Glucanase

I HC THI NGUYN
TRNG I HC S PHM
---------------------------------
NGUYN VN TUN
TUYN CHN, NUI CY CHNG ASPERGILLUS AWAMORI

SINH TNG HP ENDO--1,4-GLUCANASE V NH GI
TNH CHT L HA CA ENDO--1,4-GLUCANASE
LUN VN THC S KHOA HC SINH HC
THI NGUYN- 2009
S ha bi Trung tm Hc liu i hc Thi Nguyn
http://www.lrc-tnu.edu.vn
I HC THI NGUYN
TRNG I HC S PHM
---------------------------------
NGUYN VN TUN
TUYN CHN, NUI CY CHNG ASPERGILLUS AWAMORI

SINH TNG HP ENDO--1,4-GLUCANASE V NH GI
TNH CHT L HA CA ENDO--1,4-GLUCANASE
Chuyn ngnh: Sinh hc thc nghim

M s: 60. 42. 30
LUN VN THC S KHOA HC SINH HC
NGI HNG DN KHOA HC:
TS. Quyn nh Thi

Thc hin ti: Vin Cng ngh Sinh hcVin Khoa hc v Cng ngh Vit Nam
THI NGUYN - 2009
LI CM N!
Trc ht ti xin gi li cm Su sc ti TS. Quyn nh Thi, Trng
phng Cng ngh Sinh hc Enzyme, Ph vin trng Vin Cng ngh Sinh
hc, Vin Khoa hc v Cng ngh Vit Nam, nh hng nghin cu,
hng dn th nghim, sa lun vn v to mi iu kin v ho cht cng
nh trang thit b nghin cu ti hon thnh lun vn.
Ti xin chn thnh cm n tp th cn b Phng Cng ngh Sinh hc
Enzyme, Vin Cng ngh Sinh hc gip ti tn tnh trong sut qu trnh
lm lun vn.
Ti xin chn thnh cm n Khoa Sinh hc, Khoa Sau i hc, trng
i hc S phm, i hc Thi Nguyn cng cc thy c gio nhit tnh
ging dy v to iu kin cho ti hon thnh kho hc.
Ti cng xin chn thnh cm n B mn Ho- Sinh hc, Ban ch nhim
khoa Khoa hc C bn, Trng i hc Nng Lm, i hc Thi Nguyn
to iu kin cho ti i hc.
Cui cng, ti xin chn thnh cm n nhng ngi thn trong gia nh
v bn b gip v ng vin ti trong sut thi gian hc tp.
Thi Nguyn thng 9 nm 2009

Hc vin
Nguyn Vn Tun
CC CH VIT TT
APS
Ammonium persulphate
BSA
Bovine serum albumin
CMC
Carboxyl methyl cellulose
cs
Cng s
DNA
Deoxyribonucleic acid
DEAE
Diethylaminoethyl
i chng
EDTA
Ethylene diamine tetraacetic acid
IU
International unit
kb
Kilo base
kDa
Kilo Dalton
LB
Luria and Bertani
Marker
Nxb
Nh xut bn
OD
Optical density
PCR
Polymerase chain reaction
rRNA
Ribosome ribonucleic acid
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
TBE
Tris base-Boric acid-EDTA
TE
Tris-EDTA
v/v
Volume/volume
w/v
Weight/volume
MC LC
M U ................................................................................................................ 1
CHNG 1. TNG QUAN TI LIU ............................................................... 3
1.1
KHI NIM V CELLULASE V ENDO--1,4-GLUCANASE ............ 3
1.2
NGUN GC V PHN LOI ENDO--1,4-GLUCANASE .................. 4

1.2.1 Ngun gc ............................................................................................. 4
1.2.2 Phn loi................................................................................................ 6
1.3
CU TRC CA ENDO--1,4-GLUCANASE .......................................... 7

1.3.1 Cu trc bc mt.................................................................................... 7
1.3.2 Cu trc khng gian ............................................................................... 9
1.3.3 Cu trc ca trung tm xc tc v vng lin kt c cht......................... 9
1.4
C CH XC TC CA ENDO--1,4-GLUCANASE ........................... 11
1.5
NG DNG CA ENDO--1,4-GLUCANASE ....................................... 12

1.5.1 Trong cng nghip thc phm ............................................................. 12
1.5.2 Trong cng nghip sn xut thc n gia sc ......................................... 13
1.5.3 Trong cng nghip sn xut dung mi hu c...................................... 15
1.5.4 Trong cng nghip sn xut giy v bt giy ....................................... 15
1.5.5 Trong cng nghip sn xut cht ty ra .............................................. 16
1.5.6 Trong cng ngh x l rc thi sn xut phn bn vi sinh .................... 16
1.6
NH HNG CA IU KIN MI TRNG N KH NNG
SINH TNG HP ENDO--1,4-GLUCANASE ....................................................... 17

1.7
TNH CHT L HA CA ENDO--1,4-GLUCANASE ...................... 20
1.8
MT S NGHIN CU C LIN QUAN VIT NAM ...................... 22
CHNG 2. NGUYN LIU V PHNG PHP ........................................ 25

2.1
NGUYN LIU V HA CHT ............................................................... 25

2.1.1 Chng nm .......................................................................................... 25
2.1.2 Thit b th nghim .............................................................................. 25
2.1.3 Ha cht .............................................................................................. 26
2.1.4 Mi trng .......................................................................................... 26
2.1.5 Dung dch v m................................................................................ 27

2.2
PHNG PHP ........................................................................................... 28

2.2.1 Nui cy vi sinh vt ............................................................................. 28
2.2.2 Xc nh hot tnh endoglucanase ........................................................ 28
2.2.3 Kho st nh hng ca mt s yu t ln kh nng sinh tng hp
endoglucanase ................................................................................................ 30
2.2.4 Tinh sch enzyme ................................................................................ 32
2.2.5 in di SDS-PAGE ............................................................................. 33
2.2.6 Xc nh tnh cht l ha ca endoglucanase ....................................... 34
2.2.7 Xc nh hm lng protein tng s ..................................................... 35
2.2.8 Cc phng php sinh hc phn t ...................................................... 36
2.2.9 X l s liu ........................................................................................ 41
CHNG 3. KT QU V THO LUN ....................................................... 42

3.1
TUYN CHN V PHN LOI CHNG ASPERGILLUS AWAMORI
SINH TNG HP ENDOGLUCANASE CAO ........................................................ 42

3.1.1 Tuyn chn .......................................................................................... 42
3.1.2 Phn loi chng nm si da vo phn on gene 28S rRNA .............. 43
3.2
TI U CC IU KIN SINH TNG HP ENDOGLUCANASE .... 45

3.2.1 Kh nng sinh tng hp endoglucanase theo thi gian ......................... 45
3.2.2 Nhit nui cy ................................................................................ 47
3.2.3 nh hng ca nng c cht cm ng ............................................ 48
3.2.4 nh hng ca ngun carbon v nng ngun carbon ...................... 50
3.2.5 nh hng ca ngun nitrogen v nng ngun nitrogen ................. 52
3.2.6 pH mi trng nui cy ban u .......................................................... 54
3.3
TINH SCH ENDOGLUCANASE ............................................................ 55

3.3.1 Tinh sch qua ct sc k lc gel sephadex G-100 ................................ 55
3.3.2 Tinh sch qua ct sc k trao i ion DEAE ........................................ 56
3.4
NHNG TNH CHT L HA CA ENDOGLUCANASE ................. 57

3.4.1 nh hng ca nng c cht .......................................................... 57
3.4.2 Nhit phn ng ti u ..................................................................... 58

3.4.3 pH phn ng ti u .............................................................................. 60
3.4.4 bn nhit ................................................................................... 61
3.4.5 bn pH ........................................................................................... 63
3.4.6 nh hng ca dung mi hu c ......................................................... 64
3.4.7 nh hng ca ion kim loi................................................................. 65
3.4.8 nh hng ca mt s cht ty ra...................................................... 67
KT LUN V KIN NGH ............................................................................. 69
TI LIU THAM KHO ................................................................................... 71
PH LC ............................................................................................................. 79
DANH MC CC BNG
Bng 2.1. Cc thit b chnh c s dng trong th nghim ................................. 25
Bng 2.2. Danh sch ha cht chnh c s dng trong th nghim ..................... 26
Bng 2.3. Danh sch dung dch v m s dng trong th nghim ......................... 27
Bng 2.4. Thnh phn gel in di bin tnh protein ............................................... 33
Bng 3.1. Hot tnh endoglucanase ca 26 chng A. awamori ............................... 43
Bng
3.2.
Trnh
nucleotide
on
gene
28S
rRNA
chng
A. awamori VTCC-F-099 ...................................................................................... 79

Bng 3.3. Kh nng sinh tng hp endoglucanase theo thi gian .......................... 79
Bng 3.4. nh hng ca nhit nui cy .......................................................... 80
Bng 3.5. nh hng nng c cht cm ng .................................................... 80
Bng 3.6. nh hng ca ngun carbon ................................................................ 50
Bng 3.7. nh hng ca nng li ng ............................................................ 80
Bng 3.8. nh hng ca ngun nitrogen ............................................................. 52
Bng 3.9. nh hng ca nng ammonium acetate .......................................... 81
Bng 3.10. nh hng ca pH mi trng nui cy ban u ................................ 81
Bng 3.11. Hot tnh endoglucanase cc phn on qua ct Sephadex G-100 ....... 81
Bng 3.12. Hot tnh endoglucanase cc phn on qua ct sc k DEAE ............ 82
Bng
3.13.
Tm
tt
qu
trnh
tinh
sch
endoglucanase
chng
A. awamori VTCC-F-099 ...................................................................................... 57

Bng 3.14. nh hng ca nng c cht n hot tnh endoglucanase ............. 82
Bng 3.15. nh hng ca nhit phn ng n hot tnh endoglucanase .......... 82
Bng 3.16. nh hng ca pH hn hp phn ng n hot tnh endoglucanase .... 83
Bng 3.17. bn nhit ca endoglucanase ..................................................... 83
Bng 3.18. bn pH ca endoglucanase ............................................................. 84
Bng 3.19. bn pH theo thi gian ca endoglucanase ...................................... 85
Bng 3.20. nh hng ca dung mi hu c n hot tnh endoglucanase............ 86
Bng 3.21. nh hng ca ion kim loi ................................................................ 66
Bng 3.22. nh hng ca mt s cht ty ra n hot tnh endoglucanase ........ 86
DANH MC CC HNH
Hnh 1.1. S thy phn lin kt -1,4-O-glucoside ca cellulase ........................ 3
Hnh 1.2. Trnh t amino acid tng ng vi cu trc bc 2 ca Cel12A t mt s
chng vi sinh vt .................................................................................................... 8
Hnh 1.3. M hnh cu trc khng gian (A); S trung tm xc tc (B)
ca Cel12A t H. grisea ......................................................................................... 9
Hnh 1.4. Cu trc vng CBD ca Cel12A t Humicola grisea ............................. 10
Hnh 1.5. C ch thy phn phn t cellulose (A) v phc h cellulose (B) ca cc
enzyme thuc phc h cellulase ............................................................................. 11
Hnh 2.1. ng chun nng glucose............................................................... 30
Hnh 2.2. Quy trnh tinh sch endoglucanase t chng A. awamori VTCC-F-099 . 33
Hnh 2.3. ng chun Bradford dng BSA lm chun ........................................ 36
Hnh 3.1. Hot tnh endoglucanase ca 26 chng A. awamori nghin cu ............. 42
Hnh 3.2. in di DNA tng s (A); Sn phm PCR (B): vi khun DNA
tch chit t chng A. awamori VTCC-F-099; Sn phm ct vector ti t hp bng
XbaI v XhoI (C). ................................................................................................. 44
Hnh 3.3. Cy phn loi chng A. awamori VTCC-F-099 ..................................... 45
Hnh 3.4. Kh nng sinh tng hp endoglucanase theo thi gian ca chng
A. awamori VTCC-F-099 ..................................................................................... 46
Hnh 3.5. nh hng ca nhit nui cy n kh nng sinh tng hp
endoglucanase ca chng A. awamori VTCC-F-099 ............................................. 48
Hnh 3.6. nh hng ca nng CMC n kh nng sinh tng hp endoglucanase
ca chng A. awamori VTCC-F-099 .................................................................... 49
Hnh 3.7. nh hng ca nng li ng n kh nng sinh tng hp
Hnh 3.8. nh hng ca nng ammonium acetate n kh nng sinh tng hp
endoglucanase ca chng A. awamori VTCC-F-099.............................................. 53
Hnh 3.9. nh hng ca pH mi trng nui cy n kh nng sinh tng hp

Hnh 3.10. in di trn gel polyacrylamide sn phm tinh sch endoglucanase
t chng A. awamori VTCC-F-099 qua ct Sephadex G-100 ............................... 56
Hnh 3.11. in di sn phm tinh sch endoglucanase trn gel
polyacrylamide....... ............................................................................................... 57
Hnh 3.12. nh hng ca nng c cht ln hot tnh endoglucanase t chng
A. awamori VTCC-F-099 ...................................................................................... 58
Hnh 3.13. nh hng ca nhit phn ng ln hot tnh endoglucanase t chng
A. awamori VTCC-F-099 ...................................................................................... 60
Hnh 3.14. nh hng ca pH phn ng ln hot tnh endoglucanase t chng
A. awamori VTCC-F-099 ...................................................................................... 61
Hnh 3.15. nh hng ca nhit ln bn endoglucanase t chng
A. awamori VTCC-F-099 ...................................................................................... 62
Hnh 3.16. nh hng ca pH ti bn endoglucanase t chng
A. awamori VTCC-F-099 ...................................................................................... 63
A. awamori VTCC-F-099 theo thi gian ................................................................ 64
Hnh 3.18. nh hng ca dung mi hu c ln hot tnh endoglucanase t chng
A. awamori VTCC-F-099. ..................................................................................... 65
Hnh 3.19. nh hng ca cht ty ra ln hot tnh endoglucanase t chng
A. awamori VTCC-F-099. ..................................................................................... 68
M U
Cellulose l mt thnh phn quan trng cu to nn lp thnh t bo

thc vt. l mt loi polysaccharide c cu trc phc tp. Vic phn hy
cellulose bng cc tc nhn l ha gp nhiu kh khn, lm nh hng n
tc ca nhiu qu trnh sn xut cng nghip.
Endo--1,4-glucanase (3.2.1.4) l mt trong ba loi enzyme thuc h
enzyme thy phn cellulose (cellulase). Enzyme ny tham gia phn ct
ngu nhin cc lin kt -1,4 glucoside t bn trong cc phn t cellulose v
mt s loi polysaccharide tng t khc to thnh cc oligosaccharide.
Endo--1,4-glucanase c th c sn xut bi rt nhiu loi vi sinh vt
nh vi khun, nm men, nm mc. Ngoi ra, endo--1,4-glucanase cn c
thc vt, ng vt nguyn sinh v mt s loi ng vt khng xng sng
khc. Cc enzyme c ngun gc khc nhau s c cu trc khc nhau. iu
ny dn ti s khc nhau v hot tnh xc tc v iu kin phn ng ti u
ca cc enzyme.
Hin nay, vic s dng cc enzyme nh endo--1,4-glucanase
trong mt s ngnh cng nghip nh: cng nghip thc phm, cng nghip
ch bin thc n gia sc, cng nghip giy, bt git, sn xut dung mi hu
c v x l mi trng l rt quan trng, mang li nhiu gi tr to ln.
Tuy nhin, nhng enzyme v ch phm c lin quan c s dng
trong cc ngnh cng nghip Vit Nam v mt s nc hin nay ch yu
c nhp khu t nc ngoi vi gi thnh cao. V th, vic nghin cu, sn
xut ra cc ch phm enzyme c ngun gc t nhin ang l mt i hi cp
thit i vi nhiu quc gia trn th gii trong c Vit Nam. Nc ta l
mt nc sn xut nng nghip nn ngun nguyn liu dng sn xut cc
enzyme nh endo--1,4-glucanase l rt phong ph. Xut pht t nhng l do
trn v tnh hnh nghin cu ti Vit Nam, chng ti tin hnh thc hin
ti: Tuyn chn, nui cy chng Aspergillus awamori sinh tng hp
endo--1,4-glucanase v nh gi tnh cht l ha ca endo--1,4glucanase vi cc mc tiu: (1) Tuyn chn cc chng Aspergillus awamori
sinh tng hp endo--1,4-glucanase cao v tm mi trng nui cy thch hp
to endo--1,4-glucanase ngoi bo, (2) Tinh sch c endo--1,4glucanase v nh gi mt s tnh cht l ha ca n.
CHNG 1. TNG QUAN TI LIU
2.1 KHI NIM V CELLULASE V ENDO--1,4-GLUCANASE

Cellulase l nhm enzyme thy phn c kh nng ct mi lin kt
-1,4-O-glucoside trong phn t cellulose v mt s c cht tng t khc.
l mt phc h gm nhiu loi enzyme khc nhau v c xp thnh
3 nhm c bn: endo--1,4-glucanase hay carboxymethyl cellulase (CMCase)
(EC 3.2.1.4), exo--1,4-glucanase hay cellobiohydrolase (EC 3.2.1.91) v
-glucosidase hay -D-glucoside glucohydrolase (EC 3.2.1.21) [30], [39].
Mi loi enzyme tham gia thy phn c cht theo mt c ch nht nh v
nh c s phi hp hot ng ca cc enzyme m phn t c cht c
thy phn hon ton to thnh cc sn phm n gin nht.
Endo--1,4-glucanase l loi enzyme tham gia xc tc phn ng
thy phn cc lin kt -1,4 glucoside bn trong cc phn t cellulose v
mt s c cht tng t khc thnh cc sn phm n gin hn (cc
oligosaccharide).
Hnh 1.1. S thy phn lin kt -1,4-O-glucoside ca cellulase
2.2 NGUN GC V PHN LOI ENDO--1,4-GLUCANASE

2.2.1 Ngun gc
Endo--1,4-glucanase c th c tng hp t rt nhiu ngun khc
nhau trong t nhin, trong ch yu c ngun gc t vi sinh vt. Trong
t nhin c rt nhiu chng vi khun, x khun, nm mc v mt s loi nm
men c kh nng sinh tng hp endo--1,4-glucanase.
Nm mc l mt trong nhng i tng vi sinh vt c kh nng
sinh tng hp endo--1,4-glucanase mnh nht. Nhiu chng nm mc thuc
cc chi Aspergillus, Trichoderma, Penicillium, Phanerochaete c
nghin cu l c kh nng sinh tng hp endo--1,4-glucanase mnh nh:
Aspergillus niger [25], [26], A. flavus, A. fumigatus [27] A. terreus [29]
Trichoderma reesei [53], Penicillium persicinum, P. brasilianum [33],
Penicillium sp. [10], Phanerochaete chrysosporium [34].
Bn cnh nm mc, vi khun cng c xem l mt trong nhng i
tng c kh nng sinh tng hp endo--1,4-glucanase kh phong ph. Nhiu
loi vi khun hiu kh c nghin cu l c kh nng sinh tng hp
endo--1,4-glucanase mnh nh Acidothemus cellulobuticus [22], Bacillus
pumilis [32], Cellulomonas flavigena, C. udai [20], [21], Pseudomonas
fluoressens [42]. Khng ch c cc vi khun hiu kh m mt s vi khun
k kh cng c kh nng sinh tng hp endo--1,4-glucanase mnh nh
Clostridium [57].
Nhiu chng x khun thuc chi Actinomyces, Streptomyces cng c
kh nng sinh tng hp endo--1,4-glucanase mnh nh: Actinomyces griseus
[13], Streptomyces reticuli [61].
Ngoi ra, endo--1,4-glucanase cn c sinh tng hp thc vt

Arabidopsis [23], ng vt nguyn sinh v mt s ng vt khng xng
sng khc nh mi [62], ng vt thn mm nh Mytilus edulis [63].
Trong s cc ngun sinh enzyme trn th vi sinh vt c xem l ngun
cung cp enzyme vi nhiu u im ni bt v c tnh cht c o vt xa
so vi enzyme c ngun gc t ng vt, thc vt. V th, chng c
s dng rng ri trong qu trnh sn xut cc ch phm enzyme.
Trc ht, vi sinh vt l ngun nguyn liu v tn sn xut enzyme
vi s lng ln. y cng l ngun nguyn liu m con ngi ch ng to
ra c. Chu k sinh trng ca vi sinh vt ngn (t 16-100 gi). Vi sinh vt
sinh trng, pht trin vi tc cc k nhanh chng, khi lng li nh,
kch thc b, nhng t l enzyme trong t bo tng i ln nn quy trnh
sn xut ch phm enzyme kh d dng, hiu sut thu hi cao. Hn na,
enzyme t vi sinh vt c hot tnh rt mnh, vt xa cc sinh vt khc. i
vi mt s trng hp c th dng 100% sinh khi vi sinh vt lm ngun
enzyme.
Vi sinh vt rt nhy cm i vi tc ng ca mi trng, thnh phn
dinh dng nui chng cng nh mt s tc nhn l ha, c hc khc. Do
c th thay i nhng iu kin nui cy chn nhng chng cho
hm lng enzyme ng k vi hot tnh xc tc cao. C th ni rng, vi
vi sinh vt, ngi ta c th iu khin s tng hp enzyme d dng hn trn
cc i tng khc tng lng enzyme c tng hp hoc tng hp
nh hng enzyme. Tuy vy, trong qu trnh chn ngun nguyn liu t
vi sinh vt, cn lu mt s vi sinh vt c kh nng sinh c t c
bin php x l thch hp.
2.2.2 Phn loi

Theo y ban danh php ca Hip hi Ha sinh v Sinh hc phn t
Quc t, cellulase thuc lp 3 (hydrolase): cc enzyme xc tc phn ng
thy phn, t 2 (glycosidase): thy phn cc lin kt glycoside, nhm 1:
thy phn lin kt O- v S-glycoside (International Union of Biochemistry
and Molecular Biology, 1992; http://afmb.cnrs-mrs.fr/ pedro/CAZY/db.html).
Cellulase l mt phc h gm nhiu loi enzyme khc nhau. Ty theo
quan im ca tng tc gi m cc enzyme thuc phc h cellulase c xp
thnh cc nhm khc nhau. Trc y, cellulase c chia lm hai nhm:
nhm enzyme C1 v nhm enzyme Cx. Cc enzyme C1 c kh nng thy phn
si cellulose t nhin, c tnh c hiu khng r rng. Cc enzyme Cx c
chia thnh hai loi: exo -1,4-glucanase (3.2.1.21) xc tc cho phn ng ct
t gc glucose t u khng kh ca chui cellulose; endo -1,4-glucanase
(3.2.1.4) hot ng ty tin hn, xc tc cho phn ng thy phn lin kt
bn trong phn t cellulose [7].
Hin nay, cellulase c chia lm ba dng: dng 1 l endoglucanase
hoc
1,4--D-glucan-4-glucanohydrolase
hay
carboxylmethylcellulase
(CMCase) (EC 3.2.1.4), dng 2 l exoglucanase bao gm 1,4--D-glucan

glucanohydrolase
(cn
gi
cellodextrinase)
(EC
3.2.1.74)
1,4--D-glucan cellobiohydrolase (cellobiohydrolase) (EC 3.2.1.91), dng 3 l

-glucosidase hoc -glucoside glucohydrolase (EC 3.2.1.21). Endoglucanase
xc tc cho phn ng thy phn cc lin kt bn trong phn t cellulose.
Exoglucanase thy phn cc lin kt u kh v u khng kh ca phn t
cellulose. -glucosidase thy phn cc phn t cellodextrin v cellobiose
thnh glucose [30], [39], [45].
Cc cellulase c ngun gc khc nhau c sp xp thnh cc h.

Trc y, da vo cu trc bc mt ca phn t protein enzyme, cellulase
c chia lm 6 h: A, B, C, D, E v F [35]. Sau , cellulase c chia
thnh 9 h: A, B, C, D, E, F, G, H v I da vo cu trc ca tm xc tc [31].
Hin nay, cc cellulase c sp xp trong 12 h: 5, 6, 7, 8, 9, 10, 12, 44, 45,
48, 61 v 74 [45].
2.3 CU TRC CA ENDO--1,4-GLUCANASE
2.3.1 Cu trc bc mt
Endoglucanase t cc ngun gc khc nhau c thnh phn cu to v
cu trc khc nhau. S khc nhau th hin trc ht s a dng v khi
lng phn t, thnh phn v trt t sp xp ca cc amino acid trn chui
polypeptide. Chng A. oryzae KBN616 c kh nng sinh tng hp hai loi
endoglucanase l Cel A v Cel B. Phn t protein Cel A gm 239 amino acid,
trng lng phn t 31 kDa, c xp vo h cellulase H. Trong khi ,
Cel B c xp vo h cellulase C vi chiu di 416 amino acid v
khi lng phn t 53 kDa [43]. Phn t endoglucanase t A. aculeatus
bao gm 410 amino acid vi khi lng phn t khong 43,7 kDa [59]. Cc
endoglucanase t chng A. niger Z10 c khi lng phn t 83 kDa v
50 kDa [25], t A. terreus l 25 kDa [29], t Trichoderma reesei l 48 kDa v
55 kDa [40], [47], t Bacillus sp. D04 c khi lng 35 kDa [56].
Sandgren v cs (2003) c nhng nghin cu chi tit v cu trc
Cel12A thuc h 12 (GH 12) endoglucanase t nm chu nhit
Humicola grisea. Cel12A ca H. grisea l mt chui polypeptide gm 224
amino acid cu to nn cc chui , v cc vng ni (Hnh 1.2) [55].
Hnh 1.2. Trnh t amino acid tng ng vi cu trc bc 2 ca

Cel12A t mt s chng vi sinh vt [55].
2.3.2 Cu trc khng gian

S khc nhau v cu trc ca cc enzyme cn th hin s sp xp
trong khng gian cc chui polypeptide, cc trung tm xc tc v cc vng
lin kt c cht. Chnh nh s khc nhau v cu trc khng gian ca
cc protein enzyme dn ti s khc nhau v cc tnh cht ha l ca chng.
Phn t Cel12A ca H. grisea cu to bi 1 chui , 2 chui (A v
B). Chui -A c cu to bi 6 di xon (A1-A6), chui -B c cu to
bi 9 di xon (B1-B9). Cc di xon c ni vi nhau bi cc vng ni,
c 4 vng ni ln 29-32, 40-47, 92-96, v 165-169 ni tng ng cc
di xon : B2-A2, A2-A3, A5-B5 v A6-B7 (Hnh 1.3A) [55].
2.3.3 Cu trc ca trung tm xc tc v vng lin kt c cht
Trung tm xc tc ca Cel12A t H. grisea gm 2 amino acid l
glutamic acid E-120 v E-205 (Hnh 1.3B), cn Cel12A t Trichoderma
reesei l E-116 v E-200 [55].
Hnh 1.3. M hnh cu trc khng gian (A); S trung tm xc tc

(B) ca Cel12A t H. grisea [55]
Vai tr lin kt c cht khi tham gia xc tc thy phn cellulose ca cc

cellulase c thc hin nh cc vng lin kt c hiu cellulose bind domain
(CBD). Cc CBD c th nm trong trung tm hot ng ca enzyme hoc
khng. Ty thuc vo tng loi enzyme m vng lin kt ny c cu to
ha hc v cu trc khng gian khc nhau. Cel12A ca H. grisea c 3 vng
lin kt vi s tham gia ca cc amino acid, trong cystein (C175, C206,
C216) ng vai tr trung tm. CBD1 vi C175 lm trung tm nm trn
di xon -A6 lin kt yu vi cc amino acid T85, I123, A144, F173, I177,
v F180 bng tng tc Van der Walls. Lin kt gia cc amino acid trong
CBD1 c kch thc 3,5 n 4,1 (Hnh 1.4A). CBD2 vi C206 lm
trung tm nm trn di xon -B4 lin kt vi cc amino acid Q34, W52,
W54, S63, P65, Y91, A98 v T204. Lin kt gia cc amino acid trong CBD2
c kch thc 3,3 n 4,9 v CBD2 cha trung tm hot ng (Glu 205)
(Hnh 1.4B). CBD3 vi C216 lm trung tm nm trn di xon -A4 lin kt
vi cc amino acid W48, V87, W89, F214 v F219. Lin kt gia cc
amino acid trong CBD3 c kch thc 3,3 n 4,8 (Hnh 1.4C) [55].
Hnh 1.4. Cu trc vng CBD ca Cel12A t Humicola grisea [55]
10
2.4 C CH XC TC CA ENDO--1,4-GLUCANASE
Mi dng enzyme trong phc h cellulase tham gia thy phn phn t
c cht theo mt c ch ring. Tuy nhin, nhng enzyme ny thng
phi hp hot ng thy phn hon ton phn t c cht thnh sn phm
n gin nht l glucose.
Endo--1,4-glucanase tham gia thu phn cc lin kt -1,4 glucoside
bn trong cc phn t cellulose v mt s loi polysaccharide tng t khc.
Sn phm phn ct l cc oligosaccharide. Exoglucanase thy phn cc
lin kt u kh v u khng kh ca phn t c cht, gii phng cc
oligosaccharide, cellobiose v glucose. -Glucosidase thy phn cc phn t
cellodextrin v cellobiose to thnh cc phn t glucose [45], [58] (Hnh 1.5).
Hnh 1.5. C ch thy phn phn t cellulose (A) v phc h cellulose

(B) ca cc enzyme thuc phc h cellulase
11
2.5 NG DNG CA ENDO--1,4-GLUCANASE

Cc cellulase ni chung v endo--1,4-glucanase ni ring c
ng dng rng ri trong nhiu ngnh cng nghip nh: cng nghip
thc phm, cng nghip sn xut thc n gia sc, cng nghip sn xut
dung mi hu c, sn xut cht ty ra, cng nghip giy v bt giy, c bit
trong cng ngh x l rc thi sn xut phn bn vi sinh.
2.5.1 Trong cng nghip thc phm
S dng cc ch phm enzyme c th coi l mt trong nhng phng
hng tin b c trin vng nht ca sn xut nc qu v nc ung khng
cn. Dch qu sau khi p chit thng cha cc thnh phn t bo tht qu v
cc cht s c bn cht polysaccharide lm cho dch c nht cao v mu
c. Glucanase thng c s dng ph v thnh t bo, thy phn cc
polysaccharide lm gim nht ca dch qu to thun li cho qu trnh tch
chit v lm trong. Glucanase kt hp vi cc hemicellulase, pectinase trong
ch phm enzyme Viscozyme 120L c ng dng ch yu x l ph v
mng t bo u tng [14]. Trong qu trnh sn xut nc c rt thng s
dng endoglucanase x l giai on dch ha to ra nhiu pectin hn.
Trong cng ngh sn xut bia, dch ln men ngoi cc thnh phn
ng, protein cn c mt lng khng nh cc phn t khi lng cao nh
cellulose v -glucan, lm nh hng xu n qu trnh lc v cht lng sn
phm. Ngi ta thng s dng -glucanase loi b nhng thnh phn
ny. Cc ch phm nh Finizym 200L gm cc cellulase t A. niger,
-glucanase t Bacillus subtillis, Disporotrichum dimorphosporum c
s dng trong sn xut bia.
Ngoi ra, endoglucanase cn c s dng trong cng ngh sn xut
bnh m, bnh bisqui v thc phm chc nng. Glucanase t Humicola
12
insolens c ng dng trong sn xut bnh m, lm tng mm v xp cho

bnh m. Glucanase t Trichoderma reesei, A. niger c ng dng trong sn
xut fructooligosaccharide. y l mt trong s cc oligosaccharide chc
nng (prebiotic) c sn xut b sung vo khu phn n. Prebiotic c
nhiu li ch v mt dc phm cng nh c nh hng tt n sc khe. Khi
c b sung vo c th ngi v ng vt, prebiotic c nhiu tc ng sinh
l quan trng nh: ngn cn qu trnh xm nhp ca cc vi sinh vt gy bnh,
bo v cc chc nng ca gan, lm gim lng cholesterol trong huyt thanh,
tng kh nng min dch ca c th, khng li bnh ung th, cn tr s
pht trin ca cc vi sinh vt trong khoang ming [38], [64]. Cc ph ph
phm nng nghip nh li ng, b ma, b sn, v tru l c cht l tng cho
vic sn xut cc oligosaccharide. Cc enzyme tt nht s dng cho qu trnh
ny ch yu c ngun gc t nm mc. Hin nay, nhu cu s dng cc
oligosaccharide cc nc trn th gii l rt ln. Ti Nht Bn, nhu cu
hng nm v cc oligosaccharide vo khong 1000-2000 tn/nm [48].
2.5.2 Trong cng nghip sn xut thc n gia sc
Chn nui l mt nhnh quan trng ca ngnh nng nghip Vit Nam.
Thu nhp t chn nui hng nm t 20% tng gi tr kinh t ca ngnh
nng nghip. Chn nui khng nhng cung cp cc loi thc phm nh trng,
tht, sa cho th trng m cn cung cp ngun phn bn rt hu ch cho
ngnh trng trt. y cng l ngun thu nhp ng k gp phn xa i
gim ngho, nng cao i sng cho ngi dn Vit Nam. Tuy nhin, ngnh
nng nghip ni chung v chn nui ni ring ang gp phi nhiu kh khn.
Mt trong nhng vn cn c gii quyt hin nay l lm th no
nng cao nng sut vt nui, tng hiu qu chn nui m vn m bo an ton
cho mi trng xung quanh, c bit l sc khe con ngi.
13
Trc tnh hnh , nhiu nhm gii php c a ra v mt trong

nhng gii php c nhiu nh khoa hc quan tm l s dng cc loi thc
n chn nui c ngun gc sinh hc hoc bn sinh hc nh sn xut v b
sung cc ch phm enzyme vo trong thc n chn nui. B sung ch phm
enzyme vo thc n chn nui mt mt lm tng kh nng tiu ha thc n v
hp th cht dinh dng, t lm tng tc sinh trng pht trin, nng
cao cht lng vt nui. Mt khc, nh tc dng ca enzyme m thc n c
tiu ha trit , khai thc ti a ngun nng lng vn c, tit kim chi ph
chn nui, ng thi lm gim lng phn thi ra ngoi, gp phn quan trng
trong vic gim thiu nhim mi trng.
Thc n gia sc, gia cm c ch bin t cc loi ng cc c cha
nhiu cellulose v glucan. Nhng thnh phn ny thng khng c tiu ha
trit , lm tng nht ca dch d dy. Do chng hn ch s hp thu
cc cht dinh dng, lm gim kh nng tiu ha ca ng vt. B sung
-glucanase vo thc n s lm tng kh nng phn gii cc hp cht trn,
gii phng glucose v cc oligosaccharide, lm gim nht, tng kh nng
hp thu v chuyn ha thc n [14]. Nhiu nghin cu cho thy, khi b sung
glucanase c lp hoc kt hp vi cc enzyme khc vo thc n chn nui
lm tng ng k tc tng trng v gim thiu bnh tt cho vt nui.
Omogbenigun v cs (2004) cho thy, khi b sung t hp ch phm glucanase
v mt s enzyme khc (amylase, invertase, protease, phytase, xylanase)
x l thc n cho ln con 25 ngy tui c tc dng nng cao kh nng
tiu ha so vi i chng l khu phn c s khng b sung enzyme nh sau:
kh nng tiu ha tinh bt tng 87-94%, cc polysaccharide khc
tng 10-18%, phytate tng 59-70% [52].
Tim nng ng dng to ln ca glucanase trong chn nui thu ht s
quan tm ca nhiu cng ty ch bin thc n gia sc, gia cm. Mt s ch
14
phm l t hp cc enzyme khc nhau c th k n nh: Rovazyme G2

(DSM Nutritional Products Ltd, Thy S) l t hp ca 3 loi enzyme
(cellulase, -glucanase v xylanase); Roxazyme G (DSM Nutritional Products
Ltd, Thy S) l t hp ca 7 loi enzyme (cellulase, glucanase, protease,
amylase, pectinase, xylanase, hemicellulase); Natugrain (tp on BASF,
c) l hn hp ca endoxylanase v endoglucanase; Rhodizyme-CF
(Rampart-Power Bangladesh Ltd) l t hp ca 5 loi enzyme (cellulase,
amylase, protease, lipase, pectinase). Ch phm SSF ca M hin ang bn ti
th trng Vit Nam l t hp ca 6 loi enzyme: -glucanase (200 BGU/g),
phytase (1000 PU/g), -amylase (30 FAU/g), pectinase (4000 AJDU/g),
protease (700 HUT/g) v xylanase (100 XU/g) [3].
Vit Nam c mt s nghin cu v ng dng enzyme trong
chn nui. Chu Th Thanh Bnh v cs (2002) nghin cu ng dng cc
chng nm men trong ch bin b thi t hoa qu giu cht s lm thc n
cho gia sc [2].
2.5.3 Trong cng nghip sn xut dung mi hu c
S dng glucanase x l nguyn liu giu cellulose, glucan trc khi
ln men lm tng hiu sut thu hi dung mi ln trung bnh l 1,5%. Nhiu
ch phm enzyme c s dng trong ngnh cng nghip ny nh
neutrase 0,5L c cha -glucanase s dng trong cng nghip sn xut
ethanol. ng thi, nhiu chng vi sinh vt k kh trong chi Clostridium
sinh tng hp glucanase c s dng trong cng ngh ln men sn xut dung
mi hu c, acetic acid [24], sn xut acetone, butanol v isopropanol [28].
2.5.4 Trong cng nghip sn xut giy v bt giy
Trong cng nghip sn xut giy v bt giy, nguyn liu ban u c
nghin c hc v x l ha hc cc si g c tch ring khi nhau
15
chuyn thnh bt giy cha cc si v bt mn. Trong quy trnh sn xut giy
cn loi b lignin khi bt giy, cn cellulose th c gi li. Phng php
thng thng l b sung dung dch chlor hoc chlor diocide. y l mt
phng php tn km v thng gy nhim mi trng do thnh phn chlor
tn d trong nc thi. V th, trong nhng nm gn y mt gii php mi
c a ra thay th phng php truyn thng, l s dng cc
ch phm enzyme trong c glucanase x l bt giy.
Glucanase thng c b sung vo cng on nghin bt giy lm
thay i nh cu hnh ca si cellulose, tng kh nng nghin v tit kim
khong 20% nng lng cho qu trnh nghin c hc. ng thi x l
glucanase trc khi x l ha cht nghin bt ha hc s lm ph v lp v
ngoi ca g, lm tng kh nng khuch tn ca ha cht vo pha trong g,
tng hiu qu kh lignin [14], [15]. c bit trong cng ngh ti ch giy,
glucanase c s dng ty mc in bm trn giy. K thut ny m ra
trin vng y ha hn cho ngnh cng nghip sn xut giy v bt giy
ti sinh [37].
2.5.5 Trong cng nghip sn xut cht ty ra
Cng vi protease, lipase, amylase, cellulase v hemicellulase c
ng dng sn xut cht ty ra. Cellulase s thy phn cc t si ca si
vi l ni bm ca cc phn t bi bn, loi b chng khi qun o. Tnh n
nm 2002 c nhiu cellulase c sn xut dng cho bt git nh:
endoglucanase v exoglucanase t Thermomyces lanuginous [51].
2.5.6 Trong cng ngh x l rc thi sn xut phn bn vi sinh
Vic s dng enzyme quan trng nht trong cng tc bo v mi trng
l s dng enzyme trong x l cht thi, chuyn cc cht thi thnh sn phm
c ch, hoc trong cng ngh ti s dng ph thi, chuyn cc ph thi
16
thnh sn phm c ch. Trong nhiu nm qua c th gii v Vit Nam, cc

chng vi sinh vt sinh tng hp enzyme phn hy cellulose c ng dng
rt c hiu qu x l rc thi sinh hot.
Nm 1999, Nguyn Lan Hng v cs phn lp v tuyn chn c
cc chng vi khun v x khun c hot tnh cellulase, sau b sung vo b
rc thi rt ngn c chu k x l rc thi sinh hot t 5-7 ngy. Nhiu
chng vi khun, x khun v nm c nghin cu v ng dng c hiu
qu trong qu trnh x l rc thi Vit Nam [1], [5], [8], [12].
Nhiu ch phm vi sinh trong c cha h sinh vt sinh tng hp
cellulase c nghin cu v sn xut x l rc thi. Trong ,
ch phm Micromix 3 khi b sung vo b rc thi c thi kh rt ngn
c 15 ngy , gim mt na thi gian ln men so vi i chng. ng thi,
lng mn to thnh khi x l rc bng ch phm Micromix 3 cao hn 29%
v cc cht dinh dng cao hn 10% so vi i chng. Sn phm ca qu
trnh x l rc thi c phi trn v b sung thm mt s vi sinh vt c ch
c nh m to thnh phn bn vi sinh, c s dng rng ri trong nng
nghip gp phn nng cao nng sut cy trng, gim thiu c ngun v
nguy c gy nhim mi trng [1].
Ngoi ra, c nhng nghin cu nh hng ca cellulase n nm
gy bnh cy trng nh cellulase ca Trichoderma harzianum, t ng
dng sn xut thuc bo v thc vt sinh hc [6].
2.6 NH HNG CA IU KIN MI TRNG N KH
NNG SINH TNG HP ENDO--1,4-GLUCANASE
Trong t nhin, a s cc loi vi sinh vt sng hoi sinh, phn gii cc
ngun hp cht hu c c sn trong mi trng thnh cc cht dinh dng
cn thit cho qu trnh sinh trng v pht trin ca mnh. Kh nng
17
sinh trng, pht trin cng nh kh nng sinh tng hp cc enzyme chu s
tc ng ca nhiu yu t mi trng nh: nhit nui cy, pH mi trng,
ngun carbon, ngun nitrogen.
Ngun carbon: cc loi vi sinh vt sinh tng hp endoglucanase c th
s dng nhiu ngun carbon khc nhau ty thuc c im ca tng loi. C
loi ch thch hp vi mt hoc mt s t ngun carbon, c loi th khng i
hi nghim ngt m c kh nng s dng nhiu ngun carbon khc nhau.
Ngun carbon c th n gin nh cc loi ng n, ng i hoc
phc tp nh glucan, tinh bt, cellulose.
Nghin cu ca Tng Th Chnh v cs (1999) cho thy, ngun carbon
thch hp cho sinh trng v sinh tng hp cellulase ca cc chng vi khun
chu nhit phn lp t b rc thi l glucose v CMC. Ngun carbon
thch hp cho s sinh trng v sinh tng hp cellulase ca cc chng x
khun CD6-9 l tinh bt, chng CD9-9 l CMC v saccharose, chng CD5-12
l lactose [5]; cc chng Actinomyces griseus l b ma hoc mn ca. Ngun
carbon thch hp i vi cc chng x khun u m l v lc hoc rm [12].
i vi cc chng nm mc, ngun carbon thch hp cho qu trnh sinh
tng hp cellulase v mt s loi enzyme khc l cc ngun carbon t nhin,
c bit l cc ph ph phm nng nghip. Theo Acharya v cs (2008), ngun
carbon thch hp nht cho cc chng A. niger sinh tng hp endoglucanase l
mn ca [18]. Cn theo kt qu nghin cu ca Ojumu v cs (2003), chng A.
flavus Linn isolate NSPR 101 c kh nng sinh tng hp cellulase khi s
dng mn ca, b ma hay li ng lm ngun c cht, trong mn ca c
xem l ngun c cht ti u.
Cc loi Penicillium pinophilum, P. persicinum, P. brasilianum
sinh tng hp cellulase mnh nht i vi ngun cellulose t nhin, cn i
18
vi ngun carbon xylan hoc birchwood xylan th kh nng sinh tng hp rt

km [33]. Theo kt qu nghin cu ca Trnh nh Kh (2006), ngun carbon
thch hp nht cho chng Penicillium sp. DTQ-HK1 l rm [10].
Ngun nitrogen: cc loi vi sinh vt khc nhau c nhu cu khc nhau
i vi ngun nitrogen. Nhn chung, cc loi u c kh nng s dng c
ngun nitrogen v c v hu c nhng mc ng ha ty thuc tng loi.
Cc chng vi khun v x khun chu nhit sinh tng hp cellulase mnh nht
trong mi trng cha ngun nitrogen l peptone v cao nm men [5].
Ngun nitrogen urea v u nnh nh hng mnh m n qu trnh
sinh tng hp cellulase ca chng A. niger NRRL-363 vi hot tnh cellulase
tng s t 46 n 76 IU/g [11]. Nghin cu ca Narasimha v cs (2006) cho
thy, cc chng A. niger c th s dng nhiu ngun nitrogen khc nhau nh:
urea, peptone, ammonium nitrate. Trong , urea l ngun nitrogen tt nht
cho kh nng sinh tng hp cc cellulase ca cc chng nm ny [49].
Nhit nui cy: Cn c vo s thch nghi nhit sinh trng, cc
loi vi sinh vt c chia lm 3 nhm: cc loi a lnh v chu lnh; cc loi
a m v cc loi chu nhit. Cc loi a lnh c kh nng sinh trng trong
iu kin di 15C, cc loi a m thng sinh trng pht trin tt
khong nhit 20-37C; cn cc loi chu nhit c kh nng sinh trng v
pht trin tt nhit cao trn 50C.
Khi nghin cu cc chng vi khun v x khun chu nhit phn lp
t b rc thi cho thy, cc chng ny sinh tng hp cellulase mnh nht
iu kin nhit 45-55C v c th chu c nhit 65-80C [5].
Nghin cu ca Nguyn Lan Hng v cs (2003) cng cho thy, cc chng
vi khun v x khun a nhit sinh tng hp cellulase cao nht nhit
50C [9]. Chng x khun Actinomyces griseus c kh nng sinh tng hp
19
cellulase ti u nhit 58C [13]. Trong khi , cc chng nm mc N1

v N2 c nhit sinh tng hp cellulase ti u l 31 v 34C [8]. Acharya
v cs (2008) cho rng, cc chng A. niger tng hp cellulase mnh nht khi
ln men trong iu kin 28C, pH 4,0-4,5 [18].
pH mi trng nui cy ban u: pH mi trng ban u nh hng
quan trng n kh nng sinh tng hp cellulase ca cc chng vi sinh vt.
Ty thuc vo tng loi, tng chng m pH mi trng ban u thch hp l
acid, trung tnh hay kim. Cc chng vi khun v x khun chu nhit sinh
tng hp cellulase thch hp vi pH mi trng ban u l 8,0 [5]; cn chng
x khun Actinomyces griseus thch hp vi pH mi trng ban u l 6,7
[13]. i vi cc chng nm mc N1 v N2 sinh tng hp cellulase ti u
mi trng c pH ban u l 4,5 v 5,5 [8]; cc chng A. niger sinh tng hp
cellulase mnh nht trong khong pH mi trng ban u t 6,0-7,0 [19].
Ngoi nhng yu t trn, tc sinh trng v kh nng sinh tng hp
cc loi enzyme ca cc vi sinh vt cn chu s tc ng ca nhiu yu t
khc nh: thi gian nui cy, tc lc v sc kh, lng ging c tip vo
ban u.
2.7 TNH CHT L HA CA ENDO--1,4-GLUCANASE
Ty thuc vo cu trc v ngun gc ca enzyme, hot tnh enzyme t
mnh nht nhit , pH nht nh. bn i vi nhit v pH hay mc
v tnh cht chu nh hng ca cc cht ty ra, dung mi hu c hay
ion kim loi cng c s khc nhau.
nh hng ca nhit : vn tc phn ng do enzyme xc tc
ch tng ln khi tng nhit trong mt gii hn nht nh, cha nh hng
n cu trc enzyme. Hot tnh enzyme t cc i nhit thch hp,
khong nhit thch hp ca nhiu enzyme vo khong 40-50C. nhit
20
cao, enzyme b bin tnh lm hot tnh gim mnh hoc mt hot tnh; cn
nhit thp di 0C, hot tnh enzyme b gim nhiu nhng li c th
phc hi khi a v nhit thch hp [4].
Cellulase t A. niger NRRL-363 hot ng mnh nht 50C [11];
trong khi cellulase t A. niger Z10 l 40C [25], cn hot tnh xc tc ca
endoglucanase III t Trichoderma reesei t ti a 55C [46]. Theo
kt qu nghin cu ca Kiamoto v cs (1996), cc endoglucanase t chng A.
oryzae KBN616 hot ng tt nht trong di nhit 45-55C [43].
nh hng ca pH: Kh nng hot ng ca enzyme cn ph thuc
vo pH mi trng phn ng. Ty thuc vo bn cht ca enzyme m pH
thch hp enzyme hot ng c th trung tnh, kim hoc acid. Theo
nghin cu trc y cho thy, pH ti u cho hot ng ca cellulase t
A. niger NRRL-363 l 5,5 [11]; ca cellulase t A. niger Z10 l 4,5 v 7,5
[25]; ca endoglucanase III t Trichoderma reesei l 4,0-5,0 [46]; ca
endoglucanase t A. oryzae KBN616 l 4,0 v 5,0 [43]. Khi nghin cu nh
hng ca pH ln hot tnh endoglucanase t chng nm a acid A. terreus
M11, Gao v cs (2008) cho rng enzyme ny c kh nng hot ng trong di
pH 2-5, trong pH 2 l tt nht [29].
nh hng ca ion kim loi: Cc ion kim loi c th km hm hoc
hot ha s hot ng ca cc enzyme. Cc ion kim loi nng nng nht
nh c th gy bin tnh v km hm khng thun nghch enzyme. Sharma v
cs (1995) nhn thy, ion Ca2+ lm tng hot tnh cellulase ca Bacillus sp.
D04 ln 40% so vi i chng; cn Mg2+ lm gim nh (hot tnh cn li
92%) v Zn2+ c ch mnh hot tnh enzyme ny (hot tnh cn li bng 37%
so vi i chng) [56]. Theo nghin cu ca Gao v cs (2008), endoglucanase
t A. terreus M11 b gim 77% hot tnh khi vi Hg2+ (2 mM), 59% khi
vi Cu2+ (2 mM) [29].
21
Ngoi ra, cc dung mi hu c, cc cht ty ra cng nh hng mnh

m n hot tnh ca enzyme. Ty thuc vo bn cht ca cc cht trn cng
nh bn cht ca enzyme m tnh cht v mc nh hng ti hot ng
ca enzyme l khc nhau. Nghin cu ca Trnh nh Kh (2006) trn chng
Penicillium sp. DTQ-HK1 cho thy, cc dung mi hu c methanol, ethanol,
isopropanol v acetone u c ch hot ng ca cellulase, c bit l
n-butanol c ch mnh nht, hot tnh cellulase ch cn 33-63%. Cc cht
ty ra tween 20, tween 80, SDS v triton X-100 u lm gim hot tnh
cellulase mc khc nhau, trong SDS lm gim mnh hot tnh
cellulase ch cn 18-34% [10].
2.8 MT S NGHIN CU C LIN QUAN VIT NAM
Cho n nay, Vit Nam c kh nhiu tc gi nghin cu v
cellulase ni chung v endo--1,4-glucanase ni ring. Cc nghin cu c
tin hnh trn nhiu phng din khc nhau lin quan n loi enzyme ny.
Vn mi sinh ngy cng tr nn trm trng trn phm vi ton cu.
Vit Nam, lng rc thi ra ngoi mi trng ngy cng ln, nguy c nhim
mi trng nhiu ni l rt cao. Vic s dng bin php vi sinh hc
trong x l rc thi v ang mang li nhiu gi tr to ln. Tuy nhin,
vic s dng cc vi sinh vt c sn trong t nhin x l rc thi, thi gian
thng ko di gy nn tnh trng nhim mi trng, tn nhiu din tch v
cng sc. x l rc trit hn, gim gi thnh v thi gian x l,
ngoi vic to iu kin thch hp cho vi sinh vt pht trin tt th vic tuyn
chn cc vi sinh vt c kh nng sinh trng nhanh, hot tnh phn gii mnh,
chu c nhit cao b sung vo cc b rc l mt trong nhng hng
nghin cu v ang c nhiu nh khoa hc quan tm.
Nm 1999, Tng Th Chnh v cs tin hnh nghin cu cc iu kin
ln men v nh hng ca cc yu t mi trng n kh nng sinh tng hp
22
cellulase ca mt s chng vi khun a nhit c phn lp t b rc thi,

nhm tm ra iu kin ti u nht cho kh nng sinh tng hp cellulase, ng
dng vo vic x l rc thi cha nhiu cellulose. Kt qu cho thy, cc
chng vi sinh vt nghin cu c kh nng chu c nhit 80C, nhit
ln men ti u t 45C n 55C, pH mi trng ban u thch hp nht l 8.
Ngun carbon tt nht cho sinh trng v sinh tng hp cellulase ca cc
chng vi khun nghin cu l glucose v CMC; ngun nitrogen l peptone v
cao nm men. Cc tc gi cng nghin cu ng thi ca qu trnh
sinh tng hp cellulase v kt qu cho thy, thi gian tch ly cao nht
48 gi ln men [5].
Nguyn c Lng v cs (1999) tin hnh nghin cu kh nng
sinh tng hp cellulase ca Actinomyces griseus. Qua nghin cu tc gi thy
rng kh nng sinh tng hp cellulase ca A. griseus l cao v
kh nng ny t ti u 58C, pH ban u l 6,7, m ban u l 55% vi
thi gian nui cy l 72 gi. Qua nghin cu tc gi cng thy rng
ngun lignocellulose thch hp l b ma hoc mn ca [13]. Cng trong nm
ny, Phm Th Ngc Lan v cs tin hnh nghin cu v tuyn chn c
mt s chng x khun a m phn lp t mn rc mt s ni c kh nng
phn gii cellulose mnh. Trong s 195 chng x khun nghin cu th
cc chng x khun phn lp c t cc mu rm mc v t chn ng rm
c kh nng phn gii cellulose v CMC mnh nht [12].
Trong s cc loi vi sinh vt, nm si l mt trong nhng i tng c
kh nng sinh tng hp cellulase cao. Vic tm ra cc chng nm si c kh
nng phn hy cellulose cao v ti u iu kin sinh tng hp cellulase ca
chng ang l vn c nhiu tc gi quan tm. Nm 1999, ng Minh
Hng nghin cu tuyn chn c hai chng nm si c kh nng phn
23
gii cellulose cao. Tc gi cng nghin cu v tm ra c mt s iu

kin ti u cho kh nng sinh tng hp cellulase ca hai chng nm ny [8].
Nm 2003, Hong Quc Khnh v cs nghin cu kh nng sinh tng
hp v c im ca cellulase t chng A. niger NRRL-363. Qua nghin cu,
tc gi tm ra c mt s thng tin v iu kin c bn cho s tng hp
cellulase ca chng ny trn mi trng tru xay v mt s cht thi
cng nghip nh mt r ng [11].
Bng cch tuyn chn cc chng vi sinh vt chu nhit, phn gii
cellulose cao, L Kim Bng v cs (1999) xy dng c quy trnh
cng ngh sn xut ch phm Micromix 3 b sung vo b rc thi. Nghin
cu cho thy, khi ch phm ny c b sung vo b rc thi c thi kh
rt ngn c 15 ngy , mt na thi gian ln men so vi b i chng
b sung ch phm VSV-xen. Lng mn to thnh ca b b sung
ch phm Micromix 3 cao hn 29%, v cc cht dinh dng cao hn 10% so
vi b i chng [1].
24
CHNG 2. NGUYN LIU V PHNG PHP
3.1 NGUYN LIU V HA CHT

3.1.1 Chng nm
Hai mi su chng A. awamori do Vin Vi sinh vt v Cng ngh
Sinh hc - i hc Quc Gia H Ni cung cp c s dng tuyn chn,
nui cy chng vi sinh vt sinh tng hp endoglucanase cao nht. T thu
enzyme lm nguyn liu cho cc nghin cu tip theo.
3.1.2 Thit b th nghim
Bng 2.1. Cc thit b chnh c s dng trong th nghim
Thit b
Hng sn xut (quc gia)
B n nhit
Trung Quc
Box cy LB-1234
Vit Nam
Cn phn tch BL 150S
Sartorius (Thy S)
H thng chp nh gel
Wealtec (M)
H thng in di ngang
Mupid (Nht bn)
L vi sng
SamSung (Hn Quc)
My ly tm lnh Mikro 22R
Hettich (c)
My quang ph UV-2500
LaboMed Inc (M)
My PCR Mastercycler personal
Eppendorf (c)
My chun pH 827 pH Lab
Metrohm (Thy S)
My lc nui cy
Certomat HK (c)
Ni kh trng ES-315
Tomy (Nht Bn)
T lnh 4C, -20C, -80C
Toshiba, Sanyo (Nht Bn)
25
3.1.3 Ha cht
Bng 2.2. Danh sch ha cht chnh c s dng trong th nghim
Ha cht
Hng sn xut (quc gia)
3,5-dinitrosalicylic acid (DNS)
Fluka
Cao nm men
Difco (M)
DEAE
Heldelberg (c)
Peptone
Merck (c)
SDS
Sigma (M)
Sephadex G100
Pharmacia (M)
Sodium dodecylsulfate
Sigma (M)
Triton X-100
Merck (c)
Tween-20
BioBasic Inc (M)
Tween-80
BioBasic Inc (M)
CMC
Prolabo (Php)
3.1.4 Mi trng
Mi trng Czapek: 0,2% (w/v) NaNO3, 0,1% K2HPO4, 0,05%
MgSO4, 0,05% KCl, 2% Saccharose, pH 6,5. Mi trng c b sung
2% agar.
Mi trng MT1 theo Mandles (g/l): urea 0,3; (NH4)2SO4 1,4;
KH2PO4 2; CaCl2 0,3; MgSO 4.7H2O 0,3; peptone 1; cao nm men 10;
tween 80 1; CMC 10; cc yu t vi lng 0,1% (v/v) bao gm cc mui:
FeSO 4 18 mM; MnSO4 6,6 mM; ZnSO4 4,8 mM, CoCl2 15 mM. pH 7,0
[36], [60].
Mi trng LB (Luria-Bertani): 1% (w/v) peptone; 0,5% (w/v) cao nm
men; 1% (w/v) NaCl; pH 7,0, kh trng, vi mi trng c b sung
26
2% (w/v) agar. i vi vic chn chng mang plasmid, mi trng c

b sung 100 g/ml ampicillin.
3.1.5 Dung dch v m
Bng 2.3. Danh sch dung dch v m s dng trong th nghim
Thnh phn, nng
Dung dch
Dung dch Bradford gc
100 ml 95% ethanol; 350 mg serva blue G; 200 ml

88% phosphoric acid
m Bradford th nghim
425 ml H2O; 15 ml 95% ethanol; 30 ml 88%

phosphoric acid; 30 ml dung dch bradford gc
m in di protein (bin
tnh)
25 mM Tris-HCl; 192 mM glycine; 0,1% SDS,

pH 8,4
m KP (K2HPO4)
0,1 M K2HPO4, pH 6,5
m tra mu protein (bin 10% SDS; 0,05% brommophenol blue; 50%

tnh) 5x
glycerol; 10% 2 mecaptho ethanol pha trong m
0,312 M Tris-HCl, pH 6,8
Dung dch DNS (1000 ml) 205,4 g KNaC4H4O6.4H2O; 4,15 g Na2S2O5; 5,3 g
DNS; 9,9 g NaOH; 3,8 ml phenol
Dung dch I
m Tris HCl 50 mM, pH 8,0; 50 mM NaCl
Dung dch II
m Tris HCl 50 mM, pH 8,0; 1000 mM NaCl
Dung dch III
m 80 mM phosphate, pH 7,5
Dung dch A
m 1,5 M Tris-HCl, pH 8,8
Dung dch APS
10% ammonium persulfate
Dung dch B
m 0,5 M Tris-HCl, pH 6,8
Dung dch C
30% acrylamide; 0,8% bis-acrylamide
Dung dch D
10% SDS; 25 mM EDTA
Dung dch nhum gel

polyacrylamide
0,1% (w/v) coomassie brilliant blue; 30% (v/v)

methanol; 10% (v/v) acetic acid
Dung dch ra PAGE
30% (v/v) methanol; 10% (v/v) acetic acid
Dung dch protease K
20 g/ml protease K
27
Dung dch RNase
10 g/ml RNase
Dung dch ampicillin gc
100 mg/ml ampicillin
Dung dch nhum gel

agarose
0,1 g/ml ethidium bromide
m TE
100 mM Tris- HCl; 1 mM EDTA, pH 8,0
m tra mu DNA 6x
0,09% (w/v) brommophenol blue; 0,09% (w/v)

xylene xyanol (FF); 60% (v/v) glycerol
m TBE 10x
108 g Tris base; 55 g boric acid; 40 ml 0,5 M EDTA

pH 8,0
m cell lysis
10 mM Tris, 100 mM EDTA, 2% (w/v) SDS, pH 8,0
3.2 PHNG PHP

3.2.1 Nui cy vi sinh vt
Nui cy hot ha nm si
Cc chng A. awamori ly t cc ng ging c nui cy trn
mi trng Czapek lng iu kin 30C, pH 6,5, lc 200 vng/pht trong
72 gi.
Nui cy nm thu sinh khi enzyme
Cc chng A. awamori sau khi hot ha c nui cy chm trong
mi trng MT1 vi 0,5% CMC lm c cht, 30C, lc 200 vng/pht. Sau
96 gi nui cy, dch canh trng c ly tm loi sinh khi t bo, thu dch
enzyme th.
3.2.2 Xc nh hot tnh endoglucanase
nh tnh hot tnh endoglucanase
Hot tnh endoglucanase c xc nh tng i theo phng php
khuch tn enzyme trn a thch. a thch cha 0,5% c cht CMC trong
m 100 mM potassium phosphate pH 6,5, dy khong 0,5 cm c c l
28
ng knh 0,5 cm. 50 l dch enzyme vo l ri 12 gi 4C cho enzyme

khuch tn u xung quanh. Sau tip 8 gi 37C, ly ra nhum bng
dung dich 1% lugol v o ng knh vng phn gii c cht.
Hot tnh tng i ca enzyme t l thun vi kch thc ng knh vng
phn gii c cht: Dhalo = D - d (vi D l ng knh vng ngoi v d l
ng knh l) [7].
Xc nh hot endoglucanase
Hot endoglucanase c xc nh chnh xc da vo lng
ng kh to thnh sau phn ng bng phng php o quang ph
theo Miller (1959) [47].
Tin hnh
ng th nghim (lp li 3 ln): 0,1 ml dch enzyme c ht vo ng
nghim, thm 0,4 ml dung dch 0,5% CMC trong m 100 mM potassium
phosphate, pH 6,5. Hn hp c lc u v 50C trong 20 pht, sau
b sung ngay 1,25 ml dung dch thuc th DNS v un si cch thy 5 pht.
ng i chng: 0,1 ml dch enzyme c ht vo ng nghim, thm
1,25 ml dung dch thuc th DNS trong 5 pht, b sung tip 0,4 ml dung
dch 0,5% CMC. Hn hp c lc u v 50C trong 20 pht, sau ly
ra v un si cch thy 5 pht.
Hn hp sau phn ng c o hp th quang ph bc sng
540 nm. Kt qu o hp th quang ph c i chiu vi th chun
nng glucose suy ra lng ng kh tng ng c gii phng ra.
Dng ng chun: Glucose c pha trong nc ct vi nng
chun khc nhau t 0,04-0,18 mg/ml. 2 ml dung dch glucose chun c o
hp ph bc sng 540 nm nh trn. Sau , mi tng quan gia
hp ph v nng glucose c xy dng bng phn mm Microsoft Excel.
29
Kt qu cho thy, ng nng chun glucose tuyn tnh trong di

nng 0,04-0,18 mg/ml (Hnh 2.1).
ng chun c phng trnh: y = 2,6411x - 0,0799. Trong :
y - hp ph bc sng 540 nm (OD540 nm); x - nng glucose (mg/ml)
0.5
y = 2,6411x - 0,0799
0.4
OD 540 nm
R = 0.997
0.3
0.2
0.1
0
0.04
0.08
0.12
0.16
0.2
Glucose (mg/ml)
Hnh 2.1. ng chun nng glucose

n v hot : mt n v hot quc t (IU) c nh ngha l
lng enzyme lm thy phn c cht (CMC) thnh ng kh tng ng
1 mol glucose trong mt pht iu kin th nghm.
3.2.3 Kho st nh hng ca mt s yu t ln kh nng sinh tng hp
endoglucanase
3.2.3.1 Thi gian nui cy
Chng A. awamori VTCC-F-099 c nui cy trong mi trng MT1
c bn cho sinh tng hp endoglucanase, dch canh trng c thu
sau nhng khong thi gian nui cy khc nhau: t 24-240 gi v xc nh
hot tnh endoglucanase.
30
3.2.3.2 nh hng ca nhit mi trng

xc nh nhit nui cy ti u, chng nm nghin cu c nui
trong mi trng MT1 c bn cho sinh tng hp endoglucanase, lc
200 vng/pht 25C, 28C; 30C, 32C v 37C; pH 7,0. Dch canh trng
c thu sau 96 gi nui cy v xc nh hot tnh endoglucanase.
3.2.3.3 nh hng ca nng c cht cm ng
C cht vi mt nng nht nh thng ng vai tr l cht cm ng
cho qu trnh sinh tng hp loi enzyme tng ng. xc nh kh nng
cm ng ca c cht CMC n kh nng sinh tng hp endoglucanase, chng
A. awamori VTCC-F-099 c nui cy chm trong mi trng MT1 c bn
pH 7,0, lc 200 vng/pht 30C, CMC c b sung vo mi trng vi
cc nng khc nhau t 0,2-4%. Sau 96 gi nui cy, thu dch ni v
xc nh hot tnh endoglucanase.
3.2.3.4 nh hng ca ngun carbon v nng ngun carbon
Chng A. awamori VTCC-F-099 c nui cy chm trong mi trng
MT1 c bn vi 2% CMC lm ngun c cht cm ng, nui lc
200 vng/pht 30C, pH 7,0 v ngun cao nm men c thay th bng cc
ngun carbon khc (v c ph, glucose, v lc, v qut, li ng, b ma,
saccharose, lactose, v cm tru, mn ca, x da) vi cng nng . Dch
ni canh trng c thu sau 96 gi xc nh hot tnh endoglucanase
ngoi bo.
Sau khi chn c li ng l ngun carbon thch hp nht cho
kh nng sinh tng hp endoglucanase, li ng c b sung vo mi trng
vi cc nng khc nhau t 0-5% (w/v) xc nh nng ti u.
31
3.2.3.5 nh hng ca ngun nitrogen

MT1 c bn vi 2% CMC lm ngun c cht cm ng, 3% li ng lm
ngun carbon, nui lc 200 vng/pht 30C, pH 7,0 v cc ngun nitrogen
(ammonium sulphate, urea, peptone) c thay th bng cc ngun nitrogen
khc vi cng nng . Dch ni canh trng c thu sau 96 gi nui cy
xc nh hot tnh endoglucanase ngoi bo.
3.2.3.6 nh hng ca pH mi trng
MT1 b sung 2% CMC lm ngun c cht cm ng, 3% li ng lm
ngun carbon v 0,3% ammonium acetate lm ngun nitrogen, nui lc
200 vng/pht 30C, pH ban u thay i t 3,0-8,0. Dch ni mi trng
c thu 96 gi xc nh hot tnh endoglucanase ngoi bo.
3.2.4 Tinh sch enzyme
Dch enzyme sau khi c ly tm 10000 vng/pht trong 10 pht, c
a ln ct sc k lc gel Sephadex G-100 vi tng th tch 10 ml. Ct c
kch thc 2,6 x 60 cm c cn bng vi m 50 mM potassium phosphate,
pH 7,5. Tc dng chy l 25 ml/gi. Thu 20 phn on, th tch
mi phn on 2 ml. Hm lng protein v hot tnh enzyme c xc nh
trong mi phn on.
Dch enzyme sau khi qua ct sc k lc gel Sephadex G-100, chn
nhng phn on c hot tnh endoglucanase cao c a tip ln ct sc k
trao i ion DEAE vi tng th tch 12 ml. Ct c kch thc 2,6 x 60 cm
c cn bng vi m 50 mM Tris-HCl, pH 8,0; 50 mM NaCl, thi mu
bng m 50 mM Tris-HCl, pH 8,0; 1000 mM NaCl. Tc dng chy l
32
20 ml/gi. Thu 20 phn on, th tch mi phn on 2 ml. Hm lng

protein v hot tnh enzyme c xc nh cho mi phn on. Qu trnh
tinh sch endoglucanase c th c tm tt nh s hnh 2.2.
Dch enzyme th
Ly tm 10000 vng/10 pht
Dch trong
Ct Sephadex G100
Ct DEAE
Enzyme tinh sch

Hnh 2.2. Quy trnh tinh sch endoglucanase t chng A. awamori VTCC-F-099
3.2.5 in di SDS-PAGE
Khi lng phn t tng i ca enzyme tch chit v tinh sch
ca cc dch enzyme c nh gi bng phng php in di trn gel
polyacrylamide theo Laemmli [44]. Thnh phn gel in di c th hin nh
bng 2.4.
Bng 2.4. Thnh phn gel in di bin tnh protein
Thnh phn
Gel tch (l)
Gel co (l)
H2O
1940
1180
Dung dch A
1500
Dung dch B
500
Dung dch C
2500
300
Dung dch D
60
20
APS 10%
24
10
TEMED
6031
2015
Tng
33
Bn in di c chy vi cng dng in 18-20 mA cho gel

co v 25-28 mA cho gel tch.
3.2.6 Xc nh tnh cht l ha ca endoglucanase
3.2.6.1 nh hng ca nng c cht
tm nng c cht thch hp dng xc nh hot tnh
endoglucanase, phn ng c thc hin vi cc nng c cht t 0,2-2%
CMC pha trong m 100 mM potassium phosphate, pH 6,5. Hot tnh enzyme
c xc nh sau 20 pht phn ng 50C.
3.2.6.2 Xc nh nhit ti u v bn nhit
Hn hp phn ng ca dch enzyme (0,002 mg protein) vi c cht
1,2% (w/v) CMC pha trong m potassium phosphate pH 6,5 c cc
nhit khc nhau trong khong 30-70C, trong 20 pht tm nhit phn
ng ti u.
xc nh bn nhit ca endoglucanase t chng nm nghin cu,
dch enzyme tinh sch (0,002 mg protein) c cc nhit khc nhau
t 30C n 70C, sau cc khong thi gian: 6; 12; 24; 36; 48; 60 v 72 gi.
Hot tnh cn li ca enzyme c xc nh theo phng php nu trn vi
thi gian phn ng l 20 pht nhit phn ng ti u.
3.2.6.3 Xc nh pH phn ng ti u v bn pH
xc nh pH phn ng ti u cho phn ng xc tc ca enzyme
nghin cu, dch enzyme tinh sch (0,002 mg protein) c vi c cht
CMC pha trong m 100 mM sodium acetate, pH t 3,5-5,5 v 100 mM
potassium phosphate, pH t 6,0-8,0, hn hp phn ng c 50C trong
20 pht.
34
nh gi bn pH ca enzyme nghin cu, trc ht

endoglucanase (0,002 mg protein) c trong cc m c pH thay i trong
khong 3,5-8,0, trong 2 gi 37C. Sau , chn ra khong pH thch hp
tip tc xc nh bn pH theo thi gian: 6, 12, 24, 36, 48, 60, 72 gi.
3.2.6.4 nh hng ca dung mi hu c
nh gi nh hng ca dung mi hu c ln hot tnh
endoglucanase, dch enzyme c vi 10-30% cc loi dung mi hu c:
acetone, ethanol, isopropanol, methanol v n-butanol nhit 37C. Sau
2 gi , hot tnh cn li ca enzyme c xc nh.
3.2.6.5 nh hng ca mt s cht ty ra
Dch enzyme (0,002 mg protein) c vi 0,5-2% cht ty ra tween
20, tween 80, SDS v triton X-100 nhit 37C. Sau 2 gi, hot tnh cn
li c xc nh nh gi nh hng ca chng ln hot tnh
endoglucanase.
3.2.6.6 nh hng ca ion kim loi
nh gi tnh cht v mc nh hng ca ion kim loi ln
hot tnh endoglucanase, dch enzyme (0,002 mg protein) c cng vi
mui ca cc ion kim loi: Ag+, Ca2+, Co2+, Cu2+, Fe3+, K+, Mg2+, Ni2+, Zn2+
v EDTA cc nng t 5-15 mM 37C. Sau 2 gi, hot tnh cn li ca
enzyme c xc nh.
3.2.7 Xc nh hm lng protein tng s
Hm lng protein c xc nh theo phng php Bradford.
Phng php ny da trn nguyn tc: cc protein khi phn ng vi
Coomassie Brilliant Blue s hnh thnh phc hp mu c kh nng hp th
nh sng mnh nht bc sng 595 nm, cng mu t l thun vi
35
nng protein trong dung dch. Hm lng protein tng s c xc nh

da vo th chun protein t dung dch albumin huyt thanh b (Hnh 2.3).
0.6
OD 595 nm
y = 2,5x - 0,0169
R = 0.993
0.4
0.2
0.0
0
0.05
0.1
0.15
0.2
0.25
Hm lng protein (mg/ml)
Hnh 2.3. ng chun Bradford dng BSA lm chun

3.2.8 Cc phng php sinh hc phn t
3.2.8.1 Tch chit DNA tng s ca nm si
T bo nm si c nui cy trong mi trng Czapek 30C, lc
200 vng/pht, sau 72 gi dch canh trng c ly tm 10.000 vng/pht
trong 10 pht. Ta t bo c nghin nhanh, nh trong ci s cha nitrogen
lng, sau c b sung 600 l m cell lysis v lc nh. B sung 65 l
lysozyme, 37C trong 45 pht, cho 25 l protease K 55C
trong 2-3 gi. B sung tip hn hp chloroform/isoamyl alcohol (24:1) vi
th tch tng ng, trn u trong 5 pht, sau ly tm 12.000 vng/pht
trong 15 pht. Ht 500 l dch ni cha DNA sang ng eppendorf mi v
b sung 500 l isopropanol. Hn hp c o nh v ta DNA -20C
36
trong 2-3 gi. Ta sau khi ly tm c lm kh bng giy thm v b sung

500 l 70% ethanol ra DNA, loi mui v isopropanol. Sau khi ly tm
12.000 vng/pht trong 10 pht, ta c lm kh, ha trong 40 l m TE
v bo qun -20C [54].
3.2.8.2 Khuch i gene bng PCR
on gene 28S rRNA ca chng A. awamori VTCC-F-099 c
khuch i bng k thut PCR, s dng cp mi NL1 (5-GCATATCAA
TAAGCGGAGGAAAAG-3); NL4 (5-GGTCCGTGTTTCAAGACGG-3).
Hn hp phn ng PCR bao gm: 1 l DNA khun; 0,5 l mi
mi loi; 1,2 l 25 mM MgCl2; 1 l Taq polymerase; 1,6 l 2,5 mM dNTP;
2 l m PCR, b sung nc ct ln tng th tch 20 l. Phn ng thc hin
theo chu trnh nhit: 95C/5 pht; 35 chu k: 95C/1 pht, 50C/1 pht,
72C/1 pht; 72C/10 pht. Sn phm PCR c in di trn gel
0,8% agarose.
3.2.8.3 Gn dnh DNA
Phn ng gn dnh sn phm PCR vo vector tch dng pJET 1.2/blunt
c thc hin theo protocol ca hng Fermentas. Hn hp bao gm:
2 l H2O; 5 l m gn dnh 2x; 0,5 l DNA blunting enzyme; 1,5 l
sn phm PCR. Hn hp c trn u v 70C trong 15 pht. Sau
, ly ra b sung tip 0,5 l vector pJET 1.2/blunt v 0,5 l T4 -DNA
ligase. Phn ng gn dnh c thc hin 22C trong 2-3 gi to plasmid
ti t hp.
37
3.2.8.4 Bin np plasmid ha hc

Plasmid c bin np vo t bo kh bin E. coli DH5 theo
phng php sc nhit v ha hc. Mt khun lc E. coli DH5 c cy
chuyn vo 3 ml mi trng LB, lc 200 vng/pht 37C qua m. 100 ml
mi trng LB c tip ging vi 1 ml dch t bo nui qua m,
nui lc 200 vng/pht 37C. Khi OD600 nm t 0,4-0,6 (khong 2-3 gi
nui cy), dch nui cy c thu li v t vo trong lnh 1 gi, ri ly tm
6000 vng/pht trong 10 pht 4C. Ta t bo c ha u trong 100 ml
dung dch 100 mM CaCl2 lnh v trng, 15 pht v ly tm nh trn.
Ta t bo li c ha u trong 40 ml dung dch 100 mM CaCl2 lnh v
trng, khong 1 gi v ly tm 4000 vng/pht trong 10 pht. Ta
t bo c ha vo 500 l dung dch 100 mM CaCl2 lnh, v trng cha
15% glycerol. Dch hn hp t bo v glycerol c chia nh 40 l vo cc
ng eppendorf, dng bin np ngay hoc bo qun -80C.
Nm l plasmid ti t hp c trn vi 40 l dch t bo kh bin,
20-30 pht 4C. Hn hp c gy sc nhit 45 giy 42C. Sau
t ngay vo lnh, 5 pht ri b sung 250 l mi trng LB kh
trng. Sau khi nui lc hn hp 200 vng/pht trong khong 45-60 pht
37C vi 50-200 l dch t bo bin np c cy chi trn mi trng LB
c c cha cht khng sinh ampicillin, qua m 37C.
3.2.8.5 Tch chit DNA plasmid
Mi khun lc bin np pht trin trn a thch c cy vo ng
nghim cha 1,5 ml LB lng b sung 0,1% (v/v) ampicillin, nui lc
qua m 37C, 200 vng/pht. Ta t bo t 1,5 ml dch nui cy (ly tm
6000 vng/pht trong 5 pht) c lc rung khong 30 giy trong 150 l
38
dung dch I thnh dch ng nht, 15-20 pht nhit phng. Sau ,
thm 150 l dung dch II vo hn hp v o trn nh, tip tc b sung
150 l dung dch III vo hn hp v o trn nh. Hn hp c b sung
450 l dung dch chloroform/isoamyl alcohol (24:1), lc rung v ly tm
13.000 vng/pht trong 10 pht. 450 l pha trn c ht sang ng mi,
b sung 320 l isopropanol, lc nh v 5 pht -80C ta DNA plasmid.
Ta DNA plasmid (ly tm 15 pht vi 13.000 vng/pht) c ra vi 500 l
70% ethanol 4C loi mui v isopropanol v c lm kh trong khng
kh nhit phng. Cui cng ta DNA plasmid c ha trong m TE
pH 8,0 hoc nc kh ion v trng, phn tch in di, ct bng enzyme
ct hn ch kim tra phn on chn hoc bo qun -20C [54].
Sau , plasmid ti t hp mang on gene cn tch dng c bin
np li vo t bo E. coli DH5, tch chit v c tinh sch theo protocol:
b sung 0,25 l RNase vo dch cha plasmid, 37C trong 1-2 gi, ly ra
b sung 450 l H2O kh ion v o u hn hp. Tip , b sung 450 l
chloroform/isoamyl alcohol (24:1), o u trong 5 pht. Sau khi ly tm
13.000/pht trong 15-20 pht, dch ni c ht sang ng eppendorf mi v
b sung 500 l isopropanol cng vi 30 l dung dch 3 M CH3COONa.
Hn hp c -20C trong 15-20 pht, sau ly ra ly tm
13000 vng/pht trong 20 pht thu ta. Ta c ra li 2 ln bng
70% ethanol. Sau khi ta c ra sch v lm kh s c ha trong 40 l
H2O. Mu plasmid tinh sch c s dng gii trnh t nucleotide [54].
3.2.8.6 Ct DNA bng enzyme ct hn ch
DNA plasmid ti t hp c ct bng cp enzyme ct hn ch XbaI v
XhoI trong 2 gi 37C kim tra phn on chn. Hn hp phn ng
gm: 3 l DNA plasmid ti t hp; 0,25 l enzyme ct hn ch mi loi; 2 l
39
m Tango Y+; b sung nc kh ion n tng th tch 10 l. Sau phn ng,

sn phm ct c in di trn gel 0,8% agarose kim tra.
3.2.8.7 in di gel agarose
Nguyn l ca phng php ny l da vo c tnh ca DNA l
i phn t tch in m, d trong mi trng c in trng, cc phn t
DNA c kch thc khc nhau di chuyn t cc m sang cc dng vi
tc khc nhau. Bn gel agarose thch hp cho vic lm gi phn tch
cc phn t DNA. in di DNA hoc plasmid c thc hin trn gel
0,8% (w/v) agarose. Agarose c pha trong m 1x TBE, vi sng cho
agarose tan hon ton. Sau ngui xung khong 60C v vo khay
in di ci lc sn. Khi gel ng n nh th g lc ra, t vo bung
in di v tra mu. Sau khi chy in di xong, bn gel c ly ra khi khun
v nhum trong EtBr khong 3-5 pht, EtBr c th ci xen vo trong DNA v
pht sng di nh sng tia cc tm, do c th pht hin ra s hin din
ca DNA.
3.2.8.8 Gii trnh t nucleotide
Sau khi tinh sch plasmid ti t hp, phn on chn c gii trnh t
nucleotide theo nguyn l ca phng php Sanger ci tin da trn cc
dideoxynucleotide. DNA polymerase xc tc gn cc deoxynucleotide vo
u 3-OH mch n DNA ang tng hp, khi gp dideoxynucleotide
nh du hunh quang khng c nhm 3-OH phn ng tng hp s b dng
li. Kt qu l hn hp sn phm sau phn ng bao gm cc polynucleotide
c kch thc hn km nhau mt nucleotide v c pht hin bng mu d
hunh quang trn my c trnh t nucleotide t ng ABI PRISM 3100
Avant Genetic Analyzer. Kt qu c trnh t c phn tch, x l
bng phn mm DNAStar so snh trnh t nucleotide.
40
3.2.9 Phng php x l s liu

Phn mm Microsoft Excel c s dng x l s liu thu c
trong qu trnh thc nghim v tnh gi tr ca cc tham s thng k.
Chng trnh DNAstar c s dng phn tch trnh t nucleotide,
so snh trnh t nucleotide v xy dng cy phn loi chng nm nghin cu.
41
CHNG 3. KT QU V THO LUN
4.1 TUYN CHN V PHN LOI CHNG ASPERGILLUS

AWAMORI SINH TNG HP ENDOGLUCANASE CAO
4.1.1 Tuyn chn
Hai mi su chng A. awamori c nui cy chm trong mi trng
nui cy c bn cho sinh tng hp endoglucanase, 30C, lc 200 vng/pht,
sau 96 gi thu dch enzyme th. S dng phng php khuch tn enzyme
trn a thch nh tnh hot tnh endoglucanase v phng php o hot
endoglucanase chn c chng A. awamori VTCC-F-099 c kh nng
sinh tng hp endoglucanase cao nht trong s 26 chng A. awamori
nghin cu (Hnh 3.1, Bng 3.1). Nh vy, chng A. awamori VTCC-F-099
c chn ti u cc iu kin nui cy v tinh sch, nh gi tnh cht
l ha ca endoglucanase.
Hnh 3.1. Hot tnh endoglucanase ca 26 chng A. awamori nghin cu

42
Bng 3.1. Hot tnh endoglucanase ca 26 chng A. awamori

Chng
Hot tnh
IU/ml
Dhalo
(mm)
A. awamori VTCC-F-014
0,42
12,5
0,38
Hot tnh
Chng
IU/ml
Dhalo
(mm)
0,33
27,0
12,5
0,33
19,0
0,45
15,0
0,23
15,5
0,46
13,5
0,38
9,5
0,38
15,0
0,34
4,0
0,42
18,0
0,40
13,0
0,51
29,0
0,39
14,0
0,45
17,5
0,41
15,5
0,43
15,5
0,48
26,0
0,33
15,0
0,36
19,0
0,47
18,5
0,39
15,0
0,49
28,0
0,46
16,0
0,44
14,0
0,46
14,5
4.1.2 Phn loi chng nm si da vo phn on gene 28S rRNA

Sn phm tch chit DNA tng s ca chng A. awamori VTCC-F-099
c in di kim tra trn gel 0,8% agarose. Kt qu hnh 3.2A cho thy
DNA tng i sch, khng b gy v lng m bo cho cc nghin cu
nhn dng v c trnh t. Phn ng PCR c tin hnh vi cp mi NL1 v
NL4, sau khi in di kim tra, sn phm thu c c kch thc khong hn
600 bp (Hnh 3.2B).
Sn phm PCR sau khi c gn vo vector pJET 1.2 v bin np vo
t bo E. coli DH5 c tch plasmid v ct kim tra bng cp enzyme
ct hn ch XbaI v XhoI. Kt qu nhn c mt on DNA c
kch thc khong hn 600 bp (Hnh 3.2C).
43
bp
1500
1000
750
500
3000
2000
1500
1000
750
500
250
250
614 bp
Hnh 3.2. in di DNA tng s (A); Sn phm PCR (B): vi khun

DNA tch chit t chng A. awamori VTCC-F-099; Sn phm ct vector ti
t hp bng XbaI v XhoI (C). M: Marker.
Plasmid ti t hp c mang on gene mong mun c tch vi s
lng ln v lm sch theo protocol ca Sambrook v Russell (2001) c
trnh t nucleotide. on gene 28S rRNA ca cc chng nm si nghin cu
c xc nh trnh t nucleotide trn my gii trnh t t ng ABI PRISM
3100 Avant Genetic Analyzer. on gene 28S rRNA ca chng A. awamori
VTCC-F-099 c chiu di 614 bp (Bng 3.2).
So snh trnh t on gene 28S rRNA ca chng nm nghin cu vi
mt s trnh t gene 28S rRNA ca nm si cng b trn GenBank cho
thy, chng A. awamori VTCC-F-099 c quan h h hng gn gi vi mt s
i din thuc chi Aspergillus. Phn tch s liu bng phn mm DNAStar
cho thy chng A. awamori VTCC-F-099 c tng ng 99,5% vi chng
A. awamori VTCC-F-296 (GQ903327), 96,9% vi chng A. flavus IFM
54306 (AB363745), 96,6% vi chng A. flavus UWFP 570 (AY216669),
96,1% vi chng A. flavipes UWFP 1022 (AY216668). Trnh t on gene
28S rRNA ca chng A. awamori VTCC-F-099 c ng k trong
Genbank vi m s l GQ892590.
44
Af 45
Af 69
Aa 27
Aa 90
Af 68
1.8
0
Nuc leotide S ubs titutions (x100)
Hnh 3.3. Cy phn loi chng A. awamori VTCC-F-099

Af 45: Aspergillus flavus IFM 54306 (AB363745); Af 69: A. flavus UWFP
570 (AY216669); Aa 27: A. awamori VTCC-F-296 (GQ903327); Aa 90:
A. awamori VTCC-F-099 (GQ892590); Af 68: A. flavipes UWFP 1022
(AY216668)
4.2 TI U CC IU KIN SINH TNG HP ENDOGLUCANASE

4.2.1 Kh nng sinh tng hp endoglucanase theo thi gian
nh gi kh nng sinh tng hp endoglucanase theo thi gian,
chng A. awamori VTCC-F-099 c nui cy trong mi trng MT1
30C, lc 200 vng/pht. Dch canh trng nui cy c thu cc thi gian
khc nhau xc nh hot tnh endoglucanase. Kt qu cho thy, kh nng
sinh tng hp endoglucanase ca chng A. awamori VTCC-F-099 tng dn t
24 gi n 96 gi nui cy. Ti thi im 96 gi chng ny sinh tng hp
endoglucanase mnh nht, hot tnh t 0,55 IU/ml. Sau , hot tnh
endoglucanase gim dn trong cc khong thi gian nui cy tip theo. Sau
120 gi nui cy hot tnh l 0,39 IU/ml, t 71% v n 240 gi hot tnh l
0,33 IU/ml, t 60% so vi hot tnh cc i (Hnh 3.4, Bng 3.3).
45
Hot tnh endoglucanase tng i (%)
120
80
40
0
0
24
48
72
96
120 144 168 192 216 240 264
Thi gian nui cy (gi)
Hnh 3.4. Kh nng sinh tng hp endoglucanase theo thi gian ca

chng A. awamori VTCC-F-099
Kt qu nghin cu ca chng ti cng ph hp vi mt s kt qu
nghin cu trong nc cng nh trn th gii. Theo kt qu nghin cu ca
mt s tc gi trc y cho thy, cc chng thuc Trichoderma harzianam,
Trichoderma spp, Phanerochaete chrysosporium v A. niger sinh tng hp
endoglucanase cao nht sau 96 gi nui cy vi hot tnh enzyme tng ng
l 1,88; 1,53; 2,40 v 0,096 IU/ml [18], [41]. Cc chng nm si phn lp t
rc thi sinh tng hp cellulase mnh nht sau 144 gi nui cy [8], cn ba
chng nm si thuc cc loi Penicillium pinophinum, P. persicinum,
P. brasilianum c kh nng sinh tng hp cellulase mnh nht sau 110-140
gi nui cy vi hot tnh khong 0,4 IU/ml [33], chng Penicillium sp.
DTQ-HK1 sinh tng hp cellulase mnh nht sau 120 gi nui cy [10].
Tng Th Chnh v cs (1999) khi nghin cu mt s chng vi khun v
x khun chu nhit phn lp t b rc thi cho thy, cc chng vi khun
46
sinh tng hp cellulase mnh nht sau 48 gi nui cy, cn cc chng

x khun sinh tng hp cellulase mnh nht sau 48-72 gi nui cy [5].
Nh vy, cc chng nm si thng sinh tng hp endoglucanase chm hn
so vi cc chng vi khun v x khun.
4.2.2 Nhit nui cy
Chng A. awamori VTCC-F-099 c nui cy trong mi trng MT1,
lc 200 vng/pht trong 96 gi, cc nhit khc nhau: 25, 28, 30, 32 v
37C nh gi nh hng ca nhit mi trng nui cy ln kh nng
sinh tng hp endoglucanase ca chng nm ny v tm ra nhit nui cy
ti u. Kt qu trn hnh 3.5 cho thy, hot tnh endoglucanase tng dn trong
di nhit 25C-30C, nhit ti u cho kh nng sinh tng hp
endoglucanase ca chng nm nghin cu l 30C, hot tnh t 0,56 IU/ml.
cc nhit cao hn, hot tnh endoglucanase li gim dn, 37C ch cn
0,44 IU/ml, t 78% so vi hot tnh cc i (Hnh 3.5, Bng 3.4).
Khi nghin cu trn cc chng vi khun v x khun chu nhit
phn lp t b rc thi, Tng Th Chnh v cs (1999) thy rng, cc chng
ny sinh tng hp cellulase mnh nht iu kin nhit 45-55C v c th
chu c nhit 65-80C [5]. Nghin cu ca Nguyn Lan Hng v cs
(1999) cng cho thy, cc chng vi khun v x khun a nhit sinh tng hp
cellulase cao nht nhit 50C [9]. Chng x khun thuc Actinomyces
griseus c kh nng sinh tng hp cellulase ti u nhit 58C [13].
Theo Acharya v cs (2008), cc chng A. niger sinh tng hp cellulase mnh
nht khi ln men trong iu kin 28C, pH 4,0-4,5 [18]. Kt qu nghin cu
ca Trnh nh Kh (2006) trn chng Penicillium sp. DTQ-HK1 cho thy,
chng ny sinh tng hp cellulase mnh nht khi nui cy trong iu kin
30C [10]. Nh vy, chng A. awamori VTCC-F-099 l mt chng a m v
47
trong cc nhit kho st, nhit ti u cho kh nng sinh tng hp
endoglucanase l 30C.
120
80
40
0
20
25
30
Nhit nui cy
35
40
(oC)
Hnh 3.5. nh hng ca nhit nui cy n kh nng sinh tng hp

endoglucanase ca chng A. awamori VTCC-F-099
4.2.3 nh hng ca nng c cht cm ng
C cht vi mt nng nht nh thng ng vai tr l cht cm ng
cho qu trnh sinh tng hp mt loi enzyme tng ng. xc nh kh
nng cm ng ca c cht CMC n kh nng sinh tng hp endoglucanase,
chng nm nghin cu c nui cy trong mi trng c bn cho sinh tng
hp endoglucanase, CMC c b sung vi cc nng khc nhau:
t 0,2-4%, sau 96 gi thu dch canh trng v xc nh hot tnh
endoglucanase. Kt qu cho thy, kh nng sinh tng hp endoglucanase
ca chng A. awamori VTCC-F-099 thay i r rt khi nng CMC trong
mi trng thay i. Hot tnh endoglucanase tng dn trong di nng
48
t 0-2% CMC, t cc i ti nng 2% (0,81 IU/ml). Sau , khi nng

CMC tip tc tng vt qu 2% th hot tnh endoglucanase li gim dn.
nng 3% CMC th hot tnh endoglucanase cn khong 61% (0,49 IU/ml)
v nng 4% hot tnh ch cn 0,42 IU/ml, t khong 52% so vi
hot tnh khi t cao nht (Hnh 3.6, Bng 3.5). Nh vy, CMC vi nng
2% c vai tr cm ng tt nht kh nng sinh tng hp endoglucanase ngoi
bo ca chng A. awamori VTCC-F-099.

120
80
40
0
0
Nng CMC (%)
Hnh 3.6. nh hng ca nng CMC n kh nng sinh tng hp

Khi nghin cu chng Penicillium sp. DTQ-HK1, Trnh nh Kh
(2006) thy rng, chng ny sinh tng hp cellulase mnh nht khi 0,5%
CMC c b sung vo mi trng nui cy [10].
49
4.2.4 nh hng ca ngun carbon v nng ngun carbon

Chng A. awamori VTCC-F-099 c nui cy trong mi trng MT1
cha 2% CMC, cao nm men c thay th bng mt s ngun carbon khc,
30C, lc 200 vng/pht, sau 96 gi. Kt qu cho thy, kh nng
sinh tng hp endoglucanase ca chng nm nghin cu khi s dng cc
ngun carbon khc nhau c s sai khc ng k. Trong , hot tnh enzyme
ny cao nht khi li ng c s dng lm ngun carbon thay cho cao nm
men (0,87 IU/ml), tip theo l b ma (0,75 IU/ml) v glucose (0,70 IU/ml)
(Bng 3.6). Nh vy, vi mc ch tm ngun nguyn liu sn c, nht l
ngun ph ph phm nng nghip th li ng c xem l ngun carbon
thay th thch hp cho chng A. awamori VTCC-F-099 sinh tng hp
endoglucanase.
Bng 3.6. nh hng ca ngun carbon
Hot tnh endoglucanase
Ngun carbon
IU/ml
B ma
0,75 0,146
86
Cao nm men
0,65 0,166
74
Glucose
0,70 0,134
80
Lactose
0,28 0,084
32
Li ng
0,87 0,158
100
Mn ca
0,39 0,101
45
X da
0,54 0,129
62
Sucrose
0,60 0,019
69
V c ph
0,64 0,127
74
V cm tru
0,57 0,052
65
V lc
0,51 0,026
59
V qut
0,65 0,166
74
50
Theo mt s nghin cu trc y, cc chng nm mc sinh tng hp

cc enzyme thuc nhm cellulase mnh trong nhng mi trng c
ngun carbon t nhin nh mn ca, b ma, li ng [18], [50]. mt s
chng Penicillium c kh nng sinh tng hp endoglucanase v -glucosidase
mnh trong mi trng c ngun c cht cellulose t nhin cn i vi
ngun c cht l xylan yn mch v birchwood xylan th hot tnh rt thp
[33]. Chng Penicillium sp. DTQ-HK1 sinh tng hp cellulase mnh nht khi
0,6% rm c s dng nh mt ngun carbon [10].
Sau khi tm c ngun carbon thay th thch hp l li ng, chng ti
tin hnh ti u nng ngun carbon thay th trn. Kt qu nghin cu cho
thy, hot tnh endoglucanase c s thay i ng k khi thay i nng
li ng trong mi trng nui cy. Trong di nng kho st (0,5-5%) th
nng li ng thch hp nht cho kh nng sinh tng hp endoglucanase ca
chng nm nghin cu l 3%, hot tnh t 0,90 IU/ml (Hnh 3.7, Bng 3.7).
120
80
40
0
0
Nng li ng (%)
Hnh 3.7. nh hng ca nng li ng n kh nng sinh tng hp

51
Theo nghin cu ca mt s tc gi th li ng khng ch thch hp cho

kh nng sinh tng hp cc enzyme thuc nhm cellulase m cn l ngun c
cht cm ng cho mt s loi vi sinh vt sinh tng hp mt s enzyme khc
nh xylanase [16], [17].
4.2.5 nh hng ca ngun nitrogen v nng ngun nitrogen
Kt qu kho st nh hng ca cc ngun nitrogen trong mi trng
nui cy ln kh nng sinh tng hp endoglucanase ca chng
A. awamori VTCC-F-099 cho thy, kh nng sinh tng hp endoglucanase
ca chng nm nghin cu khi nui trong mi trng c cha cc ngun
nitrogen khc nhau c s khc nhau ng k. Trong s cc ngun nitrogen
c kho st th ammonium acetate c xem l ngun nitrogen thch hp
nht cho chng A. awamori VTCC-F-099 sinh tng hp endoglucanase
(4,88 IU/ml) (Bng 3.8). y l mt nguyn liu kh d kim nn c chn
l ngun nitrogen thay th cho cc ngun nitrogen c trong mi trng
c bn MT1.
Bng 3.8. nh hng ca ngun nitrogen
Ngun nitrogen
Ammonium acetate
Ammonium nitrate
Ammonium sulphate
Bt c
Bt u tng
Casein
Peptone
Urea

IU/ml
4,88 0,129
3,91 0,104
4,19 0,124
4,10 0,118
3,51 0,115
3,44 0,128
3,67 0,105
3,87 0,077
100
80
86
84
72
71
75
79
Nghin cu ca Tng Th Chnh v cs (1999) cng cho thy, cc ngun

nitrogen hu c (peptone, cao nm men v bt u tng) thch hp cho
52
qu trnh sinh trng v sinh tng hp cellulase ca cc chng vi khun

chu nhit [5]. Bt u tng cng l ngun nitrogen nh hng mnh ti
kh nng sinh tng hp cellulase ca chng A. niger RNNL-363 [11]. Nghin
cu ca Trnh nh Kh (2006) cho thy, chng Penicillium sp. DTQ-HK1
sinh tng hp cellulase mnh nht khi 1,6% bt u tng c s dng nh
ngun nitrogen trong qu trnh ln men [10].
Sau khi tm c ngun nitrogen thay th thch hp l ammonium
acetate, chng ti tin hnh nghin cu nh hng ca nng ngun
nitrogen ny n kh nng sinh tng hp endoglucanase ca chng nm
nghin cu.
120
80
40
0
0
0.1
0.2
0.3
0.4
0.5
0.6
Nng ammonium acetate (%)
Hnh 3.8. nh hng ca nng ammonium acetate n kh nng sinh

tng hp endoglucanase ca chng A. awamori VTCC-F-099
Kt qu nghin cu cho thy, hot tnh endoglucanase ca chng nm
nghin cu c s thay i ng k khi thay i lng amonium acetate trong
thnh phn mi trng. Hot tnh enzyme tng nh trong di nng
53
0,1-0,25%, sau tip tc tng mnh v t cc i ti 0,3% amonium acetate

(4,97 IU/ml). Tip theo, khi nng amonium acetate tip tc tng vt qu
0,3% th hot tnh endoglucanase li gim dn. nng 0,35% ammonium
acetate th hot tnh endoglucanase l 4,03 IU/ml, t 81% v nng
0,5% ammonium acetate th hot tnh ch cn 3,51 IU/ml, t 71% so vi
hot tnh enzyme khi t cc i (Hnh 3.8, Bng 3.9).
4.2.6 pH mi trng nui cy ban u
pH mi trng nui cy ban u l mt trong nhng yu t c nh
hng ln n tc sinh trng v kh nng sinh tng hp cc enzyme ca
cc loi vi sinh vt. Chng A. awamori VTCC-F-099 c nui cy trong mi
trng MT1 cha 2% CMC, 3% li ng, 0,3% ammonium acetate c iu
chnh pH t 3,0 n 8,0 bng cc dung dch HCl/NaOH, lc 200 vng/pht,
30C, trong 96 gi. Kt qu c th hin nh hnh 3.9 v bng 3.10.

120
80
40
0
2
pH
Hnh 3.9. nh hng ca pH mi trng nui cy n kh nng

sinh tng hp endoglucanase ca chng A. awamori VTCC-F-099
54
Kt qu trn hnh 3.9 v bng 3.10 cho thy, kh nng sinh tng hp
endoglucanase tng dn trong di pH 3,0-6,5, t cc i ti pH 6,5
(5,22 IU/ml). Sau , khi pH tip tc tng vt qua pH 6,5 th hot tnh
enzyme bt u gim dn. pH 7 hot tnh l 4,84 IU/ml, t 93% v n
pH 8 th hot tnh enzyme ch l 4,57 IU/ml, t 88% so vi hot tnh cc i.
Nh vy, pH 6,5 l ti u cho chng A. awamori VTCC-F-099 sinh tng hp
endoglucanase.
Nghin cu ca ng Minh Hng (1999) trn mt s chng nm si
cho thy, kh nng sinh tng hp cellulase ca chng mnh nht trong khong
pH 4,5-6,0 [8]. Cc chng A. niger sinh tng hp endoglucanase mnh nht
trong khong pH mi trng ban u t 6,0-7,0 [19]. Trong khi , cc chng
vi khun v x khun chu nhit sinh tng hp cellulase thch hp vi pH
mi trng ban u l 8,0 [5].
4.3 TINH SCH ENDOGLUCANASE
4.3.1 Tinh sch qua ct sc k lc gel sephadex G-100
Dch enzyme sau khi c ly tm 10000 vng/pht trong 10 pht, c
a ln ct sc k lc gel sephadex G-100 vi tng th tch l 10 ml. Tc
dng chy l 25 ml/gi. Thu 20 phn on, th tch mi phn on 2 ml.
Kt qu kim tra hot tnh endoglucanase ca cc phn on (Bng 3.11)
cho thy, endoglucanase tp trung cc phn on t 6 n 16. in di
hnh 3.10 cho thy, cc phn on vn cn kh nhiu bng protein,
tuy nhin xut hin mt bng protein m c khi lng di 35 kDa.
55
kDa
166
66
45
35
Hnh 3.10. in di trn gel polyacrylamide sn phm tinh sch

endoglucanase t chng A. awamori VTCC-F-099 qua ct Sephadex G-100
(1-5: cc phn on 7-11 qua ct sephadex G-100; 6: Marker; 7: dch
enzyme th)
4.3.2 Tinh sch qua ct sc k trao i ion DEAE
Nhng phn on qua ct sephadex G-100 c hot tnh endoglucanase
cao (phn on 6-16) c a tip ln ct sc k trao i ion DEAE vi tng
th tch 12 ml. Tc dng chy l 20 ml/gi. Thu 20 phn on, th tch mi
phn on 2 ml. Kt qu kim tra hot tnh endoglucanase ca cc phn on
cho thy, cc phn on t 3 n 7 c hot tnh ring cao hn hn so vi cc
phn on khc (Bng 3.12). Cc phn on c hot tnh cao: t phn on 3
n phn on 7 c in di kim tra trn gel 12,5% polyacrylamide.
in di hnh 3.11 cho thy, enzyme thu c cc phn on trn l
kh sch. Cc phn on trn u c duy nht mt loi protein c khi lng
phn t khong 32 kDa. Enzyme thu c c sch 3,08 ln so vi
dch enzyme th ban u (Bng 3.13).
56
Bng 3.13. Tm tt qu trnh tinh sch endoglucanase t chng

Bc tinh sch
Hot tnh
endoglucamase
Protein tng s
(mg protein/ml)
IU/ml
IU/mg protein
tinh
sch (ln)
Hiu sut
thu hi (%)
Dch th
0,79
16,69
21,19
100
Sc k lc gel
Sephadex G-100
0,28
6,30
22,44
1,06
76
Sc k trao i
ion DEAE
0,02
1,19
65,30
3,08
kDa
66
45
35
Endoglucanase
(32 kDa)
25
18
14
Hnh 3.11. in di sn phm tinh sch endoglucanase trn gel

polyacrylamide (1: dch enzyme th; 2: marker; 3-7: cc phn on 3-7
qua ct DEAE).
4.4 NHNG TNH CHT L HA CA ENDOGLUCANASE

4.4.1 nh hng ca nng c cht
Khi tng nng c cht th vn tc phn ng tng theo ngha l
hot tnh ca enzyme tng. Tuy nhin, vn tc phn ng t cc i mt
gii hn nng c cht nht nh, nu tip tc tng nng c cht, c th
vn tc phn ng li gim xung [4].
57
120
80
40
0
0
0.5
1.5
2.5
Nng CMC (%)
Hnh 3.12. nh hng ca nng c cht ln hot tnh endoglucanase

t chng A. awamori VTCC-F-099
kho st nh hng ca nng c cht CMC n hot tnh xc tc
ca endoglucanase, phn ng c thc hin nhit 50C, pH 6,5 vi
nng c cht t 0,2-2% CMC. Kt qu cho thy, hot tnh endoglucanase
tng tuyn tnh khi tng nng c cht trong di t 0,2 n 1,2% CMC, t
cc i nng 1,2% CMC. Sau , nng c cht tip tc tng vt qu
1,2% CMC th hot tnh endoglucanase li gim, ch t 75% so vi hot tnh
cc i nng 1,4% CMC v nng 2% CMC th hot tnh ch bng
43% so vi hot tnh cc i (Hnh 3.12, Bng 3.14). Nh vy, nng
c cht 1,2% CMC l ti u cho phn ng xc tc ca endoglucanase t
chng A. awamori VTCC-F-099.
4.4.2 Nhit phn ng ti u
Nhit phn ng nh hng mnh m n hot tnh xc tc ca
enzyme, mi enzyme c mt nhit phn ng thch hp. Trong gii hn
58
nhit cha lm bin tnh enzyme, hot tnh enzyme tng khi nhit tng.
Tuy nhin, khi nhit tng qu gii hn th hot tnh ca enzyme li gim.
kho st nhit ti u ca endoglucanase t chng A. awamori VTCCF-099, phn ng c thc hin cc nhit khc nhau t 30-70C vi
nng c cht ti u 1,2% CMC pha trong m 100 mM potassium
photphate, pH 6,5.
Khi tng nhit t 30 n 50C, hot tnh tng i ca
endoglucanase cng tng dn t 42% n 100%. Sau , khi nhit tip
tc tng vt qu 50C th hot tnh endoglucanase li gim dn. nhit
70C hot tnh ch cn 32% so vi hot tnh cc i (Hnh 3.13, Bng 3.15).
Nguyn nhn c th do khi nhit tng cao lm t gy mt s
lin kt yu trong phn t protein enzyme, lm thay i cu trc ca
phn t ny, c bit l cu trc trung tm hot ng ca enzyme, t
nh hng ti hot tnh xc tc ca enzyme. Nh vy, nhit thch hp
cho phn ng phn gii c cht CMC ca endoglucanase t A. awamori
VTCC-F-099 l 50C, y l mt enzyme a nhit. Chng A. awamori
VTCC-F-099 v endoglucanase ca n c th c s dng vo mt s qu
trnh cng nghip khng i hi iu kin nhit qu cao nh cng nghip
sn xut thc n chn nui, cng nghip x l rc thi, cng nghip giy.
Nhng nghin cu trc y cho thy, Cellulase ca A. niger NRRL363 hot ng mnh nht 50C [11]; trong khi cellulase ca A. niger Z10
l 40C [25], cn hot tnh xc tc ca endoglucanase III t Trichoderma
reesei t cao nht 55C [46]. Theo kt qu nghin cu ca Kitamoto
v cs (1996), cc endoglucanase t chng A. oryzae KBN616 hot ng
tt nht trong di nhit 45-55C [43].
59
120
80
40
0
20
30
40
50
Nhit (oC)
60
70
80
Hnh 3.13. nh hng ca nhit phn ng ln hot tnh

endoglucanase t chng A. awamori VTCC-F-099
4.4.3 pH phn ng ti u
Enzyme rt nhy cm vi pH ca mi trng, do pH c nh hng
rt ln vi tc ca phn ng enzyme. Mi enzyme c mt tr s pH
thch hp cho s hot ng ca n. nh hng ca pH i vi phn ng
enzyme c th do nhiu tc dng khc nhau: pH c th nh hng n tc
phn ng enzyme do tc ng vo trng thi ion ha ca phn t enzyme,
nht l cc nhm hot ng ca enzyme. tm ra khong pH ti u cho
hot ng ca endoglucanase t A. awamori VTCC-F-099, phn ng gia
enzyme v c cht c thc hin nhit ti u 50C, vi nng c cht
ti u 1,2% CMC pha trong cc m sodium acetate v potassium phosphate
c pH thay i trong khong 4,0-8,0.
60
120
80
40
0
3
6
pH
Hnh 3.14. nh hng ca pH phn ng ln hot tnh endoglucanase t

chng A. awamori VTCC-F-099
Kt qu nghin cu cho thy, hot tnh endoglucanase tng mnh
trong khong pH 4,0-5,0. Hot tnh tng i t 17% pH 4,0 v t ti a
pH 5,0 (4,08 IU/ml). Sau , hot tnh tng i gim dn xung cn 82%
pH 5,5 v gim mnh xung 37% pH 8,0 (Hnh 3.14, Bng 3.16).
pH thch hp i vi cellulase t A. niger RNNL-363 l 5,5 [11]; ca
cellulase t A. niger Z10 l 4,5 v 7,5 [25]. Nh vy, endoglucanase t chng
A. awamori VTCC-F-099 hot ng tt trong di pH 5,0-5,5 (100-82%),
thuc loi endoglucanase a acid yu.
4.4.4 bn nhit
Hu nh tt c cc enzyme u b bin tnh t nhiu v nh hng ti
hot tnh di tc ng ca nhit . Nhit c th lm cho mt s lin kt
hydro trong mng li lin kt hydro tham gia vo vic gi vng cu trc
61
ca enzyme b t gy. Mc nh hng ty thuc vo nhit cao

hay thp v thi gian . nh gi bn nhit ca endoglucanase t chng
A. awamori VTCC-F-099, dch enzyme tinh sch c cc nhit khc
nhau trong khong 30-70C, sau nhng khong thi gian nht nh hot tnh
cn li ca enzyme c xc nh.
120
80
40
20
40
Thi gian (gi)
60
80
Hnh 3.15. nh hng ca nhit ln bn endoglucanase t chng

A. awamori VTCC-F-099. (): 30C; (): 40C; (): 50C; (): 60C;
(): 70C
Kt qu trn hnh 3.15 v bng 3.17 cho thy, hot tnh endoglucanase
t chng A. awamori VTCC-F-099 u gim tuyn tnh theo thi gian v
nhit cng cao th hot tnh gim cng mnh. Sau 24 gi 30C v
40C hot tnh enzyme gim nh, hot tnh tng i cn 94% v 85%.
50C hot tnh tng i cn 79% sau 24 gi v gim mnh sau 36 gi ,
cn 70C hot tnh gim mnh ch sau 6 gi . Nh vy, enzyme
nghin cu tng i bn di nhit 30-50C, c th bo qun nhit
62
phng v ng dng trong mt s qu trnh cng nghip khng i hi nhit

qu cao nh cng nghip sn xut giy v bt giy, cng nghip sn xut
thc n cho vt nui, cng nghip x l rc thi.
4.4.5 bn pH
Do pH mi trng c nh hng n bn ca protein enzyme nn
vic la chn m c pH thch hp bo qun enzyme l rt quan trng.
y, bn ca endoglucanase c kho st trong cc m sodium acetate
vi di pH 3,5-5,5 v m potassium phosphate vi di pH 6,0-8,0. Sau 2 gi
vi m 37C, hot tnh enzyme c xc nh.
160
120
80
40
0
C
3.5
4.5
5.5
6.5
7.5
pH

Kt qu nh hnh 3.16 v bng 3.18 cho thy, sau 2 gi ,
endoglucanase t chng A. awamori VTCC-F-099 bn trong di pH 4,0-6,0
(m acetate pH 4,0-5,5; m phosphate pH 6,0). Hot tnh enzyme sau khi
vi cc m c pH 4,0-6,0 gim khng ng k, thm ch hot tnh cn tng
nh sau khi vi m acetate pH 4,5-5,5.
63
Tip theo, m sodium acetate pH 4,0-5,5 v potassium phosphate

pH 6,0 trong s cc pH thch hp cho endoglucanase t chng
A. awamori VTCC-F-099 c chn tip tc kho st bn pH ca
enzyme nghin cu theo thi gian. Kt qu cho thy, hot tnh endoglucanase
c duy tr kh tt trong m sodium acetate pH 4,5-5,5. Sau 24 gi ,
hot tnh tng i gim khng qu 20% so vi nhm i chng (Hnh 3.17,
Bng 3.19). Nh vy, m sodium acetate pH 4,5-5,5 thch hp duy tr
hot tnh endoglucanase trong qu trnh bo qun.
120
80
40
0
0
12
24
36
48
Thi gian (gi)
60
72
84

A. awamori VTCC-F-099 theo thi gian (): pH 4; (): pH 4,5; ():
pH 5,0; (): pH 5,5; (): pH 6,0
4.4.6 nh hng ca dung mi hu c
Dung mi hu c nh hng ln n hot tnh ca enzyme. Khi thm
dung mi hu c vo dung dch enzyme s lm gim hng s in mi
ca dung dch, lm tng lc ht tnh in gia cc phn t protein [14].
64
kho st nh hng ca cc dung mi hu c, dch enzyme c

cng cc dung mi hu c vi cc nng khc nhau 37C. Sau 2 gi,
hot tnh cn li c xc nh. Kt qu cho thy, c 4 dung mi hu c
tt c cc nng kho st u c nh hng c ch hot ng ca
endoglucanase, trong ethanol lm hot tnh endoglucanase gim mnh
nht. Sau 2 gi vi 30% (v/v) ethanol 37C, hot tnh tng i
ca endoglucanase gim ch cn 53% so vi nhm i chng (Hnh 3.18,
Bng 3.20). Nh vy, ethanol l dung mi hu c lm c ch mnh nht v
acetone l dung mi c ch yu nht hot tnh endoglucanase trong 4
dung mi hu c kho st.

120
80
40
0
C
Act
BtOH
EtOH
IsOH
MtOH
Dung mi hu c
Hnh 3.18. nh hng ca dung mi hu c ln hot tnh endoglucanase

t chng A. awamori VTCC-F-099. (): 0%; (): 10%; (): 20%; (): 30%;
Act: Acetone; BtOH: n-Butanol; EtOH: Ethanol; IsoOH: Isopropanol; MtOH:
Methamol.
4.4.7 nh hng ca ion kim loi
nh gi nh hng ca cc ion kim loi n hot tnh
endoglucanase, dch enzyme c thm vo cc ion kim loi khc nhau
vi nng 5, 10 v 15 mM. Sau 2 gi 37C hot tnh endoglucanase cn
65
li c xc nh v so snh vi mu i chng. Kt qu c th hin

nh bng 3.21
Bng 3.21. nh hng ca ion kim loi
Kim loi
Nng (mM)
Ag+
Hot tnh endoglucanase sau 2 gi (%)

5
10
15
74
70
43
2+
97
95
90
Co 2+
99
96
79
Cu 2+
126
137
148
EDTA
146
153
155
Fe 2+
129
120
107
K+
92
86
87
Mn 2+
70
63
54
Ni 2+
96
93
74
Zn 2+
94
79
78
Ca
Kt qu trn bng 3.21 cho thy, tnh cht v mc nh hng ca

cc ion kim loi i vi hot tnh ca endoglucanase t chng A. awamori
VTCC-F-099 l khc nhau. Mt s ion kim loi (Fe2+, Cu2+) v EDTA lm
tng nh hot tnh enzyme. Khi nng cc ion trn thay i th hot tnh ca
endoglucanase cng thay i theo cc chiu hng khc nhau. Khi nng
Cu2+ v EDTA tng t 5 mM ln 15 mM th hot tnh enzyme tng i cng
tng tng ng t 126% ln 148% (i vi Cu2+), t 146% ln 155% (i vi
EDTA). Trong khi , khi nng ca Fe2+ tng t 5 mM ln 15 mM th
hot tnh tng i ca enzyme li gim t 129% xung cn 107%.
Ngc li, cc ion kim loi khc (Ag+, Ca2+, Co2+, K+, Mn2+, Ni2+,
Zn2+) li lm gim hot tnh xc tc ca enzyme nghin cu vi cc mc
66
khc nhau. nng thp, cc ion Ca2+, K+, Co2+, Ni2+, Zn2+ lm gim nh
hot tnh xc tc ca endoglucanase. Khi nng cc ion ny cng tng,
hot tnh enzyme gim cng mnh. Ring ion Ag+ v Mn2+ lm c ch r rt
hot tnh endoglucanase. nng 15 mM, hot tnh tng i ch t 43%
(i vi Ag+) v 54% (i vi Mn2+) so vi mu i chng.
4.4.8 nh hng ca mt s cht ty ra
Mt s loi cht ty ra c c tnh hot ng b mt nn khi c
cng enzyme s lm nh hng n cu trc v hot ng ca enzyme.
nh gi nh hng ca cht ty ra ti hot tnh endoglucanase, dch
enzyme c cng cc dung dch cht ty ra (Tween 20, Tween 80, SDS,
Triton X-100) cc nng khc nhau t 0,5; 1,0; 1,5; 2%. Sau 2 gi
37C hot tnh cn li c xc nh.
Kt qu nghin cu th hin hnh 3.19 v bng 3.22 cho thy, cc
cht ty ra trn u c nh hng c ch hot ng xc tc ca enzyme
nghin cu. tween 20 nng 0,5-1,0% nh hng yu n hot tnh
endoglucanase. Hot tnh tng i gim cn 90-78% so vi i chng sau
khi enzyme cng vi 0,5-1% (v/v) Tween 20. nng 1,5-2% Tween 20,
hot tnh tng i ch cn 75-61%. Tween 80 lm gim hot tnh
endoglucanase t 83 xung 64% khi tng nng t 0,5 ln 2%.
Triton X-100 lm gim mnh hot tnh t 82 xung 59% so vi i chng khi
tng nng t 0,5-2%. Ring i vi SDS, th hot tnh endoglucanase b c
ch r rt. Nng SDS cng cao th hot tnh tng i cng gim, ngay
nng rt thp 0,5% hot tnh ch t 45% cn nng cao 2%
th hot tnh ch cn 16% so vi i chng.
67
120
80
40
0
C
Tween 20
Tween 80
SDS
Triton X-100
Cht ty ra
Hnh 3.19. nh hng ca cht ty ra ln hot tnh endoglucanase t chng

A. awamori VTCC-F-099. (): 0%; (): 0,5%; (): 1%; (): 1,5%; (): 2%.
Thng SDS c ch rt mnh hot ng ca enzyme ni chung do c
kh nng hnh thnh mt lp in tch m xung quanh phn t protein enzyme
v lm thay i cu trc ca phn t enzyme. Do , enzyme mt kh nng
lin kt v phn gii c cht. Khi nghin cu nh hng ca cc cht ty ra
trn ti hot tnh xc tc ca cellulase t chng Penicillium sp. DTQ-HK1,
Trnh nh Kh (2006) cng thy rng c 4 cht trn u l cht c ch
hot ng ca cellulase. Trong , SDS l cht c ch mnh nht, hot tnh
enzyme gim xung cn 18% sau khi vi 2% SDS trong 2 gi 37C [10].
68
KT LUN V KIN NGH
KT LUN
1. Chng Aspergillus awamori VTCC-F-099 c kh nng sinh tng hp
endoglucanase cao nht trong s 26 chng A. awamori c kho st.
Chng ny c nh loi bng ch th gene 28S rRNA. Trnh t
nucleotide on gene 28S rRNA ca chng ny c ng k
trong GeneBank vi m s GQ892590.
2. Chng A. awamori VTCC-F-099 sinh tng hp endoglucanase mnh
nht sau 96 gi nui cy trong mi trng MT1 pH 6,5, lc
200 vng/pht 30C. Nng c cht cm ng ti u l 2% CMC,
ngun carbon v nitrogen thch hp l li ng (3%) v
ammonium acetate (0,3%).
3. tinh sch c endoglucanase c khi lng phn t khong 32 kDa
t chng A. awamori VTCC-F-099 bng phng php s dng kt hp
ct sc k lc gel sephadex-G100 v ct sc k trao i ion DEAE.
Hiu sut thu hi endoglucanase khong 7%, sch 3,1 ln.
4. Nhit v pH phn ng ti u ca endoglucanase t chng
A. awamori VTCC-F-099 l 50C v 5,0. Enzyme ny bn trong di
nhit 30-50C v di pH 4,5-5,5. cc nng 5 mM, 10 mM,
15 mM hot tnh endoglucanase u tng nh di nh hng ca cc
ion kim loi: Fe2+, Cu2+ v EDTA. Ngc li, cc ion Ag+, Ca2+, Co2+,
K+, Mn2+, Ni2+, Zn2+ lm hot tnh endoglucanase gim. c bit l Ag+
lm hot tnh endoglucanase gim mnh, ch cn 43% nng
15 mM sau 2 gi 37C. Cc dung mi: acetone, ethanol, methanol,
iso propanol v n-butanol u lm gim hot tnh endoglucanase.
69
c bit, dung mi 30% methanol lm hot tnh endoglucanase

gim mnh, cn 52% sau khi 2 gi 37C. Cc cht ty ra kho st
u lm gim hot tnh xc tc ca endoglucanase. c bit l SDS
lm gim mnh hot tnh enzyme, ch cn 20% sau khi vi 2% SDS
trong 2 gi 37C.
KIN NGH
1. Th nghim ln men lng ln vi cc ngun c cht sn c, r tin
nh: li ng, bt giy, bt u c, bt tng.
2. Ti u quy trnh tch chit lng ln endoglucanase t mi trng
nui cy.
3. Nhn dng, biu hin gene m ha endoglucanase, xy dng quy trnh
sn xut enzyme ti t hp.
4. To ch phm enzyme b sung vo thc n chn nui, nng cao
cht lng ca vt nui.
70
TI LIU THAM KHO
Ti liu ting Vit

1.
L Kim Bng, L Gia Hy, Tng Th Chnh, Phan Tuyt Minh, L

Thanh Xun, Trn Quang Huy, o Ngc Quang, Phm Th Cc
(1999), S dng vi sinh vt c hot tnh phn gii cellulose cao nng
cao cht lng phn hy rc thi sinh hot v nng nghip. Bo co
khoa hc, Hi ngh Cng ngh Sinh hc ton quc, Nxb Khoa hc v
K thut, H Ni: 546-551.
2.
Chu Th Thanh Bnh, Nguyn Ln Dng, Lng Thy Dng (2002),

"Phn lp, tuyn chn v nghin cu cc chng nm men c kh nng
phn gii cellulose nhm ng dng trong x l b thi hoa qu lm thc
n chn nui." Tp ch Di truyn hc v ng dng, 2: 34-36.
3.
B Nng nghip v Pht trin Nng thn (2006), Danh mc thc n

chn nui, nguyn liu thc n chn nui c nhp khu vo Vit
Nam. S 01/2006/Q-BNN.
4.
Phm Th Trn Chu, Phan Tun Ngha (2006), Enzyme v ng dng,

Nxb Gio dc.
5.
Tng Th Chnh, L Kim Bng, L Gia Hy (1999), Nghin cu sn xut

cellulase ca mt s chng vi sinh vt u nhit phn lp t b rc
thi. Bo co khoa hc, Hi ngh Cng ngh Sinh hc ton quc, Nxb
Khoa hc v K thut, H Ni: 790-797.
6.
Cao Cng, Nguyn c Lng (2003), Kho st qu trnh cm ng

enzyme chitinase v cellulase ca Trichoderma harzianum nh hng
ca hai enzyme ny ln nm bnh Sclerotium rolfsii. Bo co khoa hc,
Hi ngh Cng ngh Sinh hc ton quc, Nxb Khoa hc v K thut,
H Ni: 321-324.
71
7.
Nguyn Ln Dng, Nguyn ng c, ng Hng Min, Nguyn

Vnh Phc, Nguyn nh Quyn, Nguyn Phng Tin, Phm Vn Ty
(1976), Mt s phng php nghin cu vi sinh vt tp 2 v 3, Nxb
Khoa hc v K thut, H Ni.
8.
ng Minh Hng (1999), Nghin cu cc yu t nh hng n kh

nng sinh tng hp cellulase ca mt s chng vi sinh vt x l rc.
Bo co khoa hc, Hi ngh Cng ngh Sinh hc ton quc, Nxb Khoa
hc v K thut, H Ni: 333-339.
9.
Nguyn Lan Hng, Hong nh Ha (2003), H vi khun c hot tnh

thy phn tinh bt, protein, cellulose hoc du lu trong qu trnh
phn hy cht thi hu c. Bo co khoa hc, Hi ngh Cng ngh Sinh
hc ton quc, Nxb Khoa hc v K thut, H Ni: 288-291.
10. Trnh nh Kh (2006), Tuyn chn, nui cy chng vi sinh vt sinh

tng hp cellulase v nh gi tnh cht l ha ca cellulase. Lun vn
Thc s Khoa hc, Trng i hc Khoa hc T nhin, i hc
Quc Gia H Ni.
11. Hong Quc Khnh, Ng c Duy, Nguyn Duy Long (2003),
Kh nng sinh tng hp v c im cellulase ca Aspergillus niger
RNNL-363. Bo co khoa hc, Hi ngh Cng ngh Sinh hc
ton quc, Nxb Khoa hc v K thut, H Ni: 304-307.
12. Phm Th Ngc Lan, Phm Th Ha, L Kim Bng (1999), Tuyn chn
mt s chng x khun c kh nng phn gii cellulose t mn rc.
Bo co khoa hc, Hi ngh Cng ngh Sinh hc ton quc, Nxb
Khoa hc v K thut, H Ni: 177-182.
13. Nguyn c Lng, ng V Bch Hnh (1999), Kh nng sinh tng
hp cellulase ca Actinomyces griseus. Bo co khoa hc, Hi ngh
Cng ngh Sinh hc ton quc, Nxb Khoa hc v K thut, H Ni:
804-809.
72
14. ng Th Thu, L Ngc T, T Kim Anh, Phm Thu Thy, Nguyn

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PH LC
Bng 3.2. Trnh t nucleotide on gene 28S rRNA chng A. awamori
VTCC-F-099
GCATATCAAT
CCTCAGTAAC
AAAGCTGGCT
GATGCTTTGG
GGCCGTCAGA
TCCGTGCCCG
TGGGAATGCA
GCTAAATACT
GATCGAAAGA
GCACGTGAAA
TCGCCCGCGG
CCCCGTGGGC
AGGCCCCTGG
CAGGGGTGCA
TCGGCACGGA
TTGAAACACG
Trnh t nucleotide
AAGCGGAGGA AAAGAAACCA
GGCGAGTGAA GCGGCAAGAG
CCTTCGGAGT CCGCATTGTA
GTGCGGCCCC CGTCTAAGTG
GAGGGTGAGA ATCCCGTCTT
TGTAAAGCTC CTTCGACGAG
GCTCTAAATG GGTGGTAAAT
GGCCGGAGAC CGATAGCGCA
TGAAAAGCAC TTTGAAAGGA
TTGTTGAAAG GGAAGCGCTT
GGTTCAGCCG GCATTCGTGC
GGGCCAGCGT CGGTTTGGGC
AATGTAGTGC CCTCCGGGGC
ATGCGGCCAG CCTGGACCGA
CGCTGGCATA ATGGTCGTAA
GACC
V tr
ACCGGGATTG
CTCAAATTTG
ATTTGCAGAG
CCCTGGAACG
GGGCGGGGTG
TCGAGTTGTT
TTCATCTAAA
CAAGTAGAGT
GAGTTAAACA
GCGACCAGAC
CGGTGTACTT
GGCCGGTCAA
ACCTTATAGC
GGAACGCGCT
ACGACCCGTC
40
80
120
160
200
240
280
320
360
400
440
480
520
560
600
614
Bng 3.3. Kh nng sinh tng hp endoglucanase theo thi gian

Thi gian (gi)
OD1
OD2
OD3
24
48
72
96
0,30
0,39
0,39
0,45
0,31
0,38
0,39
0,43
120
144
168
192
216
240
0,28
0,27
0,21
0,23
0,22
0,22
0,30
0,26
0,30
0,23
0,29
0,24
79
0,31
0,38
0,39
0,44
IU/ml
0,41 0,002
0,49 0,006
0,49 0,002
0,55 0,012
%
74
89
91
100
0,29
0,26
0,25
0,23
0,25
0,23
0,39
0,36
0,35
0,33
0,35
0,33
71
66
64
60
64
60
0,008
0,002
0,043
0,001
0,036
0,011
Bng 3.4. nh hng ca nhit nui cy

Nhit (C)
25
28
30
32
37
OD1
OD2
OD3
0,21
0,33
0,47
0,40
0,33
0,21
0,34
0,49
0,38
0,34
0,25
0,35
0,41
0,39
0,34

IU/ml
%
0,32 0,003
56
0,44 0,013
78
0,56 0,045
100
0,50 0,007
88
0,44 0,007
78
Bng 3.5. nh hng nng c cht cm ng

Nng CMC (%)
OD1
OD2
OD3
0
0,2
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
0,09
0,11
0,16
0,52
0,63
0,62
0,47
0,38
0,32
0,32
0,05
0,07
0,22
0,55
0,65
0,73
0,47
0,39
0,32
0,32
0,05
0,08
0,16
0,51
0,63
0,71
0,48
0,38
0,32
0,32

IU/ml
%
0,15 0,023
19
0,18 0,021
22
0,27 0,035
34
0,64 0,020
79
0,75 0,010
93
0,81 0,056 100
0,58 0,005
72
0,49 0,006
61
0,42 0,003
52
0,42 0,002
52
Bng 3.7. nh hng ca nng li ng

Li ng (%)
OD1
OD2
OD3
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
0,44
0,54
0,60
0,41
0,41
0,53
0,34
0,38
0,38
0,22
0,58
0,49
0,70
0,58
0,60
0,78
0,65
0,60
0,54
0,42
0,53
0,97
0,71
0,79
0,94
1,01
0,83
0,80
0,70
0,64
80

IU/ml
%
0,65 0,067
70
0,78 0,263
87
0,79 0,058
88
0,71 0,186
79
0,76 0,269
85
0,90 0,238 100
0,72 0,251
81
0,71 0,208
79
0,65 0,159
73
0,53 0,211
60
Bng 3.9. nh hng ca nng ammonium acetate

Ammonium acetate (%)
OD1
OD2
OD3
0,10
0,15
0,20
0,25
0,30
0,35
0,40
0,45
0,50
0,55
0,28
0,30
0,30
0,31
0,39
0,34
0,29
0,27
0,24
0,24
0,21
0,23
0,27
0,32
0,34
0,26
0,28
0,28
0,26
0,24
0,25
0,25
0,28
0,35
0,44
0,31
0,30
0,28
0,27
0,26

IU/ml
%
3,44 0,038
69
3,58 0,039
72
3,86 0,014
78
4,25 0,023
85
4,97 0,050 100
4,03 0,040
81
3,90 0,012
78
3,74 0,001
75
3,51 0,012
71
3,40 0,011
68
Bng 3.10. nh hng ca pH mi trng nui cy ban u

pH
OD1
OD2
OD3
3,0
3,5
4,0
4,5
5,0
5,5
6,0
6,5
7,0
7,5
8,0
0,26
0,32
0,34
0,36
0,37
0,38
0,39
0,42
0,38
0,37
0,36
0,33
0,34
0,35
0,38
0,40
0,41
0,42
0,46
0,42
0,40
0,40
0,23
0,26
0,31
0,32
0,33
0,33
0,36
0,37
0,34
0,30
0,31

IU/ml
%
3,67 0,051
70
4,05 0,042
78
4,34 0,021
83
4,58 0,030
88
4,71 0,036
90
4,79 0,040
92
4,93 0,030
94
5,22 0,047 100
4,84 0,040
93
4,62 0,051
88
4,57 0,048
88
Bng 3.11. Hot tnh endoglucanase cc phn on qua ct Sephadex G-100

Phn on
3
4
5
6
7
8
9
10

IU/ml
1,79
1,83
2,01
4,02
4,89
4,52
4,58
7,21
IU/mg protein
213,71
169,95
183,17
330,37
288,36
149,94
78,66
109,57
Phn on
11
12
13
14
15
16
17
18

IU/ml
6,66
6,81
6,57
6,46
4,03
3,70
2,70
2,14
IU/mg protein
80,07
82,83
87,70
106,64
72,49
93,57
103,29
224,31
Bng 3.12. Hot tnh endoglucanase cc phn on qua ct sc k DEAE

Phn on
3
4
5
6
7
8
9
10

IU/ml
0,76
0,73
0,82
1,53
1,30
1,13
1,07
1,78
IU/mg protein
79,74
56,86
55,75
71,65
62,10
57,80
56,16
57,59
Phn on
11
12
13
14
15
16
17
18

IU/ml
2,01
1,91
1,91
1,62
1,01
0,98
0,60
0,49
IU/mg protein
48,75
43,89
40,60
34,68
23,12
30,99
24,09
24,49
Bng 3.14. nh hng ca nng c cht n hot tnh endoglucanase

CMC (%)
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0

(IU/mg protein)
84,15 0,005
119,71 0,007
179,43 0,008
260,87 0,010
295,95 0,013
377,49 0,060
283,28 0,021
232,33 0,009
173,19 0,006
163,45 0,006
%
22
32
48
69
78
100
75
62
46
43
Bng 3.15. nh hng ca nhit phn ng n hot tnh endoglucanase

Nhit
30
37
40
45
50
55
60
65
70

(IU/mg protein)
45,37 0,005
60,67 0,025
93,11 0,004
95,35 0,001
109,09 0,004
64,95 0,006
50,85 0,001
45,76 0,003
34,65 0,004
82
%
42
56
85
87
100
60
47
42
32
Bng 3.16. nh hng ca pH hn hp phn ng n hot tnh endoglucanase

(IU/mg protein)
37,87 0,009
144,65 0,002
226,68 0,002
184,88 0,007
118,54 0,005
95,25 0,004
90,58 0,003
82,20 0,072
84,54 0,010
Nhit
4,0
4,5
5,0
5,5
6,0
6,5
7,0
7,5
8,0
%
17
64
100
82
52
42
40
36
37
Bng 3.17. bn nhit ca endoglucanase

Nhit

Thi gian (gi)
(IU/mg protein)
30
40
0
6
12
24
36
48
60
72
6
12
24
410,23
399,02
388,99
385,77
382,75
285,43
258,34
236,23
368,43
355,86
346,61
0,005
0,001
0,008
0,004
0,002
0,004
0,001
0,006
0,007
0,001
0,005
36
48
60
72
6
12
24
314,85
277,92
254,15
228,53
361,13
324,98
302,38
0,006
0,006
0,007
0,005
0,001
0,004
0,025
83
100
97
95
94
93
70
63
58
90
87
84
77
68
62
56
88
79
74
50
60
70
36
210,12 0,001
48
60
72
6
12
24
36
48
60
150,88
130,33
124,09
255,61
179,33
131,79
91,36
65,83
59,60
0,024
0,005
0,006
0,014
0,009
0,006
0,001
0,008
0,001
72
6
12
24
36
48
60
72
38,94
202,22
143,77
112,50
54,92
26,57
23,94
23,35
0,001
0,002
0,010
0,007
0,002
0,003
0,001
0,000
51
37
32
30
62
44
32
22
16
15
9
49
35
27
13
6
6
6
Bng 3.18. bn pH ca endoglucanase

pH
i chng
6,5
3,5
4,0
4,5
5,0
5,5
6,0
6,5
7,0
7,5
8,0
Acetate
Phosphate
(IU/mg protein)
234,57
56,48
266,53
322,93
347,29
285,43
241,78
229,21
245,38
223,27
210,21
84
0,008
0,028
0,008
0,020
0,004
0,004
0,011
0,008
0,020
0,004
0,004
%
100
24
114
138
148
122
103
98
105
95
90
Bng 3.19. bn pH theo thi gian ca endoglucanase

pH
4,0
4,5
5,0
5,5
6,0
Thi gian (gi)

0
6
12
24
36
48
60
72
0
6
12
24
36
48
60
72
0
6
12
24
36
48
60
72
0
6
12
24
36
48
60
72
0
6
12
24
36
48
60
72

(IU/mg protein)
259,02 0,002
217,52 0,031
166,28 0,005
105,38 0,001
73,33 0,001
67,88 0,004
54,24 0,004
36,70 0,002
322,35 0,016
310,56 0,014
269,06 0,005
267,21 0,006
264,19 0,004
185,66 0,000
122,34 0,009
104,90 0,004
310,76 0,008
301,21 0,002
281,14 0,004
277,53 0,001
275,00 0,004
202,42 0,006
149,52 0,008
116,68 0,003
253,47 0,004
244,02 0,001
217,62 0,008
210,02 0,005
203,59 0,004
174,26 0,006
175,82 0,002
173,58 0,007
209,43 0,001
198,72 0,001
192,58 0,000
143,09 0,008
137,73 0,006
136,56 0,005
125,45 0,005
115,42 0,010
85
%
100
84
64
41
28
26
21
14
100
96
83
83
82
58
38
33
100
97
90
89
88
65
48
38
100
96
86
83
80
69
69
68
100
95
92
68
66
65
60
55
Bng 3.20. nh hng ca dung mi hu c n hot tnh endoglucanase

Dung Mi
i chng
Acetone
n- Butanol
Ethanol
isopropanol
Methanol
Nng (%)
0
10
20
30
10
20
30
10
20
30
10
20
30
10
20
30

(IU/mg protein)
372,91 0,016
349,04 0,031
333,75 0,004
325,95 0,004
287,28 0,003
225,12 0,025
213,53 0,003
229,60 0,001
198,52 0,001
195,79 0,002
365,02 0,050
276,27 0,044
258,24 0,004
307,64 0,001
271,30 0,011
266,82 0,004
%
100
94
89
87
77
60
57
62
53
53
98
74
69
82
73
72
Bng 3.22. nh hng ca mt s cht ty ra n hot tnh endoglucanase

Cht ty ra Nng (%)
i chng
Tween 20
Tween 80
SDS
Triton X-100
0,0
0,5
1,0
1,5
2,0
0,5
1,0
1,5
2,0
0,5
1,0
1,5
2,0
0,5
1,0
1,5
2,0

(IU/mg protein)
213,53 0,004
191,22 0,008
167,83 0,004
160,43 0,006
131,30 0,008
177,48 0,001
160,43 0,004
144,55 0,003
136,56 0,001
97,01 0,005
83,17 0,001
51,70 0,001
33,48 0,004
175,73 0,004
147,67 0,002
134,32 0,003
125,56 0,010
86
%
100
90
79
75
61
83
75
68
64
45
39
24
16
82
69
63
59
87

Glucanase

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I HC THI NGUYN

TUYN CHN, NUI CY CHNG ASPERGILLUS AWAMORI

LUN VN THC S KHOA HC SINH HC

THI NGUYN- 2009

S ha bi Trung tm Hc liu i hc Thi Nguyn

TUYN CHN, NUI CY CHNG ASPERGILLUS AWAMORI

Chuyn ngnh: Sinh hc thc nghim

NGI HNG DN KHOA HC:

TS. Quyn nh Thi

S ha bi Trung tm Hc liu i hc Thi Nguyn

Thi Nguyn thng 9 nm 2009

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Bovine serum albumin

Carboxyl methyl cellulose

Ethylene diamine tetraacetic acid

Luria and Bertani

Polymerase chain reaction

Ribosome ribonucleic acid

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tris base-Boric acid-EDTA

S ha bi Trung tm Hc liu i hc Thi Nguyn

KHI NIM V CELLULASE V ENDO--1,4-GLUCANASE ............ 3

NGUN GC V PHN LOI ENDO--1,4-GLUCANASE .................. 4

CU TRC CA ENDO--1,4-GLUCANASE .......................................... 7

NG DNG CA ENDO--1,4-GLUCANASE ....................................... 12

NH HNG CA IU KIN MI TRNG N KH NNG

SINH TNG HP ENDO--1,4-GLUCANASE ....................................................... 17

TNH CHT L HA CA ENDO--1,4-GLUCANASE ...................... 20

MT S NGHIN CU C LIN QUAN VIT NAM ...................... 22

CHNG 2. NGUYN LIU V PHNG PHP ........................................ 25

NGUYN LIU V HA CHT ............................................................... 25

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2.1.5 Dung dch v m................................................................................ 27

PHNG PHP ........................................................................................... 28

CHNG 3. KT QU V THO LUN ....................................................... 42

TUYN CHN V PHN LOI CHNG ASPERGILLUS AWAMORI

SINH TNG HP ENDOGLUCANASE CAO ........................................................ 42

TI U CC IU KIN SINH TNG HP ENDOGLUCANASE .... 45

TINH SCH ENDOGLUCANASE ............................................................ 55

NHNG TNH CHT L HA CA ENDOGLUCANASE ................. 57

S ha bi Trung tm Hc liu i hc Thi Nguyn

3.4.2 Nhit phn ng ti u ..................................................................... 58

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A. awamori VTCC-F-099 ...................................................................................... 79

A. awamori VTCC-F-099 ...................................................................................... 57

S ha bi Trung tm Hc liu i hc Thi Nguyn

S ha bi Trung tm Hc liu i hc Thi Nguyn

Hnh 3.9. nh hng ca pH mi trng nui cy n kh nng sinh tng hp

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Cellulose l mt thnh phn quan trng cu to nn lp thnh t bo

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CHNG 1. TNG QUAN TI LIU

2.1 KHI NIM V CELLULASE V ENDO--1,4-GLUCANASE

Hnh 1.1. S thy phn lin kt -1,4-O-glucoside ca cellulase

S ha bi Trung tm Hc liu i hc Thi Nguyn

2.2 NGUN GC V PHN LOI ENDO--1,4-GLUCANASE

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Ngoi ra, endo--1,4-glucanase cn c sinh tng hp thc vt

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2.2.2 Phn loi

(CMCase) (EC 3.2.1.4), dng 2 l exoglucanase bao gm 1,4--D-glucan

1,4--D-glucan cellobiohydrolase (cellobiohydrolase) (EC 3.2.1.91), dng 3 l

S ha bi Trung tm Hc liu i hc Thi Nguyn