B GIO DC V O TO

TRNG C KINH T CNG NGH TPHCM

KHOA CNG NGH SINH HC
*****************

GVGD: ThS. L Thanh Hi

TPHCM, thng 03 nm 2013

MC LC
LI NI U ............................................................................................................................ 1
NI QUY THC TP ............................................................................................................... 2
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 ................................................................................................... 3

2.3.

Tin hnh .................................................................................................................. 3

2.4.

Kt qu ..................................................................................................................... 4

BI 2: PHN NG BIURE ...................................................................................................... 5

1/

Nguyn tc ...................................................................................................................... 5

2/

Thc hnh ....................................................................................................................... 5

2.1.

Nguyn liu v ha cht ........................................................................................... 5

2.2.

Dng c v thit b ................................................................................................... 5

2.3.

Tin hnh .................................................................................................................. 5

2.4.

Kt qu ..................................................................................................................... 5

BI 3: XC NH IM NG IN CA PROTEIN CASEIN........................................ 6
1/

Nguyn tc ...................................................................................................................... 6

2/

Thc hnh ....................................................................................................................... 6

1.1.

Nguyn liu v ha cht ........................................................................................... 6

1.2.

Dng c v thit b ................................................................................................... 6

1.3.

Tin hnh .................................................................................................................. 6

1.4.

Kt qu ..................................................................................................................... 6

BI 4: SO SNH TC DNG XC TC CA ENZYME VI XC TC V C ............ 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 5: TNH C HIU CA ENZYME ............................................................................... 9

1/

Nguyn tc ...................................................................................................................... 9

2/

Thc hnh ....................................................................................................................... 9

2.1.

Nguyn liu v ha cht ........................................................................................... 9

-i-

2.2.

Dng c v thit b................................................................................................... 9

2.3.

Tin hnh ................................................................................................................. 9

2.4.

Kt qu ................................................................................................................... 10

BI 6: TC DNG CA CHT KCH THCH V CHT KM HM .............................. 11

1/

Nguyn tc ................................................................................................................... 11

2/

Thc hnh .................................................................................................................... 11

2.1.

Nguyn liu v ha cht ........................................................................................ 11

2.2.

Dng c v thit b................................................................................................. 11

2.3.

Tin hnh ............................................................................................................... 11

2.4.

Kt qu ................................................................................................................... 12

BI 7: XC NH HOT TNH CA ENZYME LIPASE .................................................. 13

1/

Nguyn tc ................................................................................................................... 13

2/

Thc hnh .................................................................................................................... 13

2.1.

Nguyn liu v ha cht ........................................................................................ 13

2.2.

Dng c .................................................................................................................. 13

2.3.

Tin hnh ............................................................................................................... 13

2.4.

Tnh kt qu ........................................................................................................... 14

BI 8: KHO ST HOT TNH ENZYME ASCOBATOXIDASE .................................... 15

1/

Nguyn tc ................................................................................................................... 15

2/

Thc hnh .................................................................................................................... 15

2.1.

Nguyn liu v ho cht ........................................................................................ 15

2.2.

Dng c .................................................................................................................. 15

2.3.

Tin hnh ............................................................................................................... 15

2.4.

Cch tnh kt qu: .................................................................................................. 16

BI 9: XC NH HM LNG PROTEIN TRONG DCH CHIT T QU DA

(PHNG PHP LOWRY) ................................................................................................... 17
1/

Nguyn tc ................................................................................................................... 17

2/

Thc hnh .................................................................................................................... 17

2.1.

Nguyn liu v ho cht ........................................................................................ 17

2.2.

Dng c v thit b................................................................................................. 17

2.3.

Tin hnh ............................................................................................................... 17

2.4.

Tnh kt qu ........................................................................................................... 19

BI 10: KHO ST HOT TNH ENZYME BROMELIN ................................................. 20

1/

Nguyn tc ................................................................................................................... 20

2/

Thc hnh .................................................................................................................... 20

2.1.

Dng c .................................................................................................................. 20

2.2.

Ho cht ................................................................................................................. 20
- ii -

2.3.

Cch tin hnh ........................................................................................................ 20

2.4.

Kt qu ................................................................................................................... 21

BI 11: XC NH HOT TNH ENZYM -AMYLASE THEO PHNG PHP SMITH

V ROE ................................................................................................................................... 22
1/

Nguyn tc .................................................................................................................... 22

2/

Thc hnh ..................................................................................................................... 22

2.1.

Nguyn liu v ho cht ......................................................................................... 22

2.2.

Dng c v thit b ................................................................................................. 22

2.3.

Cch tin hnh ........................................................................................................ 23

2.4.

Tnh kt qu............................................................................................................ 25

- iii -

LI NI U
Gio trnh Thc hnh cng ngh protein enzyme c bin son nhm phc v cho mn
hc thc hnh cng ngh protein enzyme ca sinh vin chuyn ngnh cng ngh thc phm
v sinh hc ng dng Khoa Cng ngh Sinh hc Trng Cao ng Kinh t Cng ngh
TPHCM. Gio trnh c bin son da trn c s tham kho cc ti liu v sch th nghim
v protein enzyme trong v ngoi nc.
Trong ti liu ny gm 11 bi thc hnh nhm gip sinh vin lm quen vi cc phng tin
v quy tc an ton th nghim, s dng dng c, thit b v thc hnh th nghim nh tnh,
nh lng acid amin, protein, enzyme t n gin n phc tp.
Cc bi th nghim ny gip sinh vin nm vng tnh cht ca protein enzyme v hiu r
hn cc kin thc l thuyt hc t mn hc Cng ngh Protein Enzyme.

Tc gi
ThS. L Thanh Hi

-1-

NI QUY THC TP
1. Sinh vin phi c mt ti phng th nghim ng gi, nu ngh hc phi c l do chnh
ng, phi xin php gio vin.
2. Sinh vin phi c k, nm vng ni dung bi th nghim nh v chun b sn bn bo
co kt qu th nghim trc khi th nghim.
3. Trong phng th nghim phi mc o blouse, rt thn trng khi s dng dung mi d chy
n v ha cht c hi.
4. p dng tr im 20% tng bi th nghim trong cc trng hp sau: khng chun b
trc, khng c o blouse, i tr qu 10 pht khng gi trt t, tc phong khng nghim
tc, vi phm ni quy phng th nghim, thao tc cu th khng thn trng.
5. Khi lm th nghim phi trt t, cn thn, gi sch ni lm th nghim, tit kim ha cht,
lm v hay gy h hng thit b th phi bi thng.
6. Sau khi th nghim, sinh vin phi ra sch dng c, sp xp li dng c, ha cht ng
ch, lau sch bn th nghim v bn giao cho cn b ph trch phng th nghim.
7. Cui bui sinh vin phi np bo co kt qu li cho gio vin hng dn.

-2-

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 imino 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
o Dung dich A: 27,8 g NaH2PO4 ho tan trong 1000ml
o Dung dch B: 53.05g Na2HPO4 .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
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)

2.2. Dng c v thit b

-

Bnh nh mc 100ml
Pipette 1ml
Pipette 10ml
Erlen 100ml
Burette 25ml
Becher 100ml

: 1 bnh
:2
:2
: 6 bnh
:1
: 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%.
Cho vo bnh th hai: 20ml dung dch c pH 9,2; 5 git bromthymol blue 0,04%
v 3 git phenolphtalein 0,5%

Khi dung dch trong bnh 1 c mu xanh lc nht, dung dch trong bnh 2 c mu tm xanh.
Mu ca cc dung dch trn gi trong bnh kn c th bn trong na thng.

-3-

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

=

  0,0007 100 1000

20 

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

-4-

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%

Dung dch NaOH 10%
Ure
Dung dch lng trng trng 1%

: 10ml
: 10ml
: 2 3g
: 10ml

2.2. Dng c v thit b

-

ng nghim
Pipette 1ml
Pipette 5ml
Becher 50ml

: 04 ng
: 02 pipette
: 01 pipette
: 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 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 CuSO4 1%, lc u.
2.4. Kt qu
Chp hnh, ghi mu v vit phng trnh phn ng ca 2 phn ng trn.

-5-

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 (CH3COOH) 0,1N

: 100ml
Dung dch natri acetate (CH3COONa) 0,1N
: 100ml
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
Pipette 1ml
Bnh nh mc 100ml
Becher 100ml
Ni cch thy

: 10 ng
: 02 pipette
: 01 bnh
: 03 cc
: 01 ci

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 1. B tr th nghim xc nh im ng in ca protein casein

TT ng nghim

01

02

03

04

05

CH3COOH 0,1N (ml)

0,1

0,2

1,0

4,0

8,0

Nc ct (ml)

8,9

8,8

8,0

5,0

1,0

Dung dch casein 0,4% (ml)

1,0

1,0

1,0

1,0

1,0

pH

5,6

5,3

4,7

4,1

3,8

Mc kt ta
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.

-6-

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
Dung dch HCl 1%
Nc ct
Dung dch h tinh bt 10%

: 5ml
: 5ml
: 5ml
: 10ml

2.2. Dng c v thit b

-

ng nghim
Pipette 1ml
Ni un cch thy
T m
Becher 50ml

: 04 ng
: 02 pipette
: 01 ci
: 01 ci
: 03 cc

2.3. Tin hnh

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

ng th nht: 1ml nc ct
ng th hai: 1ml dung dch enzyme amylase
ng th ba: 1ml dung dch enzyme amylase
ng th t: 1ml dung dch HCl 1%

Sau t cc ng nghip th nht v th hai nhit phng, ng th ba vo t m 60oC.

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 v
quan st hin tng trong cc ng nghim

-7-

Thc hnh Cng ngh Protein - Enzyme

2.4. Kt qu
Chp hnh v ghi kt qu vo bng sau:
Bng 2. Bng kt qu th nghim so snh tc dng xc tc ca enzyme vi xc tc v c

Nhit

ng nghim

C cht

Xc tc

Tinh bt

Khng

30

Tinh bt

Amylase

30

Tinh bt

Amylase

60

Tinh bt

HCl

100

( C)

Phn ng
Lugol

Nhn xt v tc dng ca tng loi xc tc v nh hng ca nhit .

-8-

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
Dung dch HCl 1%
Nc ct
Dung dch h tinh bt 10%

: 5ml
: 5ml
: 5ml
: 10ml

2.2. Dng c v thit b

-

ng nghim
Pipette 1ml
Ni un cch thy
T m
Becher 50ml

: 04 ng
: 02 pipette
: 01 ci
: 01 ci
: 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 37oC trong 15 pht. Ly ra, cho vo ng nghim th nht v th

hai mi ng 02 git thuc th Lugol v t vo b iu nhit ang si trong 2 pht th ly cc
ng nghim ra, lm ngui ti nhit phng.
Cho ng th ba v th t mi ng 02 git thuc th Folin.

-9-

Thc hnh Cng ngh Protein - Enzyme

2.4. Kt qu
Chp hnh v ghi kt qu vo bng sau:
Bng 3. Bng kt qu th nghim xc nh tnh c hiu ca enzyme

ng nghim

C cht

Xc tc

Tinh bt

Amylase

Tinh bt

Bromelin

Casein

Amylase

Casein

Bromelin

Nhn xt v tnh c hiu ca enzyme.

- 10 -

Phn ng th

Kt qu

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
Dung dch NaBr 0,5%
Dung dch CuSO4 0,5%
Nc ct
Dung dch h tinh bt 1%
Thuc th Lugol
Thuc th Fehling

: 5ml
: 5ml
: 5ml
: 5ml
: 20ml
: 5ml
: 5ml

2.2. Dng c v thit b

-

ng nghim
Pipette 5ml
Pipette 1ml
B iu nhit
Becher 50ml

: 03 ng
: 01 pipette
: 01 pipette
: 01 thit b
: 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: 1ml nc ct
ng th hai: 1ml dung dch NaBr 0,5%
ng th ba: 1ml dung dch CuSO4 0,5%

Sau cho vo c ba ng mi ng 1ml dung dch enzyme amylase. Lc u v vo t m

37oC trong 15 pht. Ly ra, cho vo mi ng 2 git thuc th Lugol, lc u.
Tip tc cho vo mi ng 5ml dung dch Fehling v t vo b iu nhit ang si trong 2
pht, lm ngui ti nhit phng

- 11 -

Thc hnh Cng ngh Protein - Enzyme

2.4. Kt qu
Chp hnh v ghi kt qu vo bng sau:
Bng 4. Bng kt qu th nghim xc nh tc dng ca cht hot ha v kim hm hot tnh ca enzyme amylase

ng nghim

C cht

Xc tc

Tinh bt

Amylase

Tinh bt

Amylase

Tinh bt

Amylase

Phn ng th

Kt qu

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

- 12 -

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
Sa ti
KOH 0,1N
Cn 96%
Phenolphtalein 1%
Hn hp nc - glycerin
H2SO4 0,1N chun
Vi mn
Giy lc
Phu lc

: 4g
: 100ml
: 100ml
: 50ml
: 15ml
: 40ml (t l 3 nc : 1glycerin)
: 100ml
:1
:4

2.2. Dng c
-

Erlen 100ml
B iu nhit
Buret 25ml
Bercher 100ml
Pipet 5ml
Pipet 10ml
Ci s
Bp in

:7
:1
:1
:4
:2
:2
:1
: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 -2 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.

- 13 -

Thc hnh Cng ngh Protein - Enzyme

Tin hnh th nghim 2 ln. Ly kt qu l gi tr trung bnh ca 2 ln th nghim

Cch xc nh h s hiu chnh k:
-

Chun 10ml dung dch KOH 0,1N bng dung dch H2SO4 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:

  = 24 24
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:
 = 1 2   100
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

- 14 -

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 Na2HPO4 .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
l sau:
Dung dich A: 39 ml + dung dich B: 61ml thm nc va 200ml.

2.2. Dng c
-

Ci s
Becher 50ml
Becher 100ml
Erlen 100ml
Bnh nh mc 50ml
Erlen 250ml
Buret 25 ml
Pipet 5ml
Pipet 10ml
Giy lc
Phu lc

:1
:2
:2
:3
:2
:4
:1
:2
:2
:4
: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
-

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.

- 15 -

Thc hnh Cng ngh Protein - Enzyme

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 KIO3 0,01N. Lc u,
yn 5 pht ri chun bng dung dch Na2S2O3 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 Na2S2O3 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:
=

1 2   0,088


Trong
X

: Hot ca enzyme ascorbatoxidase (UI)

V1

: Th tch Na2S2O3 dng erlen th nghim

V2

: Th tch Na2S2O3 dng erlen i chng

: 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)

: Thi gian phn ng (pht)

- 16 -

Thc hnh Cng ngh Protein - Enzyme

BI 9: XC NH HM LNG PROTEIN TRONG DCH CHIT T

QU DA (PHNG PHP LOWRY)
1/ Nguyn tc
Hu ht cc protein u cha Tyrosin v Tryptophan. Hm lng ca nhng acid amin ny
ty thuc vo tng loi protein. V vy nhng protein cng mt loi vi nhau c hm lng
cc acid amin ny ging nhau.
Khi cho cc protein tc dng vi thuc th Folin s to thnh mt phc cht c mu. Cng
mu ca cht ny t l vi hm lng Tyrosin v Tryptophan (tng ng vi hm lng
protein). V vy c th dng phng php so mu xc nh hm lng protein.
2/ Thc hnh
2.1.
2.2.
2.3.

Nguyn liu v ho cht

Da (thm) xanh
: 1 tri
Dung dch albumin 0,1%
: 100ml
Dung dch A:
: 100ml
(Cn 2g Na2CO3 ha tan trong NaOH 0,1N va 100ml)
Dung dch B:
: 100ml
(Cn 0,5g CuSO4.5H2O ha tan trong dung dch citrat natri 1% va 100ml)
Dung dch C
: 50ml
(L hn hp ca 2 dung dch A v B theo t l 49:1 (ch pha dng trong ngy))
Thuc th Folin
: 0,2ml
Dng c v thit b
ng nghim
Pipet 1ml
Pipet 10ml
My o mt quang
Curvet
Erlen 100ml
Becher 250ml
My xay sinh t
Vi mn
Giy lc
Phu lc

: 20
:2
:1
:1
: 20
:4
:2
:2
:1
:4
:2

Tin hnh

Chun b dch protein mu t qu da: Chn tri thm xanh ti, gt b v ngoi, ct nh
v xay nt trong my xay sinh t. Lc qua tm vi mn v vt nc. Lc bng giy lc thu
dch lc trong. Dch lc l dung dch protein mu.

- 17 -

Thc hnh Cng ngh Protein - Enzyme

Xy dng ng chun albumin:

Bng 5. B tr th nghim xc nh ng chun albumin

ng nghim s

Nng protein (g/ml)

50

100

150

200

250

Dung dch albumin 0,1%

(ml)

0,5

1,0

1,5

2,0

2,5

Nc ct (ml)

10

9,5

9,0

8,5

8,0

7,5

T mi nng khc nhau ht ra 0,4 ml cho vo cc ng nghim

khc nhau
Dung dch protein (ml)
Dung dch C (ml)

0,4

0,4

0,4

0,4

0,4

0,4

0,2

0,2

2,4

2,4

Lc u v yn nhit phng 5 pht

Thuc th Folin (ml)

0,2

0,2

0,2

0,2

Lc u v yn nhit phng 10 pht

Nc ct (ml)

2,4

2,4

2,4

2,4

o mt quang bc sng 500 nm

V th biu din s bin thin ca mt quang (OD) theo nng protein chun
(g/ml) vi trc tung l OD, trc honh l nng protein.

- 18 -

Thc hnh Cng ngh Protein - Enzyme

Xc nh hm lng protein trong mu

Bng 6. B tr th nghim xc nh hm lng protein trong mu da

ng nghim s

Dung dch protein mu (ml)

0,4

0,4

0,4

0,4

Dung dch C (ml)

0,2

0,2

0,2

0,2

2,4

2,4

2,4

2,4

Lc u v yn nhit phng 5 pht

Thuc th Folin (ml)
Lc u v yn nhit phng 10 pht
Nc ct (ml)
o mt quang bc sng 500 nm
sao cho gi tr OD nm trong khong 0,05 0,25
Tin hnh th nghim 2 ln. Kt qu l gi tr trung bnh.
2.4.

Tnh kt qu

T ng chun so snh mt quang ca ng nghim cha mu protein. T suy ra hm

lng protein ca nguyn liu l a (g/ml)
Lng protein (M) c trong 1 g nguyn liu c tnh theo cng thc:
=

 10!3 "
# $%&'(/1#


Trong
a

: hm lng protein (g/ml dung dch)

: h s pha long

: khi lng nguyn liu ly phn tch (g)

- 19 -

Thc hnh Cng ngh Protein - Enzyme

BI 10: KHO ST HOT TNH ENZYME BROMELIN

1/ Nguyn tc
Bromelin l 1 enzyme thuc loi protease c nhiu trong tri thm. kho st hot tnh ca
bromelin, ngi ta cho enzyme ny thu phn cc c cht protein (hemoglobin, casein) v
do cc peptid c gii phng ra c cha Tyrosin bng cch cho tc dng vi thuc th Folin.
2/ Thc hnh
2.1.

Dng c

Becher 100ml, 250ml, 500ml

Chu thu tinh

Pipet 0,2ml, 1ml, 2ml, 5ml

My ly tm

Phu lc

Quang ph k

ng nghim
2.2.

Ho cht

Dung dch NaOH 0,5N 1N

Dung dch HCl 2N 0,2N

Dung dch acid tricloracetic 5%

Dung dch Tyrosin mu: 45mg pha trong 100ml dung dch HCl 0,2N

Dung dch m KH2PO4 1M: 13,6g pha vi nc ct thnh 100ml

Dung dch hemoglobin 2%: cn 2g Hb + 36g ure + 8ml dung dch NaOH 1N ho trong
40ml nc ct. 25oC trong 60 pht, thm 10ml dung dch m KH2PO4 1M, iu
chnh li pH 6 vi dung dch HCl 2N. Thm nc y ti vch mc 100ml. Bo qun
trong t lnh.

Thuc th Folin
2.3.

Cch tin hnh

Chit dch nc thm cha enzyme

Chn tri thm xanh ti, gt b v ngoi, ct nh v xay nt trong my xay sinh t. Lc
qua tm vi mn v vt nc. Nc thm em ly tm 6000 vng/pht trong 10 pht. Ly
dch bn trn c cha enzyme bromelin.

- 20 -

Thc hnh Cng ngh Protein - Enzyme

Kho st hot tnh enzyme bromelin:

Thc hin 3 loi ng nghim (mi loi lm 3 ng ly trung bnh)
Bng 7. B tr th nghim kho st hot tnh ca enzyme bromelin

ng nghim
Dung dch Hb 2% trong m
photphat

ml

Tyrosin

Mu

Khng

2,5

2,5

2,5

35oC trong 5 pht

Dung dch tricloracetic 5%

ml

Dung dch HCl 0,2N

ml

0,5

0,5

Dung dch tyrosin mu

ml

0,5

Dung dch nc tri thm c

cha enzyme bromelin

ml

0,2

Lc u yn 35oC trong 10 pht

Dung dch tricloracetic 5%

ml

Dch nc thm cha enzyme

bromelin

ml

0,2

0,2

Lc u mi ng v yn trong khong 10 pht 25oC, em lc.

Ht vo cc ng nghim khc mi ng 2,5ml dch lc, 5ml dung dch NaOH 0,5N v 1ml
thuc th Folin. Lc k, yn khong 15 pht v em c mt quang di sng
578nm hay 620nm.
2.4.

Kt qu

Hot tnh ca enzyme bromelin c biu th l s g tyrosin sinh ra do s thu phn Hb ca

enzyme c trong 1ml dung dch hay 1mg hn hp cha bromelin trong 1 pht.
Hot tnh ca enzyme bromelin:
450 +", 1
1

.//0
+"10
2
Vi

1 : ml dung dch tyrosin

2 : ml dung dch cha enzyme

- 21 -

Thc hnh Cng ngh Protein - Enzyme

BI 11: XC NH HOT TNH ENZYM -AMYLASE THEO

PHNG PHP SMITH V ROE
1/ Nguyn tc
Hot tnh -amylase biu th kh nng ca enzym amylase xc tc phn ng thy phn tinh
bt n dextrin trong 1 pht 50oC v c th hin bng s n v ca enzym trong 1g mu.
Khi phn ng thy phn tinh bt xy ra, lng tinh bt cn li cha c thy phn s to
phn ng mu vi Iod v c so mu bng my quang ph bc sng 620 nm.
2/ Thc hnh
2.1.

Nguyn liu v ho cht

La ny mm

: 100 ht

Dung dch m Sorensen 1/15M, pH = 5,6

: 100ml

(Trn chung 5 ml dung dch Na2HPO4 1/15M v 95 ml dung dch KH2PO4 1/15M)
-

Dung dch Na2HPO4 1/15M

: 5ml

(Ha tan 23,876 g Na2HPO4.12H2O trong nc va 1 lt)

-

Dung dch KH2PO4 = 1/15M

: 95ml

(Ha tan 2,2681 g KH2PO4 trong nc va 250 ml))

-

Dung dch tinh bt 1%

: 25ml

(1 g tinh bt trong 100 ml dung dch m pH = 5,6)

-

Dung dch HCl 1N

: 25ml

(85 ml dung dch HCl m c trong nc thnh 1 lt)

-

Dung dch NaCl 3%

: 25ml

(3 g NaCl trong nc thnh 100 ml)

-

Thuc th Lugol

: 25ml

(0,5 g I2 v 5 g KI trong nc thnh 200 ml)

2.2.

Dng c v thit b

Ci s

:1

ng nghim

: 20

ng ong 50ml

:1

B iu nhit

:1

Pipet 1ml

:2

Pipet 10ml

:2

Becher 100ml

:3

Erlen 100ml

:3

My quang ph

:1

Curvet

:6

- 22 -

Thc hnh Cng ngh Protein - Enzyme

Giy lc

:4

Phu lc

:3

2.3.

Cch tin hnh

Ly trch amylase: cho khong 100 ht la ny mm cho vo ci s nghin nhuyn vi 3-5 ml

nc ct. Tip tc nghin v thm dn n ht 50ml nc ct ha tan enzym. Lc dch
bng giy lc, thu dch lc c cha enzym amylase.

- 23 -

Thc hnh Cng ngh Protein - Enzyme

Tin hnh th nghim theo bng sau:

Bng 8. B tr th nghim xc nh hot tnh ca enzyme amylase

ng nghim
Dung dch ha cht
ng mu (t)

ng i chng (o)

Nc ct (ml)

Dch chit -amylase (ml)

Dung dch m (ml)

Dung dch tinh bt 1% (ml)

Dung dch NaCl 3% (ml)

0,5

0,5

Dung dch HCl 1N (ml)

em 50oC trong 30 pht

Dung dch HCl 1N (ml)

Nc ct (ml)

5,5

5,5

Thuc th Lugol (ml)

0,5

0,5

Lc u hn hp trong ng nghim ri mang i so mu bc sng

620 nm sao cho gi tr OD nm trong khong 0,05 0,25
Tin hnh th nghim lp li 3 ln. Kt qu l gi tr trung bnh.

- 24 -

Thc hnh Cng ngh Protein - Enzyme

2.4.

Tnh kt qu

Hot ca enzyme amylase:

1=

+"0 +"   "

./
+"2 

Trong :
ODo

: mt quang ca ng i chng.

ODt

: mt quang ca ng mu th.

: lng tinh bt ban u tham gia phn ng (mg).

: thi gian phn ng (pht).

: h s pha long mu enzym.

- 25 -