3

Li ni u

Trong c th sng, hu nh khng c mt qu trnh ha hc no li khng c lin quan mt thit n cc qu trnh sinh hc do cc enzyme xc tc. C th ni rng s sng gn lin vi enzyme. Ngoi mt nhm nh phn t RNA c hot tnh xc tc, cc enzyme u c bn cht protein; chng bo m cho cc qu trnh chuyn ha cc cht trong c th sng tin hnh vi tc nhp nhng, cn i, theo nhng chiu hng xc nh. Enzyme hc (Enzymology) l mt mn hc c v tr then cht trong ha sinh ang pht trin mnh m v xm nhp vo rt nhiu ngnh khoa hc, c bit thu ht s ch ca cc nh sinh hc v sinh y hc v nhng hiu bit c bn v enzyme c lin quan mt thit n sinh hc phn t v y hc phn t v l nhng kin thc c bn rt quan trng ca sinh hc v sinh y hc. Gio trnh nhm cung cp cho sinh vin cc ngnh, chuyn ngnh lin quan n sinh hc trong i hc Hu nhng kin thc c bn v enzyme - cht xc tc sinh hc. Cun sch c bin son theo chng trnh gio dc i hc c i hc Hu ph duyt, bi tp th tc gi trng i hc Khoa hc, i hc Hu. Sch ny cng c th dng lm ti liu tham kho cho sinh vin cc trng khc, nhng ngi chun b thi tuyn sau i hc cng nh cc cn b nghin cu lin quan. Cc tc gi xin cm n nhng ng nghip gp nhiu kin b ch trong qu trnh bin son. c bit cc tc gi xin chn thnh cm n GS.TSKH L Don DinGim c Trung tm T vn u t Nghin cu pht trin Nng thn Vit Nam (INCEDA), Ch tch Hi Ha sinh Vit Nam nhn phn bin v cho rt nhiu nhng li khuyn qu bu nhm hon thin gio trnh. Vi thi gian bin son v kinh nghim cn hn ch, cun sch cn cha tht y v chc chn khng th trnh khi nhiu thiu st. Chng ti rt mong nhn c nhiu kin ng gp ca cc bn ng nghip, sinh vin v bn c ln xut bn sau s c hon thin hn.

Thay mt cc tc gi Ch bin PGS.TS. Qu Hai

Mc lc
Trang

Li ni u Chng 1
1.1. 1.2. 1.2.1. 1.2.2. 1.2.3. 1.2.4. 1.3. 1.4. 1.5. M u nh ngha enzyme Lc s nghin cu enzyme Giai on 1 Giai on 2 Giai on 3 Giai on 4 Phng hng nghin cu enzyme Nhng vn cn cp khi nghin cu enzyme Vn nghin cu enzyme nc ta Ti liu tham kho

3 7
7 7 7 8 9 12 14 16 17 18

Chng 2
2.1. 2.2. 2.2.1. 2.2.2. 2.2.3. 2.2.4. 2.2.5. 2.3. 2.3.1. 2.3.2.

Phng php nghin cu enzyme

Nhng nguyn tc chung khi nghin cu enzyme Tch v lm sch (tinh ch) enzyme Chn ngun nguyn liu Chit rt enzyme Cc phng php tch tng phn protein enzyme Kt tinh protein enzyme nh gi tnh ng th ca protein enzyme Hot enzyme Phng php xc nh hot enzyme n v hot enzyme Ti liu tham kho

19
19 21 21 26 28 38 39 41 41 41 4 43

Chng 3
3.1. 3.2.

Cch gi tn v phn loi enzyme

Cch gi tn enzyme Phn loi enzyme

44
44 44

3.2.1. 3.2.2.

Cc lp enzyme Cc phn ng enzyme Ti liu tham kho

44 46 51

Chng 4
4.1. 4.2. 4.3. 4.4. 4.5. 4.5.1. 4.5.2. 4.5.3. 4.5.4. 4.6. 4.7.

Cu trc phn t enzyme

Bn cht ha hc ca enzyme Thnh phn cu to ca enzyme Cu trc bc 4 ca enzyme Trung tm hot ng ca enzyme Phng php thm d v pht hin cc nhm chc nng trong trung tm hot ng ca enzyme Phng php dng cht c ch Phng php nh du bng c cht c hiu hoc coenzyme Xc nh tr s pK ca cc nhm hot ng Nghin cu cu trc phn t Cc dng phn t ca enzyme Phc hp multienzyme Ti liu tham kho

52
52 53 54 56 57 58 59 60 60 61 62 63

Chng 5
5.1. 5.2. 5.2.1. 5.2.2.

Tnh c hiu ca enzyme

Khi nim chung Cc hnh thc c hiu c hiu kiu phn ng c hiu c cht Ti liu tham kho

64
64 64 64 64 68

Chng 6
6.1. 6.2.

C ch tc dng ca enzyme
C ch ca phn ng c xc tc ni chung C ch ca xc tc enzyme Ti liu tham kho

69
69 69 73

Chng 7
7.1. 7.2. 7.2.1. 7.2.2. 7.2.3. 7.2.4. 7.2.5. 7.2.6. 7.2.7

ng hc Enzyme
ngha ca vic nghin cu ng hc enzyme ng hc cc phn ng enzyme nh hng ca nng enzyme nh hng ca nng c cht [S] nh hng ca cht km hm (inhibitior) nh hng ca cht hot ha (activator) nh hng ca nhit nh hng ca pH Cc yu t khc Ti liu tham kho

74
74 74 74 75 79 9 87 88 89 91

Chng 8
8.1 8.2 8.2.1 8.2.2

Sinh hc enzyme
S phn b enzyme trong t bo iu ha hot v s lng ca enzyme trong t bo iu ha hot enzyme iu ha sinh tng hp enzyme Ti liu tham kho

92
92 94 94 101 108

Chng 9
9.1. 9.1.1. 9.1.2. 9.2. 9.2.1. 9.2.2. 9.2.3.

Cng ngh enzyme v ng dng

Cng ngh enzyme Enzyme vi cng ngh sinh hc Cng ngh sn xut enzyme ng dng ng dng trong y dc ng dng trong ha hc ng dng trong cng nghip Ti liu tham kho

109
109 109 109 111 111 112 113 116

Chng 1

M u
1.1. nh ngha enzyme
Trong c th sng (cc t bo) lun lun xy ra qu trnh trao i cht. S trao i cht ngng th s sng khng cn tn ti. Qu trnh trao i ca mt cht l tp hp cc quy lut ca rt nhiu cc phn ng ha hc khc nhau. Cc phn ng ha hc phc tp ny c lin quan cht ch vi nhau v iu chnh ln nhau. Enzyme l cc hp cht protein xc tc cho cc phn ng ha hc . Chng c kh nng xc tc c hiu cc phn ng ha hc nht nh v m bo cho cc phn ng xy ra theo mt chiu hng nht nh vi tc nhp nhng trong c th sng. Chng c trong hu ht cc loi t bo ca c th sng. Chnh do nhng tc nhn xc tc c ngun gc sinh hc nn enzyme cn c gi l cc cht xc tc sinh hc (biocatalysators) nhm phn bit vi cc cht xc tc ha hc. Enzyme hc l khoa hc nghin cu nhng cht xc tc sinh hc c bn cht protein. Hay ni cch khc, enzyme hc l khoa hc nghin cu nhng tnh cht chung, iu kin, c ch tc dng v tnh c hiu ca cc enzyme.

1.2. Lc s nghin cu enzyme

Do enzyme hc c coi nh ct sng ca ha sinh hc nn phn ln cc nghin cu ha sinh t trc n nay u lin quan nhiu n enzyme. V s pht trin ca hc thuyt enzyme, c th chia thnh 4 giai on: - Giai on 1: trc th k th XVII - Giai on 2: t th k XVII n na u th k XIX - Giai on 3: t gia th k XIX n 30 nm u ca th k XX - Giai on 4: t nhng nm 30 ca th k XX n nay. 1.2.1. Giai on 1 Trc th k XVII ngi ta bit s dng cc qu trnh enzyme trong i sng song ch c tnh cht kinh nghim thc t v thng qua hot ng ca vi sinh vt. l cc qu trnh ln men ru, mui da, lm tng v nc chm... thi k ny ngi ta cha hiu v bn cht enzyme v cc qu trnh ln men.

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1.2.2. Giai on 2 giai on ny cc nh bc hc tin hnh tm hiu bn cht ca cc qu trnh ln men. Thi k ny khi qut hin tng ln men nh l hin tng ph bin trong s sng v enzyme l yu t gy nn s chuyn ha cc cht trong qu trnh ln men. Vo nhng nm 1600 ca th k XVII, Van Helmont l ngi u tin c gng i su tm hiu bn cht ca qu trnh ln men. Van Helmont nhn thy thc cht ca s tiu ha l s chuyn ha ha hc ca thc n v gii thch c ch ca n vi s so snh n vi qu trnh ln men ru. Danh t ferment (t ch Latinh fermentatio - s ln men) c Van Helmont dng ch tc nhn gy ra s chuyn bin cc cht trong qu trnh ln men ru. Vo na cui th k th XVIII, nh t nhin hc ngi Php l Raumur cng nghin cu bn cht ca s tiu ha. Nh t nhin hc ny cho chim qu en nut nhng ming tht t sn trong ng kim loi c thnh c c sn v buc vo dy thp. Sau vi gi khng thy g trong ng. Hin tng ny thc y s nghin cu thnh phn dch tiu ha tm hiu kh nng tiu ha ca dch d dy. Sau th nghim ny mt thi gian, vo nm 1783, nh bc hc ngi l Spalanzani lp li th nghim bng cch ly dch d dy trn vi tht mi v thy c hin tng ha tan xy ra. Vo u th k XIX, cc nh nghin cu tch c cc cht gy ra qu trnh ln men. Nm 1814 Kirchoff, vin s Saint Petercburg pht hin nc chit ca mm i mch c kh nng chuyn ha tinh bt thnh ng nhit thng. y l cng trnh u tin thu c ch phm amylase dng dung dch v lch s enzyme hc thc s c xem nh bt u t y. Mi chn nm sau (nm 1833), hai nh khoa hc ngi Php l Payen v Pessoz chng minh cht c hot ng phn gii tinh bt thnh ng c th tch c dng bt. Th nghim c tin hnh bng cch cho etanol vo dch chit ca la i mch ny mm th thy xut hin kt ta. Kt ta c hnh thnh ny c kh nng chuyn ha tinh bt v nu un kt ta ny s mt tc dng chuyn ha. Danh t diastase (t ch Latinh diastasis - phn ct) l do Payen v Persoz dng gi enzyme amylase lc by gi.

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Tip ngi ta cng tm ra v tch c nhiu enzyme khc nh enzyme phn gii protein ca dch tiu ha trong d dy nh Pepsin (Emberle v Shwan) - nhng nh khoa hc ngi c, nm 1836)... Sau , l thuyt xc tc ra i. Nm 1835, nh khoa hc Berzelius c quan im cho rng tng tc phn ng l hin tng xc tc. y l mt quan im ng. Song tht ng tic l nh khoa hc ny coi cc cht xc tc ny hot ng c l do " lc sng" khng theo s iu khin ca con ngi. y l quan im duy tm, siu hnh lm tr tr s pht trin ca khoa hc nht l nh hng su sc n sh pht trin ca ngnh enzyme hc. 1.2.3. Giai on 3 Giai on t gia th k XIX n 30 nm u ca th k XX. giai on ny mt s lng rt ln cc enzyme dng ha tan c tch chit. Trong thi k ny, c hai trng phi u tranh vi nhau: l trng phi Pasteur - nh bc hc v i ngi Php v trng phi Liebig - nh bc hc ni ting ngi c. * Trng phi Pasteur: Nm 1856 Pasteur cp n bn cht ca qu trnh ln men. ng cho rng khng th tch cc enzyme khi t bo. Tc dng v tnh cht ca enzyme gn lin vi s sng ca t bo v qu trnh ln men ru l kt qu hot ng sng ca t bo nm men ch khng phi l kt qu ca tc dng ca enzyme. ng tin hnh th nghim v nhn thy nu mt dung dch hu c, v d dung dch glucose trong bnh kh trng th khng xy ra qu trnh ln men ru. Chnh v suy ngh y, Pasteur chia cc enzyme thnh 2 loi: "enzyme c t chc" v "enzyme khng c t chc". Theo ng, cc "enzyme c t chc" l nhng enzyme khng th tch khi t bo, khi tch chng s b mt tc dng xc tc nh cc enzyme ca cc t bo nm men thc hin qu trnh ln men ru; cn cc "enzyme khng c t chc" l cc enzyme c th thc hin tnh xc tc ca n ngoi c th nh cc enzyme c trong dch tiu ha (v d Pepsin trong d dy, amylase trong tuyn nc bt, trong mm thc...) Quan im sai lm ny ca Pasteur thng tr ngnh enzyme hc trong mt thi gian di. Nm 1878 Kuhne ngh dng danh t "ferment" (t ting Latinh: fermentatio = ln men) gi cc "enzyme c

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t chc" v gi cc cht chit c tc dng xc tc cho phn ng ha hc l cc enzyme (t ch Hy Lp: en = bn trong, zyme = men ru, tc l " trong nm men" gi cc enzyme "khng c t chc". Danh t enzyme c xut pht t y. * Trng phi Liebig: Chng li quan im trn ca Pasteur, Liebig (trc c c Berzelius) cho rng c th khng c hot ng ca cc t bo vi sinh vt cng c qu trnh ln men. iu c ngha l ng coi enzyme nh l mt cht ha hc gy nn hiu qu tng t nh cc cht xc tc, tc dng c trong v ngoi t bo, khng ph thuc vo hot ng sng ca vi sinh vt. Nhng nm 1871 Liebig tht bi v thc nghim khng chng minh c quan im trn ca mnh . Cc th nghim c tin hnh bng cch ly dch chit t t bo nm men nghin nt u khng c tc dng gy ln men ru. Cng vo nm1871 Manatxein l mt bc s ngi Nga dng ct thch anh nghin cc t bo nm men v thu c dch chit khng cha t bo c kh nng bin i ng thnh ru. Nhng nhng quan st ny khng c ai ch ti. Chnh v vy, quan im siu hnh ca Pasteur hn ch kh nhiu s pht trin ca ngnh enzyme hc. n nm 1897, H. Bchner - mt nh khoa hc ngi c nhn c dch chit nm men bng cch phn hu t bo hon thin hn. Trong th nghim ny, cc t bo nm men c nghin nt hon ton cng vi bt thy tinh, sau c p bng p sut cao. Dch chit thu c khng cha t bo vn c kh nng gy ra qu trnh ln men (chuyn ha glucose thnh ru). iu chng t qu trnh ln men ru khng phi l kt qu ca hot ng sng ca t bo nm men m l kt qu tc dng ca cc enzyme vn c trong cc t bo. Do , quan im sai lm v enzme "c t chc" v enzyme "khng c t chc" m thc cht l v bn cht ca enzyme n lc ny mi hon ton b nh , m ra mt thi k pht trin mi ca ngnh enzyme hc. Cng t khng c s phn bit v ni dung gia thut ng "ferment" v "enzyme". C th ni rng, cng trnh ca Bchner nh du mt bc ngot quan trng trong lch s pht trin ca enzyme hc. Sau , nhiu loi enzyme trong c th sng c tm ra. V vy vic phn loi v gi tn cc enzyme mt cch thng nht cng cn thit. Nm 1883, Duyclo, nh bc hc Php ra nguyn tc phn loi enzyme theo c cht (substrate) do chng bin i v thm ui tn cng "ase" vo. V d enzyme phn gii tinh bt (amilun) l amylase. Tuy vy, trong thc t cn tn ti nhiu ngoi l v thut ng, v d nhng tn gi enzyme pepsin, trypsin, catalase trc y vn c dng.

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thi k ny, da vo thnh tu ca ha hc, c bit l ha l v ha keo, cc nh khoa hc hng vo vic nghin cu cc tnh cht ha v l hc ca enzyme cng nh hon thin cc phng php lm thun khit enzyme. Giai on quan trng nht trong thi k ny l cc cng trnh ca nh bc hc v i ngi c E. Fisher. ng t nn mng cho nhng khi nim hin i v tnh c hiu ca enzyme, v s tng tc khng gian gia enzyme v c cht. Gi thuyt ni ting ca ng l gia enzyme v c cht kt hp vi nhau nh " kha vi cha kha". Ri nhng nghin cu ca Bach v Palladin v cc enzyme xy ha kh to nn c s cho vic xy dng hc thuyt xy ha kh sinh hc. Trong thi gian ny ngi ta cng pht hin ra c tnh tc dng thun nghch ca enzyme (anilepski, 1894), cc coenzyme cng c pht hin (Harden v Young, 1906). H l nhng ngi khm ph ra rng, dch chit t bo nm men cha hai loi cht cn thit cho qu trnh ln men l "zymase" v "cozymase". H nhn thy dch chit t bo nm men mt hot tnh xc tc nu b thm tch hoc b un ln n 50 oC. Nhng dch chit b thm tch khng hot ng s hot ng khi c trn vi dch b un nng khng hot ng. Nh vy hot ph thuc vo s c mt ca hai loi cht: thnh phn khng bn vi nhit (heat - labile); khng c th thm tch c (c gi l zymase) v mt phn on bn vi nhit (heat - stable), c th thm tch c (c gi l cozymase). Ngy nay chng ta bit rng "zymase" bao gm tt c enzyme, cn "cozymase" bao gm cc ion kim loi, ATP, ADP v cc coenzyme nh NAD+. Thi gian ny ngi ta cng hiu bit c tc dng km hm v hot ha ca mt s enzyme (Sorensen 1909). Vo u th k XX, pht sinh ra c s ng hc trong tc ng ca enzyme da vo nhng nghin cu ca nh bc hc Anh l Brown v nh bc hc Php l Henri. n nm 1913, Michaelis v Menten pht trin cc cng trnh trn v nu ln thuyt ng hc ca s xc tc enzyme. Sau i chin th gii ln th nht nh bc hc ni ting ngi c l Willstatter c rt nhiu cng hin trong vic tm hiu bn cht ha hc ca enzyme. l cng trnh khoa hc 5 nm ca ng v cc cng s (1922) nhm lm thun khit enzyme bng phng php hp th chn lc. Qua t nhn xt thy l nhng giai on cui ca qu trnh lm thun khit enzyme, thng b mt i nhng cht cha c bit no do, enzyme b mt tnh xc tc, cho php Willstatter nu ln ln u tin gi thuyt v enzyme hai cu t (enzyme hai thnh phn). Nhm hot ng (coenzyme, coferment, agon) ch c kh nng xc tc khi kt hp vi

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phn protein c hiu (apoferment, apoenzyme, feron = protein) n xc nh cc c tnh ca enzyme v ng vai tr ch o trong vic th hin tc dng xc tc ca enzyme. Willstatter coi feron (protein) l cht tr ch c tc dng g. Agon l cht c hp ph trn cht ny. V vo nm 1926, trong mt dp thuyt trnh, ng cho rng enzyme khng thuc mt trong cc hp cht bit, tc l enzyme khng phi l protein, khng phi l glucid, m chng l nhng "cht c bit". chnh l quan nim sai lm ca Willstatter. ng l ngi tm ra c nhiu phng php lm sch enzyme cng nh lm sng t nhiu tnh cht c hiu enzyme. Nhng mc ch chnh l lm sng t bn cht ha hc ca enzyme thi ng li khng t c. Ngy nay ngi ta quan nim nu l enzyme hai thnh phn th phn coenzyme quy nh kiu phn ng v chu trch nhim lm bn. Cn apoenzyme quy nh tnh c hiu ca enzyme cng nh tng hiu sut xc tc. Coenzyme + apoenzyme = (holo) enzyme = (enzyme hon chnh) Coferment + apoferment = (holo) ferment Coenzyme ch dng ch phn khng phi protein ca enzyme trong trng hp khi n d tch khi phn apoenzyme khi cho thm tch qua mng bn thm v c th tn ti c lp. Phn khng phi protein ca enzyme c gi l nhm ngoi hay nhm "prostetic" khi n lin kt cht ch vi phn protein ca enzyme. 1.2.4. Giai on 4 Bn cht ha hc ca enzyme ch c xc nh ng n t sau khi kt tinh c enzyme. Nm 1926 nh ha sinh M tr tui Sumner (39 tui) thnh cng trong vic chng minh protein c kt tinh t ht u tng l cht ging enzyme xc tc cho phn ng thy phn ur. y cng chnh l enzyme u tin c kt tinh. Bn nm sau (1930) M Northrop tch c pepsin dng tinh th, v vo nm 1931 Northrop v Kunitz cng tch c trypsin dng tinh th. Trong thi k ny J.B.S Hardane vit quyn "Enzymes". Mc d lc bn cht phn t ca Enzyme hu nh vn cn l b mt, nhng tc gi a ra d on tuyt vi v vai tr ca cc tng tc v lin kt yu gia enzyme v c cht trong c ch hot ng ca enzyme. iu ny vn gi nguyn tnh thi s trong thi i ca chng ta.

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Cc cng trnh ca Sumner v Northrop m ra mt chng mi trong lch s pht trin ca enzyme hc hin i. Nhng kt qu t c cho php xc nh c mt cch dt khot bn cht ha hc ca enzyme l protein. Phi ni rng bn cht ha hc ca phn ln enzyme l protein v nh ngha c tnh cht kinh in v Enzyme phi xem li t sau pht hin ca T. R. Cech nm 1981. Cech pht hin mt RNA c hot tnh xc tc nh enzyme v gi l ribozyme (xut pht t cc tn ribose v enzyme). Ribozyme xc tc cho qu trnh chuyn ha tin cht. RNA thng tin (pre - m RNA) thnh m-RNA. Do enzyme khng nht thit phi l protein! y l mt pht minh c ngha rt ln. Tc gi ca pht minh ny c gii Nobel nm 1989. Cho n nay khong 100 ribozyme c bit. C th ni rng, nhng cng trnh ni trn m mn cho giai on th t ca lch s pht trin enzyme hc ko di cho n hin nay. T gia th k th XX, nht l thi gian gn y enzyme hc pht trin rt mnh. Nh ng dng cc phng php mi, hin i nh: in di, sc k, quang ph, ng v phng x... cho php nghin cu cu trc cng nh c ch tc dng ca nhiu enzyme, c ch ca qu trnh sinh tng hp enzyme v s iu ha hot ng ca enzyme trong t bo. Ngi ta xc nh c cu to ca coenzyme. xc nh c mi lin h ca enzyme v cc vitamin (nhiu vitamin l thnh phn cu to ca coenzyme v phn ln cc vitamin tan trong nc l thnh phn cu to ca cc coenzyme). Ngi ta cng xc nh c cc enzyme xc tc cho cc qu trnh trao i cht nh: h thng enzyme ng phn, Embden Meyerhof - Parnas nm 1933, h thng enzyme ca chu trnh Kreps Szent Gyorgy nm 1937 (chu trnh citric acid), chu trnh ornithrin trong trao i cht ca protein nm 1932 (Krebs - Henseleit). Nh nhng phng php mi trong vic tch v lm sch enzyme, ngi ta xc nh c vai tr rt quan trng ca kim loi trong s xc tc ca enzyme v tc dng hot ha ca chng. xc nh c s phn b ca cc enzyme trong t bo. nghin cu c ch tc dng cng nh cu to cc protein enzyme. Bng phng php Rhengen, ngi ta nghin cu cu trc ca ca phn t enzyme, nh cu trc ca ribonuclease (1960, Stein). Trong vng hn 40 nm tr li y nghin cu cc enzyme sinh tng hp nh nucleotide phosphorylase (Greenberg Marago, 1955), DNA - polymesase ( Kornberg,1956), RNA - polymesase (Spieglman, Hurwist,

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1958 - 1961) v cc nghin cu v iu ha sinh tng hp protein enzyme ca Jacob, Monod (1961). T nm 1961 pht hin ra isoenzyme trong c th l enzyme xc tc c th tn ti di nhiu dng khc nhau, xc tc trong cng mt c th, cho mt phn ng, c sai khc mt s tnh cht nh di ng in di. Nm 1969 ngi ta tng hp c enzyme u tin l ribonuclease (Denkewalter v Hirschmann, Gutte v Merrifield). y l enzyme gm 124 amino acid, bn vi nhit, c th un nng ln 800C vi thi gian ngn. C th tng hp enzyme bng hai phng php khc nhau: - Tng hp tng peptid ring bit ri sau ni li vi nhau. - Dng cht gi (polymer): cm ln trn ny mt gc amino acid, sau cm tip 123 gc amino acid khc. Vic tng hp ny thnh cng trong 3 tun bao gm 11931 giai on, 369 phn ng. nu ln c mt phng php mi v tng hp enzyme. y ngi ta dng phng php t ng ha, khi c 124 gc amino acid th chui polypepid t tch ra. iu ny cho thy mi khi chui polypeptid c la chn theo mt trt t ng n th c th t un cong trong khng gian. y chnh l kh nng t t chc Nhng thnh tu nghin cu c bn v enzyme l c s pht trin cc nghin cu ng dng enzyme trong thc t. Trong my chc nm cui ca th k XX v u th k XXI, ngi ta ch nghin cu vic ng dng enzyme. Ngi ta tn dng cc nguyn liu giu enzyme tch enzyme, dng ch phm enzyme ny ch bin cc nguyn liu khc nhau hoc s dng vo mc ch khc nhau. nhiu nc hnh thnh ngnh cng ngh enzyme, hng nm sn xut hng trm tn ch phm enzyme phc v cho cc ngnh sn xut khc nhau v cho y hc.

1.3. Phng hng nghin cu enzyme

Ngnh enzyme hc tri qua mt thi k pht trin kh di. Nh nhng phng php vt l, ha hc, con ngi t c nhng thnh tu rc r trong vic nghin cu bn cht ca enzyme. K t khi cc nh khoa hc x vo vic tm kim bn cht ca enzyme; t suy ngh cho rng s xc tc l do "lc sng" (Berzelius, 1835) qua vic coi enzyme ch hot ng c khi cn trong c th sng (Pasteur, 1856) n vic khng nh mt cch dt khot bn cht ha hc ca enzyme l protein (Sumner, Northrop (1926, 1930) v n gn y vi pht hin RNA c

15
hot tnh xc tc ca enzyme v c gi l ribosyme (Cech, 1981) v xem enzyme khng nht thit phi l protein, chng t s pht trin y si ng ca ngnh enzyme hc. C th ni enzyme hc l mt mn hc c v tr then cht trong ha sinh. Mn hc ny ang pht trin mnh m v xm nhp vo rt nhiu ngnh khoa hc, n ang l i tng nghin cu ca cc nh ha l, ha sinh, l sinh... v c bit thu ht s ch ca cc nh sinh hc v sinh y hc v nhng hiu bit c bn v enzyme cng nh v s xc tc sinh hc c lin quan mt thit vi sinh hc phn t v y hc phn t l nhng kin thc c bn rt quan trng ca sinh hc v sinh y hc. Bi vy, hin nay hng nghin cu v phm vi ca nhng vn enzyme hc c th c tm tt nh sau: 1) Vi mc ch xc nh cu trc phn t ca chng, ngi ta ang c gng hon thin nhng phng php tch v tinh ch enzyme. Nh vy c th nhn c cc ch phm enzyme c tinh khit cao c th dng cho vic nghin cu nhng tnh cht c bn v c th s dng trong y hc. Cc phng php c th tin hnh l: - Sc k i lc: gi li cht cn thit v cho sang qu trnh phn hp ph. - Sc k hp ph la chn: c tin hnh trn cellulose, sephadex. 2) Nghin cu iu kin v tc tc ng ca cc enzyme cng nh nh hng ca cc yu t vt l v ha hc i vi hot ng ca enzyme. 3) Lm sng t bn cht ca qu trnh xc tc ca enzyme v c ch tc dng ca n. y cn xem xt mi lin quan gia cu trc v chc nng ca protein enzyme c kh nng xc tc (v d trong mt s trng hp xem trung tm hot ng ca enzyme ch no tng hp phn m bo chc nng ca n: papain trung tm hot ng ca enzyme c nhm SH 1/3 phn t, v vy ch cn tng hp 1/3 phn t enzyme l cho mc ch ca mnh. 4) Nghin cu sinh hc enzyme. iu c ngha l phi tm hiu s to thnh enzyme trong t bo sng, tc dng iu chnh hot ng ca enzyme, vai tr ca chng trong vic thc hin cc chc nng sinh l khc nhau c th sng. Cn phi xem s phn b ca enzyme trong t bo, qua thy c mi lin h gia chc nng v cu to gia cc thnh phn t bo. Ngoi ra cng cn nghin cu mi quan h hp tc gia cc enzyme trong t bo xem quy lut tc dng ca enzyme. ng thi

16
cng cn nghin cu s tin ha ca enzyme lin quan vi s pht sinh v tin ha ca s sng. 5) Nghin cu tnh c hiu ca cc enzyme. 6) Nghin cu ci tin phng php v k thut thc nghim mi ca ha l, sinh hc vo nghin cu enzyme thc y s pht trin ca enzyme hc. 7) Nghin cu enzyme ng dng trong thc t nhm mc ch h gi thnh, tng bn ca ch phm. chnh l mc ch cui cng ca enzyme hc. thc hin c mc ch ny, cn phi c hng gii quyt: - Ci to ngun nguyn liu vi sinh vt l ngun nguyn liu tt. - Chn phng php tch. - Dng lp li (enzyme khng tan) T nm 1950 c nhiu cng trnh nghin cu to cc ch phm enzyme khng tan bng cch gn enzyme vo cc cht khng ha tan nh thy tinh, cellulose, nilon... Nh dng khng tan nn c th s dng lp li nhiu ln mt lng enzyme xc nh, v vy nng cao hiu qu s dng enzyme. (V d trong cng nghip dt ch phm amylase ca cc vi khun Bac. subtilis, Bac. mesentericus, Bac. diastaticus, Bac. amylosolvens... c tnh u vit l chu nhit cao, dng trong r h vi (ty lp h bt trn vi, to iu kin tt, d dng khi nhum, ty vi sau ny, nhng tn dng tip th ngi ta lin kt vi bt thy tinh to thnh cc ch phm enzyme khng tan). Vic ng dng enzyme amylase ( - amylase v glucoamylase) em li nhng thay i c bn trong k thut sn xut ng tinh bt. So vi phng php acid, phng php thy phn bng enzyme c nhng u im hn hn, lng glucose thu c cao hn (5 - 10%), cho php loi tr kh nng to thnh cc sn phm ph c v ng, yu cu v thit b n gin, kt qu cho php thu c glucose vi hiu sut cao, cht lng tt v gi thnh r hn.

1.4. Nhng vn cn cp khi nghin cu enzyme

Thng thng khi nghin cu enzyme ngi ta thng xc nh bn ca ch phm enzyme. Mun vy, cn xem xt nhng im sau y: 1) Tnh cht phn t protein enzyme Trc ht phi xem n tnh cht l hc. l hnh dng phn t im ng in, bn ca enzyme vi pH, nhit .

17
K n phi xem tnh cht ha hc ca phn t enzyme. chnh l cu trc phn t enzyme. 2) Tnh cht xc tc ca phn t enzyme. y phi xem bn cht ca phn ng, tnh c hiu ca enzyme. Phi ch n cu to ca trung tm hot ng cng nh mi lin quan gia cu trc v chc nng ca n. Tnh cht ca enzyme: chnh l cc tnh cht ng hc ca enzyme. 3) Tnh cht sinh hc ca enzyme chnh l s phn b ca enzyme trong t bo, s sinh tng hp protein enzyme cng nh nh hng ca s thiu ht enzyme trong c th sng. Ngoi ra y cn ch n ,mi lin h gia enzyme nghin cu v cc enzyme khc, cc tnh cht min dch cng nh to thnh hin tng cm ng enzyme.

1.5. Vn nghin cu enzyme nc ta

Hu nh mi phn ng ho hc trong c th sng u cn phi c vai tr xc tc ca enzyme - cht xc tc sinh hc. Chnh v vy, cc nghin cu v enzyme thu ht s quan tm ca cc cn b ho sinh hc, sinh hc thc nghim v nhiu nh nghin cu cc lnh vc lin quan khc. Cc nghin cu nhm theo hng tch, tinh sch enzyme, to cc ch phm c sch khc nhau, nghin cu cu trc, mi lin quan gia cu trc v hot tnh sinh hc ca enzyme, kh nng ng dng enzyme trong thc t. Nghin cu v cng ngh enzyme c tin hnh bi nhiu tc gi nh s dng ph tng ca l m sn xut pancrease, pepsin, trypsin... s dng mm m sn xut amylase. c nhng th nghim cng ngh nh sn xut amino acid t nhng tm bng protease, bt protein tht bng bromelain t t da, ln men ru bng enzyme c nh trn ct. Cng c nhng nghin cu s dng peroxydase, cyt-P450 trong ch to biosensor v thuc pht hin cht c... Trong lnh vc y dc, vic nghin cu su v c ch tc dng ca mt s enzyme nhm mc ch kin to nn mt s thuc dng iu tr mt s bnh c bit l to ra mt s ch phm thuc chng suy dinh dng tr em, ng thi tin hnh sn xut i tr. y l mt ng gp rt thit thc v kp thi trong vic phng chng suy dinh dng nc ta.

18 TI LIU THAM KHO

Ti liu ting Vit
1. Nguyn Hu Chn, 1983. Enzyme v xc tc Sinh hc. Nxb Y hc, H Ni. 2. Phm Th Trn Chu, Trn Th ng, 2000. Ha sinh hc. Nxb Gio dc, H Ni. 3. Ngc Lin, Phm Th Trn Chu, 1972. Enzyme I, II. i hc Tng hp, H Ni. 4. Nguyn Tin Thng, Nguyn nh Huyn, 1998. Gio trnh sinh ha hin i. Nxb Gio dc, H Ni. 5. Nguyn Xun Thng, o Kim Chi, Phm Quang Tng, Nguyn Vn ng, 2004. Ha sinh hc. Nxb Y hc, H Ni. 6. L Ngc T, La Vn Ch, Phm Trn Chu, Nguyn Ln Dng, 1982. Enzyme vi sinh vt. Nxb KH&KT, H Ni. 7. L Ngc T (ch bin), L Vn Ch, ng Th Thu, Phm Quc Thng Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din, 2000. Ha sinh Cng nghip, Nxb KH&KT, H Ni.

Ti liu ting nc ngoi

1. Bermeyer H. U, Bermeyer J. and Grasel M. (editors). 1983. Methods of enzymatic analysis. Vol II. Verlag chemie Weinheim. 2. Lehringer A. L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004. 3. Pelmont J., 1993. Enzymes. Presses universitaires de grenobe. 4. Stryer L., 1981. Biochemistry. W.H.Freeman and company. San Francisco. 5. Biochemical information, 1973. Boehringer Mannheim GmbH. Biochemica

19

Chng 2

Phng php nghin cu enzyme

C hai loi phng php dng nghin cu cc phn ng enzyme. Mt trong hai phng php l la chn cc mu sau nhng thi gian nht nh v o s bin i ca phn ng enzyme. Qua hng lot im ring bit nhn c s xy dng c ng biu din ca cc bc phn ng. Phng php khc l tin hnh quan st bn thn hn hp phn ng theo tin trnh xy ra v c th xy dng c mt s ln nhng thay i, hoc da vo cc phng php t ng ghi li. Chng ta s nhn c nhng ng biu din lin tc cc bc pht trin ca phn ng enzyme. phng php u, ngi ta thng o nng c cht hoc nng sn phm ca phn ng. Nu phn ng tng thi c hai cch va nu trn c th c s dng o hot ng ca enzyme. Trong mi trng hp xc nh tc , ngi ta phi nhn c t nht l 3 im: mt im thi im khng, im th hai khong thi gian nht nh tri qua, im th 3 khong thi gian ln gp hai ln khong trc. T c th kim tra c s ng n ca phn ng enzyme trong khong thi gian quan st. Ni chung, phn ln cc phng php c s dng nghin cu cc phn ng enzyme l loi phng php nghin cu lin tc v n c mi ngi a dng hn.

2.1. Nhng nguyn tc chung khi nghin cu enzyme

Nh phn u ni n, enzyme l nhng cht xc tc sinh hc c bn cht protein v rt khng n nh. Trong nhng iu kin bt li, chng rt khng bn, c th d dng b bin tnh (denaturation) v b mt hot . Do , khi lm vic vi enzyme, phi lun lun ch trnh nhng iu kin d lm mt hot ca n. Thng thng phn ln cc enzyme hot ng c vng pH trung tnh hoc gn nh trung tnh (pH = 7 2). V vy cc yu t acid mnh, kim mnh d gy bin tnh enzyme. Nhng ion kim loi nng nh ch, ng, thy ngn... v cc iu kin v nhit cao cng thng lm mt hot enzyme. c bit l khi tch v lm sch enzyme, cn tin hnh nhit thp. Nhit thng

20

dng cho cc cng vic ny thng thng t 0 0C n 50C. i vi cc enzyme khng bn, cc cng on lm sch c th c tin hnh nhit thp hn (t - 50C n - 200C). Trong cc trng hp ny, ngi ta hay s dng cc hn hp lnh nh nc vi CO2 hoc nc vi mui NaCl, hoc thm ch ngi ta dng c hn hp nc vi sulfuric acid m c... V d v mt s hn hp lm lnh c trnh by trn bng 2.1.
Bng 2.1. Hn hp lm lnh Thnh phn hn hp Nc : mui Nc : H2SO4 m c T l 100:33 (3:1) 100: 25 (4:1) Nhit t c - 21,30C - 20,00C

Nh trn ni, nhiu enzyme b mt hot tnh cc dung dch c pH < 5 hoc pH > 9, tuy rng c mt s ngoi l nh pepsin bn trong acid. Do , ty thuc mi loi enzyme, song nn ch trnh pH qu acid hoc qu kim. Khi iu chnh pH ca dung dch m c cha enzyme cn phi thm t t v rt thn trng cc acid hoc kim. V khi thm ha cht iu chnh pH th nn tin hnh 00C. Khi lm vic vi enzyme cng cn ch trnh to bt v nhiu enzyme b bin tnh (mt hot tnh) mt phn cch hai pha nc v kh. trnh vic to bt c th xy ra, ngi ta thng rt dung dch enzyme theo thnh ng thy tinh v khng c lc. C khi vic tch tng phn enzyme bng bt d lm mt hot tnh enzyme. V vy, khc phc tnh trng ny, ngi ta thng thm ammonium sulfate di dng dung dch bo ha ca n. Trong khi x l cc mu th nghim nh ct, thi, xay nh cc mu thc vt v ng vt (v d l cy, tht, cc c quan ni tng...) khng dng cc dng c dao ko dng c xay han r trnh tc dng ca cc ion kim loi nng nh (Cu, Pb, Fe...) m dng dng c inox.. Khi dng cc dung mi hu c nh aceton, alcol kt ta enzyme cn tin hnh nhit thp. Tch kt ta enzyme bng cch ly tm lnh tt hn lc lnh v tin hnh nhanh hn. mt s trng hp, khi tch v lm sch enzyme c hin tng gim dn hot , v vy cn phi lm th nghim nhanh. Tt nht l thc hin th nghim lin tc, khng ngt qung. V d tch chit cc enzyme chng oxy ha (antioxidant enzyme) ty th trong vng 6h v o lun nu khng th mt hot tnh. Cn microsome th tin trnh c th ko di hn vn

21

khng nh hng n hot cc enzyme chng oxy ha v cc enzyme oxy ha kh. Mt iu cn ch na l trong khi tin hnh xc nh hot ca cc enzyme, nu xc nh trong khong nhit no th tt c cc thnh phn ca hn hp phn ng phi c gi nhit y. Lc ny nht thit phi dng my n nhit ( thermostate). Khi hn hp phn ng t c nhit cn thit th mi tin hnh o. pH trong qu trnh ny cng phi c gi n nh bng dung dch m v phi m bo chnh xc ca pH: nhng phn ng to acid th phi thm kim vo v ngc li. m bo kt qu tin cy, trnh sai s nhiu, phi ly tht chnh xc lng dch enzyme. Ngi ta thng dng loi pipette khng chia hoc sau ny dng cc loi micropipette. Trong khi th nghim cn ch trnh nh ri enzyme vo dung dch nghin cu. V d ang lm th nghim vi amylase chng hn th khng ni chuyn nhiu. Khi c ch phm enzyme, cn bo qun chng nhit thp. Mt s enzyme n nh dung dch m c ca ammonium sulfate. Trong trng hp ny, ngi ta gi cc kt ta dng huyn ph trong dung dch ammoni sulphate bo ha v ly ch phm ra bng cch ly tm. Trong iu kin phng th nghim, vic sy kh ch phm enzyme s lm mt hot enzyme hon ton. Nhng iu kin chn khng nu sy kh nhit thp hoc dng phng php ng kh (lyophilization) th c th duy tr c hot ng bnh thng ca chng.

2.2. Tch v lm sch (tinh ch) enzyme

2.2.1. Chn ngun nguyn liu Enzyme l nhng cht xc tc sinh hc, c nhiu trong c th sng. Vic iu ch chng bng phng php ha hc vi s lng ln l vic lm rt kh khn v y tn km nu khng mun ni l iu khng tng, nn ngi ta thng thu nhn chng t cc ngun sinh hc. Mc d enzyme c trong tt c cc c quan, m ca ng vt thc vt cng nh trong t bo vi sinh vt, song vic tch enzyme p ng yu cu v mt kinh t ch c th tin hnh khi nguyn liu c cha mt lng ln enzyme cng nh cho php thu c enzyme vi hiu sut cao v d dng tinh ch chng. Vic phn b ca enzyme trong t bo cng khng ng u, trong mt loi t bo cng c th c nhiu enzyme ny song khng c enzyme khc. Lng enzyme li thay i ty theo giai on sinh trng pht trin

22

ca sinh vt v ty theo loi nn chng ta phi chn ngun nguyn liu thch hp cho vic chit rt v tinh ch enzyme. C ba ngun nguyn liu sinh hc c bn: cc m v c quan ng vt, m v c quan thc vt, t bo vi sinh vt. Trong tt c cc nguyn liu c ngun gc ng vt th tuyn tu, mng nhy d dy, tim... dng tch enzyme rt thun li. Dch tu tng c cha amylase, lipase, protease, ribonuclease v mt s enzyme khc. T ngn t ca d dy b ngh ngi ta c th thu nhn ch phm renin lm ng sa trong sn xut fomat. Ngi ta cng sn xut pepsin t d dy ng vt. Nhng khc vi pepsin, renin c kh nng ng t sa cao m khng thy phn su sc casein. Renin l ch phm enzyme c gi tr ln trong cng nghip. thc vt: thng thng enzyme hay c mt cc c quan d tr nh ht, c, qu. C quan d tr giu cht g th nhiu enzyme chuyn ha cht y. V d trong ht cy thu du c nhiu lipase, trong ht u tng c nhiu enzyme urease. Thc ny mm cha nhiu - amylase, c khoai lang li c nhiu - amylase. Ngi ta thu c mt s ch phm enzyme thy phn nh papain, bromelain, fixin t thc vt bc cao. Papain thu c t mu nha u xanh, bromelain thu c t cc b phn (l, thn, qu) cy da, cn fixin c tch t dch p thn v l cy Ficus. Qua cc ngun nguyn liu ng, thc vt chnh c th t chit xut cc ch phm enzyme, chng ta thy rng hai ngun nguyn liu ny khng th dng sn xut cc ch phm enzyme vi quy m ln bi cc nhc im sau y: - Chu k sinh trng ca chng di - Ngun nguyn liu ny khng ci to c. - Nhiu nguyn liu dng lm thc phm (dng n) khng th dng lm nguyn liu sn xut vi quy m ln cc ch phm enzyme nhm tho mn cc nhu cu ca nn kinh t quc dn. Dng vi sinh vt lm ngun nguyn liu sn xut cc ch phm enzyme c nhiu u im ni bt v c tnh cht c o vt xa so vi ngun nguyn liu t ng vt, thc vt, cng nh s khc phc c mi kh khn v hn ch trn. Trc ht vi sinh vt l ngun nguyn liu v tn sn xut enzyme vi s lng ln. y cng l ngun nguyn liu m con ngi

23

ch ng to ra c. Chu k sinh trng ca vi sinh vt ngn (t 16 - 100 gi) v vy c th nui cy hng trm ln trong nm. Enzyme vi sinh vt c hot tnh rt mnh, vt xa cc sinh vt khc. V vy ch cn mt lng nh enzyme c th chuyn ha mt lng ln c cht. S liu tnh ton cho bit, trong vng 24 gi, vi sinh vt c kh nng chuyn ha mt lng thc n gp 30 - 40 ln so vi trng lng c th chng. Trong khi , h enzyme ca con ln trn 50 kg ch c th chuyn ha c vi kg thc n trong ngy. H enzyme vi sinh vt v cng phong ph. Vi sinh vt c kh nng tng hp nhiu loi enzyme khc nhau, trong c nhng enzyme ng, thc vt khng tng hp c. V d cellulase, raxemase...Phn ln cc thc n nui vi sinh vt li d kim v gi r. Nhiu vi sinh vt cho enzyme thng c kh nng pht trin trn cc mi trng n gin, gi r, d kim nh cc ph liu ca cc ngnh sn xut. Hn na, c th dng nhng nguyn liu khng phi thc phm, nhng dung dch mui v c nui vi sinh vt. V vy dng vi sinh vt lm ngun thu enzyme s mang li gi thnh r, thi gian nhanh v hiu qu kinh t cao. Vi sinh vt sinh sn pht trin vi tc cc k nhanh chng, khi lng li nh, kch thc b, nhng t l enzyme trong t bo tng i ln nn quy trnh sn xut ch phm enzyme kh d dng, hiu sut thu hi cao. Lng enzyme c th c sn xut ra trong mt thi gian ngn. i vi mt s trng hp c th dng 100% sinh khi vi sinh vt lm ngun enzyme. Vi sinh vt rt nhy cm i vi tc ng ca mi trng, thnh phn dinh dng nui chng cng nh mt s tc nhn l ha, c hc khc. Do c th thay i nhng iu kin nui cy chn ging to nhng chng t bin cho ta hm lng enzyme ng k vi hot tnh xc tc cao. C th ni rng, nh ngun enzyme vi sinh vt, ngi ta c th iu khin s tng hp enzyme d dng hn cc ngun nguyn liu khc tng lng enzyme c tng hp hoc tng hp nh hng enzyme. Tuy vy trong qu trnh chn ngun nguyn liu t vi sinh vt, cn lu mt s vi sinh vt c kh nng sinh c t c bin php x l thch hp. Ni chung cc vi sinh vt mun c s dng lm ngun nguyn liu tch enzyme cn phi tho mn cc iu kin sau: - Kh nng tng hp enzyme mnh trong mt thi gian ngn. - D tch enzyme v khng sinh c t.

24

C mt iu l th l: trong iu kin bnh thng, vi sinh vt ch tng hp ra mt lng enzyme va cho hot ng sinh l c th ca chng ( thng c gi l s tng hp enzyme "bn th"). Nu khi tng hm lng mt s cht hoc thm mt s cht mi vo mi trng nui cy, c bit l c cht ca enzyme, th s tng hp enzyme tng ng tng ln mt cch ng k, khc thng c khi cn tng hp enzyme mi: hin tng trn gi l s cm ng sinh tng hp enzyme. Cht gy nn s cm ng sinh tng hp gi l cht cm ng. S tng hp mt lng ng k enzyme gi l siu tng hp enzyme. thu c ngun enzyme di do t vi sinh vt, cn phi nui cy chng. C hai phng php nui cy vi sinh vt thu enzyme: phng php nui cy b mt v phng php nui cy b su hay l phng php ni v phng php chm. Trong phng php nui cy b mt, ngi ta cho vi sinh vt pht trin v bao ph trn b mt cc hot cht dinh dng rn, c lm m, dng lm mi trng (cm go, cm np, cm m, bp xay nh...). mi trng xp ngi ta trn thm mt lng nh mt ca... Sau khi nui thi gian vi sinh vt tng hp enzyme mi trng c sy nh, nghin nh. Ch phm thu c dng rn - th. Mun c ch phm tinh khit phi qua giai on tch v tinh ch enzyme. Khc vi phng php nui cy b mt, trong phng php nui cy b su ngi ta cho vi sinh vt pht trin trong mi trng lng. Nguyn liu chnh v ph bin l dch ng glucose, fructose, maltose, saccharose... dch thy phn cellulose, tinh bt... Ngun nit hu c thng dng l nc chit bp, chit malt, dch t phn nm men. Cn chn pH ph hp vi chng vi sinh vt v s tng hp enzyme theo mong mun. Sau khi nui, ta thu c canh trng lng - dng th. lm tng lng enzyme vi sinh vt chng ta cn ch tuyn la v chn ging cc chng vi sinh vt c hot tnh enzyme cao, tng hp c enzyme cn thit v vi s lng nhiu. Cc chng c phn lp theo phng php thng thng ch tng hp mt lng nh enzyme (enzyme bn th), do cn tin hnh gy t bin bng cc phng php sinh hc, l, ha hc... to chng c kh nng siu tng hp enzyme. Vi sinh vt sau khi c tuyn chn, cn c nhn ging v nui trong iu kin ti u chng sinh trng tt, tng hp nhiu enzyme.

25

Ngoi ra cn phi chn mi trng v thnh phn mi trng dinh dng c nh hng trc tip n s sinh trng v tng hp enzyme ca vi sinh vt. Trong thnh phn mi trng phi c cc cht m bo c s sinh trng bnh thng ca vi sinh vt v tng hp enzyme. c bit lu l tng s tng hp enzyme ngi ta thng da vo hin tng cm ng. V nu nh trong thnh phn mi trng c cc cht cm ng th cht hay sn phm phn gii ca n s km hm hoc lm yu tc dng km to ca cht km hm nhm bo m kh nng sinh tng hp enzyme cho khng b cn tr. Cht cm ng tng hp enzyme cho thm vo mi trng nui thng l c cht tng ng ca enzyme cn tng hp. V d: Mun tch - amylase nm mc (Asp. Oryzae), ngi ta cho vo mi trng nui cy tinh bt, maltose, isomaltose, oligosaccharid... c cha lin kt - 1,6 glucozid. Mun tch pectinase Asp. Niger, ngi ta cho thm vo mi trng pectin. i vi hemicellulase th cht cm ng l hemicellulose; cn i vi proteinase cht cm ng c hiu lc l protein, bt u nnh, lng, sng nghin nh ( Actinomyces fradiae). Cht cm ng cng c th l nhng cht ging c cht v nhng sn phm thy phn ca chng. V d: thay cho protein th peptid v thay cho tinh bt th erithrodextrin u c tc dng cm ng. C nhiu yu t nh hng i vi mi trng nui cy. Nhit nui cy thng thng t 25 - 300C. Tr s pH ban u ca mi trng (ch yu mi trng nc) cng c th gy nh hng no n s to thnh enzyme, nhng khi cng cn tnh n kh nng bin i nhanh chng ch s bi vi sinh vt. Thng thng i vi - amylase, pH ti u cho s sinh tng hp (pH = 7 - 8) khc vi pH ti u cho hot ng ca n (pH = 4,7 - 4,9). Cc enzyme ng ha khc ca nm mc nh glucoamylase th pH ti u cho s sinh tng hp v cho hot ng l chung nhau (4,5 - 5,0). thng kh cng rt cn thit cho vic sinh tng hp enzyme. V vy mi trng b mt ngi ta thng thm cht xp nh tru vo, cn mi trng b su (mi trng dch th) , th ngi ta thng lc (nu enzyme cn lc th vic ny cc k quan trng). m cng rt quan trng (ch c tc dng nui cy b mt), ph thuc vo thnh phn mi trng b mt. Mt iu cn ni thm na l enzyme thng cha cc t bo sinh vt gi l cc enzyme trong t bo (intracellular), nhng n cng c

26

th c cc sinh vt tit ra mi trng sng. l cc enzyme ngoi t bo (extracellular). Enzyme vi sinh vt thng chit l enzyme ngoi bo.

27

2.2.2. Chit rt enzyme Mun tm hiu ton b hot ng sng ca c th sinh vt, chng ta phi bit bn cht ca nhng bin i ha hc xy ra trong tng m t bo. iu ch thc hin c khi chng ta tch c cc t bo ra khi cc m v chit rt cng nh lm sch cc enzyme cha trong chng. T cc dng enzyme tinh khit thu c chng ta c th nghin cu su sc c ch tc dng, tnh c hiu trong hot ng xc tc ca chng. Ty theo nhng c tnh ring bit ca tng loi enzyme m la chn phng php lm sch cho thch hp. Trong qu trnh tinh ch enzyme, mc du trnh t v cc th thut cc bc c th thay i , song vn c nhng nguyn tc chung. Nh chng ta bit, trong c th sinh vt, enzyme c trong t bo cht v cc cu t (nhn, microsome, ty th, lysosome...) ca t bo. T bo c bao bc bng mt lp mng. Lp mng ny vi khun i khi rt bn v dy. Ngi ta cn thy nhiu enzyme lin kt rt cht ch vi cc cu t ca t bo. Cc phn t enzyme khng c kh nng i qua mng ca t bo v mng ca cc cu t ca t bo. Do c th chit rt cc enzyme ni bo, bc u tin l phi ph v cu trc ca cc t bo c cha enzyme v chuyn chng vo dung dch. C th ph v cu trc ca cc t bo bng cc bin php c hc nh nghin vi bt thy tinh hoc ct thch anh, lm ng ha bng thit b ng ha (homogenizator). Thit b c chy thy tinh gn vi mt mt quay v c th iu chnh c tc quay theo yu cu. Cc t bo gia chy thy tinh v thnh ci s b ph hy. vic ph v c hiu qu m thc vt, trc khi nghin ngi ta thng thi nh mu vo ngn hoc cho trng nc (v d nh i vi mu ht kh). Cn cc m ca ng vt nh gan hoc thn, khi chit enzyme ngi ta cn ct b cc m lin kt. Mun tch c cc enzyme trong cc cu t ca t bo, ngi ta cn phi dng cc yu t vt l v ha hc khc nhau nh sng siu m, dng cc dung mi hu c nh butanol, aceton, glycerin, ethyl acetate... v cht detergent. Cc ha cht c tc dng tt cho vic ph v cc cu t ca t bo v trong cc c quan ny thng cha m. Sau khi ph v cc cu trc ca t bo, enzyme c chit bng nc ct, bng cc dung dch m thch hp hoc cc dung dch mui trung tnh.

28

C mt s yu t nh hng n qu trnh chit rt cn lu . Trc ht l nhit . trnh mt hot tnh hoc thm ch v hot, cn chit rt v tin hnh kt ta enzyme nhit thp (t 3 n 5 0C). Cc thao tc phi nhanh. Mt s cht in ly lm tng qu trnh chit rt enzyme nh NaCl, ZnCl2, CaCl2. Tc dng ca chng cn ph thuc vo phng php dng khi chit rt. V d nh nu dng my nung th c ba cht trn u c tc dng. Nu ch lng th ch NaCl c tc dng. V vy cn dng cht in ly thch hp. V d khi chit rt amylase, nu cho thm NaCl 0,1 - 0,2 % vo dung dch chit rt th hiu sut chit rt tng ln 30%. Ngi ta cn nhn thy, nu thm vo dch chit CaCl2 0,2% s lm cho kt ta enzyme tt hn v cu trc ca kt ta cng tt hn. Trong qu trnh chit rt enzyme cc i tng ng, thc vt, c trng hp cn c mt cht mu lm nh hng n vic lm sch hoc xc nh hot enzyme. Trong trng hp ny ngi ta cn cho thm vo cht kh loi mu. Mu ca hemoglobin hng cu hoc ca chlorophyll v mt s cht mu khc l c th b loi tr bi hn hp ethanol, chloroform vi t l thch hp. Hot enzyme superoxide dismutase (SOD) - mt enzyme chng xy ha c th xc nh sau khi loi mu khi dch chit enzyme. cc mu t ng vt c sc t melanin mu nu. Ngi ta thng loi mu trn ct nha trao i ion bng cch cho dch enzyme qua ct hoc lc. Khi qua ct, cht mu b gi li v enzyme khng b gi. V d ngi ta hay dng DEAE - cellulose (Diethylamino ethyl - cellulose) hoc than hot tnh. Trong qu trnh ny phi ch kim tra pH. Sau khi loi mu cn kim tra li hot ca enzyme. Trong dch chit, ngoi enzyme cn c cc protein cu trc, cc cht cao phn t khc nhau nh polysaccharid nucleic acid, cc cht c phn t nh nh ng monose, cc cht lipid, mui khong... loi chng phi s dng phi hp nhiu bin php khc nhau. loi b mui khong v cc loi ng... l cc tp cht c phn t lng thp, ngi ta thng dng phng php thm tch (dialysis) i nc hay i cc dung dch m long hoc bng cch lc qua gel sephadex. Cch lm thm tch nh sau: cho dung dch enzyme vo ti colodion hoc cellophane (thng thng ngi ta dng cellophane tt hn), sau t c ti vo nc ct hoc dung dch m pha long (nh m phosphate c pH = 7, nng 0,01M chng hn). Mng cellophane l mng bn thm, c kch thc l cho cc cht c phn t nh xuyn v i qua vo cc dung dch m long theo nh lut khuch tn. Cn li trong mng l cc cht protein c phn t ln. (Hnh 2.1)

29

Hnh 2.1. Thm tch loi mui (NH4)2SO4 trong kt ta protein

loi b cc protein tp (protein cu trc, protein tr) v cc cht c phn t lng cao khc ngi ta hay dng kt hp cc phng php khc nhau: phng php bin tch 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 phng php sc k (sc k hp ph, sc k trao i ion), in di, phng php lc gel. Phng php bin tch chn lc nh tc dng ca nhit hoc pH ca mi trng ch dng i vi trng hp cc enzyme bn vi nhit hoc bn vi acid. Th thut c tin hnh nh sau: dch enzyme c gi 50 70 C hay pH = 5 trong mt thi gian xc nh. Protein b bin tnh c loi b bng cch lc hoc ly tm
0

2.2.3. Cc phng php tch tng phn protein enzyme Protein l cc cht lng tnh,v vy trong cc dung dch acid v kim chng s b phn ly nh sau: kim Protein - COOH acid acid Protein - NH2 kim mt ch s pH xc nh, mi phn t protein c mt in tch tng s no y m ln ca n ph thuc vo s lng cc nhm tch in dng v tch in m. Kt qu l ch s nng ion hydro c nh, cc protein khc nhau trong hn hp s c tng in tch khc nhau. protein - NH+3 protein - COO- + H+

30

Nhiu phng php dng tch cc hn hp protein u da vo c tnh ny. Cc phn t protein mang in tch tng s (dng hoc m) cng du y nhau ra xa nn d tan vo dung dch. Mi mt protein c mt tr s pH nht nh m tng s in tch m v in tch dng trong phn t bng khng. Tr s pH gi l im ng in. im ng in, ha tan ca protein l thp nht, protein rt d b kt ta.Da vo tnh cht ny, ngi ta c th tch tng phn cc protein enzyme trong hn hp... cc phng php tch tng phn ny, ngi ta c th s dng phng php kt ta thun nghch bng mui hoc cc dung mi hu c, phng php sc k ct. Ni chung t kt qu tt, ngi ta thng phi hp hai phng php vi nhau. 2.2.3.1. Dng mui (NH4)2 SO4 tch enzyme 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). Ngoi ra nng (NH4)2SO4 cn thit kt ta enzyme khc nhau th khc nhau nhiu. V d: Protease ca nm mc d b kt ta 70% ca (NH4)2 SO4 bo ha hon ton, cn amylase ca mm la b kt ta 50% bo ha ca dung dch mui ny. iu ni ln tnh kt ta la chn ca (NH4)2SO4 cao hn cc mui khc. Thng dng hai dng bt hoc bo ha - Khi dng bt: Ngi ta cho tng t mt vo dch chit enzyme. Cch cho cng nh hng ln n lng kt ta ban u ca enzyme. Khi cho mui vo dch chit cn phi c my khuy t m bo s ha tan ca mui. - Khi dng dung dch bo ha: Trong nhiu sch v phng php nghin cu ngi ta a ra bng tnh s lng mui cn thit pha cc dung dch c bo ha khc nhau nhng nhit nht nh. Khi nim v s phn trm ca bo

31

ha hon ton c cp n. Nh v d trn ni, enzyme c th b kt ta 50% (0,5) hoc 70% (0,7) ca bo ha hon ton ca (NH 4)2 SO4. Khi cho dung dch (NH4)2 SO4 vo dch chit enzyme th nng (NH4)2 SO4 khng tng t ngt. Sau khi kt ta xong ngi ta thng lng khong 2h hoc qua m, mc ch l to kt ta hon ton ( phng php dng dung mi hu c th khng cn lu). Kt ta c ly ra bng cch ly tm hoc lc qua phu Buckner. Khi ha tan kt ta li ngi ta thng thm ion Ca++ lm bn (CaCl2 hoc Ca(COOH)2). giai on loi mui, ngi ta dng phng php thm tch nh c trnh by phn trc. Thi gian thm tch thng l 24 - 28h, nc thay cng nhiu cng nhanh cng tt. C th loi mui bng cch lc qua gel sephadex G25 l dn sut ca dextran. u th ca phng php ny l tin hnh vi thi gian ngn (khong 30 '), nn khng lm mt hot enzyme. Mui c trng lng phn t b b gi li, cc enzyme c trng lng phn t ln xung trc (xem phng php lc gel 2.2.3.4.a) Giai on tip theo l lm ng kh thnh bt trng. Chuyn trng thi t dch nc sang trng thi kh m khng qua trng thi lng. tin li ngi ta a ra cng thc cch tnh lng (NH 4)2 SO4 cho vo dung dch c bo ha cho trc (S1) t n mt bo ha cn thit (S2) Ty theo trng thi (NH4)2 SO4 cho thm vo dung dch chit enzyme, m c cng thc tnh ton khc nhau. - i vi (NH4)2 SO4 dng bt 0,515 x V (S2 - S1) x (g) = 1 - 0,272 S2 Trong V l th tch dung dch S1, S2 l bo ha cho trc v bo ha cn t v d S1= 0,5 v S2= 0,7 chng hn. Ngi ta cng c th dng bn ton (nomogram) chiu v xc nh c lng (NH4)2 SO4 thm vo dung dch enzyme t c

32

mt bo ha nht nh. Hoc c th i chiu bng c sn. Lng (NH4)2 SO4 a vo dung dch c bo ha nht nh c khc nhau ty thuc nhit th nghim. - i vi (NH4)2 SO4 dng dung dch bo ha. Th tch (tnh theo ml) ca dung dch bo ha cn cho vo 100ml dung dch c bo ha ban u S1 t n mt bo ha S2 cn thit c tnh theo cng thc sau: 100 (S2 - S1) V (ml) = 1 - S2 2.2.3.2. Dng dung mi hu c Phng php ny c tin hnh da trn c s: ha tan ca protein ph thuc vo s tng tc ca cc nhm tch in trong phn t protein vi cc phn t nc. S tng tc (cn gi l s hydrate ha) s b gim xung khi thm vo dung dch enzyme cc dung mi hu c. Dung mi hu c thng dng l ethanol, isopropanol, acetone hoc hn hp cc loi ru. phng php ny cng ch tin hnh nhit thp (t 50C tr xung). Dng dung mi hu c c th tin hnh tch phn on di 00C v c th n - 200C, nh vy n c tc dng tt n n nh ca protein enzyme. Khi c kt ta, ch ly nhanh kt ta ra khi dung mi bng cch dng my li tm. Phng php ny c li th l khng cn loi mui, nhng c nhc im l hay c mu. 2.2.3.3. Dng nhit Cng c th dng nhit loi b cc protein enzyme tp ra khi dch chit enzyme. Tuy vy phng php ny t c dng v him enzyme bn vi nhit. 2.2.3.4. Cc k thut 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

33

tip tc lm sch bng phng php sc k ct. Phng php sc k (chromatography) l do hai ch "chroma" l mu sc v " grapho" l vit, ngha l "vit bng mu". Thu ban u, ngi ta s dng phng php sc k tch cc cht mu v ch sau ny ngi ta mi p dng cho vic tch cc cht khng mu. a. Phng php dng cht ry phn t (lc gel - gel filtration) C s ca phng php lc gel l da vo s khc nhau v kch thc hnh dng v phn t lng ca enzyme c trong hn hp tch chng ra. m bo cho vic tch enzyme c tt, cht ry phn t phi l cht tr, khng phn 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). Gel sephadex l cht tho mn cc yu t trn. Sephadex l ch phm dextran do cc loi vi sinh vt khc nhau l Leuconostoc to ra khi chng c nui cy trn mi trng cha saccharose. Trng lng phn t ca dextran c th t ti hng triu v ln hn. Phn t dextran bao gm cc chui do cc gc glucose to thnh cc lin kt glucsid 1,6. Sephadex nhn t dextran bng cch x l ha hc (do tc dng ca epichlohidrin) to ra cc li phn nhnh c lin kt ngang gi l "sng phn t" v cht ny tr thnh khng tan trong nc. S lin kt ngang to ra cng nhiu, kch thc ca l sng phn t cng nh. Phng php lc trn sephadex c tin hnh nh sau: cho sephadex vo ct thy tinh di v cn bng bng dung dch m c pH nht nh. Sau cho dung dch enzyme ln ct. Khi lc v chit bng dung mi thch hp, cc phn t c trng lng phn t nh ( y l cc mui) s khuych tn chm chp qua cc l nh ca cc ht Sephadex b trng phng, cn cht c trng lng phn t ln hn ( trng hp ny l protein enzyme ) khng c kh nng i vo m lch nhanh qua cc ht sephadex v s ri xung trc, s c chit nhanh ra khi ct (Hnh 2.2. v hnh 2.3.). V vy ta c th tch c cht c trng lng phn t cao hn ra khi cht c phn t lng nh. Hng Sephadex (Pharmacia) ca Thu in tung ra th trng cc loi sephadex c kch thc khc

34

nhau c k hiu t G10 n G200. S k hiu nhm ch ra mc nhn (ht) nc ca chng. V d G10 ch khi trng phng th 1g gel kh nhn 1ml nc (1ml/g)

mui protein

Hnh 2.2. Hot ng ca lc phn t sephadex

Cc sephadex c k hiu khc nhau t G10 n G200 phc v trong vic lc phn t cho php cc cht c trng lng phn t khc nhau lt vo cc ngng sau y: Cc loi sephadex G. 10 G. 15 G. 25 G. 50 G. 75 G. 100 G. 150 G. 200 ht tinh (F) ht th (C) ht tinh (F) ht th (C) 3.000 - 70.000 4.000 - 150.000 5.000 - 400.000 5.000 - 800.000 1500 - 30.000 Trng lng phn t 0 - 700 0 - 1.500 100 - 5000

35

Cc gel lc phn t c sn xut trong 4 c ht cng trong mt vng lc phn t: ht th (coarse), ht trung bnh (medium), ht mn (fine), ht siu mn, rt mn (superfine). Cac phn t ln khng th i vao cac hat Sephadex

Hat Sephadex

Cac phn t nho i vao bn trong cac hat Sephadex

Hnh 2.3. Tch cc phn t theo kch thc bng sc k lc gel

S chnh lch nhiu v phn t lng ca cc enzyme (12700 1.000.000) cho php ngh rng tch v lm sch enzyme, phng php lc gel sephadex l phng php c nhiu trin vng. Ni c t lin kt ngang tch cht c trng lng phn t ln v ngc li. Ngi ta cn s dng sephadex loi mui thay cho qu trnh thm tch. Cng nhm cht ry phn t c ngun gc polysaccharid, l ch phm dextran nh sephadex (pharmacia) cn c Molselect (Reanal) - l sn phm ca Hungary c ng dng nhiu trong nghin cu. C th dng lm c c cc cht c trng lng phn t ln nh protein, peptid, loi mui khi protein enzyme (dng nhanh hn so vi thm tch), lc gel tch theo trng lng phn t (nh protein huyt thanh) hoc tch cc sn phm protein c hnh thnh di tc dng ca enzyme phn ct (nh - G - globulin b ct bi papain).

36

Cc Molselect cng c k hiu t G10 n G200 phc v trong vic lc phn t cho php cc cht c trng lng phn t khc nhau lt vo cc ngng sau y: Cc loi Molselect G - 10 G - 15 G - 25 G - 50 G - 75 G - 100 G - 200 Trng lng phn t <700 <1500 100 - 5000 500 - 10.000 1.000 - 50.000 1.000 - 100.000 1.000 - 200.000

Ngoi nhm cht ry phn t l ch phm dextran cn c nhm cht ry phn t l ch phm gel acrylamide bao gm Biogel (Bio - Rad) v Acrilex (Reanal). Acrilex gel l loi copolime, sn phm ca Hungary c to ra t acrylamide v N, N' - methylen bis - acrylamide. Cc acrilex gel c k hiu t P - 2 n P -300 dng tch cc cht c trng lng phn t trong ngng t 100 n 300.000. C th s dng vng pH t 2 11. Cht th ba l agarose loi sulphate. Hay ph bin l loi sepharose (pharmacia). Ngi ta thng dng cht ny tch cc phn t c trng lng ln hn 106. Tm li bng phng php lc phn t ngi ta c th tch cc cht c trng lng phn t khc nhau c trong hn hp (nh polymer, polysaccharid, nucleic acid , protein) ra. Ngi ta c th dng k thut ny loi mui thay cho qu trnh thm tch. V hn th na, trong qu trnh tinh ch protein enzyme, chng cn c s dng c c dung dch protein enzyme.Vic sc k, lc phn t protein hoc chit xut protein thng thu c dung dch long, nu khng c c dung dch cho ph hp i vi cc nghin cu tip theo th khng dng c. Molselect G 25 rt thch hp cho vic c c cc dung dch long ca cc cht c trng lng phn t ln nh protein, peptid. Bng cch trn vi dung dch protein long, Moltelect s nhn nc v cht c trng lng phn t nh, nh vy dung dch protein c c c m khng c s thay i v pH v lc ion ca n. b. Phng php sc k trao i ion

37

Phng php sc k trao i ion da vo s khc nhau v in tch tng s ca cc protein enzyme. Hay ni cch khc, phng php ny c da trn c s ca phn ng trao i ion gia protein c tan trong nc hoc dung dch m long v cc tc nhn trao i ion. Tc nhn (hay nguyn liu) trao i ion c th l cht nha c tch nhm sinh ion hoc l cht ionit. y l nhng cht gi tr, khng tan trong nc, c bn cht l cellulose hoc cht gel dextran c li phn nhnh (Sephadex, Molselect) hoc l cht nha polystirol. Cht gi th ny thng kt hp vi cc nhm ion ha. Cc cht trao i ion c cht gi l cellulose, sephadex, molselect thng thng c dng tch protein enzyme, cn cc cht trao i ion c cht gi l polystirol (v d nh Dowex, Amberlite) ch dng tch cc peptid c trng lng phn t nh hn. * Cc cht trao i ion c cht gi cellulose Cationit CM - cellulose (carboxylmetyl - cellulose) - l mt dn xut este ca cellulose. Cellulose - O - CH 2 COOH. Khi phn li cho ra COO-. y l cht trao i cation. Trn nhng cationit, th cc protein kim c tha nhng nhm amin v nhng nhm kim khc c hp ph. S hp ph trn cc cationit c tin hnh vi nhng dung dch long pH 1,5 6,5. (Cc protein kim c cha cc amino acid diamino - mono carboxylic nh lys, Arg, His) Anionit DEAE - cellulose (diethylamino - ethyl - cellulose) l dn xut este ca cellulose.) C2H5 Cellulose - O - CH2 - CH2 - N C2H5 Trong H2O n c phn ly: Cellulose - O - C2H4 - N - (C2H5)2 + H2O C2H5 Cellulose - O - C2H4 - N+ H y l cht trao i anion Cc anionit c p dng phn tch cc protein acid c tha nhng nhm carboxyl t do. S hp ph protein trn nhng ionit nh vy C2H5 + OH -

38

c tin hnh vi nhng dung dch m c lc ion thp (0,005 - 0,1M) pH 7,5 - 8,5, (cc protein acid c cha cc amino acid monoamin). Trong hn hp cht ionit vi dung dch m c pH tng ng, cc cht ionit ni trn tr nn tch in,. V vy trn b mt lp cht gi s hnh thnh mt lp in tch c du ph thuc vo kiu nhm chc ha hc ca n. Nu thm protein - enzyme vo dung dch m th cc phn t enzyme mang in tch s b cc nhm tch in tri du ca cht trao i ion ko li. Khi dng mt dung dch m phn hp ph c pH khc hoc khi thm mt loi ion khc c lc ion ln hn th phn t protein enzyme s b y ra khi cht trao i ion. Khi tin hnh phn hp ph, thng ngi ta thm vo cc ion Na+ v Cl- trong NaCl c nng tng dn theo bc thang hay theo gradient. Cc phn t protein enzyme no c in tch tng s nh th s b y ra trc do lc lin kt vi cht trao i ion yu. Cn nhng protein enzyme no c lin kt vi ionit ln hn th s b y ra bng mt lc ion ca mui ln hn. Nh vy, bng cch ny, chng ta c th tch c tng phn cc loi protein enzyme. Vic tch tng phn c la chn tt nht l khi tng dn nng cc ion thay th. Nh nng ion ca mui tng dn (gradient) ngi ta c th rt ra t ct cc loi protein enzyme khc nhau. C th thu nhn dch chit enzyme protein sau khi qua ct bng my thu phn on t ng. Theo th t tng phn dch thu c, ngi ta tin hnh nh lng protein theo cc phng php Lowry hay phng php o quang ph v xc nh hot ca enzyme. Ngoi vic dng mui NaCl cho cc ion Na+ v Cl-, ngi ta c th dng cc loi mui khc nh KCl, Na3PO4. Nu cht gi l sephadex th chng ta c cht trao i ion sephadex. l cc loi DEAE - sephadex v CM - sephadex. u im ca loi ny l va tch c protein enzyme v kch thc v v in tch tng s ca cc protein enzyme. Trng hp CM - sephadex trn cht gi sephadex c gn nhm COO- O - CH2 - COOH phn ly COO - (mang in tch m) y l cht trao i cation. c. Phng php dng cht hp ph

39

Nhiu protein v enzyme gn mt cch chn lc vo cc cht hp ph nht nh nh silicagel, bentonite, - aluminium hydroxid, hydroxyapatite v nh vy, chng c th c lm sch vi hiu sut cao. Phng php hp ph chn lc - hp ph trong th tch (thm cht hp ph trc tip vo dch enzyme) hoc trn ct (sc k hp ph) c dng ph bin trong vic tch v lm sch enzyme. Cht hp ph ch yu thng c dng l hydroxyapatite cho hiu qu phn tch c bit cao. Hp ph chn lc enzyme c th thc hin bng mt trong hai cch : cht hp ph hoc hp ph protein tp hoc hp ph enzyme. Qu trnh hp ph thng c tin hnh 00C. Bng cch thay i pH hoc lc ion ca dung mi thch hp, cc enzyme c hp ph c th c chit khi cht hp ph. d. Phng php dng cht hp ph c hiu sinh hc hay l phng php sc k i lc (affinity chromatography) C s ca phng php ny l ngi ta gn nhng phn t (cht) vo cht mang (cht gi) rn bng lin kt cng ha tr m protein enzyme cn tch s tng tc c hiu vi n. Nhng cht c th l c cht (Substrate) hoc cht c ch (inhibitor) cnh tranh. Hay ni cch khc dng cht ch c kh nng lin kt c hiu vi mt enzyme hoc protein m ngi ta nghin cu. Cht mang th rn c th l bt k mt loi no phc v cho lc gel nh sephadex, nhng ngi ta hay s dng nht l gel sepharose. trn ct cha c cht c nh pH v lc ion ph hp, ch c enzyme no c kh nng chuyn ha c cht mi gn vo, cc protein khc th chy xung ct. Bng cch thay i pH v lc ion ph hp hoc c th bng cch thm c cht c ha tan vo th c th tch c enzyme khi ct trng thi sch. 2.2.4. Kt tinh protein enzyme y l phng php c hiu tt nht tch tng phn protein enzyme giai on tinh ch cui cng. Khi protein enzyme c lm tinh khit hon ton, trong nhng trng hp ring bit, ngi ta c th tin hnh kt tinh chng. Mt iu cn ch l protein enzyme trng thi tinh th khng th c coi l bng chng v s tinh khit. Cc tinh th protein enzyme kt tinh ln u i khi c sch khng vt qu 50% v c th cha cc protein enzyme khc. Ngi ta thng tin hnh kt tinh protein enzyme trong dung dch

40

(NH4)2SO4. Qu trnh kt tinh c th tin hnh t t ko di vi ngy thm ch hng tun nu mun nhn c cc tinh th tt. Thng thng l thm mui (NH4)2SO4 vo dung dch protein enzyme kh m c cho n khi lm vn c nh nhng dung dch. Sau t dung dch vo mt ni, ng thi tng rt t t nng mui trong dung dch. C th tin hnh tng nng mui theo nhiu cch, thm dung dch mui m c hn vo dung dch protein enzyme theo tng git, thm mui qua mng bn thm hoc c th cho bay hi chm chp dung dch protein enzyme. Trong qu trnh kt tinh c th thay i ch s pH hoc nhit . kt tinh protein enzyme c d dng, nhng giai on trc , ngi ta thng tch tng phn cc protein enzyme bng cc dung mi hu c. iu ny c l lin quan n vic cc cht c bn cht lipid b loi ra khi dung dch protein enzyme to iu kin tt cho qu trnh kt tinh. 2.2.5. nh gi tnh ng th ca protein enzyme Khi nhn c mt protein enzyme trng thi kt tinh, ngi ta phi th li mc tinh khit hay tnh ng th ca n. ng th ca ch phm protein enzyme phi c kim tra bng mt s phng php da trn nhng nguyn l khc nhau. Trong mt s trng hp protein enzyme c coi l ng th khi ly tm, nhng li c th phn chia thnh mt s isoenzyme bng phng php in di trn gel. Chnh v vy, nu dng nhiu loi phng php khc nhau kim tra sch ca protein m kt qu u cho l ng th th protein c th c cng nhn l tinh khit. Nhng phng php kim tra tnh ng th hay dng l xy dng th v ha tan, in di v siu ly tm. - Phng php kim tra tnh ng th (hoc cn gi l tnh ng nht) ca protein n gin v nhy nht l xy dng ng biu din v ha tan. Cch lm nh sau: Trong hng lot mu dng mt th tch khng i mt loi dung mi (nc hoc dung dch mui) lc vi nhng s lng enzyme khc nhau. Sau lc v xc nh s protein trong dch lc. Cui cng xy dng ng th. Trong nhng loi mu u, tt c cc protein thm vo b ha tan v s lng protein thm vo bng s lng protein c trong dch lc hay dch ly tm. Kt qu nhn c biu din l mt ng thng. Sau dung dch t c bo ha. Nu protein em ha tan l tinh khit ngha l ng nht th khi thm protein trong dch lc s khng tng ln v ng biu din c mt im un.

41

Nu trong mu c mt protein th hai th sau khi t c bo ha i vi protein t ha tan hn, loi protein th hai cn c th ha tan c na.
S lng protein trong dch lc

B Hnh 2.4. ng biu din ha tan protein

Kt qu l c mt im bo ha th hai v ng biu din c hai im un. Nu dch chit (hn hp) c nhiu protein enzyme th s c A: Protein n th nhiu im un. Phng php ny c Northrop v Kunitz s dng rt c kt qu. Nay vn cn ng dng nhiu. B: Hn hp 2 protein - Phng php th hai xc nh ng th ca protein enzyme l phng php in di. Phng php in di l ng dng tnh cht lng tnh ca protein, da trn c protein cho thm ca cc tiu phn ch phm S lng s dch chuyn protein enzyme mang in trong in trng. em ch phm protein enzyme in di pH v lc ion nht nh. Nu trn in di c mt nh th chng t protein enzyme l n th. Nu c hai nh chng t c hai protein enzyme trong ch phm . - Phng php siu ly tm: y cng l phng php rt quan trng xc nh tnh ng th ca protein enzyme. Phng php c thc hin nh sau: Dng lc ly tm rt ln bng cch tng s vng quay ly tm ln hng nghn, hng vn vng trong mt pht. Vi tc ly tm rt ln, ngi ta c th tch ra c cc phn t enzyme c trng lng phn t khc nhau.

42

Tc kt ta ca protein enzyme trong my siu ly tm c xc nh bng trng lng phn t ca n. Khi dng quay th cc phn t protein li khuych tn vo dung dch. Bi vy, cn phi quan st tc lng trong qu trnh siu ly tm. Chnh v vy, trong cc siu ly tm, ngi ta gn mt thit b quang hc c bit, v cc ng nh sng ca kt qu phn tch ln mt mn. Nu trong dung dch c mt loai protein enzyme (c mt trng lng phn t) th trn mn sng s cho ng th c mt nh. Nu lm vic vi hai protein enzyme th s c hai nh v.v...

2.3. Hot enzyme

2.3.1. Phng php xc nh hot enzyme Khc vi trong ha hc phn tch bnh thng, trong enzyme hc, ngi ta khng nh lng enzyme mt cch trc tip m thng xc nh gin tip thng qua xc nh hot ng (cn gi l hot ) ca enzyme. Trong phn ng c enzyme xc tc, s hot ng ca enzyme c biu hin bng cch lm thay i cc tnh cht vt l, ha l cng nh tnh cht ha hc ca hn hp phn ng. Theo di nhng bin i c th bit c chnh xc mc hot ng ca enzyme thng qua xc nh c cht b mt i hay lng sn phm c to thnh trong phn ng. xc nh hot ca enzyme cc dch chit hoc ch phm ngi ta thng dng cc phng php vt l hoc ha hc. Cc phng php, so mu, o kh, o phn cc, o nht, chun ... c dng ph bin trong nghin cu nh lng cc phn ng enzyme. C th chia ra ba nhm phng php sau: 1. o lng c cht b mt i hay lng sn phm c to thnh trong mt thi gian nht nh ng vi mt nng enzyme xc nh. 2. o thi gian cn thit thu c mt lng bin thin nht nh ca c cht hay sn phm vi mt nng enzyme nht nh. 3. Chn nng enzyme nh th no trong mt thi gian nht nh thu c s bin thin nht nh v c cht hay sn phm. 2.3.2. n v hot enzyme Hi ngh quc t v ha sinh enzyme a ra khi nim n v enzyme quc t (hoc n v enzyme tiu chun) vo nm 1961. n v hot enzyme (U) l lng enzyme c kh nng xc tc lm chuyn ha 1 micromole (1mol) c cht sau mt pht iu kin tiu chun.

43

1 U = 1mol c cht (10-6 mol)/ pht. T nm 1972 ngi ta li a thm khi nim Katal (Kat) - Katal (Kat) l lng enzyme c kh nng xc tc lm chuyn ha 1 mol c cht sau mt giy iu kin tiu chun 1 Kat V = 6.107 U 1U = 1 microkatal 60

i vi ch phm enzyme, ngoi vic xc nh mc hot ng cn cn phi nh gi sch ca n. i lng c trng cho sch ca ch phm enzyme l hot ring. - Hot ring ca mt ch phm enzyme l s n v enzyme/ 1mg protein (U/mg) cng c th 1g ch phm hoc 1 ml dung dch enzyme. Thng thng hm lng protein c xc nh bng phng php Lowry. Khi bit khi lng phn t ca enzyme th c th tnh hot phn t. - Hot phn t l s phn t c cht c chuyn ha bi mt phn t enzyme trong mt n v thi gian. Hot phn t ln (cn gi l con s chuyn ha hoc con s vng: turnover number) c ngha l phn ng c xc tc xy ra rt nhanh. Nh vy, hot phn t chnh l kh nng xc tc: hot phn t cng cao th kh nng xc tc cng ln. V d ngi ta xc nh c hot phn t cao ca mt s enzyme tinh khit nh catalase (5,6 x 106) acetyl - cholinesterase (3,0 x 106), -amylase (1,2 x 106). Cng cn ch rng trong mt s trng hp nh ngha v n v hot enzyme trn khng th p dng c. Nu cn thit chng ta s a ra cc iu kin th nghim tng i hoc nh ngha ca cc n v khc. ni c nhiu hn mt mi lin kt ca phn t c cht b tn cng th mt n v hot enzyme l lng enzyme c kh nng xc tc lm chuyn ha mt micro - ng lng ca nhm lin quan sau 1 pht iu kin xc nh. ni c hai phn t ging nhau phn ng vi nhau th 1 n v hot l lng enzyme xc tc cho s chuyn ha ca 2 mol c cht sau 1 pht.

44

TI LIU THAM KHO

Ti liu ting Vit
1. Nguyn Hu Chn, 1983. Enzyme v xc tc Sinh hc. Nxb Y hc, H Ni. 2. Phm Th Trn Chu, Trn Th ng, 2000. Ha sinh hc. Nxb Gio dc, H Ni. 3. Ngc Lin, Phm Th Trn Chu, 1972. Enzyme I, II. i hc Tng hp, H Ni. 4. Nguyn Tin Thng, Nguyn nh Huyn, 1998. Gio trnh sinh ha hin i. Nxb Gio dc, H Ni. 5. Nguyn Xun Thng, o Kim Chi, Phm Quang Tng, Nguyn Vn ng, 2004. Ha sinh hc. Nxb Y hc, H Ni. 6. L Ngc T, La Vn Ch, Phm Trn Chu, Nguyn Ln Dng, 1982. Enzyme vi sinh vt. Nxb KH&KT, H Ni. 7. L Ngc T (ch bin), L Vn Ch, ng Th Thu, Phm Quc Thng Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din, 2000. Ha sinh Cng nghip, Nxb KH&KT, H Ni.

Ti liu ting nc ngoi

1. Bermeyer H. U, Bermeyer J. and Grasel M. (editors). 1983. Methods of enzymatic analysis. Vol II. Verlag chemie Weinheim. 2. Lehringer A. L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004. 3. Pelmont J., 1993. Enzymes. Presses universitaires de grenobe. 4. Stryer L., 1981. Biochemistry. W.H.Freeman and company. San Francisco. 5. Biochemical information, 1973. Boehringer Mannheim GmbH. Biochemica.

44

Chng 3

Cch gi tn v phn loi enzyme

3.1. Cch gi tn enzyme
Trong thi gian u khi ngnh enzyme hc cha pht trin, ngi ta thng gi tn enzyme mt cch ty tin, ty theo tc gi. V d nh cc tn pepsin, trypsin, chimotrypsin hin nay vn c dng gi l tn thng dng. Sau , ngi ta thng gi tn enzyme bng cch ly tn c cht c hiu ca enzyme cng thm ui t ase. V d urease l enzyme tc dng vo ure, proteinase l enzyme tc dng vo protein, lipase l enzyme tc dng vo lipid, amylase l enzyme tc dng vo tinh bt (amidon). i vi cc nhm enzyme cng xc tc mt loi phn ng, ngi ta ly tn ca phn ng enzyme thm ui t ase, v d nhng enzyme xc tc s oxy ha c gi l oxydase, nhng enzyme kh hydrogen c gi l dehydrogenase ... Tn gi y , chnh xc theo quy c quc t - tn gi h thng ca enzyme c gi theo tn c cht c hiu ca n cng vi tn ca kiu phn ng m n xc tc, cng thm ui ase, v d enzyme xc tc cho s thy phn ure (carbamid): H2N - C - NH2 + H2O CO2 +2NH3

O
c tn h thng l Carbamid - amidohydrodase (Tn thng dng l urease)

3.2. Phn loi enzyme

Mc ch ca phn loi enzyme l nhn mnh mt cch chnh xc v tng qut, mi quan h v nhng iu ging nhau ca mt loi enzyme. 3.2.1. Cc lp enzyme Tiu ban v enzyme (The enzyme Commission. EC) c t chc bi Hi ha sinh quc t (The internationl Union of Biochemistry, IUB)

45

a ra cch phn loi thng nht da trn cc loi phn ng v c ch phn ng. Theo cch phn loi ny th enzyme c chia ra lm su lp ln nh s t 1 n 6. Cc s th t ny l c nh cho mi lp. Su lp enzyme theo phn loi quc t gm c: 1. Oxydoreductase: Cc enzyme xc tc cho phn ng oxy ha - kh. Trong nhm ny c tt c cc enzyme c cc tn thng thng bit nh dehydrogenase, oxydase, cytochromreductase v peroxydase. Trong cc phn ng do chng xc tc xy ta s vn chuyn hydrogen, s chuyn electron, s oxy ha bi oxy phn t, bi hydrogen peroxide hoc bi cc cht oxy ha khc. 2. Transferase: Cc enzyme xc tc cho phn ng chuyn v. Cc transferase do bn cht ca nhng gc m chng vn chuyn c th tham gia vo cc qu trnh trao i cht rt khc nhau. Trong lp transferase bn cnh transaminase v methyltransferase cn c cc kinase khc nhau (xc tc ch yu cho s vn chuyn ca gc phosphate t hp cht cao nng ti cht khc, mt phn ln cc enzyme trc kia gi l mutase v mt vi loi synthetase, v d cc enzyme tng hp DNA v RNA). 3. Hydrolase: Cc enzyme xc tc cho phn ng thy phn. Trong lp ny c cc enzyme phn gii este (v d lipid), glucozid, amid, peptid, protein. 4. Lyase: Cc enzyme xc tc cho phn ng phn ct khng cn nc, loi nc to thnh ni i hoc kt hp phn t nc vo ni i. Thuc vo lp ny c cc enzyme c gi l hydratase, aldolase, decarboxylase cng nh mt s desaminase. 5. Isomerase: Cc enzyme xc tc cho phn ng ng phn ha. Tnh cho n cng th chng xc tc cho nhng phn ng chuyn cc nhm khc nhau bn trong phn t. Trong lp ny khng nhng c nhng enzyme chuyn ha cc ng phn hnh hc v ng phn quang hc (nh alaninracemase) m c cc enzyme xc tc cho cc phn ng v d s chuyn ha aldose thnh cetose (glucosophosphate isomerase, trc kia gi l phosphohexoisomerase) hoc bin i v tr ca lin kt este bn trong phn t (v d phosphoglucomutase) 6. Ligase: Cc enzyme xc tc cho phn ng tng hp c s dng lin kt giu nng lng ATP. v.v...

46

y cn ch thm l cc enzyme phn ct c phn loi vi tn lyase. Nu cn bng chuyn dch v pha tng hp th enzyme cng c th c gi l synthase. Ngc li chng ta gi cc enzyme xc tc cho phn ng kt hp 2 phn t c s tham gia ca ATP hoc cc nucleotide triphosphate tng t hoc c s dng mi lin kt giu nng lng l synthetase. Tn gi theo h thng phn loi ca lp ny l ligase trnh s i tro vi tn synthase ni trn. Mi lp (class) li c chia thnh nhiu lp ph (sub-class) v phn lp ph (sub-sub-class), ri sau th t ca enzyme trong phn lp ph (cng c ti liu phn chia theo: loi (lp), t, nhm v th t enzyme). Nh vy, mi enzyme trong h thng c phn loi v t tn theo m 4 ch s biu th phn ng xc tc: con s u ch lp, s th hai ch lp ph, s th ba ch phn lp ph, s th t ch r s bc th t ca enzyme. V d, enzyme xc tc cho phn ng: Ethanol + NAD+ acetaldehyde + NADH + H+ c tn gi l alcohol dehydrogenase (ADH), tn quc t theo kha phn loi l: Alcohol: NAD oxydoreductase, EC 1.1.1.1 Trong , m s 1 u tin biu th tn lp enzyme l oxydoreductase (lp 1); m s 1 th hai biu th lp ph 1: tc dng ln nhm CH - OH ca cc cht cho; m s 1 th ba biu th phn lp ph 1: cht nhn l NAD hay NADP v m s 1 cui cng ch s th t ca enzyme. Nh vy, trong cch gi h thng ca enzyme ADH trn c tn ca c cht v ca coenzyme cng nh tn ca qu trnh chuyn ha ha hc c xc tc vi tn cng ase. Sau tn ca enzyme l s ca n theo danh sch cc enzyme do tiu ban v enzyme ra (enzyme commission, EC). 3.2.2. Cc phn ng enzyme 3.2.2.1. Lp enzyme oxydoreductase Lp enzyme ny gm 14 lp ph, xc tc cho cc phn ng oxy ha kh. Phn ng oxy ha tng ng vi s tch in t ra khi c cht, phn ng kh l phn ng thu nhn in t v thng i km vi nhau. Qu trnh tng qut c th biu th nh sau:

47

AKh Box + e Akh + Box

Aox + e Bkh Aox + Bkh

Trong AKh l c cht A dng kh, Aox l c cht A dng oxy ha, e l in t, Box l c cht B dng oxy ha, B Kh l c cht B dng kh. Cc enzyme thuc lp ny l nhng enzyme 2 thnh phn c cc coenzyme nh NAD+, NADP+, FMN, FAD, hem... Ngoi kiu phn loi chnh thc theo quy c quc t, thng thng ngi ta phn bit cc enzyme lp ny thnh cc lp ph nh dehydrogenase, oxydase, oxygenase v peroxydase. - Dehydrogenase: xc tc cho phn ng tch H trc tip t c cht v chuyn n NAD+. NADP+, FMN, FAD. - Oxydase: Xc tc cho qu trnh chuyn in t n oxy do hot ha oxy lm cho n c kh nng kt hp vi proton c trong mi trng. - Oxygenase: xc tc cho phn ng kt hp trc tip oxy vo phn t ca hp cht hu c (thng l cc cht c vng thm). C th phn bit hai loi: oxygenase v hydroxylase. Oxygenase xc tc cho phn ng kt hp ton b phn t oxy cn hydroxylase ch kt hp mt na phn t oxy (thng dng OH) vo hp cht hu c. - Peroxydase: cc peroxydase in hnh v catalase c coenzyme l hem, xc tc cho phn ng oxy ha cc cht hu c khi c H2O2. 3.2.2.2. Lp enzyme transferase Lp ny gm tm lp ph. Cc enzyme lp ny cng l nhng protein phc tp, bn cht ha hc ca cc coenzyme rt khc nhau, ty theo bn cht ca nhm c chuyn v. y l lp cc enzyme chuyn nhm (khng phi hydrogen) gia hai c cht, t c cht A sang c cht B. A-R+B B-R+A Trong A - R l c cht A c mang nhm R, B - R l c cht B c mang nhm R. Cc enzyme ny xc tc s vn chuyn cc nhm monocarbon, nhm alkyl, nhm glucosyl, cc nhm c phosphore, cc nhm cha lu hunh.

48

- Acyltransferase: Cc enzyme ny xc tc cho phn ng chuyn nhm acyl thng l thng qua coenzyme A, to thnh phc CoAS ~ acyl. - Glucosyltransferase: xc tc cho phn ng vn chuyn gc ng (hexose, pentose) t cht cho n cc cht nhn khc nhau, thng gp nht l nhm OH ca mt gc saccharide khc hoc cc gc phosphate, nguyn t N ca nhn d vng. Thuc lp ph ny cn c cc enzyme phosphorylase, l cc enzyme vn chuyn glucosyl n gc phosphate hoc t gc phosphate i. Aminotransferase: cc enzyme ny c coenzyme l pyridoxal phosphate xc tc cho phn ng chuyn v nhm amin. Cc phn ng quan trng nh chuyn thun nghch nhm amin ca amino acid n - cetoacid. - Phosphotransferase: Hu ht cc phn ng chuyn gc phosphoryl thng c ATP tham gia vi tnh cht l cht cho, gc phosphate c chuyn t ATP (hoc c th l NTP khc) n nhm hydroxyl ca alcol hoc saccharide. Cc enzyme ny thng c lip v Kinase (v d nh hexokinase) Thuc phosphotransferase cn c phosphomutase, xc tc cho phn ng chuyn phosphate ni phn t. 3.2.2.3. Lp enzyme hydrolase Lp enzyme ny bao gm 10 lp ph, xc tc cho phn ng thy phn, phn ng ny lm t lin kt ng ha tr gia hai nguyn t ca phn t c cht gn cc phn t ca phn t H2O vo cc ha tr c to nn do s t lin kt k trn. C th c biu th nh sau: A - B + H2O A - H + B - OH Trong A - B l phn t c cht. Cc phn ng do enzyme lp ny xc tc lun c nc tham gia. c im khc l cc hydrolase thng khng cn coenzyme cho hot ng xc tc ca chng. Mt s hydrolase ph bin c vai tr quan trng i vi qu trnh tiu ha nh amylase, peptide hydrolase, lipase... - Amylase xc tc cho qu trnh thy phn tinh bt, glycogen v cc polysaccharide tng t. C 3 loi amylase khc nhau v tnh c hiu tc dng i vi lin kt glucoside v mt s tnh cht khc. - amylase phn gii cc lin kt 1,4 - glucoside gia chui mch polysaccharide, v vy cng gi l endo - amylase to thnh cc dextrin phn t thp.

49

- amylase xc tc phn ng thy phn lin kt 1,4 - glucoside k t u khng kh to thnh ch yu l maltose v dextrin phn t ln. Glucoamylase xc tc cho phn ng thy phn cc lin kt 1,4 1,6 -glucoside bt u t u khng kh ca chui polysaccharide. Sn phm ch yu c to thnh di tc dng ca enzyme ny l glucose v dextrin. - Peptide hydrolase xc tc cho phn ng thy phn lin kt peptide to thnh peptide phn t thp, amino acid. Cc peptide hydrolase khc nhau c tnh c hiu khc nhau i vi lin kt peptide. Mt s enzyme phn gii cc lin kt peptide gia chui mch polypeptide gi l endo peptide hydrolase hay proteinase (pepsine, trypsin, chimotrypsin...); mt s khc li thy phn cc lin kt u mt ca chui mch, gi l exo peptide hydrolase hay peptidase. - Lipase xc tc cho phn ng thy phn triglycerid to thnh cc acid bo t do v glycerol (thy phn ln lt tng lin kt este) 3.2.2.4. Lp enzyme lyase Lp enzyme ny gm 5 lp ph, xc tc cho vic phn gii tch ra khi c cht mt nhm no cng vi vic to thnh lin kt i hoc kt hp vi cc ni i. C th biu th nh sau: X A Y B A=B + X-Y

Lp ny c nhng enzyme tc ng vo cc lin kt C - C, C - O, C - N, C - S, C - Halogen Pyruvat decarboxylase xc tc cho phn ng loi CO2 khi phn t pyruvic acid, to thnh aldehyde tng ng l acetaldehyde. Coenzyme ca n l thiamine pyrophosphase. Enzyme ny c mi quan h cht ch vi pyruvat dehydrogenase. Fumarathydratase xc tc cho phn ng tch thun nghch phn t H2O khi malic acid, to thnh fumaric acid (c mt ni i) 3.2.2.5. Lp enzyme isomerase Lp enzyme gm 5 lp ph, xc tc cho s bin i ln nhau ca cc loi ng phn quang hc, ng phn hnh hc hay ng phn v tr. Trong qu trnh ny c s sp xp li trong phn t c cht. C th biu th nh sau:

50

X A

Y B

Y A

X B

UDP - glucose - 4 - epimerase xc tc cho s chuyn ha tng h phc tp gia galactose v glucose, tc l lm xoay nhm OH xung quanh nguyn t carbon v tr th t ca ng galactose. Enzyme c coenzyme NAD+, xc tc cho phn ng: UDP - galactose UDP - glucose. D - glucose - 6 - phosphate - cetoisomerase xc tc cho phn ng chuyn ha ln nhau gia D - glucose - 6 - P v fructose - 6 - P 3.2.2.6. Lp enzyme ligase Lp enzyme ny xc tc cho nhng phn ng kt hp hai phn t vi nhau nh nng lng ca mt lin kt giu nng lng trong ATP hay mt hp cht tng t v thng km theo s loi b cc phn t ca mt phn t nc. Thuc v lp enzyme ny c 4 lp ph v chng thng to nn cc lin kt C - O, C - S, C - N, C - C. Cc enzyme ligase xc tc cho vic to thnh aminoacyl - tRNA t amino acid v tRNA giai on u tin trong s sinh tng hp protein. Ngoi ra chng cn xc tc cho s sinh tng hp amiono acid, to ra nhng dn cht acyl - CoA... Pyruvatcarboxylase xc tc cho phn ng carboxyl ha acid pyruvic acid to thnh oxaloacetic acid. Enzyme ny c cha nhm ph l biotin, cn acetyl - CoA v Mg++ cho phn ng xc tc.

51

TI LIU THAM KHO

Ti liu ting Vit
1. Nguyn Hu Chn, 1983. Enzyme v xc tc Sinh hc. Nxb Y hc, H Ni. 2. Phm Th Trn Chu, Trn Th ng, 2000. Ha sinh hc. Nxb Gio dc, H Ni. 3. Ngc Lin, Phm Th Trn Chu, 1972. Enzyme I, II. i hc Tng hp, H Ni. 4. Nguyn Tin Thng, Nguyn nh Huyn, 1998. Gio trnh sinh ha hin i. Nxb Gio dc, H Ni. 5. Nguyn Xun Thng, o Kim Chi, Phm Quang Tng, Nguyn Vn ng, 2004. Ha sinh hc. Nxb Y hc, H Ni. 6. L Ngc T, La Vn Ch, Phm Trn Chu, Nguyn Ln Dng, 1982. Enzyme vi sinh vt. Nxb KH&KT, H Ni. 7. L Ngc T (ch bin), L Vn Ch, ng Th Thu, Phm Quc Thng Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din, 2000. Ha sinh Cng nghip, Nxb KH&KT, H Ni.

Ti liu ting nc ngoi

1. Bermeyer H. U, Bermeyer J. and Grasel M. (editors). 1983. Methods of enzymatic analysis. Vol II. Verlag chemie Weinheim. 2. Lehringer A. L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004. 3. Pelmont J., 1993. Enzymes. Presses universitaires de grenobe. 4. Stryer L., 1981. Biochemistry. W.H.Freeman and company. San Francisco. 5. Biochemical information, 1973. Boehringer Mannheim GmbH. Biochemica.

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Chng 4

Cu trc phn t enzyme

Enzyme c kh nng v hiu lc xc tc rt ln, c tnh c hiu rt cao. m bo cho chc nng ca enzyme l cht xc tc sinh hc, cu trc ca enzyme phi rt tinh vi v phc tp.

4.1. Bn cht ha hc ca enzyme

T gn mt th k trc y, cc nh khoa hc x vo vic xc nh bn cht ha hc ca enzyme. Cho n nay, c th ni rng, ngoi nhm nh phn t RNA c hot tnh xc tc, tuyt i a s enzyme c bn cht l protein v s th hin hot tnh xc tc ph thuc vo cu trc bc 1, 2, 3 v 4 ca phn t protein v trng thi t nhin ca chng. Thc t l bn cht ha hc ca enzyme ch c xc nh ng n t sau khi kt tinh c enzyme. enzyme u tin nhn c dng tinh th l urease ca u tng (Sumner, 1926), tip theo l pepsin v trypsin (Northrop v Kunitz, 1930, 1931). Sau nhng tc gi khc cng kt tinh c mt s enzyme khc v c bng chng xc nhn cc tinh th protein nhn c chnh l cc enzyme. Kt qu nghin cu tnh cht ha l ca enzyme cho thy enzyme c tt c cc thuc tnh ha hc ca cc cht protein v hnh dng phn t: a s enzyme c dng hnh cu (dng ht). T l gia trc di v trc ngn ca phn t vo khong 1 - 2 hoc 4 - 6. V khi lng phn t: cc enzyme c khi lng phn t ln, thay i rt rng t 12000 dalton n 1.000.000 dalton hoc ln hn. V d ribonuclease c khi lng phn t l 12700, glutamat dehydrogenase c khi lng phn t l 1.000.000. a s enzyme c khi lng phn t t 20.000 n 90.000 hoc vi trm nghn. Do kch thc phn t ln, cc enzyme khng i qua c mng bn thm. Enzyme tan trong nc, khi tan to thnh dung dch keo; chng cng tan trong dung dch mui long, glycerin v cc dung mi hu c c cc khc. Enzyme khng bn v d dng b bin tnh di tc dng ca nhit cao. Enzyme b bin tnh th mt kh nng xc tc. Mc gim hot tnh ca enzyme tng ng vi mc bin tnh ca protein trong ch phm. Kim, acid mnh, kim loi nng cng lm cho enzyme bin tnh. Cng nh protein, enzyme cng c tnh cht lng tnh.

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4.2. Thnh phn cu to ca enzyme

Cng nh protein, enzyme c th l protein n gin hoc protein phc tp. Trn c s , ngi ta thng phn enzyme thnh hai nhm: enzyme mt thnh phn (enzyme mt cu t) v enzyme hai thnh phn (enzyme hai cu t). Trng hp enzyme l mt protein n gin gi l enzyme mt thnh phn. Trng hp enzyme l mt protein phc tp ngha l ngoi protein n gin cn c mt nhm ngoi no khng phi protein gi l enzyme hai thnh phn. Phn protein ca enzyme hai thnh phn c gi l apoprotein hay apoenzyme, cn phn khng phi protein gi l nhm ngoi hoc coenzyme. Phn khng phi protein thng l nhng cht hu c c hiu c th gn cht vo phn protein hoc c th ch lin kt lng lo v c th tch khi phn protein khi cho thm tch qua mng. Coenzyme l phn khng phi protein ca enzyme trong trng hp khi n d tch khi phn apoenzyme khi cho thm tch qua mng bn thm v c th tn ti c lp. Phn khng phi protein ca enzyme c gi l nhm ngoi hay nhm prosthetic, khi n lin kt cht ch vi phn protein ca enzyme bng lin kt ng ha tr. Mt phc hp hon chnh gm c apoenzyme v coenzyme c gi l holoenzyme. Mt coenzyme khi kt hp vi cc apoenzyme to thnh cc holoenzyme khc nhau xc tc cho qu trnh chuyn ha cc cht khc nhau nhng ging nhau v kiu phn ng. Coenzyme trc tip tham gia phn ng xc tc, gi vai tr quyt nh kiu phn ng m enzyme xc tc v lm tng bn ca apoenzyme i vi cc yu t gy bin tnh. Cn apoenzyme c tc dng nng cao hot tnh xc tc ca coenzyme v quyt nh tnh c hiu ca enzyme. Cc coenzyme thng l cc dn xut ca cc vitamin ha tan trong nc. Cn ch l s phn bit coenzyme v nhm ngoi ch l tng i, v kh c th c mt tiu chun tht rnh mch phn bit lin kt cht ch v lin kt khng cht ch, nht l trong nhng nm gn y, ngi ta chng minh rng, nhiu coenzyme cng kt hp vo apoenzyme ca chng bng lin kt ng ha tr. Do , ngy nay ngi ta t ch n s phn bit coenzyme v nhm ngoi. Ngoi ra, trong thnh phn cu to, rt nhiu enzyme c cha kim loi. Thuc loi enzyme hai thnh phn gm c hu ht cc enzyme ca cc lp 1, 2, 4, 5, 6. Cc enzyme thy phn (lp 3) thng l enzyme mt thnh phn c cha ion kim loi hoc i hi ion kim loi lm cofactor (ng yu t).

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4.3. Cu trc bc 4 ca enzyme

Trong nhiu trng hp, cc chui polypeptide c cu trc bc ba c th kt hp vi nhau to thnh phn t enzyme c cu trc bc bn. Nh vy cu trc bc bn l cch sp xp c trng trong khng gian ca cc chui polypeptide ring bit trong phn t enzyme. n nay ngi ta xc nh rng s ln cc enzyme trong t bo u c cu trc bc bn. Cc enzyme c cu trc bc bn l enzyme olygomer v polymer do nhiu n v nh cu to nn, mi n v nh l do mt chui polypeptide. Cc n v nh trong mt phn t enzyme c th ging nhau, nhng cng c th khc nhau v cu to v chc nng, hoc cng c th mt s ging nhau, mt s khc nhau. Nhng enzyme do nhiu n v nh cu to nn cn c gi l cc enzyme polymer v cc n v nh c gi l protomer (cc n v nh cn c gi l cc mnh hoc tiu phn di n v) So vi cc enzyme monomer, cc enzyme c cu trc bc bn c nhng im sai khc sau y: - C trng lng phn t tng i ln, vo khong hn 100.000 - Phn t thng cha mt vi trung tm hot ng, c khi c n 3,4 trung tm hot ng. - Kh nng tng tc ca mt trung tm hot ng vi c cht s ph thuc vo trng thi chc nng ca cc trung tm hot ng khc. Trong mt s trng hp, mi tiu phn c mt trung tm hot ng nhng s tng tc gia cc tiu phn s nh hng n cu hnh khng gian ca trung tm hot ng trn mi tiu phn, do nh hng n hot ng xc tc ca enzyme. Trong mt s trng hp khc, cc nhm nh chc ca trung tm hot ng li nm trn cc tiu phn khc nhau, do hot ng ca enzyme ch th hin khi c s kt hp ng n gia cc tiu phn. Nh vy, enzyme c cu trc bc bn c tnh t chc ca mt h thng hp tc cao. - L iu kin cn thit xut hin tnh cht allosteric ca enzyme. Cn ni thm rng, enzyme allosteric (enzyme d lp th, d khng gian) l enzyme m cht trao i c th lm nh hng (c ch hoc hot ha) ln tc dng ca chng. Hnh nh hin tng d lp th (allosteric) bt u xy ra trc ht cc enzyme c xy dng nn t mt s tiu n v v hiu ng d lp th c nh hng n bn ca lin kt gia cc tiu n v ny (xem thm phn enzyme d lp th).

55

- Gm cc tiu phn di n v: a s cc enzyme c cu trc bc bn cha t 2 - 4 protomer, mt s enzyme khc cha t 6 - 8 protomer. V d enzyme catalase c trng lng phn t 252.000, cha 6 mnh di n v, mi mnh c phn t lng l 42.000. Mt s enzyme cha n 12 protomer v d nh arginine carboxylase, oxaloacetate carboxylase. - S sp xp ca cc mnh di n v trong phn t enzyme thng c tnh cht i xng cao. C 4 kiu chnh, c biu th hnh di y.

Hnh 4.1. S sp xp ca cc tiu phn trong enzyme c cu trc bc bn

- Cc tiu phn tng tc vi nhau bng cc kiu lin kt khc nhau. Trong a s trng hp nh tng tc k nc, lin kt hydrogen, mi s trng hp khc nh lin kt disulfide (v d glucoseoxydase ca Asp.niger) hoc cu polypeptide (v d nh enzyme leucin-s-RNAsynthetase). Cu polypeptide ny c vai tr quan trng vi tnh c hiu ca enzyme, khi mt n s thay i tnh cht phn ng. Cn lu l bn ca tng tc gia cc tiu phn ph thuc vo kiu lin kt gia chng. V vy, di tc dng ca cc ha cht khc nhau, cc enzyme c th b phn ly thun nghch thnh cc mnh di n v. V d lin kt hydrogen b ph v di tc dng ca ur, clorua guanidin chloride nng cao; tng tc k nc b ph v di tc dng ca mt s dung mi hu c nh dioxan, ethylen clorhydrin v.v... v mui trung ho nng rt cao. bn cu trc bc bn ca phn t enzyme ph thuc vo t l gia tng s th tch cc gc acid k nc (VK) vi tng s th tch cc gc amino acid a nc (V) trong phn t enzyme. Nu VK/V > 1, cu to bc bn ca phn t kh bn vng; ngc li nu VK/V < 1, khng to thnh cu trc bc bn hoc nu c th cng khng bn vng. V d, cytochrome C, ribonuclease d dng to thnh cu trc olygomer, nhng cu trc ny d dng b phn ly ngay c khi lc qua gel sephadex. Tuy nhin t l VK/V ca cc enzyme c cu trc bc bn khng phi lun

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lun ln hn t s VK/V ca cc enzyme monomer, tri li trong mt s trng hp c th bng hoc b hn. V d enzyme polymer phosphorylase b c t l VK/V ging vi - chymotrypsine (1,04) v nh hn t l VK/V ca lysozyme. (1,08) S hnh thnh cu trc bc bn l bc u tin trn con ng hnh thnh cc h thng t chc cu trc di t bo.

4.4. Trung tm hot ng ca enzyme

Ton b cu trc khng gian ca phn t enzyme c vai tr quan trng i vi hot tnh xc tc ca enzyme. Tuy nhin, hot ng ca enzyme lin h trc tip vi mt phn xc nh trong phn t enzyme. Trung tm hot ng ca enzyme l phn ca phn t enzyme trc tip kt hp vi c cht, tham gia trc tip trong vic to thnh v chuyn ha phc cht trung gian gia enzyme v c cht to thnh sn phm phn ng. Trung tm hot ng bao gm nhiu nhm chc nng khc nhau ca amino acid, phn t nc lin kt v nhiu khi c c cofactor hu c (coenzyme) v v c. cc enzyme mt thnh phn, trung tm hot ng thng bao gm mt t hp cc nhm chc nng ca amino acid khng tham gia to thnh trc chnh ca si polypeptide. V d nhm - SH ca cysteine - OH ca serine, threonine v tyrosine, - NH2 ca lysine, -COOH ca glutamic acid, aspartic, vng imidazol ca histidine, indol ca tryptophan, nhm guanidin ca arginine. Cc nhm ny c th xa nhau trong mch polypeptide nhng li gn nhau trong khng gian, c nh hng xc nh trong khng gian cch nhau nhng khong cch nht nh sao cho chng c th tng tc vi nhau trong qu trnh xc tc. V d: trung tm hot ng ca - chymotrypsin bao gm nhm hydroxyl ca Ser - 195, imidazol ca His - 57 v nhm carboxyl ca Asp 102. Cc gc ny kh xa nhau trong chui polypetide nhng gia cc nhm chc nng ca chng ch cch nhau t 2,8 - 3,0 . Trung tm hot ng ca cc enzyme hai thnh phn thng bao gm nhm ngoi (vitamin, ion kim loi ...) v cc nhm chc nng ca cc amino acid phn apoenzyme. S tng ng v cu hnh khng gian gia trung tm hot ng v c cht c hnh thnh trong qu trnh enzyme tip xc vi c cht.

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Theo quan nim ca Fisher th trung tm hot ng ca enzyme c hnh thnh sn vi mt cu to nht nh ch cho php c cht c cu to tng ng kt hp vo. Do c th v s tng ng nh kha vi cha kha(Hnh 4.2.a).

C cht +
Vng hot ng Phc hp ES Enzyme

C cht +

Phc hp ES Enzyme

a.

b.

Hnh 4.2 M hnh Fisher (a) v m hnh Koshland (b)

Thuyt ny tuy cng gii thch c mt s hin tng nhng khng gii thch tha ng c nhiu kt qu thu c trong thc nghim. V vy, Koshland a ra mt gi thuyt khc hp dn v t nh hn. Theo thuyt ny th c im ca vng trung tm hot ng l rt mm do v linh hot, cc nhm chc nng ca trung tm hot ng ca enzyme t do cha t th sn sng hot ng, khi tip xc vi c cht, cc nhm chc nng trong phn trung tm hot ng ca phn t enzyme thay i v tr trong khng gian, to thnh hnh th khp vi hnh th ca c cht (Hnh 4.2.b). Cng v vy, ngi ta gi m hnh ny l m hnh tip xc cm ng hoc khp cm ng. Gia c cht v trung tm hot ng to thnh nhiu tng tc yu, do c th d dng b ct t trong qu trnh phn ng gii phng enzyme v sn phm phn ng. Trung tm hot ng ca cc enzyme c cu trc bc 4 c th nm trn mt phn di n v hoc bao gm cc nhm chc nng thuc cc phn di n v khc nhau.

4.5. Phng php thm d v pht hin cc nhm chc nng trong trung tm hot ng ca enzyme
y l vic kh khn v phc tp, phi s dng hng lot phng php khc nhau. Khi xc nh c vai tr quan trng ca mt nhm no i vi hot tnh enzyme, cha c ngha l nhm thuc trung tm

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hot ng ca enzyme, bi v c nhiu nhm chc nng ch lm nhim v duy tr cu trc khng gian hot ng cho phn t enzyme. Mun thm d, pht hin v xc nh cc nhm chc nng ca phn t enzyme, ngi ta thng dng cc phng php vt l, ha hc, xc nh hng s ion ha ca cc nhm chc nng v tt nht l kt hp vi vic nghin cu cu trc phn t ca enzyme v ca trung tm hot ng. Cc phng php vt l c kh nng ph hu mt cch c hiu cc nhm chc nng ca enzyme thng khng nhiu, v kh c th chn c cc tc nhn ch ph hu mt s nhm ha hc nht nh m li khng lm nh hng n ton b cu trc ca phn t enzyme. Chnh v vy, thng thng ngi ta kha, ph hu, hoc nh du cc nhm chc nng bng cc thuc th ha hc nh cht c ch c hiu, c cht hoc coenzyme . 4.5.1. Phng php dng cht c ch Ty trng hp c th dng cc cht c ch c hiu khc nhau, v d: - i vi mt s enzyme oxy ha kh c nhm hot ng l ion st ngi ta dng xyanua (cyanide CN) pht hin vai tr ca ion st i vi hot tnh ca enzyme v CN kt hp vi ion st lm cho enzyme mt kh nng vn chuyn in t. V d CN c ch enzyme cytochromoxydase. - Mt s enzyme cn c ion kim loi tham gia vo qu trnh xc tc hoc gi n nh cu trc phn t enzyme. thm d pht hin c tnh cn kim loi ca chng, ngi ta dng cht kt hp kim loi nh EDTA (Ethylen diamino tetraacetate) hoc orthophenantrolin... Nu l enzyme cn kim loi th s mt hot tnh. - thm d vai tr nhm - S - CH 3 ca methionine i vi hot tnh ca enzyme th oxy ha nhm ny thnh sunfoxit tng ng: - S - CH3 O v enzyme s mt kh nng xc tc. - Vai tr ca nhm - SH ca Cysteine i vi hot ng ca enzyme c xc nh bng cch cho phn ng vi mui kim loi nng hoc dn cht ca chng v d PCMB (parachloromercuri - benzoate) hoc cho phn ng vi iodoacetate. Di tc dng ca cc cht nu, nhm SH ca enzyme s b kha v enzyme mt hot ng. - pht hin nhm imidazol ca Histidine i vi hot tnh ca enzyme ngi ta ph hu nhm ny bng phng php oxy ha quang hc

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vi s c mt ca xanh metylen hoc cho phn ng vi iodoacetate. Khi nhm chc ny b ph hu hoc b kha th enzyme u mt hot tnh. - tm hiu vai tr ca nhm - NH2 ca lysine c th cho phn ng vi fluordinitro benzen (FDNB) to thnh dinitrophenyl (DNP) mu vng. FDNB phn ng vi cc nhnh bn ca amino acid khc nh imidazol, phenol, thiol th to ra dn cht DNP khng mu. Cng c th c ch nhm - NH2 bng cch cho phn ng vi nhng yu t oxy ha. - Vai tr ca nhm - OH ca serine i vi hot tnh ca enzyme c thm d bng cch cho tc dng vi cht c ch c hiu l diisopropylfluor-phosphate (DFP). DFP s kha nhm - OH ca serine v enzyme mt hot tnh. Trong mt s trng hp cc nhm chc nng tham gia vo c ch xc tc c th c pht hin d dng hn so vi cc nhm ha hc khc cng loi, l nh kh nng phn ng tng ln do v tr c bit ca chng trong phn t enzyme. 4.5.2. Phng php nh du bng c cht c hiu hoc coenzyme - Nhiu ngi cho rng dng c cht c hiu nh du cc nhm chc nng l hp l nht song cng gp nhiu kh khn v phc hp c to thnh thng khng vng bn (phc hp enzyme - c cht d dng b phn ly ngc chiu, phc hp enzyme - sn phm ca phn ng cng phn ly vi tc cao). Tuy vy, bng phng php thc nghim khn kho ngi ta tch c cc sn phm trung gian ca phn ng kt hp vi enzyme bng lin kt ng ha tr vng bn. V d nh ngi ta tch c phosphoserine nh du trong trng hp ca phosphoglucosemutase qu trnh phn ng vn chuyn phosphore, hoc phc hp enzyme vi sn phm trung gian ca triosephosphate dehydrogenase. - Phng php nh du bng coenzyme cng c s dng v c gi tr trong mt s trng hp. Nh ngi ta nghin cu trung tm hot ng ca cytochrome C bng cch dng coenzyme heme lm cht nh du cng nh dng pyridoxal phosphate lm cht nh du nghin cu mt s enzyme cn coenzyme ny lm yu t phi hp. Cn lu l ch c th dng coenzyme, c cht hay cht ging c cht nh du nhng nhm chc nng trong trung tm hot ng ca enzyme khi no lin kt ha hc gia cht nh du vi nhng nhm ny

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vng bn hoc c bin thnh nhng lin kt vng bn bng phng php thch hp. Mt khc, khi c cht hoc coenzyme kt hp vi enzyme lm cho enzyme khng b c ch bi cc thuc th c hiu c th do enzyme hoc c cht kt hp vi cc nhm chc nng, hoc cng c th lm ngn cch mt cch c hiu gia cc nhm chc nng ca enzyme v thuc th. Nh vy, cc trng hp nu, cc nhm chc nng u c kt lun bng cch suy lun gin tip ch khng phi bng cc kt qu thc nghim trc tip. 4.5.3. Xc nh tr s pK ca cc nhm hot ng Enzyme l mt phn t protein c hiu c nhiu nhm ha hc c th b ion ha, nn enzyme c th tn ti di nhiu trng thi ion ha khc nhau; nhng thng ch c mt dng ion ca phn t enzyme c kh nng th hin hot tnh xc tc, trong chnh trng thi ion ha ca cc nhm hot ng ca enzyme c lin quan trc tip n qu trnh xc tc. Trng thi ion ha ca cc nhm khc khng c lin quan trc tip n c ch xc tc ca enzyme thng khng nh hng n hot tnh ca enzyme. Trng thi ion ha ca enzyme, ca c cht v ca phc hp enzyme-c cht chu nh hng trc tip ca pH ca mi trng phn ng. Do nghin cu nh hng ca pH i vi hot tnh enzyme c th tm c tr s pK ca nhm hot ng ca enzyme (K = hng s ion ha, pK = - logK), Vi tr s ca pK c th suy on cc nhm hot ng ca enzyme. Cng cn ch rng, phng php xc nh tr s pK ch l nhng ch dn s b v cn phi phi hp vi nhiu phng php khc. 4.5.4. Nghin cu cu trc phn t Cc phng php ha hc c hiu hoc xc nh tr s pK ch l nhng thng tin cn thit suy on v cha chng minh v vai tr ca cc nhm hot ng. V vy cn phi nghin cu cu to ha hc v cu trc khng gian ca phn t enzyme v c bit l ca trung tm hot ng. Bng cch ct b dn cc gc amino acid ca phn t enzyme ngi ta chng minh hot enzyme ch ph thuc vo mt s b phn nht nh ca phn t enzyme.V d papain l enzyme thy phn protein, khi b ct b 2/3 s gc amino acid vn cn hot ng, trung tm hot ng nm u nhm - COOH.

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4.6. Cc dng phn t ca enzyme

Trong cu trc phn t ca enzyme, tnh cht tinh vi v phc tp khng ch gii hn phm vi tng phn t, t thnh phn cu to v cc bc cu trc cho n cu to ca trung tm hot ng cng vi vai tr ca cc nhm chc nng m cn th hin tnh a dng ca cc phn t enzyme. Tnh a dng ca nhiu enzyme khc nhau c pht hin cc c th sng khc nhau t ngi, ng vt, thc vt n vi sinh vt. Ngi ta thy rng c nhng enzyme xc tc cng mt phn ng ha hc v c cng tnh c hiu c cht nhng c ngun gc khc nhau nn th hin nhiu tnh cht khc nhau. Aldolase c ngun gc t nm men c nhiu tnh cht khc vi aldolase ca m ng vt; pepsin, trypsin, chymotrypsin, xanthin - oxydase v lysozyme cng c nhng dng phn t khc nhau. Nhiu enzyme tng t nhau thu c t cng mt loi m nhng ca nhng loi khc nhau cng c nhng tnh cht khc bit nhau: - amylase ca dch nc bt v dch ty ca ngi th ging nhau, nhng chng khc vi - amylase thu c t ty ln v ha tan, v pH thch hp v mt s tnh cht khc. Nhng enzyme c ngun gc t nhng m khc nhau ca cng mt loi, tuy xc tc cng mt loi phn ng ha hc, nhng khc nhau rt r rt v tnh c hiu c cht nh trng hp ca nhng cholinesterase. Cc enzyme xc tc nhng phn ng chuyn ha ging nhau trong cc t bo ca nhiu m khc nhau c tnh cht c hiu c quan, v d lactat dehydrogenase ca c tim v c xng khc nhau r rt v tc di chuyn in di v nhiu tnh cht khc. Ngay trong mt m hay mt c quan, cng tn ti nhng dng phn t khc nhau: trong c tim t nht cng c hai dng phn t ca lactat dehydrogenase c tc di chuyn in di khc nhau, t nm men c th tch ra c bn dng phn t ca phosphoglyceraldehyde dehydrogenase. Trong mt t bo, mt enzyme no cng c th c nhng dng phn t khc nhau tn ti trong cc b phn khc nhau ca t bo, v d aspartat aminotransferase c dng phn t trong ty lp th khc vi dng phn t ca enzyme ny bo tng. Nh vy, tnh a dng ca cc phn t enzyme c th th hin nhiu mc , t cc loi khc nhau n cc m hay c quan khc nhau ca cng mt c th v ngay c cc b phn khc nhau ca cng mt t bo. Cc dng phn t khc nhau ca cng mt enzyme, tuy cng c chc nng xc tc mt phn ng ha hc ging nhau nhng v cu trc phn t ca chng u t nhiu c khc nhau, do chng c nhng tnh cht khc nhau v ha hc, vt l, min dch ... thm ch ngay c ng hc v tnh c hiu ca phn ng enzyme.

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Theo kin ngh chnh thc ca y ban thng trc v enzyme ca Hi Ha sinh Quc t, danh t isoenzyme c dng ch nhng dng phn t khc nhau ca mt enzyme tn ti trong mt loi; ngoi danh t ny, danh t isozyme cng c quen dng. Theo mt s tc gi, khi phn loi cc dng phn t khc nhau ca enzyme, phi phn bit gia isoenzyme v heteroenzyme. Danh t isoenzyme ch dnh cho nhng dng phn t ca mt enzyme c ngun gc t cng mt c quan v m cng nh phi c cng hot ng xc tc nh nhau; danh t heteroenzyme dnh cho nhng dng phn t cng c hot ng xc tc ging nhau nhng c th c ngun gc t cc c quan hoc loi khc nhau. Trong cc enzyme a dng trnh by, cc isoenzyme lactat dehydrogenase c nghin cu c it k do chng c ngha v mt chn on bnh. Chng c 5 dng phn t tetrame c cng trng lng phn t nh nhau c th phn chia c ring r bng in di thnh cc cu t rt khc nhau.

4.7. Phc hp multienzyme

Trong c th sng, ngoi cc enzyme polyme - nhng enzyme c cu trc bc bn, do nhiu n v nh cu to nn; cn tn ti h thng nhiu enzyme hay cn gi l h thng multienzyme. l nhng h thng gm cc enzyme c lin quan vi nhau, xc tc cho dy chuyn phn ng ca mt qu trnh trao i xc nh, trong sn phm ca phn ng do enzyme trc xc tc l c cht ca enzyme xc tc cho phn ng tip theo. C th minh ha h thng gm 3 enzyme E 1, E2, E3 xc tc cho dy chuyn phn ng nh sau:
E1 E2 E3

ABCD Trong s ny, A l c cht ca E1, B l sn phm ca phn ng do E1 xc tc nhng li l c cht ca E2 v.v... Cc h thng nhiu enzyme trong t bo c mc t chc phc tp khc nhau. Cc enzyme trong h thng nhiu enzyme c th tn ti ring r dng ha tan, khng lin kt vi nhau hoc c th kt t vi nhau, lin kt vi nhau kh bn to thnh phc hp nhiu enzyme khi tch ring khi phc hp enzyme, s mt hot tnh xc tc. Ngoi ra, mt s h thng

63

enzyme c th lin kt vi thnh phn cu to ca t bo nh mng ribosome. Cc enzyme ca chui h hp xc tc cho qu trnh chuyn in t t c cht n oxy, c gn cht vo mng trong ca ty th v thc cht l mt phn cu trc ca ty th. Ngi ta gp cc phc hp multienzyme trong nhiu qu trnh chuyn ha: qu trnh kh carboxyl oxy ha ca pyruvic acid to thnh acetyl coenzyme A, qu trnh tng hp acid bo ngoi ty lp th nm men, loi c v, loi chim..., qu trnh tng hp nhng peptide c hot tnh khng sinh nh gramicidine v tyrocidin, qu trnh tng hp tryptophan v.v...

TI LIU THAM KHO

Ti liu ting Vit
1. Nguyn Hu Chn, 1983. Enzyme v xc tc Sinh hc. Nxb Y hc, H Ni. 2. Phm Th Trn Chu, Trn Th ng, 2000. Ha sinh hc. Nxb Gio dc, H Ni. 3. Ngc Lin, Phm Th Trn Chu, 1972. Enzyme I, II. i hc Tng hp, H Ni. 4. Nguyn Tin Thng, Nguyn nh Huyn, 1998. Gio trnh sinh ha hin i. Nxb Gio dc, H Ni. 5. Nguyn Xun Thng, o Kim Chi, Phm Quang Tng, Nguyn Vn ng, 2004. Ha sinh hc. Nxb Y hc, H Ni. 6. L Ngc T, La Vn Ch, Phm Trn Chu, Nguyn Ln Dng, 1982. Enzyme vi sinh vt. Nxb KH&KT, H Ni. 7. L Ngc T (ch bin), L Vn Ch, ng Th Thu, Phm Quc Thng Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din, 2000. Ha sinh Cng nghip, Nxb KH&KT, H Ni.

Ti liu ting nc ngoi

1. Bermeyer H. U, Bermeyer J. and Grasel M. (editors). 1983. Methods of enzymatic analysis. Vol II. Verlag chemie Weinheim. 2. Lehringer A. L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004. 3. Pelmont J., 1993. Enzymes. Presses universitaires de grenobe. 4. Stryer L., 1981. Biochemistry. W.H.Freeman and company. San Francisco. 5. Biochemical information, 1973. Boehringer Mannheim GmbH. Biochemica.

64

Chng 5

Tnh c hiu ca enzyme

5.1. Khi nim chung
Do cu trc l ha c bit ca phn t enzyme v c bit l ca trung tm hot ng m enzyme c tnh c hiu rt cao so vi nhng cht xc tc thng thng khc. Mi enzyme ch c kh nng xc tc cho s chuyn ha mt hay mt s cht nht nh theo mt kiu phn ng nht nh. c tnh tc dng la chn cao ny gi l tnh c hiu hoc tnh chuyn ha ca enzyme. Tnh c hiu l mt trong nhng c tnh c bn quan trng nht ca enzyme.

5.2. Cc hnh thc c hiu

C th phn bit hai kiu c hiu: c hiu kiu phn ng v c hiu c cht. 5.2.1. c hiu kiu phn ng Phn nhiu mi enzyme u c tnh c hiu vi mt loi phn ng nht nh. Nhng cht c kh nng xy ra nhiu loi phn ng ha hc th mi loi phn ng y phi do mt enzyme c hiu xc tc. V d, amino acid c kh nng xy ra phn ng kh carboxyl, phn ng kh amin bng cch oxy ha v phn ng vn chuyn nhm amin, v vy mi phn ng y cn c mt enzyme c hiu tng ng xc tc theo th t l decarboxylase, aminoacid oxydase v aminotransferase. 5.2.2. c hiu c cht Mi enzyme ch xc tc cho s chuyn ha mt hoc mt s cht nht nh. Mc c hiu c cht ca cc enzyme khc nhau khng ging nhau, ngi ta thng phn bit thnh cc mc nh sau: - c hiu tuyt i Mt s enzyme hu nh ch xc tc cho phn ng chuyn ha mt c cht xc nh v ch xc tc cho phn ng y m thi. V d: Urease, arginase, glucoseoxydase v.v... i vi cc enzyme ny, ngoi cc c cht c hiu ca chng l ure, arginine, - D - Glucose (theo th t tng ng) chng cng c th phn gii mt vi cht khc nhng vi vn tc thp hn nhiu. Chng hn nh urease, ngoi ure n

65

cn c th phn gii hydroxyure nhng vi tc thp hn 120 ln. Nh vy urease c th xc tc cho hai phn ng sau: H2N \ C=0 + H2O CO2 + 2NH3 urease / H2N ure HOHN \ C = 0 + H2O / H2N hydroxyure

urease

NH2OH + NH3 + CO2 hydroxyamin

i vi trng hp glucose oxydase: enzyme ny c trong cc loi nm mc, c kh nng oxy ha c hiu -D-glucose thnh gluconic acid -D-glucose gluconic acid Enzyme ny c kh nng phn gii 10 c cht song vi kh nng nh hn nhiu. V d: nu coi tc oxy ha tng i acid -D-glucose l 100% th .D.glucose ch bng 0,64 % (ngoi ra maltose 0,19%, D.galactose 0,14%). Hnh nh trong trng hp c hiu tuyt i, cu trc trung tm hot ng ca enzyme tng ng rt cht ch vi cu trc ca c cht n mc ch mt sai khc nh v cu trc ca c cht cng lm cho enzyme khng xc tc c. Nhng enzyme c tnh c hiu tuyt i thng c dng nh lng chnh xc c cht ca n. - c hiu nhm tuyt i Cc enzyme ny ch tc dng ln nhng cht c cng mt kiu cu trc phn t, mt kiu lin kt v c nhng yu cu xc nh i vi nhm nguyn t phn lin kt chu tc dng. V d: maltase thuc nhm glucosidase ch xc tc cho phn ng thy phn lin kt glucoside c to thnh t nhm OH glucoside ca - glucose vi nhm OH ca mt monose khc. Glucose oxydase

66

- c hiu nhm tng i Mc c hiu ca cc enzyme thuc nhm ny km hn nhm trn. Enzyme c kh nng tc dng ln mt kiu lin kt ha hc nht nh trong phn t c cht m khng ph thuc vo cu to ca cc phn tham gia to thnh mi lin kt . V d lipase c kh nng thy phn c tt c cc mi lin kt este. Aminopeptidase c th xc tc thy phn nhiu peptid - c hiu quang hc (c hiu lp th) Hu nh tt c cc enzyme u c tnh c hiu khng gian rt cht ch, ngha l enzyme ch tc dng vi mt trong hai dng ng phn khng gian ca c cht. Enzyme ch tc dng vi mt trong hai dng ng phn quang hc ca cc cht. V d phn ng kh nc ca malic acid to thnh fumaric acid di tc dng ca fumarathydratase ch xy ra i vi L malic acid m khng tc dng ln D - malic acid : COOH CH COOH | || fumarate HO CH HOOC CH | hydratase CH2 | COOH L-malic acid Fumaric acid Enzyme cng th hin tnh c hiu ln mt dng ng phn hnh hc cis hoc trans. V d: enzyme fumarathydratase ch tc dng ln dng trans ca fumaric acid m khng tc dng ln dng cis to thnh L malic acid : COOH | fumarate HOOC CH HO CH || hydratase | CH2 CH COOH | COOH fumaric acid (dng trans) L-malic acid

67

Trong t nhin cng c cc enzyme xc tc cho phn ng chuyn ha tng h gia cc cp ng phn khng gian tng ng. V d, lactatracemase ca vi khun xc tc cho phn ng chuyn ha ln nhau gia D v L lactic acid, aldo - 1 - epimerase xc tc cho phn ng ng phn ha - D - glucose thnh - D - Glucose, maleinat cis - trans isomerase ca vi khun xc tc cho phn ng ng phn ha gia maleic acid (dng cis) v fumaric acid (dng trans)v.v... Cc enzyme ny c vai tr quan trng khi sn xut cc cht dinh dng bng phng php ha hc, v chng c th chuyn cc cht t dng c th khng th s dng c thnh dng c th hp th. Enzyme cn c kh nng phn bit c 2 gc i xng trong phn t ging nhau hon ton v mt ha hc. V d, hai nhm - CH2OH trong phn t glycerin, glycerophosphatkinase xc tc cho phn ng chuyn v gc phosphate t ATP n C3 ca glycerin (ch khng phi C1).

68

TI LIU THAM KHO

Ti liu ting Vit
1. Nguyn Hu Chn, 1983. Enzyme v xc tc Sinh hc. Nxb Y hc, H Ni. 2. Phm Th Trn Chu, Trn Th ng, 2000. Ha sinh hc. Nxb Gio dc, H Ni. 3. Ngc Lin, Phm Th Trn Chu, 1972. Enzyme I, II. i hc Tng hp, H Ni. 4. Nguyn Tin Thng, Nguyn nh Huyn, 1998. Gio trnh sinh ha hin i. Nxb Gio dc, H Ni. 5. Nguyn Xun Thng, o Kim Chi, Phm Quang Tng, Nguyn Vn ng, 2004. Ha sinh hc. Nxb Y hc, H Ni. 6. L Ngc T, La Vn Ch, Phm Trn Chu, Nguyn Ln Dng, 1982. Enzyme vi sinh vt. Nxb KH&KT, H Ni. 7. L Ngc T (ch bin), L Vn Ch, ng Th Thu, Phm Quc Thng Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din, 2000. Ha sinh Cng nghip, Nxb KH&KT, H Ni.

Ti liu ting nc ngoi

1. Bermeyer H. U, Bermeyer J. and Grasel M. (editors). 1983. Methods of enzymatic analysis. Vol II. Verlag chemie Weinheim. 2. Lehringer A. L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004. 3. Pelmont J., 1993. Enzymes. Presses universitaires de grenobe. 4. Stryer L., 1981. Biochemistry. W.H.Freeman and company. San Francisco. 5. Biochemical information, 1973. Boehringer Mannheim GmbH. Biochemica.

69

Chng 6

C ch tc dng ca enzyme
6.1. C ch ca phn ng c xc tc ni chung
Vn tc phn ng ha hc c xc nh bi gi tr nng lng hot ha tc l mc nng lng cc cht tham gia phn ng phi t c ct t lin kt cn thit v hnh thnh cc lin kt mi. Nng lng hot ha cng ln th vn tc phn ng cng chm v ngc li. Do lm gim nng lng hot ha phn ng, cc cht xc tc c tc dng thc y vn tc phn ng ha hc. V d, bt platin l mt cht xc tc ha hc c s dng rng ri. V cc cht tham gia phn ng trn b mt platin u c chuyn sang trng thi c kh nng phn ng cao hn. Do vy nng lng hot ha s nh hn v tc phn ng s cao hn. Nh vy, trong cc phn ng c xc tc, cht xc tc lm gim nng lng hot ha ca phn ng ha hc, c ngha l n ch tham gia vo cc phn ng trung gian m khng ng vai tr l cht tham gia phn ng. Sau phn ng, cht xc tc li phc hi v trng thi ban u tip tc xc tc.

6.2. C ch ca xc tc enzyme
Hu nh tt c cc bin i ha sinh trong t bo v c th sng u c xc tc bi enzyme pH trung tnh, nhit v p sut bnh thng trong khi a s cc cht xc tc ha hc khc li ch xc tc nhit v p sut cao. Chnh nh vic to c mi trng c hiu (bi trung tm hot ng ca enzyme lin kt vi c cht) c li nht v mt nng lng thc hin phn ng m enzyme c c nhng kh nng c bit nu trn. Trong phn ng c s xc tc ca enzyme, nh s to thnh phc hp trung gian enzyme - c cht m c cht c hot ha. Khi c cht kt hp vo enzyme, do kt qu ca s cc ha, s chuyn dch ca cc electron v s bin dng ca cc lin kt tham gia trc tip vo phn ng dn ti lm thay i ng nng cng nh th nng, kt qu l lm cho phn t c cht tr nn hot ng hn, nh tham gia phn ng d dng.

70

Nng lng hot ha khi c xc tc enzyme khng nhng nh hn rt nhiu so vi trng hp khng c xc tc m cng nh hn so vi c trng hp c cht xc tc thng thng. V d trong phn ng phn hy H2O2 thnh H2O v O2 nu khng c cht xc tc th nng lng hot ha l 18 Kcal/mol, nu c cht xc tc l platin th nng lng hot ha l 11,7 Kcal/mol, cn nu c enzyme catalase xc tc th nng lng hot ha ch cn 5,5 Kcal/mol. Nhiu dn liu thc nghim cho thy qu trnh to thnh phc hp enzyme c cht v s bin i phc hp ny thnh sn phm, gii phng enzyme t do thng tri qua ba giai on theo s sau. E + S ES P + E [Trong E l enzyme, S l c cht (Substrate), ES l phc hp enzyme - c cht, P l sn phm (Product) - Giai on th nht: enzyme kt hp vi c cht bng lin kt yu to thnh phc hp enzyme - c cht (ES) khng bn, phn ng ny xy ra rt nhanh v i hi nng lng hot ha thp; - Giai on th hai: xy ra s bin i c cht dn ti s ko cng v ph v cc lin kt ng ha tr tham gia phn ng. - Giai on th ba: to thnh sn phm, cn enzyme c gii phng ra di dng t do. Cc loi lin kt ch yu c to thnh gia E v S trong phc hp ES l: tng tc tnh in, lin kt hydrogen, tng tc Van der Waals. Mi loi lin kt i hi nhng iu kin khc nhau v chu nh hng khc nhau khi c nc. Vi phng php nghin cu bng tia X v phng php ha hc ngi ta lm sng t cch thc gn c cht v c ch hot ng ca mt s enzyme nh lysozyme, chymotrypsin, carboxypeptidase A v.v... Sau y s gii thiu chi tit hn c ch phn ng ca carboxypeptidase A. Carboxypeptidase A (EC 3.4.17.1) thuc nhm peptidhydrolase, xc tc cho s thy phn lin kt peptid, phn ng xy ra vi vn tc ln nu amino acid u C l amino acid thm. enzyme ny cng thy phn lin kt este. Carboxypeptidase A c khi lng phn t 34,3. KDa cha 1 mol Zn/1 mol E. Zn tham gia trong hot ng xc tc ca enzyme. Khi thay th Zn bng cc kim loi ha tr hai khc lm thay i hot v c th c

71

tnh c hiu ca enzyme. Trong phn t enzyme, Zn gn b mt phn t, tng tc vi gc His - 69, His - 196 v Glu - 72. Cc gc amino acid c vai tr xc tc trong trung tm hot ng ca enzyme l: Arg - 145, Tyr - 248 v Glu - 270. C ch phn ng xc tc ca Carboxypeptidase A c xc nh trn c s kt qu nghin cu phn ng ca n vi dipeptid glycyltyrosine. Qu trnh phn gii lin kt peptid c th c phn thnh cc bc sau: - To thnh phc ES: Khi tip xc vi c cht, cc nhm trong trung tm hot ng ca enzyme thay i v tr trong khng gian. Nhm guanidin ca Arg - 145 cng nh nhm carboxyl ca Glu - 270 dch chuyn 2, nhm hydroxyl ca Tyr - 248 dch chuyn 12 t ch gn trn b mt phn t chuyn vo trong n vng gn vi lin kt peptid ca c cht. Tng tc gia cc nhm chc ca trung tm hot ng vi glycyltyrosine nh sau: (Hnh 6.1)
Vng khng phn cc trong phn t enzyme

Hnh 6.1. S biu din tng tc gia glycyltyrosine vi cc nhm chc nng trong trung tm hot ng ca carboxypeptidase A (c cht vit nt m)

- Nhm carboxyl t do ca c cht kt hp vi nhm tch in dng ca Arg - 145 ca enzyme qua lin kt ion. - Nhm NH trong lin kt peptide ca c cht to thnh lin kt hydrogen vi nhm - OH ca Tyr - 248.

72

- Oxy trong nhm - CO - ca lin kt peptide tng tc vi Zn, cn carbon trong nhm - CO - ny tng tc vi nhm carboxyl ca Glu - 270 qua phn t nc. - Ct t lin kt gii phng sn phm. Nguyn t Zn phn cc lin kt - CO, tng tnh i in t ca nguyn t carbon, do lm tng tng tc ca n vi nc hoc vi nhm i nhn ca phn t protein enzyme. Gc Glu - 270 hot ha phn t nc, nhm - OH c to thnh tn cng trc tip vo nguyn t cacbon ca - CO - (trong lin kt peptide ca c cht), lin kt peptid b ko cng ra v b t. Gc Tyr - 248 nhng hydrogen cho nhm NH trong lin kt pepti cho c cht, gii phng sn phm u tin l tyrosine ca c cht v acyl enzyme (hnh 6.2).

Hnh 6.2. C ch phn ng xc tc ca carboxypeptidase A

Sau khi lin kt peptide b ct t, trng thi ion ha ca cc nhm acid v base b bin i tng ng vi pH mi trng, Tyr - 248 kt hp vi proton, tr v trng thi ban u.

73

TI LIU THAM KHO

Ti liu ting Vit
1. Nguyn Hu Chn, 1983. Enzyme v xc tc Sinh hc. Nxb Y hc, H Ni.
2. Phm Th Trn Chu, Trn Th ng, 2000. Ha sinh hc. Nxb Gio dc, H Ni.

3. Ngc Lin, Phm Th Trn Chu, 1972. Enzyme I, II. i hc Tng hp, H Ni. 4. Nguyn Tin Thng, Nguyn nh Huyn, 1998. Gio trnh sinh ha hin i. Nxb Gio dc, H Ni. 5. Nguyn Xun Thng, o Kim Chi, Phm Quang Tng, Nguyn Vn ng, 2004. Ha sinh hc. Nxb Y hc, H Ni. 6. L Ngc T, La Vn Ch, Phm Trn Chu, Nguyn Ln Dng, 1982. Enzyme vi sinh vt. Nxb KH&KT, H Ni. 7. L Ngc T (ch bin), L Vn Ch, ng Th Thu, Phm Quc Thng Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din, 2000. Ha sinh Cng nghip, Nxb KH&KT, H Ni.

Ti liu ting nc ngoi

1. Bermeyer H. U, Bermeyer J. and Grasel M. (editors). 1983. Methods of enzymatic analysis. Vol II. Verlag chemie Weinheim. 2. Lehringer A. L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004. 3. Pelmont J., 1993. Enzymes. Presses universitaires de grenobe. 4. Stryer L., 1981. Biochemistry. W.H.Freeman and company. San Francisco. 5. Biochemical information, 1973. Boehringer Mannheim GmbH. Biochemica.

74

Chng 7

ng hc Enzyme
7.1. ngha ca vic nghin cu ng hc enzyme
Nghin cu ng hc enzyme l nghin cu nh hng ca cc yu t: nng c cht, enzyme, pH mi trng, nhit , cc cht km hm n tc phn ng do enzyme xc tc. Vic nghin cu ng hc enzyme s cho ta bit c cc vn sau y: - C th bit c c ch phn t ca s tc ng ca enzyme. - Cho php ta hiu bit c mi quan h v mt lng ca qu trnh enzyme. - Thy c vai tr quan trng c v mt l lun ln thc tin: khi la chn cc n v hot ng enzyme ngi ta cn phi bit nhng iu kin tt nht i vi hot ng ca enzyme, cng nh cn phi bit c cc yu t nh hng n hot ng ca chng. - L iu kin cn thit thc hin tt cc bc tinh ch enzyme, v ngi ta cn phi kim tra v mt lng bng cch xc nh c h thng hot ng ca ch phm enzyme trong cc giai on tinh ch.

7.2. ng hc cc phn ng enzyme

7.2.1. nh hng ca nng enzyme Trong iu kin d tha c cht, ngha l [S] >>[E] th tc phn ng ph thuc vo [S], v= K[E] c dng y=ax. Nh ngi ta o [E] bng cch o vn tc phn ng do enzyme xc tc. C nhiu trng hp trong mi trng c cha cht km hm hay hot ha th vn tc phn ng do enzyme xc tc khng ph thuc tuyn tnh vi [E] . v

[E]
Hnh 7.1. S ph thuc ca vn tc phn ng vo [E]

75

7.2.2. nh hng ca nng c cht [S] Ta kho st trng hp n gin nht: ch mt c cht k1 E + S k-1 Gi v1 l vn tc ca phn ng to thnh phc cht ES. Gi v-1 l vn tc ca phn ng to phn ly phc cht ES to thnh E v S. Gi v2 l vn tc ca phn ng to thnh E v P (sn phm). v1 = k1[E][S] v-1 = k-1[ES] v2 = k2[ES] Khi h thng t trng thi cn bng ta c: k-1[ES]+k2[ES] = k1[E][S] (k-1+k2)[ES] = k+1[E][S] (2) Gi E0 l nng ban u: [E0]=[E]+[ES]=>[E]=[E0]-[ES] (3) Thay tr s [E] t (3) vo (2) ta c: (k-1+k2)[ES] = k1([E0]-[ES]) [S] k1 [E0] [S] [ES] = -------------k-1+ k2+k1[S] Nu t Km= k-1+k2/ k1 (Km: gi l hng s Michalis Menten) Ta c: [ES] = [E0][S]/ Km+[S] Mt khc vn tc phn ng to thnh sn phm P l: V = k2[ES] ES k2 E + P (1)

76

Thay [ES] bng gi tr trn ta thu c: k2[E0] [S] v = ----------------- (4) Km + [S] Qua y ta thy nng enzyme cng cao th vn tc phn ng enzyme cng ln. Vn tc t cc i khi ton b enzyme lin kt vi c cht, ngha l: Vmax= k2[E0] Thay vo phng trnh (4) ta c: [S] v = Vmax ------------- (5) Km+ [S] Phng trnh (5) gi l phng trnh Michelis Menten Km gi l hng s Michelis Menten c trng cho mi enzyme. Km c trng cho i lc ca enzyme vi c cht, Km c tr s cng nh th i lc ca enzyme vi c cht cng ln, ngha l vn tc ca phn ng do enzyme xc tc cng ln.

Hnh 7.2. Bin thin vn tc phn ng theo nng c cht

77

Khi tng [S] th v phn ng tng, tng [S] n mt gi tr no th v t n gi tr vmax v s khng tng na nu ta vn tip tc tng [S]. Khi Km = [S] th v0 =1/2 Vmax Nm 1934. Lineweaver v Burk, trn c s ca phng trnh (5) nghch o bin thnh dng ng thng y = ax+b, n c ngha ln i vi vic nghin cu km hm enzyme.

1/v

1/Vmax -1/Km 1/[S]

Hnh 7.3. S ph thuc ca tc phn ng vo nng c cht theo Lineweaver-Burk

Trong nhiu phn ng do enzyme xc tc c 2 hay nhiu c cht, v d hexokinase xc tc phn ng: ATP + glucose hexokinase ADP + glucose 6 phosphate C ch enzyme xc tc cho phn ng 2 c cht c th nh sau: a/ C ch to phc 3 thnh phn S2

78

b/ C ch khng to phc 3 thnh phn

y l trng hp c cht th 2 (S2) ch kt hp vo enzyme ( trng thi E) sau khi P1 c to thnh. Vn tc ca phn ng trong trng hp ny c th c phn bit qua hng s Michalis-Menten i vi mi c cht. Qua nghin cu ng hc cho thy: Enzyme
(No)

C cht

Km(mM)

Phn ng 2 c cht (bisubstrate) thng vn chuyn 1 nguyn t hay 1 nhm chc t c cht ny n c cht khc. Khi cho S2 khng i, ng biu din tc trong c hai trng hp

79

(a): to phc 3 thnh phn

(b): khng to phc 3 thnh phn

7.2.3. nh hng ca cht km hm (inhibitior) L cht c tc dng lm gim hot hay lm enzyme khng cn kh nng xc tc bin c cht thnh sn phm. N c th l cht km hm thun nghch hay bt thun nghch. Km hm thun nghch (reversible inhibition) c th l cnh tranh (competitive), phi cnh tranh (uncompetitive) hay hn tp (mixed). * Cch 1: Km hm cnh tranh (Competitive inhibition) Trong trng hp km hm cnh tranh l c cht v cht km hm u tc dung ln trung tm hot ng ca enzyme, Cht km hm chon ch ca c cht enzyme.

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Hnh 7.4. Kiu km hm cnh tranh (competitive inhibition)

Khi c cht d tha, nng cht km hm thp th c th loi b tc dng ca cht km hm, cn nng c cht thp v nng cht km hm cao th li c tc dng km hm hon ton. 1/v= (Km/Vmax) 1/S +1/Vmax = 1+[I]/KI

1/v [I]

khng c cht km hm 1/Vmax 1/[S]

Hnh 7.5. S ph thuc ca tc phn ng vo nng c cht theo Lineweaver - Burk khi c km hm canh tranh

Ngi ta thy km hm nh vy phn ln gia cht km hm v c cht c s tng ng v mt ha hc. v d: malic acid c cu trc gn ging vi succinic acid nn km hm cnh tranh enzyme succinatdehydrogenase, l enzyme xc tc cho s bin i succinic acid thnh fumaric acid.

81

Trng hp c bit ca km hm cnh tranh l km hm bng sn phm. Trng hp ny xy ra khi mt sn phm phn ng tc dng tr li enzyme v chon v tr hot ng phn t enzyme. ng thng c cht km hm th c xin ln hn v ct trc tung mt im l 1/Vmax * Cch 2: Km hm phi cnh tranh (Uncompetitive inhibition) c trng ca kiu km hm ny l cht km hm ch lin kt vi phc hp ES, m khng lin kt vi enzyme t do.

1/v=(Km/Vmax)1/[S] + /Vmax 1/v [I]

1/Km

khng c cht km hm

82

1/[S]
Hnh 7.6. S ph thuc ca tc phn ng vo nng c cht theo Lineweaver - Burk khi c km hm phi cnh tranh

* Cch 3: Km hm hn tp (Mixed inhibition)

Trong , cht km hm khng nhng lin kt vi enzyme t do m cn lin kt vi c phc hp ES to thnh phc hp EIS khng to c sn phm P. Hin tng km hm ch ph thuc vo nng cht km hm. Tc cc i o c khi khng c mt cht km hm l cao hn khi c mt cht km hm. Gi tr Km thay i khng ging nh trong trng hp cnh tranh. Tng t nh trn ta c phng trnh : 1/v = (Km/Vmax)1/[S] +/vmax 1/v [I]

khng c cht km hm

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1/[S]
Hnh 7.7. S ph thuc ca tc phn ng vo nng c cht theo Lineweaver - Burk khi c km hm hn tp

Cc gi tr , c nh ngha nh trn. Trng hp = gi l khng cnh tranh (noncompetitive).

Bng 7.1. nh hng ca kiu km hm ln Vmax v Km Cnh tranh (Competitive) Phi cnh tranh (Uncompetitive) gim gim Hn tp (Mixed) gim tng Khng cnh tranh (Noncompetitive) gim khng nh hng

Vmax khng nh hng Km tng

Trng hp km hm enzyme bng nng cao ca c cht gi l km hm c cht nh km hm urease khi nng ure cao, ngoi ra cn c cc enzyme khc nh lactatdehydrogenase, carbonxypeptidase, lipase, pyrophotphatase, photphofructokinase (i vi ATP). Nguyn nhn ca nhng hin tng ny cn cha c bit r. c th l: + Tn ti nhiu trung tm lin kt vi c cht bng cc i lc khc nhau. Khi nng c cht thp th enzyme c th ch lin kt vi mt phn t c cht, cn khi nng c cht cao n lin kt vi nhiu c cht dn n hnh thnh phc hp ES khng hot ng. + C cht cng c th c lin kt nh nhng v tr c bit ca enzyme. l mt nhm enzyme quan trng (enzyme d lp th) bn cnh trung tm xc tc cn c trung tm iu chnh. + C cht c th lin kt vi mt cht hot ha v bng cch ny n tch khi E. + C cht c th chon ch (ngn cn) mt cofactor hay mt coenzyme. + C cht c th nh hng n ion lc ca mi trng v qua lm mt i tnh chuyn ha ca enzyme. Km hm bt thun nghch (irreversible inhibition) Nhiu trng hp, cht km hm c tc dng bt thun nghch. i khi kh phn bit gia thun nghch v bt thun nghch v cht km

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hm bt thun nghch c th hiu nh cht km hm thun nghch khng cnh tranh (noncompetitive). Nhn chung hiu qu km hm ph thuc cc yu t: nng cht km hm, nng enzyme , thi gian tc dng. Sau y ta xt cc c ch tng tc bt thun nghch trong iu kin nng [I]>>[E]. 1/ Trng hp 1

(1) (2) T (2) suy ra [E] th vo (1), ng thi thay [I] bng [I0] :

Trong Ly tch phn ta c

Bng cch ly tch phn phng trnh tc , c th nhn c biu thc m t s bin i nng cht phn ng hay sn phm theo thi gian. iu ny cc k hu dng trong vic xc nh hng s tc v bc phn ng. 2/ Trng hp 2

Mt khc:

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Nng enzyme t do s l:

Thay vo trn v ng thi thay [I] bng [I0] :

Trong

Tng t nh trn ta c:

3/ Trng hp 3 Enzyme v I nhanh chng tng tc to phc thun nghch ES, sau tip tc to phc bt thun nghch

Thay [E] vo trn ta c

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4/ Trng hp 4

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7.2.4. nh hng ca cht hot ha (activator) L cht lm tng kh nng xc tc chuyn ha c cht thnh sn phm. Thng thng l nhng cation kim loi hay nhng hp cht hu c nh cc vitamin tan trong nc. V d: Mg++ hot ha cc enzyme m c cht c phosphoryl ha nh pyrophosphatase (c cht l pyrophosphate), adenosintriphosphatase (c cht l ATP). Cc cation kim loi c th c tnh c hiu, tnh i khng v tc dng cn tu thuc vo nng . Tnh cht hot ha ca cc cation kim loi: + Mi cation kim loi hot ha cho mt kiu phn ng nht nh. + Cation kim loi c tnh c hiu tng i hay tuyt i. + Cation kim loi c th c s i khng ion. + Ph thuc nng cation kim loi . + Cation kim loi lm thay i pH ti thch. + Ph thuc bn cht cation kim loi. 7.2.5. nh hng ca nhit Ta c th tng vn tc ca mt phn ng ha hc bng cch tng nhit mi trng, hin tng ny tun theo quy lut Vant -Hoff. iu ny c ngha khi tng nhit ln 100C th tc phn ng tng ln 2 ln. Hot

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Nhit (C)
Hnh 7.8. nh hng ca nhit ln hot enzyme

i vi phn ng do enzyme xc tc cng c th p dng c quy lut ny nhng ch trong mt phm vi nht nh,v bn cht enzyme l protein.Khi ta tng nhit ln trn 40-500C xy ra qu trnh ph hy cht xc tc. Sau nhit ti thch tc phn ng do enzyme xc tc s gim. Nh tn ti nhit ti u ngi ta phn bit phn ng ha sinh vi cc phn ng v c thng thng. Mi enzyme c mt nhit ti thch khc nhau, phn ln ph thuc ngun cung cp enzyme, thng thng trong khong t 40-60 0C, cng c enzyme c nhit ti thch rt cao nh nhng enzyme ca nhng chng a nhit. 7.2.6. nh hng ca pH S phn li khc nhau ca mt phn t protein cc gi tr pH khc nhau lm thay i tnh cht ca trung tm lin kt c cht v hot ng phn t enzyme, dn n gi tr xc tc khc nhau ph thuc vo gi tr pH. Nh bit mi enzyme c mt pH ti thch, mi enzyme c ng biu din nh hng pH ln vn tc ca phn ng do enzyme xc tc c dng nh hnh 7.9:

Hnh 7.9. nh hng pH ln hot enzyme

nh hng ca gi tr pH n tc dng enzyme c th do cc c s sau: a/ Enzyme c s thay i khng thun nghch phm vi pH cc hp.

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b/ hai sn ca pH ti thch c th xy ra s phn ly nhm prosthetic hay coenzyme. c/ Lm thay i mc ion ha hay phn ly c cht. d/ Lm thay i mc ion ha nhm chc nht nh trn phn t enzyme dn n lm thay i i lc lin kt ca enzyme vi c cht v thay i hot tnh cc i. Nh xc nh Vmax v Km ph thuc gi tr pH cho php nhn nh li bn cht ca cc nhm tham gia vo lin kt c cht v qu trnh t xc tc. 7.2.7. Cc yu t khc + nh sng: C nh hng khc nhau n tng loi enzyme, cc bc sng khc nhau c nh hng khc nhau, thng nh sng trng c tc ng mnh nht, nh sng c tc ng yu nht. nh sng vng t ngoi cng c th gy nn nhng bt li, enzyme trng thi dung dch bn hn khi c kt tinh dng tinh th, nng enzyme trong dung dch cng thp th cng km bn, tc ng ca tia t ngoi s tng ln khi nhit . V d: di tc ng ca tia t ngoi nhit cao, enzyme amylase nhanh chng mt hot tnh. + S chiu in: in chiu vi cng cng cao th tc ng ph hy cng mnh. Tc ng s mnh hn i vi dch enzyme c nng thp. C th do to thnh nhng gc t do, t tn cng vo phn ng enzyme. + Sng siu m: Tc ng rt khc nhau i vi tng loi enzyme, c enzyme b mt hot tnh, c enzyme li khng chu nh hng. * Nhn xt chung: bn ph thuc vo trang thi ca tn ti enzyme, cng tinh khit th enzyme cng km bn, dch cng long th bn cng km, tc ng ca mt s ion kim loi trong dch vi nng khong 10-3M nh Ca++ lm tng tnh bn. Enzyme allosteric (d lp th, d khng gian) Cho n nay, ngi ta m t enzyme m hot tnh enzyme ph thuc nng c cht khng c dng hyperbol m c dng sigmoid l enzyme d lp th (hnh 7.10).

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i vi enzyme ny, khi nng c cht thp th tc phn ng tng chm, sau tip tc tng nng th tc nhanh chng t gi tr cc i. Nh ta bit, enzyme tun theo ng hc Michalis-Menten th 1 hay nhiu c cht cng ch lin kt vo 1 v tr trn phn t enzyme, iu ny s dn n enzyme bo ha c cht. Cn enzyme c ng cong tc sigmoid ch xut hin khi enzyme l mt oligomer, nn c th lin kt vi nhiu phn t c cht. iu ny c ngha l trn enzyme d lp th c nhiu trung tm lin kt, mi monomer c 1 trung tm lin kt.

Hnh 7.10. Bin thin vn tc phn ng theo nng c cht

Ngi ta cho rng, trong trng hp ny c tnh hp tc gia cc v tr lin kt c cht trong phn t enzyme oligomer. Cc enzyme oligomer ny c Monod gi l enzyme d lp th (allosteric). ng cong tc sigmoid c th b cht iu ha (modulator) y v pha tri hay phi. Cht iu ha dng tc lm tng i lc ca enzyme allosteric vi c cht, ngc li l cht iu ha m. Cc cht iu ha c th lm nh hng khc nhau n cc thng s ng hc, lm thay i gi tr ring l mt trong hai gi tr Km hay Vmax .

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Hnh 7.11. Minh ha khi c modulator

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TI LIU THAM KHO

Ti liu ting Vit
1. Phm Th Trn Chu, Trn thi ng. 1999. Ho sinh hc, NXB Gio dc, H Ni. 2. Qu Hai. 2004. Chuyn enzyme, Ti liu lu hnh ni b Trng HKH Hu. 3. Trn Thanh Phong. 2004. Chuyn enzyme, Ti liu lu hnh ni b Trng HKH Hu.

Ti liu ting nc ngoi

1. Bergmeyer H. U. 1968. Methods of enzymatic analysis, translated from the third German edition, Academic Press, New York. 2. Copeland R. A. 2000. Enzymes,copyright by Wiley-VCH, Inc. 3. Gilbert H. F. 1992. Basic concepts in biochemistry, Copyright by the Mcgraw- Hill companies, Inc. 4. Lehninger A. L. 2004. Principles of Biochemistry, 4th Edition. W.H Freeman.

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Chng 8

Sinh hc enzyme
8.1. S phn b enzyme trong t bo
Nh trnh by phn trc, enzyme c trong tt c cc c th ng vt, thc vt v vi sinh vt. Tuy vy, s phn b enzyme khng ng u gia cc loi, cc t bo m v c quan khc nhau. Ngi ta thy c nhng enzyme tn ti hu ht mi m mi t bo: Nh cc enzyme xc tc cho qu trnh ng phn, sinh tng hp protein, nucleic acid. Mt s enzyme khc ch c trong mt s c quan ring bit, v d nh pepsin ch c trong d dy. l enzyme c bit, c trng cho mt m. Mt khc, cng mt enzyme c trong cc m khc nhau hoc thm ch cc b phn khc nhau ca cng mt loi t bo cng c th khc nhau v lng v c khi c v cht. Hm lng enzyme trong mt m hoc mt c quan nht nh cn ph thuc vo nhiu yu t khc nh: Giai on sinh trng v pht trin, trng thi sinh l ca t bo, cc yu t bn ngoi v.v... Mi loi cu trc di t bo ca c th bc cao nh nhn t bo, ty lp th, lysosome, h thng li ni cht nguyn sinh vi cc ht ribosome... u c cu trc v chc nng ring vi nhng h enzyme c hiu. Nhng enzyme ny hoc ho tan trong dch lng, hoc gn cht vo cc mng ca cc cu trc . Do cu trc c bit nh vy ca t bo, enzyme c phn b thnh tng ngn c hiu. S khu tr v sp t cc enzyme mt cch hp l trong cc cu trc ca t bo lm cho cc phn ng enzyme c tnh cht nh hng, c phi hp tc dng vi nhau v to ra nhng h thng phn ng dy chuyn lin tc, nhp nhng v n khp vi nhau. Trong nhn t bo c th thy cc enzyme thuc cc nhm khc nhau xc tc cho cc qu trnh khc nhau. l cc enzyme nicotinicmono-nucleotide adenylyl transferase, 5-nucleotidase, NAD(P) nucleosidase, arginase, ATP-ase v mt s enzyme khc. Ni chung trong nhn cha nhiu enzyme lin quan n qu trnh trao i nucleotide, trong cc enzyme tham gia cc qu trnh trao i cc hp cht c tnh cht cha kha. Nhng enzyme trong nhn t bo thng c mt vi lng rt nh. Vic nghin cu nhng enzyme ny thng gp nhiu kh khn v

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trong qu trnh thao tc, mt s enzyme c th thot ra hoc hp thu vo nhn t bo. Trong ty lp th c cha hu ht cc h enzyme c lin quan n qu trnh chuyn ha nng lng v cng c coi l nhng nh my cung cp nng lng. Trong cc h enzyme ca ty lp th, trc ht phi k n h enzyme ca chui h hp t bo v ca qu trnh phosphoryl ha to ATP. Mc du ngi ta c pht hin cytochrome ngoi ty lp th, nhng oxydase th ch thy trong ty lp th. Ngoi nhng enzyme k trn, trong ty lp th cn c h enzyme cyclophorase bao gm ton b cc enzyme ca chu trnh Krebs. Cng c th tm thy mt s enzyme ca chu trnh ny trong bo tng nh isocitrate dehydrogenase, malate dehydrogenase... nhng h thng enzyme hon chnh ca chu trnh ny th ch tm thy trong ty lp th. Ngoi ra, ngi ta cng tm thy cc h enzyme ko di acid bo, cc h enzyme phn gii acid bo v nhiu enzyme khc trong ty lp th. Cch sp t cc h enzyme k trn trong ty lp th c lin quan cht ch vi nhau m bo cho cc qu trnh chuyn ha phi hp nhp nhng vi nhau. Trong lysosome c th pht hin nhiu enzyme loi thy phn (hydrolase) c tc dng ph v nhiu loi phn t ln nh nucleic acid, protein, cht bo v nhiu loi phn t ln khc nh mucopolysaccharide... thnh nhng phn t nh c kh nng c chuyn ha di tc dng ca cc enzyme ca ty lp th. Bnh thng enzyme c bc kn trong mng lipoprotein ca lysosome v do khng c tc dng vi cc cht trong bo tng. Khi mng lysosome b v hoc b tn thng, cc h enzyme ca n c gii phng ra, s lm tiu hy c t bo. Cc ht ribosome dnh trn h thng li ni cht nguyn sinh, l ni xy ra qu trnh sinh tng hp protein, do c cc h enzyme ca qu trnh sinh tng hp protein. Bo tng l phn lng ca t bo c cha rt nhiu loi enzyme, c tt c cc enzyme xc tc cho qu trnh ng phn hay cho cc qu trnh phn gii glucose. Nhiu cng trnh nghin cu chng minh rng qu trnh ng phn xy ra ch yu bo tng. Nhiu enzyme ca qu trnh ny c th dnh mng ngoi ca h thng li ni cht nguyn sinh, c nhng enzyme gn su vo mng ca h thng ny. Sn phm pyruvate ca qu trnh ng phn c vn chuyn qua mng vo ty lp th tip tc kh cacboxyl bng cch oxy ha thnh acetyl CoA tham gia vo chu trnh Krebs.

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Cc cu trc mng trong t bo c tnh thm chn lc, nhiu cht chuyn ha khng qua c mng ca ty lp th, v d nh oxaloacetate, isocitrate, NADH + H+, NADPH + H+. Chnh tnh thm chn lc ny lm tng thm tnh c hiu ca cc ngn trong t bo. Cng cn lu rng, trong t bo sng nguyn vn, enzyme thng cha hot ng ti mc ti a v nhng iu kin trong c th cng cha phi l nhng iu kin thch hp nht cho s hot ng ca enzyme. y chnh l nhng iu kin quan trng gip cho c th iu ho cc qu trnh chuyn ha.

8.2. iu ha hot v s lng ca enzyme trong t bo

Nh chng ta bit, cc b phn ca t bo hot ng rt nhp nhng, bo m hot ng sng bnh thng ca t bo. Trong t bo c hng nghn phn ng khc nhau to nn ton b qu trnh chuyn ha ca mt cht c th ( to nn mt sn phm no ) gm mt chui cc phn ng do nhiu enzyme (mt h thng enzyme) xc tc: A B C .... Z
E1 E2 E3

Theo s ca chui phn ng trn th cht A c th bin thnh sn phm cui cng l cht Z phi nh h thng nhiu enzyme E1, E2, E3... xc tc cc phn ng k tip nhau. Cht Z lun lun c sn xut vi s lng ph hp vi nhu cu ca t bo: khi thiu, dy chuyn phn ng tng ln, khi tha, dy chuyn phn ng t ng gim. Thc t mi mt qu trnh xy ra theo ng nhu cu ca t bo khng qu tha ngay c trong iu kin dinh dng y . Nhng hin tng c th c gii thch bng hai c ch: iu ha hot ca chnh phn t enzyme trong chui phn ng (bn thn enzyme hot ng bnh thng hay gim hot ng do b c ch) v iu ha sinh tng hp cc enzyme (s lng cc enzyme c sn xut ra t hay nhiu) Nhng qu trnh ny c lin quan mt thit vi nhau v iu chnh ln nhau. 8.2.1. iu ha hot enzyme Trong t bo, nhiu loi enzyme thng khng s dng ht kh nng xc tc ca chng. Cc iu kin bnh thng trong t bo khng phi l nhng iu kin enzyme c th biu hin hot ng ti a, v cn phi duy tr mt d tr nng lc ca enzyme lm tng tc ca qu trnh chuyn ha khi c nhu cu cn thit ca t bo. Nh vy trong t bo c nhng tc nhn iu ha hot ca enzyme v to ra kh nng bin i

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thch ng ca cc qu trnh chuyn cc enzyme trong mt chui phn ng khng c s ng u v tr s hot . Nhng tc nhn hoc yu t nh hng ca mi trng ni bo c th chia thnh ba loi chnh. Loi th nht l cc yu t khng c hiu ca mi trng phn ng nh pH, th nng oxy ha kh, lc ion, nhit ... Loi th hai l cc hp cht c tc dng c hiu vi trung tm hot ng do s ph hp v cu tr khng gian. l cc cht tham gia phn ng enzyme nh c cht, coenzyme, cc cht vn chuyn trung gian hoc l cc cht hot ha hay cht c ch enzyme. Loi th ba l cc hp cht c tc dng c hiu nhng khng tham gia v mt ha hc vo phn ng do enzyme xc tc v thng khng ging v mt khng gian vi cc cht tham gia phn ng. l cc cht c tc dng d lp th thuc v nhm ny, bao gm cc sn phm cui cng ca mt s chui chuyn ha (c bit l h thng ng ha) mt s cht chuyn ha khc v cng c th c mt s hormone. y ta xt n s iu ha hot enzyme theo cc c ch d lp th (allosteric) v c ch thay i cn bng gia hai dng hot ng v khng hot ng ca enzyme. iu ha theo c ch d lp th c thc hin khi sn phm chuyn ha cui cng ca mt dy phn ng ha hc xc tc bi nhiu enzyme c th tc dng hot ha hay c ch ln enzyme xc tc phn ng u tin l mt enzyme d lp th. Enzyme d lp th (allosteric enzyme) c tn gi t ting Hi Lp l allos c ngha l khc v stereos c ngha lp th, khng gian. l nhng enzyme c tc dng iu chnh c hiu trn nhng v tr then cht ca mng li chuyn ha, c bit l nhng qu trnh sinh tng hp. Trong phn t ca enzyme d lp th ngoi trung tm hot ng lm chc nng xc tc, cn c mt s v tr khc c th tng tc vi cc cht khc gi l trung tm allosteric (trung tm iu ha, trung tm d lp th, trung tm d trung gian). Cc cht kt hp vo cc trung tm ny gi l cc cht iu ha allosteric (cht iu ha d lp th). Enzyme d lp th thay i hot xc tc thng qua s hay i cu hnh khng gian ca phn t enzyme, ca trung tm hot ng khi gn vi cc cht iu ha d lp th. Nu lm tng hot gi l cht iu ha dng; nu lm gim hot gi l cc cht iu ha m. Cc cht iu ha ny kt hp vi enzyme nhng khng b chuyn ha di tc dng ca enzyme m n kt hp. Enzyme d lp th thng l nhng enzyme c cu trc bc bn, do cc n v nh cu to nn; trong phn t thng c 2 hay mt s trung tm hot ng, c th kt hp vi 2 hay mt s phn t c cht. C cht

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c th thc hin chc nng ca cht iu ha - iu ha homotropic (ng hp, ng hng). Cc cht iu ha c cu trc khc c cht - iu ha heterotropic (d hp, d hng). Tuy nhin phn ln cc enzyme d lp th thuc kiu hn hp ng v d hp ngha l hot ng ca chng c th c iu ha nh c cht v cc cht trao i khc. Trong c ch iu ha enzyme thng ni n hin tng c ch ngc (feed back inhibition). C ch ca s c ch ngc chnh l mt loi c ch iu ha d lp th m thng thng sn phm cui cng ca qu trnh phn ng l cht iu ha d lp th m. Do hin tng c ch ngc c gi l c ch d lp th. Khi enzyme cn c tc dng hot ha ca mt cht iu ha dng chuyn thnh dng enzyme hot ng th hin tng c gi l s hot ha d lp th. c ch d lp th l nguyn tc rt ph bin i vi cc qu trnh chuyn ha v c cc c im sau y: - C ch c ch ngc xy ra cc chui phn ng dn n s tng hp mt cht no (cht ny c dng tng hp cc i phn t: v d cc amino acid isoleucine v histidine c dng tng hp protein, nucleotide c dng tng hp nucleic acid) - Sn phm cui cng c tc dng c ch d lp th. - Sn phm cui cng thng ch tc dng c hiu v trc tip ln enzyme u tin l enzyme d lp th. - Sn phm cui cng (cht c ch ngc) c cu trc ha hc khc vi c cht ca phn ng m n c ch, v vy tc dng c ch ca n khng phi do cnh tranh vi c cht , m do n lm thay i cu dng khng gian ca enzyme khin enzyme khng tip nhn c c cht. (Hnh 8.1)

Hnh 8.1. Hiu ng d lp th m a. Enzyme (E) tip nhn c cht (S) b. Yu t d lp th A gn vo trung tm d lp th, cu trc enzyme (trung tm hot ng ca enzyme) thay i v khng tip nhn c c cht; hot gim.

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C ch c ch ngc c th c biu th theo s sau: A E1 B E2 C E3 .... Z

(-)

Nh vy, mi khi sn phm cui cng Z c tng hp tng ln vt qu nhu cu ca t bo th n c ch enzyme u tin E1 khin cho phn ng u tin A B gim i v do , mc d cc enzyme sau l E2, E3... khng b c ch (vn c kh nng hot ng bnh thng), nhng chng khng c cc c cht B, C... chuyn ha, kt qu l ton chui phn ng b gim st, s tng hp sn phm cui cng Z gim i. Khi t bo thiu cht Z, enzyme E1 khng b c ch nn phn ng u tin li xy ra v c chui phn ng cng vy: kt qu l s tng hp cc cht Z tng ln p ng nhu cu ca t bo. V d, E.Coli isoleucine l sn phm chuyn ha cui cng ca chui phn ng chuyn ha ca threonine; phn ng u tin ca chui phn ng do enzyme d lp th threonine dehydratase xc tc, khi lng isoleucine tng cao (do c tng hp nhiu) th n c ch enzyme ca phn ng u tin theo c ch d lp th (hnh 8.2)
L- Threonine Threonine dehydratase () - ketobutyrate - aceto oxybutyrate - - dihydroxy - methyl valerale L. Isoleucine Hnh 8.2: S c ch threonine dehydratase bi Isoleucine theo c ch c ch ngc, (-): c ch.

qu trnh sinh tng lng amino acid thng xy ra c ch ny. trng hp sinh tng hp nucleotide pyrimidine cytidin triphosphate (CTP) cng tng t nh trn. Aspartate transcarbamoylase (thng c vit tt l ATC - ase v c tn quc t l aspartate carbamoyltransferase) ca E.Coli xc tc cho phn ng chuyn v carbamoyl t carbamoylphosphate n aspartic acid l phn ng u tin trong qu trnh tng hp CTP. Chnh CTP l sn

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phm cui cng ca qu trnh sinh tng hp, l cht iu ha m c hiu ca ATC - ase, CDP v CMP khng c tc dng vi enzyme ny. ATC c cht iu ha dng l ATP hoc AMP, cht ny lm o ngc tc dng c ch ca CTP. Kt qu nghin cu cho thy enzyme c trng lng phn t (TLPT) l 300.000 nhng c th phn ly thnh hai n v xc tc nh nhau v ba n v iu ha nh nhau. Mi mt n v xc tc c TLPT khong 100.000 v do ba chui polypeptide to nn, mi chui c TLPL 33.000 c gi l chui C. Mi n v xc tc c ba trung tm xc tc, mi n v iu ha c hai chui polypeptide c gi l chui R c TLPT 17.000 v c mt nguyn t Zn2+ kt hp vo mi chui R. Mi n v iu ha c th kt hp hai phn t cht iu ha CTP, mi chui mt R. Khi phn t enzyme b phn ly, cc n v xc tc tch khi cc n v iu ha, hot xc tc ca enzyme vn cn nhng enzyme khng b km hm bi CTP. CTP ch c tc dng km hm phn ng enzyme khi no c s kt hp gia cc n v xc tc vi cc n v iu ha thnh phn t enzyme y . Hin tng ny c gii thch l do lc y s kt hp ca CTP vo n v iu ha s ko theo s bin i dng khng gian ca n v xc tc cng nh ca ton b phn t enzyme theo hng khng c li cho hot xc tc, do lm gim hot enzyme. Nh vy, s tng hp nn sn phm cui cng c iu ha mt cch hon ton t ng da trn s c ch hoc gii c ch i vi enzyme c sn trong t bo. l c ch iu ha nhanh v n tc ng trc tip trn hot ca enzyme. C ch iu ha c ch ngc rt c li i vi t bo v n lm ngng s sn xut tha cc sn phm cui cng, do tit kim c nng lng v cc nguyn liu dng tng hp nn sn phm . Trong vic iu ha hot ng enzyme theo c ch thay i cn bng gia hai dng enzyme hot ng v khng hot ng, trc ht phi k n qu trnh hot ha zymogen. Phn ln cc enzyme c tng hp trong c th thnh nhng phn t enzyme c hot tnh, nhng c nhng enzyme nh cc protease ca ty tng, d dy cng nh cc protease xc tc cho qu trnh ng mu thng c tng hp nn qua mt dng trung gian cha c hot tnh xc tc gi l zymogen hoc proenzyme. l nhng tin cht to thnh enzyme, ch khng phi l enzyme thc s. Nhng cht ny khng c hot tnh, phi tri qua mt qu trnh bin i, sp xp li cu trc phn t mi tr thnh enzyme hot ng c. Qu trnh chuyn ha pro-enzyme thnh enzyme gi l qu trnh hot ha, c thc hin nh s t xc tc hoc

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do cc enzyme khc xc tc. Qu trnh hot ha zymigen c mt s c im chung nh sau: - L qu trnh thy phn gii hn protein (limited proteolysis), ct t mt s lin kt peptide gn u N ca phn t zymogen. on peptide c to thnh c th b loi ra hoc vn gn vi phn cn li ca phn t nh cc cu disulfide. - Khi lin kt peptide b ct t thng lm thay i cu hnh khng gian ca phn t theo hng c li cho hot ng xc tc, to thnh phn t enzyme. - Hiu sut hot ha zymogen ph thuc vo iu kin hot ha, nng zymogen, bn cht v nng ca enzyme xc tc cho qu trnh hot ha, nhit , pH v mt s yu t khc. ngi v nhiu loi ng vt c v, cc enzyme thy phn protein (protease) trong ng tiu ha u c tng hp ra di dng tin cht ca enzyme. V d pepsinogen do nhng t bo chnh ca tuyn d dy tng hp nn v l tin cht ca pepsin, chymotrysinogen v trypsinogen ca tuyn ty theo th t l tin cht ca chymotrysin v trypsin. Cc cht ny u ch c hot ha thnh dng enzyme hot ng sau khi tit vo lng ng tiu ha. Pepsinogen c hot ha thnh trypsin di tc dng ca chnh trypsin hoc enterokinase, cn chymotrypsinogen c hot ha di tc dng ca trypsin v chymotrysin. Hin tng tng hp ra cc zymogen c mt ngha sinh hc quan trng. C th ni rng, cc protease trong ng tiu ha c tng hp qua giai on trung gian nh vy chnh l mt c ch t bo v ca c th. V nu khng nh vy th chnh cc tuyn tng hp nn cc loi enzyme ny s b tiu hy bi chnh nhng enzyme do chng tng hp nn. Hot enzyme cng c iu ha nh s bin i ln nhau gia cc dng hot ng v khng hot ng qua nhng thay i ng ha tr v cu trc phn t ca chng. V d enzyme glycogen phosphorylase m c v gan c iu ha hot bng cch gn thm (hoc ly i) nhm phosphate. Enzyme ny xc tc phn ng b gy phn t polysacharide glycogen thnh nhng glucose-1 - phosphate. (Glucose)n + Pi (glucose)n-1 + Glucose-1 - P Enzyme ny tn ti di hai hng l phosphorylase a (dng hot ng) v phosphorylase b (dng khng hot ng). Phosphorylase a l mt

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protein olygomer vi hai n v cu to, mi n v c mt gc serine c phosphoryl ha nhm hydroxyl. Nhng nhm phosphate ny l cn thit cho hot ng xc tc ca enzyme v c th chu phn ng thy phn bi enzyme phosphorylase - phosphatase. Phosphorylase a + 2H2O phosphorylase phosphatase phosphorylase b + 2Pi.

Cc nhm phosphate b loi b lm cho phosphorylase a tr thnh phosphorylase b. Cc phn t enzyme ny hot ng rt km hoc khng hot ng trong qu trnh ct glycogen so vi phosphorylase a. Nhng phn t phosphorylase b khng hot ng ny c th c ti hot ha tr thnh phosphorylase a c hot tnh cao, di tc dng ca phosphorylasekinase. Phn ng enzyme ny xc tc s phosphoryl ha cc gc serine ca phosphorylase nh cc phn t ATP. 2ATP + phosphorylase b phosphorylase kinase 2ADP + phosphorylase a

Nh vy, qu trnh iu ha hot ca glycogen phosphorylase c thc hin bng cch bin i ng ha tr. (Hnh 8.3)

Hnh: 8.3. iu ha enzyme phosphorylase nh qu trnh phosphoryl ha

Cng vi kiu iu ha d lp th bi cc enzyme d lp th, cc enzyme iu ha ng ha tr p ng vi nhng s bin i v trng thi chuyn ha ca mt t bo hoc m trong nhng thi gian tng i ngn:

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nhng enzyme d lp th, tnh bng giy, cn nhng enzyme iu ha ng ha tr thng tnh bng pht. 8.2.2. iu ha sinh tng hp enzyme Nh trn ni, hiu ng d lp th m (c ch ngc) v hiu ng d lp th dng (hot ha enzyme d lp th) c tc dng to ln trong vic iu ha nhanh chng cc qu trnh chuyn ha trong t bo v c th. Nhng khi c s thay i ln v s lng v cht chuyn ha (v d mt cht no c sn xut hoc gim st qu nhiu, s tng thm hay rt bt r rt cht dinh dng mi trng nui cy...) th hiu ng d lp th khng p ng. C c ch th hai phi hp: c ch iu ha sinh tng hp enzyme. y l c ch chm v phi qua nhiu khu trung gian (tc ng ln hot ng ca gen v qua ln s tng hp protein - enzyme). C ch ny chm song rt kinh t: tit kim c nguyn liu tng hp protein - enzyme. Trong c th thng tn ti hai loi enzyme, loi th nht l enzyme thng trc hay enzyme c cu (constitutive enzymes), l nhng enzyme tham gia thnh phn c bn ca hot ng t bo, gm tt c cc loi enzyme xc tc qu trnh chuyn ha ca t bo v lc no cng c trong t bo, loi th hai l enzyme cm ng (inductive - enzyme) bnh thng c lng rt t, khng ng k, chng s c tng ln nhanh chng khi a vo mi trng cht xc nh. iu ha sinh tng hp enzyme c thc hin theo kiu cm ng v km hm v c bit nhiu h thng procaryote (t bo vi khun v thc khun th). B gen ca mt vi khun bao gm nhim sc th c nht gm 3,8 triu i nucleotide c kh nng m ha hn 3000 protein khc nhau trong trng hp ca E.Coli. Cc vi sinh vt thng thch nghi d dng i vi nhng bin i trong thnh phn ca mi trng dinh dng nh hin tng tng hp cm ng ca enzyme. Khi xut hin trong mi trng mt c cht mi (i vi nhng mi trng tng i ngho), khng cha nhng cht dinh dng thng thng, (v d nh glucose) th s tnh tng hp enzyme trong t bo tng ln nhanh chng t ngt do hin tng cm ng, (gi l s tng hp cm ng). Vi s lng enzyme c tng ln, c cht mi ny s c bin ha nhanh chng thnh mt dng d ng ha hn. Chng hn khi cho thm tryptophan vo mi trng nui cy E.Coli th enzyme D-serindeaminase c tng hp tng ln 200 ln, trong khi hm lng ca L-serindeaminase chi tng 4 ln, cn L-threonindeaminase th khng thay i. Khi thm L.threonine cng vo mi trng y, s tng hp L-threonindeaminase chim u th. Cn ch , cc enzyme c chc

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nng phn gii (d ha) thng l cc enzyme cm ng. S cm ng ch thy cc mi trng khng cha ngun carbon d ng ha hn: ngay c khi s cm ng bt u, ta vn c th ngn cn hin tng ny bng cch cho thm glucose vo mi trng, ngi ta gi l hiu ng glucose. Nh vy, s cm ng ca cc enzyme l mt c ch d b bo m cho s chuyn ha ca t bo c th thc hin c mt cch bnh thng khi c nhng thay i khng thun li trong thnh phn ca mi trng. S cm ng c tnh a hng v tnh hp ng, c im ny c th hin ch: mt cht cm ng c th cng mt lc gy ra s tng hp cm ng ca mt vi enzyme. Chng hn cht cm ng -galactosidlactose hoc cc cht tng t ca n) ngoi kh nng gy ra s tng hp cm ng enzyme -galactosidase cn ng thi gy ra s tng hp cm ng hai cht xc tc na l galactosidpermease (c tc dng chuyn c cht qua mng t bo) v galactosidtransferase; c ba loi protein-enzyme ny c tng hp cm ng song song, vi t l khng thay i. Ni cch khc, s tng hp cm ng ba protein-enzyme ny c thc hin c tnh cht hp ng v s lng. Ngoi hin tng cm ng va ni trn, ngi ta cn pht hin ra hin tng km hm s tng hp enzyme khi thm cc cht chuyn ha nht nh vo mi trng nui cy vi khun. Khi cho thm vo mi trng mt nng valine tng i cao, s lm ngng s tng hp cc enzyme tham gia vo qu trnh sinh tng hp amino acid ny. Nng cc enzyme hin c s gim i v chng b pha long v b thoi ha dn trong qun th cc t bo ang mc. Nu thm cc amino acid khc nh methionine, tryptophan, arginine... cng nh mt s baz nit nh uracyl, cytosin, adenin, guanin cng gy ra s km hm chn lc nh trn. Nh vy cc enzyme lm chc nng tng hp (ng ha) thng b km hm, mc d c mt s trng hp enzyme phn gii (d ha), v d nh phosphatase kim cng b km hm nh trn. Hin tng km hm cng c tnh cht a hng, khi cho thm vo mi trng mt cht chuyn ha gy km hm, th s tng hp tt c cc enzyme trong h thng tng hp tng ng u b ngng li ng thi mc km hm s tng hp ca tt c cc enzyme sinh tng hp ra n, bt u t Nacetylglutamatreductase: histidine km hm tt c cc enzyme tng hp ra n t phosphoribosyl-ATP-pyrophosphorylase: uracil v cytosine km hm tt c h thng enzyme tng hp ra chng bt u t aspartat carbamyl transferase. Do ta thy rng, nhng gii hn ca mt n v iu ha di truyn thng trng vi cc gii hn ca h thng enzyme. iu xc nhn li mt ln na v quan nim cho rng h thng enzyme l mt n v chc nng ca s chuyn ha.

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Hin tng km hm ch xy ra vi nng kh cao ca cc cht km hm, khi nng gim xung th s tng hp cc enzyme tng ng li c phc hi, l s gii km hm. Ta c th ni rng, s lng ca cc enzyme trong h thng c kim tra mt cch thun nghch bng nng ca sn phm cui cng ca chng, ng thi c im ca s iu chnh ny l s lin h ngc m tnh. S tng nng sn phm s km hm s tng hp cc enzyme ny. Nh c c ch ny, cc t bo trnh c s tiu ph cc nguyn liu to nng lng v to hnh dng cho s tng hp cc enzyme, khi m cc sn phm tng ng c trong mi trng. Hin tng gii km hm, ging nh hin tng cm ng. Mc d b ngoi i lp nhau, nhng hin tng km hm v hin tng cm ng c lin quan su sc vi nhau. nh hng i khng ca ornithine v arginine i vi s tng hp ornithincarbamyltransferase thuc h thng arginine l mt chng minh r rng v tnh ng nht ny. Sn phm cui cng ca h thng l arginine c tc dng km hm s tng hp enzyme ny, cn c cht ca enzyme l ornithine th cnh tranh vi arginine, lm gim tc dng ca arginine, do to ra hin tng gii km hm hoc hin tng tng hp cm ng enzyme. Kt qu l s lng enzyme trong t bo c quyt nh bi tng quan cc nng ni bo ca c cht v ca sn phm ca h thng. Trn c s nhng hiu bit hin i v c ch tng hp protein cng vi nhng kt qu nghin cu v hin tng cm ng v km hm trong s tng hp enzyme, ngi ta xy dng c mt h thng quan nim v c ch iu ha tng hp enzyme. S tng hp enzyme, nh ta bit, c thc hin trn cc ht ribosome, cu trc c hiu ca phn t enzyme l do nhng m hiu di truyn ca RNA thng tin (m-RNA) quyt nh. Cc m hiu di truyn trn m.RNA c sao chp t phn DNA nhim sc th tng ng (nhng gen cu trc hay cistron). S truyn t thng tin cu trc t DNA n RNA c thc hin nh vai tr ca DNA-RNA polymerase v chu s kim tra ca cc "gen iu ha" (regulator,R). Sn phm ca cc gen ny c gi l cht km hm, (repressor, R), c kh nng kha s truyn t thng tin cu trc t DNA n m.RNA. Trong s iu ha tng hp mt h thng enzyme ton vn do mt nhm gen cu trc chi phi, cht km hm khng phn ng vi tng gen cu trc ni trn m ch phn ng vi mt b phn c vai tr quyt nh trong vic truyn t thng tin c gi l "gen tc ng (operator, O). Gen ny thng nm ch bt u ca nhm "gen cu trc" (Structural gene, cistron,S) cng vi cc "gen cu trc", "gen iu ha" v "khi ng (promotor, P) to thnh mt n v iu ha c lp gi l operon. Ngi ta cho rng, gen tc ng l im m u cho "vic c" thng tin cu trc ca

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ton operon: v vy khi cht km hm c kt hp vo gen ny s c tc dng kha s truyn t thng tin ca c operon v c ch s tng hp ton b b h thng enzyme tng ng. Tc dng qua li gia cht km hm vi gen tc ng l ty thuc vo nng ni bo ca cc cht chuyn ha c lin quan. Trong cc h thng enzyme cm ng, khi khng c c cht, cht km hm s tr thnh hot ng v c tc dng kha gen tc ng, c ch s tng hp enzyme, nhng khi c mt c cht th cht ny c tc dng lm mt hot tnh ca cht km hm, lm cho n khng cn tc dng kha gen tc ng na v nh vy s tng hp enzyme s c thc hin tc thi mnh m. Ngi ta gi l s tng hp cm ng ca enzyme, hin tng ny c th c coi l mt hnh thc c bit ca s gii km hm. (Hnh 8.4) a. Khng c cht cm ng R mRNA P AP O S1 S2 S3 DNA

repressor, R' b. C cht cm ng R mRNA P AP o S1 S2 S3 DNA mRNA

A repressor, R'

E1

E2 B D

E3 C

Cht cam ng
Hnh 8.4. C ch iu ha cm ng sinh tng hp enzyme

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Ghi ch: A, B, C, D c cht v cc sn phm ca chui phn ng do enzyme E1, E2, E3 xc tc; AP: RNA polymerase. Cc k hiu khc c gii thch trong bi.

Trong cc h thng enzyme b km hm cht km hm (sn phm ca gen u ha) ch c kh nng kha gen tc ng khi sn phm cui cng ca h thng cc phn ng enzyme tng ln n mc qu tha, trong trng hp ny sn phm cui cng c vai tr nh mt cht ng km hm (corepressor) (hnh 8.5)
a. Khng c cht ng km hm (corepressor)

R mRNA

P AP

S1

S2

S3

DNA mRNA

E1 A repressor, R'

E2 B D

E3 C Cht ng km hm

b. C cht ng km hm (corepressor)

R mRNA

P AP

S1

S2

S3

DNA

repressor, R'

Cht ng km hm

Hnh 8.5: C ch km hm sinh tng hp enzyme bi sn phm cui cng. (ghi ch nh hnh 8.4)

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Khi nng sn phm gim xung thp, cht km hm tr nn mt hot tnh v tch ra khi gen tc ng, lm cho s truyn t nhng thng tin cu trc tr li hot ng bnh thng, v nh vy s tng hp enzyme c gii km hm. Ngi ta gi iu ha sinh tng hp enzyme theo kiu cm ng v km hm trn thuc loi iu ha m tnh. Nhiu dn liu thc nghim cho thy cc gen bo m sinh tng hp mt s enzyme cm ng xc tc cho qu trnh phn gii khng ch chu s kim tra theo c ch cm ng m cn chu s kim tra theo c ch khc nh tc dng ca AMP vng (AMPv, cycle-AMP, c-AMP), gi l km "hm phn gii" (catabolic repression) AMPv c tc dng kch thch qu trnh sao chp m ca cc operon phn gii. Hin tng ny c nghin cu nhiu i vi operon lactose. Theo nhiu tc gi, tc dng kch thch ca AMPv i vi qu trnh sao chp m c thc hin nh mt protein c bit lm trung gian gi l protein nhn AMPv hay cn gi l protein hot ha gen phn gii (catabolite gene activator protein CAP). Khi AMPv kt hp vi CAP to thnh phc hp c tc dng hot ha promotor lm cho RNA-polymerase d dng kt hp vi n bt u qu trnh sao chp m, nh vy AMPv c tc dng lm tng cng qu trnh sao chp. Kiu iu ha operon phn gii theo c ch ny cng c gi l kiu iu ha dng tnh. Nh vy, operon lactose chu s iu ha di truyn kp: iu ha m tnh thc hin nh cht cm ng thng qua repressor (tnh cht m th hin ch s iu ha xy ra khi khng c cht cm ng, repressor kt hp vi operator ngn cn qu trnh sao chp); iu ha dng tnh thc hin bng con ng iu ha xc nh s tng hp CAP cn thit m bo qu trnh sao chp. Trong s iu ha m tnh, mt cht c ch kin kt vi phn t DNA phi b loi ra trc khi phin m c th xy ra. Trong iu ha dng tnh, mt phn t cht tc ng phi lin kt vi DNA. Mt h thng cng c th c iu ha bng c hai cch dng tnh v m tnh, trong trng hp , h thng l "m" khi cht iu ha dng tnh c gn vi DNA v cht iu ha m tnh khng c lin kt vi DNA. Trong h thng iu ha m tnh, mt cht c ch c mt trong t bo v cn tr s phin m. Mt cht i lp vi cht c ch phin m gi l cht cm ng cho php m u s phin m. Trong h thng iu ha dng tnh, mt phn t cht tc ng (c th l protein, phn t nh hay phc hp phn t) hot ha mt im m u. S iu ha dng tnh v

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m tnh khong loi tr ln nhau, v th mt h thng, c c ch iu ha dng tnh v m tnh u c s dng; hai loi cht iu ha p ng c nhng iu kin khc nhau c trong t bo. S iu ha dng tnh v m tnh c p dng cho h thng phn gii v cho c chu trnh tng hp. Trn y l c ch iu chnh nhng t bo vi khun: nhng c th bc cao, c ch c nhng im khc v c phn phc tp hn. Mt s cng trnh thc nghim ch r ra rng, nhng t bo ca c th bc cao, nhng protein kt hp vi DNA trong nhim sc th c vai tr trong s iu chnh ny. Trn thc t trong phng th nghim ngi ta thy histon ng vai tr cht c ch trong vic sao chp thng tin di truyn. C th loi tr s c ch ny bng cch phosphoryl ha cc histon di tc dng ca hai loi protein-kinase, mt loi c iu chnh v mt loi khng c iu chnh bi AMP vng. Ngi ta cng k n vai tr ca cc protein acid ca chromatin, cc cht ny hot ha s sao chp. Cc hormon steroid cng c vai tr iu ha trn h gen, v d cortison lm tng cng tng hp mt s enzyme bng cch tng s tng hp nhng mRNA tng ng. S iu chnh cng xy ra trong qu trnh dch m. S iu chnh ny tc ng n nhng bin i trn mRNA (nh cng thm poly A, loi b nhng mnh khng mang m di truyn) v n s kt hp mRNA trn ribosom cng nh nhng giai on khc nhau ca s dch m di truyn t mRNA sang cc phn t enzyme. C ch phn t ca cc tc dng iu ha k trn cng ngy cng c b sung chi tit hn. Ta c th rt ra kt lun l: trong t bo c nhng c ch iu chnh rt phc tp v rt c hiu qu, m bo cho s lin h thng tin cht ch gia b my di truyn ca t bo v cc qu trnh chuyn ha vt cht trong t bo. Nh cc c ch ny m nng ni bo ca cc phn t nh c th kim tra s tng hp cc phn t enzyme. Ni mt cch khc, cc phn t nh ny (c cht v cht chuyn ha) c th iu khin c s lng v cht lng ca cc h thng enzyme trong t bo v do iu khin c c tnh ca nhng bin i chuyn ha ring ca chng.

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TI LIU THAM KHO

Ti liu ting Vit
1. Nguyn Hu Chn, 1983. Enzyme v xc tc Sinh hc. Nxb Y hc, H Ni.
2. Phm Th Trn Chu, Trn Th ng, 2000. Ha sinh hc. Nxb Gio dc, H Ni.

3. Ngc Lin, Phm Th Trn Chu, 1972. Enzyme I, II. i hc Tng hp, H Ni. 4. Nguyn Tin Thng, Nguyn nh Huyn, 1998. Gio trnh sinh ha hin i. Nxb Gio dc, H Ni. 5. Nguyn Xun Thng, o Kim Chi, Phm Quang Tng, Nguyn Vn ng, 2004. Ha sinh hc. Nxb Y hc, H Ni. 6. L Ngc T, La Vn Ch, Phm Trn Chu, Nguyn Ln Dng, 1982. Enzyme vi sinh vt. Nxb KH&KT, H Ni. 7. L Ngc T (ch bin), L Vn Ch, ng Th Thu, Phm Quc Thng Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din, 2000. Ha sinh Cng nghip, Nxb KH&KT, H Ni.

Ti liu ting nc ngoi

1. Bermeyer H. U, Bermeyer J. and Grasel M. (editors). 1983. Methods of enzymatic analysis. Vol II. Verlag chemie Weinheim. 2. Lehringer A. L., 2004. Principle of Biochemistry, 4th Edition. W.H Freeman, 2004. 3. Pelmont J., 1993. Enzymes. Presses universitaires de grenobe. 4. Stryer L., 1981. Biochemistry. W.H.Freeman and company. San Francisco.
5. Biochemical information, 1973. Boehringer Mannheim GmbH. Biochemica.

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Chng 9

Cng ngh enzyme v ng dng

9.1. Cng ngh enzyme
9.1.1. Enzyme vi cng ngh sinh hc Enzyme c xem nh l mt k thut quan trng ca cng ngh sinh hc do c cc chc nng sau: - Enzyme l cht xc tc cho mi bin i vt cht trong cng ngh sinh hc. - Enzyme v nhiu hot cht sinh hc khc l sn phm ca cng ngh sinh hc. Chng c th dng lm cng c mi ca cng ngh sinh hc, hay s dng trong cc lnh vc khc . - Enzyme c xem l thuc th c tnh chuyn ha cao m khng c enzyme th cc qu trnh cng ngh sinh hc khng th ti u ha c 9.1.2. Cng ngh sn xut enzyme Trong sn xut ch phm enzyme, cn ch n nhng yu t: 9.1.2.1. Ngun enzyme 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. 9.1.2.2. Cch thu nhn + Chn i tng: Phi da vo c rng sinh hc ca i tng.i vi vi sinh vt cn ch n khu chn ging , vn di truyn ging , kh nng sinh trng v pht trin ca ging , c tnh sinh l ha sinh ca ging. + Cc phng php nui cy: - Mi trng nui cy: Ty chng chn mi trng thch hp, thnh phn dinh dng phi ph hp vi sinh trng pht trin, c bit l cc yu t cn thit cho qu trnh sinh tng hp protein. Cn nm vng c ch iu ha c nhng thay i thch nghi.

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- Phng php nui cy b mt; l nui cy trn gi th rn vi hm lng nc thp khong 15-20%. Ngoi thnh phn dinh dng l protein, tinh bt, khong c th trn cc cht lm xp thong kh. Ty chng khng ch nhit , pH mi trng , m, thi gian nui cycho t hiu qu sinh tng hp enzyme cao nht. - Phng php nui cy chm: l nui cy trong mi trng dch th, hm lng cht kh ti a t 25-30%, thng t 10-15%. Ngoi protein, tinh bt, khongcn c th b sung kch thch t. Cng nh trn, ty chng khng ch nhit , pH mi trng, m, thi gian nui cy cho t hiu qu sinh tng hp enzyme cao nht. Vi hai phng php trn, mi loi c u khuyt im ring. Nui cy b mt thng cho hiu sut cao, d g b tng phn nu b nhim , nhc im l tn mt bng nhiu, kh t ng ha. Phng php nui cy chm d t ng ha, phi loi b hon tan khi b nhim. + Thu nhn ch phm enzyme: i vi canh trng b mt hay cc i tng thc vt, c th ng ha nu cn, sau dng dung dch m hay nc ct chit rt enzyme ra khi canh trng b mt ta c dch chit enzyme . i vi canh trng b su ch cn lc b sinh khi l c dch chit tng t trn. Sau c th dng cc tc nhn kt ta thun nghch nh aceton, ethanol, mui trung tnh c ch phm enzyme dng sch hn. T ch phm sch ny, bng k thut in di, lc gel ta c th tch tng phn c enzyme tinh khit hn.Ty mc ch s dng ta to ra ch phm thch hp. nng cao gi tr s dng, hin nay ngi ta thng to ra ch phm enzyme gi l enzyme khng tan. Enzyme khng tan Hu ht cc enzyme trong c th u dng lin kt vi mng cn c cht i qua mng enzyme chuyn ha n thnh sn phm. Trong cng nghip thng s dung enzyme dng ha tan, thng ch s dng c mt ln v l l do ngi ta to ra enzyme khng tan. to ra enzyme khng tan c nhiu phng php khc nhau nh phng php hp ph vt l, phng php a enzyme vo khun gel, phng php cng ha tr ca enzyme v cht mang.

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Phng php hp ph vt l: l phng php hp ph ln b mt cht mang. Cht mang nh cm, than hot tnh, bt thy tinhNhc im ca phng php l enzyme d ha tan tr li, lin kt lng lo, khi chu tc ng lc ion ln d b nh ra. Phng php a enzyme vo khun gel: enzyme d nh v trong gel, mng li cht trng hp cng nh enzyme s c gi cht hn. y l cch c dng kh ph bin. Phng php cng ha tr ca enzyme v cht mang: da vo i lc gia enzyme v cht mang to phc gia enzyme - cht mang bng lin kt cng ha tr. y cng l phng php c dng ph bin.

9.2. ng dng
Hin nay, vic sn xut ch phm enzyme cc loi v ang pht trin mnh m trn qui m cng nghip. Thc t c hng nghn ch phm enzyme bn trn th trng th gii, cc ch phm ny c khai thc v tinh ch c mc tinh khit theo tiu chun cng nghip v ng dng. Cc ch phm enzyme ph bin nh amylase, protease, catalase, cellulase, lipase, glucoseoxydase Ch phm enzyme khng ch c ng dng trong y hc m cn c ng dng trong nhiu lnh vc cng nghip khc nhau, trong nng nghip, trong ha hc " ngha ca vic s dng enzyme trong cc lnh vc thc t khng km so vi ngha ca vic s dng nng lng nguyn t". 9.2.1. ng dng trong y dc Enzyme c mt v tr quan trng trong y hc. c bit l cc phng php nh lng v nh tnh enzyme trong ha hc lm sng v phng th nghim chn on. Do , hin nay trong y hc xut hin lnh vc mi gi l chn on enzyme, c nhim v: - Phn tch xc nh nng c cht nh glucose, ure, cholesterol vi s h tr ca enzyme . - Xc nh hot tnh xc tc ca enzyme trong mu sinh vt. - Xc nh nng c cht vi s h tr ca thuc th enzyme nh du. Dng enzyme nh lng cc cht, phc v cng vic xt nghim chn on bnh, v d dng kim tra glucose nc tiu rt nhy.

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Urease nh lng ure Dng enzyme lm thuc v d protease lm thuc tc nghn tim mch, tiu m vt thng, lm thng ng h hp, chng vim, lm thuc tng tiu ha protein, thnh phn ca cc loi thuc dng trong da liu v m phm Trong y hc cc protease cng c dng sn xut mi trng dinh dng nui cy vi sinh vt sn xut ra khng sinh, cht khng c Ngoi ra ngi ta cn dng enzyme protease c c v tinh ch cc huyt thanh khng c cha bnh. Amylase c s dng phi hp vi coenzyme A, cytocrom C, ATP, carboxylase ch thuc iu tr bnh tim mch, bnh thn kinh, phi hp vi enzyme thy phn cha bnh thiu enzyme tiu ha. 9.2.2. ng dng trong ha hc Cho n nay, vic ng dng enzyme trong ha hc l do enzyme c cm ng cao i vi nhit , pH v nhng thay i khc ca mi trng. Mt trong nhng ng dng ch phm enzyme ng c ch nht trong thi gian gn y l dng cht mang gn phc enzyme xc tc cho phn ng nhiu bc. V d tng hp glutathion, acid bo, alcaloid, sn xut hormoneCng bng cch to phc, ngi ta gn vi sinh vt s dng trong cng ngh x l nc thi, sn xut alcohol, amino acid Trong nghin cu cu trc ha hc, ngi ta cng s dng enzyme, v d dng protease nghin cu cu trc protein, dng endonuclease nghin cu cu trc nucleic acid Dng lm thuc th trong ha phn tch. 9.2.3. ng dng trong cng nghip Vic s dng enzyme trong cng nghip l a dng, phong ph v t c nhiu kt qu to ln. Th nhn thng k s b sau y v cc lnh vc dng protease ta c th thy c s a dng: cng nghip tht, cng nghip ch bin c,cng nghip ch bin sa, cng nghip bnh m, bnh ko, cng nghip bia, cng nghip sn xut sa kh v bt trng, cng nghip hng phm v m phm, cng nghip dt, cng nghip da, cng nghip phim nh, cng nghip y hcVi amylase, c dng trong sn xut bnh m, cng nghip bnh ko, cng nghip ru, sn xut bia, sn xut mt,glucose, sn xut cc sn phm rau, ch bin thc n cho

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tr con, sn xut cc mt hng t qu, sn xut nc ngt, cng nghip dt, cng nghip giyTrong phm vi gio trnh ny chng ta ch cp n vic ng dng ch phm enzyme trong mt s lnh vc. 9.2.3.1. ng dng trong 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. 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. 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.

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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. 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.

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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. 9.2.3.2. ng dng trong cng nghip dt Trong cng nghip dt, ch phm amylase c dng r h vi trc khi ty trng v nhum. Amylase c tc dng lm vi mm, c kh nng nhng t, ty trng v bt mu tt. R h bng enzyme khng nhng nhanh, khng hi vi, mao dn tt m cn m bo v sinh, do tng c nng sut lao ng. Trong sn xut t tm, ngi ta dng protease lm sch si t. Vi cng on x l bng enzyme sau khi x l bng dung dch x phng s gip la c tnh n hi tt, bt mu ng u v d trang tr trn la. 9.2.3.3. ng dng trong cng nghip thuc da Trong cng nghip da, enzyme protease c dng lm mm da, lm sch da, rt ngn thi gian, trnh nhim mi trng. Vic x l c tin hnh bng cch ngm da trong dung dch enzyme, hay pht dch enzyme ln b mt da. Enzyme s tch cc cht nhn v lm t mt s lin kt trong phn t collagen lm cho da mm hn. Thc t cho thy khi x l da bng ch phm protease t vi sinh vt c th rt ngn thi gian lm mm v tch lng xung nhiu ln. iu quan trng l cht lng lng tt hn khi ct. So vi phng php ha hc th vic x l bng enzyme c s lng lng tng 20-30%. Lng khng cn x l thm sau khi ngm trong dch enzyme. 9.2.4. ng dng trong nng nghip C th s dng cc loi ch phm enzyme khc nhau chuyn ha cc ph liu, c bit l cc ph liu nng nghip ci to t phc v nng nghip. Nht hng nm sn xut hng vn tn ch phm cellulase cc loi dng trong nng nghip. C ch phm cha c cellulase, hemicellulase, protease v amylase. Cng ngh ny kh ph bin nhiu quc gia. nc ta vic dng enzyme vi sinh vt gp phn trong sn xut phn hu c ang c khai thc thay th cho phn ha hc.

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Tm li, c th ni rng, vic nghin cu ng dng cc ch phm enzyme ngy cng c ch trng cc lnh vc khc nhau. Trong 20 nm cui th k XX v cc nm du ca th k XXI cc enzyme khc nhau c ng dng. Vit Nam bc u c nhiu nghin cu ng dng cc enzyme trong ch bin nng sn, thc phm, nht l trong lnh vc sn xut bia, ru, ch bin tinh bt (Vin cng nghip thc phm, Vin cng ngh sinh hc cng ngh thc phm, i hc Bch khoa H Ni). Vic nghin cu cc enzyme phc v nng nghip, cng nghip cng c quan tm v c cc kt qu ng khch l. V d, ch phm enzyme mi ra i phc v nng nghip E2001 c tc dng tng ph nhiu t, tng nng sut cy trng. c cc nghin cu ng dng protease trong sn xut ru bia, rt ngn thi k ln men cng nh sn xut nc mm ngn ngy bng cng ngh enzyme protease. Enzyme amylase cng c nghin cu ng dng rng ri trong sn xut ng bt, maltodextrin, nha glucose, siro, glucose fructose quy m cng nghip.

TI LIU THAM KHO

Ti liu ting Vit
1. Qu Hai. 2004. Chuyn enzyme, Ti liu lu hnh ni b Trng HKH Hu. 2. Trn Thanh Phong. 2004. Chuyn enzyme, Ti liu lu hnh ni b Trng HKH Hu. 3. Trn Thanh Phong. 2005. Chuyn Cng ngh ha sinh, Ti liu lu hnh ni b Trng HKH Hu. 4. Nguyn Xun Thnh (ch bin), L Vn Hng, Phm Vn Ton. 2003. Cng ngh vi sinh vt trong sn xut nng nghip v x l mi trng, Nxb Nng nghip, H Ni. 5. L Ngc T (ch bin), L Vn Ch, ng Th Thu, Phm Quc Thng Nguyn Th Thnh, Bi c Hi, Lu Dun, L Don Din, 2000. Ha sinh Cng nghip, Nxb KH&KT, H Ni.

Ti liu ting nc ngoi

1. Copeland R. A. 2000. Enzymes,copyright by Wiley-VCH, Inc. 6. Lehninger A. L. 2004. Principles of Biochemistry, 4th Edition. W.H Freeman.