B GIO DC V O TO

TRNG CAO NG KINH T - CNG NGH TP.HCM

KHOA CNG NGH SINH HC

BO CO THC HNH

TH CNG NGH PROTEIN-ENZYME

GVHD: ThS. L Thanh Hi
CHUYN NGNH: Cng Ngh Sinh Hc ng Dng
NIN KHA: 2012 2015
SVTH: Nhm 3 (C8UD1)
Hunh Th Mai Tho
Bi Th Bch Thy
Phm Th Kim Ngn
Lm Vn Tr
L Phc Thin
Nguyn Th Hong Yn
Thnh Ph H Ch Minh
Thng 10/2014
MC LC

Thc hnh Cng ngh Protein - Enzyme

BI 1: NH LNG NIT ACID AMIN BNG PHNG PHP CHUN FORMOL

(PHNG PHP SORENSEN)............................................................................3
1/ Nguyn tc................................................................................................3
2/ Thc hnh................................................................................................3
2.1.

Nguyn liu v ha cht.......................................................................3

2.2. Dng c v thit b...............................................................................4

2.3. Tin hnh...........................................................................................4
2.4. Kt qu..............................................................................................5
BI 2: PHN NG BIURE.................................................................................7
1/ Nguyn tc...............................................................................................7
2/ Thc hnh...............................................................................................7
2.1. Nguyn liu v ha cht........................................................................7
2.2. Dng c v thit b...............................................................................7
2.3. Tin hnh...........................................................................................7
2.4. Kt qu..............................................................................................8
BI 3: XC NH IM NG IN CA PROTEIN CASEIN..........................10
1/ Nguyn tc.............................................................................................10
2/ Thc hnh.............................................................................................10
1.1. Nguyn liu v ha cht......................................................................10
1.2. Dng c v thit b.............................................................................10
1.3. Tin hnh.........................................................................................11
1.4. Kt qu..............................................................................................11
BI 4: SO SNH TC DNG XC TC CA ENZYME VI XC TC V C........13
1/ Nguyn tc..............................................................................................13
2/ Thc hnh...............................................................................................13
2.1. Nguyn liu v ha cht........................................................................13
2.2. Dng c v thit b...............................................................................13
2.3. Tin hnh..........................................................................................14
2.4. Kt qu.............................................................................................14
BI 5: TNH C HIU CA ENZYME.............................................................17
1/ Nguyn tc.............................................................................................17
2/ Thc hnh.............................................................................................17

GV: ThS. L Thanh Hi

Thc hnh Cng ngh Protein - Enzyme

2.1. Nguyn liu v ha cht......................................................................17

2.2. Dng c v thit b.............................................................................18
2.3. Tin hnh.........................................................................................18
2.4. Kt qu.............................................................................................18
BI 6: TC DNG CA CHT KCH THCH V CHT KM HM....................21
1/ Nguyn tc.............................................................................................21
2/ Thc hnh.............................................................................................21
2.1. Nguyn liu v ha cht......................................................................21
2.2. Dng c v thit b..............................................................................22
2.3. Tin hnh.........................................................................................22
2.4. Kt qu............................................................................................23
BI 7: XC NH HOT TNH CA ENZYME LIPASE.....................................25
1/ Nguyn tc.............................................................................................25
2/ Thc hnh.............................................................................................25
2.1. Nguyn liu v ha cht......................................................................25
2.2. Dng c...........................................................................................25
2.3. Tin hnh.........................................................................................26
2.4. Tnh kt qu.....................................................................................27
PHN T THIT K TH NGHIM XC NH NHIT TI THCH CA ENZYME
ASCOBATOXIDASE.......................................................................................29
1/ Nguyn tc...............................................................................................29
2/ Thc hnh...............................................................................................29
2.1. Nguyn liu v ho cht.........................................................................29
2.2. Dng c.............................................................................................29
2.3. Tin hnh...........................................................................................30
2.4. Cch tnh kt qu:................................................................................31
BI 8: KHO ST HOT TNH ENZYME ASCOBATOXIDASE..........................34
1/ Nguyn tc.............................................................................................34
2/ Thc hnh.............................................................................................34
2.1. Nguyn liu v ho cht......................................................................34
2.2. Dng c...........................................................................................35
2.3. Tin hnh.........................................................................................35
2.4. Cch tnh kt qu:..............................................................................36

GV: ThS. L Thanh Hi

Thc hnh Cng ngh Protein - Enzyme

MC LC HNH
Hnh 1.1 Dung dch Ph 9,2 (bn tri), pH 7,0 (phi)......................................6
Hnh 1.2: Bnh mu, bnh pH 9,2 v bnh kim chng (t tri sang phi) sau
qu trnh chun bng dung dch NaOH ...................................................7
Hnh 2.1: ng 1 (phi, Biure), ng 2 (tri, albumin)...................................10
Hnh 3.1 Th nghim im ng in ca casein t tri sang phi (1,2,3,4,
v 5)...........................................................................................................14
Hnh 4.1: ng 1,2,3,4 (t tri sang phi) sau qu trnh lm ngui 30 pht 17
Hnh 5.1: ng 1,2,3 v 4 t tri sang phi sau qu trnh th nghim..........21
Hnh 6.1: ng 1,2 v 3 t tri sang phi sau qu trnh th nghim.............26
Hnh7.1: Kt qu sau chun ca i chng v mu...............................30
Hnh 8.1: Trc chun v sau chun bng Na2S2O3 0,01N................35
Hnh 9.1: Trc chun v sau chun .................................................39
MC LC BNG
Bng 3.1 B tr th nghim xc nh im ng in ca casein................13
Bng 4.1 Bng kt qu th nghim so snh tc dng xc tc ca enzyme
vi xc tc v c.........................................................................................16
Bng 5.1 Bng kt qu th nghim xc nh tnh c hiu ca enzyme. . .20
Bng 6.1 Bng kt qu th nghim xc nh tc dng ca cht hot ha v
kim hm hot tnh ca enzym amylase....................................................25
Bng 7.1: Cch tin hnh th nghim.........................................................32
Bng 8.1: Nhit v hot ca Acid Ascobatoxidase sau qu trnh th
nghim.......................................................................................................33
Bng 8.2: pH v hot ca enzyme ascobatoxidase sau qu trnh th
nghim.......................................................................................................34

GV: ThS. L Thanh Hi

Thc hnh Cng ngh Protein - Enzyme

BI 1: NH LNG NIT ACID AMIN BNG PHNG PHP CHUN

FORMOL (PHNG PHP SORENSEN)
1/ Nguyn tc
Cc aldehyde d kt hp vi nhm amin. Khi cho formaldehyde tc dng vi acid
amin, nhm amin b methylen ha to thnh dn xut methylen amino acid. Hp
cht to thnh c tnh acid mnh hn acid amin t do, cc nhm carboxyl ca chng
d dng nh phn bng kim, qua gin tip tnh c lng nit amin ca cc
acid amin c trong dung dch.
2/ Thc hnh
2.1. Nguyn liu v ha cht
- Nc mm

: 1ml

- Dung dch NaOH 0,05N

: 100ml

- Dung dch HCl 0,05N

: 30ml

- Formol trung tnh

: 10ml

- Bromthymol blue 0,04%

: 20ml

- Phenolphtalein 0,5%

: 20ml

- Dung dch m phosphate pH = 7

: 50ml

Dung dich A: 27,8 g Na2HPO4 ho tan trong 1000ml

Dung dch B: 53.05g Na2HPO4 .7H2O hoc 71.1g Na2HPO4.12H2O pha trong
1000ml
pha dung dch m phosphate pH =7 ht dung dch A v dung dch B theo t l
sau: Dung dich A: 39 ml + dung dich B: 61ml thm nc va 200ml.
Dung dch m phosphate pH = 9,2

: 50ml

(Ha tan 456g kali hydro phosphate ngm 3 phn t nc (K2HPO4.3H2O) trong
1000ml nc ct).

GV: ThS. L Thanh Hi

Thc hnh Cng ngh Protein - Enzyme

2.2. Dng c v thit b

- Bnh nh mc 100ml

: 1 bnh

- Pipette 1ml

:2

- Pipette 10ml

:2

- Erlen 100ml

: 6 bnh

- Burette 25ml

:1

- Becher 100ml

: 4 ci

2.3. Tin hnh

Chun b thang mu c pH 7 v pH 9,2
Ly 2 erlen 100ml:
- Cho vo bnh th nht: 20ml dung dch c pH 7,0 v 5 git bromthymol blue
0,04%
c mu xanh lc nht
- Cho vo bnh th hai: 20ml dung dch c pH 9,2; 5 git bromthymol blue 0,04%
v 3 git phenolphtalein 0,5%
c mu tm xanh (mu xanh nh tm)
Mu ca cc dung dch trn gi trong bnh kn c th bn trong na thng.

Hnh 1.1 Dung dch Ph 9,2 (bn tri), pH 7,0 (phi)

GV: ThS. L Thanh Hi

Thc hnh Cng ngh Protein - Enzyme

Xc nh hm lng nit amin trong nc mm:

Nc mm l mt dung dch thy phn protein c th nh lng nit bng phng
php chun formol. Do nc mm c mu ti cn phi pha tht long mi c th so
mu ca cht ch th.
Ly 1ml nc mm cho vo bnh nh mc 100ml, dng nc ct nh mc thnh
100ml, lc u.
Ly vo bnh erlen c cng dung tch vi 2 bnh mu chun 20ml dch mu pha long
t bnh nh mc, thm 5 git bromthymol blue. Nu dung dch c mu xanh dng th
thm tng git HCl hay H2SO4 0,05N; nu dung dch c mu vng th thm tng git
NaOH 0,05N cho n khi dung dch c mu ng vi mu ca bnh 1 c pH 7,0.
Thm 3 git phenolphtalein 0,5%; 4ml formol trung tnh ri chun bng NaOH
0,05N cho n khi hn hp c mu ng vi mu ca dung dch trong bnh 2 c pH 9,2.
Song song tin hnh lm th nghim kim chng, thay dung dch nghin cu bng nc
ct.

Hnh 1.2: Bnh mu, bnh pH 9,2 v bnh kim chng (t tri sang phi) sau qu
trnh chun bng dung dch NaOH 0,05N
GV: ThS. L Thanh Hi

Thc hnh Cng ngh Protein - Enzyme

2.4. Kt qu
S gram nit acid amin c trong 1 lt nc mm:
x=

( 2.40,334 ) x 1 x 0,0007 x 100 x 1000

= 7,231(g)
20 x 1

Trong :
x: lng gram nit acid amin c trong 1 lt nc mm
a: s ml dung dch NaOH 0,05N dng chun dung dch th nghim
b: s ml dung dch NaOH 0,05N dng chun dung dch kim chng
T: h s hiu chnh nng ca dung dch NaOH em dng so vi nng chun
V: s ml nc mm cho vo bnh nh mc
0,0007: s gram nit vi 1ml NaOH 0,05N

GV: ThS. L Thanh Hi

Thc hnh Cng ngh Protein - Enzyme

BI 2: PHN NG BIURE
1/ Nguyn tc
y l phn ng thng dng pht hin lin kt peptide (-CO-NH-). Phn ng
xy ra i vi cc cht cha t hai lin kt peptide tr ln. Phn ng ny dng
nh lng protein bng cch lp th chun vi cc dung dch protein chun c
nng xc nh nh k thut so mu. Nng protein ti thiu nh lng
chnh xc l 10mg/ml.
Ty thuc vo gc R m mu phn ng c th l mu xanh tm, tm hoc hng.
2/ Thc hnh
2.1. Nguyn liu v ha cht
- Dung dch CuSO4 1%

: 20ml

- Dung dch NaOH 10%

: 20ml

- Ure

: 2 3g

- Dung dch lng trng trng 1%

: 40ml

2.2. Dng c v thit b

- ng nghim

: 04 ng

- Pipette 1ml

: 02 pipette

- Pipette 5ml

: 01 pipette

- Becher 50ml

: 03 cc

2.3. Tin hnh

Ly ng nghim kh, sch cho vi tinh th ure, un nh ti nng chy, kh cng to
thnh biure khi c NH3 bay hi, nhn bit bng mi hoc th bng giy qu o pH.
ngui thm vo 2 ml dung dch NaOH 10%, lc u cho tan ht v thm 2 3
GV: ThS. L Thanh Hi

Thc hnh Cng ngh Protein - Enzyme

git dung dch CuSO4 1%. Ly ng nghim th hai cho 1ml dung dch lng trng
trng, 1ml dung dch NaOH 10%, thm 2 3 git CuSO 4 1%, lc u.

mu tm
2.4. Kt qu
Chp hnh, ghi mu v vit phng trnh phn ng ca 2 phn ng trn.

Hnh 2.1: ng 1 (phi, Biure), ng 2 (tri, albumin)

ng 1 (BIURE): Khi un ure trn ngn la n cn th ta thy tinh th ure tan,
bay hi mt t ri kt tinh li (kh NH3 bay hi). Nhng sau khi cho 1-2ml dung
dch NaOH 10% vo th ta thy tinh th ure tan ra to thnh dung dch lng khng
mu. Khi lc ri cho thm dung dich CuSO 4 1% vo th dung dch ha mu hng
nht.
Acide cianuric
Urea un

nh NaOH 10%

NH3 (bay hi)

cianuric c trung ha)

Biure (2 phn t Ure kt hp to thnh)

lm xut hin lin kt

peptide (-CO-NH-). Trong mi trng kim Biure +CuSO4 cho phc mui Cu c
mu tm hng

GV: ThS. L Thanh Hi

10

Thc hnh Cng ngh Protein - Enzyme

ng 2: Trong dung dich lng trng trng (Albumin) cng c lin lt peptide nn
cng c phn ng Biure to phc hp mui Cu vi polypeptide c mu tm xanh.
Trong mi trng kim mnh, lin kt peptide trong phn t protein phn ng vi
CuSO4 to thnh phc cht mu tm hoc m tm . Ty theo di ca mch
peptide m mu ca phc s bin thin t xanh tm n tm .

GV: ThS. L Thanh Hi

11

Thc hnh Cng ngh Protein - Enzyme

BI 3: XC NH IM NG IN CA PROTEIN CASEIN
1/ Nguyn tc
Cc phn t protein l cc polymer c tnh in ly lng cc. Trong dung dch, khi
pH thay i n phn ly to thnh cc nhm tch in dng v cc nhm tch in
m khc nhau. i vi mi protein s c mt gi tr pH xc nh m ti tng s
in tch m bng tng s in tch dng, khi phn t protein trung ha v in,
pH gi l im ng in ca protein. Ti im ng in, dung dch protein
khng bn, d b kt ta.
2/ Thc hnh
1.1. Nguyn liu v ha cht
- Dung dch acid acetic (CH3COONa) 0,1N

: 200ml

- Dung dch natri acetate (CH3COONa) 0,1N

: 50ml

- Dung dch casein 0,4%: Cn 0,4g casein cho vo becher 100ml v thm 20ml
dung dch CH3COONa 0,1N. t ln ni cch thy un nng ha tan hon ton.
Chuyn sang bnh nh mc 100ml v nh mc ti 100ml bng dung dch
CH3COONa 0,1N.
- Nc ct

: 50ml

1.2. Dng c v thit b

- ng nghim

: 10 ng

- Pipette 1ml

: 02 pipette

- Bnh nh mc 100ml

: 01 bnh

- Becher 100ml

: 03 cc

- Ni cch thy

: 01 ci

GV: ThS. L Thanh Hi

12

Thc hnh Cng ngh Protein - Enzyme

1.3. Tin hnh

Ly dung dch CH3COOH 0,1N v nc vo 5 ng nghim theo th t nh bng
sau:
Lc u, sau thm vo mi ng 1ml dung dch casein v theo di s bin i ca
chng trong mi ng nghim.
Bng 3.1 B tr th nghim xc nh im ng in ca casein

1.4. Kt qu
ng nghim c nhiu kt ta nht l im ng in ca Casein
Chp hnh v ghi li kt qu xc nh pH ng in ca Casein

GV: ThS. L Thanh Hi

13

Thc hnh Cng ngh Protein - Enzyme

Hnh
im ng in ca casein t tri sang phi (1,2,3,4, v 5).

Mc kt ta
ng 1: Kt ta nht
ng 2: Kt ta ht
ng 3: Kt ta nhiu
ng 4: Kt ta nhiu nht
ng 5: Kt ta trung bnh
Kt lun: Vy im ng in ca protein Casein l pH = 4,1.

GV: ThS. L Thanh Hi

14

3.1 Th nghim

Thc hnh Cng ngh Protein - Enzyme

BI 4: SO SNH TC DNG XC TC CA ENZYME VI XC TC V

C
1/ Nguyn tc
Cht xc tc l cht lm cho phn ng xy ra nhanh hn nhng khng b tiu hao
trong qu trnh phn ng. Enzyme l cc cht xc tc ca cc h thng sinh hc.
Chng c kh nng xc tc c bit, thng mnh hn nhiu so vi cc cht xc tc
tng hp. Tc dng xc tc ca chng mang tnh c hiu cao i vi c cht, lm
tng ng k tc cc phn ng ha hc xy ra trong mi trng nc iu kin
nhit v pH n ha.
2/ Thc hnh
2.1. Nguyn liu v ha cht
- Enzyme amylase

: 50ml

- Dung dch HCl 1%

: 50ml

- Nc ct

: 10ml

- Dung dch h tinh bt 10%

2.2. Dng c v thit b
- ng nghim

: 50ml

: 04 ng

- Pipette 1ml

: 02 pipette

- Ni un cch thy

: 01 ci

- T m
- Becher 50ml

GV: ThS. L Thanh Hi

: 01 ci
: 03 cc

15

Thc hnh Cng ngh Protein - Enzyme

2.3. Tin hnh

Ly 4 ng nghim ghi s, mi ng 2ml dung dch tinh bt 10%. Sau thm
vo:
- ng th nht

: 10ml nc ct

- ng th hai

: 10ml dung dch enzyme amylase

- ng th ba

: 10ml dung dch enzyme amylase

- ng th t

: 10ml dung dch HCl 1%

Sau t cc ng nghip th nht v th hai nhit phng, ng th ba vo t

m 600C. t ng th t vo ni cch thy ang si. Sau 10 pht ly cc ng
nghim ra, cho vo mi ng 5ml thuc th Lugol. t c 4 ng vo ni cch thy
trong 2 pht. Ly ra lm ngui vquan st hin tng trong cc ng nghim.
2.4. Kt qu
Chp hnh v ghi kt qu vo bng sau:
Bng 4.1
th nghim so
xc tc ca
xc tc v c

Bng kt qu
snh tc dng
enzyme vi

Hnh 4.1: ng 1,2,3,4 (t tri sang phi) sau qu trnh lm ngui 30 pht
GV: ThS. L Thanh Hi

16

Thc hnh Cng ngh Protein - Enzyme

Nhn xt phn ng Lugol:

ng 1: Cho ra phc xanh c mu m nht.
ng 2: Cho ra phc c mu xm nht c s phn cch r rng (lp di mu trng
c).
ng 3: Cho ra phc c mu xm.
ng 4: Cho ra phc xm trong c s phn cch (lp di mu trng trong).
Gii thch
ng 1: Mu xanh lam xut hin khi thuc th lugol tip xc tinh bt, bi v thnh
phn chnh ca thuc th lugol l it v kali-it (KI). Do cu to dng mch xon
c l rng tinh bt hp thu iod cho mu xanh c trng iu kin thng, khi c
tc dng nhit th mt mu do iod tch ra khi tinh bt trng thi t do dn n
I2 thng hoa lm mt mu xanh, lm lnh c mu xanh tr li l do cn mt s iod
bm trn thnh ng nghim hp thu trn phn t tinh bt lm mu xanh xut hin
tr li.
ng 2,3:
Thy phn
Tinh bt
Amylase

Tinh bt + Lugol (iod, KI) cho mu xanh

(Glucose, Maltose, Maltodextrin, Acrodextrin) + Lugol
Amylodextrin + Lugol cho mu tm
Erythrodextrin + Lugol cho mu tm
o
ng 4: 100 C Tinh bt s b thy phn bi HCl. Sau lng tinh bt cn d
tip tc phn ng vi Lugol cho ra phc c mu xanh.
Tt c cc phn ng sinh ha trong t bo sinh vt u c thc hin trong iu
kin n ha, khng i hi nhit cao (Enzyme xc tc v c Amylase) nhng tc
phn ng vn mnh hn so vi Enzyme xc tc hu c (HCl) phi cn nhit
thch hp th phn ng mi xy ra.

GV: ThS. L Thanh Hi

17

Thc hnh Cng ngh Protein - Enzyme

BI 5: TNH C HIU CA ENZYME

1/ Nguyn tc
Tnh cht c hiu l biu hin kh nng xc tc ca enzyme i vi c cht nht
nh. Enzyme c tnh c hiu rt cao. Tnh cht c hiu ca enzyme cho thy s
khc bit rt ln gia enzyme vi cc cht xc tc khc. Mi loi enzyme ch c
kh nng xc tc cho s chuyn ha mt hay nhiu cht nht nh theo mt kiu
phn ng nht nh. Tnh cht c hiu ca enzyme biu hin mt s kiu c
hiu sau:
c hiu kiu phn ng: mi enzyme ch xc tc cho mt kiu phn ng chuyn
ha nht nh trong cc kiu phn ng nh phn ng oxy ha kh, phn ng
chuyn b, phn ng thy phn
c hiu kiu c cht: c 3 kiu c hiu c cht
- c hiu tuyt i: mi enzyme ch xc tc i vi mt c cht nht nh
- c hiu tng i: mi enzyme ch xc tc i vi mt kiu lin kt ha hc
nht nh
- c hiu quang hc (c hiu lp th): mi enzyme ch xc tc i vi mt dng
ng phn quang hc ca c cht (cis hoc trans)
2/ Thc hnh
2.1. Nguyn liu v ha cht
- Enzyme amylase

: 5ml

- Nc ct

: 5ml

- Dung dch h tinh bt 5%

: 10ml

GV: ThS. L Thanh Hi

18

Thc hnh Cng ngh Protein - Enzyme

- Casin 2%

: 20ml

2.2. Dng c v thit b

- ng nghim

: 04 ng

- Pipette 1ml

: 02 pipette

- Ni un cch thy

: 01 ci

- T m

: 01 ci

- Becher 50ml

: 03 cc

2.3. Tin hnh

Ly 4 ng nghim ghi s, sau thm vo:
- ng th nht: 2ml dung dch h tinh bt 5% + 1ml dung dch enzyme amylase
- ng th hai: 2ml dung dch h tinh bt 5% + 1ml dung dch enzyme bromelin
- ng th ba: 2ml dung dch casein 2% + 1ml dung dch enzyme amylase
- ng th t: 2ml dung dch casein 2% + 1ml dung dch enzyme bromelin
Lc u v vo t m 600C trong 15 pht. Ly ra, cho vo ng nghim th nht
v th hai mi ng 02 git thuc th Lugol.Cho ng th ba v th t mi ng 02
git thuc th Folin.
2.4. Kt qu
Chp hnh v ghi kt qu vo bng sau:

Bng 5.1 Bng kt qu th nghim xc nh tnh c hiu ca enzyme

GV: ThS. L Thanh Hi

19

Thc hnh Cng ngh Protein - Enzyme

Nhn xt v tnh c hiu ca Enzyme.

Hnh 5.1: ng 1,2,3 v 4 t tri sang phi sau qu trnh th nghim.

Gii thch:
GV: ThS. L Thanh Hi

20

Thc hnh Cng ngh Protein - Enzyme

ng 1: : Mu xanh lam xut hin khi thuc th lugol tip xc tinh bt, bi v thnh
phn chnh ca thuc th lugol l it v kali-it (KI). Do cu to dng mch xon
c l rng tinh bt hp thu iod cho mu xanh c trng iu kin thng.
Enzyme amylase

Glucose

Tinh bt

Maltose
Thy phn

Maltotetrose
Dextrin phn t thp

Enzyme amylase thy phn lin kt -1,4-glycoside ca tinh bt thnh cc dextrin

ln n nh, cui cng l glucose.
ng 2: Enzyme bromelin khng tc dng vi tinh bt. S d th nghim c mu
xanh l do (tinh bt + Lugol (iod, KI)) cho mu xanh c trng.
ng 3: Enzyme amylase khng thy phn Casein.
ng 4: Enzyme bromelin thy phn c cht casein. Qu trnh thy phn s b s
cho ra Polypeptide v acid amin.

GV: ThS. L Thanh Hi

21

Thc hnh Cng ngh Protein - Enzyme

BI 6: TC DNG CA CHT KCH THCH V CHT KM HM

1/ Nguyn tc
Cc cht c tc dng lm tng hot tnh ca enzyme gi l cc cht hot ha
enzyme. Cc cht hot ha c th l nhng anion, cc ion kim loi (t th 11 n
th 55 trong bng h thng tun hon), cc cht hu c c cu trc phc tp. Cc
cht hot ha thng lm nhim v chuyn nhm hydrogen hoc nhng cht c kh
nng ph v mt s lin kt trong phn t tin enzyme hoc cc cht c tc dng
phc hi cc nhm chc nng trong trung tm hot ng ca enzyme.
Cc cht km hm hot ng ca enzyme thng l cc cht c mt trong phn ng
enzyme, lm gim hot tnh enzyme nhng khng b enzyme lm thay i tnh cht
ha hc, cu to ha hc v tnh cht vt l ca chng. Cc cht gy km hm hot
ng ca enzyme bao gm cc ion, cc phn t v c, cc cht hu c v c
protein.
- Cht km hm cnh tranh: l nhng cht c cu trc tng t nh cu trc ca c
cht. Chng thng l cht kim hm thun nghch. Chng c kh nng kt hp vi
trung tm hot ng ca enzyme v chim ly v tr ca c cht trong trung tm
hot ng.
- Cht km hm khng cnh tranh: nhng cht ny khng chim trung tm hot
ng ca enzyme m lin kt vi v tr ngoi trung tm hot ng lm thay i cu
trc khng gian ca phn t enzyme theo chiu hng bt li cho hot ng xc
tc.
2/ Thc hnh
2.1. Nguyn liu v ha cht
- Enzyme amylase
GV: ThS. L Thanh Hi

: 50ml
22

Thc hnh Cng ngh Protein - Enzyme

- Dung dch NaBr 0,5%

: 50ml

- Dung dch CuSO4 0,5%

: 50ml

- Nc ct

: 50ml

- Dung dch h tinh bt 1%

: 50ml

- Thuc th Lugol

: 50ml

- Thuc th Fehling

: 50ml

2.2. Dng c v thit b

- ng nghim

: 03 ng

- Pipette 5ml

: 01 pipette

- Pipette 1ml

: 01 pipette

- B iu nhit

: 01 thit b

- Becher 50ml

: 03 cc

2.3. Tin hnh

Cho vo 03 ng nghim mi ng 5ml dung dch h tinh bt 1%, sau cho vo:
- ng th nht: 10ml nc ct
- ng th hai: 10ml dung dch NaBr 0,5%
- ng th ba: 10ml dung dch CuSO4 0,5%
Sau cho vo c ba ng mi ng 10ml dung dch enzyme amylase. Lc u v
vo t m 370C trong 15 pht. Ly ra, cho vo mi ng 2 git thuc th Lugol,
lc u.

GV: ThS. L Thanh Hi

23

Thc hnh Cng ngh Protein - Enzyme

Tip tc cho vo mi ng 2ml dung dch Fehling v t vo b iu nhit ang si

trong 2 pht, lm ngui ti nhit phng.
2.4. Kt qu
Chp hnh v ghi kt qu vo bng sau:
Bng 6.1 Bng kt qu th nghim xc nh tc dng ca cht hot ha v
kim hm hot tnh ca enzym amylase

Nhn xt v tc dng ca cht hot ha v cht kim hm n hot tnh ca enzyme

amylase.

GV: ThS. L Thanh Hi

24

Thc hnh Cng ngh Protein - Enzyme

Hnh 6.1: ng 1,2 v 3 t tri sang phi sau qu trnh th nghim.

Nhn xt:
ng 1: Xut hin mu xanh nht nht => tc hot ha ca cht hot ha yu v
nc ct khng phi l cht km hm hay kch thch cho qu trnh thy phn tinh
bt.
ng 2: Xut hin mu xanh trung bnh =>dung dch NaBr c tc dng kch thch
cho phn ng thy phn mc trung bnh.
ng 3: Xut hin mu xanh m nht =>dung dch CuSO 4 c tc dng km hm
lm phn ng thy phn chm li.

GV: ThS. L Thanh Hi

25

Thc hnh Cng ngh Protein - Enzyme

BI 7: XC NH HOT TNH CA ENZYME LIPASE

1/ Nguyn tc
Lipase l enzym thuc nhm thy phn, phn nhm esterase xc tc phn ng
thy gii cc ni ester gia glycerine v cc acid bo trong glyceride.
Nh hot ng ca lipase, cc acid bo c gii phng lm tng chua ca mi
trng phn ng. Lng acid s c chun bng kim v ch s hot ca
enzym s l lng kim cn trung ha cc acid bo mi c hnh thnh.
2/ Thc hnh
2.1. Nguyn liu v ha cht
- Tu tng ng vt

: 4g

- Sa ti

: 100ml

- KOH 0,1N

: 100ml

- Cn 96%

: 50ml

- Phenolphtalein 1%

: 15ml

- Hn hp nc h glycerin

: 40ml (t l 3 nc : 1glycerin)

- H2SO4 0,1N chun

: 100ml

- Vi mn

:1

- Giy lc

:4

- Phu lc
2.2. Dng c
- Erlen 100ml
GV: ThS. L Thanh Hi

:7
26

Thc hnh Cng ngh Protein - Enzyme

- B iu nhit

:1

- Buret 25ml

:1

- Bercher 100ml

:4

- Pipet 5ml

:2

- Pipet 10ml

:2

- Ci s

:1

- Bp in

:1

2.3. Tin hnh

Chun b ch phm lipase t tu tng ng vt: Cn 1g ty tng ng vt, cho vo
ci nghin 5ml hn hp nc glycerin (t l 3:1), nghin k t 3 5 pht. Sau ,
li cho thm 5ml hn hp nc glycerin v nghin tip 1 h2 pht. Lc vt hn
hp qua vi mn, sau lc li qua giy lc, thu dch lipase lm th nghim
Xc nh hot tnh ca enzyme lipase:
Ly 02 erlen 100ml, cho vo mi bnh 10ml sa ti
- Erlen 1 (erlen th nghim): cho vo 2 ml lipase, lc u, t vo b iu nhit
370C 400C trong 1 gi
- Erlen 2 (erlen i chng): un si sa trong bnh, sau cho ngay 2ml lipase vo
v un si tip 5 pht. ngui n nhit phng. Sau t vo b iu nhit
370C 400C trong 1 gi. Sau 1 gi ly erlen ra, thm vo mi bnh 8ml cn 96%,
vi git phenolphtalein, chun c hai erlen bng KOH 0,1N.
Tin hnh th nghim 2 ln. Ly kt qu l gi tr trung bnh ca 2 ln th nghim

GV: ThS. L Thanh Hi

27

Thc hnh Cng ngh Protein - Enzyme

Cch xc nh h s hiu chnh k:

- Chun 10ml dung dch KOH 0,1N bng dung dch H 2SO4 0,1N chun, thuc
th mu l phenolphtalein cho n khi dung dch va mt mu hng th dng li
- Xc nh lng H2SO4 0,1N chun cn dng
- Tin hnh th nghim 3 ln. Ly kt qu l gi tr trung bnh ca 3 ln th nghim
2.4. Tnh kt qu
H s hiu chnh k c tnh nh sau:
- Tnh nng KOH thc t:

- H s hiu chnh k:

Trong :
CKOHt : Nng dung dch KOH thc t
VKOHt : Th tch dung dch KOH thc t
CKOHp : Nng dung dch KOH pha
VKOHp : Th tch dung dch KOH pha
Hot lipase: c biu th bng s ml KOH dng chun acid bo to
thnh t s thy phn lipid c trong 1 lt sa v c tnh theo cng thc sau:

GV: ThS. L Thanh Hi

28

Thc hnh Cng ngh Protein - Enzyme

X= (V1 - V2) x k x 100/3

X= (3,3- 2,6) x 1 x 100/3 = 23,3333 UI
Trong :
X

: Hot ca enzyme lipase (UI)

V1

: S ml KOH 0,1N dng chun erlen th nghim (erlen 1)

V2

: S ml KOH 0,1N dng chun erlen i chng (erlen 2)

: H s hiu chnh ca dung dch KOH (k = 1).

Hnh7.1: Kt qu sau chun ca i chng v mu.

GV: ThS. L Thanh Hi

29

Thc hnh Cng ngh Protein - Enzyme

PHN T THIT K TH NGHIM XC NH NHIT TI THCH CA

ENZYME ASCOBATOXIDASE
1/ Nguyn tc
Ascobatoxidase l enzym thuc lp oxi ho kh (oxireductase) c nhiu trong cc
loi rau qu
ti v nhiu nht lp v qu. Enzym ny oxi ho acid ascorbic thnh acid
dehydroascorbic.
Trong thnh phn ca enzym ny c s hin din ca ion Cu2+.
2/ Thc hnh
2.1. Nguyn liu v ho cht
- Da chut

: 100g

- Dung dch m photphat 0,15M pH7

: 200ml

- Acid ascorbic (nng 1mg/ml)

: 100ml

- Dung dch KI 10%

: 150ml

- Dung dch HCl 5%

: 250ml

- Dung dch h tinh bt 1%

: 100ml

- Dung dch KIO3 0,01N

: 250ml

- Dung dch Na2S2O3 0,01N

: 1000ml

- Cch pha dung dch m phosphate pH 7:

o Dung dich A: 289 g NaH2PO4 ho tan trong 80ml
o Dung dch B: 8,53g Na2HPO4.12H2O pha trong 120 ml.
o pha dung dch m phosphate pH =7 ht dung dch A v dung dch B theo t
l sau: Dung dich A: 80ml + dung dich B: 120ml.
2.2. Dng c
- Ci s

:1

- Becher 50ml

:2

- Becher 100ml

:2

GV: ThS. L Thanh Hi

30

Thc hnh Cng ngh Protein - Enzyme

- Erlen 100ml

:3

- Bnh nh mc 50ml

:2

- Erlen 250ml

:4

- Buret 25 ml

:1

- Pipet 5ml

:2

- Pipet 10ml

:2

- Giy lc

:4

Mu

Nhit

Acid

KI

HCl

H tinh

KIO3

(Bnh

chn

ascorbic

10%

5%

bt

0,01N

erlen)

(0C)

(ml)

(ml)

(ml)

(git)

(ml)

300C
400C
450C
500C
600C

10
10
10
10
10

5
5
5
5
5

10
10
10
10
10

10
10
10
10
10

(un
1
2
3
4
5
Phu lc

si)
1
2
3
4
5

10
10
10
10
10
:2

2.3. Tin hnh

Chit tch enzym ascobatoxidase: Cn 30g da chut (b ht) cho vo ci s,
nghin vi 10 15ml dung dch m phosphat 0,15M pH 7. Dch chit c cho
vo bnh nh mc 50ml v thm dung dch m n vch nh mc. Lc u nhiu
ln v yn trong 1 gi. Lc thu dch lc. Dch lc l ch phm enzym
ascobatoxidase.
Kho st nhit ti thch enzym ascobatoxidase:
Bng 7.1: Cch tin hnh th nghim

GV: ThS. L Thanh Hi

31

Thc hnh Cng ngh Protein - Enzyme

Lc u, yn 5 pht ri chun bng dung dch Na 2S2O3 0,01N n khi mt

mu xanh.
Xc nh h s hiu chnh ca Na2S2O30,01N bng dung dch KIO3 0,01N
chun (tng t nh cch xc nh h s hiu chnh ca dung dch KOH 0,1N)
2.4. Cch tnh kt qu:
Hot tnh ca enzym ascobatoxidase c hin th bng s mg acid ascorbic b oxi
ho di tc ng ca enzym c trong 1g nguyn liu trong thi gian 1 pht.
Hot tnh ca enzym c tnh theo cng thc:

Trong
X : Hot ca enzyme ascorbatoxidase (UI)
V1 : Th tch Na2S2O3dng erlen th nghim
V2 : Th tch Na2S2O3dng erlen i chng
k : H s hiu chnh ca dung dch Na2S2O30,01N
0,088 : S mg acid ascorbic tng ng vi 1 ml dung dch Na 2S2O30,01N
m : Khi lng nguyn liu chit enzym (g)
t : Thi gian phn ng (pht) 0C
Bng 8.1: Nhit v hot ca Acid Ascobatoxidase sau qu trnh th
nghim.

Nhit (0C)

30

40

45

50

60

Hot (UI)

65

85

100

95

60

GV: ThS. L Thanh Hi

32

Thc hnh Cng ngh Protein - Enzyme

OPTIMUM TEMPERATURE
120
100
80
60

ACTIVITY (U/mL)

40
20
0
30

35

40

45

50

55

60

TEMPERATURE 0C

th biu din nhit ti thch ca Enzyme Ascobatoxidase l nhit (450C)

Bng 8.2: pH v hot ca enzyme ascobatoxidase sau qu trnh th
nghim

Ph

Hot

85

55

25

GV: ThS. L Thanh Hi

33

Thc hnh Cng ngh Protein - Enzyme

OPTIMUM pH
100
90
80
70
60
RELATIVE ACTIVITY (%)

50
40
30
20
10
0
4

4.5

5.5

6.5

7.5

8.5

pH

th biu din pH ti thch ca Enzyme Ascobatoxidase l pH= 6.

Hnh 8.1: Trc chun v sau chun bng Na2S2O3 0,01N.

GV: ThS. L Thanh Hi

34

Thc hnh Cng ngh Protein - Enzyme

BI 8: KHO ST HOT TNH ENZYME ASCOBATOXIDASE

1/ Nguyn tc
Ascobatoxidase l enzym thuc lp oxi ho kh (oxireductase) c nhiu trong cc
loi rau qu ti v nhiu nht lp v qu. Enzym ny oxi ho acid ascorbic
thnh acid dehydroascorbic. Trong thnh phn ca enzym ny c s hin din ca
ion Cu2+.
2/ Thc hnh
2.1. Nguyn liu v ho cht
- Da chut

: 100g

- Dung dch m photphat 0,15M pH7

: 150ml

- Acid ascorbic (nng 1mg/ml)

: 100ml

- Dung dch KI 10%

: 50ml

- Dung dch HCl 5%

: 100ml

- Dung dch h tinh bt 1%

: 50ml

- Dung dch KIO3 0,01N

: 150ml

- Dung dch Na2S2O3 0,01N

: 100ml

- Cch pha dung dch m phosphate pH 7:

o Dung dich A: 27,8 g NaH2PO4 ho tan trong 1000ml
o Dung dch B: 53.05g Na 2HPO4 .7H2O hoc 71.1g Na2HPO4.12H2O pha trong
1000ml
o pha dung dch m phosphate pH =7 ht dung dch A v dung dch B theo t
GV: ThS. L Thanh Hi

35

Thc hnh Cng ngh Protein - Enzyme

l sau: Dung dich A: 39 ml + dung dich B: 61ml thm nc va 200ml.

2.2. Dng c
- Ci s

:1

- Becher 50ml

:2

- Becher 100ml

:2

- Erlen 100ml

:3

- Bnh nh mc 50ml

:2

- Erlen 250ml

:4

- Buret 25 ml

:1

- Pipet 5ml

:2

- Pipet 10ml

:2

- Giy lc

:4

- Phu lc

:2

2.3. Tin hnh

Chit tch enzym ascobatoxidase: Cn 30g da chut (b ht) cho vo ci s,
nghin vi 10 15ml dung dch m phosphat 0,15M pH 7. Dch chit c cho
vo bnh nh mc 50ml v thm dung dch m n vch nh mc. Lc u nhiu
ln v yn trong 1 gi. Lc thu dch lc. Dch lc l ch phm enzym
ascobatoxidase.
Kho st hot tnh enzym ascobatoxidase

GV: ThS. L Thanh Hi

36

Thc hnh Cng ngh Protein - Enzyme

- Ht 10 ml dung dch enzym cho vo becher 100ml. Sau cho thm 10ml dung
dch acid ascorbic. Lc u, yn nhit phng 30 pht. Tip theo un si trc
tip trn bp ngng phn ng, ri cho vo bnh nh mc 50 ml v lm y bng
nc ct.
- Ly 10ml dch trong bn trn cho vo erlen 250ml, thm 5ml dung dch KI 10%,
10 ml dung dch HCl 5%, 10 git h tinh bt 1% v 10 ml dung dch KIO 3 0,01N.
Lc u, yn 5 pht ri chun bng dung dch Na 2S2O3 0,01N n khi mt
mu xanh.
- Lm mt mu i chng tng t nh trn vi 10ml dung dch enzym c
un si trong 2 pht v lm lnh.
- Tin hnh th nghim 2 ln. Kt qu l gi tr trung bnh
Xc nh h s hiu chnh ca Na 2S2O3 0,01N bng dung dch KIO3 0,01N chun
(tng t nh cch xc nh h s hiu chnh ca dung dch KOH 0,1N)
2.4. Cch tnh kt qu:
Hot tnh ca enzym ascobatoxidase c hin th bng s mg acid ascorbic b oxi
ho di tc ng ca enzym c trong 1g nguyn liu trong thi gian 1 pht.
Hot tnh ca enzym c tnh theo cng thc:

X=

( 58,652 ) x 1 x 0.088
=0.58 UI
1 x1

Trong
X : Hot ca enzyme ascorbatoxidase (UI)
GV: ThS. L Thanh Hi

37

Thc hnh Cng ngh Protein - Enzyme

V1 : Th tch Na2S2O3 dng erlen th nghim

V2 : Th tch Na2S2O3 dng erlen i chng
k : H s hiu chnh ca dung dch Na2S2O3 0,01N
0,088 : S mg acid ascorbic tng ng vi 1 ml dung dch Na2S2O3 0,01N
m : Khi lng nguyn liu chit enzym (g)
t : Thi gian phn ng (pht)

Hnh 9.1: Trc chun v sau chun

GV: ThS. L Thanh Hi

38