Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

LI M U
Ngy nay, cng vi s pht trin mnh m ca cng ngh sinh hc, cc ch phm enzyme c sn xut cng nhiu v c s dng trong hu ht trong cc lnh vc nh: ch bin thc phm, nng nghip, chn nui, y t Hng nm lng enzyme c sn xut trn th gii t khong trn 300.000 tn vi gi tr trn 500 triu USD, c phn phi trong cc lnh vc khc nhau. . Phn ln enzyme c sn xut quy m cng nghip u thuc loi enzyme n cu t, xc tc cho phn ng phn hy. Khong 75% ch phm l enzyme thy phn c s dng cho vic thy phn c cht t nhin . Protease l enzyme c s dng nhiu nht hin nay trong mt s ngnh sn xut nh: ch bin thc phm (ng t sa lm pho mat, lm mm tht, b sung lm tng cht lng sn phm trong sn xut bia, x l ph ph phm trong ch bin thc phm), sn xut cht ty ra, thuc da, y t, nng nghip Qua nhiu nm, vic gia tng s dng vi sinh vt nh l mt ngun cung cp protease ci thin ng k hiu qu sn xut v sn phm c to ra nhiu hn. Tuy nhin gi thnh ch phm protease cn kh cao, do cng

Cc phng php tinh sch enzyme

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

hn ch vic s dng rng ri enzyme trong san xut. Cc ch phm thu c sau qu trnh nui cy sn xut enzyme cha phi l ch phm c tinh khit cao v protein ch chim 20-30% .V vy, vic nghin cu ci tin phng php tch v tinh ch enzyme nhm thu c ch phm c tinh khit cao rt cn thit. tch v tinh ch enzyme ni ring v protein ni chung thng c mt lot phng php ha-l v ha hc khc nhau. C th chia lm nm nhm phng php sau: - Phng php bin tnh chn lc - Phng php kt ta phn an - Phng php hp th chn lc - Phng php sc k - Phng php tch h hai pha nc Cho n nay khng c phng php tch v lm sch chung cho cc enzyme. xy dng phng php tch v lm sch mt enzyme no d cn bit la chn v phi hp mt cch c hiu qu nht cc bin php tch v lm sch khc nhau.

PHN I: TNG QUAN

Cc phng php tinh sch enzyme 2

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

I/Khi nim enzyme - Enzyme l nhng protein c kh nng xc tc cho cc phn ng ho hc vi mc c hiu khc nhau nhit tng i thp. - Enzyme c trong tt c cc t bo sng, l cc cht xc tc sinh hc. - Enzyme c y cc tnh cht ca cht xc tc, nhng enzyme c hiu sut xc tc ln hn tt c cc cht xc tc hu c v v c khc.Enzyme khng nhng c th xc tc cho cc phn ng xy ra trong c th sng, m sau khi tch khi h thng sng, nhng iu kin nht nh chng vn gi c hot tnh xc tc. Nh vy,c th ni mt cch khi qut, enzyme l nhng phn t ln tn ti trong t nhin, hay c tng hp c kh nng xc tc cho mt hay nhiu phn ng vi mc c hiu khc nhau nhit v p sut bnh thng. II/Nhng tiu chun tinh sch ca enzyme xc nh xem protein c chit tch ra c phi l enzyme tinh khit khng hay cn cha nhng tp cht protein khng hat ng, ngi ta s dng mt s phng php ch yu da trn vic nghin cu nhng tnh cht vt l ca protein.

Cc phng php tinh sch enzyme

Gvhd :Ths ng Th Ngc Dung -

Ha Sinh Thc phm

Lm lng (kt ta) dung dch protein trong phn quay phn tch ca

my siu ly tm: pht hin mt nh ca protein th l protein thun nht. Tuy nhin mt s protein tuy khc nhau nhng c cng trng lng phn t v c hnh dng tiu phn ( hoc c khi c s b ph ca cc nh hng ca khi lng v hnh dng) th c hng s lng trng nhau. V th khi siu ly tm, chng c th lng vi tc nh nhau v cho mt nh chung.
-

in li ( phng php ranh gii di ng): phng php ny rt hiu

qu. Tuy nhin kh m phn bit c nhng protein ging nhau v tr s di ng in li. Dung dch enzyme khi c in li khu vc trong jela tinh bt hoc thch ch ra mt nh cn i th l du hiu ca enzyme thun nht. Th nhng khi hm lng nhng protein tp nh(<=1%) th kh pht hin ra ng. Xc nh hat ring, mt quang hc ring trong nhng nh

quang ph c trng cng hm lng cc nhm ngai c hiu.

-

Bin php kh nhy cm xc nh mc thun nht ca enzyme

( cng nh protein khc) l kt hp s phn tch in li v nhng phn ng min dch ha hc c hiu, c bit in di trn thch theo grabar.
-

Bin php nghin cu c trin vng nhng tng i t nhy v

thun nht ca protein l s xc nh ha tan ca n theo norchrop.

Cc phng php tinh sch enzyme 4

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Thng xt thun nht ca enzyme, ngi ta da trn s so snh nhng kt qu thu c t mt vi phng php khc nhau. c bit, nhng ch phm enzyme cng tinh khit th cng d hng v khi bo qun trong cc dung dch 40C u nhanh chng mt hot tnh. V th, khi ti kt tinh enzyme lp li ta thng thy c s gim hot ring v cc tinh th b nhim bi phn enzyme b c ch.

Cc phng php tinh sch enzyme

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

PHN II TCH CHIT& TINH SCH ENZYME

I. Cc iu cn lu khi tch chit v tinh sch enzyme C 3 kh khn trong khi tch enzyme khi t bo cn phi ht sc lu . - Enzyme c trong t bo sinh vt vi lng khng ln so vi cc thnh phn khc. - Enzyme l cht hu c khng bn, chng rt d b bin tnh khi b tc ng ca cc yu t bn ngoi. - Enzyme l prtin. Prtin enzyme lun lun i cng nhng loi prtin khng phi enzyme nhng li c tnh cht l ho rt ging prtin enzyme. Do , rt d mt hot tnh sinh hc khi b tch ra khi t bo v c th.Vic mt hot tnh c th do nhit cao, s thu phn bi cc enzyme proteolytic, tc dng ca pH thp, cc cht oxy ho hay cht kh, cc cht gy bin tnh,cc cht c ch bt thun nghchTuy nhin, cc enzyme khc nhau c th nhy cm vi nhng tc nhn nu trn cc mc rt khc nhau. Trong sn xut ch phm enzyme th vic gi c hot tnh ca enzyme l mt trong nhng yu cu hng u.Thng thng qua mi bc tinh sch

Cc phng php tinh sch enzyme

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

th mt phn enzyme cng nh hot ng ca ch phmb mt i, gi thnh cao ln do chiph v thit b, nguyn liu, cng sc v do mt hot . Vi enzyme dng trong mc ch cng nghip, khng phi lc no cng cn ph phm c tinh sch cao,trong mt s trng hp c th dng ph phm enzyme dng th. hn ch ti a s gim hot enzyme trong qu trnh tinh sch th thng cn chn dung dch chit rt enzyme thch hp, va cho hiu sut chit sut enzyme cao, va khng lm bin tnh enzyme. Cc bc tinh sch thng c tin hnh nhit thp ( khong 40C hoc thp hn, tu yu t gy kt ta) va hn ch s bin tnh enzyme cng nh tc dng phn gii ca cc enzyme proteolytic c mt trong dch chit.i vi nhiu enzyme,c th b sung cc cht c ch ca enzyme proteolytic vo dch chit hn ch s thu phn i vi cc enzyme quan tm. Cc enzyme proteolytic c phn thnh 4 lp l: + protease serine + protease cystein + protease axit + protease cha kim loi. Trn c s ca 4 lp protease ny, nhi ta cng cha cc cht c ch ca cc enzyme ny theo 4 lp tong ng. Tuy vy, do s ph bin ca 2 loi
Cc phng php tinh sch enzyme 7

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

protease serine v protease cystein, nn vic b sung vo dch chit protein enzyme cc cht c ch ca hai loi protease ny l ph bin. Trong mt s trng hp, i vi cc enzyme nhy cm vi cc phn ct proteolytic, th c 4 loi cht c ch u c s dng. B sung cc yu t lm bn ( nh CaCl 2, hay MgCl2 nng 2-5mM, glycerol 10-20%...) cht chng oxy ho, xy dng quy trnh tinh sch n gin t bc nht cng l nhng cch hn ch s gim hot ng ca enzyme. i vi mt s enzyme bn nhit th c th dng bc x l nhit ( 70-800C, trong vng 15-30 pht) ngay giai don u va hn ch tc dng phn ct proteolytic, va loi b mt s protein khng monh mun khc. Tng t c th dng bc x l axit 9 a pH xung 3-4) giai on u i vi cc enzyme bn pH thp. III. Thu nhn ch phm enzyme th Ngoi tr mt s trng hp enzyme quan tm l enzyme ngoi bo t vi sinh vt ( enzyme c tit vo mi trng trong qu trnh nui cy t bo) v cc enzyme ngoi bo t cc dch ca ng vt v thc vt, cc trng hp cn li, bc u tin trong nghin cu enzyme l phi thu nhn dch chit enzyme t nguyn liu. thu dch chit m hoc t bo cha enzyme, trc ht phi ph v t bo.

Cc phng php tinh sch enzyme

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

thu nhn enzyme ni bo, ngi ta phi ph v t bo bng nhiu phng php.Cc phng php ph v t bo thng c s dng ph v t bo nh: + Phng php c hc. Nghin bi, nghin c cht tr nghin nh bt thu tinh, ct thch anh, ng ho bng thit b ng ho + Phng php vt l nh dng sng siu m + Phng php ho hc nh dng cc loi dung mi, butylic, aceton, glycerol, ethylacetate. Sau khi ph v t bo, ngi ta thng s dng nc, dung dch m, dung dch mui trung tnh tch enzyme ra khi sinh khi x l theo nhng phng php trn. Dch chit thu c,ngi ta gi l ch phm enzyme th. S d gi l ch phm enzyme th v trong ch phm ny,ngoi enzyme ra cn c nc, protein khng phi enzyme cc thnh phn ca t bo. Ch phm thu c cc bc ban u ny thng cha rt nhiu protein v cc hp cht khng mong mun, nng enzyme quan tm thp. Dch chit c th c c c nhit thp lm tng nng enzyme, hoc c b sung tc nhn gy kt ta protein enzyme loi b mt s cht khng mong mun, sau ho tan enzyme trong mt th tch nh dung dch m thch hp.
Cc phng php tinh sch enzyme 9

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

kt ta protein cho mc ch tinh sch, ngi ta thng dng dung mi hu c nh ethanol hay aceton hoc mui trung tnh. Cc dung mi hu c khi thm vo dung dch nc s lm gim hng s in mi ca dung dch, do lm thay i tnh tan ca protein v dn n lm kt ta protein. Nng ca cc dung mi gy kt ta protein thng t 80% (V/V) tr ln. S kt ta c th c tnh chn lcmt phn, ngha l cc protein khc nhau c th c kt ta bng cc nng khc nhau ca dung mi. Dung mi hu c sau c th c loi bng cch cho bay hoi trong chn khng, hay thm ch trong hkng kh. kt ta enzyme t dch chit th, ngi ta cng c th dng mt s mui trung tnh, thng dng nht l ammonium sulfatese, do mui ny c mc ho tan rt cao trong nc, t lm mt hot tnh enzyme, thm ch cn c tc dng lm bn enzyme. Mt s im na ca mui ammonium sulfate l kh nng kt ta chn lc protein, do s loi b c nhiu protein khng mong mun trong dch chit. Lu khi s dng mui ammonium sulfate l phi b sung vo dung dch enzyme mt cch t t, kt hp khuy u trnh kh nng tng qu nhanh cc b ca nng mui, lm mt di tnh kt ta chn lc ca n, thm ch c th lm bin tnh mt s enzyme km bn. Hn ch ca phng php s dng ammonium sulfate chnh l mt nhiu thi gian, do nng ammonium sulfate cao nn t trng ca
Cc phng php tinh sch enzyme 10

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

dung mi ln, kt ta c cc protein cn phi ly tm tc cao v nhit thp, sau cn loi mui khi ch phm khi chuyn sang bc tinh sch tip theo. IV/Cc phng php tch chit v tinh sch enzyme *Khi nim Vic lm sch enzyme chnh l vic loi protin khng phi l enzyme, nc,cc thnh phn khc ca t bo khi enzyme. Cng vc ny hon ton khng d dng. Do , vic lm sch enzyme i hi ngi lm nghin cu v enzyme, ngoi s hiu bit v enzyme ra cn phi nm vng rt nhiu thao tc k thut khc nhau trnh gy mt tnh hot ng ca enzyme. Trong dch chit, ngoi enzyme cn cha cc protein tp v nhiu cht khc, loi b chng phi s dng phi hp nhiu bin php khc nhau. loi b mui v cc tp cht c phn t lng thp thng dng cc bin php thm tch i vi nc hay i vi cc dung dch m long hoc bng cch lc qua gel sephadex. loi b cc protein tp v cc tp cht c phn t lng cao khc, thng dng kt hp nhiu bin php khc nhau: phng php bin tnh chn lc nh tc dng ca nhit hoc pH ca mi trng, phng php kt ta phn on bng mui trung tnh hoc cc dung mi hu c, cc hpng php sc k( sc k hp ph, sc k trao i ion),in di, phng php lc gel.
Cc phng php tinh sch enzyme 11

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

1/Phng php bin tnh chn lc Phng php ny da trn s tc ng ca nhit , pH lm bin tnh cc loi protein tp. Phng php ny ch thch hp vi enzyme chu axt v bn nhit.Ngi ta a nhit ca dung dch enzyme ln 50-70 0C v pH <=5. iu kin ny nhng enzyme khng bn nhit v khng chu c pH thp s b bin tnh.Sau , bng phng php ly tm hoc phng php lc loi b cc thnh phn khng phi l enzyme m ta mong mun. 2/Phng php kt ta phn an Phng php kt ta phn on bng (NH4)2 SO4 da trn c s s khc nhau v kh nng kt ta ca cc protein enzyme mt nng mui (tnh theo phn % nng bo ha) xc nh c dng ph bin loi b bc u protein tp ca cc dch enzyme. Cc loi mui c th c dng l (NH4)2 SO4, Na2 SO4, MgSO4 ... ngi ta nhn thy mui (NH4)2 SO4 l tt nht v n khng lm hi m lm n nh (lm bn) hu ht cc loi enzyme. Loi mui ny li r v ph bin. ha tan ca n li rt ln (bo ha 767g/l 250C). Phng php ny ch dng vi nhng enzyme t b bin tnh bi cc dung mi c chn dng. gi cho enzyme khi b mt kh nng hot ng, qu trnh kt ta phi c thc hin nhit thp (-50C).

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

3/Phng php hp ph chn lc Ngi ta c th cho cht hp ph trc tip vo dung dch enzyme, hoc cho dung dch enzyme chy qua mt ct, trong ct c cha cht hp ph. Cht hp th c s dng rng ri l hyrocyapatite. Ngi ta c th thc hin mt trong hai cch sau: hoc l hp ph enzyme hoc l hp ph protein tp. trnh lm bin tnh enzyme, ngi ta thng thc hin qu trnh hp ph nhit 00C.Sau khi hp ph, ngi ta li tin hnh chit enzyme (qu trnh phn hp ph) bng cc dung mi thch hp. 4/Phng php sc k * Phng php sc k trao i ion Da trn c s ca phn ng trao i ion gia protein tan trong nc hoc dung dch m long v tc nhn trao i ion, ngi ta p dng phng php sc k trao i ion lm sch enzym. Ngi ta thng s dng cc tc nhn trao i ion sau: - Nha trao i ion
-

Dn

xut

ester

ca

cellulose

nh

carboxymethyl-cellulose,

diethylamino-ethyl-cellulose cellulose.

(DEAE-cellulose), triethylamino-ethyl-

Ngi ta thng s dng dietylaminoetylxelluloza (DEAE-cellulose) hn c. u im ca DEAE-cellulose l vi dch chit thch hp ngoi tp
Cc phng php tinh sch enzyme 13

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

cht protein cn c kh nng loi b cc tp cht axit nucleic.iu cho php khng cn dng n cc bin php nhm loi b axit nucleic kh v phc tp nh kt ta protamin hoc streptomixin sunfat thng c dng trc y. Sephadex l dn xut ca polysaccharide dextran.Trong cht ny, cc phn t ca chng thng lin kt vi nhau bi cc lin kt ngang nh tc dng ca epiclohydrin, tao thnh sng phn t. S lng lin kt ngang to thnh cng nhiu, kch thc ca l sng phn t cng nh.Khi tin hnh lc, cc cht phn t c khch thc nh s khuch tn vo bn trong cc ht sephadex c ngm trong dung dch m, cn cc phn t c kch thc ln hn khng c kh nng i vo cc ht s c chit nhanh ra khi ct. Vy c s ca phng php lc gel sephadex da vo s khc nhau v kch thc, hnh dng v phn t lng ca cc cht c trong hn hp.Phng php ny c p dng rng ri trong k thut enzyme. * Phng php sc k ct Dch chit enzyme c loi b phn ln cc protein tp nhng vn cha m bo ng nht cn thit. Do dch chit enzyme c tip tc lm sch bng phng php sc k ct. Phng php dng cht ry phn t (lc gel - gel filtration). m bo cho vic tch enzyme c tt, cht ry phn t phi l cht tr, khng phn
Cc phng php tinh sch enzyme 14

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

ng vi protein enzyme. Cht ny cng khng ha tan v tng i bn vi cc yu t v c hc cng nh sinh hc. Ngoi ra cht c s dng cho mc ch lc phn t phi l cht khng c tnh n hi (khng co) v phi l cht a nc (hydrophyl). 5/Phng php tch h hai pha nc. C s k thut ny da vo s phn b khc nhau ca hn hp trong dung dch hai pha khng ha tan vo nhau. Cc h hai pha c trng bng cch trn cc h dung mi vo nhau to thnh hai pha ring bit. H dung mi c s dng trong phng php ny c th l polymer/polymer nh: polyethylene glycol (PEG) v dextran hoc polymer/mui nh PEG v potassium phosphate, sodium sulphate, sodium phosphate,... Cc protein v mnh v t bo c kh nng ha tan khc nhau gia hai pha, v vy phng php ny c th c dng cho c hai trng hp: phn tch protein khi mnh v t bo v phn chia cc enzyme trong sut qu trnh tinh sch protein. H hai pha nc c xem l phng php tt cho vic phn tch v tinh sch cc hn hp, v phn tch cht lng c mt khc nhau d dng hn phn tch cc cht rn ra khi cc cht lng. So vi cc phng php tinh sch khc th h hai pha nc c mt s u im hn nh: c ha tan trong nc ca hai pha ln (70-80%), t tinh sch cao, hiu sut cao, d dng s dng quy m ln v c bit polymer c ti s dng.
Cc phng php tinh sch enzyme 15

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

S phn tch t hiu qu cao ty thuc vo cc thng s nh khi lng phn t v in tch ca i tng nghin cu, nng v khi lng phn t ca cc polymer, nhit , pH, thi gian, lc ion ca hn hp v s hin din ca cc loi mui a ha tr nh phosphaste hoc sulphate... 6/ Ngoi ra cn c cc phng php : - Phng php dng cht hp ph c hiu sinh hc - Phng php sc k i lc (affinity chromatography) Hin nay, cha c mt phng php chun no tht s c hiu qu trong vic lm sch enzyme.Do , vic lm sch ch tht s c hiu qu khi ta bit la chn cc phng php ring,kt hp vi nhau, to ra h thng phng php ni tip vi nhau mt cch hi ha nht.

Cc phng php tinh sch enzyme

16

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

V/Quy trnh sn xut 1/T ngun thc vt a.Enzyme bromel Bromelin l tn gi chung cho nhm enzyme thc vt cha nhm sulfhydryl, c kh nng phn gii protein c thu nhn t h bromeliaceae, c bit l cy da (thn v tri). Enzyme bromelin c th thu c t trong thn, trong phn tht qu v trong chi qu. Ty theo ngun thu nhn m ngi ta phn bit bromelin thn hay bromelin qu v bromelin thn l nhm c gi tr kinh t. mi b phn khc nhau trn cy da, bromelin c pH ti u khc nhau v c cu to khc nhau. Bromelin chim 50% protein trong qu da. N c kh nng thy phn kh mnh v hot ng tt pH t 6-8. Bromelin c hot tnh xc tc s phn gii protein tng t nh papain trong m u hay ficin trong cy thuc h Sung. Enzyme bromelin c trng lng phn t khong 33.000 DA, ln gp 1,5 ln so vi papain. Tht qu da ch c hot tnh enzyme bromelin khong t 3 thng trc khi chn, trong hot tnh bromelin cao nht l khong 20 ngy tc khi chn. Khi tri chn, hot tnh bromelin gim xung nhng khng mt hn.

Cc phng php tinh sch enzyme

17

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Bromelin cn c th thu c t trong thn da(trung bnh c th thu c 3,6 kg bromelin t 378l nc rt ra t thn cy da.

Cc phng php tinh sch enzyme

18

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Vic thu nhn v tinh sch bromelin c th thc hin theo s sau:
Qu/thn/chi da

Xay nhuyn, lc

Dch lc

Ly tm B Dch ly tm

Kt ta

Ch phm brommeli n

Sy kh

Tinh sch

Sn phm enzym tinh khit Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Thu dch enzyme th Qu da hoc thn c xoay nhuyn, vt k, lc b b v thu dch lc, ly tm dch lc vi tc 6.000 vng/pht trong 10 pht lai b cht x s thu c dch chit c cha bromelin. Cc phng php tch bromelin * Phng php kt ta enzyme i vi enzyme protein cng nh cc protein enzyme khc, c th thu c enzyme, iu cn thit l phi ph v lp nc lin kt xung quanh phn t ny bng cch b sung vo dung dch protein enzyme cc dung mi hoc cc ho cht a nc c i lc vi nc mnh hn protein li ko nc ra khi phn t protein , gip cho protein kt ta xung. Cc dung mi thng c s dng kt ta bromelin l acetone v ethanol, cn cc ha cht khc nh cc mui trung tnh nng cao cng c th kt ta c enzyme. Heinicker v Gortner dng acetone kt ta bromelin t thn v tri. Bromelin trong dung dch nc rt nhy cm vi cc dung mi hu c do s kt ta t hiu qu cao thng tin hnh qu trnh kt ta trong iu kin lnh. Nh vy, nc p t thn v tri da phi c lm lnh 0-40C v aceton tinh khit 200C.Ethanol cng c s dng kt ta protein v hn hp bromelin-ethanol cng phi gi iu kin nhit lnh. Sau khi
Cc phng php tinh sch enzyme 20

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

thu c kt ta, ta bromelin phi c ra bng aceton v lm kh tht nhanh bromelin khng b bin tnh. c th thu nhn enzyme, vic u tin l phi ph v t chc m c cha enzyme bng cc nghin m thu dch chit c cha bromelin ri sau dng cc tc nhn khc nhau kt ta bromelin. Nhng yu t chnh nh hng n hiu sut v cht lng ca ch phm enzyme l: - Nng ca ammonium sulfate. - pH ca dch chit.
-

Nhit kt ta.

- Thi gian tip xc ca tc nhn m v dch chit. * Phng php hp ph C th s dng kaolin hay betonite hp ph bromelin. Cch lm: cho kaolin kh ( hoc ngm trng n ) vo dung dch nc da sau ly tm vi t l 25 mg kaolin/ml dung dch nc da. Khuy u bng my khuy t, sau ly tm thu ta. Ta c gi l bromelinkaolin. * Phng php siu lc S dng mng FS 50PP c gi tr lai tr 30.000, din tch mng 336 cm2.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Dch da sau ly tm c cho qua siu lc, trong qu tr2inh ny thm nc ct vo tinh sch v thu c dung dch c c. Thu nhn ta bng cch s dng acetone hoc sy thng hoa dung dch c c. Thu nhn bromelin bng phng php s dng cacboxyl methyl cellulose (CMC). Phng php tch bromelin bng CMC cho php thu c bromelin dng bt trng c hat tnh cao, thi gain nhanh v n gin hn so vi cc phng php khc. Phng php tinh sch enzyme bromelin Enzyme bromelin c tinh sch bng phng php thm tch, lc qua sephadex, phng php sc k. * Tinh sch bng phg php thm tch. Cn 1g enzyme th, pha trong 10ml dung dch m sodium phosphate 0,03M c pH7,2. Sau khi tt c enzyme ho tan th cho hn hp vo ti cellophane ri t ti vo cc c cha 1L dung dch m sodium phosphate 0,03M, pH 7,2. Ti cellophane c chun b bng cch trc khi dng c trng y bng dung dch m tng t nh trn. Tin hnh thm tch trong 6 gi v c sau 2gi thay dung dch m bn ngoi 1 ln. Dung dch m pha ngoi ti c khuy lin tc bng 1 my khuy t. Trong qu trnh thm tch, lm gim s bin tnh ca enzyme do nhit, ngi ta thng tin hnh tinh sch enzyme nhit thp.
Cc phng php tinh sch enzyme 22

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

* Tinh sch bng cch lc qua sephadex. - Tinh sch bng cch lc qua sephadex G-50. - Tinh sch bng cch lc qua sephadex G-10. * Tinh sch bromelin bng phng php sc k. Bromelin thn c cho chy sc k trn Duolite CS101 pH 6,05 cho 2 phn on khc nhau v in tch vi hot tnh xc tc phn gii BAEE, casein v glucagon. Bromelin thn v bromelin qu th c cho chy sc k gel trn sephadex G-75, tip theo s dng phng php sc k trao i ion trn sulfo ethyl sephadex ch cho mt phn on protein n c hot tnh phn gii protein. b/ Enzyme papain : c tch t nha tri u xanh, papain thuc nhm enzyme thu phn c ngun gc thc vt c nghin cu nhiu nht v tnh cht v c ch hot ng. papain l mt sulfhydryl protease, u tin oc tch ra bi Bals v cng s ca ng, sau bi Kimmel v Smith t nha kh. Thu nhn nha u . iu kin ly nha. Cy: ang sinh trng tt, khng b su bnh, thn cy mp mp cho nha nhiu.
Cc phng php tinh sch enzyme 23

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Qu: ly nhng qu ang cn xanh, v qu mn, c trng lng t 0,31kg l tt nht. Thi gian ly nha: sng sm, kt thcs vo gia bui sng. Khi m khng kh thp, dng nha chy chm v c, kh thu nhn. -Ma kh: nha t, c, nng protein cao. -Ma ma: nha nhiu, long, nng protein thp. Tin hnh :
-

Nha u ly qu xanh cn trn cy.

- Dng dao inox c u nhn rch vi ng theo qu ch ng knh qu to nht. - Hng ly nha chy ra bng l thu tinh mu ming rng trong 4-6 pht, sau khi ly nha xong y np kn gi trong ti. - Bo qun lnh: - Khi ly nha cn trnh khng cho cc cht bn hoc cng trng ln vo.
-

Khng nn trn nha kh vi nha ti v n lm gim cht lng. Khi tin hnh cc thao tc vi nha ti, trnh khng cho nha tip xc vo da v n s gy bng. ng thi cng khng nn nha tip xc vi cc dng c lm t kimloi nng nh st, ng v n lm bin mu v gim hot tnh enzyme.

Cc phng php tinh sch enzyme

24

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

- Nha ti khng bn, v th nn sy kh ti 5% m. Phng php thu nhn papain tinh khit. - Ho tan nha u , mc ch l loi b cc cht nha, cn, tp ln - Loi b cc cht khng tan pH 9. - Qu trnh phn on bng ammodium sulfute. - Qu trnh phn on bng NaCl. - Kt tinh. - Ti kt tinh.
-

Sy chn khng.

Phng php tinh sch enzym tinh khit. * Phng php hp ph c chn lc. thc hin phng php ny ngi ta cho dch enzyme chy t t qua ct cht hp ph. Khi , ty theo kh nng tng tc ca cht hp ph vi tng loi enzyme, cc enzyme khc nhau s c hp ph trn cht hp ph vi kh nng khc nhau. Sau dnf cc dung dch m thch hp chit rr enzyme ra khi ct. Phng php hp ph thng dng lm c dung dch enzyme. C th hp ph chn lc cc enzyme theo 2 cch:

Cc phng php tinh sch enzyme

25

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

- Hp ph m tnh: nu enzyme khng c hp th, phng php x l ny c th xem nh l dng cht hp th tch nhng hp cht tp ra khi dung dch enzyme. -Hp ph dng tnh: nu enzyme c hp ph, n s c tch ra khi nhng cu t khc trong dung dch v sau c ra gii khi cht hp ph bi dung dch m pH kim v cc dung dch m khc c kh nng ra gii enzyme. Sau khi li tm loai cc hp cht khng tan, dung dch enzyme c th thm tch loi dch m. Mun s hp ph enzyme tt nht cn phi tun theo nhng nguyn tc chung sau y: - Hm lng protein trong dung dch khng vt qu 1%. - T l ca gel( trng lng kh) i vi protein thng l 20:1. - Mi trng hi axit pH=5-6. - Nng cht in li( mui) thp trong dung dch. S tn ti ca bt k mt lng mui no cng u nh hng n qu trnh hp ph. Qu trnh c tin hnh tun t nh sau: -Cho vo dung dch enzyme lin tip nhng lng thch hp gel. -Trn u. -Quay lng. -Thu hi tng phn gel trc khi cho phn tip theo vo.
Cc phng php tinh sch enzyme 26

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

* Phng php ct sc k: qu trnh tinh ch enzyme t hiu qu cao nht, ngoi nhng phng php thng thng ph bin nh kt ta enzyme bng mui hoc cc dung dch hu c t cc dung dch, phng php thp ph chn lc trn jela v c. Sc k trao i ion l mt phng php hiu qu chit tch enzyme tinh khit. qu trnh phn tchc th da trn s hp ph, trao i ion hoc hiu ng ry phn t, nhng trn thc t, hu nh cc qu trnh phn tch u chu nh hng ca c 3 qu trnh trn. Qu trnh da trn phn ng trao i ion gia cc protein tan trong nc hoc nhng dung dch m long vi nhng cht trao i in khc nhau, protein c to bng cch to cc lin kt tnh in gia nhng phn tnh in trn b mt cht hp ph. Thng thng, cho dung dch ch phm enzyme vo trong dung dch m vi ct trao i in c cn bng -> dung dch m gii hp ph bng dung dch in gii (gradient mui) vi nng tng dn chy qua cc ct, cc in ca dung dch ra s y ra khi nha, cc enzyme va lin kt vi nha. Khi , enzym no c i lc vi nha km nht s d b y ra khi nha nht. Do , ln lt cc enzyme

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

khc nhau s c chit ra khi ct theo tng phn chit khc nhau, trong c phn cha enzyme cn thu vi nng cao nht. Lc hp ph ca mt cht trao i in c xc nh bi : -Tnh cht ha l ca protein. -c tnh ca nhng nhm tch in trong cht trao i in. -Nhng iu kin hp ph protein. Do khng th ra trc nhng iu kin cn thit tch tt nht nhng enzyme ra khi nhng protein khc bng cht trao i ion m phi ty vo trng hp, nhng iu kin cn phi c nh thc nghim la chn. Khi phn tch, qu trnh tin hnh tun t nh sau: -Ly mt th tch V dung dch ( khng qu 1/3 th tch ct) cho chy mt phn qua ct lai mui ra khi dung dch protein. -Protein chy ra khi ct vo mt th tch dung dch hi ln hn so vi th tch ca n lc a vo ct . -Ngai ra, cc ct cha sephadex G-75, G- 100 v G-200 cng xy ra s phn tch tng phn cc protein ty theo khi lng phn t ca chng. Do , vic lai tr nhng cht kt tinh ra khi dung dch, cc ct cha sephadex t G-75 n G-200 c th s dng phn tch tng phn protein da theo kch thc phn t.
Cc phng php tinh sch enzyme 28

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

* Phng php in di L phng ph vt l c gi tr phn tch cc hn hp protein, enzyme v cng l phng php xc nh s tinh khit c cc ch phm protein.Gn y,phng php ny c p dng nhng giai an tinh ch enzyme cui cng. Nguyn tc: C nhiu phng php in ly khc nhau.Trong tinh ch enzyme c hai phng c p dng: Phng php rang gii chuyn ng: c p dng ch yu xc nh im ng in v tinh khit cng tnh thun nht ca ch phm enzyme. Phng php in di khu vc trn giy v trong khi hoc trong ct c cha cc cht lm y: c p dng chit xut s b v tinh khit enzyme. iu kin cn thit tin hnh in di: pH, nng dung dch m, in th ca dng in v thi gian th nghim c la chn qua thc nghim v tnh cht ca enzyme nghin cu. * Phng php kt tinh Khi enzyme t n ting khit ph hp n c th kt tinh.Tuy nhin kt tinh khng c ngha l ch phn enzyme tinh
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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

khit.Ngi ta xc nh rng nhng tinh th u tinh ca protein hay enzyme thng c tinh khit khng qu 50% v c th cha nhiu loi enzyme khc nhau. Hat ring ca tinh th tng khi cc enzyme ti kt tinh v qu trnh ti kt tinh cn c lp li nhiu ln cho n khi hat ring tng n mt gi tri cao nht. Phng php kt tinh v ti kt tinh c ng dng khi tinh ch enzyme nh l mt phng php phn an protein c gi tr. V th tt nht nn xem qu trnh kt tinh nh l mt phng php c th ca qu trnh phn an, v c s dng nhng giai an tinh ch cui cng. S kt tinh enzyme thng c tin hnh t cc ammonium sulfate. Enzyme thng c kt tinh theo nhiu phng php khc nhau.V d: nh t cc dung mi h c .Khi nhng phng php ny tht bi ngi ta c th kt tinh mui kim lai nng ca enzym. Chng hn, phosphopyruvate hydratase c kt tinh nh mui thy ngn khng hat ng, t cc enzyme hat ng c th c thu nhn thng qua s lai b kim lai bng qu trnh thm tch i vi dung dch ammonium sulfate cha ammonia v cyanyte. c/ Enzyme Ficin : Ficin l 1 protease c tm thy trong nha ca cy thuc h Ficus (sung, v), huc h Moraceae, b Urticales.
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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Hu ht cc b phn ca cy c cha nha bao g cao su 2,4%, resin, albumin, cerin, ng, malic acid, rennin, proteae, diatase, Ngi ta nhn thy rng hot tnh ca enzyme ficin cao khi nha c thu nhn vo bui sng sm. Do , nha sung thng c thu vo sng sm, sy kh dng trong cng ngh thc phm. Phng php thu nhn ficin th t cy sung: T l v qu: l v qu sau khi hi v c ra s, ct nh v ngm trong nc ri xay nhuyn.Lc qua vi thng, loi b xc v thu ly dch lc. Dch lc c xc nh em i xc nh hot tnh enzyme bng phng php Kunitz, t xc nh hot tnh ca enzyme ficin cha trong dch lc. T nha thn: nha sung c thu vo bui sng t 6-8 gi. Nha sung c bo qun lnh ngn ng c nhit thp hn 00C. Hot tnh ca protease trong nha ti c xc nh bng phng php Kunitz. Mt s phng php chit tch enzyme: * Phng php chit tch enzyme da vo tnh ho tan: -Ta phn on enzyme bng dung mi hu c. -Kt ta enzyme bng mui ammonium sulfate.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

S ch dch nha ti t cy sung Ficus rasemosal thnh ch phm enzyme ficin - Dch nha sung sau khi thu c pha theo t l 1g dch nha trong 2ml nc ct. -Khuy u dung dch bng my khuy t trong 5 pht enzyme ho tan hon ton trong nc. -Ly tm hn hp vi tc 3000 vng /pht trong thi gian 10 pht cao su ng c li v lng xung. Loi b phn lng pha di, thu dch ni bn trn. -Dng phng php ta phn on enzyme bng ethanol 96% thu ly cc phn on v xc nh hot tnh enzyme trong cc phn on. -Nhng phn on c hot tnh enzyme c pha lc gel loi b cc tp cht trong cc phn on enzyme. -Cc phn on sau khi qua ct gel s c xc nh li hot tnh, trn vi tinh bt vi t l 1:1 ri sy kh nhit 40 0C. Ch phm enzyme thu c dng bt s bo qun nhit 0-400C.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Vic thu nhn ficin t nha sung c th c thc hin theo s sau
Dch nha sung

Pha nc ct(t l 1g dch nha: 2 ml nc)

Khuy bng my khuy t trong 5 pht

Ly tm tc 3000 vng/pht, 10 pht Loi b ta Thu dch bn trn Ta phn on bng ethanol Thu ly cc phn on Xc nh hot tnh cc phn on

Thu cc phn on c hot tnh v a lc gel

Thu phn on Xc nh hot tnh cc phn on n vi tinh bt Sy kh 400C Ch phm enzyme

Bo qun nhit lnh 0-40C

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Cc phng php tinh sch enzyme Ficin: * Phng php lc gel: Cch lm: + Nhi ct: ngm gel sephadex G75 trong dung dch NaN3 0,02% trong 24 gi gel trng n han ton. - Cho dung dch m vo ct. - Cho mt lp gn thy tinh vo ct, gel t t vo ct n chiu cao cn thit. Trong qu trnh gel vo ct phi khuy u gel c ng nht v phi ch l lun lun c mt lp dung dch m trn mt ngn cn hin tng kh gel. t ln gel mt lp giy lc. - ct n nh trong vi gi, ra ct bng dung dch m phosphate. + Np mu: cn mu v ho tan mu vo 3-5 ml dung dch m pH 6,1 sao cho nng protein khong 10 mg/ml. - Np mu vo ct, sau khi ht mu vn tip tc cho dung dch m qua ct. - Thu mu vo ng nghim, mi ng nghim cha 2,5ml. Mi phn on c thu trong thi gian 5-10 pht, em cc phn on i o OD 280nm. Dn chung nhng phn on no c s hin din ca enzyme o hot tnh v xc nh hm lng enzyme.
Cc phng php tinh sch enzyme 34

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

* Phng php in dimini-gel SDS polyacrylamide: Cch lm: + Chun b ho cht: - Dung dch acrylamide - m gel phn tch Tris-HCl 1,5M ; pH 8,8. - Dung dch SDS(10%). - Dung mi pha mu. - m in di. - Dung dch nhum protein. - Dung dch ra mu. + Tin hnh: - Chun b mu: ho tan mu kh dung dch trong dung mi pha mu c nng protein khong 1mg/ml dung dch pha mu. un cch thu hn hp trong 5 pht, ngui, cho vo cc ging ca enzyme. - Chun b gel phn tch(10ml) m gel phn tch: 2,5ml; Dung dch acrylamide: 4,15ml; Amonium persulfate; t tinh th; nc ct: 3,2ml SDS 10%: 0,1ml TEDMED: 3,3microlit

Cc phng php tinh sch enzyme

35

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Lc nh cho tan ht ri dng pipette a gel phn tch vo khong gia hai tm knh cho n lc gel cao khong 8cm ( ch khng c bt kh). Cho mt lp nc ct ln gel, ch gel ng li. - Chun b gel gom m gel gom: 0,5ml; Dung dch acrylamide: 0,3ml; Acnomium persulfate: t tinh th; Lc nh cho tan ht. Sau khi gel phn tch ng li, ht b phn nc ct trn ri dng pipette a gel gom vo khun. Lc ny a lc vo khun to ging. Khi gel gn ng, nh nhng ly lc ra ri lp khun gel vo in di. Tin hnh in di: cho dung dch in di vo 2 bung in di. Bm t t 5-10 microlit mu vo cc ging. chy in di. Sau khong 1 gi, khi thy vch mu ch th di chuyn xung n gn y ca gel th ngt in v tin hnh nhum gel. Nhum gel: ngm gel trong dung dch thuc nhum protein, lay ng nh cho thuc nhum ngm vo gel(1-4 gi). Chuyn gel sang dung dch ra mu, thay dung dch vi ln n khi gel trong sut v cc nc ct 1,18ml SDS 10%: 20 microlit TEDMED: 1microlit

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

vch mu xanh xut hin ti cc v tr protein. Chp hnh v ghi nhn kt qu.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

2.T ngun ng vt Cc enzym thu t ng vt nh: rennet, pepsin, catalase, lipase, trypsin, pancreatin v lysozyme ng vt. * Pepsin Thu nhn pepsin Phng php 1: D dy b ra sch tch mng nhy nghin v ngm nc c butanol 5% trnh nhim khun sau ch ha bng etanol pepsin kt ta. Phng php 2 : Nim mc d dy bo qun nhit lnh v tch ra khi vt mi m dng HCl hay H3PO4, H2SO4 vi nng thch hp chit tch v hot ha thnh pepsinogen thnh pepsin. Dung dch pepsin kh s c dn qua mt cht kt ta cholesterol. Cht kt ta thu c sau khi x l bng ester s thu li cht cn c thnh phn cu to l pepsin. Tinh sch pepsin Phng php kt tinh ca Northrop

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Dung dich pepsin + MgSO4

Cht kt ta (ra dung dich MgSO4) Ha tan bng NaOH +NaOH +H2SO4 Lm lnh ti kt ta trong acid

Pepsin tinh khit

Cc phng php tinh sch enzyme

39

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Dung dich pepsin + MgSO4

Cht kt ta (ra dung dich MgSO4) Ha tan bng NaOH +NaOH +H2SO4 Lm lnh ti kt ta trong acid

Pepsin tinh khit

*Rennin Phng php thu nhn: Phng php 1 : -D dy b xay nhuyn -HCl 0.2% -Cho 0.7 acid boric trn tng V Phng php 2: -D dy b xay nhuyn -Nc mui 10%

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

- Chnh HCl pH =4 -Cho 0.7 acid boric trn tng V. Gi nhit 35-40 0 C. Ly ra lc qua vi mng thu c rennin Phng php tinh sch

Dich rennet + NaCl v gelatin (li tm 5.000vng/pht) Cht kt ta (rennet dng bt) Dim tch vi mui trung tnh + NaCl Dung dich bo ha

Li tm, lc, sy kh Rennet tinh khit

3.T ngun vi sinh vt: So vi protease ng vt v thc vt, protease vi sinh vt c nhng c im khc bit. Trc ht h protease vi sinh vt l mt h thng rt phc tp bao gm nhiu enzyme rt ging nhau v cu trc, khi lng v hnh dng phn t nn rt kh tch ra di dng tinh th ng nht. Cng do l phc h gm nhiu enzyme khc nhau nn protease vi sinh vt thng c tnh c hiu rng ri cho sn phm thu phn trit v a dng.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Cc loi enzym trn th trng c trch ra t vi khun hay nm nh cc loi: Bacillus, Aspergillus v Trichoderma. S chung tch enzym

Nm mc nghin nt+H2 O Thit b khuych tn

Dung dch nc giu emzim t VSV

B sung thm mm mc hoc VSV cn chit tch emzyme

Dung dich cha emzym

Tho b nm mc c chit rt

Khuch tn bng nhit 25 28 O C

Dich thu khng cha tp cht

NG DNG ENZYME PROTEASE

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

- Protease c s dng rng ri trong nhiu ngnh cng nghip nh cng nghip thc phm, cng nghip nh, cng nghip dc phm v nng nghip 1. Cng nghip thc phm. Protease vi cng nghip thc phm: Vic s dng trong ch bin lm mm tht l ng dng c tnh truyn thng. Nhn dn ta t rt lu dng thm nu canh tht b; dng rau sng l chui cht, v kt hp thc n nhiu tht; u trong chng to bnm thc cht l s dng papain, bromelain, fixin. Ngi Nga cn dng protease t ht u tng ny mm lm mm tht. Ngoi kh nng phn gii lm mm tht, to thc n d tiu ha, cng ngh sn xut cc loi dch thy phn giu protein c p dng mt cch c hiu qu tnh nng ca protease. - Trong cng nghip ch bin tht : protease c dng lm mm tht nh s thy phn mt phn protein trong tht, kt qu lm cho tht c mt mm thch hp v c v tt hn. Protease c s dng lm mm tht v tng hng v tht. (ngm tht vo dinh dng protease pH v nhit xc nh- phng php ny ph bin v thun li nht; Tm hn hp lm mm tht (enzyme, mui, bt ngt). Tim dung dch enzyme vo tht; tim dung

Cc phng php tinh sch enzyme

43

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

dch enzyme vo con vt trc khi git m). S dng protease sn xut dch m: t Streptomyces fradiae tch c ch phm keratineza thu phn c keratin rt c gi tr sn xut dch m t da, lng v. Nu dng axit thu phn s mt i hon ton cc axit amin cha lu hunh, nu dng kim thu phn s b raxemic ho (chuyn dng L sang D lm gim gi tr sinh hc ca axit amin). thu phn su sc v trit protein (trong nghin cu, ch to dch truyn m y t) cn dng cc protease c tnh c hiu cao v tc dng rng, mun vy ngi ta thng dng phi hp c 3 loi protease ca 3 loi: vi khun, nm mc, thc vt vi t l tng cng 1- 2% khi lng protein cn thu phn. u im ca vic thu phn protease bi enzyme l bo ton c vitamin ca nguyn liu, khng to ra cc sn phm ph, khng lm sm mu dch thu phn. - Trong ch bin thu sn: Khi sn xut nc mm (v mt s loi mm) thng thi gian ch bin thng l di nht, hiu sut thu phn ( m) li ph thuc rt nhiu a phng, phng php gi nn, nguyn liu c. Nn hin nay quy trnh sn xut nc mm ngn ngy c hon thin trong s dng ch phm enzyme thc vt (bromelain v papain) v vi sinh vt rt ngn thi gian lm v ci thin hng v ca nc mm. Tuy nhin vn cn mt s tn ti cn phi hon thin thm v cng ngh.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

- Trong cng nghip sa: protease c dng trong sn xut phomat nh hot tnh lm ng t sa ca chng. Protease t mt s vi sinh vt nh A. candidus, P. roquerti, B. mesentericus, c dng trong sn xut pho mat . - Trong cng nghip sn xut bnh m, bnh quy... protease lm gim thi gian trn, tng do v lm nhuyn bt, to xp v n tt hn. - Trong sn xut bia: ch phm protease c ngha quan trng trong vic lm tng bn ca bia v rt ngn thi gian lc. Protease ca A. oryzae c dung thy phn protein trong ht ng cc, to iu kin x l bia tt hn . 2. Cc ngnh cng nghp khc : - Trong cng nghip da: protease c s dng lm mm da nh s thy phn mt phn protein ca da, ch yu l collagen, thnh phn chnh lm cho da b cng. Kt qu loi b khi da cc cht nht v lm cho da c mm do nht nh, tnh cht c hon thin hn sau khi thuc da. Trc y, lm mm da ngi ta dng protease c phn lp t c quan tiu ha ca ng vt. Hin nay, vic a cc protease tch t vi khun (B. mesentericus, B. subtilis), nm mc (A. oryzae, A. flavus) v x khun (S. fradiae, S. griseus, S. rimosus...) vo cng nghip thuc da em li nhiu kt qu v dn dn chim mt v tr quan trng. - Trong cng nghip dt: Proteinase vi sinh vt c s dng lm sch t tm, ty t nhn to (cc si nhn to c bng cc dung dch cazein,
Cc phng php tinh sch enzyme 45

Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

gelatin) si c c bng, d nhum. 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 ho cht ty trng. Ngoi ra, protease cn c ng dng rng ri trong rt nhiu ngnh khc nh: - iu ch dch m thy phn dng lm cht dinh dng, cht tng v trong thc phm v sn xut mt s thc n king. - Protease ca nm mc v vi khun phi hp vi amylase to thnh hn hp enzyme dng lm thc n gia sc c tiu ha cao, c ngha ln trong chn nui gia sc v gia cm. - iu ch mi trng dinh dng ca vi sinh vt sn xut vaccine, khng sinh, - Sn xut keo ng vt, cht git tng hp git ty cc cht bn protein, sn xut m phm,

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

NG DNG CC LOI ENZYME KHC TRONG CNG NGH THC PHM

Enzyme l mt cng c ch bin cc ph liu ca cng nghip thc phm thnh thc n cho ngi v vt nui. Ngi ta cn khai thc tnh ng t nh ca renin, pepsin vo cng nghip thc phm nh trong sn xut phomat. Pectinase vi cng nghip thc phm: Pectinase c dng trong mt s ngnh cng nghip thc phm sau: - Sn xut ru vang. - Sn xut nc qu v nc ung khng c ru. - Sn xut cc mt hng t qu: qu c c, mt. - Sn xut nc gii kht. - Sn xut c ph. Ch phm pectinase c s dng trong sn xut nc qu t cc nguyn liu qu nghin hay lm trong nc qu p. Bi v khi c pectin th khi qu nghin s c trng thi keo, do khi p dch qu khng that ra c. Nh pectinase m nc qu trong sut, d lc, hiu sut tng.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Pectinase cn gp phn chit rt cc cht mu, tanin v cc cht ha tan khc, do lm tng cht lng ca thnh phm. Nhng nghin cu khi p nho c x l bng pectinase khng nhng lm tng hiu sut m cn lm tng mu sc. Trong sn xut mt nh, mt ng nh pectinase m dch qu c nng m c hn. Cellulase vi cng nghip thc phm: Cellulose l thnh phn c bn ca t bo thc vt, v vy n c mt trong mi loi rau qu cng nh trong cc nguyn liu,ph liu ca cc ngnh trng trt v lm nghip. Nhng ngi v ng vt khng c kh nng phn gii cellulose. N ch c gi tr lm tng tiu ha, nhng vi lng ln n tr nn v ch hay cn tr tiu ha. Ch phm cellulase thng dng : - Tng cht lng thc phm v thc n gia sc. - Tng hiu sut trch ly cc cht t nguyn liu thc vt. ng dng trc tin ca cellulase i vi ch bin thc phm l dng n tng hp thu, nng cao phm cht v v v lm mm nhiu loi thc phm thc vt. c bit l i vi thc n cho tr con v ni chung cht lng thc phm c tng ln.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Mt s nc dng cellulase x l cc loi rau qu nh bp ci, hnh, c rt, khoai ty, to v lng thc nh go. Ngi ta cn x l c ch, cc loi to bin Trong sn xut bia, di tc dng ca cellulase hay phc h citase trong c cellulase, thnh t bo ca ht i mch b ph hy to iu kin tt cho tc ng ca protease v ng ha. Trong sn xut agar-agar, tc dng ca ch phm cellulase s lm tng cht lng agar-agar hn so vi phng php dng acid ph v thnh t bo. t bit l vic s dng ch phm cellulase tn thu cc ph liu thc vt em thy phn, dng lm thc n gia sc v cng ngh ln men. Nhng ng dng ca cellulase trong cng nghip thc phm c kt qu rt tt. Tuy nhin hn ch ln nht l rt kh thu c ch phm c cellulase hot cao. Amylase vi cng nghip thc phm: Ch phm amylase c dng ph bin trong mt s lnh vc ca cng nghip thc phm nh sn xut bnh m, glucose, ru , bia... Trong sn xut bnh m, ch phm amylase lm thay i hon tan cht lng ca bnh m c hng v, mu sc, xp...Ch phm amylase sch cho cht lng bnh m tt hn dng phc hp vi protease.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

Trong sn xut bnh ko ngi ta thng dng maltose l sn phm thy phn tinh bt bng amylase v glucose bng glucoamylase. Chnh glucoamylase, l yu t lm tng hiu sut trong sn xut ru. Trong sn xut bia, vic s dng amylase c trong cc ht ny mm thay th malt gp phn ng k trong vic gim gi thnh.

Cc phng php tinh sch enzyme

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Gvhd :Ths ng Th Ngc Dung

Ha Sinh Thc phm

KT LUN

C th thu nhn enzyme t ng vt nh trypsin, chimotrypsin, t thc vt nh papain ca u , amylase ca i mch. Nhng enzyme vi sinh vt l ngun ph bin v gi thnh c ngha kinh t nht. Protease cn thit cho cc sinh vt sng, rt a dng v chc nng t mc t bo, c quan n c th nn c phn b rt rng ri trn nhiu i tng t vi sinh vt (vi khun, nm v virus) n thc vt (u , da...) v ng vt (gan, d dy b). Hin cho n nay khng c phng php tch sch enzyme chung. xy dng phng php v tinh sch enzyme no cn bit phi hp mt cch c hiu qu nht cc bin php tch v lm sch enzyme khc nhau. Mc d trong vic xy dng c phng php mi t c nhiu thnh tu vn tch v lm sch enzyme thn khit vn gp nhiu kh khn, phc tp.

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