, ng l thot ca ct v cho dd m lm y 20% th tch ct - Rt u dd gel vo ct thnh mt dong di chuyn nh, trnh bn gel m bo vic nhi ct u v trnh bi bot kh - Khi lp nn hnh thnh trong ct t 2-5cm, m kha u ra ca ct cho n khi ct c np y gel - Khi ct np y gel, kha u ra ca ct v gn Flow Adaptor. M kha u ra ca ct ri v cho dd m vi th tch gp 2 ln th tch lp nn - ng u ra ca ct v iu chnh Flow Adaptor xung lp nn gel. Np mu vo trn b mt lp nn bng bm hoc tim mu qua Flow Adaptor.

Chun bi mu
- Mu chy sc k phi sch ( khng bi nhim bn v khng c cc ht rn), hoa tan hon ton trong dd m. - Loc mu qua milipore (mng loc 0,4m) s lm gia tng bn/ thi gian s dng ca ct. Nu v tnh cht ca mu m khng loc c th em ly tm mu

Thu v xc inh mu tch c

- Dich ra khi ct c o d hp th 280nm bng Detector trong h sc k v c th hin hp th di dng sc bng phn mm LP Dataview trn my tnh. - Dich c thu t ng bng my thu mu (Collector), mi phn on 2ml

3. Hot tnh ca bromeline:

C 3 hot tnh khc nhau: peptidase, amidase v esterase. Bromeline thn c nhiu c cht t nhin v c th thy phn c c cht t nhin, c cht tng hp. Kh nng phn gii cc c cht t nhin ca bromeline: C cht Hot tnh phn gii casein (UI/mg)
Bromeline thn Bromeline qu xanh
4,0

Bromeline qa chn
3,0

Casein

7,4

 Kh nng phn gii cc c cht nhn to ca

Bromeline:
Hot tnh phn gii Benzoyl-L-Arginine amide(BAA) ca bromeline:

C cht

Hot tnh phn gii (UI/mg)

Bromeline thn
Bromeline qu xanh 9,1 Bromelin qu chn

BAA

3,7

7,2

Hng s Michaelis vi cc c cht tng hp khc nhau * *: iu kin to=5oC, pH=6,0

BAA BAEE BAEM

Bromeline thn

1,2x10-3

1,7x10-1

3,2x10-2

4.CC YU T NH HNG N HOT ENZYME:

nh hng ca nng enzyme nh hng ca nng c cht nh hng ca cc cht km hm nh hng ca cc cht hot ha nh hng ca nhit nh hng ca nng mui nh hng ca pH Ngoi cc yu t trn hot enzyme cn ph thuc vo nhiu yu t khc nh: nh sng (c bit l tia t ngoi), sng siu m, tia bc x Do n nh hng ti tc phn ng enzyme

nh hng ca nng enzyme:

Khi nng enzyme thp, c cht cao th tc phn ng h thuc tuyn tnh vo nng enzyme Khi nng enzyme tng th tc phn ng thy phn tng ti mt gii hn nht inh th khng tng na

nh hng ca nng c cht:

Nng c cht tng th tc phn ng tng nhng ch tng ti mt mc gii hn nht inh. Gi s phn ng ch c mt c cht S,Enzyme E xc tc chuyn ha S thnh P th xy ra phn ng : E + S V1,K1 ES V3,K3 E+P
V2,K2

 Phng trnh ph thuc gia vn tc phn ng v nng c cht: - phng trnh michaelis - menten
V =
Vmax * [S]

Trong : Km : Hng s michaelis - menten. - Phng trnh lineaweaver v burk: 1 Km 1 1 + V = Vmax [S] Vmax Phng trnh ny l phng trnh n gin c chuyn t phng trnh michaelis - menten

Km + [S]

 Hemoglobin: kh nng phn gii ca

bromeline mnh hn papain 4 ln Casein: hot tnh ca bromeline tng t nh papain BAA, BAEE: kh nng thy phn ca bromeline yu hn papain

So snh hot tnh mt s loi protease trn c cht Azocasein

A280nm
0.6
1 0.4 2 0.2
1. Pronaza 2. Bromelain 3. Chymotripsin

3
0 0.01 0.02 0,03 0.04 mg enzyme

 nh hng bi nhit :
Nhit nh hng n hot tnh enzyme, nng enzyme, nng c cht, dng tn ti ca enzyme VD: + dich chit qu (pH=3,5), khi tng nhit ln 600C th bromelin vn con hot tnh. + 5oC, pH=4-10 th enzyme gi hot tnh ti a trn casein trong vong 24h. + 55oC, pH=6,1 th enzyme bi mt 50% hot tnh trong vng 20 pht. + Qu trnh ng kh lm mt 27% hot tnh.

 nh hng ca pH: pH l yu t quan trong nht nh hng n hot tnh xc tc ca enzyme. pH ti thch ca bromeline khng n inh m ty thuc vo nhit , thi gian phn ng, bn cht v nng c cht, tinh sch ca enzyme, bn cht ca dung dich m, s hin din ca cht tng hot. VD: Enzyme bromeline tinh sch ch con li 6070% hot tnh Bromeline c bin pH rng 3-10, nhng pH ti thch ca enzyme l t 5-8 ty thuc vo c cht.

S nh hng ca pH i vi hot tnh ca ch phm bromelain:

Hot tnh (%) 100 .................................................................................. .................................................................................. 80

60
40 2 0 5

2
1

1-Casein 2-Azocasein

10

pH

Kh nng thy phn ca bromelain vi cc loi protease khc:

A280-366nm
1.6
1 1.2 2 0.8 0.4
1-Casein 2-Azocesein 3-Albumin

3
0

30

60

90

120

Thi gian(pht)

nh hng ca cc cht km hm:

Cht km hm l nhng ion,phn t v c,hu clm gim hoc mt hot tnh ca enzyme C 2 loi cht km hm: - Cht km hm cnh tranh: l nhng cht km hm thun nghich enzyme,c cu trc tng t cu trc c cht,nn c kh nng cnh tranh km hm vi c cht trung tm hot ng ca enzyme - Cht km hm khng cnh tranh:lm thay i cu trc khng gian ca phn t enzyme theo hng khng c li cho hot tnh xc tc ca enzyme.

nh hng ca cc cht hot ha:

L nhng cht anion,cc ion kim loi ,cht hu cc tc dng lm tng hot tnh xc tc ca enzyme trong gii hn nng xc inh

Bng 1 : nh hng ca cht hot ha n bromelin

Tn ho cht
L. xystein Dithiothreitol

Nng
1.10-2 1.10-2

Hot tnh ca bromelin

100 95.5

KCN
Thioglycolic axit

1.10-2
1.10-3

96
99

Bng 2: nh hng ca mt s cht c ch hot ng ca bromelin Tn cht c ch Khng c cht c ch N-chloromercuribenzoat HgCl2 Iodoaxetat Nng _ 4.10-2 1,44.10-2 2.10-2 Hot tnh ca bromelin 100 16 0 0

 nh hng ca cc ion kim loi:

Cc ion kim loi gn vo cc trung tm hot ng ca enzyme, lm thay i hot tnh enzyme. Mui thy ngn c nh hng quan trong n hot tnh ca enzyme v mc km hm thay i theo nng ca mui Ngoi ra con c nhng cht c tc ng c ch bromelin do chng kt hp vi nhm S-H- ca trung tm phn ng ca enzyme.

 nh hng bi trng thi v iu kin bo qun:

Hot tnh (UI/mg protein) Dich chit Nguyn liu ti Bo qun 4oC trong 5 ngy Sy kh 4oC 1.600 830 1.888 ng kh 1.150 630 1.360

 Mt s cht c ch hot ng bromelain

Tn cht c ch Khng c cht c ch
N-chloromercuribenzoat

Nng (M)
-

Hot tnh bromelain

100

4.10-2

16

HgCl2

1,44.10-2

Iodoaxetat

2.10-2

3.Xc inh hm lng protein theo Lowry

a.Nguyn tc: -cc protein u cha 2 amino acid: Tyrosin v Tryptophan, hm lng ca nhng a.a ny ty thuc vo loi protein. Vy nhng protein cng mt loi vi nhau s cha hm lng amino acid ny nh nhau - Protein tc dng vi thuc th folin to thnh phc cht c mu. Mu ny t l vi hm lng Tyrosin v Tryptophan (hm lng protein). V th dng phng php so mu xc inh hm lng protein.

b. Ha cht: Na2CO3 , NaOH 0,1% Citrate natri 1% Albumin tinh khit Thuc th folin Dung dich Albumin 0,1%: cn 0,1g Albumin pha long vi nc ct thnh 100ml dung dich Dung dich A: cn 2g Na2CO3 ha tan trong NaOH 0,1N thnh 100ml Dung dich B:cn 0,5g CuSO4.5H2O ha tan trong citrate natri 1% thnh 100ml Dung dich C:pha hn hp dung dich A v B theo t l 49:1 (ch pha dng ngay trong ngy)

c. Cc bc tin hnh:
dng ng chun Albumin: - Pha dung dich Albumin cc nng :0, 100, 200, 300, 400, 500, 600, 700 g/ml theo bng:
S ng nghim Hm lng protein g/ml Dung dich Albumin 0,1% Nc ct ml
0 1 2 3 4 5 6 7

100

200

300

400

500

600

700

10

 Ht 0,5ml dung dich albumin 0,1% c cc nng 0, 100, 200, 300, 400, 500, 600, 700 g/ml cho vo cc ng nghim sch sy kh v nh s th t sn Thm vo 2ml dung dich C, lc u v yn nhit phong trong 10 pht Thm tip 0,2ml thuc th folin, lc u trong 5-10 pht, thm nc ct cho 5ml em o hp th bc sng 550nm V biu biu din s bin thin ca mt quang (OD) theo hm lng protein chun g/ml

xc inh hm lng protein trong mu enzyme protease:

- Ht 0,5ml dd enzyme protease (Bromelin trch t da dng th c pha long trong m phosphate (pH=7) 0,1M cho vo ng nghim sch sy kh - Thm vo 2ml dd C, lc u v yn nhit phng trong 10 pht - Thm tip 0,2 ml thuc th folin, lc u trong 5-10 pht, thm nc ct cho 5ml - em o hp th bc sng 550nm

tnh ton: T ng chun, so snh mt quang cha mu ca ng nghim protein, suy ra hm lng protein ca 0,5ml dd enzyme protease pha long. T tnh c: - Hm lng protein c trong 1ml dd enzyme pha long (mg protein/ml dd enzyme protease pha long) - Hm lng protein c trong 1g enzyme protease ban u (mg protein/g enzyme protease ban u)

4.Xc nh hot Enzyme protease

Phng php Anson ci tin Nguyn tc : protease -Protein peptide +acid amin - inh lng sn phm to thnh bng phn ng mu vi thuc th Folin- Ciocalteau.Da vo thi chun inh lng tyrosin tng ng vi lng sn phm thy phn di tc dng ca Enzyme.Hot ng ca ch phm c biu din bng n vi hot enzyme protease.

-Mt n vi hot enzyme protease l lng enzyme trong 1 pht 30oC,pH thch hp c th thy phn protein (casein hoc Hb) to thnh cc sn phm hoa tan trong TCA (tricloacetic acid) v cho phn ng mu vi thuc th Folin-Ciocalteau tng ng vi 1 mol tyrozin . Cch pha dung dich tiu chun Tyrosin (1mol/1ml) -Cn chnh xc 18,12mg tyrosin tinh khit, kh hoa tan trong dd HCl 0,2N . -Cho vo bnh inh mc 100ml v thm vo HCl 0,2N vo cho ti vch inh mc. -T dung dich ny pha long tip tc bng dd HCl 0,2N nhn c dd c cha 0,01mol/ml n 0,05mol/ml.

Lp th tiu chun Tyrosin:

T dung dich pha long trn, ly 1ml cho vo ng nghim,thm 5ml dd Na2CO3 0,4M lc u, thm 1ml Folin pha long 5 ln. 30 nhit phong v so mu bng my so mu V thi chun: trc honh ghi s mol Tyrosin,trc tung ghi s oc trn my

Tin hnh
Ly 2 ng nghim (1 ng th nghim,1 ng kim tra). ng th nghim: 1ml enzyme protease (30oC),1ml dd c cht (30oC ) gi ng10 pht 30oC .cho ngay 5ml TCA 5%lc u ,gi 30 pht 30oC . Loc ,ly 1ml dich loc cho vo ng nghim khc .thm vo 5ml dd Na2CO3 0,4M lc u,thm 1ml thuc th folin (pha long 5 ln),lc u,gi 30 pht nhit phng. So mu trn my so mu UV-VIS bc sng 660nm

 ng kim tra:
Cho 2ml dd c cht (30oC), cho 5ml dd TCA5% lc u ri cho 1ml dd enzyme .cc giai on tip theo lm tng t ng th nghim. Tnh kt qu: Ly s liu oc trn my ca mu th nghim v mu kim tra.Da vo thi tiu chun tnh lng mol tng ng. S n vi hot (HP) ca 1ml dich enzyme c tnh:

Trong :
- T: thi gian enzyme tc dng vi c cht (10 pht) - X: s th tch ton b hn hp (7ml)

III. ng Dng
Trong cng ngh thc phm:
L cng c xc inh cu to,kh nng hoa tan v cc
tnh cht khc ca thit c. Bin i protein thit c thnh dich thy phn giu acid amin ph hp cho s tiu ha ca ngi hay lm thc n cho gia sc. Sn xut cc ch phm protein c.

Thy phn protein c:

Sn xut cc ch phm c m c t cc loi c r tin v nhng thnh phn khng ngon ca c. Sn xut dich thy phn protein c bng enzyme thc vt thm vo nc mm.

Lm mm tht
Mt phn bromelin c kh nng thy phn 1000 phn thit. Thy phn gan bo lm cao gan ,thuc b.

Trong y hc:
Lm tng h min dich gip cho ngi bi ung th gim c di cn.(liu dng 200 -300mg bromelin/kg kt hp vi x tri hoc ha tri) Nga cao huyt p,lot,gin tnh mch,ph phi,huyt khi. Cha au tim( do lm tan mu t gy au tim). Bi ln vt thng,vt bng lm tan cc m hoi t,chng t huyt,gim ph n. Cha ri lon tiu ha,gip tiu ha cht m tt,tng kh nng khng.

 Phi hp vi khng sinh trong iu tri cc bnh nhim trng. iu tri bong gn,cng c,au v nhc c. Bromelin trong da l hp cht thin nhin c kh nng ngn c ch protease HIV Sn xut cc sn phm chc nng c tc dng tng cng min dich v kh nng khng ca c th,tng kh nng phong mt s bnh.ng thi h tr trong iu tri cc bnh nan y,him ngho.

Dc phm c hot lc cao

Cc loi dc phm cha bromelin

Bt enzyme bromelin dng cho ngi n king

Bt enzyme bromelin b sung vo thc n cho gia sc(bo,nga )

Trong cc lnh vc khc

S dng bromelin thu nhn cht c ch protease:
Bromelin c s dng thu nhn cc cht c ch protein c cha nhm SH. Hin nay trong cng nghip v y t thng s dng rng ri cc cht c ch ny. Phng php thu c cht c ch protein dng tinh sch,cht c ch ny tc ng mnh ln enzyme nh papain,fixin.

Nguyn liu

m phosaphate (pH:4,6)

m phosphate pH:7,6-7,8

Nghin

Qua ct cha bromelin c inh

0,01 M NaOH c cha 0,1M NaCl pH11,512

Loc

Ly tm

Thu cht c ch

S dng trong qu trnh ng t sa: Bromelin ang c quan tm s dng vo mc ch ch bin cc sn phm t sa nh hot tnh ng t casein tng t rennin Enzyme bromelin gip cho s kt ta casein tt hn

 Trong phn t casein c nhiu nhm phosphate v nhng nhm k nc, cc phn t polyme c hnh thnh t casein rt c bit v bn. Nhng phn t ny c cu to t hng trm v hng nghn nhng phn t n l v hnh thnh nn dung dich keo, to nn mu trng ca sa. Nhng phc cht ny c goi l cc micelle casein.

 Phn a nc ca casein c cha nhm carbohydrate, nhm ny nh bn ngoi ca cc micelle phc hp, gip cc micelle bn vng, chng li s kt t. Casein v cc nhm carbonhydrate ca n r rng rt quan trong trong sn xut phomat. c s dng trong cng on u tin ca qu trnh sn xut phomat, enzyme bromelin s loi b carbonhydrate ca casein ra khi b mt ca micelle. Do cc micelle s mt i kh nng ha tan v lin kt vi nhau to thnh sa ng.

Kt lun
Protease mang li nhng ng dng to ln cho i sng con ngi:
Khc phc khuyt im t nhin ca nguyn liu. Nng cao gi tri thng phm ca nguyn liu. L cng c ca qu trnh chuyn ha trong dy chuyn ch bin thc phm.Tham gia vo cc giai on chuyn ha chnh,cc giai on hon thin sn phm.

S dng protease kt hp mt cch hp l v hiu qu vi cc phng php khc l cn thit c hiu sut cao,m rng quy m sn xut.

Nhm 3:
1. 2. 3. 4. 5. 6. 7. 8. 9. Phan Thi Huyn Trn_50131765 Nguyn Thi Tho Trang_50131697 Nguyn Thi ngoc_ Trinh Nh Ngoc Phan Thi Ngoc Yn Nguyn Anh Thi Nguyn Hong Thc Linh Nguyn Thy Anh Nguyn Vn Tin