B CNG THNG

TRNG I HC CNG NGHIP TP.HCM

VIN CNG NGH SINH HC - THC PHM

MN: HA SINH THC PHM

TI: PHNG PHP TCH CHIT, XC NH HOT V
NG DNG CA PROTEASE
GVHD: NGUYN TH TRANG

NHM 2.
STT TN
MSSV
1
Hong Th Thanh Nga
14063011
2
Nguyn Th Lin
14049971
3
Nguyn Th Ngn
14080081
4
Nguyn Th Tho Nguyn 14047721

Nhm trng

Tp.hcm, Ngy 18 thng 9 nm 2015

2014-2015
1

M u
Ngy nay vi s pht trin mnh m ca Cng Ngh sinh hc,
cc ch phm enzyme c sn xut nhiu v c s dng hu ht
trong cc lnh vc nh: ch bin thc phm, nng nghip, chn nui, y
t...Hng nm lng enzyme c sn xut trn th gii t khong trn
300.000 tn vi trn 500 triu USD, c phn phi trong cc lnh vc
khc nhau.
Trong , Protease l enzyme c s dng nhiu nht hin nay
trong mt s ngnh sn xut nh; ch bin thc phm (ng t sa lm
cho phomt,lm mm tht, b sung tng cht lng sn phm trong
sn xut bia. X l ph ph phm trong ch bin thc phm...(sn xut
cht ty ra, thuc da, y t, nng nghip...)
Phn 1 - Phng php tch chit enzyme protease
tch v tinh ch enzyme ni chung l protease ni ring th c
mt s phng php ha - l khc nhau. C th chia lm ba nhm chnh
sau:
- Phng php kt ta
- Phng php sc k
- Phng php phn tch h hai pha nc
Protease cn thit cho cc sinh vt sng, rt a dng v chc nng
t mc t bo, c quan c th nn c phn b rt rng ri trn
nhiu i tng t vi sinh vt ( vi khun, nm, virus) n thc vt ( u
, da,..) v ng vt (gan, d dy b,..). So vi protease ng vt v
thc vt, protease vi sinh vt c nhng c im khc bit. Trc ht h
protease vi sinh vt l mt h thng rt phc tp bao gm nhiu enzyme
rt ging nhau v cu trc, khi lng v hnh dng phn t nn rt kh
tch ra di dng tinh th ng nht.

Cng do l phc h gm nhiu enzyme khc nhau nn protease vi sinh

vt thng c tnh c hiu rng ri cho sn phm thy phn trit v
a dng.
Phn 2 - Thu nhn v lm sch enzyme protease t vi sinh vt v
thc vt
2.1 Thu nhn v lm sch enzyme protease t vi sinh vt.
Ngun thu protease vi sinh vt ch yu l vi khun, nm mc v
x khun.
Qu trnh sn xut cc ch phm enzyme vi sinh vt bao gm cc
giai on ch yu:
Tuyn chn v ci tao ging vi
Sinh vt

Phng php bo qun ging

Vi sinh vt

Mi trng nui cy vi sinh

Tng hp enzyme

Tch v lm sch ch phm

enzyme

Tuy nhin do thu c canh trng v nng enzyme thp nn

khi tch thu hi enzyme s c gi thnh cao. Tn in nng cho khuy
trn, nu khng bo m v trng s b nhim hng lot, ton b gy tn
thng ln.
2.2 Tch v lm sch ch phm enzyme.
3

Mt iu cn ni thm na l enzyme thng cha cc t bo vi

sinh vt gi l cc enzyme trong t bo (intracellular), nhng n cng c
th c cc vi sinh vt tit ra mi trng sng. l cc enzyme ngoi
t bo (extracellular). Enzyme vi sinh vt thng chit l enzyme ngoi
bo.
Cc phn t enzyme khng c kh nng i qua mng t bo v
mng ca cc cu t ca t bo. Do c th chit rt cc enzyme ni
bo, bc u tin l phi ph v cc cu trc ca cc t bo c cha
enzyme v chuyn chng vo dung dch.
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, lm ng ha bng
thit b ng ha (homogenzizator).
Mun tch c cc enzyme trong cc cu t ca t bo, ngi ta
cn phi dng n cc yu t vt l v ha hc khc nhau nh sng siu
m. Dng cc dung mi hu c nh buthanol, aceton, glycerin, ethyl
acetate... v cht detergent. 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.
Sau khi ph v cc cu trc ca t bo, enzyme c chit
bng nc ct, bng cc dung dch m thch hp hoc bng cc dung
dch mui trung tnh.
C mt s yu t nh hng n qu trnh chit rt cn lu .
Trc ht l nhit . trnh mt hot tnh hoc thm ch v hot,
cn chit rt v tin hnh kt ta enzyme nhit thp (t 3 n 50C).
Cc thao tc phi nhanh. Mt s cht in li 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.
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 cc
lc qua gel sephadex.
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 loi b cc protein tp (protein cu trc, protein tr) v cc

cht c phn t lng cao khc ngi ta hay dng kt hp cc phng
php khc nhau: phng php bin tch chn lc nh tc dng ca nhit
hoc pH ca mi trng, phng php kt ta phn on bng mui
trung tnh hoc cc dung mi hu c,cc phng php sc k( sc k
hp ph, sc k trao i ion) in li, phng php lc gel.
Mc ch yu cu: cc ch phm enzyme c s dng cc dng
khc nhau theo mc tinh khit( hot ring). Trong mt s trng
hp, canh trng nui cy vi sinh vt c cha enzyme c s dng trc
tip di dng th khng cn tch tp cht nu chng khng gay nh
hng ng k n sn phm v qui trnh cng ngh sau ny( v d: sn
xut ru, nc chm thc vt, da). Cng c khi ngi ta cn s dng
ch phm enzyme tinh khit trong cng nghip dt, cng nghip mch
nha, y hc, nghin cu khoa hc.
Enzyme ni chung rt d gim hot tnh di tc dng ca cc tc
nhn bn ngoi do khi tch v tinh ch enzyme trnh s bin hnh
protein nh hng ln n hot tnh enzyme cn tin hnh nhanh chng
nhit thp, pH thp khng c mt cc cht gy bin hnh
enzyme.
2.2.1 Phng php kt ta
Phng php kt ta phn on bng (NH4)2SO4 da trn c s s
khc nhau v kh nng kt ta ca cc protein enzyme mt nng
mui (tnh theo % nng bo ha) xc nh c dng ph bin
loi b bc u protein tp ca cc dung dch enzyme cc loi mui c
th c dng l (NH4)2SO4, Na2SO4, MgSO4... Ngi ta nhn thy
mui (NH4)2SO4 l tt nht v n khng lm hi m lm n nh (lm
bn) hu ht cc loi enzyme. Loi mui ny li r v ph bin. ha
tan ca n li rt ln (bo ha 767 g/l 250C).
2.2.2 Phng php sc k
Cc k thut sc k ct
5

Dch chit enzyme c loi b phn ln cc protein tp nhng

vn cha m bo ng nht cn thit. Do dch chit enzyme c
tip tc lm sch bng phng php sc k ct.
Phng php dng cht ry phn t (lc gel - lc filtration)
C s ca phng php lc gel l da vo s khc nhau v kch
thc hnh dng phn t lng ca enzyme c trong hn hp tch
chng ra.
m bo cho vic tch enzyme c tt, cht ry phn t phi
l cht tr, khng phn ng vi protein enzyme. Cht ny cng khng
ha tan v tng i bn vi cc yu t v c hc cng nh sinh hc.
Ngoi ra cht c s dng cho mc ch lc phn t phi l cht khng
c tnh n hi(khng co) v phi l cht a nc (hydrophyl).
Phng php sc k trao i ion
Phng php sc k trao i ion da vo s khc nhau din tch
tng s ca cc protein enzyme. Hy ni cch khc, phng php ny
da trn c s phn ng trao i ion gia protein c tan trong nc
hoc dung dch m long v cc tc nhn trao i ion. Tc nhn ( hay
nguyn liu) trao i ion c th l cht nha c tch nhm sinh ion hoc
l cht ionit. y l nhng cht gi tr, khng tan trong nc, c bn
cht l cellulose hoc cht gel dextran c li phn nhnh ( Sephadex,
Molselect) hoc l cht nha polystiron. Cht gi th ny thng kt hp
vi cc nhm ion ha. Cc cht trao i ion c cht gi l cellulose,
sephadex, molselect thng thng c dng tch protein enzyme,
cn cc cht trao i ion c cht gi l polystirol ( v d nh Dowex,
Amberlite) ch dng tch cc peptid c trng lng phn t nh hn.
Anionit DEAE - cellulose (diethylamino - ethyl - cellulose) l dn
xut este ca cellulose.
C2H5
C2H5
Cellulose - O CH2 - N
C2H5
6

Trong H2O n c phn li:

Cellulose C2H4 N (C2H5) + H2O
C2H5
+ OH
Cellulose O C2H4 N+
C2H5
H
y l cht trao i anion
Nu cht l sephadex th th chng ta c cht trao i ion sephadex.
l cc loi DEAE sephadex v CM sephadex. u im ca loi
ny l va tch c protein enzyme v kch thc v v din tch tng
s ca cc protein enzyme. Trng hp CM sephadex trn cht gi
sephadex c gn nhm COO- O CH2 - COOH
Phn ly
COO (mang in tch)
y l cht trao i cation.
Ngoi ra, c cc phng php:
7

- Phng php dng chy ph

- Phng php dng cht ph c bit hiu sinh hc hay l
phng php sc k i lc ( afinity chromatography).
2.2.3 Phng php tch h hai pha nc
C s k thut ny da vo s phn b khc nhau ca hn hp
trong dung dch hai pha khng ha tan vo nhau. Cc h hai pha c
trng bng cch trn cc h dung mi vo nhau to thnh hai pha
ring bit. H dung mi c s dng trong phng php ny c th l
polymer/polymer nh: polyethylene glycol (PEG) v dextran hoc
heejpolymer/mui nh PEG v patasium phosphate, sodium phosphate,
sodium sulphate
Cc protein v mnh v t bo c kh nng ha tan khc nhau gia
hai pha, v vy phng php ny c th c dng cho c hai trng
hp: phn tch protein khi mnh v t bo v phn chia cc enzyme
trong sut qu trnh tinh sch protein.
H hai pha nc c xem l phng php tt cho vic phn tch
v tinh sch cc hn hp, v phn tch cht lng c mt khc nhau d
dng hn phn tch cht rn ra khi cht lng.
So vi cc phng php tinh sch khc th h hai pha nc c mt s u
im hn nh:
+ C ha tan trong nc ca hai pha ln (70 80,5).
+ t tinh sch cao, hiu sut cao, d dng s dng quy
m ln.
+ V c bit polymer c ti s dng.
S phn tch t hiu qu cao ty thuc vo cc thng s nh khi
lng phn t v in tch ca i tng nghin cu, nng v khi
lng phn t ca cc polymer, nhit , pH, thi gian, lc ion ca hn
hp v s hin din ca cc loi mui a ha tr phosphate hoc
sulphate

Phn 3 - Xc inh hot protease

Cch xc nh hot ca protease t qu u v nm mc
Aspergillus oryzae.
3.1. Ha cht v dng c
- Qu u , nm mc Aspergillus oryzae.
- Dung dch tartaric acid 1%; dung dch trichloacetic acid 0,3M; dung
dch casein 2%; dung dch m Britton v Robinson (acetic acid,
phosphoric acid, boric acid); dung dch HCl 0,2N; dung dch tyrosine 1
mM; dung dch Folin.
- Dng c thy tinh (bnh tam gic, ng nghim, pipete)
- T m, bp un cch thy
- My so mu.
3.2. Nguyn tc
Dng dch chit qu c enzyme thy phn protein. Sau dit
enzyme v kt ta protein cha b thy phn bng trichloacetic acid.
nh lng sn phm c to thnh trong phn ng bng phn ng
mu vi thuc th Folin, kt qu phn tch da vo th chun
tyrosine.
n v hot protease l lng enzyme phn gii protein to
thnh cc sn phm ha tan trong trichloacetic acid tng ng vi mt
micromol tyrosine (0,181 mg) trong mt pht, 300C.
Protease ca dch chit qu c xc nh pH 7,2 0,2.
3.3. Cc bc tin hnh
a. Chun b dung dch m Britton v Robinson pH 7,2
- Dung dch A: Acetic acid 0,04M (2,32 ml pha thnh 1000 ml),
phosphoric acid 0,04M, boric acid 0,04M.
- Dung dch B: 8g NaOH ha tan trong nc ct v dn nc n 1000
ml.
- Dung dch m Britton v Robinson c pH khc nhau ph thuc vo t
l pha th tch dung dch A v B. Pha m pH 7,2: ly 100 ml dung dch
A v 55 ml dung dch B, dn nc ct n 200 ml.
b. Dung dch casein 2%
9

Ha tan 2g casein vo 90 ml m Britton v Robinson pH 7,2.

Nu pH ca dung dch thp hn 7,2 th dng vi git NaOH 1N iu
chnh, dn m n 100 ml.
c. Dung dch enzyme
- Cn 1g u , nghin vi m Britton v Robinson pH 7,2.
dch nghin vo bnh nh mc loi 100 ml. Dng m ra ci chy,
nc ra cho vo bnh nh mc. B sung m n vch ngn. Lc u,
lc ly dch trong.
- Chun b dch enzyme t dch ln men vi nm A. oryzae : Nui
cy A. oryzae trong mi trng dch th CzapekDox v trng
(saccharose 30g; NaNO3 3,5g; K 2 HPO 4 1,5g; MgSO4 0,5g; KCl 0,5g;
FeSO 4 0,01g ri thm 20 ml sa c hoc 10g gelatin, hoc casein,
nc ct 1000 ml) trong iu kin nhit phng trn my lc 200
vng/pht sau 72 gi thu ly dch lc.
d. Tin hnh phn ng
Cho vo 2 ng nghim, mi ng 20 ml c cht (casein), t vo
chu n nhit 300C. Sau 10 pht cho vo mi ng nghim 20 ml dung
dch enzyme (cng 300C), lc trn u v 10 pht 300C. Sau
cho vo mi ng nghim dung dch trichloacetic acid 0,3M; lc trn
nhanh protein d v cc hp cht cao phn t khc kt ta, gi cc
ng nghim 300C thm 20 pht na ri lc ly dch. Ly 1 ml dch lc
v 5 ml dung dch Na2 CO 3 0,5M cho vo ng nghim, trn u v thm
1 ml thuc th Folin. Sau 20 pht phn ng, dung dch c mu xanh da
tri, mang o trn my so mu.
Th nghim i chng thay dung dch enzyme bng 20 ml nc
ct.
So snh kt qu ca th nghim v i chng.
e. Xy dng ng chun tyrosine
Pha dung dch gc tyrosine c nng 103 (1 mM): cn
181,12 mg tyrosine, ha tan trong dung dch HCl 0,2N ri nh mc n
100 ml bng HCl 0,2N. T dung dch gc pha long thnh dy nng
dung dch nh sau:
Tyrosine(ml)
HCl

0,1
4,9

0,2
4,8

0,4
4,6

0,6
4,4

0,8
4,2

1,0
4,0

1,5
3,5
10

0,2N(ml)
Nng
tyrosine
(mM)

0,02

0,44

0,08

0,12

0,16 0,20 0,30

Mi dung dch trn ly 1 ml cho vo 7 ng nghim, cho thm

vo mi ng 5 ml Na2 CO 3 0,5M v 1 ml thuc th Folin, lc u.
Mu i chng thay 1 ml dung dch tyrosine bng 1 ml nc ct. Gi
hn hp phn ng trong 20 pht, sau o trn my so mu bc
sng 720 nm. Ly s liu trung bnh ca hai ng nghim dng ng
chun tyrosine vi trc honh l nng tyrosine (e) tnh theo
micromol/ml, trc tung l mt quang (OD).
3.4. Tnh kt qu
3.4.1. inh tnh hot protease
+ Kim tra hot protease mi trng cha casein hoc
gelatin tinh khit: Dng mi trng thch a CzapekDox thm 10g
gelatin, dng khoan ng khoan b thi thch ri cho vo 1 ml dch
nui A. oryzae trn, sau 24 gi dng thuc th HCl 1% hoc dung dch
NH
( 4 )2 SO 4

bo ha ln b mt mi trng thch a, nu A. oryzae

sinh ra protease th s c mt vng trong sut xung quanh l khoan do

cc protein b phn gii, vng khng b phn gii vn c mu trng
c.
Cn c vo ng knh vng phn gii xc nh hot
protease.
3.4.2. inh lng hot protease
Hot protease (Hdp) tnh bng n v/gam hay n v/ml
theo cng thc:
Hdp=

OD41000
a10w

Trong : OD - mt quang o c ca mu
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4 - T l th tch ca hn hp phn ng vi dung dch enzyme. Sau khi

thm trichloacetic acid:
2 ml c cht ( tyrosine ) +2 ml enzyme+ 4 ml TCA
=4
2 ml enzyme

A - ng lng tyrosine xc nh theo ng chun (mt quang m

1 micromol tyrosine c trong 1 ml dung dch chun, khi phn ng cho
thuc th Folin.)
10 - Thi gian thy phn c cht (pht)
W - Lng ch phm enzyme (ly thy phn c cht tyrosine tnh
bng mg c trong 1 ml dung dch enzyme)
1000 - H s chuyn sang gam ch phm.
3.5. Nhng im cn lu
Khi tin hnh th nghim cc dng c phi c v sinh sch
s.
Cch xc nh nh tnh hot protease t A. oryzae c th
dng phng php c l v dng thuc th l

NH
( 4 )2 SO 4

bo ha

pht hin vng thy phn protein.

Chn qu (u cn xanh) mi thu hoch, khng b dp nt, h hng.
iu kin thy phn: Nng protein l 1% (nng c cht trong
dung dch), thy phn trong vng 10 pht nhit 300C. Trc khi
tin hnh phn ng thy phn, dung dch c cht v dung dch enzyme
u phi a n 300C. Lm ngng phn ng bng cch cho mt lng
dung dch trichloacetic acid 0,3M bng th tch hn hp phn ng, trn
u, gi 300C trong 20 pht, lc ly dch. Lng enzyme cn cho
phn ng sao cho mt quang ca dung dch khi so mu nm trong
phm vi 0,20,6.

Phn 4 - ng dng ca protease

12

4.1. Trong cng nghip thit

-Trong cng nghip tht, c th dng cc ch phm protease lm
mm tht.
- Ch phm enzyme lm mm tht thng l papain hay hn
hp ca papain vi protease ca vi sinh vt.
- Ty theo tnh cht tht m s dng cc ch phm.
- Cht lng cc loi tht c nng cao.
- Thi gian chn ca tht rt i nhiu ln.
- K thut lm mm tht khng phc tp lm.C th:
+ Ngm tht trong phc hp emzyme pH, nhit , nng thch
hp.
+ Ri ln b mt tht cht lm mm dng bt c cha enzyme v
cht ph gia.
+ Tim dung dich vo m.
+ a dung dch enzyme vo mu ng vt trc khi git tht.
4.2. Trong cng nghip sa
- Protease c kh nng ng t sa.
- Tnh cht ny t lu c ng dng vo thc phm.
- ng dng chnh: sn xut phomat.
+ Renin ng vai tr quan trng trong cng ngh ny
+ Ngi ta ang c tm chng vi sinh vt c tnh cht tng t
hoc thay th mt phn renin.
-

4.3. Trong cng nghip thuc da

Cht quan trong nht ca da l collagen, di tc dng ca protase,
cc cht nhn tch ra, mt s lin kt si collagen b ph hy. Kt
qu l da thu c mm nht nh.
Trc y thng dng protease thu t ng tiu ha ng
vt ,ngy nay hiu qu hn c l cc enzyme thu c t c vi
khun v nm mc.
Rt ngn thi gian lm mm, tch long xung nhiu ln , cht
lng tt hn ,s lng lng thu c nhiu, khng x l thm
Ngi ta c th ngm nguyn liu da trong dung dch enzyme
trong dung dch enzyme hoc pht h enzyme ln b mt .
13

4.4. Trong sn xut t tm

- Qa trnh lm sch t si t nhin tng i phc tp v kh khn.
- Si sau khi ko kn thng c khong 30% xerixin. Mun tch
xerixin phi nu trong dung dch x phng. Mt lng nh xerixin
nm li trn la s lm gim n hi ca la.
- tch xerixin cn li ngi ta thng dng ch phm protease
t nm mc, vi khun.
4.5 - Trong sn xut cht ty ra
- Phn hy vt bn dng protein trn vi
- C tc dng ty ra ging nh mu, lng trng, sa, nc rau,
u, nc st thc n,.... V ch c hot tnh trong dung dch git.
4.6-Trong sn xut hng phm, m phm
- Hin nay mt s nc sn xut nhng loi kem cha enzyme
xoa mt, xoa tay, co ru.
- Di tc dng ca protease trong kem,
Cc biu b da cht tch ra, da non v mi s xut hin trn b mt, s
pht trin lng, tc chm li.
4.7-Trong y hc
- Protease c s dng sn xut cc mt trng dinh dng
hn hp c protein dng trong nui cy vi khun v cc vi sinh vt khc.
- Dng cc ch phm protease c c v tinh ch cc huyt
thanh khng c.
- ng dng cha mt s bnh ng tiu ha.

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