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Enzyme Hoc PDF
Enzyme Hoc PDF
Mc lc
Trang
Li ni u
Chng 1
M u
1.1.
nh ngha enzyme
1.2.
Lc s nghin cu enzyme
1.2.1.
Giai on 1
1.2.2.
Giai on 2
1.2.3.
Giai on 3
1.2.4.
Giai on 4
12
1.3.
14
1.4.
16
1.5.
Vn nghin cu enzyme nc ta
17
18
Chng 2
19
2.1.
19
2.2.
21
2.2.1.
21
2.2.2.
Chit rt enzyme
26
2.2.3.
28
2.2.4.
38
2.2.5.
39
Hot enzyme
41
2.3.1.
41
2.3.2.
n v hot enzyme
41
2.3.
4
43
Chng 3
44
3.1.
Cch gi tn enzyme
44
3.2.
44
3.2.1.
Cc lp enzyme
44
3.2.2.
Cc phn ng enzyme
46
51
Chng 4
52
4.1.
Bn cht ha hc ca enzyme
52
4.2.
53
4.3.
Cu trc bc 4 ca enzyme
54
4.4.
56
4.5.
57
4.5.1.
58
4.5.2.
59
4.5.3.
Xc nh tr s pK ca cc nhm hot ng
60
4.5.4.
60
4.6.
61
4.7.
Phc hp multienzyme
62
63
Chng 5
64
5.1.
64
5.2.
64
5.2.1.
64
5.2.2.
c hiu c cht
64
68
Chng 6
C ch tc dng ca enzyme
69
6.1.
C ch ca phn ng c xc tc ni chung
69
6.2.
C ch ca xc tc enzyme
69
73
Chng 7
ng hc Enzyme
74
7.1.
74
7.2.
ng hc cc phn ng enzyme
74
74
7.2.2.
7.2.3.
79
7.2.4.
7.2.5.
87
7.2.6.
nh hng ca nhit
nh hng ca pH
7.2.7
Cc yu t khc
89
91
Chng 8
Sinh hc enzyme
92
8.1
92
8.2
94
8.2.1
iu ha hot enzyme
94
8.2.2
101
108
Chng 9
109
9.1.
109
9.1.1.
109
9.1.2.
109
ng dng
111
9.2.1.
ng dng trong y dc
111
9.2.2.
ng dng trong ha hc
112
9.2.3.
113
116
7.2.1.
9.2.
75
88
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.
8
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.
9
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
10
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.
11
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
12
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.
13
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,
14
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.
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.
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.
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.
19
Chng 2
20
T l
Nhit t c
Nc : mui
100:33 (3:1)
- 21,30C
Nc : H2SO4 m c
100: 25 (4:1)
- 20,00C
21
22
23
24
25
26
27
28
29
Protein - COOH
acid
acid
protein - NH+3
Protein - NH2
kim
30
31
32
33
34
mui
protein
G. 10
0 - 700
G. 15
0 - 1.500
G. 25
ht tinh (F)
100 - 5000
ht th (C)
G. 50
ht tinh (F)
1500 - 30.000
ht th (C)
G. 75
3.000 - 70.000
G. 100
4.000 - 150.000
G. 150
5.000 - 400.000
G. 200
5.000 - 800.000
35
Hat Sephadex
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
G - 10
<700
G - 15
<1500
G - 25
100 - 5000
G - 50
500 - 10.000
G - 75
1.000 - 50.000
G - 100
1.000 - 100.000
G - 200
1.000 - 200.000
37
+ OH C2H5
38
39
40
41
B
Hnh 2.4. ng biu din ha tan protein
42
43
= 6.107 U
1U
1
microkatal
60
44
44
Chng 3
O
c tn h thng l Carbamid - amidohydrodase (Tn thng dng
l urease)
45
46
47
AKh
Aox + e
Box + e
Bkh
Akh + Box
Aox + Bkh
B-R+A
48
A - H + B - OH
49
A=B
+ X-Y
50
UDP - glucose.
51
52
Chng 4
53
54
55
56
57
C cht
+
C cht
+
Vng hot ng
Phc hp ES
Phc hp ES
Enzyme
Enzyme
a.
b.
58
59
60
61
62
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
64
Chng 5
65
urease
-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.
66
67
68
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.
[E]
Hnh 7.1. S ph thuc ca vn tc phn ng vo [E]
75
+ S
k2
ES
E + P (1)
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)
76
77
1/v
1/Vmax
-1/Km
1/[S]
hexokinase
78
C cht
Km(mM)
(No)
79
80
= 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
81
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
khng c cht km hm
83
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
Hn tp
(Mixed)
gim
gim
gim
Km
gim
tng
khng nh hng
tng
84
(1)
(2)
T (2) suy ra [E] th vo (1), ng thi thay [I] bng [I0] :
Trong
Ly tch phn ta c
Mt khc:
85
Nng enzyme t do s l:
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
86
4/ Trng hp 4
87
88
Nhit (C)
Hnh 7.8. nh hng ca nhit ln hot enzyme
89
90
91
92
92
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
93
94
E2
E3
95
96
97
E1
(-)
E2
E3
....
- aceto oxybutyrate
L. Isoleucine
Hnh 8.2: S c ch threonine dehydratase bi Isoleucine theo c ch
c ch ngc, (-): c ch.
98
99
(glucose)n-1 + Glucose-1 - P
100
phosphorylase
phosphatase
phosphorylase b + 2Pi.
phosphorylase
kinase
2ADP + phosphorylase a
101
102
103
104
S1
S2
S3
DNA
S1
S2
S3
DNA
AP
mRNA
repressor, R'
b. C cht cm ng
R
P
AP
mRNA
mRNA
A
repressor, R'
E1
E2
B
E3
C
D
Cht cam ng
Hnh 8.4. C ch iu ha cm ng sinh tng hp enzyme
105
S1
S2
S3
DNA
AP
mRNA
mRNA
E1
E2
B
E3
C
D
repressor, R'
Cht ng km hm
b. C cht ng km hm (corepressor)
R
mRNA
repressor, R'
S1
S2
S3
DNA
AP
Cht ng km hm
106
107
108
109
Chng 9
110
111
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.
112
113
114
115
116