B khoa hc v cng ngh

Vin Cng ngh thc phm

301 Nguyn Tri, Thanh Xun, H Ni




BO CO TNG KT
KHOA HC VA KY THUT



ti cp nh nc
NGHIN CU NG DUNG CNG NGH ENZYM
TRONG CH BIN MT S NNG SN THUC PHM
M s : KC 04 07

Ch nhim ti cp nh nc : PGS. TS. Ng Tin Hin







ti nhnh cp nh nc

Nghin cu cng ngh sn xut mt s loi du bo
bng lipaza
Ch nhim ti nhnh cp nh nc : PGS. TS. ng th Thu






H Ni, 10 2004
Bn tho vit xong thng 09 2004


Ti liu ny c chun b trn c s kt qu thc hin ti cp nh nc,
m s : KC 04 - 07.


Danh sch ngi thc Hin

STT H v tn C quan cng tc Mc
1 ng Th Thu i Hc Bch Khoa HN
2 Quyn nh Thi Vin CNSH TTKHTN v
CNQG

3 Phng Th Thu i Hc Bch Khoa HN
4 Hong Lan i Hc Bch Khoa HN
5 Bin Cng i Hc Bch Khoa HN
6 Thiu Linh Thu i Hc Bch Khoa HN
7 Nguyn Th Snh i Hc Bch Khoa HN

















Tm tt ni dung

Lipase (EC 3.1.1.3) l enzym c ng dng trong nhiu ngnh nh
cng nghip thc phm, cng nghip ho hc, cng nghip m phm, cng
nghip da, trong y dc v cc ngnh cng nghip khc do c kh nng
xc tc thu phn triglyxerit thnh di, mono glyxerit hoc glyxerol v cc
axit bo nh hot ng trn b mt phn pha du nc. Trong cng nghip
du v cht bo, vic s dng lipase rt ph bin. C khong hn 100
loi lipase khc nhau c dng chuyn i lipit thnh cc cht khc
Hin nay, Vit Nam s dng mt lng ln lipase trong ngnh
cng nghip thc phm, cng nghip ty ra, ho hc, y hc song cc
ch phm lipase ang s dng phn ln l nhp khu. Do vy, mc ch
ca ti tp trung nghin cu thu nhn v s dng hiu qu lipase t hai
i tng l nm men Candida rugosa v Bacillus sp
Phng php nghin cu
- Vi sinh vt c nui cy theo phng php nui chm c 2 chng
Candida rugosa v Bacillus sp trn my lc lc n nhit Shellab v trn
thit b ln men 2 lt.
- Xc nh hot lipase bng phng php chun .
- C nh lipase trn nylon 6 bng lin kt ng ho tr.
- Tch v tinh ch lipase bng etanol v sc k trao i ion DEAE
cellulose.
- Bng phng php ton hc thc nghim ti u mi trng nui cy
theo m hnh bc 1 ca Box Wilson.
- So snh sn phm phomat c ch bin c b sung lipase v khng b
sung lipase bng phng php cm quan thc phm.
ti thu c nhng kt qu chnh sau:
- Tm c iu kin nui cy ti u cho sinh tng hp lipase t Candida
rugosa cho mt lt l:
Mi trng nui gm: KH2PO4: 15 g; K2HPO4:5,5 g;
(NH4)2SO4:4g; MgSO4:1g; FeCl3:10m g; inositon: 0,004mg; biotin:
0,008mg; Thiamine.HCl: 0,2mg; Du oliu: 1%; axit palmitic: 1.2 g;
H2O: 1000ml;
S t bo nm men cy ban u: 2,6.10
6
tb/ml canh trng;
Thi gian nui: 40 gi
- Tm c iu kin nui cy ti u cho sinh tng lp lipase t Bacillus
sp l: mi trng BMGY vi thnh phn: (1% (w/v) cao nm men, 2%
(w/v) bacto-peptone, 100 mM kali phosphate, pH 7.0, 4 x 10-5% (w/v)
biotin v 1% (v/v) methanol) 30C lc 220 vng/pht
- Nng cn thch hp cho kt ta thu lipase l 90%. Lipase sau kt ta
cn cho chy sc k trn sc k cho thy lipase sau khi qua ct trao
i ion DEAE - cellulose xut hin 3 pick. Tin hnh xc nh hot
lipase cc phn on. Kt qu cho thy ch c pick 1 v pick 3 c hot
tnh lipase (69UI/ml v 77UI/ml) v Lip1 v Lip3 c khi lng phn t
l 58 v 62 kDa. Lipase sau cc bc lm sch: kt ta etanol, sc k
trao i ion DEAE Cellulose thu c ch phm c hot ring
55,16 U/mg protein, mc tinh sch l 12,5 ln; hiu sut thu hi l
37,06%.
- kho st c cc iu kin ti u c nh lipase trn Nylon-6
l: nng HCl cht mang l 3,5N; glutaraldehit l 10%, nng lipase
em c nh l 1mg/ml (550U/ml); s dng dung dch m phosphat pH
7,7.
- xc nh mt s c tnh ca lipase c nh trn Nylon-6
Nhit ti u l 70C, bn vng nhit 40 - 60C, sau 18 gi
hot cn li l 52,4 66,7%.
pH ti u l 7,0; bn vng pH 7 - 7,5; sau 18 gi hot cn 50,6
- 57%.
- xc nh mt s c tnh ca lipase tan t Candida rugosa
Nhit ti u l 60C, bn vng nhit 40 - 50C, sau 18 gi
hot cn li l 50,2 - 59%.
pH ti u l 8,0; bn vng pH 7,5 - 8,0, sau 18 gi hot cn
53,9 - 62%.
im ng in ca lipase l pI = 7,5
nh hng ca cc ion kim loi n hot tnh ca lipase:
- Ion Pb
2+
c ch mnh (lm gim hot ti 33,26%)
- Cc ion Cu
2+
, Fe
3+
, Zn
2+
c ch yu (lm gim hot tnh 13%)
- Cc ion Na
+
, K
+
khng nh hng.
- Cc ion Mg
2+
, Ca
2+
c kh nng hot ho lipase.
- Mt s c tnh lipase t Bacillus sp
Tnh c hiu c cht i vi tributyrin l cao nht: 101 U/ml.
Nhit ti u l 70C (91,1 U/ml).
Lipase c bn nhit, sau 24 gi hot tnh vn cn 63%.
pH 9,5 l thch hp nht cho phn ng, hot tnh t 108,1 U/ml.
pH 9,0 m Tris-HCl v pH 10,0 m glycine khng nh hng ti
bn ca lipase, hot tnh cn li vn t 117%.
Ca
2+
tng hot tnh lipase ln 10-12%, ngc li K
1+
, Mg
2+
, Fe
3+
gim
hot tnh lipase t 11-42%.
Cc dung mi hu c u gim hot tnh lipase. Acetone, 2-propanol
v ethanol gim mt na, methanol gim mt phn t.
Cc cht ty nh hng ln hot tnh lipase. Triton X-100 tng hot
tnh ln 63%. Tween 20 gi nguyn hot tnh (107%). Tween 80 gim
mt na. Cn SDS km hm hot tnh lipase 3%.
- kho st kh nng thu phn du u tng ca lipase c nh thu
c du c ch s axit t 80,5.
- S dng lipase trong sn xut phomat kt qu cho thy phomat c b
sung lipase lm tng cc ch tiu cht lng cm quan (mu sc,
hng thm,...)so vi sn phm phomat khng b sung lipase


MC LC
M u..........................................................................................
TNG QUAN TI LIU ...................
1.1. Cc ngun thu lipase ....................................................
1.2. cc yu t nh hng n kh nng sinh tng hp
lipase............................................................................................................
1.2.1. nh hng ca thnh phn mi trng dinh dng.........
1.2.2. nh hng ca iu kin nui.............................................
1.3. Cu to ca lipase: .............
1.3.1. Khi lng phn t..
1.3.2. Trnh t axit amin, cu trc khng gian..
1.4.Mt vi c tnh ca lipase .
1.4.1.Cht hot ho v cht km hm
1.4.2. Tnh c hiu c cht
1.4.3. C ch ng hc xc tc ca lipase
1.4.4. Nhit v pH hot ng ca lipase
1.5. Tch tinh ch lipase
1.6. Cc phng php c nh lipase .
1.6.1. Phng php gn enzym bng lin kt ng ho tr
1.6.2. Phng php gi enzym trong khun gel
1.6.3. Phng php hp ph vt l trn cc cht mang c
cha hoc khng cha in tch.
1.7. ng dng ca lipase
1.7.1. Trong cng nghip sn xut cc cht ty ra.......
1.7.2. Trong cng nghip thc phm.
1.7.3. Trong cng nghip dc v ho cht.......................
1.7.4. Trong cng nghip thuc da
1.7.5 Trong cng nghip m phm.

NGUYN LIU V PHNG PHP .................
2.1. Nguyn liu...
1
2
2
3
3
5
5
5
6
8
8
9
11
12
12
14
14
15

16
16
17
17
18
19
19

20
20
2.1.1. Chng vi sinh vt..
2.1.2. Cht mang Nylon-6..
2.1.3. Ho cht
2.1.4. Thit b s dng.
2.1.5. Mi trng nghin cu.
2.2. Phng php nghin cu
2.2.1. Phng php vi sinh vt ...
2.2.2. Phng php ho sinh..

KT QU V THO LUN.................
3.1. Tm iu kin nui cy thch hp thu lipase
3.1.1. Tm iu kin nui cy thch hp thu lipase t Candida
rugosa............................................................................................
3.1.1.1. Kho st nh hng ca thi gian nui ti sinh tng hp
lipaza.................................................................................................
3.1.1.2. Kho st nh hng ca tc lc n qu trnh sinh
tng hp lipaza................................................................................
3.1.1.3. Kho st nh hng ca ngun cacbon n sinh tng
hp lipaza.....................................................................................
3.1.1.4. Kho st nh hng ca nng axit palmitic n
sinh tng hp lipaza.....................................................................
3.1.1.5. Ti u ho iu kin nui chm Candida rugosa
3.1.2.Tm iu kin nui thu lipase t Bacillus sp
3.2. Tch tinh ch lipase t Candida rugosa
3.2.1. Kho st nng etanol thch hp kt ta lipase
3.2.2. Tinh sch lipase bng phng php sc k trao i
ion trn h thng FPLC ......................................................
3.3. Kho st cc iu kin ti u c nh lipase t Candida
rugosa trn Nylon-6.
3.3.1. nh hng ca nng HCl x l ht n kh nng
c nh enzym lipase..
20
20
20
20
21
22
22
22

25
25

25

25

25

26
26

27
31
32
32

34

35
36
3.3.2. nh hng ca nng glutaraldehit n kh nng
c nh enzym..
3.3.3. nh hng ca nng enzym n kh nng c nh
enzym...
3.3.4. nh hng ca dung dch m n kh nng c nh
enzym lipase..........
3.4. Xc nh mt s c tnh ca Lipase ....................................
3.4.1. Mt s c tnh ca lipase t do v c nh t Candida
rugosa............................................................................................
3.3.1.1. nh hng ca nhit ti hot lipase t Candida
rugosa........................................................................................
3.4.1.2. nh hng ca nhit ti tnh bn lipase t do v c
nh...............................................................................................
3.4.1.3. nh hng ca pH ti hot lipase t do v c nh
3.4.1.4. nh hng ca pH ti tnh bn lipase t do v c nh
3.4.1.5. nh hng ca ion kim loi ti hot lipase t do
3.4.1.6. Xc nh im ng in ca lipase ...............................
3.4.2. Mt s c tnh ca lipase Bacillus-sp..............................
3.5. KHO ST KH NNG NG DNG LIPASE t
Candida rugosa...............................................................................
3.5.1. Kho st kh nng thu phn du vi lipase c nh
3.5.2. Kho st kh nng s dng lipase tan trong sn xut
phomat............................................................................................

KT LUN.......................................................................
TI LIU THAM KHO...............................................


36

37

38
40

40
40

41
43
45
47
49
50

55
55

56
59
62




M u
Lipase l enzym xc tc thu phn triglyxerit thnh di, mono
glyxerit hoc glyxerol v cc axit bo nh hot ng trn b mt phn pha
du nc. Lipase (EC 3.1.1.3) l enzym linh hot c th s dng xc tc
nhiu loi phn ng khc nhau, c kh nng chu kim, chu nhit. Chnh v
th, lipase c ng dng rng ri trong cng nghip nh cng nghip thc
phm, cng nghip ho hc, cng nghip m phm, cng nghip da, trong
y dc v cc ngnh cng nghip khc[2].
Lipase c phn b c ng vt, thc vt v vi sinh vt, song
trong cng nghip ngun thu lipase ln nht l t vi sinh vt. Trong c
nm mc, nm men, vi khun v x khun u c kh nng tng hp lipase.
Vo nhng nm cui th k XX, tng sn lng cc loi enzym ton
th gii vo khong 1300 tn/nm[3]. Th trng th gii v enzym cng
nghip trong nm 1994 c tnh khong 1 t la M, trong lipase
chim 7% (70 triu) [sheldon, 1996]. Trong cng nghip du v cht bo,
vic s dng lipase rt ph bin. C khong hn 100 loi lipase khc
nhau c dng chuyn i lipit thnh cc cht khc[13].
Hin nay, Vit Nam s dng rt ph bin lipase trong ngnh cng
nghip thc phm, cng nghip ty ra, ho hc, y hc v cn mt lng
ln lipase, song cc ch phm lipase ang s dng phn ln l nhp khu.
Do vy, ti tp trung nghin cu thu nhn v s dng hiu qu lipase t
hai i tng l nm men Candida rugosa v Bacillus sp





Tng quan
1.1. CC NGUN THU LIPASE (EC.3.1.1.3)
Lipase c phn b rng trong cc loi vi sinh vt, ng vt v c
thc vt.
ngi v ng vt c xng sng, nhiu lipase kim sot s thu
phn, s hp th, s to thnh cht bo v chuyn ho lipoprotein. Lipase
ng vt chia lm 3 nhm da trn v tr v hot ng ca chng: lipase
thc phm, lipase m v lipase sa.
thc vt, lipase c tm thy m d tr ca ht du, ht ng
cc trong qu trnh ny mm ca ht. Hu ht cc lipase khng hot ng
trong thi k ng ng, ngoi tr lipase tm thy ht u caston[16].
Lng ln lipase trong cng nghip c sn xut t cc loi vi sinh
vt: vi khun, nm mc, nm men[10].
Vi khun: Lipase ngoi bo ca Streptomyces rimous R6554 W c
tch t dch lc canh trng bng sc k ct. Streptomyces l vi khun t
Gram (+) th hin kh nng c bit i vi s tng hp cc cht trao i
th cp v s dng lipase thu phn ngoi bo ph hu nguyn liu hu
c trong mi trng sng t nhin. Tuy nhin cho n nay, ch c mt vi
lipase Streptomyces c xc nh hot tnh[10]. Ngoi ra, ngi ta
cng tm thy lipase c trong mt s loi vi khun khc nh
Pseudomonas cepacia c hot tnh ngay nhit cao, trong mi trng
kim v k nc[5].
Nm mc: Lipase c tm thy mt s loi nh: Aspergillus,
Rhizopus (tch t qu da), Rhizopus oryzae (phn lp t du da),
Pythiumnltimum (trong dung dch hu c, khng c nh tng), v Mucor
sp humicola lamuginosa[6].
Nm men: Nm men Candida rugosa l mt ngun sn xut lipase
quan trng. Cc c tnh xc tc cu trc ho sinh ca lipase trong loi nm
men Candida rugosa c cng b trn nhiu ti liu[7]. C t nht 7
ging lipase t Candida rugosa trong c 4 ging c xc nh c
tnh v 3 ging c dng sn xut enzym thng phm.
1.2. cc yu t nh hng n kh nng sinh tng hp lipase
C nhiu yu t nh hng n sinh tng hp enzym. Nhng nhng c
im v tnh cht, sinh l, sinh ho ca vi sinh vt c ngha quyt nh
hn c. V vy vic tuyn chn chng c hot lc tng i trong t
nhin sau tng cng kh nng sinh tng hp cao ca chng bng
phng php gy t bin lm thay i ngun gen trung tm nh cc yu t
ho hc, vt l,c mt ngha v cng quan trng.
Khng phi tt c mi vi sinh vt u c kh nng sinh tng hp enzym
nh nhau, ngay c nhng chng cng chi, cng loi cng c th rt khc
nhau v lng enzym do chng sn sinh ra. V th vic tuyn chn cc vi
sinh vt c kh nng to nhiu enzym cn phi lun kt hp cht ch vi
vic kim tra hot lc ca hng chc, hng trm chng vi sinh vt tm ra
nhng chng c kh nng sinh tng hp enzym cao nht.
Ngoi vic tun chn chng vi sinh vt c kh nng sinh tng hp cc h
enzym c hot cao cn phi tin hnh nui cy chng trong cc iu
kin ti u nhm m bo kh nng sinh tng hp v duy tr hot lc
enzym ca chng.
1.2.1. nh hng ca thnh phn mi trng dinh dng
Thnh phn mi trng l nhn t c tc ng quan trng n hot ng
cng nh qu trnh tng hp enzym ca vi sinh vtj, mun sinh trng, pht
trin c cn phi c cc hot cht cha C, N, H v O y . Ngoi ra vi
sinh vt cn c cung cp cc cht khong Mg, Ca, S, P, Fe, K v mt s
cht cm ng c trng ca tng enzym.
nh hng ca ngun cacbon
Cc ngun cacbon khc nhau s nh hng khc nhau ln s pht trin v
sn sinh enzym ca mi sinh vt. E. Dalmau v cng s (2000) nghin
cu s nh hng ca ngun cacbon n sinh tng hp lipase ca chng
Candida rugosa bng phng php nui bn lin tc cho thy cc
hydratcacbon v cc axit khng lin quan n cht bo th khng lm tng
s sn xut lipase. Hiu sut thu c enzym cao nht vi cc ngun
cacbon l lipit hoc axit bo. S kt hp 2 loi c cht hydratcacbon v axit
bo khng ci thin s sinh tng hp lipase. Glucose km hm s sinh tng
hp lipase, ngc li, tween 80 kch thch s tng hp lipase.
nh hng ca ngun nit
Nit l yu t nh hng ln n s sinh tng hp ca nhiu enzym, v nit
l ngun cung cp vt liu (nguyn t nit) cho qu trnh sinh tng hp
protein enzym trong qu trnh pht trin.Silbel F. v cng s (2002) nghin
cu nh hng ca ngun cacbon v nit n s sinh tng hp lipase ca
chng Candida rugosa cho thy trong nhng ngun cacbon v nit nghin
cu th hot lipase ca canh trng nui t c cao nht (5,58 U/ml)
khi mi trng nui cha ngun nit l cao nm men v proteose-pepton
vi ngun cacbon l du oliu. V hot t thp nht khi mi trng nui
cy cha ngun nit l trypton v ngun cacbon l lactose. Tuy nhin nh
hng ca ngun nit n s tng hp lipase ca cc chng khc nhau l
khc nhau, do vy trong nghin cu ca Melisa E. L. Gutara v cng s cho
thy khi ln men b mt chng Penicillium simplicissimum th nh hng
ca ngun nit n sinh tng hp lipase l khng ng k.
nh hng ca ngun khong
Cc nguyn t a lng v vi lng c nh hng ln ti s sinh trng v
sinh tng hp lipase ca vi sinh vt. Theo cng b ca Rohit Sharma v
cng s (2001) cho thy Mg c nh hng n bn nhit ca enzym,
nu thiu Mg trong mi trng nui th s sinh tng hp lipase b gim
xung. Phospho nh hng trc tip ti sinh sn ca nm mc v cc vi
sinh vt. V vy mun tng cng s tng hp lipase cn cung cp y
lng phospho y lng cn thit ca nguyn t ny. Lu hunh kch
thch s to thnh lipase nm men, ngun lu hunh dng l (NH
4
)
2
SO
4
.
Khi chn mi trng dinh dng nui vi sinh vt cn ch n thnh
phn, cht lng v s tng quan v khi lng gia cc cu t. Cc
nghin cu trc y cho thy c kh nng tng cng sinh tng hp
enzym ln hng chc ln v hn na bng cch thay i thnh phn mi
trng mt cch chun xc, T l ti u gia cc cu t chnh, tuyn chn
v cho thm cc cht cm ng c hiu lc, b sung lng cn thit cc cht
khong cng nh cc yu t kch thch sinh trng.
1.2.2. nh hng ca iu kin nui
iu kin nui c nh hng ln n qu trnh sinh trng v pht trin ca
vi sinh vt, iu kin nui y bao gm cc yu t m, pH, thng
kh, tc lc, nhit mi trng
pH mi trng
pH ban u ca mi cng nh hng khng nh ti s pht trin v kh
nng to enzym. Jau-Yann Wu (2000) nghin cu s sinh tng hp lipase
cho thy hot lipase c tng hp cao nht pH 8,5.
thng kh
Trong qu trnh sinh trng vi sinh vt tiu th 25-35% cht dinh dng
ca mi trng v thi ra mt lng ln nhit v CO
2
. V vy phi h nhit
bng thng gi vi khng kh v trng c m tng i gn 100%, ch
thng kh v trng c th lin tc hoc gim on hoc thng kh t
nhin. Trong mi trng nui cy chm phi m bo sao cho oxy ho
tan khng thp hn 20% bo ho (F. Valero, 1998).
Mt t bo
S lng t bo vi sinh vt cng c nh hng ti sinh tng hp lipase ca
nm men Candida rugosa. Trong cng mt mi trng nui, nu s lng
t bo qu t s lm ko di pha n nh v pha logarit v nh hng n s
sinh tngr hp lipase, ngc li khi s lng t bo ban u qu nhiu pha
logit cha hon thnh th mi trng cn kit ngun dinh dng v vy
lipase cng c tng hp t hn. Do vy ta cn phi nghin cu tm mt
t bo ban u thch hp cho kh nng sinh tng hp lipase cao.
1.3. CU TO CA LIPASE
1.3.1. Khi lng phn t
Cng nh cc enzym khc, lipase c th thu t m, c quan ng,
thc vt v vi sinh vt. Cc lipase u c nhng tnh cht chung, song
chng cng khc nhau v mt s tnh cht nh: tnh c hiu c cht, mc
phn gii c cht, khi lng phn t, thnh phn axit amin v iu kin
hot ng. S khc nhau ny khng nhng gia cc loi m ngay c gia
cc chng trong cng mt loi[7].
Khi lng phn t ca lipase dao ng kh nhiu gia cc chng vi
sinh vt: Candida rugosa c 2 ng phn l LipA (58kDa) v LipB
(62kDa); Geotrichum candidum khong 60.000 Da[34], Aspergillus niger
97.000 Da, Penicillium crustosum 29.300 Da, C.Paralipolytica 55.900 Da.
Awai v cng s cng tch c t P.cyclopum hai lipase c khi lng
phn t 27 kDa v 30 kDa. Lipase c sn xut bi P.camemberti l mt
gluco-protein c khi lng phn t l 38 kDa.
1.3.2. Trnh t axit amin
Thnh phn axit amin ca lipase t cc ngun khc nhau cng khc
nhau, nhng nhiu trnh t axit amin c suy ra t trnh t ca lipase tch
dng, t cho thy c nhng im tng ng gia nhng cu trc
khng gian ca lipase. Chng hn nh trnh t ca 20 axit amin u ca
lipase t ng vt c v ging vi vng N- ca lipase t P.camemberti v
ging mt phn vi lipase t Humicola lanuginosa v Aspergillus oryzae
nhng khc nhau so vi lipase t P.Cyclopium lipase 1. So snh tip u N
tip theo ca lipase 1 t P. expansium v P.solium cho thy ngoi 16 ca
20 axit amin u l khc nhau cn li l nh nhau. Nhng axit amin cui
c 15 trong s 20 axit amin lng cn l do lipase P.expansium v
P.solium[11].
i vi lipase t Candida rugosa th s sp xp trnh t N ca
nhng protein th hin s tng ng vi lipase 2 v 3 nhng khng tng
ng vi lipase 1[13]. S quyt nh trnh t N- trn 19 axit amin ch l s
tng ng cao vi nhng lipase tng t .
Ni chung c rt t nhng trnh t ging nhau hon ton, ngoi tr
nhng trnh t quanh vng trung tm hot ng. Khi so snh trnh t axit
amin vng quanh trung tm hot ng ca nhm gm 3 enzym l protease,
lipase, cutinase vi enzym esterrase, ngi ta thy s tn ti ca trnh t
bo th GxGxG v tr serin. Ngoi l duy nht bit vi trnh t bo th
trn l s thay th ca alanin cho glyxin th 2 trong subtilisin. Khng mt
trnh t bo th no c tm thy quanh vng gc histidin hoc a.aspastic
(glutamic) ca trung tm hot ng tr trng hp ca cc enzym tng
ng t cc loi h hng rt gn.

MELALALSLI ASVAAAPTAT LANGDTITGL NAIINEAFLG 40
IPFAEPPVGN LRFKDPVPYS GSLDGQKFTS YGPSCMQQNP 80
EGTYEENLPK AALDLVMQSK VFEAVSPSSE DCLTINVVRP 120
PGTKAGANLP VMLWIFGGGF EVGGTSTFPP AQMITKSIAM 160
GKPIIHVSVN YRVSSWGFLA GDEIKAEGSA NAGLKDQRLG 200
MQWVADNIAA FGGDPTKVTI FGESAGSMSV MCHILWNDGD 240
NTYKGKPLFR AGIMQSGAMV PSDAVDGIYG NEIFDLLASN 280
AGCGSASDKL ACLRGVSSDT LEDATNNTPG FLAYSSLRLS 320
YLPRPDGVNI TDDMYALVRE GKYANIPVII GDQNDEGTFF 360
GTSSLNVTTD AQAREYFKQS FVHASDAEID TLMTAYPGDI 400
TQGSPFDTGI LNALTPQFKR ISAVLGDLGF TLARRYFLNH 440
YTGGTKYSFL SKQLSGLPVL GTFHSNDIVF QDYLLGSGSL 480
IYNNAFIAFA TDLDPNTAGL LVKWPEYTSS SQSGNNLMMI 520
NALGLYTGKD NFRTAGYDAL FSNPPSFFV 549
Hnh 1. Trnh t axit amin ca lipase t Candida rugosa
1.3.3. Cu trc khng gian
c rt nhiu th nghim c trin khai nhm tm hiu cu trc
ca lipase, trong cu trc ba chiu ca lipase c coi l c s hiu
c phn ng ng hc, c hiu c cht, tin on nhng c im sinh
ho v hot ng ca lipase ti b mt phn pha du nc. Lipase t
Rhizomun miehei l enzym thu phn u tin c cu trc bc 3 c xc
nh bi tinh th hc tia X[16].
Dng phng php tia X pht hin cu trc /. Lin kt song
song lm cu ni vi cu trc xon c hnh thnh cu trc phc tp.
Cc cu disunfua c tm thy trong phn t lipase c l nhm lm n
nh cu trc gp np ca enzym. Trong cu trc ny protein hnh cu cha
mt nhn k nc to nn mt chui khng cc cnh c ngha quan
trng cho s n nh phn t. Bn cnh cn c vi nhm c cc v
nhng phn t c lin kt. S c mt ca nhng phn t nc lm
tng lc lin kt ca Vander Wall v lc phn tn trong nhn, v vy s n
nh v cu trc khng gian ca phn t enzym c tng ln[16].


Hnh 2. Cu trc khng gian ca lipase t Candida rugosa [21]
Nhm quan trng nht to thnh trung tm hot ng ca lipase l
b ba AspHisSer. Lipase tham gia vo v tr xc tc vi proteinase v
esterase. i lp vi proteinase, trung tm xc tc ca n khng phn b
b mt ngoi m nm di chui xon b mt. V tr hot ng ca mi
lipase u c Ser, His v Asp (hoc Glu) v hon ton b che khut bi mt
on np cu to bi mt hoc hai chui xon. B mt mang Trypsin hon
ton khng cc v tc ng qua li vi u k nc bao quanh trung tm
hot ng. Trong hu ht cu trc lipase, u serin hot ng trong chui
pentapeptit c trnh t Gly-X
1
-Ser-X
2
-Gly cu trc cng c tm thy
protease serin (Boel v cng s, 1998).
1.4. MT VI C TNH CA LIPASE
1.4.1. Cht hot ho v cht km hm
Phn ng do enzym xc tc ph thuc vo nhiu yu t, trong
cht hot ho v cht km hm cng nh hng ti vn tc phn ng v
hot enzym trong phn ng. Hot enzym c th b thay i di tc
dng ca mt s cht c bn cht ho hc khc nhau.
* Cht hot ho enzym
Cht ny lm tng hot xc tc ca enzym. Cc cht ny c bn
cht ho hc khc nhau, c th l cc anion, cc ion kim loi hoc cc cht
hu c c cu to phc tp hn.
Cc cht hot ng cation nh cc mui amon c a vo lm
tng kh nng thu phn du oliu ca lipase t Mucor [Shamkant Patkar v
Fredrick Bjorkling, 1992].
Didier Rotticci v cng s chng minh rng lipase t Candida
antaractica c th c hot ho bi cc cht hot ng b mt. Trong mi
trng c cht l axetat p-nitrophenyl c cha cht hot ng ion SDS
(Sodium dodecyl sulfate) th tc phn ng tng ln. Chng t SDS c
tc dng trong vic tng cng tc phn ng ca lipase[9].
Tuy nhin tc dng kch hot ch gii hn nhng nng xc nh,
vt qu gii hn ny c th lm gim hot ca enzym.
* Cht km hm enzym
Cc cht lm gim hot ca enzym gi l cht km hm (c ch-
Inhibitor), k hiu l I. Cc cht ny c th l nhng ion, cc phn t v c,
hu c, k c cc protein. Cc hp cht km hm hot tnh lipase c th
bng cch thay i cu trc lipase hoc tc ng vo tnh cht b mt.
C ch hot ng km hm lipase bi protein v polypeptit vn cha
c nghin cu r rng. gii thch hot ng ca s km hm ny c
hai gi thuyt c a ra: (1) cht km hm protein c hp ph ln b
mt v gy ra s thay i cc tnh cht ca b mt ; (2) S tc ng qua
li trc tip ca protein vi lipase v cnh tranh vi c cht. Ngi ta lm
nghin cu v km hm lipase t tu tng nga v lipase t Rhizopus
arrhizus , R.delemar vi mt lot cc protein ph bin gm : myoglobin,
serum albumin, ovalbumin, meltin. Tt c cc protein ny km hm lipase
t tu tng nga khi khng c mt colipase v mui mt. Mt iu th v l
lipase t R.delmar b km hm bi cc protein ny trong khi lipase t R.
Arrhizus khng h b nh hng bi cc protein [24].
1.4.2. Tnh c hiu c cht
* c im ca ngun c cht
Lipase (EC.3.1.1.3) l enzym xc tc trong qu trnh thu phn cc
triglyceride khng trn nc mt phn cch nc v lipit. Cc este ca
axit cacboxinic mch ngn d tan trong nc v c thu phn t t vi
th tch nc ln v c th thu phn hon ton. Cc cht to thnh ca qu
trnh thu phn l 1,2- hoc 2,3- diglyxerit; 2-monoglyxerit; 1- hoc 3-
monoglyxerit v glyxerol. Da vo cc iu kin ca phn ng thu phn
t c n cn bng v cc thnh phn trung gian chui thu phn
c tch ra nh s kt tinh, chng ct hoc sc k[16].
Lipase l cht xc tc thu phn chui triglyxerit di hoc cc este
metyl alcohol ca mt chui di cc axit bo
lipase
Triglyxerit + H
2
O ------> iglyxerit + axit bo

* Thu phn cc lin kt este gia glyxerol v cc axit bo
Lipase l mt cht xc tc sinh hc c s dng vo nhiu mc
ch khc nhau. Trn th gii p dng thnh cng lipase t cc ngun
khc nhau xc tc cho s thu phn, s tng hp cht bo v s trao i
nhm ca nhng cht este[13]. Tnh cht c th kh ph bin vi tt c
cc lipase l phn ng ca chng trong mi trng khng ng nht, trong
lipase hu nh ch th hin chc nng ring mt phn pha du
nc[16]. Lipase c s dng rt nhiu trong tng hp ho hu c vi vai
tr l mt nhm enzym c kh nng phn gii hydro t triacylglyxerol, trn
b mt phn pha du nc[7].
Gn y ngi ta pht hin c lipase t cy ci du c tnh cht
xc tc s thu phn triolein nhanh hn khong 70 ln so vi s thu phn
ca tri-linolein di nhng iu kin khng c nc, lipase ny xc tc s
este ho axit oleic bng butanol[14].
Lipase xc tc phn ng thu phn du khng tan thnh cc sn
phm ho tan; phn ng ny cng c th xy ra theo chiu ngc li trong
lipase xc tc to thnh acyl glyxerol t glyxerol v axit bo.
1.4.3. C ch ng hc xc tc ca lipase
Theo cc nghin cu lipase c nhm serin, histidin v axit aspastic
trung tm hot ng ca n. Vi cc c cht khng tan trong nc, hot
tnh ca lipase t c cc i ch khi n c phn tn vo gia b mt
phn pha du nc. Qu trnh c gi l qu trnh hot ho phn
pha[24] (hnh 3)

S
E
is
* E
s
* E
s
*S
-------------------------------------------------------------------Mt phn pha

S
w

E E* E*S
w
Nc


P
w

Hnh 3. M hnh hot ng ca lipase trn c cht ho tan v khng tan
trong nc. S thay i hnh th ca lipase trong qu trnh hot ng xc
tc. M hnh ny do Verger xut (1980).

Trong :
E: Lipase ho tan khng hot ng
E*: Lipase ho tan dng hot ng
E
s
*: Lipase hot ng c hp ph
E
is
*: Lipase khng hot ng c hp ph
S
w
: C cht tan trong nc
S: C cht khng tan trong nc
E*S
w
v E
s
*S: Phc hp lipase- c cht
Khi khng c mt nc hoc khi c nc vi lng nh, phn ng
este ho v phn ng este c u tin. Trong trng hp ny, nhng c
tnh nh tc xc tc v tnh c hiu ph thuc nhiu vo iu kin
phn ng v bn cht c cht [Paul Woolley & Steffen B. Petersen, 1992].
1.4.4. Nhit v pH hot ng ca lipase
Nhit v pH l hai yu t nh hng rt nhiu n hot ng cng
nh tnh bn ca lipase. Lipase thu t cc ngun khc nhau chu nh hng
bi nhit v pH khc nhau.
Theo Marija Ablamic v Iwanalescie, lipase tng hp t chng
Streptomyces riromus R6554W hot ng mnh nht di nhit 50-
60C, pH ti u l 9,5. iu kin pH v nhit khng thch hp, lipase
u b km hm hot ng v gim tnh bn. enzym pH 9,5; nhit
23C sau 24 gi hot gim 5%, pH 10-11 hot gim 55%. nhit
30C hot gim cn 25%, 90C hot cn 30% so vi hot
lipase ti 55C[17].
Lipase t chng S.riromus bn cng nhit 20-50C v mt hot
tnh nhanh vng nhit cao hn 70C[17].
Lipase t chng Penicillium cyclopium c pH ti u trong khong
4,5-7,0[10]. Lipase t nm Pythium ultium c pH hot ng iu kin ti
u l 7,5-8,5. Cn i vi lipase t chng Rhizopus oryae c pH ti u l
7,5[6].
a s lipase hot ng nhit 50-60C, tuy nhin c mt vi
chng hot ng nhit thp hn. Chng Penicillium cyclopium,
Pythium ultium v Rhizopus oryae chu nhit km, s b c ch khi nhit
ln ti 50C[6].
Lipase t Candida rugosa n nh trong mi trng kim pH t 7-
10 v c hot mnh nht pH 8,5 [Sharon v cng s, 1998].
1.5. TCH TINH CH LIPASE
Tu vo mc ch s dng m ngi ta tinh sch enzym cc mc
khc nhau. c c enzym thun khit hn, ngi ta tin hnh tinh
ch enzym t ch phm enzym th. C nhiu phng php tinh ch:
Phng php kt ta da trn tnh ho tan, cc phng php sc k trao i
ion da trn tch in ca enzym; phng php lc gel da vo khi
lng phn t, kch thc enzym; cc phng php sc k i lc da vo
v tr lin kt c hiu ca enzym. Hin nay, vic tch tinh ch enzym c
tin hnh da trn s kt hp ca nhiu phng php. Cc phng php
sc k thng c s dng hn c v n cho php thu nhiu ch phm
enzym c thun khit cao. Vn tch enzym thun khit vn cn gp
phi mt s kh khn do lng enzym c trong t bo t nhiu c ln tp
cht v lun c ng thi vi cc protein khc cng c cc tnh cht l ho
ging nhau, enzym li khng bn v d b mt kh nng xc tc do tc
ng ca cc yu t bn ngoi[4].
i vi cc enzym ni bo do cc phn t enzym khng c kh nng
i qua mng t bo do cn phi ph v cu trc ca t bo bng cc bin
php c hc (nghin vi bt thu tinh hoc ng ho bng thit b ng
ho) hoc ph v t bo bng tc dng ca cc dung mi hu c trong sng
siu m Sau khi ph v t bo enzym c chit ri loi tp cht ri mi
c tch tinh ch bng cc phng php khc nhau.
Lipase c nhiu nh khoa hc trn th gii nghin cu nhm
lm tinh sch hn v thng c thc hin qua nhiu ct. M.
Kambourova v cng s tin hnh tinh sch lipase t Bacillus
stearothermophilus MC7 qua lc gel t tinh sch l 1,56 v hiu sut
thu hi l 75%; qua ct Sephadex G-20 t tinh sch l 16,47, hiu sut
thu hi l 60%; qua ct DEAE- xenlulo th tinh sch l 19,25 v hiu
sut thu hi l 10,2%. Qua kim tra tinh sch bng chy in di protein
bin tnh trn SDS-PAGE xc nh c khi lng phn t l 66kDa
(2003)[20].
Lipase t Pseudomonas mendocina PK-12CS c tinh sch qua ct
sc k trao i anion DE-52 t c tinh sch l 240,99 v hiu sut
thu hi l 14,78%. Qua kim tra tinh sch bng chy in di protein
bin tnh trn SDS-PAGE xc nh c khi lng phn t l 80kDa
(2003)[33].
R. K. Saxena v cng s tch v tinh sch lipase t Aspergillus
carneus qua ct tng tc k nc Octyl Sepharose t c mc tinh
sch l 24,1 v hiu sut thu hi l 38,4% (2003)[25].
i vi lipase t Candida rugosa, M. A. Pernas v cng s tin
hnh tinh sch qua ct DEAE- Sephacel t tinh sch l 2,3 v hiu sut
thu hi l 4,9%. Qua kim tra tinh sch bng chy in di protein bin
tnh trn SDS-PAGE xc nh c khi lng phn t l 60 kDa
(2000)[15].
1.6. CC PHNG PHP C NH LIPASE
Enzym c nh l nhng enzym c gn ln trn nhng cht mang
khng ho tan hoc cc enzym c lin kt li vi nhau to i phn t
enzym khng tan. Vic s dng enzym c nh c ngha to ln nh c
th s dng nhiu ln mi lng enzym em li hiu qu kinh t cao. Dng
enzym c nh ln vo trong sn phm sau ngi ta c th tch ra khi
sn phm d dng nn khng lm nh hng n mu sc, mi v v cht
lng thc phm. S dng enzym c nh c th lm ngng phn ng
nhanh chng bng cch ly ch phm ra khi phn ng khi cn thit. Chnh
v vy m enzym c nh c s dng rng ri v ngi ta cng tm ra
c rt nhiu phng php c nh enzym.
1.5.1. Phng php gn enzym bng lin kt ng ho tr
C nh enzym bng lin kt ng ho tr vi cc polyme c
hot ho l mt trong nhng phng php c nh enzym ph bin nht,
m bo lin kt vng chc ca enzym vi cht mang. Bn cht ca
phng php ny l enzym c gn vi cht mang thng qua cu ni.
Cu ni ny c kch thc khng ln v c hai u, mt u ni vi cht
mang polyme, u cn li ni vi enzym. V d glutaraldehit cng hay
c s dng lm cu ni gn enzym v n cha hai nhm aldehit hai
u ca phn t. Hai nhm ny gi tr pH trung tnh s phn ng vi cc
nhm NH
2
t do. Nh vy, mt u ca glutaraldehit c gn vo cht
mang cn u kia s c gn vo enzym[4].
Cht mang thng c s dng l cc polyme khc nhau nh:
celluloza, agaroza, alginic axit, chitin, collagen, keratin v cc dn xut ca
chng; hoc s dng cc polyme tng hp nh polyme ca acrylic axit,
polyurethan, cc dn xut N-vinylpyrolidon v cc loi polyme khc[4].
Da trn c s l thuyt , Carneiro-da-Cunha v cng s c
nh lipase t Candida rugosa ln mng xenluloza v dn xut ca
celluloza, s dng tc nhn hot ho l cacboiimit (HN=C=NH) thu c
kt qu c nh trn mng celluloza axetat hot ng l 0(mols
-1
m
-2
);
trn Textile cotton tissue l 1002(mols
-1
m
-2
) (1999)[18].
S dng phng php ny, theo P. Padmini c th c nh lipase trn
ht polyamid (Nylon-6). Nylon-6 c l hng nh c s dng lm cht
mang cho vic c nh lipase. Trong qu trnh c nh s dng
glutaraldehit 10% , nng HCl 3,5N x l ht v nng enzym l
1mg/ml trong m photphat 7,7[23].
1.6.2. Phng php gi enzym trong khun gel
Nguyn tc ca phng php ny l gi enzym vo trong khun gel
bng cch trng hp ho gel khi c mt ng thi gel v enzym. Sau khi
kt thc qu trnh trng hp ca gel th enzym c gi trong cc khun
gel .
Gel thng s dng l gel polyacrylamit hoc agaroza, Na-alginat.
C nhiu phng php gi enzym vo khun gel: gi di dng ht
(vin); gi enzym trong cc l nh ca gel dng si; gi enzym trong bao
vi th (Microcapsul); phng php tin polyme.
c nh lipase t Candida rugosa theo phng php ny, ngi
ta gn enzym vo trong khun gel di dng ht. Monome c dng
y l Na alginat. Alginat t lu c dng to mng gel c nh t bo
v enzym xc tc cc phn ng trong cc dung mi ho tan. Mng gel Ca
alginat c tc dng lm cho enzym n nh hn, bn nhit hn khi n bc
trong cc khun gel. Lipase sau khi c c nh trong cc ht alginat c
th c xc tc phn ng lin tc hoc c th ti s dng nhiu ln cho
hiu qu kinh t cao[28]. OConnell v J. Varley da trn phng php
ny c nh lipase t Candida rugosa ln mng kh CGAs (Colloidal
Gas Aphrons) thu c hiu sut c nh l 80% (2001)[22].
1.6.3. Phng php hp ph vt l trn cc cht mang c cha hoc
khng cha in tch
Phng php c nh enzym c s dng ph bin nht l phng
php hp ph vt l khng thng qua lin kt ho tr. Qu trnh thc hin
hp ph kh n gin: cht hp ph v enzym c trn ln vi nhau trong
mt khong thi gian cho php s hp ph xy ra nh tng tc ca cc
lin kt ion, k nc, hydrogen v lc Van der Wals. Tuy nhin phng
php ny c nhc im l qu trnh gii hp ph enzym d dng xy ra
bi s thay i pH, nhit v thnh phn ion[4].
Nu cht mang khng c l xp, enzym c hp ph trn b mt
cht mang. Nu cht mang c l xp, enzym s c hp ph nh lin kt
ion. Cc cht mang v c thng c s dng l nylon, cc loi chitin ca
v tm, mai cua, bt san h, than hot tnh, than ci, gch, , si, ct
Cht mang hu c thng s dng l dn xut celluloza.
Lipase c th c c nh trn ht Amberlite-XAD16 bng phng
php hp ph vt l ln b mt ht[35].
Theo phng php ny, lipase t Candida rugosa cng c th c
c nh trn dung mi hu c metanol cho hiu sut c nh l 12,6%;
etanol hiu sut t 15,2% v axeton hiu sut l 16,3% (Sol Montero v
cng s)[29].
Cng da trn phng php hp ph vt l ln cht mang, Tien-
Chieh Hung v cng s tin hnh c nh lipase t Candida rugosa trn
Chitosan s dng glutaraldehit lm tc nhn hot ho (2003)[31].
1.7. NG DNG CA LIPASE
Lipase c ng dng rng ri trong cng nghip sn xut bt git,
tng hp cc cht hu c, trong cng nghip ch bin sa v pho mt, cng
nghip sn xut dc phm, ho cng, m phm v cc cng nghip khc.
Ngoi ra lipase c s dng rng ri trong cc hot ng bin i sinh
hc trong qu trnh thu phn cng nh qu trnh tng hp v kim sot
c cc bc trong cng nghip giy, bt giy.
1.7.1. Trong cng nghip sn xut cc cht ty ra
Hin nay th trng ln nht cho cc loi lipase l vic s dng n
nh nhng cht cu thnh trong cc loi bt git cng ngh git kh.
Ngy nay lipase c dng trong bt git thu phn cc vt du m m
con ngi tit ra qun o, chn m trong iu kin nhit thp v pH
cao vi nhng cht ho hc c hot tnh b mt [Gormen & Malmos,
1991]. Mt s lipase dng thng phm c s dng trong ngnh
thuc ty l: Lipase humicola ca Novo Nordisk, lipolase hoc lipase
P.glumace ca Unilever [Estell v cng s, 1985] c s dng trong
ngnh cng nghip ty ra. Nhng nhng lipase dng lm bt git cho
nhng h gia nh gi n nh vi cc protease v hot ng c hiu qu
trong iu kin kim.
1.7.2. Trong cng nghip thc phm
Trong cng nghip thc phm ngi ta s dng lipase ci bin
cc triacyglycerol, nng cao cht lng cc loi du thc vt r tin, cht
lng thp bng cch thay th cc axit bo no bi cc axit bo khng no,
trong dung mi him nc.
V d: Lipozyme (Novo) c dng sn xut b Cocoa tng
ng t du c v cc axit stearic s dng trong cng nghip ch bin
hp [Bjorkling, 1991].
i vi thc phm c ch bin t sa, lipase c s dng rng
ri lm tng mi v, lm tng chn ca phomat, hoc ch bin nhng
sn phm nh phomat v phn gii lipit [Bech, 1992]. Nhng axit bo t
do mch ngn (ch yu l C4, C6) c thu phn ra do tng lipase vo
sa bo dn n s tng hng v c trng cho sa. Trong khi nu cc
axit bo mch trung bnh (C12, C14) c to ra s c xu hng to ra mi
v ta nh x phng cho sn phm. Thm vo nhng axit bo t do ny
c th tham gia vo cc phn ng ho hc n gin vi vi sinh vt dn n
vic tng hp nhng gia v c mi thm khc nhau. Nhng ngun lipase
dng cho vic tng hng v phomat trn m ca nhng loi ng vt
nhai li (d non, cu, b). Mt s ch phm lipase c s dng lm tng
mi v v lm chn phomat c thu t mt s chng nh: M.miehei,
A.niger hay A. oryzae.
Lipase c s dng lm tng nhng cht bo nh vic chuyn
este ho ti mt triglycerit c gi tr cao, b cocoa, c mt hn hp 1,3-2-
oleoyl-glycerol cha no (palmitic, streraic v axit oleic) c th c ch
xut t loi du r tin chit xut tng hp thn cy c (1,3-dipalmitoy 1-
2-oleoyl-glycerol) vi axit tritearrin hay axit stearic ln lt l 1, 3 s
lipase c trng [Mastuao v cng s, 1981], trong khi phng php
chuyn i este ho ho hc di nhng iu kin kim c th dn n s
to thnh sn phm mang tnh ngu nhin v to thnh sn phm ph
khng mong mun. Bng vic s dng phng php chun b ging khc
nhau (axit palmitic hu nh khng c trong hai v tr vi 1, 3- dioleoyl-
2palmitoyl-glycerol) c th c thay th bng nhng cht thay th sa
bo c chit xut phn nhiu t cy c, du palmitoyl glycerides vi
nhng axit bo khng no [King & Padly, 1990]. Triglycerides cng vi axit
octanoic v axit decanoic v tr 1,3- v mt axit bo cn thit v tr 2- c
th c sn xut theo cch ny dnh cho nhng bnh nhn khng dch
tu hoc km hp th.
1.7.3. Trong cng nghip dc v ho cht
Trong nhng nm gn y enzym ngy cng c s dng nhiu v
rng ri trong y hc, sn xut mt s thuc v dng trc tip cha
bnh. nhiu nc trn th gii, c bit Nht Bn hng nm ngi ta
sn xut mt lng ln ch phm enzym vi sinh vt c tinh khit cao
trong c mt ca lipase.
Trong dc phm lipase c ng dng rng ri trong qu trnh sn
xut thuc cha bnh cho ngi v cc loi ho cht bo v thc vt (thuc
tr su, tr c)[19]. Gn y hn, trong lnh vc ho hc tinh khit, ngi
ta dng lipase to cc cht trung gian tng hp qua qu trnh thu
phn lp th chn lc, ng dng trong sn xut thuc tr su Pyretinoidic.
Ngoi ra trong phng th nghim cc lipase cng c nh ho hc hu c
dng lm cng c tng hp mt s cht khc nh phn gii Raxemic
epoxieste nh lipase ca Rhizopus arhisus [J.A Laitte (Php)]. Vi mong
mun c th sn xut mt loi thuc lm gin ng mch vnh, ngi ta
da vo tnh cht chn lc thu phn ca lipase trong mi trng dung mi
hu c.
Cc lipase c th xc tc este ho, chuyn i este ho v este ho
tng tc trong cc dung mi hu c vi lng nc thp [Kazlaus kas v
Bornscheuer, 1998; Godberg v cng s, 1989], ng dng chnh ca cc
lipase trong ho hu c l s phn gii cc hn hp enantiomeric. C mt
vi nhm phn t lin quan n s truyn tnh trng du hiu lm gia tng
nhng cn bnh d ng, vim l nhng lipit hay lipit aqnalogues. S truyn
tn hiu gia v trong ni bo sinh vt lin quan gn gi vi nhng loi
lipit n l nh phosphatidylinositol, lipase cng nh photpholipase u c
th c s dng tng hp trn phm vi ln.
1.7.4. Trong cng nghip thuc da
Qu trnh thuc da yu cu phi b lp cht bo di da. S dng
phi hp lipase vi cc enzym thu phn khc nh protease m ra con
ng mi cho cng nghip thuc da. Mt qu trnh enzym mi cho sn
xut t da sng n thuc da bao gm vic git, r lng v ra sch trong
b nc c pH khong 8-13 cha mt enzym a kim. R rng cn s
dng phi hp lipase vi cc protease kim hay trung tnh v mt cht hot
ng b mt khc. Ti Nga c bng sng ch s dng ch phm enzym
thuc da cu vi cc c tnh u vit hn nh bn, co gin tng
v gim cng[36].
1.7.5. Trong cng nghip m phm
Lipase c ng dng nhiu trong cng nghip m phm sn xut
cc cht hot ng b mt v cc hp cht thm. Nhng thnh phn ch
yu trong m phm v nc hoa c iu ch bng phn ng ester ho
glycerol c xc tc bi lipase. Vic chuyn ester ho ca 3,7- dimethyl
4,7- octadien- 1- ol bi lipase t nhiu ngun vi sinh vt khc nhau iu
ch oxide hoa hng l mt hng liu quan trng trong sn xut nc
hoa[36].
NGUYN LIU V PHNG PHP NGHIN CU
2.1. NGUYN LIU
2.1.1. Chng vi sinh vt
Candida rugosa nhn t Vin bo tng ging vi sinh vt Vin Cng
ngh sinh hc v Cng ngh Thc phm- i hc Bch Khoa- H Ni.
Chng Bacillus sp nhn t Phng Cng ngh Gene ng vt - Vin
Cng ngh Sinh hc
2.1.2. Cht mang Nylon-6: ca hng Sigma- M (d= 1.084)
Nylon-6 l cht nha k thut ni ting l c cng cao v sc
khng cao i vi cht ho hc v du m. Mc d vy, Nylon-6 c nhit
chuyn bin nha thp v nhit bin dng thp. l iu cn thit
trn ln Nylon-6 vi cc polyme khc to thnh cc cht c kh nng
chu nhit cao(1996)[26].
Nylon-6 c l hng nh c s dng lm cht mang cho vic c
nh lipase. N cung cp cc nhm amin hot ng mnh cui phn t
polyme, c th to phn ng vi nhm lin kt glutaraldehit [8].
H
2
-N-(CH
2
)
5
-CO-[-NH-(CH
2
)
5
-CO-]
n
-NH-(CH
2
)
5
-COOH
Nylon-6
2.1.3. Ho cht
Du oliu, cao nm men, pepton, glucoza, dch chit malt, KH
2
PO
4
,
K
2
HPO
4
, (NH
4
)
2
SO
4
, MgSO
4
.7H
2
O, FeCl
3
, CaCl
2
, NaOH, Inositon, Biotin,
thiamine.HCl, Axit palmitic, cn 99%, glutaraldehit, ht Nylon-6, v mt
s ho cht cn dng khc.
2.1.4. Thit b s dng
- T cy vi sinh vt (clearn benchop)
- Ni hp tit trng
- My o mu quang in UV-Vis
- My o pH 900 Prescisa
: Nht
: Nht
: Thu in
: Thu S
- My khuy t gia nhit (Magnetic stirrer)
- My lc n nhit Shellab
- My lc ht chn khng
- My ly tm lnh allegra 64R
- H thng sc k ct FPLC
V mt s thit b thng thng khc.
: c
: M
: c
: M
: Thu in
2.1.5. Mi trng nghin cu:
* Mi trng hot ho chng Candida rugosa (g/l)
- Dch chit malt
- Cao nm men
- Pepton
- Glucoza
: 3g
: 3g
: 5g
: 10g
B sung nc ct n tng th tch l 1000ml. Kh trng hn hp
iu kin 1 atm, 110C trong 30 pht.
* Mi trng gi ging Candida rugosa: thnh phn ging mi trng
hot ho
* Mi trng sinh tng hp lipase t Candida rugosa (g/l)
- KH
2
PO
4

- K
2
HPO
4

- (NH
4
)
2
SO
4

- MgSO
4
.7H
2
O
- FeCl
3

- Inositon
- Biotin
- Thiamine.HCl
- Axit palmitic
- C cht du oliu 1%
:15g
: 5,5g
: 4g
: 1g
: 10mg
: 0,004mg
: 0,008mg
: 0,2mg
: 1,2g

B sung nc ct n tng th tch l 1000ml. Kh trng hn hp
iu kin 1 atm, 110C trong 45 pht.
Mi trng nui cy Bacillus (BMGY): (1% (w/v) cao nm men, 2%
(w/v) bacto-peptone, 100 mM kali phosphate, pH 7.0, 4 x 10
-5
% (w/v)
biotin v 1% (v/v) methanol) 30C lc 220 vng/pht.
2.2. PHNG PHP NGHIN CU
2.2.1. Phng php vi sinh vt
Hot ho chng nm men Candida rugosa trong mi trng hot
ho nhit 30C, tc lc 200 vng/pht trong 24 gi.
Sau tin hnh nui cy sinh tng hp lipase bng phng php
nui cy chm trn mi trng sinh tng hp nhit 30C, tc lc
200 vng/pht trong 40 gi.
2.2.2. Phng php ho sinh
Phng php c nh lipase trn Nylon-6 [23]
* Nguyn l
S dng phng php gn enzym ln cht mang bng lin kt ng
ho tr. Da trn hot enzym trc v sau khi c nh, xc nh c
hiu sut c nh enzym.
* Tin hnh
Ly 30g ht Nylon-6 c lc trong 600ml dung dch HCl 3,5N
45C trong 15 pht (tc lc 200 vng/pht). Sau nhng ht ny c
tch ra bng my lc chn khng v ra nhiu ln bng nc ct.
Sau c b sung 150ml glutaraldehit 10% v lc vi tc 200
vng/pht 30C trong 30 pht. Nhng ht ny li c tch ra bng my
lc chn khng.
Sau khi hot ho, nhng ht ny c trn vi 100ml dung dch
enzym (1mg/ml) trong m phosphat pH=7,7; ri lc 45C trong 120
pht (tc lc 200 vng/pht).
Sau hn hp c 4C qua m. Ri c ra nhiu ln
bng nc ct, mi ln 20ml loi nhng enzym khng c gn kt.
Phng php xc nh hot lipase bng chun [32]
* Nguyn l
Da trn kh nng xc tc thu phn lipit ca lipase, xc nh lng
axit bo to ra trong 15 pht bng lng NaOH cn b sung duy tr pH
c nh. Hiu s gia lng NaOH sau v trc khi b sung enzym c
trng cho hot tnh xc tc ca lipase.
* Tin hnh:
Ly 10ml du n + 200ml nc ct + 1% Gum arabic -> nh tan
bng my xay sinh t trong 5 pht, to huyn ph.
Ly 20ml dung dch huyn ph cho vo cc thu tinh t ln my
khuy t, iu chnh nhit ti 65C.
Thm 470 l dung dch CaCl
2
o pH v iu chnh pH ti 8,3 (chun
bng NaOH 10mM). Sau khi n nh pH v nhit , o t thu phn
ca dung dch c cht trong 15 pht gi pH lun lun bng 8,3 (lng
NaOH tiu tn l a ml).
Sau thm 20 l dung dch lipase vo hn hp c cht v chun
bng NaOH 10mM t pH= 8,3 v bt u tnh lng tiu hao NaOH
trong 15 pht gi pH lun lun = 8,3 (lng NaOH tiu tn l b ml).
* n v hot lipase
Mt n v hot lipase (U) c xc nh nh l lng enzym c
kh nng chuyn ho to 1 micromol axt bo trong thi gian 1 pht.
Hot lipase trong 1ml dch ni c tnh theo cng thc:
(b-a)*0,01
Hot lipase = -------------- U/ml
t*v
Vi t: thi gian o t thu phn (pht)
v: Th tch enzym (ml)
a: Lng NaOH 10mM tiu tn khi cha c enzym (l)
b: Lng NaOH 10mM tiu tn khi c enzym (l)
0,01: h s chuyn i nng

Phng php tch tinh ch lipase bng sc k trao i ion DEAE
cellulose [15]
* Nguyn l:
Da trn phng php sc k trao i ion, chnh l s trao i ion
ca protein tch in vi ion ca nha trao i ion. Trong nhm tch
in (+) l cht mang trao i vi cc ion (-) v do chng c gi l
cc cht trao i anion v ngc li chng c gi l cht trao i cation.
* Tin hnh:
Dch enzym sau kt ta phn on bng etanol nng 60% c
cho qua ct DEAE cellulose vi iu kin tch phn on nh sau: ct
c cn bng trong m Tris-HCl 25mM, pH 7,5; tc chy 1 ml/pht
vi gradien nng 0-0,25M NaCl, mi phn on thu mu l 1ml/ng.
mi phn on o hm lng protein, xc nh hot lipase bit c
phn on tch lipase tt nht.
KT QU V THO LUN
3.1. Tm iu kin nui cy thch hp thu lipase
3.1.1. Tm iu kin nui cy thch hp thu lipase t Candida rugosa
3.1.1.1. Kho st nh hng ca thi gian nui ti sinh tng hp lipaza
Chng nm men c nui cy trn mi trng MT2, vi cc iu
kin: nhit nui l 30
0
C, tc lc 150 vng/pht. Kho st vi 6 thi
gian nui khc nhau: 24, 30, 36, 42, 48, 54 gi. Sau mi mc thi gian
trn dch nui cy c ly ra v xc nh hot . Kt qu c th hin
bng 1.
Bng 1: nh hng ca thi gian nui n sinh tng hp lipaza
Thi gian (gi) Hot lipaza (U/ml)
24 2
30 5.2
36 7.5
42 8.33
48 6.9
54 3.9
Kt qu nghin cu cho thy, chng nghin cu c kh nng sinh
tng hp lipaza ngay t nhng gi u nui cy v t cc i sau 42 gi
nui, cho hot lc 8.33 U/ml.
3.1.1.2. Kho st nh hng ca tc lc n qu trnh sinh tng
hp lipaza
Th nghim c tin hnh 4 tc lc khc nhau: 100, 150,
200, 250 vng/pht trn mi trng sinh tng hp lipaza nhit 30
o
C,
thi gian nui 42h. Kt qu th hin bng 2
Bng 2. nh hng ca tc lc n qu trnh sinh tng hp lipaza
Tc lc (v/p) Hot lipaza
100 5
150 8,33
200 4
250 3
Kt qu bng trn cho thy qu trnh sinh tng hp lipaza t cc
i tc lc 150 vng/pht.
3.1.1.3. Kho st nh hng ca ngun cacbon n sinh tng hp
lipaza
Chng ti tin hnh kho st vi 5 ngun cacbon khc nhau trn
mi trng sinh tng hp lipaza nhit 30
o
C, tc lc 150
vng/pht, c cht 1% du oliu, thi gian nui 42h. Kt qu th hin
bng 3
Bng 3. nh hng ca ngun cacbon n sinh tng hp lipaza
Ngun cacbon ( 2g/l) Hot lipaza (U/ml)
Glucoza 8,67
Galactoza 12,30
Tween 80 6,67
Axit Palmitic 18,00
Glyxerol 12,67
Qua bng trn cho thy hot lipaza t c cao nht trn ngun
cacbon l axit palmitic, cc ngun cacbon khc nh glyxerol v galactoza
cng cho hot tng i cao. Vy ngun cacbon thch hp nht cho
sinh tng hp lipaza ca chng nghin cu l axit palmitic. Kt qu ny
cng ph hp vi kt qu nghin cu ca tc gi E.Dalmau (2000) khi
nghin cu nh hng ca cc ngun cacbon khc nhau n sinh tng
hp lipaza cu chng candida rugosa.
3.1.1.4. Kho st nh hng ca nng axit palmitic n sinh tng
hp lipaza
Tin hnh nui cy chng nm men trn mi trng sinh tng hp
lipaza vi cc iu kin thch hp tm c trn, thay i hm lng
axit palmitic trong mi trng (g/l): 0,5; 1; 1,5; 2; 2,5; 3. Sau 42 h nui
cy xc nh hot trong dch canh trng thu c kt qu trong bng
4 sau y.
Bng 4. nh hng ca nng axit palmitic
Nng axit palmitic (g/l) Hot lipaza (U/ml)
0.5 12.3
1 15.83
1.5 17.67
2 13.67
2.5 9.33
3 5.67
T kt qu trn ta thy nng axit palmitic cng nh hng nhiu
ti kh nng sinh tng hp lipaza. Nng axit palmitic 1,5 g/l cho hot
cao nht l 17.67 (UI/ml).
3.1.1.5. Ti u ho iu kin nui chm Candida rugosa
T cc iu kin kho st c trn chng ti tin hnh ti u
ho bc 1 iu kin nui chng Candida rugosa theo 3 yu t sau:
X
1
: axit palmitic (g/l)
X
2
: S t bo nm men (tb/ ml )
X
3
: thi gian (gi)
Khi y hm mc tiu l Y: hot lipaza (U/ml)
Cc mc th nghim
Mc th nghim X
1
X
2
X
3

Mc gc 1.5 4 42
Khong bin i 0.5 2 1
Mc trn 2 6 46
Mc di 1 2 38

Lp ma trn th nghim v kt qu

S
j
2

0
.
6
2

2
.
7
4

1
.
1
3

0
.
1
5

0
.
2
9

0
.
3
6

0
.
1
1

0
.
6
5

=
6
.
0
5
Y
t
b

1
0
.
1
2

1
5
.
5

1
2
.
6
2

1
5
.
0
3

1
5
.
2
5

1
5
.
2
1

1
7
.
4
4

1
8
.
1

=
1
1
9
.
2
7
Y
2

1
0
.
6
7

1
4
.
3
3

1
1
.
8
7

1
4
.
7
6

1
4
.
8
7

1
5
.
6
3

1
7
.
6
7

1
7
.
5
3

Y
1

9
.
5
6

1
6
.
6
7

1
3
.
3
7

1
5
.
3

1
5
.
6
3

1
4
.
7
8

1
7
.
.
2

1
8
.
6
7

T
h

c
3
4

3
4

3
4

3
4

4
2

4
2

4
2

4
2

X
3

M
-

-

-

-

+

+

+

+

T
h

c 2

2

6

6

2

2

6

6

X
2

M

-

-

+

+

-

-

+

+


T
h

c

0
.
0
5

0
.
1
5

0
.
0
5

0
.
1
5

0
.
0
5

0
.
1
5

0
.
0
5

0
.
1
5

X
1

M

-

+

-

+

-

+

-

+

S
T
N

1

2

3

4

5

6

7

8


X l thng k cc kt qu thc nghim thu c
Kim tra ng nht ca ma trn theo chun Cochran
Nu G
b
> G
tt
th ma trn ng nht
G
tt
= max Sj
2
= 0.45
Sj
2

G
b
tra bng = 0.6798
G
b
> G
tt
nn cc s liu c o cng mt chnh xc nh
nhau v c th chp nhn c
Xc nh cc h s ca m hnh theo cng thc
N
y
b

=
b
0
=14.91 b
1
=1.05 b
2
=0.88 b
3
=1.59
Phng trnh hi quy c dng : y = 14.91 + 1.05x
1
+ 0.88x
2
+ 1.59x
3

Kim tra s c ngha ca cc h s
|b
i
| t.S
b
= 2.12*0.31 = 0.6572
vi t l chun student tra bng (p=0,05; f=16) = 2.12
ta thy cc h s |b
o
|,|b
1
|,|b
2
|,|b
3
| u 0.6572
Tt c cc h s trn u c ngha
Vy phng trnh hi qui cui cng l:
y = 14.91 + 1.05x
1
+ 0.88x
2
+ 1.59x
3

Kim tra s thch ng ca phng trnh hi quy theo chun
Fisher
STT M hnh Ytt Ytn (Ytt-Ytn)
2
1 14.91 - 1.05 - 0.88 - 1.59 11.39 10.12 1.61
2 14.91 + 1.05 - 0.88 - 1.59 13.49 15.5 4.04
3 14.91 - 1.05 + 0.88 - 1.59 13.15 12.62 0.28
4 14.91 + 1.05 + 0.88 - 1.59 15.25 15.03 0.05
5 14.91 - 1.05 - 0.88 + 1.59 14.57 15.25 0.46
6 14.91 + 1.05 - 0.88 + 1.59 16.67 15.21 2.31
7 14.91 - 1.05 + 0.88 + 1.59 16.33 17.44 1.23
8 14.91 + 1.05 + 0.88 + 1.59 18.43 18.1 0.11
3.93

B - N
ytn) - (ytt
S

= t
) St , y min(S
) St , y (S max
Ftt

=

S
t
= 3.93 / 4 = 0.98 F
tt
= 0.98/ 0.38
= 2.58
F
b
= 3.01 tra bng vi f
1
= N - B = 4
f
2
= N ( k -1) = 8
Trong N: s th nghim
B: s h s c ngha
k: s ln lp li
F
tt
F
b
M hnh trn l thch ng
Ti u ho theo Box - Wilson

i
= X
0
- cn di = cn trn - X
0

b
1

1
= 1,05*0,5 = 0.525
b
2

2
= 0,92*2 = 1.84
b
3

3
= 1,61*4 = 6.44 t gi tr ln nht nn ta chn X
3
lm X
cs

Chn bc nhy ca X
3
l
3
= 2

1
= (b
1

1
/ b
3

3
).
3
= 0,2

2
= (b
2

2
/ b
3

3
).
3
= 0.6
Kt qu ti u theo Box - Wilson
STT X
1
X
2
X
3
Hot
1 1 2 38 16.17
2 1.2 2.6 40 18.67
3 1.4 3.2 42 11.67
4 1.6 3.8 44 9.33
5 1.8 4.4 46 6.67

T kt qu trn cho thy qu trnh sinh tng hp enzim lipaza ca
chng nm men Candida rugosa t hiu qu tt nht th nghim th 2
vi iu kin:
Hm lng axit palmitic: 1.2 g/l
S t bo nm men : 2,6.10
6
tb/ml canh trng
Thi gian nui: 40 gi
3.1.2.Tm iu kin nui thu lipase t Bacillus sp
Chng ti nui cy chng vi sinh trong 6 mi trng khc nhau v
thnh phn (bng 5) v xc nh hot tnh lipase trong dch ni sau 120
gi nui cy. Kt qu cho thy hot enzym t cao nht khi nui cy
trong mi trng BMMY (67 U/ml canh trng). Mi trng ny s
c chn cho cc nghin cu tip theo.
Bng 5. Hot tnh lipase Bacillus- sp nui cy
6 mi trng khc nhau
Mi trng
(50 ml)
Thnh phn Hot sau 120
gi nui cy
(U/ml)
A Bt ng: 0,8g. Bt u tng: 1,6 g.
Nc da: 50 ml. Methanol 1%.
3,2
B Bt ng: 0,8g. Bt u tng: 1,6 g.
Nc da: 50 ml. Methanol: 1%.
Biotin:
5,1
C Bt ng: 0,8g. Bt u tng: 1,6 g.
Nc my: 50 ml. Methanol: 1%.
3,4
D Bt ng: 0,8g. Bt u tng: 1,6 g.
m K-phosphate: 50 ml. Methanol:
1%.
10,2
E Bt ng: 0,8g. Bt u tng: 1,6 g.
m K-phosphate: 50 ml. Methanol:
1%. Biotin
8,3
BMMY 67,0
Th nghim tip sau y nhm kho st nh hng camt s c cht
cm ng ti kh nng sinh tng hp lipase. Mi trng c bn BMMY
c b sung mt s loi du hoc gum arabic (bng 2).
Tin hnh nui vi 6 loi mi trng khc nhau, c 24 gi ly mu
xc nh hot tnh v tc pht trin ca vi khun (OD). Kt qu cho
thy cc c cht ny khng lm tng kh nng sinh tng hp enzym ca
chng nghin cu.
Bng 6. nh hng ca du v gum arabic ti kh nng sinh tng
hp lipase Bacillus-sp

Hot (U/ml) TT
Cc cht b sung
vo mi trng
BMMY
24 h 48 h 72 h 96 h 120
h
144
h
1 i chng
(BMMY)
16,0 37,2 42,0 48,4 67,0 54,0
2 1% Gum arabic 16,0 16,5 17,7 22,0 32,2 41,0
3 1% du Marvela 21,0 23,0 32,0 37,0 44,0 44,0
4 1% du Marvela
+ 1% Gum arabic
23,3 24,2 36,0 37,0 45,0 50,0
5 1% du m 19,0 21,2 38,0 42,0 42,0 42,8
6 1% du m + 1%
Gum arabic
30,0 36,0 46,0 52,0 56,0 62,0

3.2. Tch v tinh ch lipase t Candida rugosa
3.2.1. Kho st nng etanol thch hp kt ta lipaza t Candida
rugosa
Tin hnh th nghim kt ta lipaza vi cc nng khc nhau: 60, 70,
80, 90%, ly tm lnh, thu kt ta cho bc hi dung mi ta thu c lipaza
th. Kt qu xc nh hot c th hin bng 7
Bng 7. Kt ta lipaza cc nng etanol khc nhau
Nng
etanol
(%v/v)
Hot lipaza
trc khi kt ta
(UI/ml)
Hot lipaza
sau khi kt ta
cn (UI/ml)
Hiu sut
thu lipaza
(%)
57 16.73 0 0
67 16.73 0 0
76 16.73 1.33 7.95
81 16.73 5.67 21.94
86 16.73 9.83 58.76
90 16.73 12.33 73.7
91 16.73 3.33 19.9
93 16.73 0 0

0
2
4
6
8
10
12
14
57 67 76 81 86 90 91 93
nng cn( cn)
h
o

l
i
p
a
z
a

(
U
/
m
l
)
Hnh 1: kt ta enzim bng etanol
T kt qu thu c trn ta nhn thy cc nng cn trong khong
0 -67% c cho ta kt ta nhng khng c hot enzim, ch khi nng
cn t 67% tr ln l c hot v nng cn l 90% th cho ta hot
cao nht. Qua th nghim ny cho thy nng cn t 67% tr
xung ch c protein tp b kt ta v vy chng ta c th kt ta phn
on loi b bt protein tp.
3.1.2. Tinh sch lipase bng phng php sc k trao i ion-DEAE-
cellulose trn thit b FPLC
Dch enzym sau ta bng etanol c ly 15 ml cho vo ct
c cn bng trong m Tris-HCl 25mM pH 7,5; tc chy l
1,5ml/pht. Dung dch y lipase l NaCl vi gradien nng 0-0,25M;
mi phn on thu 1ml/ng.
Kt qu c biu din trn sc k hnh 2
0
10
20
30
40
50
60
70
80
1 8
1
6
2
4
3
2
4
0
4
8
5
6
6
4
7
2
8
0
8
8
9
6
1
0
4
1
1
2
1
2
0
Phn on (1ml)
A
/
2
8
0
n
m
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
H
o

(
U
/
m
l
)

0,25
1
NaC
Hnh 2. Sc k tinh sch lipase
Sc k hnh 2 cho thy lipase sau khi qua ct trao i ion
DEAE - cellulose xut hin 3 pick. Tin hnh xc nh hot lipase
cc phn on thuc cc pick. Kt qu cho thy ch c pick 1 v pick 3 c
hot tnh lipase (69UI/ml v 77UI/ml). iu ny ph hp vi nghin cu
ca M.A. Pernas (2000) [15] cng b Lip1 v Lip3 c khi lng
phn t l 58 v 62 kDa.
Kt qu cc bc tch v lm sch c th hin bng 8
Bng 8. Kt qu tch lm sch lipase

Hot lipase
Cc bc lm
sch
Tng
th tch
(ml)
Protein
tng
(mg) Tng
(U)
Ring
(U/mgPr)
Mc
sch
(ln)
Hiu
sut
(%)
Dch th 200 340 1500 4,41 (1) (100)
Sau kt ta
etanol
20 66,8 1245 18,64 4,22 83,00
DEAE -
Cellulose
8 10,08 556 55,16 12,50 37,06
Kt qu sc k hnh 2 v bng 8 cho thy lipase sau cc bc
lm sch lin tip: kt ta etanol, sc k trao i ion DEAE Cellulose
thu c ch phm c hot ring 55,16 U/mg protein, mc tinh
sch ca lipase l 12,5 ln; hiu sut thu hi l 37,06%.
3.3. KHO ST CC IU KIN TI U C NH LIPASE
t Candida rugosa trn nylon 6
3.3.1. nh hng ca nng HCl hot ho Nylon-6 n kh nng
c nh lipase
Nylon-6 l mt polyamit tch in (+). Lipase l enzym kim tnh
cha nhm (OH
-
) v vy s dng HCl hot ho cht mang, lm tng
in tch (+) l cn thit. Do vy tin hnh th nghim bng cch s dng
lipase sau kt tu etanol c nh trn Nylon-6 cc nng HCl khc
nhau vi cc iu kin n nh:
Tng lng ht Nylon-6 em x l : 3g
Nng glutaraldehit : 10%
m kali phosphat : pH 7,7
Tc lc : 200 vng/pht
Kt qu biu din bng 9
Bng 9. nh hng ca nng HCl hot ho Nylon-6 n kh nng c
nh lipase

Nng
HCl (N)
Tng th
tch nc
ra (ml)
Hot cn li
trong nc ra
(U)
Hiu sut c
nh enzym
(%)
i chng
(lipase tan)
- 550 100
3 33,5 276,4 49,75
3,5 33,5 226,1 58,90
4 33,5 245,3 55,40
4,5 33,5 259,6 52,80

Bng 9 cho thy, vi nng HCl 3,5N hiu sut c nh t
58,9% l cao nht. C th vi nng HCl thp cha kh nng hot
ho cht mang, cn vi nng cao lm thay i pH, nh hng n kh
nng lin kt lipase vo cht mang. Kt qu ny ph hp vi ti liu
cng b trc y[23].
3.3.2. nh hng ca nng glutaraldehit n kh nng c nh
lipase
Tin hnh c nh lipase vi cc nng glutaraldehit khc nhau
(8%, 10%, 12%) v cc iu kin:
Tng lng ht Nylon-6 em x l : 3g
Nng HCl : 3,5N
m kali phosphat : pH 7.7
Tc lc : 200 vng/pht
Kt qu kho st th hin bng 10




Bng 10. nh hng ca nng glutaraldehit n kh nng c nh lipase
Nng
glutaraldehit
(%)
Tng th
tch
nc ra
(ml)
Hot cn
li trong
nc ra
(U)
Hiu sut
c nh
enzym
(%)
i chng
(lipase tan)
- 550 100
8 33,5 293,1 46,7
10 33,5 209,4 61,9
12 33,5 376,8 31,5

Qua kt qu kho st cho thy nng glutaraldehit 10% cho
hiu sut c nh lipase cao nht (61,9%). Nh vt nng glutaraldehit
10% l nng thch hp lin kt enzym li vi nhau v tng lin
kt ln cht mang Nylon-6.
3.3.3. nh hng ca nng lipase n kh nng c nh
Tin hnh c nh lipase cc nng enzym khc nhau (0,5; 1; 1,5; 2
mg/ml) v c nh cc iu kin:
Tng lng ht Nylon-6 em x l : 3g
Nng HCl : 3,5N
m kali phosphat : pH 7.7
Tc lc : 200 vng/pht
Kt qu c th hin bng 11
Bng 11. nh hng ca nng enzym n kh nng c nh lipase
Nng
enzym
(mg/ml)
Tng th
tch nc
ra (ml)
Hot cn
li trong nc
ra (U)
Hiu sut
c nh (%)
0,5 (270U) 33,5 160,80 40,45
1 (550U) 33,5 224,45 59,20
1,5 (820U) 33,5 375,20 54,25
2 (1100U) 33,5 753,75 31,50
Kho st kh nng c nh lipase trn Nylon-6 vi cc nng
enzym khc nhau ta thy vi nng enzym l 1 mg/ml, hiu sut c
nh cao nht (59,2%). Kt qu kho st ny ph hp vi kt qu
nghin cu trc y[23].
3.3.4. nh hng ca dung dch m n kh nng c nh lipase
Tin hnh kho st 3 dung dch m khc nhau: m photphat,
m borat, m a nng vi cc iu kin c nh:
Tng lng ht Nylon-6 em x l : 3g
Nng HCl : 3,5N
Nng glutaraldehit : 10%
Tc lc : 200 vng/pht
Kt qu kho st th hin bng 12.
Bng 12. nh hng ca dung dch m n kh nng c nh lipase
Cc dung dch
m
Tng th
tch nc
ra (ml)
Hot cn li
trong nc ra
(U)
Hiu sut c
nh (%)
i chng
(lipase tan)
- 550 100
m phosphat 33,5 251,25 54,30
m borat 33,5 335,00 39,10
m a nng 33,5 418,75 23,86
Qua kt qu kho st vi cc loi m cho thy hiu sut c nh
vi m phosphat l cao nht (54,3%). Nh vy m thch hp cho vic
c nh lipase trn Nylon-6 l m phosphat pH 7,7. Ph hp vi kt qu
nghin cu trc y[23].
3.4. c tnh ca lipase
3.4.1. Mt s c tnh ca lipase t do v c nh t Candida rugosa
3.3.1.1. nh hng ca nhit ti hot lipase t Candida rugosa
Nhit l yu t tc ng mnh m ti hot ca enzym. Khi
nhit tng th vn tc phn ng cng tng nhng n mt gii hn no
th vn tc t cc i, nu tip tc tng th vn tc enzym s gim v
nhit cao s lm bin tnh protein gy bt hot enzym. Nhit m ti
enzym hot ng mnh nht gi l nhit ti u. Mi enzym c mt
vng hot ng khc nhau. xc nh nhit ti u ca lipase ta tin
hnh xc nh hot cc nhit 55, 60, 65, 70, 75C bng phng
php chun theo m t trong phn phng php nghin cu.
Kt qu th hin bng 9 v hnh 3
Bng 13. nh hng ca nhit ti hot lipase t do v c nh
Nhit (C) Hot lipase t do
(U/g)
Hot lipase c
nh (U/g)
40 19,5 0,5
55 27,0 2,5
60 29,7 5,5
65 27,5 10,0
70 20,5 15,0
75 16,5 11,5
80 12,5 7,5

0
5
10
15
20
25
30
35
35 40 45 50 55 60 65 70 75 80
Nhit ( C)
H
o

(
U
/
g
)
Lipaza tan
Lipaza co dinh

Hnh 3. nh hng ca nhit ti hot lipase t do v c nh
Theo hnh 3 cho thy khi nhit tng t 60C tr ln hot
lipase c nh tng dn v t cc i ti 70C (15 U/g). i vi lipase
tan, nhit 60C hot lipase t cc i (29,7U/g) v gim dn
khi nhit tng ln.
Nh vy nhit thch hp cho lipase t do v c nh hot ng
l khc nhau. Nhit ti u ca lipase c nh cao hn ca lipase t do
v lipase c nh c lp cht mang bo v nn c kh nng chu nhit.
3.4.1.2. nh hng ca nhit ti tnh bn lipase t do v c nh
Tin hnh th nghim vi lipase t do v c nh cc nhit
khc nhau: 40, 50, 60, 70
o
C vi thi gian xc nh trong 18 gi, c sau 6
gi xc nh hot mt ln.
Kt qu xc nh tnh bn nhit ca lipase t do v c nh c
th hin bng 14 v hnh 4.
Bng 14. nh hng ca nhit ti tnh bn lipase t do
Thi gian (gi)
0 6 12 18
Nhit

(
0
C)
Hot

U/g % U/g % U/g % U/g %
40 29,5 100 23,6 80,0 20,2 68,5 17,4 59,0
50 29,5 100 21,7 73,5 17,6 59,6 14,8 50,2
60 29,5 100 18,9 64,0 14,2 48,2 12,9 43,7
70 29,5 100 15,4 52,3 11,7 39,5 9,6 32,5

0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20
Thi gian (gi)
H
o

(
%
)
40 C
50 C
60 C
70 C

Hnh 4. nh hng ca nhit ti tnh bn ca lipase t do
Kt qu cho thy, i vi enzym t do vng bn nhit 40-50C,
sau 18 gi hot lipase cn 50,2 - 59% . 70C hot gim mnh
sau 18 gi ch cn 32,5%.
Bng 15. nh hng ca nhit ti tnh bn ca lipase c nh
Thi gian (gi)
0 6 12 18
Nhit

(
0
C) Hot
U/g % U/g % U/g % U/g %
40 21 100 17,8 85,5 16,0 76,2 14,0 66,7
50 21 100 15,9 75,5 13,9 66,5 12,4 59,0
60 21 100 14,0 66,7 12,0 57,1 11,0 52,4
70 21 100 13,0 61,9 10,2 48,5 8,3 39,3

0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20
Thi gian (gi)
H
o

(
%
)
40 C
50 C
60 C
70 C

Hnh 5. nh hng ca nhit ti tnh bn lipase c nh
Kt qu cho thy lipase c nh bn vng nhit 40 - 60C (sau
18 gi hot cn 52,4 66,7% so vi hot ban u). Sau 18 gi,
70
o
C hot lipase c nh cn 39,3%. Nh vy mi nc nhit , hot
lipase c nh tng khong 10% so vi lipase t do.
3.4.1.3. nh hng ca pH ti hot lipase t do v c nh
pH c nh hng kh ln n vn tc phn ng ca enzym, mi
enzym hot ng mnh trong 1 di pH khc nhau. gi tr pH m ti
enzym hot ng mnh nht gi l pH ti u. Tin hnh xc nh hot
lipase t do v c nh vi cc gi tr pH thay i t 7,0 - 8,3 vi cng c
cht du u nnh 5%, nhit 65C.
Kt qu th nghim c th hin bng 16 v biu din trn hnh 6
Bng 16. nh hng ca pH ti hot lipase
PH Hot lipase
t do (U/g)
Hot lipase
c nh (U/g)
6,5 17,5 11,5
7 21,5 15,0
7,5 25,5 12,5
8 28,5 7,5
8,5 26,5 2,5
9 21,5 1,5

0
5
10
15
20
25
30
6 6.5 7 7.5 8 8.5 9
pH
H
o

(
U
/
g
)
Lipaza tan
Lipaza co dinh
Hnh 6. nh hng ca pH ti hot lipase
Qua bng 16 v hnh 6 cho thy hot lipase c nh hot ng
mnh nht pH 7,0 (15 U/g). Khi tng pH ln trn 9,0 th hot enzym
gim ch cn 1,5 U/g.
i vi lipase t do, hot ng mnh pH 8,0 (28,5U/g). iu ny
cng ph hp vi l thuyt, lipase c c nh ln cht mang polyamit
tch in dng nn pH ti u chuyn v vng axit.
3.4.1.4. nh hng ca pH ti tnh bn lipase t do v c nh
xc nh nh hng pH ti tnh bn lipase t do v c nh, tin
hnh th nghim cc gi tr pH khc nhau: 6,5; 7,0; 7,5; 8,0 vi nhit
30C trong thi gian 18 gi. C sau 2 gi xc nh hot lipase. Kt
qu th hin bng 17 v hnh 7
Bng 17. nh hng ca pH ti tnh bn lipase tan
Thi gian (gi)
0 6 12 18 pH
Hot
U/g % U/g % U/g % U/g %
6,5 29,5 100 18,3 62,0 13,3 45,0 9,6 32,5
7 29,5 100 21,2 72,0 16,2 55,0 12,8 43,5
7,5 29,5 100 23,6 80,0 19,2 65,0 15,9 53,9
8 29,5 100 25,1 85,0 21,2 72,0 18,3 62,0

0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20
Thi gian (gi)
H
o

(
%
)
pH 6,5
pH 7
pH 7,5
pH 8
Hnh 7. nh hng ca pH ti tnh bn lipase tan
Kt qu cho thy lipase t do bn vng pH 7,5 8,0 sau 18 gi
vn cn 53,9 - 62 %.

Bng 18. nh hng ca pH ti tnh bn lipase c nh
Thi gian (gi)
0 6 12 18 pH
Hot
U/g % U/g % U/g % U/g %
6.5 21 100 14,0 66,7 9,9 47,0 8,2 39,0
7 21 100 16,7 79,5 13,8 65,7 12,0 57,0
7.5 21 100 16,0 76,2 12,2 58,2 10,6 50,6
8 21 100 12,0 57,1 8,2 39,1 6,1 29,0
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20
Thi gian (gi)
H
o

(
%
)
pH 6,5
pH 7
pH 7,5
pH 8

Hnh 8. nh hng ca pH ti tnh bn lipase c nh
Kt qu bng 18 v hnh 8 cho thy lipase c nh bn vng pH
7 - 7,5 sau 18 gi hot cn 50,6 57%. Nh vy lipase c nh vng
axit hn so vi lipase t do.
3.4.1.5. nh hng ca ion kim loi ti hot lipase t do
xc nh nh hng ca cc ion kim loi ln kh nng xc tc
ca enzym tin hnh nghin cu i vi cc ion kim loi: Ca
2+
, Na
+
, K
+
,
Cu
2+
, Zn
2+
, Fe
3+
, Mg
2+
, Pb
2+
. Tin hnh enzym vi dung dch m cha
cc ion kim loi trn vi nng 10mM trong 30 pht, iu kin nhit
phng (30C) em xc nh hot theo phng php chun so
vi mu i chng khng ion kim loi bit c mc nh hng
ca chng vi enzym lipase.
Kt qu th hin bng 19 v hnh 9

Bng 19. nh hng ca ion kim loi n hot lipase t do
Cc ion kim loi
(10
-3
M)
Hot lipase (U/ml) % hot
Mu i chng 54,0 100
Ca
2+
58,0 107,40
Mg
2+
57,5 106,48
Na
+
53,5 99,07
K
+
53,0 98,15
Cu
2+
47,0 87,03
Fe
3+
46,5 86,11
Zn
2+
46,5 86,11
Pb
2+
35,5 67,74

0
10
20
30
40
50
60
70
1 2 3 4 5 6 7 8 9
Ion kim loi
H
o
a
t

d
o

(
U
/
m
l
)

Hnh 9. nh hng ca ion kim loi n hot lipase t do

Kt qu nghin cu cho thy, cc ion kim loi Na
+
, Ca
2+
, Mg
2+
, K
+

khng nh hng nhiu n hot lipase, trong Ca
2+
v Mg
2+
cn c
chiu hng hot ho lm tng hot lipase. Cn cc ion kim loi nng
Fe
3+
, Cu
2+
, Zn
2+
c ch yu ln hot lipase khong 13-14%. Pb
2+
c
ch lipase mnh nht, n lm gim hot khong 33%.
3.4.1.6. Xc nh im ng in ca lipase t do
im ng in (pI) l pH m ti phn t protein trung ho v
in. Ti im ng in, phn t protein enzym hot ng km v d b
kt ta. im ng in ca protein enzym khng nhng c ngha l
thuyt m cn c ngha thc tin trong sn xut v vy cn phi xc
nh im ng in ca enzym bng cch o c ca dung dch
enzym khi kt ta vi cn theo Droxov (1970)
Tin hnh ly 5ml dch enzym cha 5-8mg protein enzym, 10ml
m borat cc pH khc nhau t 6,6 n 9,0. Sau cho kt ta bng
cn 96 vi nng cn l 60%. lnh trong 30 pht, ly ra o hm
lng protein bc sng = 620nm. pH no dch enzym sau ta c
c cao nht, tc l c cao nht, l im ng in pI ca
enzym nghin cu. Kt qu th hin bng 20.
Bng 20. Kt qu xc nh im ng in ca lipase t do
PH OD
620
6,6 0,577
7,0 0,631
7,5 0,704
8,0 0,688
8,4 0,615
8,8 0,566
9,1 0,454

Kt qu thu c bng 20 cho thy ti pH 7,5 dch enzym ta c
c cao nht. Vy pI ca lipase l 7,5
3.4.2. Mt s c tnh ca lipase Bacillus-sp
c hiu c cht
Lipase Bacillus c hot tnh c hiu i vi tng c cht v du n rt
khc nhau. Hot tnh i vi du olive thp nht ch t 26 U/ml, i vi
cc loi du khc nh du m tinh luyn, du nnh, Neptune, Meizan,
Nakydaco t 53-63 U/ml, i vi du Tng an t 79 U/ml v i vi
tributyrin t 101 U/ml (hnh 10).
Hnh 10. Hot lipase Bacillus
c hiu ca tng c cht. 1: Du
olive; 2: Du m (tinh luyn); 3:
Du nnh (tinh luyn); 4: Du
neptune (du c + du ht ci); 5:
Du Meizan (du olein + du ht
ci + du nnh); 6: Du Nakydaco
(du olein da + du nnh); 7: Du Tng an (du olein + du nnh
+ du canola); 8: Tributyrin (1%).
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8
Cc c cht
H
o

t

t

n
h

(
U
/
m
l
)
nh hng ca nhit
Nhit ti u ca lipase chng Bacillus chu nhit ny c xc
nh qua vic o hot tnh dch ni nhit phn ng khc nhau t
30 n 75C vi c cht l du n tng hp nng 5% v pH 8,0.
Khi nhit tng t 30 n 75C th hot tnh lipase cng tng theo vi
tc u t 31 n 91 n v/ml (hnh 11). Hot tnh t ti u nhit
70C l 91 n v/ml (hnh 11). Sau nhit 70C, hot tnh lipase c
gim cht t (89 U/ml nhit 75C). Vi biu nhit ti u ny,
lipase Bacillus thuc vo mt trong cc lipase a nhit c nghin
cu trn th gii.

0
20
40
60
80
100
30 35 40 45 50 55 60 65 70 75
Nhit (C)
H
o

t

t

n
h

(
U
/
m
l
)
Hnh 11. nh hng ca nhit n
hot lipase.




nh hng ca nhit n bn ca lipase
Di tc ng ca nhit , hot tnh lipase t nhiu u b nh hng.
Mc nh hng ty thuc vo tng loi lipase khc nhau v ty theo
khong thi gian enzym c nhit . Trong qu trnh x l
nhit, c th trc tin mt s lin kt hydro trong mng li lin kt
hydro tham gia vo vic gi vng cu trc ca lipase b t gy dn n
cu trc ca enzyme b bin i, lm cho c cht khng lin kt c vi
enzyme v khng b chuyn ha di tc dng ca enzyme. Nu x l
enzyme mt nhit qu cao, cc lin kt trong phn t enzyme s
hon ton b ph hy lm enzyme b mt hot tnh v lc ny khi a v
nhit bnh thng th hot tnh ca n khng th hi phc li c.
Thi gian lipase cng lu v nhit cng cao th hot tnh cn li
cng thp. nhit 30C, hot tnh lipase gim nh cho ti 8 gi (86%)
v sau 24 gi hot tnh cn li l 63% (hnh 12). nhit 40 v 50C,
th hot tnh - nhit tng t nhau, chng gim mnh u cho ti 6
gi (32 v 34%), sau hot tnh gim nh cho ti 24 gi (29 v
22%). i vi nhit 60 v 70C, th hot tnh - nhit cng tng
t nhau, chng gim rt nhanh ch sau 1 gi (31 v 20%) v sau
gim dn cho ti zero (70C) v 17% (60C). Kt qu ny cho thy
bn ca lipase ph thuc vo nhit v thi gian .

0
20
40
60
80
100
120
0 1 3 6 8
Thi gian (gi)
H
o

t

t

n
h

t

n
g

i

(
%
)

24
Hnh 12. nh hng ca nhit n bn lipase. Nhit 30C (X);
40C (Q); 50C (V); 60C (X); 70C (O).



nh hng ca pH n hot tnh
pH hn hp phn ng c nh hng r rt n hot tnh enzyme v n
lin quan n mc ion ha c cht v bn ca lipase. tm ra
khong pH ti u cho hot tnh lipase, xut pht t mt s thng tin v
pH ti u ca mt s chng Bacillus, chng ti bt u d tm pH t 7,0
v i dn v pha acid v kim.

Hnh 13. nh hng ca pH n hot
tnh lipase.



Hot tnh lipase t l thun vi tng pH
hn hp phn ng trong mt khong pH gii hn 5,0-9,5. pH 5,0, hot
tnh lipase l zero c ngha l lipase ny khng hot ng trong mi
trng acid. Hot tnh tng nhanh u khi pH tng ln 7,5 (76 U/ml).
Hot tnh bt u tng chm khi pH i t 7,5 (76 U/ml) ln 9,5 (108
U/ml) v t cc i ti pH 9,5. Hot tnh gim nh pH 10,0 (98 U/ml).
Di pH cn thit cho lipase hot ng l 7,5-9,5, mt loi lipase a
kim. c tnh ny cng vi kh nng thy phn tt cc loi du n khc
nhau cho thy y l mt loi enzyme rt thch hp cho ngnh cng
nghip sn xut cc cht ty ra hot ng mt mi trng c tnh kim
cao.
0
20
40
60
80
100
120
5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10
pH hn hp phn ng
H
o

t

t

n
h

(
U
/
m
l
)
nh hng ca pH n bn ca lipase
Ging nh nhit , pH cng nh hng r rt n bn lipase. Khi
pH 4, hot tnh lipase ch cn 12%, v khi pH 5-7 hot tnh cn li
mt na (48-58%) (hinh 14). Lipase gi nguyn hot tnh (92-117%) khi
mi trng pH 9,0-10 (hnh 14),
nhng khi mi trng qu kim (pH
11) hot tnh cn li ch 23%. Cng
mt pH, nhng trong cc m khc
nhau, hot tnh cn li cng khc nhau
(hnh 14). iu ny cho thy cc ion
cng nh hng ti bn lipase. Nh vy
lipase c bn cao mi trng kim v thp mi trng acid.
0
20
40
60
80
100
120
140
4 5 6 7 8 9 10 1
pH hn hp lipase
H
o

t

t

n
h

t

n
g

i

(
%
)
1
Hnh 14. nh hng ca pH n bn lipase. m acetate (X); m
phosphate (Q); m Tris-HCl (V); m glycine (X).
nh hng ca ion kim loi n hot tnh

Theo nhiu ti liu nghin cu v cu trc ca lipase cho thy lipase
khng phi l mt enzyme kim loi. Tc ng ca kim loi n lipase
trong qu trnh xc tc phn ng c th theo nhiu cch khc nhau: (a)
Tham gia vo vic gi vng n nh cu dng khng gian cn thit ca
phn t lipase m bo vic thc hin cc phn ng enzyme. (b) C tc
dng lm tng tc phn ng thng qua vic to thnh phc vi c cht.
(c) Tham gia lm cu ni kt hp c cht vi trung tm hot tnh ca
enzyme.
Kt qu cho thy cc ion kim loi ha tr I, II v III u nh hng n
hot tnh ca lipase. K
+
, Mg
2+
, Fe
3+
lm gim ng k hot tnh ca lipase
(80-89% i vi 1 mM v 57-83% i vi 10 mM) trongkhi Ca
2+
li
c nh hng tch cc n hot tnh ca lipase (tng 10-12%) (hnh 15).





Hnh 15. nh
hng ca ion
kim loi n hot tnh
lipase. Ct gch ngang: KCl; ct car: MgCl
2
; ct xy gch: CaCl
2
; ct
thang: FeCl
3
.
0
20
40
60
80
100
120
0 1 10 1 10 1 10 1 10
Nng ion (mM)
H
o

t

t

n
h

t

n
g

i

(
%
)
nh hng ca dung mi hu c
S c mt ca dung mi hu c 1% lm gim hot tnh lipase.
Acetone, 2-propanol v ethanol gim hot tnh lipase mt na, cn
methanol gim mt phn t
nh hng ca cht ty ra
Mt s cht ty ra c hot tnh b mt nh SDS, Tween 20, Tween 80,
Triton X-100 nh hng n hot tnh lipase. Triton X-100 tng hot tnh
lipase ln 63% v Tween 20 gi nguyn c hot tnh lipase (107%),
trong khi Tween 80 gim mt na, cn SDS th c ch lipase hot tnh
cn khong 3% (hnh 17).





0
20
40
60
80
100
120
140
160
180
1 2 3 4 5
Cc cht ty ra
H
o

t

t

n
h

t

n
g

i

(
%
)

Hnh 17. nh hng ca cht ty ra n bn ca lipase. 1: i chng.
2: SDS. 3: Tween 20. 4: Tween 80; 5: Triton X-100.

3.5. KHO ST KH NNG NG DNG LIPASE t Candida
rugosa
3.5.1. Kho st kh nng thu phn du vi lipase c nh
Trong cng nghip dc ngi ta thng sn xut vin nang c
cha cht bo vi ch s axit 80 100. Cng nh trong khu phn thc
n king ca nhng bnh nhn nhim m trong mu nu s dng cc loi
du n thc vt c thu phn s b ch s axit tng n 80 100 th
s lm gim hm lng m trong mu. Do vy chng ti nghin cu
kho st kh nng thu phn du u nnh ca lipase c nh.
Do lipase ch c kh nng thu phn du trn b mt lin pha v
vy chng ti tin hnh kho st s thu phn du n bng cch cho hn
hp du: nc vi cc t l khc nhau 1:1, 2:1, 1:2 chy qua ct c cha
enzym c nh 2 ln vi tc chy 15 git/pht nhit phng v c
b sung CaCl
2
5mM 2%. Sau xc nh ch s axit ca du sau khi thc
hin phn ng thu phn. Kt qu kho st th hin bng 21
Bng 21. nh hng t l du: nc n kh nng thu phn du
ca lipase
Ch s axit T l du: nc
Ln 1 Ln 2
1:1 40,32 60,14
2:1 61,30 82,50
1:2 50,49 70,14

Kt qu bng 21 cho thy vi t l du: nc l 2:1 kh nng thu phn
ca lipase l tt nht cho ch s axit l 82,5. Nn chng ti s s dng
iu kin ny cho thu phn vi lng du ln hn sau ny.
3.5.2. Kho st kh nng s dng lipase tan trong sn xut phomat
Trong qu trnh sn xut phomat t sa ti, giai on ln men
ng t phomat ngi ta s dng renin v mt s proteaza axit tnh hoc
kim tnh lm tc nhn ng t sa. Trong giai on chn c th b
sung lipase nhm lm tng hng v c trng cho phomat.
kho st kh nng s dng lipase trong qu trnh sn xut
phomat, tin hnh th nghim trn 2 mu. Mu 1: sn xut phomat c b
sung 50% renin + 50% proteaza ng t sa. Mu 2: sn xut phomat b
sung 50% renin + 50% proteaza + 0,001% lipase.
C 2 mu c chn nhit 4 10
o
C trong 6 tun. Sau
nh gi cht lng bng phng php cm quan.
nh gi bng phiu cho im (thang im 10) theo cc ch tiu
cht lng phomat. Kt qu biu din bng 22

Bng 22: Kt qu nh gi cht lng phomat bng phng php cm
quan
im Ch tiu
Mu 1
(Khng c lipase)
Mu 2
(c lipase)
Mu sc 1,14 2,21
Mi kem 4,64 5,92
Mi chua 5,85 4,57
Mi i 3,28 1,71
Mi b 3,64 5,00
Mi lactic 4,00 5,50
Mi l 1,00 0,35
V ngt 4,07 5,78
V chua 5,57 5,35
V ng 2,14 1,42
V mn 7,78 6,85
chc 4,00 5,92
tan 5,85 4,21
mn 5,78 5,28
dnh 5,28 5,42
Cm gic bo 5,00 6,28
Cht lng tng hp 6,42 7,85

Kt qu nh gi cm quan cc ch tiu ca sn phm phomat c v
khng b sung lipase cho thy cc ch tiu ca sn phm phomat c s dng
lipase u tt hn c v mu sc v hng v so vi phomat khng b sung
lipase.

KT LUN
- iu kin nui cy ti u cho sinh tng hp lipase t Candida rugosa cho
mt lt l:
Mi trng nui gm: KH2PO4: 15 g; K2HPO4:5,5 g; (NH4)2SO4:4g;
MgSO4:1g; FeCl3:10m g; inositon: 0,004mg; biotin: 0,008mg;
Thiamine.HCl: 0,2mg; Du oliu: 1%; axit palmitic: 1.2 g; H2O:
1000ml;
S t bo nm men cy ban u: 2,6.10
6
tb/ml canh trng;
Thi gian nui: 40 gi
- iu kin nui cy ti u cho sinh tng lp lipase t Bacillus sp l: mi
trng BMGY vi thnh phn: (1% (w/v) cao nm men, 2% (w/v) bacto-
peptone, 100 mM kali phosphate, pH 7.0, 4 x 10-5% (w/v) biotin v 1%
(v/v) methanol) 30C lc 220 vng/pht
- Nng cn thch hp cho kt ta thu lipase l 90%. Lipase sau kt ta
cn cho chy sc k trn sc k cho thy lipase sau khi qua ct trao
i ion DEAE - cellulose xut hin 3 pick. Tin hnh xc nh hot
lipase cc phn on. Kt qu cho thy ch c pick 1 v pick 3 c hot
tnh lipase (69UI/ml v 77UI/ml) v Lip1 v Lip3 c khi lng phn t
l 58 v 62 kDa. Lipase sau cc bc lm sch: kt ta etanol, sc k trao
i ion DEAE Cellulose thu c ch phm c hot ring 55,16
U/mg protein, mc tinh sch l 12,5 ln; hiu sut thu hi l 37,06%.
- iu kin ti u c nh lipase trn Nylon-6 l: nng HCl thch
hp cho hot ho cht mang l 3,5N; glutaraldehit l 10%, nng lipase
em c nh l 1mg/ml (550U/ml); s dng dung dch m phosphat pH
7,7.
- Mt s c tnh ca lipase c nh trn Nylon-6
Nhit ti u l 70C, bn vng nhit 40 - 60C, sau 18 gi
hot cn li l 52,4 66,7%.
pH ti u l 7,0; bn vng pH 7 - 7,5; sau 18 gi hot cn 50,6 -
57%.
- Mt s c tnh ca lipase tan t Candida rugosa
Nhit ti u l 60C, bn vng nhit 40 - 50C, sau 18 gi
hot cn li l 50,2 - 59%.
pH ti u l 8,0; bn vng pH 7,5 - 8,0, sau 18 gi hot cn 53,9
- 62%.
im ng in ca lipase l pI = 7,5
nh hng ca cc ion kim loi n hot tnh ca lipase:
- Ion Pb
2+
c ch mnh (lm gim hot ti 33,26%)
- Cc ion Cu
2+
, Fe
3+
, Zn
2+
c ch yu (lm gim hot tnh 13%)
- Cc ion Na
+
, K
+
khng nh hng.
- Cc ion Mg2+, Ca2+ c kh nng hot ho lipase.
- Mt s c tnh lipase t Bacillus sp
Tnh c hiu c cht i vi tributyrin l cao nht: 101 U/ml.
Nhit ti u l 70C (91,1 U/ml).
Lipase c bn nhit, sau 24 gi hot tnh vn cn 63%.
pH 9,5 l thch hp nht cho phn ng, hot tnh t 108,1 U/ml.
pH 9,0 m Tris-HCl v pH 10,0 m glycine khng nh hng ti
bn ca lipase, hot tnh cn li vn t 117%.
Ca
2+
tng hot tnh lipase ln 10-12%, ngc li K
1+
, Mg
2+
, Fe
3+
gim
hot tnh lipase t 11-42%.
Cc dung mi hu c u gim hot tnh lipase. Acetone, 2-propanol
v ethanol gim mt na, methanol gim mt phn t.
Cc cht ty nh hng ln hot tnh lipase. Triton X-100 tng hot tnh
ln 63%. Tween 20 gi nguyn hot tnh (107%). Tween 80 gim mt
na. Cn SDS km hm hot tnh lipase 3%.
- kho st kh nng ng dng ca lipase trong cng nghip dc, thc
phm:
T l du nc l 2:1, tc chy l 15 git/ pht, nng lipase
10U/ml du, nhit phng, thu c du u nnh c ch s axit
80,5.
Vi hm lng lipase l 0,001% b sung vo qu trnh sn xut
phomat thu c cc ch tiu cht lng sn phm tt hn sn phm
phomat khng b sung lipase c mu sc v hng v.