P.
************
GENE ALKB1, ALKB2
ENZYME N- ALKANE MONOOXYGENASE
Rhodococcus opacus B4
9/2006
P.
************
GENE ALKB1, ALKB2
ENZYME N- ALKANE MONOOXYGENASE
Rhodococcus opacus B4
GS. TS. HISAO OHTAKE
9/2006
MINISTRY OF EDUCATION AND TRAINING
NONG LAM UNIVERSITY, HCMC
FACULTY OF BIOTECHNOLOGY
OVEREXPRESS GENE ALKB1/B2 ENCODING
FOR N-ALKANE MONOOXYGENASE IN
Rhodococcus opacus B4
GRADUATION THESIS
MAJOR: BIOTECHNOLOGY
Professor Student
Pro.Dr. HISAO OHTAKE NGUYEN TRAN MINH ANH
Dr. LE DINH DON TERM: 2002 - 2006
HCMC, 09/2006
iii
TS. GS. TS. Hisao Ohtake, TS. Honda, TS. Sameshima,
TS. Yamashita
T
,
.
m 2006
iv
ALKB1, ALKB2 Rhodococcus opacus B4
N- ALKANE MONOOXYGENASE
N- ALKANE -
GS. TS. HISAO OHTAKE
Rhodococcus opacus
n- alkane monoo,
R. opacus B4.
- T.
- TR. opacus
ng .
- R. opacus.
v
TRANG
iii
iv
v
vii
viii
1. 1
1.1. 1
1.2. 1
1.2.1. 1
1.2.2. Y 1
2. 2
2.1. 2
2.2. Rhodococcus 3
2.3. Rhodococcus opacus B4 5
2.4. N- alkane monooxygenase 8
3. 11
3.1. 11
3.2. 11
3.2.1. 11
3.2.2. Plasmid 11
3.2.3. PCR primers 11
3.2.4. 12
3.3. 14
3.4. 15
3.4.1. 15
3.4.1.1. PCR 15
3.4.1.2. 15
vi
3.4.1.3. 16
3.4.2. 18
3.4.2.1. 18
3.4.2.2. 18
3.4.2.3. 18
3.4.2.4. Ligatio- vector 19
3.4.2.5. -end 19
3.4.2.6. 19
3.4.2.7. 20
3.4.2.8. X 20
4. 22
4.1. 22
4.2. 24
5. 27
5.1. 27
5.2. 27
6. 28
vii
2.1: 2
2Rhodococcus opacus B4 6
3:
alkane hydroxylase 8
22
22
23
23
24
viii
3
2.2:
-octane 9
- alkane 10
14
Rhodococcus 6
1
1.1.
Rhodococcus opacus B4
,
nhi
. ,
n- alkane
.
1.2.
1.2.1.
R. opacus alkB
1.2.2.
R. opacus n- alkane cao
2
2.1.
.
,
.
(stereo-),
(regio-)
.
),
. ,
,
.
2.1:
3
1e
, l
,
. .
,
(11). ,
.
2.2.
L
Rhodococcus. spp
1891,
,
,
,
.
rhodococci (ng
R. equi )
t h acid mycolic
. Rhodococcus
opacus ,
nhau.
,
.
4
[O]
.
oxy
.
.
. Rhodococcus
.
,
rhodococci .
alkane hydroxylase (alk)
.
i
. , ng
60
.
20 50%
, .
T,
,
sinh ra alkane.
.
,
-
dehydrogenase
.
alkane hydroxylase, trong khi
l
alkane hydroxylase C alkane hydroxylase
, o ra
(17).
alkane 10
(C
12
20
30
).
,
(+)
Corynebacterium – Nocardia – Mycobacterium – Rhodococcus
, .
AlkB1, aR. opacus
enzyme n- alkane monooxygenases
n- alkane
5
,
,
.
5,7 USD/kg (
batch) v5,9 USD/kg (
). .
2.3. Rhodococcus opacus B4
, rhodococci
.
,
. ,
.
R. opacus
. R. opacus
o
o
C,
(4 10
o
C)
(5 9). Rhodococcus opacus
, ,
, toluene, napthalene, n- alkane
n- alkane , rhodococci 1 l saccharide ng
(extra cellular polysaccharides ), ,
(Ivshina , 1982; Glazacheva , 1990).
n- alkane l, rhodococci
,
. rhodococci 100
.
Rhodococci
,
n- alkane.