Bai Giang Thuc Hanh Enzyme - 2014

TRNG I HC CNG NGHIP THC PHM TP HCM
KHOA CNSH- KTMT
B MN CNG NGH SINH HC
BI GING THC HNH
CNG NGH ENZYME

H i hc
TP. H Ch Minh, thng 4 nm 2014

Lu hnh ni b
MC LC
BI M U ..................................................................................................................... 3
1. MC CH MN HC ...................................................................................................... 3
2. CC QUI NH CHUNG .................................................................................................... 3
3. CCH PHA V S DNG DUNG DCH M.................................................................. 4
4. MT S BC C BN TRONG THU NHN V TCH CHIT ENZYME ................. 8
BI 1. NG HC V B PHN NG CA ENZYME ........................................................... 11

XC NH THNG S NG HC ENZYME SAVINASE ....................................................... 11
1. MC CH ....................................................................................................................... 11
2. THIT B - DNG C- H A CHT- MU VT ............................................................ 11
3. NI DUNG ........................................................................................................................ 12
4. BO CO ......................................................................................................................... 14
5. CCH THC NH GI ................................................................................................ 14
BI 2. TCH CHIT V XC NH HOT TNH BROMELINE ................................................. 15

1. MC CH ....................................................................................................................... 15
3. NI DUNG ........................................................................................................................ 17
4. BO CO ......................................................................................................................... 21
5. CCH THC NH GI ................................................................................................ 21
BI 3. TCH CHIT V XC NH HOT PEPSIN ........................................................... 22

BNG PHNG PHP ANSSON ....................................................................................... 22
1. MC CH ....................................................................................................................... 22
3. NI DUNG ........................................................................................................................ 23
4. BO CO ......................................................................................................................... 26
5. CCH THC NH GI ................................................................................................ 26
BI 4. NHN SINH KHI SACCHAROMYCES CEREVISIAE .................................................... 27

THU NHN ENZYME INVERTASE .................................................................................. 27
1. MC CH ....................................................................................................................... 27
2. THIT B - DNG C - H A CHT- MU VT............................................................ 27
3. NI DUNG ........................................................................................................................ 28
4. BO CO ......................................................................................................................... 30
5. CCH THC NH GI ................................................................................................ 30
BI 5. TCH CHIT V XC NH HOT TNH ENZYME INVERTASE TCH T

SACCHAROMYCES CEREVISIAE ......................................................................................... 31
1. MC CH ....................................................................................................................... 31
B mn Cng ngh sinh hc

3. NI DUNG ........................................................................................................................ 32
4. BO CO ......................................................................................................................... 35
5. CCH THC NH GI ................................................................................................ 35
BI 6. SO SNH NG HC ENZYME C NH V ENZYME T DO ................................... 36

1. MC CH ....................................................................................................................... 36
3. NI DUNG ........................................................................................................................ 37
4. BO CO ......................................................................................................................... 41
5. CCH THC NH GI ................................................................................................ 42
TI LIU MN HC .......................................................................................................... 43
PH LC PHN CNG CHUN B THC HNH .................................................................. 44
BI M U
1. MC CH MN HC
Sau khi hc xong mn hc sinh vin c kh nng:
- Chng minh, cng c l thuyt, p dng kin thc vo thc t.
-
C k nng thao tc, s dng cc thit b, dng c th nghim.
Thc hin nghim tc ni quy phng th nghim.
Hon thnh bi thc hnh theo thi gian quy nh.
2. CC QUI NH CHUNG
1. Sinh vin c v tm tt bi trc khi n lp. T son bi tham kho

trong lc lm th nghim. Khng c em bi ging vo (PTN.
2. C phiu im danh theo bui qun, khng mang b tr im v phi vit n
gio vin xc nhn c i bui hc .
3. Ngh phi c n xin php GV dy v xc nhn ca GV dy hc b. Phi vit bi
bo co ring bui b np cho GV m sinh vin ng k mn hc.
4. Vit bo co theo t, np file word cho nhm trng. GV nhn c bi bo co
sau 1 tun ca bui hc t nhm trng qua mail trc 0h ca bui k tip.
5. Kt thc mn hc 1 tun cc nhm gp 6 bi li thnh 1 file gi li cho gio vin.
Sau in ton b bi trn giy A4 np li. (SV c th cp nht li bi nu pht hin
ra li so vi bi gi cho GV)
6. a ch mail: linhdtm@cntp.edu.vn, hiendt@cntp.edu.vn, tuyendth@cntp.edu.evn.
7. SV phi xem k bi thc hnh trc khi vo PTN. u bui c kim tra bi trc
nghim, in khuyt, cu hi nh....(Lp b qu tr tin photo trong cc bui
kim tra). Nu kim tra u gi khng t s phi ra v ngay. Sau xin giy vo
lp ca phng Cng tc hc sinh Sinh vin vi l do khng chun b bi. Ri b
tr i b bui thc hnh khc. Phi c n ng ca GV dy chnh v GV dy b.
8. SV phi chu trch nhim v: trt t, an ton, dng c, ha cht v kt qu th
nghim cho bi thc tp ca mnh.
9. SV phi c mt phng th nghim (PTN) ng gi qui nh: Sng 7h, chiu 12h30
v phi c mt ti PTN sut thi gian thc hnh. n tr 10 pht khng c vo
lp. Mc o blouse khi vo phng th nghim.
10. Mi nhm c mt i din k nhn mn dng c: kim tra tnh hnh dng c
(thiu, hng, b) bo co ngay cho GVHD. SV phi ra dng c sch s trc v
sau khi th nghim. Kt thc bui thc tp mi nhm phi lau dn, lm sch ch
nhm lm th nghim, nu dng c b mt mt, h hng phi bo ngay GVHD v
mua n vo bui hc tun k tip.
11. Mi bui thc hnh, cc nhm lm theo phn cng trong bng. Nhm trc nht c
nhim v: dn v sinh, kim tra in, nc v ca trc khi ra v. Khng hon
thnh nhim v kt qu bi thc hnh t im 0
Cng vic
Bui 1
Bui 2
Bui 3
Bui 4
Bui 4
Bui 6
Dng c
1,2
Ha cht
10
3,4
Thit b
5,6
V sinh
10
7,8,9,10
12. Nhm sinh vin lm phc trnh theo yu cu ca tng bi, np cho gio vin vo bui
thc hnh k tip. Kt thc mn c thi kim tra theo yu cu gio vin.
13. Tiu chun nh gi bi tp th nghim thc hnh
- im mn hc l im trung bnh cng ca 6 bi

- Nhm trng s c cng 0,3 im thng/ im tng kt mn hc.
STT
Tiu ch nh gi
Ni dung
im ti a
Nguyn tc
0.5 im
Tin trnh th nghim
2 im
Gii thch th nghim
2 im
Kt qu
1 im
Kt lun - Kin ngh
0.5 im
Tr li cu hi cui bi
1 im
An ton, t chc
1 im
Thi gian
1 im
K nng thao tc
1 im
3. CCH PHA V S DNG DUNG DCH M

Enzyme l mt phn t sinh hc rt nhy vi s thay i pH ca mi trng,
nn vn pha v s dng dung dch m trong chit tch Enzyme l rt quan
trng. V gii hn chng trnh, nn ch trnh by cch pha v s dng nhng dung
dch m cn thit.
3.1 nh ngha pH
Gi tr ca pH ca mt dung dch c tnh theo cng thc:
pH = - log aH+
Trong :
- a H+ biu th hot ng ca ion c trong dung dch v c tnh theo biu thc:
aH = f. CH+
- Vi:
CH+ l nng H + trong dung dch c tnh theo n v ion gam /l.
f l hot H+, f 1.
Khi nng H+ trong dung dch tng th f gim v ngc li khi nng ca H+ rt
nh (dung dch long) th f = 1, lc ta s c:
CH+ = aH+
V th, trong cc dung dch long c th tnh pH gn ng theo cng thc:
pH = -log CH+
S pht trin, din tin ca nhiu qu trnh ha hc, sinh hc, sinh ha. i
hi phi nhng gi tr pH nht nh khng i hoc c thay i ch l trong mc
rt nh khng ng k. V th cn phi c cc dung dch m cho pH.
3.2. nh ngha dung dch m
Dung dch m pH l dung dch cha cc cht c tc dng gi gi tr pH trong
gii hn nht nh khi thm acid mnh hay kim mnh vo dung dch .
Cc dung dch c tnh cht ny thng l dung dch hn hp ca mt cp acid
v base lin hp, v d: CH3COOH v CH3COONa, NH4+ v NH3, H2PO4- v
HPO42-. Nhng ph bin l cc dung dch hn hp ca acid yu v base lin hp vi
n hay ngc li.
V th tc dng m ch c hiu lc ln hn trong mt phm vi nht nh ty
thuc vo bn cht ca tng acid yu v vng m hiu lc.
Tnh theo cng thc: pH = pKa 1.
Trong : pKa = - logKa vi Ka l hng s phn ly H+ ca acid trong nc.
V th mi mt hn hp m ch c tc dng trn tng thang o pH. Do
mun m cho vng pH rng phi c dung dch m ca hn hp nhiu h acid v
base lin hp. V d, dung dch m gm CH3COOH v CH3COONa c tc dng tt
trong khong pH = 3,7 5,7. Trong khi dung dch m gm NH4+ v NH3 li c tc
dng m tt trong vng pH = 8 11. Vi hn hp m gm acid H3PO4 v mui ca
n thng c tc dng m tt trong hai vng pH, tng ng vi hai hng s phn ly
acid Ka1 v Ka2 vng I c pH = 1 3,6 v vng II pH = 6 8,3.
3.3. Hiu ng pha long dung dch m
Theo cng thc pH ca dung dch m :
pH = pKa log (Ca/Cb)
Ca: l nng acid
Cb: l nng ca base lin hp
Khi pha long, s ln pha long nng Ca v Cb l nh nhau nn t s Ca/Cb
khng thay i, nhng hng s in ly ra H+ ca acid v hot ca cc ion trong
dung dch thay i. V th khi pha long s lm thay i mt t pH ca dung dch m.
S thay i pH ny c gi l hiu ng pha long ca dung dch m. Hiu ng ny

rt khc nhau i vi mi loi dung dch m. c trng cho hiu ng ny l pH1/2.
pH1/2 l gi tr pH thay i ca dung dch m khi pha long gp i bng nc ct.
Nu khi pha long pH ca dung dch tng, ta c hiu ng dng. Cn nu pH
gim ta c hiu ng m. V d khi pha long dung dch m ta c pH1/2 = -0.05, tc
l pH ca dung dch m ny gim 0.05 n v pH khi pha long gp i n.
3.4. Cch pha ch cc dung dch m
Cc ho cht khi s dng cho pha dung dch m i hi phi tinh khit. Nc ct
dng pha ch phi un si, ui ht kh CO2 trc khi dng, ng thi phi y kn
trnh kh CO2 t khng kh ha tan vo khi ngui.
Nng c trng ca cc dung dch m phi chnh xc, nn hn ch vic s
dng cc dung dch qu long hoc qu m so vi nng c trng v s gy sai s.
Cc dung dch m sau pha tt nht nn c s dng trong vng mt thng.
C nhiu dung dch m c pha vi nhiu ha cht khc nhau, tuy nhin trong
chit tch Enzyme, nhng dung dch m sau y thng c dng:
3.4.1. Hn hp m KCl v HCl
pha ch dung dch m ny ta cn dung dch KCl 0,2 M (14.9 g/l) v dung
dch HCl 0,2 M (18 ml HCl c 36%). Hn hp m ny cha 25 ml dd KCl 0,2 M v
x ml HCl 0,2 M theo bng sau:
pH 25oC
X ml HCl 0.2 M
pH1/2
1,00
67,00
+0,04
1,10
1,20
1,30
52,80
42,50
33,60
1,40
1,50
1,60
1,70
1,80
1,90
2,00
2,10
2,20
26,60
20,70
16,20
13,00
10,20
8,10
6,50
5,10
3,90
+0,13
+0,27
3.4.2. Hn hp m K2HPO4 HCl
pha ch dung dch m ny ta cn dung dch K2HPO4 0,1 M (20,42 g/l)v

dung dch HCl 0,1 M (9 ml HCl c 36%). Hn hp m ny cha 50 ml dd K2HPO4
0,1 M v X ml HCl 0,1 M theo bng sau:
pH 25oC
X ml HCl 0.1 M
pH1/2
2,20
2,30
49,50
45,80
+0,14
2,40
2,50
42,20
38,80
2,60
2,70
35,40
32,10
2,80
2,90
3,00
28,90
25,70
22,30
3,10
3,20
3,30
3,40
3,50
18,80
15,70
12,90
10,40
8,20
3,60
3,70
3,80
3,90
4,00
6,30
4,50
2,90
1,40
1,10
+0,08
+0,09
+0,04
3.4.3. Hn hp m K2HPO4- NaOH

pha ch dung dch m ny ta cn dung dch K2HPO4 0,1 M (20,42 g/l) v
dung dch NaOH 0,1M (4 gam NaOH/lt). Hn hp m ny cha 50 ml dd K2HPO4
0,1 M v X ml NaOH 0,1 M theo bng sau:
pH 25oC
X ml NaOH 0,1 M
pH1/2
4.10
4.20
4.30
1.30
3.00
4.70
+ 0.05
4.40
4.50
4.60
6.60
8.70
11.10
4.70
13.60
4.80
4.90
5.00
5.10
16.50
19.40
22.60
25.50
5.20
5.30
28.80
31.60
5.40
5.50
34.10
36.60
5.60
5.70
38.80
40.60
5.80
5.90
42.30
43.70
+ 0.06
+ 0.06
+ 0.14
3.4.4. Hn hp m KH2PO4 NaOH

pha ch dung dch m ny ta cn dung dch KH2PO4 0,1 M (12,61 g/l) v
dung dch NaOH 0,1 M (4.00 g/ lt). Hn hp m ny cha 50 ml dd KH2PO4 0,1 M
v X ml NaOH 0,1 M theo bng sau:
pH 25oC
X ml NaOH
0.1 M
5,80
5,90
6,00
6,10
6,20
3,60
4,60
5,60
6,80
8,10
6,30
6,40
6,50
6,60
6,70
6,80
6,90
9,70
11,60
13,90
16,40
19,30
22,40
25,90
pH1/2
pH 25oC
X ml NaOH
0.1 M
+0,07
7,00
7,10
7,20
7,30
7,40
29,10
32,10
34,70
37,00
39,10
7,50
7,60
7,70
7,80
7,90
8,00
40,90
42,40
43,50
44,50
45,30
46,10
+0,05
pH1/2
+0,08
+0,06
4. MT S BC C BN TRONG THU NHN V TCH CHIT ENZYME

Ty theo nhng c tnh ring bit ca tng loi enzyme m la chn phng
php lm sch cho thch hp. Trong qu trnh tinh ch enzyme, mc d trnh t v cc
th thut cc bc c th thay i, song vn c nhng nguyn tc chung.
Ph v t bo
Enzyme c trong t bo cht ca sinh vt v cc cu t (nhn, lp th, ty th,
lysosome...) ca t bo. Nhng cc phn t enzyme khng c kh nng i qua mng
ca t bo v mng ca cc cu t. Do c th chit rt cc enzyme ni bo, bc
u tin l phi ph v cu trc ca cc t bo c cha enzyme v chuyn chng vo
dung dch.
Tc nhn c hc
C th ph v cu trc ca cc t bo bng cc bin php c hc nh nghin vi
bt thy tinh hoc ct thch anh, ng ha bng thit b ng ha (homogenizator).
vic ph v c hiu qu m thc vt, trc khi nghin ngi ta thng thi nh mu
vo ngn hoc cho trng nc (v d nh i vi mu ht kh). Cn cc m
ca ng vt nh gan hoc thn, khi chit enzyme ngi ta cn ct b cc m lin kt.
Tc nhn vt l, ha hc
Mun tch c cc enzyme trong cc cu t ca t bo, ngi ta cn phi dng
cc yu t vt l v ha hc khc nhau nh sng siu m, dung mi hu c (butanol,
aceton, glycerin, ethyl acetate..) v cht detergent (cht ty). Cc ha cht c tc dng
tt cho vic ph v cc cu t ca t bo v trong cc c quan ny thng cha m.
Chit
Sau khi ph v cc cu trc ca t bo, enzyme c chit bng nc ct,
bng cc dung dch m thch hp hoc cc dung dch mui trung tnh.
Mt s yu t nh hng n qu trnh chit rt:
- Nhit : trnh mt hot tnh hoc thm ch v hot, cn chit rt v tin
hnh kt ta enzyme nhit thp (30C 50C). Cc thao tc phi nhanh.
- Mt s cht in ly lm tng qu trnh chit rt enzyme nh NaCl, ZnCl2,
CaCl2. Tc dng ca chng cn ph thuc vo phng php dng khi chit rt.
Kh mu: Trong qu trnh chit rt enzyme cc i tng ng, thc vt, c
trng hp cn c mt cht mu lm nh hng n vic lm sch hoc xc nh hot
enzyme. Trong trng hp ny ngi ta cn cho thm vo cht kh loi mu.
cc mu t ng vt c sc t melanin mu nu. Ngi ta thng loi mu trn ct
nha trao i ion bng cch cho dch enzyme qua ct hoc lc. Khi qua ct, cht mu
b gi li v enzyme khng b gi. Trong qu trnh ny phi ch kim tra pH. Sau
khi loi mu cn kim tra li hot ca enzyme.
Trong dch chit, ngoi enzyme cn c cc protein cu trc, cc cht cao phn t
-
khc nhau nh polysaccharid, nucleic acid, cc cht c phn t nh nh ng

monose, cc cht lipid, mui khong... loi chng phi s dng phi hp nhiu bin
php khc nhau.
Kt ta
Cn nng cao (960 980 ) vi t l v th tch 1 E : 3 cn hay 1 E : 4 cn.

Cc dung mi nh isopropanol, aceton vi t l dung dch Enzyme : dung mi
l 1:1,5 hay 1: 2. Ch khi ta E bng cc dung mi trn tt nht l nn nhit
thp t 30C 50C, khuy u khi cho dung mi t t vo.
Cc dung dch mui trung tnh nh NaCl, (NH4)2SO4, Na2SO4, MgSO4l tc
nhn ta tch cc. Thng thng hay dng dung dch (NH4)2SO4 v ha tan mui
ny cao (chim t 50 60 % so vi dch enzyme) khng ph thuc nhiu vo nhit ,
khng lm bin tnh enzyme. Vi pepsin ngi ta hay dng NaCl (20 25%) v mui
ny r, d tm, khng nh hng n hot tnh ca E.
Tinh sch
Sau khi kt ta E, ly tm, phn E ta lc ny cn cha nhiu tp cht nh protein,
mui, cc cht c trng lng phn t thp khc, cht mu. Nn ta cn tin hnh
tinh sch theo cc bin php sau:
- Tch cht c trng lng phn t thp (nh mui..) bng phng php thm tch
qua mng bn thm.
- Tch mt phn cc cht tp bng nng ru thp (40 45%) khi E ch yu
nm trong dung dch.
- C th ha tan E bng lng nc ti thiu, lc b cht khng tan, ri kt ta li
bng ru cao .
- Hin nay vic chit tch, tinh sch E c tin hnh vi hiu sut tng i cao
bng cc k thut hin i nh lc gel, hp ph chn lc, sc k trao i ion.
10
BI 1. NG HC V B PHN NG CA ENZYME
XC NH THNG S NG HC ENZYME SAVINASE
1. MC CH
- Xy dng phng trnh ng hc Michaelis-Menten.
- Xc nh thng s ng hc Vm, Kmax.
2. THIT B - DNG C- H A CHT- MU VT
Dng c, ha cht, thit b s dng cho bi thc hnh c k theo bng 1.1.
ng 1.1. Cc ha cht dng c thit b s dng trong bi thc hnh
(cho
nhm g m
sinh vin
A. HA CHT:
STT
TN HA CHT
QUY CCH
SL/VT
Lt
Nc ct 2 ln
Casein
Enzyme Savinase
ml
200
Folin
ml
50
Tyrosine
Giy lc
Hp
ml
200
m photphat pH =8
Dung dch casein 12mM/
m photphat pH = 8
ml
200
TCA 5%
ml
500
10
NaOH 0,5N
ml
500
11
HCl 0,2N
12
ml
500
ml
600
TN DNG C
QUY CCH
SL/VT
Cn 70
GHI CH
PTN, c lp
B. DNG C:
STT
ng nghim 18
ci
18
Pipet 10
ci
Pipet 1
ci
Pipet 5
ci
Qu bp
ci
Bnh tia
ci
a thy tinh
ci
Cc 100
ci
Cc 250
ci
GHI CH
1 t
11
10
Phu 60
ci
11
Bnh nh mc 250
Ci
QUY CCH
SL/VT
C.THIT B:
STT
TN THIT B
My o quang ph
Ci
Cn 2 s
Ci
My n nhit
Ci
My khuy t
Ci
My Votex
Ci
GHI CH
C lp
T 1: Pha dung dch casein 12 mM: Cn 2g casein vo cc pha, thm vo khong

20-30 ml dung dch NaOH 0,5N, t ln my khuy t 50-600C khuy cho casein
tan ht, vn dung dch trn my khuy t, tip tc khuy nhng khng gia nhit
na; dng HCl c nng tng ng nh vo t t n khi pH t t 7,5-8 (nh
nh HCl vo t t trnh casein b ta v pH tng qu nhanh), dng m
phosphate pH = 8 nh mc n 200 ml.
T 2: Pha dung dch m phosphate:
- Dung dch Na2HPO4 1/15M: ha tan 23,9 g Na2HPO4.12H2O trong nc va
1000 ml.
- Dung dch KH2PO4 1/15M : ha tan 9,07 g KH2PO4 trong nc va 1000
ml.
Trn 969 ml Na2HPO4 1/15M vi 31 ml KH2PO4 1/15M thu c 1000 ml dung
dch m phosphate pH = 8
T 3: Pha dung dch chun Tyrosine (1mM): 181,19 mg Tyrosine ha tan trong
HCl 0,2N 1 lt.
T 4: Pha dung dch savinase 2%: Ht 8 ml enzyme Savinase thng mi ha tan
trong 392 ml nc ct.
3. NI DUNG
Thng s ng hc ca enzyme l tc phn ng Vmax v hng s Michaelis
Km. Tc ca phn ng trong mt gii hn no ph thuc vo nng c cht c
trong mi trng. Khi enzyme cha b bo ha bi c cht th tc phn ng vn t
l thun vi nng c cht.
Do chng ti kho st bin thin vn tc phn ng thu phn casein ca
savinase bng cch thay i nng casein t 2.4 12 mM tm khong nng
casein m trong enzym cha b bo ho bi c cht.
12
Xy dng ng thng tng quan gia vn tc thu phn ca enzym v nng

c cht theo phng trnh Lineweaver Burk
1
1
K 1
M.
v v max v max S
Enzyme Savinase c sn xut bi qu trnh ln men ca cc vi sinh vt bin
i gen (Bacillus amyloliquefaciens). Sau khi kt thc qu trnh ln men, savinase
c tch chit v tinh sch t cc sn phm ln men. Enzyme ny c ng dng
trong vic sn xut bt git, nc ra chn v chng gip ty sch cc vt bn trn
qun o c ngun gc t thc n: trng, tht...hoc vt mu, bn...Savinase c tc
dng thy phn protein thnh cc peptide ngn, tan trong nc.
Enzyme Savinase hot ng trong mi trng kim cao, pH dao ng t 8 12
(ti u pH = 10), nhit hot ng ca n t 20-600C (ti u 550C), c bo
qun nhit t 3 50C.
3.1. Lp ng chun Tyrosine
Nhm c 1 t chn, 1 t l lm ng chun
ng nghim
Dd tyrosine chun (ml)
0.2
0.4
0.6
0.8
Tyrosine tng ng (mol)
0.2
0.4
0.6
0.8
HCl 0.2N (ml)
4.8
4.6
4.4
4.2
NaOH 0.5N
10
Folin pha long 3 ln
1
Lc mnh, yn trong 10 pht
o OD (Abs) ti =660 nm
Dng ng chun tyrosine biu din s tng quan gia lng tyrosine v OD.
ng 0 l ng mu, cc ng khc l ng th nghim.
3.2. Xc nh thng s ng hc ca enzyme savinase
Tin hnh th nghim

ng nghim
Casein 12 mM (ml)
m phosphate (ml)
Nng Casein (mM)
2.4
4.8
7.2
9.6
12
Savinase 2% (ml)
5
yn 50-550C trong 2 pht
TCA 5% (ml)
5
yn 15 pht, lc ly dch bn di
13
ng nghim
Dch lc
NaOH 0.5N (ml)
10
Folin pha long 3 ln

(ml)
Lc mnh, sau yn trong 10 pht

o OD (Abs)
ti =660 nm
Da vo th chun ta suy ra lng Tyrosine tng ng.

ng nghim
OD (y)
Lng tyrosine (x)
Tnh vn tc phn ng (V) cc nng c cht tng ng:
V = d(P)/dt = Lng tyrosine/thi gian.
Lp th th hin mi tng quan gia 1/S v 1/V da vo bn sau:
ng nghim
1
2
3
4
Lng casein (S)
1/S
V
1/V
S dng phn mm Excel hoc p dng phng php bnh phng cc tiu
xy dng th th hin nh hng ca nng c cht ti tc phn ng.
Tham kho bi 10 trang 66 sch Th nghim ha sinh thc phm Trn Bch
Lam NXB HQG TP HCM
4. BO CO
1. V th tng quan gia 1/S v 1/V da trn kt qu thc nghim.
2. Xc nh thng s ng hc Km v Vmax.
5. CCH THC NH GI
Theo yu cu chung mn hc.
14
BI 2. TCH CHIT V XC NH HOT TNH BROMELINE

1. MC CH
Sau khi hc xong bi sinh vin c kh nng:
-
Vit quy trnh tch chit, thu nhn dung dch enzyme bromeline th.
Xc nh hot tnh enzyme bromeline.
Vit quy trnh thu nhn enzyme bromeline dng paste.

(cho nhm g m sinh vin
A. HA CHT
TN HA CHT
STT
SL/VT
gam
100
2 Cn 90o
lit
3 Thm (da) xanh
tri
4 Vi lc
5 DD NaOH 0.5 N
ml
250
6 DD NaOH 1.0 N
ml
100
7 DD HCl 0.2 N
ml
300
8 DD HCl 2 N
ml
100
9 Folin Ciocaltue
ml
80
Acid tricloacetic TCA

10 (Cl3COOH) 5%
ml
200
11 KH2PO4 1M
ml
100
12 Hemoglobin
gam
5,0
13 Tyrosine
gam
14 Ur
gam
80
Ti colodion hoc
15 cellophane
ti
10
m phosphate c pH = 7,
16 nng 0,01M
lit
17 Nc ct 2 ln
lit
Hp
18
(NH4)2SO4
QUY CCH
Giy lc
GHI CH
PTN
HS mua
B. DNG C
STT
TN DNG C
QUY CCH
SL/VT
GHI CH
15
ng ong 50ml
Ci
ng nghim phi 18
Ci
10
Gi ng nghim
Ci
Pipep man 100 -1000l
Ci
Pipep 1ml
Ci
Pipep 2ml
Ci
Pipep 5ml
Ci
Phu thy tinh
Ci
Bnh tia nc ct
Ci
10
Dao inox
Ci
11
Qu bp
Ci
12
Bnh nh mc 100
Ci
13
Cc 100
ci
14
Bnh tam gic nt mi 100
ci
15
a thy tinh
ci
10
16
Cc thy tinh 500ml
Ci
17
Cn k 50-100
Ci
18
Cc thy tinh 1000ml
Ci
10
1 t
c lp
C.THIT B
STT
TN THIT B
QUY CCH
SL/VT
1 My o quang ph
Ci
2 Cn 2 s
Ci
3 My ly tm 6000v/p
Ci
4 My xay sinh t
Ci
5 My khy t
Ci
GHI CH
c lp
Chun b ha cht:
T 5: Pha dung dch acid Trichloroacetic 5%: Cn 5 g pha vi nc ct thnh
100ml.
T 6: Pha Dung dch m KH2PO4 1M: Cn 13.6 g pha vi nc ct thnh
100ml.
T 7: Pha dung dch Tyrosine 1/400N mu: Cn 45 mg Tyrosine pha trong
100ml HCl 0.2N.
T 8: Pha dung dch Hemoglobin 2%: Cn 2g Hb + 36g Ure + 8 ml dung dch
NaOH 1N ha trong 40 ml nc ct. 25oC trong 60 pht. Thm 10 ml dung dch
16
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.
3. NI DUNG
3.1. Nguyn tc
Bromeline l mt enzyme thuc nhm protease c nhiu trong tri thm. Chng ta
tin hnh kho st bromeline qua kh nng thy phn cc c cht protein
(Hemoglobin, Casein, Albumin.) to ra cc sn phm tan trong dung dch TCA l
cc peptide c cha Tyrosine. Vic nh lng Tyrosine bng phn ng mu vi thuc
th Folin Ciocalteu cho bit hot ng thy phn ca enzyme.
3.2. Chit dch thm c cha Enzyme
Nguyn liu: ngn (nh sinh trng), v qu, tht v li thm.
T 3: Da gt v, b li, cn khi lng m (g), xay nh bng my xay sinh t
lc qua vi mn 2 lp 1 ln, qua bng 1 ln, b cn, thu dch ly tm 4500 vng /10
pht thu V ml dch lc th c cha bromeline. Chia v cc t, mi t 50 ml thc
hin th nghim: 10ml dnh cho th hot tnh, 20ml dnh cho ta bng Ethanol, 20ml
dnh cho ta bng mui (NH4)2SO4.
Quy trnh trch ly v thu nhn bromeline dng th
Da
Lm sch
Nghin p, xay nhuyn
Lc
Ly tm 4500 v/ 10ph
B
Dch enzyme th
3.3. Phng php ta bromeline
a. Phng php ta bng dung mi hu c (Ethanol 80%, Aceton)
17
C s: ha tan ca protein ph thuc vo s tng tc ca cc nhm tch

in trong phn t protein vi cc phn t nc. S tng tc (cn gi l s
hydrate ha) s b gim xung khi thm vo dung dch enzyme cc dung mi hu c
(ethanol, isopropanol, acetone hoc hn hp cc loi ru). Tin hnh nhit thp
( 50C), c tc dng tt n n nh ca enzyme. Khi kt ta, ch ly nhanh kt
ta ra khi dung mi bng cch dng my li tm. Phng php ny li th l khng
cn loi mui, nhng nhc im l hay c mu.
ha tan ca protein trong dung dch ph thuc vo nhiu yu t, mt trong
s l hng s in mi ca dung dch. Nhn chung, nhng phn t dung mi c
hng s in mi ln (nh nc, dimethylsulphoxid) c th n nh tng tc gia
chng vi cc phn t protein v to iu kin thun li cho s ha tan ca protein
trong dung dch. Ngc li, cc dung mi vi hng s in mi nh (aceton, ethanol)
ngn cn s phn tn ca cc phn t protein trong mi trng. Do , ha tan ca
nhng phn t protein gim v xy ra kt ta do s lm gim hng s in mi hin
hu ca mi trng. iu ny c c bng cch thm mt dung mi ha tan trong
nc nh aceton vo dung dch cha protein. S ta bng aceton hoc ethanol 80% c
nhiu thun li hn v n tng i r, c sn dng tinh khit vi t cht tp nhim
gy c hay c ch i vi enzym, do nhit bay hi ca dung mi thp nn d tch
b dung mi khi ch phm enzym bng phng php sy nh bng qut gi.
Quy trnh kt ta enzym Bromeline bng cn 80%
Cn 80%, 40C
Dch enzyme th
Ta cn 4oC / 1 gi
Ly tm 4500 v/p
10 pht
Thu ta
B dch
trong
Sy kh
Bromeline
18
Lu : Phi trn cn vi dch da theo t l 4:1 (1 da : 4 cn).

Phng php ta bng mui:
Ngi ta c th dng mui (NH4)2SO4, NaCl ta protein v cc mui ny
va lm trung ha in (do cc ion tc ng tng h vi cc nhm tch in tri du),
va loi b lp v hydrat ca phn t protein. Cc protein khc nhau c th b kt ta
vi nng mui khc nhau, v vy c th dng mui tch ring cc protein ra khi
hn hp ca chng.
Quy trnh thu nhn enzym Bromeline bng mui (NH4)2SO4
Ammonium sulfate
70% bo ha
Dch enzyme th
yn, nhit
phng 30 pht
Ly tm 4500 v/p, trong
10 pht
Thu ta
B dch
trong
Thm tch
Sy kh
Bromeline
Lu : c mui (NH4)2SO4 bo ha 70%, ta phi trn vi t l 47,2g mui
trong 100ml nc thm.
Kt qu: Hy tnh hm lng protein c trong 100g nguyn liu ban u.
Phng php thm tch
loi b mui khong v cc loi ng... l cc tp cht c phn t lng
thp, ngi ta thng dng phng php thm tch (dialysis) i nc hay i cc
dung dch m long hoc bng cch lc qua gel sephadex.
Cch lm thm tch nh sau: cho dung dch enzyme vo ti colodion hoc
cellophane (thng thng ngi ta dng cellophane tt hn), sau t c ti vo
19
nc ct hoc dung dch m pha long (nh m phosphate c pH = 7, nng

0,01M chng hn). Mng cellophane l mng bn thm, c kch thc l cho cc cht
c phn t nh xuyn v i qua vo cc dung dch m long theo nh lut khuch
tn. Cn li trong mng l cc cht protein c phn t ln.
C th tng tc thm tch khi khuy dung dch ra bng my trn c hc hay
my trn t hoc l quay chm ti nh ng c khng ln. Khi thm tch cc protein
thng ngi ta tin hnh tt c cc thao tc mi trng lnh.
Hnh 1. Thm tch loi mui (NH4)2SO4 trong kt ta protein

3.3. Kho st hot tnh enzyme
Thc hin 3 ng nghim theo nh bng sau:
Dung dch
Dung dch Hb 2% trong m
phosphate
ng Tyrosin
(ml)
ng mu
(ml)
ng khng
(ml)
2.5
2.5
2.5
yn 35,50C trong 5 pht

Acid Trichlororacetic 5%
Dung dch HCl 0.2N
0.5
0.5
Dung dch Tyrosine 1/400N
0.5
Dch thm c cha enzyme

Bromeline
0.5
Lc u. yn 35o5C trong 10 pht

Dd Acid Trichlororacetic 5%
Dch thm c cha Enzyme
0.5
0.5
20
Bromeline
Lc u, yn 10 pht 250C,
Lc
Dch lc (ml)
2.5
2.5
NaOH 0.5N (ml)
Folin (ml)
2.5
Lc u, yn 15 pht
o OD (=578 hoc 660nm)
ODT =?
ODM=?
ODC=?
3.3 Kt qu
Hot tnh enzyme Bromeline c biu th l s g Tyrosine sinh ra do s thy
phn Hb ca enzym c trong 1 ml dung dch hay 1 mg hn hp cha Bromeline trong
1 pht.
Hot tnh ny c tnh theo cng thc :
X = 450 ODM 1 X1 (UI/ ml).
OD T
10
X2
Vi : X1: ml dung dch Tyrosine chun

X2: ml dung dch cha enzyme
OD T = OD T - ODC
OD M = ODM - ODC
(Tham kho trang 63 Thc tp ln sinh ha Lm Th Kim Chu)
4. BO CO
1. Nu nguyn tc kho st hot tnh Bromeline da vo mt quang?
2. Gii thch cc bc tin hnh th nghim trong bi ?
3. Xy dng cng thc tnh v gii thch ngha ca cc n v ?
5. CCH THC NH GI
Theo yu cu chung mn hc
21
BI 3. TCH CHIT V XC NH HOT PEPSIN

BNG PHNG PHP ANSSON
1. MC CH
- Tch enzyme pepsin t d dy heo.
- Xc nh hot enzyme th.
A. HA CHT
STT
TN HA CHT
QUY CCH
SL/VT
HCl 0,5%
Lt
Acetone
Lt
NaCl 25%
Lt
Thuc th Folin
ml
100
TCA 5%
ml
200
Hemoglobin 2% trong HCl
ml
200
0.06N
Dung dch chun tyrosine
ml
50
Dd NaOH 0,5 N
ml
500
HCl 0,2N
ml
500
10
Vi lc
11
D dy heo (b)
Kg
12
Sa c ng th v
13
Nc ct 2 ln
Lt
14
Giy lc
Hp
15
Giy nhm
Qun
16
CaCl2 0,01M
ml
150
GHI CH
HS mua
C lp
B. DNG C
STT
TN DNG C
QUY CCH
Dao inox + tht
Ci
Ko
Ci
Tam gic 250 nt mi
ci
ng ong 25ml
Ci
Cc thy tinh 100ml
Ci
ng nghim 18
Ci
12
SL/VT
GHI CH
Mn trc 24h
1 t
22
Phu thy tinh
Ci
Pipet 1ml
Ci
Pipet 2ml
Ci
10
Pipet 5ml
Ci
11
Pipet 10ml
Ci
12
Gi ng nghim
Ci
13
Qu bp cao su
Ci
14
Kp ng nghim
Ci
15
Bnh tia
Ci
16
a thu tinh
Ci
17
Cc thy tinh 500ml
Ci
18
Cc thy tinh 1000ml
Ci
19
Bnh nh mc 100ml
Ci
20
Chu thy tinh
Ci
C lp
Mn trc 2 ngy
C.THIT B
STT
TN THIT B
QUY CCH
SL/VT
My o quang ph
ci
Cn 2 s
ci
T m
ci
My xay sinh t c ci xay

tht
ci
GHI CH
Mn trc 2 ngy
T 9: Pha thuc th Folin: pha long 3 ln bng nc.

T 10: Dung dch chun Tyrosine 1mM: 181,19mg Tyrosine ha tan / HCl 0,2N
1 lt.
3. NI DUNG
3.1. Nguyn tc:
Pepsin c gii phng t mng nhy d dy ng vt do s t phn trong mi
trng acid long. Dung dch sau t phn c cha pepsin dng ha tan v cc thnh
phn protein khc. S tinh sch enzyme c th thc hin sau khi thu nhn c ch
phm enzyme th nh vo cc tc nhn gy ta.
Trong mi trng acid, pepsin (pepsin A, EC 3.4.23.1) phn gii c
Hemoglobin thnh Tyrosine v Tryptophane ha tan trong TCA. Xc nh hm lng
tryosine v triptophane nh vo thuc th Folin Ciocalteu.
3.2. Thu nhn dung dch enzyme
+ Chun b nguyn liu:
23
D dy b (hoc heo) mi git m, x l th sau c bo qun trong iu kin

nhit thp vi s c mt chloroform 5%. Thi gian ti a cho qu trnh bo qun l
48 gi. Cn khi lng nim mc m1 (g).
+ T phn enzyme:
Tch lp nim mc th t d dy, xay nh vi my xay sinh t trong dung dch
HCl 0.5% (t l 1:2 theo khi lng). Sau cho vo chu thu tinh ln, y kn,
yn nhit 40 420C trong b n nhit vi thi gian 24 - 28 gi.
Tin hnh lc dung dch qua vi lc (t 2- 4 ln), loi b tp cht bng cch lc
qua bng. Thu c dung dch c enzyme th c dng tin hnh th hot tnh.
+ Thu ch phm pepsin th:
Tin hnh thu kt ta pepsin bng dung dch mui NaCl 25% (hoc dung mi
aceton lnh) vi t l 1:4, cho t t dung dch mui vo kt hp vi khuy o lin tc
trnh sinh nhit trong hn hp. Thi gian t 5 10 pht, nhit khong 50C.
Tin hnh ly tm vi tc 6000/pht trong 15 pht.
Vi tc nhn ta l mui trung tnh cn qua thm tch gii phng bt lng
mui trong hn hp. Sau tin hnh sy kh bng my sy khng kh, nhit 30
400C khong 1 2 gi. i vi tc nhn ta l aceton th kh t nhin di qut gi
t 3-4 gi. Cn khi lng ch phm m2 (g).
Ch phm t nhin c dng bt mu trng hay hi vng, v nh hnh v c mi
nc tht, v hi chua. Ngi ta c th thm vo ch phm cht nn nh tinh bt hay
dextrin bo qun. iu kin gi ch phm cn kh, kn (trong l mu) v nhit
50C.
+ Hiu sut chit tch c tnh bng cng thc:
Khi lng ch phm (m2) . 100%

Khi lng nim mc thu nhn (m1)
(
3.3. Lp ng chun
Tyrosine
H
Lp 6 ng nghim theo tun t sau:
=
= (ml)
Tyrosine chun 1mM
=
HCl 0,2 N (ml)
=
ng
0,2
0,4
0,6
0,8
4,8
4,6
4,4
4,2
0,6
0,8
NaOH 0,5 N (ml)
10
= (ml)
Folin pha long 3 ln
Lng tyrosine (mol )
0,2
0,4
Lc mnh sau 5-10 pht

OD660nm
(1mol =103mmol = 106mol)

24
Dng ng chun Tyrosine biu din s tng quan gia lng Tyrosine v OD
(ng 0 l ng kim chng (KC) . Cc ng khc l ng th nghim (TN)).
3.4. Xc nh hot tnh Pepsin (hot tnh chung) trong mu ch phm

Enzyme.
Ly 2 ng nghim dn nhn v lm theo th t nh bng sau:
Cht dng
ng 1 (mu th nghim)
Hb 2% 25 C (ml)
TCA 5% (ml)
10
Enzyme pepsin (ml)
0,1
Nc (ml)
Thi gian
ng 2 (mu kim chng)
0,9
o
Gi 10 pht 35,5 C
TCA 5% (ml)
Khng cn gi 10 pht
10
Sau 30 pht em lc ly dung dch

Ht 5ml dung dch lc t hai ng nghim
chuyn sang ng nghim mi
ng nghim
NaOH 0,5 N (ml)
10
Lc mnh
Folin pha long 3

ln (ml)
3
Lc u, yn sau 5-10pht
OD = 660 nm
ODTN = ?
ODKC = ?
Tnh OD = ODTN ODKC. Da vo th chun ta suy ra lng Tyrosine .

4.4. Tnh ton
a. nh ngha n v Ansson (trang 116 Mc 6, TN CNSH tp 1- Nguyn
c Lng ch bin).
Lng Enzyme ti thiu trong iu kin chun (6 ml dch , 100mg
hemoglobin, nhit 35.5oC) thu phn hemoglobin trong mt pht to thnh sn
phm ha tan trong TCA, phn ng vi thuc th Folin to dung dch mu c OD
tng ng 1 mol Tyrosin.
b. Cng thc tnh
Hot tnh pepsin c tnh theo phng php Ansson biu th bng s n v Ansson.
mol Tyrosin *3,2 * 1,82
S n v hot Ansson =
t*v
25
a: T l th tch mu th nghim (16/5)

v: Th tch dung dch em xc nh (0,1ml)
t : Thi gian thu phn 10 pht
1,82: hng s gi tr cng mu 25OC.
3.5. Xc nh hot ng t sa
a. Nguyn tc:
Xc nh hot ng t sa ca pepsin da vo thi gian cn thit lm
ng t dung dch sa loi bo c nng xc nh. Cng thc tnh:
F = 1/(E.t)
Trong , F l hot ng t sa; E l lng enzyme tham gia (mg); t l thi
gian (pht hoc giy).
b.Tin hnh:
Cn 5g sa c ha tan trong 100 ml dung dch CaCl2 0,01N. Ht 5 ml dung
dch cho vo mt ng nghim, thm 0,5 ml dung dch enzyme pepsin, lc nh,
nhit phng. Ghi nhn thi gian t khi cho enzyme vo n khi s kt ta protein
ca sa xut hin.
4. BO CO
1. Gii thch ti sao phi chit pepsin trong mi trng acid ?
2. Tnh hiu sut thu nhn ch phm?
3. M t ch phm thu c.
4. Xc nh hot tnh pepsin theo Ansson?
5. Xy dng ng chun tyrosine?
6. Xc nh hot ng t sa F?
5. CCH THC NH GI
26
BI 4. NHN SINH KHI SACCHAROMYCES CEREVISIAE

THU NHN ENZYME INVERTASE
1. MC CH
- Pha mi trng Han sen nui cy Saccharomyces cerevisiae.
- Thc hin gi ging Saccharomyces cerevisiae.
- Nhn ging cp 1 - cp 2 Saccharomyces cerevisiae.
- Thu nhn sinh khi Saccharomyces cerevisiae.
- Quan st hnh thi ca t bo Saccharomyces cerevisiae.
2. THIT B - DNG C - H A CHT- MU VT
A. HA CHT
STT
TN H A CHT
QUY CCH
SL/VT
Nc ct 2 ln
Lt
NaOH 10%
Ml
10
HCl 10%
Ml
10
Cn 700
Lt
Cn 90
Lt
Agar
Gam
30
Bng thm
Gam
500
Bng khng thm
Gam
500
Giy pH
Thp
10
Glucose
Gam
75
11
Tripton
Gam
15
12
KH2PO4
Gam
13
MgSO4.7H2O
Gam
14
NaCl
Gam
20
15
Thuc nhum xanh

methylen 0,1%
Ml
20
GHI CH
Pha MT
Chnh pH
Kh trng
Giy bo 1kg, tick dn 100 ci, khn tay 2 ci, ko 1 ci sinh vin chun b
B. DNG C
STT
TN DNG C
1
ng nghim 18 nt vn
QUY CCH
SL/VT
GHI CH
Ci
4/ t
Lu li 1 tun
27
Tam gic 250
Ci
1/t
Tam gic 500
Ci
8 c lp
Cc thy tinh 100ml
Ci
Cc thy tinh 250ml
Ci
ng ong 25ml
Ci
Phu thy tinh
Ci
Pipet 10ml
Ci
Pipet 1ml
Ci
10
Pipet 5ml
Ci
11
Bnh tia
Ci
12
a thy tinh
Ci
13
Que cy
Ci
1 t
14
n cn
Ci
Sau 48h mn
15
Qu bp
Ci
16
Lam-lamen
Ci
que cy v n
cn
17
Cc 1000ml
Ci
C lp
QUY CCH
SL/VT
GHI CH
C . THIT B
STT
TN THIT B
T cy
Ci
Ni hp
Ci
Cn phn tch
Ci
C lp
T m, my lc
s dng lu
T m
Ci
trong 1 tun
L vi ba
Ci
My lc
Ci
Knh hin vi
Ci
1 t
Dung dch xanh methylen (methylene blue) 0,1%

- Xanh methylen: 1g
- Nc ct: 1000ml
(C th pha thnh dung dch 1% sau lc ri pha long thm 10 ln bng nc ct).
3. NI DUNG
Sinh khi nm men Saccharomyces cerevisiae l nhng t bo sng c ng dng
trong: sn xut ung ln men, sn xut bnh m, sa chua...v trong y hc nh sn
xut thuc h tr tiu ha biolactovin... Ngoi ra Saccharomyces cerevisiae cn c
28
quan tm nhiu trong lnh vc thu nhn enzyme invertase thy phn ng
sacchrose thnh ng nghch o.
3.1 Chun b pha mi trng
Thnh phn mi trng Hansen (g/l):
Glucose
MgSO4.7H2O
50
2g
Tryptone
Nc
10 g
1000 ml
KH2PO4
3g
Agar
20 g
pH
5-6
Mi trng lng Hansen khng cho agar trong thnh phn mi trng. Phn phi mi
trng vo dng c cha, dn nhn, ghi ngy gi tn ngi pha ch. Hp kh trng
mi trng nhit 1210 C, 15 pht.
Chun b:
T 1: Pha mi trng gi ging nm men: cn pha 200 ml mi trng Hansen c
(pha vo cc 250 ml, un si tan ht agar), sau phn phi cho mi t 20 ml cha
trong 2 ng nghim (10 ml/ng).
T 2: Pha mi trng nhn ging cp 1 Hansen lng: cn pha 200 ml mi trng
Hansen lng (cn cc thnh phn ha cht tr Agar vo cc 250 ml, b sung nc cho
200 ml, khuy tan, khng un), sau phn phi cho mi t 20 ml cha trong 2
ng nghim (10 ml/ng).
T 3: Pha mi trng nhn sinh khi - Hansen lng: cn pha 1000 ml mi trng
Hansen lng (cn cc thnh phn ha cht tr Agar vo cc 1000 ml, b sung nc
cho 1000 ml, khuy tan, khng un), sau phn phi cho mi t 100 ml cha
trong bnh tam gic 250 ml.
T 4: chun b 4 bnh tam gic 500 ml c cha 400 ml nc ct, y nt bng, bao
gi.
T 5: chun b 4 bnh tam gic 500 ml c cha 400 ml nc mui NaCl 0,85% (0,85 g
trong 100 ml), y nt bng, bao gi.
T 6: em ton b phn chun b hp kh trng nhit 1210 C, 15 pht.
Sau khi hp xong ly mi trng ra khi ni hp, t 6 em mi trng nhn
sinh khi, NaCl 0,85% v nc ct ngui, bo qun trong nhit phng, s
dng sau 48h.
Cc t ch s dng mi trng gi ging lm 2 ng thch nghing v 2 ng mi
trng nhn ging cp 1 cy trong bui thc hnh ny.
3.2. Cy truyn gi ging
- V sinh khu vc t bng cn 70%, chun b cy truyn.
29
Tt c cc t s dng ng ging nm men Saccharomyces cerevisiae gio vin
cung cp cy truyn vo 2 ng thch nghing chun b.

Ly mt vng que cy sinh khi nm men, cy zic zac trn ng thch nghing.
Ghi tn t/nhm ln/ngy cy vo 2 ng nghim, sau em ct lu nhit
phng trong 24 gi.
Tiu ch nh gi kt qu ca t (chp hnh bo co):

S ng nhim trn tng s ng. ng cy p.
3.3. Nhn ging cp 1
Cc t s dng ng ging nm men Saccharomyces cerevisiae (GV cung cp),
ly 2 vng que cy cho vo mi ng nghim cha 10 ml mi trng Hansen lng (mi
trng nhn ging cp 1), khuy nh. Sau nui cy 48 gi iu kin phng.
3.4. Nhn sinh khi
Cc nhm i theo lch phn cng phn ph lc
Ly ging cp 1 a ton b sinh khi sang bnh tam gic 250 cha 100 ml mi
trng Hansen lng. Sau nui cy lc 150 vng/1 pht 300C trong 72 gi.
3.5 Quan st hnh thi nm men
Quan st vt knh 40:
- Ly mt phin knh sch nh ln mt git thuc nhum (xanh methylen).
Git thuc nhum khng nn to qu (v sau s trn khi lamelle), cng khng nn
nh qu (to thnh nhiu bt kh khi y lamelle).
- Ly mt vng sinh khi nm men (t ng ging GV cung cp) ha vo git
thuc nhum. t mt cnh ca lamelle st vo pha ngoi git mu ri h t t
lamelle xung cho git mu trn u khp lamelle. Nu trn ra ngoi th dng giy lc
thm bt.
- Quan st vt knh 40 v ghi nhn kt qu. C th cn c vo mc bt mu
m nht phn bit c t bo sng v t bo cht.
Quan st vt knh 100:
- Dng que cy, pht dch nm men thnh mt lp mng trn phin knh.
- Lm kh, c nh v nhum n bng xanh methylen nh khi nhum vi khun.
- Quan st vt knh 100 v ghi nhn kt qu.
4. BO CO
1. Gi, nhn ging cp 1, nhn sinh khi c b nhim hay khng? (nh chp)
2. Hnh thi t bo nm men quan st trn vt knh 40, 100? (nh chp)
3. Tm hiu quy trnh ra t bo nm men?
5. CCH THC NH GI
30
BI 5. TCH CHIT V XC NH HOT TNH ENZYME

INVERTASE TCH T SACCHAROMYCES CEREVISIAE
1. MC CH
-
Tch chit v thu nhn invertase t sinh khi Saccharomyces cerevisiae.
Xc nh hot tnh ca invertase th bng phng php ng kh DNS.

A. HA CHT
TN HA CHT
QUY CCH
SL/VT
GHI CH
Saccharose 20g/lit
Ml
100
PTN
HCl 0,1N
Ml
100
Phenolphtalein 1%
Ml
10
Glucose chun 10mg/ml
Ml
40
Thuc th DNS
Ml
300
Nc ct 2 ln
lit
Giy nhm cun
Cun
m acetate 0.1M
pH=4.5
ml
600
Ly trc 48h
Sinh khi nm men
10
SV chun b
QUY CCH
SL/VT
GHI CH
STT
B. DNG C
STT
TN DNG C
Cc thy tinh 1000ml
Ci
Bnh nh mc 100
Ci
ng nghim phi 18
Ci
13
Gi ng nghim
Ci
Phu thy tinh
Ci
Pipet 5ml
Ci
Pipet 10ml
Ci
Pipet 1ml
Ci
Cc thy tinh 100ml
Ci
10
Bnh tia
Ci
11
a thu tinh
Ci
12
Qu bp
Ci
c lp
1 t
31
13
Tam gic 100
Ci
14
Bnh tam gic 100ml nt

mi
Ci
Mn trc 48h
C. THIT B
STT
TN THIT B
QUY CCH
SL/VT
My Votex
Ci
Bp in
Ci
10
My ph t bo bng
Ci
GHI CH
C lp
sng siu m
4
My o OD
Ci
Xoong nhm
Ci
Knh hin vi
Ci
1 t
T m c
Ci
Mn trc 48h
My ly tm
Ci
Cn phn tch 2 s
Ci
Phng th nghim chun b: Cn 5g DNS vo 300 ml nc ct vo cc, ha

tan 500C, sau thm 5ml dung dch NaOH 4M. Cui cng cho thm 150g mui
tartrat kp ha tan hon ton ri cho vo bnh nh mc bng nc ct cho 500ml,
cha trong bnh thy tinh sm mu.
Chun 3 ml thuc th DNS bng HCl 0.1N vi thuc th l phenolphtalein, nu
ht 5 - 6 ml HCl l c.
3. NI DUNG
Invertase tn khoa hc l -D-fructofuranoside fructohydrolase (EC 3.2.1.26) l
mt loi enzym thy phn saccharose thnh glucose v fructose (t l mol 1:1) cn
c gi l ng nghch o. ng nghch o c s dng kh ph bin trong
cng nghip nc gii kht v bnh ngt.
Invertase c trong ng thc vt, vi sinh vt (c bit l nm men c kh nng tng
hp invertase cao). Saccharomyces hin l vi sinh vt c quan tm nhiu nht trong
lnh vc ln men to invertase.
Invertase ca Saccharomyces gm hai loi nh sau: invertase ni bo c trng
lng phn t vo khong 135000Da v invertase ngoi bo c trng lng phn t
vo khong 270000Da.
Phn ng thy phn saccharose do invertase xc tc nh sau:
32
Invertase
Saccharose (ng khng kh)
C HOH
O
OH
OH H
H
OH
C HOH
O H HO
OH
HOH C
OH H
Glucose + Fructose (ng kh)

H
C HOH
O
OH
HO O
C HOH
H HO
OH H OH +
OH
HOH C
OH H
H OH
Lng glucose to thnh s ph thuc vo kh nng phn ct ca enzyme. Ngi ta

tin hnh nh lng glucose sinh ra sau thy phn theo nhiu phng php khc nhau
nh phng php Willsteate Schudel bit c hot ca enzyme (Trang 49Thc tp sinh ha ln - Lm Th Kim Chu, Trang 30- Thc tp sinh ha c s - Trn
M Quan).
3.1. Thu sinh khi nm men (T 1: Lm trc 48 gi theo phn cng ph lc)
- Dch nm men nhn sinh khi em i ly tm ln 1 (4500 vng/pht) trong 10
pht, loi dch trong, thu hi sinh khi nm men.
- Tip tc phi trn sinh khi nm men vi dung dch nc mui NaCl (0,85%)
theo t l 1/3 (w/w). Sau , tin hnh ly tm (4500 vng/pht) trong 10 pht thu
nhn sinh khi nm men Ra bng nc ct v trng theo t l 1/3 (w/w) v gi li
sinh khi nm men.
Bo qun sinh khi nm men 16-200C (ngn mt ca t lnh).
3.2. Ph t bo thu dch enzyme th
3.2.1. Phng php t phn (T 1: Lm trc 48 gi theo phn cng ph lc)
Trong qu trnh t phn nm men, h enzyme t phn c sn trong t bo nm
men c hot ha. Chng xc tc phn ng phn gii cc cht c trong thnh t bo
nm men v gii phng enzyme invertase ra ngoi t bo.
Cn 1g sinh khi nm men cho vo dung dch m acetate pH = 5.5 theo t l
1/6 (w/w) thc hin qu trnh t phn. trong t m 450C trong 50 gi. Sau qu
trnh t phn, mu c ly tm (5000 vng/pht) trong 10 pht tch b phn khng
tan, phn dch lng thu c l ch phm invertase th. S dng dch enzyme ny
xc nh hot tnh.
3.2.2. Phng php ph bng sng siu m
T 2: Cn 1g sinh khi nm men cho vo cc 100 c cha nc ct v trng theo t l
1/6 (w/w). Dng sng siu m (tn s ?, thi gian ?) ph v t bo. Sau qu trnh ph
t bo, mu c ly tm (5000 vng/pht) trong 10 pht tch b phn khng tan,
phn dch lng thu c l ch phm invertase th. S dng dch enzyme ny xc
nh hot tnh.
33
3.3. Xc nh hot tnh enzyme invertase

Nhm chn xc nh hot tnh dch enzyme t phn
Nhm l xc nh hot tnh dch enzyme ph sng
- Ht 4ml dung dch m acetate pH = 4,5; 4,6 ml nc ct v 0,4ml dch cha
enzyme cho vo bnh tam gic 100ml.
-
Thm vo 1ml dung dch ng Saccharose nng 2%.
- Lc u, yn cho phn ng xy ra iu kin phng trong 15 pht.

- un cch thy trong 2 pht v lm lnh di vi nc mu thy phn
dng xc nh hm lng ng glucose bng phng php DNS, t suy ra
s mol Saccharose b thy phn.
3.3.1. nh lng ng kh bng phng php acid dinitrosalicylic (DNS)
a. Nguyn tc: ng kh l cc ng cha nhm aldehyde (-CHO) hoc
ketone (-CO) nh glucose, fructose, arabinose, maltose, lactose; trong khi
saccharose, trehalose khng phi ng kh.
Phng php ny da trn c s phn ng to mu gia ng kh vi thuc
th acid dinitrosalicylic (DNS). Cng mu ca hn hp phn ng t l thun vi
nng ng kh trong mt phm vi nht nh. So mu tin hnh bc sng
540nm. Da theo th ng chun ca glucose tinh khit vi thuc th DNS s tnh
c hm lng ng kh ca mu nghin cu.
Phng trnh phn ng to mu gia ng kh v DNS acid:
b. Dng ng chun v xc nh hm lng ng

- Cho vo 6 ng nghim sch vi cc cht c th tch nh bng sau:
Ha cht
ng 1 ng 2
ng 3
ng 4
ng 5
ng 6
ng mu
Glucose 1%
0,2
0,4
0,6
0,8
1,0
Nc ct
10
9,8
9,6
9,4
9,2
Mu thy phn
Nng (%)
0
?
1
?
Ht 1ml t ng 1 n ng 6 vo 6 ng nghim tng ng khc,

ng mu gi nguyn
Thuc th DNS (ml)
3
34
Dng giy nhm bt kn u cc ng nghim

t vo ni nc ang si trong 5 pht
Lm lnh n nhit phng
OD ( = 540 nm)
o mt quang bc sng 540 nm vi ng 1 chnh OD = 0.

V ng chun glucose vi trc tung l mt quang (OD540nm), trc honh l
nng glucose.
Tm phng trnh biu din ng chun dng y = ax + b vi y = OD540nm; x =
[glucose] (%) v h s tng quan R2 nh phn mm Excel.
Ch : tin hnh cc mu dng ng chun v mu th nghim ng thi.
c. Tnh kt qu
- T phng trnh th ng chun tnh c X (mg/ml) ng kh trong
dung dch ng pha lang.
- Nhn vi h s pha lang n c lng ng trong 1 ml dung dch gc.
Chn h s pha lang n sao cho OD nm trong gii hn ng chun.
- Tnh hm lng ng trong nguyn liu (mg/g) = X *n* V/m
V = th tch dch ng gc (ml)
m = khi lng mu cn (g)
Mt n v hot tnh invertase l lng enzyme cn thit thy phn
micromol ng Saccharose trong
pht iu kin pH 4 5 v nhit 0oC.
Tnh hot tnh enzyme invertase.

4. BO CO
1. Nu cc bc thu nhn dung dch invertase ?
2. V ng chun glucose.
3. Tnh hot tnh ca Invertase?
5. CCH THC NH GI
35
BI 6. SO SNH NG HC ENZYME C NH V ENZYME T DO

1. MC CH
Sau khi hc xong bi sinh vin c kh nng:
- Xc nh thng s ng hc ca enzyme GOD t do, c nh.
- So snh gi tr Vmax, Km ca E t do v c nh.
- Nu u, nhc im phng php s dng enzyme c nh v t do.
Dng c, ha cht, thit b s dng cho bi thc hnh c k theo bng 6.1.
A. HA CHT
STT
TN HA CHT
QUY CCH
SL/VT
1 Nc ct 2 ln
lit
m acetate
2 pH =5.6
Ml
200
3 -D-Glucose
50
4 DNS
Ml
650
5 NaOH 0,1N
Ml
600
6 HCl 0,1N
Ml
400
7 Alginate
100
8 CaCl2 2%
Ml
3000
9 Bng thm
500
500
Thp
12 (NH4)2HPO4
0,4
13 MgSO4.7H2O
0,2
14 Peptone
10
15 Saccharose
80
16 KH2PO4
0,2
17 NaOH rn
40
10 Bng khng thm

11 Giy pH
GHI CH
Mi trng nui cy
B. DNG C
STT
TN DNG C
QUY CCH
Phu thy tinh
Ci
Pipet 5ml
Ci
Cc 100
Ci
SL/VT
GHI CH
1 t
36
Bnh tia
Ci
Qu bp
Ci
a thy tinh
Ci
Bm kim tim 1 cc
Ci
Mun thy tinh xc ha
Ci
cht
9
Panh kp
Ci
10
ng nghim
ci
14
11
ng ong 100
Ci
12
Pipet 5
Ci
12
13
Bnh tam gic 100ml
Ci
30
C nhm
C.THIT B
STT
TN THIT B
QUY CCH
SL/VT
1 My quang ph
Ci
2 Cn 2 s
Ci
3 My n nhit
Ci
4 My khuy t
Ci
5 My siu m
Ci
6 My ly tm
Ci
7 Ni hp
Ci
8 My lc
Ci
GHI CH
Cch pha dung dch m acetate pH 5.6

Pha dung dch A: Acetatic 0,2M: 11,55 ml CH3COOH c nh mc ti 1000ml
Pha dung dch B: Natri acetate 0,2M: 16,4g CH3COONa (27,2g CH3COONa.3H2O)
ha tan nh mc ti 1000ml
Trn 4,8ml dung dch A v 45,2 ml dung dch B c 50ml m acetate pH =5,6
3. NI DUNG
Glucose oxidase (GOD, -D-glucose: oxygen, 1-oxidoreductase, EC 1.1.3.4) l
enzym xc tc qu trnh oxi ha -D-glucose thnh acid gluconic vi oxi l cht nhn
in t v to ra hydro peroxide (H2O2). L enzym c bit n t lu, tuy nhin
gn y GOD thu ht c nhiu quan tm v nghin cu trn th gii bi nhng
ng dng quan trng trong thc phm v cc lnh vc khc nh y dc, ha hc lm
sng, cng ngh sinh hc v trong cng nghip dt. Enzym c s dng trong sn
xut cht to chua trong thc phm, lm trng bt lng trng trng, bo qun thc
phm, lm cm bin sinh hc xc nh hm lng ng glucose. Enzym c thu
37
nhn t cc ngun vi sinh vt, trong ph bin l t Aspergillus niger l nm mc c

kh nng sinh c enzym GOD ni bo v ngoi bo. Trong th nghim ny nh gi
kh nng thu nhn enzym ni bo, v hiu qu thu enzym ny cao hn so vi enzym
ngoi bo.
3.1. Hot ha nm mc ging Aspergillus niger
T 10: i lm theo phn cng ph lc
Nm mc t ng ging c hot ha trong mi trng lng
Thnh phn (g/l):
(NH4)2HPO4: 0,4
KH2PO4: 0,2
MgSO4.7H2O: 0,2
Peptone: 12
Saccharose: 80
un si mi trng 2 pht, iu chnh pH n 5,5 bng HCl 0,1 N. Ly 35 ml
mi trng cho vo bnh nn 100 ml, thanh trng 121oC, 30 pht, ngui n
nhit mi trng. Ly 3 vng que cy t ng nm mc ging cy vo mi trng.
Nui trn my lc 28-30 oC, 225 vng/pht, 24 h.
3.2. Nui cy sinh tng hp enzyme GOD
T 9: i lm theo phn cng ph lc
Tin hnh nui cy sinh tng hp enzyme trong mi trng c thnh phn
ging vi khi nui tng sinh nhng c b sung thm 40g/l CaCO3 v khng iu
chnh pH. Chuyn vo mi trng 4,5 ml dch ging nm mc sau khi tng sinh. Nui
trn my lc 28-300C, 175 vng/pht, 72 h.
3.3. Siu m trch ly enzyme ni bo
T 8: Dch sau nui cy sinh tng hp enzym c lc thu sinh khi. Cn m (g) sinh
khi nm mc. B sung 30ml m acetate pH= 5.6 v tin hnh siu m tn s
14kHz vi 8 chu k, mi chu k ko di 1 pht. Ly tm hn hp sau siu m 20 oC,
5200 vng/pht trong 15 pht v thu phn dch ni c cha enzym ni bo.
3.4. Phng php c nh Glucose oxidase
a. Nguyn tc
Vic s dng enzyme ha tan b gii hn do c ch ca sn phm cui cng, gi
tr thnh phm cao khng n nh, khng c kh nng ti s dng v kh thu hi. V
vy cc k thut c nh enzyme trn gi th to ra cc dng enzyme c nh (enzyme
khng ha tan) ang c quan tm.
b. Cch tin hnh
T 7
- Pha 100ml dung dch Alginate natri 2% (w/v) (Dung dch A)
38
Ly 100 ml dch enzyme sau khi thu nhn bng phng php ph v t bo
(Dung dch B).

Trn dung dch A v B trn vi t l 1:1 (v/v) c dung dch C
Dung dch C c 200 ml chia u cho 10 t
Cc t lm
Nh 20 ml dung dch C vo cc c cha 30ml CaCl2 2% (2g/100ml) to ht
Enzyme c nh (s dng bm tim nh cao 10-20 cm nhit phng). Dung
dch CaCl2 phi c khuy bng a thy tinh hoc t trn my khuy t t gi
cho cc ht gel va to thnh khng dnh vo nhau. Tng thi gian to gel khng qu
1% thi gian ht ngm trong dung dch CaCl2 m bo ht to thnh trong cng
mt thi gian. Ngm ht E c nh trong dung dch CaCl2 2% trong khong 30pht
tng bn ca ht. Khi dung dch alginate nh vo trong dung dch CaCl2, mt lp
v bao s hnh thnh xung quanh ht nh vo qu trnh to lin kt mng li gia
alginate v Ca2+.
Sau 30 pht vt ht E c nh ra khi dung dch CaCl2 v ra ht 3 ln bng nc
ct. Ht enzyme c nh c dng hnh cu vi ng knh trung bnh 3-4 mm.
3.5. Xy dng ng chun ca D-glucose
T 2: Pha 500ml dung dch glucose 1% s dng lm th nghim
Trong 1 nhm 2 t dng ng chun (1 chn, 1 l)
Tin hnh theo bng sau:
Ha cht
ng 1 ng 2
ng 3
ng 4
ng 5
ng 6
Glucose 1(%) (ml)
Nc ct
0,2
0,4
0,6
0,8
1,0
Nng (%)
- T cc ng nghim 1- 6 trn ly 1ml mi ng vo 6 ng nghim tng ng khc

- Thm vo mi ng 3ml thuc th DNS
- Dng giy nhm bt kn u ng nghim
- t vo ni nc ang si trong 5 pht.
- Lm lnh n nhit phng.
- o mt quang bc sng 540 nm vi mu trng pha t ng i chng.
- V ng chun glucose vi trc tung l mt quang (OD540nm), trc honh l nng
glucose. Tm phng trnh biu din ng chun dng y = ax + b vi y = OD 540nm;
x = [glucose] (mg/ml) v h s tng quan R2 nh phn mm Excel.
3.6. Xc nh hot GOD t do v GOD trong nc ra
Nguyn tc: Da trn phn ng to mu gia glucose v thuc th DNS, cng
mu ca hn hp phn ng t l thun vi nng glucose trong phm vi nht nh.
39
Da vo th ng chun vi glucose tinh khit, ta tnh c hm lng glucose c

trong mu phn tch. Cng mu o trn my quang ph ti bc sng 540 nm.
Tin hnh
ng nghim
i chng
Enzyme t do
Enzyme/ nc ra
Glucose 1% (ml)
m acetate pH= 5.6 (ml)
0.2
0.2
0.2
GOD (ml)
Nc ct (ml)
[glucose] (%)
yn trong 30 pht 30oC
Dng phn ng bng cch un cch thy trong 3 pht

Chuyn sang cc ng nghim tng ng
ng nghim
i chng
Enzyme t do
Enzyme/ nc ra
Dung dch (ml)
DNS (ml)
un si trong 5 pht
Lm lnh n nhit phng
OD 540nm
OD=ODM-ODKC
Lng glucose (y)
Xc nh hot tnh
Mt n v hot ca glucose oxidase l lng enzyme dng chuyn ha 1
mol ca glucose trong thi gian 1 pht iu kin 30oC v pH l 5.6.
3.7. Xc nh thng s ng hc
3.7.1. Xc nh thng s ng hc ca enzyme t do
Tin hnh th nghim

ng nghim
Glucose 1% (ml)
m acetate pH= 5.6
[glucose] (%)
0.2
0.4
0.6
0.8
GOD 1%(ml)
1ml
yn trong 5 pht/370C
Dng phn ng
un cch thy trong 3 pht

40

ng nghim
Dung dch (ml)
DNS (ml)
un si
5 pht
Nhit phng
Lm lnh
OD 540nm
OD=ODM-ODKC
Lng glucose (y)
Tnh vn tc phn ng
Qua tnh c 1/S v 1/V.
Tnh Vmax v Km ca enzyme t do
3.7.1. Xc nh thng s ng hc ca enzyme c nh
Tin hnh th nghim

ng nghim
Glucose 1% (ml)
m acetate pH= 5.6
[glucose] (%)
GOD c nh (g)
0.2
0.4
0.6
0.8
1
0
yn trong 5 pht/37 C
Dng phn ng
ng nghim
Dung dch (ml)
un cch thy trong 3 pht

0
1
2
3
1
DNS (ml)
un si
5 pht
Nhit phng
Lm lnh
OD 540nm
OD=ODM-ODKC
Lng glucose (y)
4. BO CO
1. Tnh gi tr Vmax, Km ca GOD t do v c nh
2. So snh, kt lun v thng s ng hc ca enzyme t do v c nh
3. Sinh vin t xy dng phng php xc nh hot ca GOD
41
5. CCH THC NH GI
i n v: 1mg = 3UI, 1ml=103 g =106 ng
B tr th nghim bi 6
Cng vic
STT
Thi gian
Nhim v
Hot ha nm mc
Chun b trc
bui thc hnh
T 10
Nui cy sinh tng hp GOD
Siu m trch ly e ni bo
Lm trong bui
thc hnh
T 8
Lp ng chun Glucose
Xc nh hot dch e th v nc ra
Cc t lm
To th gel alginate
T 7
C nh e
Cc t lm
X thng s ng hc E c nh
Cc t chn
X thng s ng hc E t do
Cc t l
T 9
T 1, t 2
Cc nhm ly s liu cho ca nhau c 9 phn vic ca bi 6 bo co.
42
TI LIU MN HC
Sch, gio trnh chnh
[1] B mn CNSH, Bi ging thc hnh cng ngh enzyme, Trng H CNTP TP
HCM, 2014.
[2] Nguyn c Lng (ch bin), Cng ngh enyme, Nh xut bn i hc quc gia
TP.HCM, 2003.
Ti liu tham kho
[3] Lm Th Kim Chu, Nguyn Thng Lnh, Vn c Chnh, Thc tp ln sinh
ha, T sch i hc Khoa hc T nhin TP.HCM, 1997.
[4] Phm Th nh Hng, 2006, K thut ha sinh, NXB HQGTP HCM.
[5] Nguyn Vn Mi, 1999, Th nghim ha sinh NXB HQGTP HCM.
[6] Trn Bch Lam, 2004, Th nghim ha sinh thc phm NXB HQG TP HCM.
[7] Nguyn c Lng, 2003, Th nghim CNSH tp , NXB HQGTP HCM.
[8] Trn M Quan, 2003, Thc tp ha sinh c s, NXB HQGTP HCM.
43
PH LC PHN CNG CHUN B THC HNH NHM C LINH
44

Bai Giang Thuc Hanh Enzyme - 2014

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Bai Giang Thuc Hanh Enzyme - 2014

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TRNG I HC CNG NGHIP THC PHM TP HCM

KHOA CNSH- KTMT

B MN CNG NGH SINH HC

BI GING THC HNH

CNG NGH ENZYME

TP. H Ch Minh, thng 4 nm 2014

BI 1. NG HC V B PHN NG CA ENZYME ........................................................... 11

BI 2. TCH CHIT V XC NH HOT TNH BROMELINE ................................................. 15

BI 3. TCH CHIT V XC NH HOT PEPSIN ........................................................... 22

BI 4. NHN SINH KHI SACCHAROMYCES CEREVISIAE .................................................... 27

BI 5. TCH CHIT V XC NH HOT TNH ENZYME INVERTASE TCH T

B mn Cng ngh sinh hc

2. THIT B - DNG C- H A CHT- MU VT ............................................................ 31

BI 6. SO SNH NG HC ENZYME C NH V ENZYME T DO ................................... 36

B mn Cng ngh sinh hc

C k nng thao tc, s dng cc thit b, dng c th nghim.

Thc hin nghim tc ni quy phng th nghim.

Hon thnh bi thc hnh theo thi gian quy nh.

1. Sinh vin c v tm tt bi trc khi n lp. T son bi tham kho

- im mn hc l im trung bnh cng ca 6 bi

Tin trnh th nghim

Gii thch th nghim

Kt lun - Kin ngh

3. CCH PHA V S DNG DUNG DCH M

S thay i pH ny c gi l hiu ng pha long ca dung dch m. Hiu ng ny

3.4.2. Hn hp m K2HPO4 HCl

B mn Cng ngh sinh hc

pha ch dung dch m ny ta cn dung dch K2HPO4 0,1 M (20,42 g/l)v

3.4.3. Hn hp m K2HPO4- NaOH

B mn Cng ngh sinh hc

3.4.4. Hn hp m KH2PO4 NaOH

4. MT S BC C BN TRONG THU NHN V TCH CHIT ENZYME

khc nhau nh polysaccharid, nucleic acid, cc cht c phn t nh nh ng

Cn nng cao (960 980 ) vi t l v th tch 1 E : 3 cn hay 1 E : 4 cn.

B mn Cng ngh sinh hc

B mn Cng ngh sinh hc

T 1: Pha dung dch casein 12 mM: Cn 2g casein vo cc pha, thm vo khong

Xy dng ng thng tng quan gia vn tc thu phn ca enzym v nng

Dd tyrosine chun (ml)

Tyrosine tng ng (mol)

HCl 0.2N (ml)

Folin pha long 3 ln

Tin hnh th nghim

Nng Casein (mM)

B mn Cng ngh sinh hc

NaOH 0.5N (ml)

Folin pha long 3 ln

Lc mnh, sau yn trong 10 pht

Da vo th chun ta suy ra lng Tyrosine tng ng.

B mn Cng ngh sinh hc

BI 2. TCH CHIT V XC NH HOT TNH BROMELINE

Vit quy trnh thu nhn enzyme bromeline dng paste.

2. THIT B - DNG C- H A CHT- MU VT

3 Thm (da) xanh

Acid tricloacetic TCA

B mn Cng ngh sinh hc

Pipep man 100 -1000l

Phu thy tinh

Bnh tam gic nt mi 100

Cc thy tinh 500ml

Cc thy tinh 1000ml

B mn Cng ngh sinh hc

m KH2PO4 1M, iu chnh li pH =6 vi dung dch HCl 2N. Thm nc y ti