B CNG THNG

TRNG I HC CNG NGHIP TP. H CH MINH

VIN CNG NGH SINH HC THC PHM

GVHD: ThS. LM KHC K

Lp: DHSH6LT
SVTH: Nhm 6
V Th Kim Chi
Nguyn Th Hiu
on Th Kim Hng Hip
V Th M Trang

ENZYME OXYDASE

GVHD: ThS. LM KHC K

MC LC
1. TNH CHT CHUNG CA OXYDASE:..................................................................4
2. NGUN THU NHN..................................................................................................6
3. VAI TR V TC DNG .........................................................................................7
3.1. Sn xut bnh nng ......................................................................................................................7
3.2. Sn xut bt trng ............................................................................................................................8
3.3. Bo qun thc phm ........................................................................................................................9
3.4. Sn xut bia, ru .........................................................................................................................10
3.5. Ch bin sa ..................................................................................................................................10
3.6. Cng ng dt ..................................................................................................................................11
3.7. M phm.........................................................................................................................................11
3.8. X l mi trng.............................................................................................................................11
3.9. nh lng thc phm ...................................................................................................................13
3.10. Y hc, chun on bnh ..............................................................................................................13

4. PHNG PHP THU NHN..................................................................................14

4.1. Phn lp vi sinh vt bng phng php cy trn mi trng c ..................................................14
4.2. Ln men sinh tng hp enzyme GOD ...........................................................................................14

5.PHNG PHP NH TNH...................................................................................15

6. PHNG PHP NH LNG ............................................................................16
6.1. Xc nh nng protein ho tan theo Lowry.................................................................................16
a. Nguyn tc......................................................................................................................................16
b. Ho cht cn dng .........................................................................................................................16
c. Cch tin hnh................................................................................................................................16
d. Xy dng ng th chun........................................................................................................17

7. PHNG PHP XC NH HOT TNH ENZYME GLUCOSE-OXIDASE..18

7.1. Xc nh hot tnh enzyme GOD bng phng php da vo ng cong chun hydrogen
peroxide.................................................................................................................................................18
7.1.1Nguyn tc:.................................................................................................................................18
7.1.2 Dng c - Ho cht....................................................................................................................19
7.1.3. Cch tin hnh..........................................................................................................................19
7.1.4. Xy dng th ng chun H2O2........................................................................................20
SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

7.1.5. Tnh kt qu..............................................................................................................................20

7.2. Xc nh hot tnh enzyme GOD bng phng php chun .....................................................20

8. PHNG PHP TINH SCH ENZYME...............................................................22

TI LIU THAM KHO.............................................................................................23

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

1. TNH CHT CHUNG CA OXYDASE:

Oxydase l enzyme thuc lp Oxydoreductase. y l enzyme xc

tc cho phn ng oxi-ha-kh.

La nhom enzyme oxy ha c cht, chuyn hoa electron n oxy. Oxydase hoat hoa
oxy, lam cho chung co kha nng kt hp vi proton co trong mi trng.

Lp enzyme nay tac dung trc tip vi oxy, to ra nc (H2O) hoc hydrogen
peroxide ( H2O2).

Enzym tiu biu l xitocromoxydase, poly phenoloxydase, ascorbatoxydase,

peroxydase, catalase, cc oxydase flavin.

S hot ng ca oxydase nh sau:

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

Hnh1 Cu trc khng gian ca glucose oxidase

Vi trng lng phn t 192.000 Dalton. Glucose oxidase l mt protein lng phn
hnh thnh t 2 tiu n v ging nhau. Mi tiu n v hoc monomer, cun vo trong 2
domain: mt domain gn vi c cht, -D-glucose, trong khi domain khc lin kt
khng ng ha tr vi mt nhn t ph, flavin adenine dinucleotide (FAD), m n s
dng nh mt tc nhn oxy ha mnh. FAD l mt thnh phn ph bin trong cc phn
ng oxy ha kh sinh hc, n nhn v cho cc in t t mt phn t.
Trong glucose oxydase, FAD hot ng nh mt cht nhn in t m lm cho n b
kh thnh FADH2; FADH2 sau b oxy ha bi cht nhn in t cui cng, phn t
oxy, oxy s b kh thnh hydrogen peroxide (H2O2).
Glucose oxidase c tit ra bi nm mc, ch yu l Aspergillus niger hay
Penicillium amagaskinense. N c phn b gia dch ngoi bo, vch t bo, v trong
cht dch nhy ca nm mc. Qu trnh tng hp gucose oxydase c th c kch thch
bi cc cht khc nhau, bao gm phn t oxy, m n kch thch s dch m enzyme.
Glucose Oxidase bn thn n khng hot ng, nhng vi s hin din ca nhng c
cht c trng, enzyme c s dng oxy ha trc tip phn t glucose hoc oxygen.

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

Nhm ngoi ca enzyme ny l FAD. Cc sn phm ca phn ng ny l gluconic acid

v hydrogen

Glucose oxydase c th oxi ha -D-glucose s dng cc cht oxi ha khc bn cnh

phn tuoxy,bao gm cc quinine v cc cht nhn 1 electron. D-glucono-1,5-lactone sau
t ng thy phn to ra gluconic acid
C6H12O6 + H2O + 1/2O2 C6H12O7 + H2O2
Glucose
gluconic acid
Phn ng ti u iu kin nhit gia 30 n 50oC ti pH gia 4.5 n 6.5.

2. NGUN THU NHN

Enzym

Ngun enzym

Glucose oxidase (EC.1.1.3.4)

Aspergillus niger.
A. Oryzae
Penicillium notatum.

Catalase (EC.1.11.1.6)
Cholesterol

Gan b, Aspergillus sp.

oxydase Nocardiaerythr

(EC.1.1.3.6)
Ascorbat

oxydase Cucurbita sp.

(EC.1.10.3.3)
Peroxidase (EC.1.11.1.7)

C ci nga

Pyruvate oxidase (EC.1.2.3.3)

Pediococcus sp.

Glycerol 3-phosphate oxidase Aerococcus viridans

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

(EC.1.1.3.21)
NADH-peroxidase

Streptococcus faccalis

(EC.1.11.1.1)
Sulfite oxidase (EC.1.8.3.1)

Gan g

Urate oxidase (EC.1.7.3.3)

Arthrobacter protophormiac

Xanthine oxidas(EC.1.1.3.22)

Sa b

3. VAI TR V TC DNG
Oxydase l enzyme ng dng nhiu trong nhiu lnh vc.

3.1. Sn xut bnh nng

Cc oxydase thng dng l Glucose oxidase, Hexose oxidase,

Peroxidase,

Lipoxygenase, polyphenol oxidase vi tc dng l: Tng lc ca gluten, m bo cht

lng bt lm bnh, ci thin mi v v mu sc ca sn phm.

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

3.2. Sn xut bt trng

Cc oxydase thng dng l Glucose oxidase, , catalase vi tc dng l: Loi

glucose ca lng trng trng trnh phn ng mailard, tng thi gian bo qun sn
phm trng.
Ngi ta thy rng nu trong bt trng c cha glucose th trong qu trnh ch
bin cng nh khi bo qun, glucose s tng tc vi protein v s b oxy ha dn to
nn nhng phm vt c mu sm v mi kh chu, lm cho cht lng thnh phm b
gim st. Trong albumin trng thng c cha 3% glucose cn trong lng cha 0,5%
glucose (so vi cht kh). V vy, trong sn xut bt trng v albumin trng, ngi ta
dng glucose oxydase oxy ha trc cc glucose , khin cho nhng qu trnh trn
khng xy ra, ng thi cng tng c thi gian bo qun cc sn phm trng.

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

3.3. Bo qun thc phm

Cc oxydase thng dng: Glucose oxidase, catalase nhm trnh thc phm khi b
oxyha, nh tht, sa, phomat
Glucose oxidase c s dng thnh cng cu ni glucose v oxy trong thc phm v
thc ung ko di thi gian bo qun cho thc phm v ung. Hydrogen peroxide
c sn sinh ra t enzyme ny hot ng nh mt cht dit vi khun rt tt
Glucose oxidase c th c s dng loi b oxy u chai nc ung trc khi
chng c ng chai.
bo qun cc sn phm dng lng, ngi ta c th ha tan vo Glucose oxidase
cng mt t glucose trc khi bao gi. Khi tt c oxy d trong sn phm s c tch
ht.
Phomai c ng gi trong mt mng c bit c tm Glucose oxidase, s ngn nga
c s xm nhp ca oxy, s bin i do oxy ha v s to mu xm lp b mt. Cn
tht nu c bc trong giy trng knh tm enzyme cng s bo v c mu sc t
nhin ca n.
Gn y, c nhng sn phm dng ti kh oxy, chng gm mt t glucose, ch
phm Glucose oxidase-catalase v dung dch m cn thit cho vo trong 1 ti polyetylen
(hay vt liu khc) ch cho oxy v khng kh i qua m khng cho nc i qua. t ti
vo trong hp, thng kn c cha cc sn phm cn bo qun. Ti s hp thu hon ton
oxy, lm cho vt liu c bo qun tt.
Sa kh, cc mt hang ko, b c th c bo qun lu nh ti kh ny, ngoi ra
cn dng bo v cc chi tit my tinh vi v cc thit b khi b han g v n mn.
SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

3.4. Sn xut bia, ru

Glucose oxidase , catalase, peroxydase, ascorbinatoxydase Ko di thi gian s dng bia,

chng s mt mu ca ru vang , v vang trng chuyn mu.
Glucose oxidase c kh nng s dng trong cng ngip lm ru, m n lm gim
cn trong ru thng qua vic loi b mt s glucose (chuyn n thnh dng D-glucono1,5-lactonea nu khng n s chuyn thnh cn. Hn na glucose oxidase c th ngn
chn s h hng ru thng qua tc ng dit vi khun ca n ln vi khun acetic acid v
vi khun lacitc acid trong sut qu trnh ln men. Enzyme ny s c b sung thm vo
ru tng hiu qu dit vi sinh vt.
Trong sn xut bia, ngi ta dng Glucose oxidase n nh thnh phn. nhit
phng bia cha c thanh trng s b c sau 10-15 ngy bo qun do nm mc v vi
khun pht trin. Nu cho mt t ch phm Glucose oxidase vo bia (1g/200-250l) th s
tch ht oxy c trong cht lng cng nh trong cc khong khng t do, kt qu l tnh
cm quan ca sn phm s c bo m trong thi gian t 50-100 ngy.

3.5. Ch bin sa
Glucose oxidase, catalase Peroxidase,Lactoperoxidase dng trong bo qun sa.

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

3.6. Cng ng dt

Glucose oxidase, catalase, Lacase dng lm trng, loi peroxide tha trn si trc khi
nhum mu, phn gii v oxy ha cht mu.

3.7. M phm
Trong thuc nhum tc, enzyme peroxydase to H 2O2 ti ch nng thp, khng
lm hi tc.

3.8. X l mi trng
Loi b H2O2 trong nc thi nhanh v c hiu qu.
Catalase (EC 1.11.1.6) xc tc phn ng c hiu phn hu H2O2 [2]. Ngoi ra,
catalase c th phn hu formaldehyde, formic acid v alcohol - Cc cht c hi vi mi
trng, c thi ra trong nc thi ca cc nh my ch bin sa, pho mt hoc cc nh
my dt, si. Catalase vi khun c tc dng tch cc trong vic ph hy chng. Trong cc
enzyme peroxidase th catalase, peroxidase v manganese peroxidase c nghin cu
nhiu phc v cho vic pht hin mt s ion kim loi c cho mi trng nh: Hg+2,
Pb+2, Cd+2, Cr+6, Mn+2 [3].
Peroxidase c ci nga (Horseradish peroxidase-HRP) c k. hiu EC 1.11.1.7, tc ng
nh catalase, xc tc phn ng c hiu. HRP l mt trong nhng enzyme c nghin
cu nhiu nht c lin quan ti phng php x l. rc thi bng enzyme. HRP c th

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

xc tc phn ng oxy ho mt ph rng cc hp cht thm c bao gm phenol,

biphenol, aniline, benzidine v cc hp cht thm d v.ng nh hydroxyquinoline v
arylamine carcinogen nh benzidine v naphthylamine.
Peroxidase c s dng ci thin qu trnh kh nc bng cn phosphate
L-galactonolactone oxidase (EC 1.1.3.24) c . ngha i vi vic x l nhim mi
trng. Enzyme ny xc tc phn ng c hiu l phn ng oxi ho L-galactono-1,4lactone thnh L-ascorbate
Cc enzyme polyphenol oxidase c kh nng xc tc cho cc phn ng xy ho cc hp
cht phenol. Cc enzyme ny c chia thnh hai phn h: tyrosinase v laccase. Hot
tnh ca c hai h u cn s c mt ca oxy phn t nhng khng cn c mt cc
coenzyme.
Tyrosinase (EC 1.14.18.1) hay polyphenol oxydase, phenolase hay catecholase xc
tc cho hai phn ng lin tip: phn ng th nht l phn ng thu phn monophenol nh
oxy phn t thnh cc o-diphenol v phn ng th hai l phn ng dehydrogen ho cc odiphenol nh oxy thnh cc o-quinon. Cc quinon thng khng bn v b polime ho
khng cn enzyme thu c cc hp cht khng tan trong nc v d dng b loi b nh
kt ta.
Laccase (EC 1.10.3.2) l mt enzyme kim loi xc tc cho phn ng oxi
hohydroquinone thnh benzoquinone
Qu tr.nh ty trng giy c p dng rng ri trong cng nghip nghin bt giy loi
b cc gc lignin trong bt giy. Cc gc ny l nguyn nhn lm cho bt giy c mu
nu v c loi b m t tn km bng cch dng cc cht ty trng nh chlorine v cc
oxid chlorine. Qu trnh ty trng to ra dch lng c mu nu en cha cc sn phm b
chlorin ho c v c kh nng gy t bin gy nguy him i vi mi trng.
Peroxidase v laccase c tc dng tch cc trong vic x l. dch lng sau qu tr.nh ty ni
trn v dng c nh ca cc loi enzyme ny trong mi trng hp u hiu qu hn so
vi dng t do.

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

3.9. nh lng thc phm

Phn tch thnh phn cholesterol, sulfite..trong thc phm.
Dng cholesterol oxydase, sau dng catalase chuyn ha tip H 2O2 c to thnh,
v xc nh formaldehyde c to thnh.
Sulfite thng dng bo qun thc phm, nng sn, lm bt hot nhiu enzyme, ngn
nga to mu nu, nu nng qu cao s gy hi. Dng sulfite oxydase, sau dng
NADP peroxydase chuyn ha H2O2 to thnh.

3.10. Y hc, chun on bnh

Lm thuc th trong ha phn tch, khng khun.
S dng glucose oxidase trong phng php o hm lng glucose trong mu
Nhng ngi bnh tiu ng cn phi kim tra hm lng glucose trong mu thng
xuyn xc nh s dao ng ca nng glucose c th tng hay gim trong mu t
a ra cch iu tr bnh hp l. Gn y, thit b cm bin sinh hc (Biosensor) c
pht trin dng cho vic o lng hm lng glucose, v glucose oxydase l mt trong
nhng enzyme ch yu m biosensor c th s dng.
S nh lng chnh xc hm lng glucose trong mu th rt quan trng trong s chun
on v iu khin s tng v gim ng huyt.
Phng php s dng thuc th trong o lng hm lng glucose trong mu
Phn ng glucose oxidase kt hp vi mt phn ng ph c s dng rng ri cho
vic xc nh glucose trong dung dch sinh hc. Nhiu phn ng b tr khc c pht
trin ci tin ton b h thng phn ng c trng hoc gi li cc c tnh vn c ca
glucose oxidase. Phng php s dng thuc th da vo 10 phn ng ch th mu ca
hydrogen peroxide m n kt hp vi 4-aminontipyrine to mt phc phenolic,
phng php ny c xut u tin bi Trinder
Phng trnh phn ng:
Glucose Oxidas
1. Glucose + O2 + H2O > Gluconic Acid + H2O2
2. H2O2 + HBA + 4-AAP> thuc nhum Quinoneimine + H2O
SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

Phng trnh 1: Glucose b oxy ha bi glucose oxydase to ra gluconic acid v

hydrogen peroxide
Phng trnh 2: Hydrogen peroxide phn ng vi HBA v 4-aminoantipyrine vi s hin
din ca enzyme Peroxidase hnh thnh nn 1 quinoneimine bt mu . Cng mu
to thnh th tng ng vi nng glucose v c th c o lng bng phng php
o mt quang bc sng gia 460-560nm.
Ch thch:
HBA = 4-hydroxybenzoic acid
4-AAP = 4-aminoantipyrine

4. PHNG PHP THU NHN

Thu nhn GOD t nm mc: chng Penicilium: Pen. Notatum, Pen.chrysoenum,
Pen.vitale v chng Aspergillus: A. niger,

4.1. Phn lp vi sinh vt bng phng php cy trn mi trng c

em vt phm (mu vt ln mc) gi nh, ha vo trong 200ml nc ct v
trng, ht 0.1 ml dch cho vo a petri c mi trng Czapek. Dng que trang tri u
khp mt thch. nhit 300C trong 3-7 ngy.
Chn khun lc ng tch bit dng que cy ly mt t VSV cy ln cc a petri khc,
lm tng t cho n khi thu c khun lc thun chng, dng que cy ly mt t bo t
khun lc cy vo ng thch nghing.

4.2. Ln men sinh tng hp enzyme GOD

Tin hnh ln men trong mi trng lng vi thnh phn (g/l): (NH4)2SO4 0.4;
KH2PO4 0.4; MgSO4.7H2O 0,1; saccharose 100; peptone 15; CaCO3 40.
un si mi trng cho tan ht, iu chnh pH 6.5 6.9. Ly 70 ml mi trng vo mi
bnh nn 250 ml, hp tit trng 1210C trong 20 pht, lm ngui xung nhit mi
trng v cy vi 3 vng que cy VSV.
SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

Bnh nn c nui 70h trn my lc vi tc 175 vng/pht. Sau ln men, canh

trng VSV c ly tm 15 pht (5200 vng/pht) tch ring phn lng v sinh khi
VSV.

5.PHNG PHP NH TNH

Nhm xc nh s hin din ca h enzyme oxidase vi sinh vt ngi ta
thc hin th nghim oxidase.
NGUYN TC:
Cytochrome oxidase trong chui chuyn in t cua h hp hiu khi vi
oxy la cht nhn in t sau cung nn chi hin din trong cac loai vi sinh
vt hiu khi hay ky khi tuy y.
Thuc th p-phenylenediamine.
Co s hin din cua cytochrome c kh trong t bao: thuc th bi oxy hoa
thanh hp cht indolphenol co mau xanh dng.

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

6. PHNG PHP NH LNG

6.1. Xc nh nng protein ho tan theo Lowry
a. Nguyn tc
Phng php ny da trn c s phc cht ng protein kh hn hp
photphomolipden photphovonphramat (thuc th Folin ciocalteu) to phc cht mu
xanh da tri c hp th cc i bc song 660nm.
Cng mu ca hn hp phn ng t l thun vi nng protein trong mt
phm vi nht nh. Da vo mc hp th quang hc ca protein chun, ta c th xc
nh c hm lng protein trong mu nghin cu.
b. Ho cht cn dng
- Dung dch A: 4g NaOH (0,1M) v 20g Na2CO3 (2%) pha trong 1000ml nc ct.
- Dung dch B: 0,5g CuSO4.5H2O (0,5%) pha trong dung dch Natri Xitrat (1%)
hoc trong dung dch Natri, Kali Tactrat 1%.
- Dung dch C: Hn hp ca hai dung dch A v B theo t l 49:1 (pha trc khi
dng).
- Thuc th folin, trc khi dng pha long hai ln sao cho axit bng 1N.
- Albumin huyt thanh b 1mg/ml
c. Cch tin hnh
- Mu th nghim: ly chnh xc 0,5ml dch cha protein vi hm lng thch hp
cho vo ng nghim, thm vo 2ml dung dch C, lc u yn trong 10 pht. Sau
thm vo hn hp trong ng nghim 0,25ml folin pha long 2 ln, lc u v yn
trong 30 pht, mu vng ca hn hp chuyn sang mu xanh da tri v t n cng
mu cc i. em so mu ca hn hp trn my so mu quang in bc sng 660nm.
Xc nh c tr s mt quang hc ca dung dch nghin cu. o trn my 3 ln lp
li v ly tr s trung bnh.

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

- Mu i chng: 0,5ml m cho vo ng nghim, thm vo 2ml dung dch C

v bc tip theo c tin hnh nh mu th nghim.
d. Xy dng ng th chun
Hm lng protein tan trong dung dch c xc nh theo phng php Lowry,
dng albumin huyt thanh b (BSA) lm cht chun.
Cn 0,01g (10mg albumin huyt thanh b) ho tan trong 10ml nc, ta c
dung dch gc c nng l 1mg/ml. Sau pha long dung dch gc bng nc ct vi
cc nng 20, 40, 60, 80, 100, 120 g/ml tin hnh xy dng th chun.
- Mu th nghim: ly chnh xc 0,5ml dung dch BSA nng pha long nh
trn cho vo ng nghim, thm vo mi ng 2ml dung dch C nhit phng 10
pht, sau cho 0,25ml thuc th folin (1N) em so mu vi bc sng 660nm.
- Mu i chng: ly 0,5 ml nc ct cho vo ng nghim, thm vo 2ml dung
dch C v cc bc tip theo c tin hnh nh mu th nghim.
Qua 3 ln lp li th nghim c th xy dng ng hi quy. Kt qu c x l
theo phng php thng k thng thng.
Ta c phng php hi quy.
y = 0,55066844x + 0,01791961

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

OD

0,6

660

0,5

nm

0,4
0,3
0,2
0,1

0
0,2

0,4

0,6

0,8

1,2

mg

Hm lng protein
Hnh 1. ng th xc nh nng Protein theo phng php Lowry
Da vo th chun, c th xc nh c hm lng protein trong mu nghin
cu.

7. PHNG PHP XC NH HOT TNH ENZYME

GLUCOSE-OXIDASE
7.1. Xc nh hot tnh enzyme GOD bng phng php da vo ng cong
chun hydrogen peroxide
7.1.1Nguyn tc:
Hot tnh enzyme Glucose oxidase c o bng cch xc nh hm lng
hydrogen peroxide (H2O2) to thnh trong phn ng chuyn ho ng glucose
thnh gluconic acid trong phn ng sau :
GOD GOD

Glucose +
SVTH: Nhm 6

O2

gluconic acid + H2O2

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

POD

H2O2+ -dianisidine

-dianisidine

+ H2 O

ng cong chun hydrogen peroxide (H2O2) c xy dng xc nh hm lng

Hydrogen peroxide (H2O2) trong phn ng .
Mt n v hot tnh l lng enzyme cn to ra 1 mol gluconic acid v H2O2 trong
mt pht iu kin pH 5.1 v nhit 250C .
7.1.2 Dng c - Ho cht
a. Dng c
ng nghim
Pipets
Quang ph k
Cuvettes
b. Ho cht & enzyme
Dung dch Hydrogen peroxide (H2O2) chun
Acid sulfuric (H2SO4)
Thuc th -dianisidine
Enzyme Peroxidase (POD)
Enzyme Glucose oxidase (GOD)
Dung dch Glucoses
7.1.3. Cch tin hnh
Cho 0.1 ml (2mg/ml) glucose oxidase vo ng nghim cha 2.9 ml dung dchglucose (1
20mM) c pha trong dung dch m acetate (pH 5.1). Phn ngc thc hin nhit
250C trong thi gian 1 3 pht .
Sau thm vo 0.1ml (60U/ml) peroxidase (POD) v 2.4mlo-dianisidine (0.21mM)
POD l cht xc tc cho phn ng gia Hydrogen peroxide (H2O2) v -dianisidine

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

( cht to mu). Phn ng c dng li bng cch thm vo 0.5ml dung dch acid
sulfuric 2.5M
o mt quang OD 530nm (OD530) v xc nh lng hydrogen
peroxide(H2O2) to ra da trn ng tng quan tuyn tnh gia gi tr OD530 v nng
chun hydrogen peroxide (H2O2).
7.1.4. Xy dng th ng chun H2O2
Pha long dung dch hydrogen peroxide (H2O2)chun theo cc nng khc nhau. C
th cho trong bng sau :
Ha cht
H2O2 (ml)
Nc ct (ml)
Nng

ng 1
0,2
9,8
0,2

ng 2
0,4
9,6
0,4

ng 3
0,6
9,4
0,6

ng 4
0,8
9,2
0,8

ng 5
1,0
9,0
1,0

T cc ng nghim trn ly 2.4ml mi ng nghim vo 5 ng nghim khc

v thm vo mi ng 2.4mlo-dianisidine ( cht to mu) vi 0.1ml (60U/ml)
peroxidase (POD) cht xc tc phn ng. Sau cho vo 0.5ml H2SO4 2.5M
dng phn ng .
o mt quang OD 530nm (OD530) v xy dng ng chun H2O2 .
7.1.5. Tnh kt qu
T phng trnh th ng chun tnh c lng H2O2 to thnh trong phn ng
enzyme .

7.2. Xc nh hot tnh enzyme GOD bng phng php chun

y l c s dng phng php ph bin xc nh hot tnh ca GOD. Trong
phng php ny, 1 ml dung dch enzyme c thm vo 25 ml m CH3COONa 60
mM pH 5.6 cha 2% -D-glucose. Hn hp c lc 1h trong khng kh 300C trn
my lc, tc 200 vng/pht. Phn ng dng li bi thm vo 20ml dung dch NaOH
0.1N. Hn hp em chun vi dung dch chun HCl 0.1N. Mu trng (khng c
SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

enzyme) thc hin tng t di iu kin th nghim . Hot tnh ca GOD xc nh

bi cng thc:
(VO V.)N.1000/60

Trong :

Vo l th tch HCl dng chun mu trng.

V l th tch HCl dng chun mu th nghim.

N l nng ca dung dch HCl chun.
Mt n v hot tnh enzyme c nh ngha l lng enzyme c th oxi ha 1.0 mol
-D-glucose thnh axit D-gluconic v H2O2 trong 1 pht pH 5.6 v nhit 300C.

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

8. PHNG PHP TINH SCH ENZYME

Mu ban u
(ph v t bo, ly tm)

Thu dch ly tm

(NH4)2SO4
Kt ta
Loi mui
Chy sc k trao i in
(trao i in m)

Loi mui
Sy kh
(nhit thp)

Sc k lc gel
Sy kh (nhit thp)

Sn phm

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

TI LIU THAM KHO

1. Ha sinh cng nghip. L Ngc T. NXB khoa hc v k thut
2. Cng ngh sinh hc tp 3 enzym v ng dng. Phm Th Trn Chu, Phan Tun
Ngha. NXB gio dc Vit Nam.
3. Cng ngh enzyme. Nguyn c Lng. NXB i hc quc gia TP.HCM.
4.

NH GI KH NNG SINH TNG HP GLUCOSE OXIDASE NGOI

BO CA CC CHNG NM MC PHN LP T MI TRNG T
NHIN. Tuyn tp Bo co Hi ngh Sinh vin Nghin cu Khoa hc ln th 7
i hc Nng nm 2010,p366-370.

5.Tng lai ng dng Enzyme trong x l ph thi. Tp ch Khoa hc i hc Quc gia

H Ni, Khoa hc T nhin v Cng ngh 23 (2007) 75-85
6. Glucose oxidase An overview. Sandip B. Bankar, Mahesh V. Bule, Rekha S.
Singhal, Laxmi Ananthanarayan. Biotechnology Advances 27 (2009) 489501
7. Factors regulating production ofglucose oxidase by Aspergillus niger.
D. G. Hatziuikolaou v B. J. Macris (1995), Elsevier Science Inc, 17, (6/1995), tr.530-34.
8. Characterization and quantitative analysis glucose oxydase.

SVTH: Nhm 6

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ENZYME OXYDASE

GVHD: ThS. LM KHC K

MC LC
1. TNH CHT CHUNG CA OXYDASE:..........Error: Reference source not found
2. NGUN THU NHN..........................................Error: Reference source not found
3. VAI TR V TC DNG..................................Error: Reference source not found
3.1. Sn xut bnh nng.......................................................................................Error: Reference source not found
3.2. Sn xut bt trng...........................................................................................Error: Reference source not found
3.3. Bo qun thc phm........................................................................................Error: Reference source not found
3.4. Sn xut bia, ru...........................................................................................Error: Reference source not found
3.5. Ch bin sa....................................................................................................Error: Reference source not found
3.6. Cng ng dt....................................................................................................Error: Reference source not found
3.7. M phm.........................................................................................................Error: Reference source not found
3.8. X l mi trng.............................................................................................Error: Reference source not found
3.9. nh lng thc phm.....................................................................................Error: Reference source not found
3.10. Y hc, chun on bnh................................................................................Error: Reference source not found

4. PHNG PHP THU NHN............................Error: Reference source not found

4.1. Phn lp vi sinh vt bng phng php cy trn mi trng c...................Error: Reference source not found
4.2. Ln men sinh tng hp enzyme GOD............................................................Error: Reference source not found

5.PHNG PHP NH TNH............................Error: Reference source not found

6. PHNG PHP NH LNG.......................Error: Reference source not found
6.1. Xc nh nng protein ho tan theo Lowry................................................Error: Reference source not found

7. PHNG PHP XC NH HOT TNH ENZYME GLUCOSE-OXIDASE

...................................................................................Error: Reference source not found
7.1. Xc nh hot tnh enzyme GOD bng phng php da vo ng cong chun hydrogen peroxide.......Error:
Reference source not found
7.2. Xc nh hot tnh enzyme GOD bng phng php chun ......................Error: Reference source not found

8. PHNG PHP TINH SCH ENZYME........Error: Reference source not found

TI LIU THAM KHO.......................................Error: Reference source not found

SVTH: Nhm 6

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