Tp ch Cng ngh Sinh hc 8(3A): 811-818, 2010

811
S DNG MA TRN PLACKETT-BURMAN V PHNG PHP P NG B MT-
THIT K CU TRC C TM NHM TI U HA SINH TNG HP LIPASE T
BACILLUS LICHENIFORMIS GBDTY1
Hunh Th Thanh Hin
1
, Trnh Th Bch Huyn
2
, Bi Hng Qun
3
1
i hc Nng Lm thnh ph H Ch Minh
2
i hc Bch Khoa thnh ph H Ch Minh
3
Vin Cng ngh sinh hc v Thc phm, i hc Cng nghip thnh ph H Ch Minh
TM TT
Vi khun Bacillus licheniformis GBDTY1 c ti u ha qu trnh nui cy nhm thu c sn lng
lipase cc i. Chng ti s dng thit k th nghim Plackett-Burman kim tra mc nh hng ca
cc yu t l ha khc nhau ln sn lng lipase. Trong , t l CaCl
2
, t l ging v t l dch chit nm men
l ba yu t c tc ng mnh nht (p<0,05). Thit k th nghim theo phng php p ng b mt (RSM)-
phng n cu trc c tm (CCD) c thc hin v tm ra gi tr ti u ca ba yu t l t l CaCl
2
dch
(1,80%), t l ging (3,30%), v chit nm men (3,52%) cho sn lng lipase t chng B.licheniformis cc i
theo m hnh l 21,459 (U/ml) sau 72h nui cy.
T kha: Lipase, Bacillus licheniformis, Ma trn Plackett -Burman, Phng php p ng b mt (RSM)-
Thit k cu trc c tm (CCD)
M DU
Lipase (EC 3.1.1.3) l mt nhm enzyme thy
phn cc triacylglycerol. Lipase c th c to ra
t nhiu chng vi khun nh Bacillus (B.
alkalophilus, B.brevis, B. amyloliquefaciens,
B.licheniformis, B.stearothermophilus) hoc
Streptomyces (S.lividans, S.murinus). Lipase cn
c th c tng hp t cc loi nm men nh
Candida, Pichia, Saccharomyces (Treichel, 2010;
Rey et al., 2003; Charles et al.; 1988). Lipase vi
sinh vt ang c ng dng trong cc lnh vc
nh: thc phm, ha hc, dc phm, m phm,
thuc da, cng nghip ty ra (Starace et al.,
1983), sn xut biodiesel (de Vieira et al., 2006),
sn xut cc polymer phn hy sinh hc, y hc
ng dng, cng nghip giy v ch to cc
biosensor (Hasan et al., 2006). Lipase t vi khun
c nhiu kh nng thng mi ha do chng n
nh, tnh chn lc cao v c hiu trn nhiu c
cht (Dutra et al., 2008). Trn th gii, nghin cu
v lipase vi sinh vt t c nhng thnh tu
ng k. Nghin cu lipase vi sinh vt tp trung
vo vi khun (Pseudomonas spp., Bacillus spp.)
(Hou, 1994; Jeger et al., 1994); nm mc
(Aspergillus spp., Rhizopus spp.); nm men
(Candida spp., Geochitrum spp., Yarrowia spp.)
(Hou, 1994; Vakhlu, Kour, 2006).
Vi khun B.licheniformis c ng dng trong
sn xut cng nghip sn xut cc enzym
protease, -amylase, penicillinase, pentosanase, -
mannonase, cycloglucosyltransferase v khng sinh.
Trnh t gen ca chng B.licheniformis DSM13
c gii m cho php nghin cu su hn qu trnh
trao i cht v cc kh nng tim n ca vi khun
ny (Veith et al., 2004). Amylase t B.licheniformis
c s dng trong thy phn tinh bt, cng nghip
dt, h dn (Gray et al., 1986; Stephens et al.,
1984). Tinh sch lipase n dng ca Bacillus
licheniformis c thc hin bi Nthangeni et al.
(2001), h ti xp loi lipase Bacillus
licheniformis, Bacillus subtilis v Bacillus pumilus
vo phn h I.4 do trng lng phn t t 19 - 20
kDa. Horani (1996) dng dng B.licheniformis
H1 sinh tng hp lipase chu nhit ln n 70
o
C,
hot tnh cao nht pH 10, pH 12 cn 65% hot
tnh. Lipase thu c pha cn bng.
Vit Nam, nhm nghin cu ca Quyn nh
Thi nghin cu tng i nhiu v lipase t phn
lp cc chng vi sinh vt sinh tng hp lipase
(Quyn nh Thi et al., 2003), ti u sinh tng hp
lipase t chng Geotrichum sp. DTQ-26.3 (Nguyn
S L Thanh et al., 2006) v t Ralstonia M1
(Quyen et al., 2007), nh gi cc tnh cht l ha
ca lipase t chng Geotrichum sp. DTQ-26.3
(Nguyn S L Thanh, Quyn nh Thi, 2007),
Hunh Th Thanh Hin et al.
812
Ralstonia M1 (Quyen et al., 2007) cho ti nhn dng
lipase (Quyen et al., 2004) v biu hin lipase ti t
hp t chng Ralstonia M1 vi mc cao E. coli
(Quyn nh Thi et al., 2004; Quyen et al., 2005).
Quyn nh Thi v ng tc gi (2007) cng
nhn dng, biu hin v nh gi mt enzyme c
hot tnh thy phn lipid l esterase EstM cng t
chng Ralstonia M1 (Quyen et al., 2007). Nm
2009, chng ti cng bt u nghin cu v lipase
(Bi Hng Qun v Nguyn c Lng, 2009). Tuy
nhin, ti u sinh tng hp lipase B.licheniformis
theo ma trn Plackett-Burman v phng php p
ng b mt - phng n cu trc c tm cha c
p dng Vit Nam.
Ti u ha qu trnh ln men xy dng m
hnh nhm thu c sn lng ln v gia tng quy
m sn xut c mt ngha quan trng trong vic
ng dng nhng nghin cu c bn lipase vo trong
cng nghip. Mt phng php thc hin hiu qu,
chi ph thp, cho php nghin cu s tng tc v
ng thi tin on c gi tr ti u ca cc yu
t- thit k th nghim ti u a yu t theo Plackett-
Burman (Plackett, Burman, 1946; Dennis, 1995)
c a ra v s dng rng ri sng lc thnh
phn mi trng trong iu kin nui cy lc (Li et
al., 2007; Liu et al., 2008). Sau bc sng lc ban
u, th nghim ti u theo phng php RSM-CCD
c dng ti u ha gi tr cc yu t ang c
nghin cu.
Trong nghin cu ny, chng ti ti u ha
cc yu t l ha trong qu trnh nui cy theo thit
k Plackett-Burman v RSM-CCD d on sn
lng lipase cc i t dch nui cy vi khun
B.licheniformis GBDTY1.
VT LIU V PHNG PHP
Mi trng nui cy v gi ging
Vi khun B.licheniformis GBDTY1 do Cng
ty TNHH Pht trin Cng ngh sinh hc ton cu
(GBD Co., Ltd) cung cp, c gi trn mi
trng thch nghing MHA 13
o
C hoc trong
glycerol 20/80
o
C.
Nh tng du - nc
Nh tng du - nc c thnh phn: 2 g arabic,
80 ml nc ct hai ln, 20 ml du n, pH 7. Hn hp
c to nh bng my Sonicator 3000 mc 75
3 W trong thi gian 10 pht. Nh tng c chun
b mi ngay trc khi th nghim v dng ht trong
ngy.
nh lng lipase (Soares et al., 1999 trch dn
bi Souza et al., 2010).
Hot tnh lipase c nh lng theo phng
php chun ca Soares et al., (1999) vi 10 ml
nh tng du - nc bng 25mM NaOH dng ch
th mu phenolphtalein. Mt n v hot tnh lipase
c nh ngha l lng enzyme cn thit gii
phng 1 mol acid bo trong 1 pht iu kin th
nghim (pH 7,0 v nhit phng).
Thit k Plackett-Burman v phng php RSM-
CCD
xc nh c cc yu t v cc mc nh
hng n sinh tng hp lipase ca vi khun
B.licheniformis GBDTY1, 11 yu t c chn l
glucose, dch chit nm men, du n, NaCl, CaCl
2
,
MgCl
2
, MgSO
4
, KH
2
PO
4
, Tween 80, pH, t l ging
lm th nghim. Th nghim c thit k theo ma
trn Plackett-Burman(Plackett, Burman 1946; Dennis,
1995) vi 11 yu t trong 12 th nghim (Bng 2)
sng lc cc yu t quan trng nh hng n sn
lng lipase (U/ml). Mc thp (-1) v cao (+1) ca 11
yu t c lit k trong bng 1.
Ba yu t chnh RSM-CCD c xc nh gi tr
ti u v c nghin cu 5 mc (-, -1, 0, +1, +)
(Bng 3) trong CCD 20 th nghim (Bng 4)
(Castillo, 2007).
Hm p ng c chn l sn lng lipase (Y,
U/ml dch nui cy). M hnh ha c biu din
bng phng trnh bc 2:
Y = b
o
+ b
1
x
1
+ b
2
x
2
+ b
3
x
3
+ b
11
x
1
2
+ b
22
x
2
2
+ b
33
x
3
2
+
b
12
x
1
x
2
+ b
23
x
2
x
3
+ b
13
x
1
x
3
Trong , b
1
, b
2
, b
3
l cc h s bc 1; b
11
, b
22
,
b
33
l cc h s bc 2; b
12
, b
23
, b
13
l cc h s
tng tc ca tng cp yu t; x
1
, x
2
, x
3
, x
11
, x
22
,
x
33
, x
12
, x
23
, x
13
l cc bin c lp. S liu c
phn tch bng chng trnh Design expert 7.0.0

ca Stat-Ease Inc. USA. T kt qu phn tch, xc

nh mc ti u ca cc yu t cho sn lng
lipase t cc i.
Tp ch Cng ngh Sinh hc 8(3A): 811-818, 2010
813
Bng 1. Cc bin trong ma trn Plackett-Burman v nh hng ca chng.
Tn yu t Gi tr ca cc yu t Mc nh hng tin cy
Glucose (%w/v) Thp (-1) Cao (+1) -0,64
a
0,0466
Dch chit nm men (%w/v) 0,2 0,8 2,67
a
0,0111
Du n (%v/v) 0 1 -2,3
a
0,0129
NaCl (%w/v) 0,5 2 2,64
a
0,0113
CaCl2 (%w/v) 0,1 0,5 3,25
a
0,0091
(NH4)2SO4 (%w/v) 0,1 1 -0,047
b
MgSO4 (%w/v) 0,1 0,2 1,60
a
0,0186
KH2PO4 (%w/v) 0,1 1 -0,77
a
0,0385
Tween 80 (%v/v) 0,1 0,3 -0,89
a
0,0332
pH 0,3 1 -2,21
a
0,0135
T l ging (%v/v) 6 9 3,09
a
0,0096
a
C ngha tin cy = 0,05;
b
Khng c ngha tin cy = 0,05.
Bng 2. Ma trn thit k th nghim Plackett-Burman.
Th
nghim
Cc bin Lipase (U/ml)
(72 h)
X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 Thc
nghim
M
hnh
1 +1 -1 +1 -1 -1 -1 +1 +1 +1 -1 +1 1,35 1,32
2 +1 +1 -1 +1 -1 -1 -1 +1 +1 +1 -1 2,07 2,04
3 -1 +1 +1 -1 +1 -1 -1 -1 +1 +1 +1 4,86 4,84
4 +1 -1 +1 +1 -1 +1 -1 -1 -1 +1 +1 1,80 1,82
5 +1 +1 -1 +1 +1 -1 +1 -1 -1 -1 +1 13,87 13,85
6 +1 +1 +1 -1 +1 +1 -1 +1 -1 -1 -1 3,43 3,43
7 -1 +1 +1 +1 -1 +1 +1 -1 +1 -1 -1 4,93 4,95
8 -1 -1 +1 +1 +1 -1 +1 +1 -1 +1 -1 3,47 3,45
9 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 +1 8,53 8,55
10 +1 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 2,31 2,35
11 -1 +1 -1 -1 -1 +1 +1 +1 -1 +1 +1 5,60 5,62
12 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 1,27 1,24
Bng 3. Nng ba yu t dng trong RSM-CCD.
Yu t Tn Phm vi nghin cu Mc
- -1 0 +1 +
x1 CaCl2 (%w/v) 0,16 - 1,84 0,16 0,5 1,0 1,5 1,84
x2 T l ging (% v/v) 3,30 - 11,7 3,30 5,0 7,5 10 11,7
x3 Chit nm men (%w/v) 0,32 - 3,68 0,32 1 2 3 3,68
Hunh Th Thanh Hin et al.
814
Bng 4. K hoch thc nghim theo RSM-CCD ti u ha sn lng lipase.
Th nghim Mi trng c bn Lipase (U/mL) - 72 h
CaCl2
(%w/v)
T l ging (%v/v) Dch chit nm men
(%w/v)
Thc nghim Suy t m hnh
1 0,5 5 1 10,38 10,426
2 1,5 5 1 15,88 15,499
3 0,5 10 1 15,79 15,424
4 1,5 10 1 16,73 17,348
5 0,5 5 3 13,59 13,54
6 1,5 5 3 19,03 18,595
7 0,5 10 3 15,37 16,300
8 1,5 10 3 19,07 18,224
9 0,16 7,5 2 12,54 12,618
10 1,84 7,5 2 17,48 18,501
11 1 3,3 2 11,75 12,188
12 1 11,7 2 16,34 16,080
13 1 7,5 0,32 15,63 15,619
14 1 7,5 3,68 18,77 18,959
15 1 7,5 2 16,29 15,56
16 1 7,5 2 15,07 15,56
17 1 7,5 2 15,19 15,56
18 1 7,5 2 15,75 15,56
19 0 7,5 2 15,65 15,56
20 1 7,5 2 15,36 15,56
KT QU
Chng vi khun c nui trong 100 ml mi
trng NB trong bnh 250 ml nhit thng vi
ch lc. Mt t bo cao nht sau 25 gi nui
cy (2,3x10
7
t bo/ ml).
Sng lc cc yu t nh hng quan trng n
sn lng lipase B.licheniformis
Ma trn Plackett-Burman thu c sn lng
lipase t 1,32 13,85 U/ml dch nui cy (Bng 2).
Gi tr nh hng ca tng yu t ln sn lng
lipase c tnh ton bng phn mm Design
expert 7.0.0 (Bng 1). Yu t no c gi tr nh
hng dng v ln s nh hng ti sn lng
lipase B.licheniformis. Ba yu t c gi tr nh hng
dng v ln s nh hng ti sn lng lipase vi
tin cy (p < 0,05) l: t l CaCl
2
, t l ging v t
l dch chit nm men. Cn c vo h s nh hng,
chng ti thy rng t l CaCl
2
c tc ng mnh
nht n sn lng lipase (3,25), tip n l t l
ging (3,09) v cui cng l t l dch chit nm
men (2,67). Cc yu t glucose, du n, NaCl, pH,
MgSO
4
, KH
2
PO
4
, Tween 80 c nh hng khng
ng k n sn lng lipase tin cy p < 0,05.
Chng ti chn t l CaCl
2
, t l ging v dch chit
nm men l nhng yu t nh hng mnh nht n
sn lng lipase cho thit k th nghim theo RSM-
CCD.
Ti u ha gi tr cc yu t cho sn lng lipase
cc i
Sau khi sng lc cc yu t chnh nh hng n
sn lng lipase, k hoch ha thc nghim c
thc hin theo RSM-CCD, x l bng Design
expert

7.0.0. Gi tr hm p ng theo thc nghim

v tin on theo m hnh c trnh by trong bng
4. Sau khi phn tch phng sai (ANOVA), phng
trnh hi quy c dng nh l mt m hnh tin
on sn lng lipase thu c. Sn lng lipase c
th c tin on t phng trnh sau:
Y = 15,56 + 1,75x
1
+ 1,16x
2
+ 0,99x
3
0,79x
1
x
2
-
0,55x
2
x
3
- 0,50x
2
2
+ 0,61x
3
2
Trong , Y l sn lng lipase (U/ml); x
1
, x
2
, x
3
ln lt l t l CaCl
2
(%w/v), t l ging v t l
Tp ch Cng ngh Sinh hc 8(3A): 811-818, 2010
815
dch chit nm men (%v/v). H s hi quy (R
2
) tnh
c l 0,9385. iu ny th hin rng c 93,85% s
liu thc nghim tng thch vi s liu tin on
theo m hnh. Gi tr R
2
ln hn 0,75 th hin m
hnh tng thch vi thc nghim c ngha l c s
tng quan rt cht ch gia cc yu t th nghim
v sn lng lipase. Gi tr R
2
tin on l 0,8130
ph hp vi R
2
iu chnh l 0,9027 ( lch 0,0897
< 0,2). T l tn hiu so vi nhiu l 19,148 > 4 ch
ra rng tn hiu y . H s bin d CV trong th
nghim mc thp l 4,51% chng t mc phn
tn sn lng lipase l thp.
Mt p ng (Hnh 1; Hnh 2) th hin s tng
tc ca tng cp yu t v t biu ny c th xc
nh c gi tr ti u ca tng yu t lm cho hm
p ng cc i. M hnh d on sn lng
lipase ti a t c (21,3495 U/ml) gi tr cc
yu t: t l CaCl
2
(1,84%), t l ging (5,32%), v
dch chit nm men (3,35%).
Trong ba yu t phn tch, a vo sn xut
qui m ln th t l ging cng gim cng tt do
chng ti ci t t l ging mc thp nht v tin
hnh x l bng phn mm v thu c 30 gii
php. Gii php 19 vi t l CaCl
2
(1,83%), t l
ging (3,30%), v dch chit nm men (3,67%) d
on thu c sn lng lipase ti a l 22,247
U/ml. Tuy nhin, do chi ph ca dch chit nm men
kh cao, nn chng ti chn gii php t l CaCl
2
dch(1,80%), t l ging (3,30%), v dch chit nm
men (3,52%) d on thu c sn lng lipase ti
a l lipase thu c vn mc cao, va p ng
c yu cu k thut va p ng yu cu v kinh
t. Sn lng lipase thu c mc d cao hn th
nghim ca Jonsson v Snygg (1974) trong mi
trng NB (6,3 U/ml) nhng vn cn thp hn so
vi th nghim ca Bayoumi v ng tc gi (2007)
(50,23 U/ml).
Hnh 2. Mt p ng lipase theo t l ging v YE. Hnh 1. Mt p ng lipase theo t l CaCl2 v t l ging.
Hunh Th Thanh Hin et al.
816
THO LUN
Cng c thit k th nghim ti u a yu t ca
Plackett-Burman v phng php p ng b mt -
phng n cu trc c tm (RSM-CCD) theo chng
ti nh gi l nhng cng c mnh trong vic sng
lc v ti u ha gi tr cc yu t lm cho hm p
ng cc i. Vic s dng cc cng c ny cng vi
phn mm chuyn dng Design expert gim c
thi gian tiu tn, gim cc th nghim ng thi c
th la chn mt trong nhng gii php ti u do
phn mm ngh.
Lipase B.licheniformis thng c kh nng chu
nhit v chu kim do c nhiu ng dng trong
thc t c bit l cng ngh cht ty ra. Chng ti
s tip tc nghin cu tinh sch v tm hiu cc tnh
cht ca lipase B.licheniformis nhm ng dng vo
thc t.
KT LUN
T 11 yu t ban u sng lc v chn c 3
yu t l t l CaCl
2
(1,80%), t l ging (3,30%), v
dch chit nm men (3,52%) d on cho sn lng
lipase t chng B.licheniformis cc i theo m hnh
l 21,459 (U/ml).
Li cm n: Chng ti xin gi li cm n n Ban
lnh o Vin Cng ngh sinh hc thc phm i
hc Cng nghip Tp.HCM to iu kin cho
chng ti thc hin ti ny.
TI LIU THAM KHO
Bayoumi RA, El-louboudey SS, Sidkey NM, v Abd-El-
Rahman MA, (2007) Production, purification and
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1752-1765.
Bi Hng Qun v Nguyn c Lng (2009) Ti u ha
sinh tng hp lipase t Pichia anomala VTCC Y0787 s
dng ma trn Plackett Burman v phng php p ng
b mt phng n cu trc c tm. Tp ch Cng ngh
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Tp ch Cng ngh Sinh hc 8(3A): 811-818, 2010
817
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OPTIMIZATION LIPASE PRODUCTION BY BACILLUS LICHENIFORMIS GBDTY1
USING DESIGN OF PLACKETTBURMAN MATRIX AND CENTRAL COMPOSITE
DESIGNS - RESPONSE SURFACE METHODOLOGY
Huynh Thi Thanh Hien
1
, Trinh Thi Bich Huyen, Bui Hong Quan
3,
1
Ho Chi Minh University of Agriculture and Forestry
2
Ho Chi Minh University of Technology
3
Institutes of Biotechnology and Food Technology, Ho Chi Minh University of Industry
SUMMARY
The bacteria Bacillus licheniformis GBDTY1 was optimized for maximum lipase production. We used the
design of optimum multifactorial experiments Plackett-Burman to estimate level effect of physical and
chemical factors on lipase production. As the result, CaCl
2
(%), inoculums size (%) and yeast extract (%) were
identified as significant factors (p<0.05). After screening, these factors were subsequently optimized using the
response surface methodology (RSM) - Central Composite Designs (CCD). Using RMS-CCD, the optimal
levels of three factors were found as follows: CaCl
2
(1.80%), inoculums size (3.30%) and yeast extract

Author for correspondence: Tel: +848. 38940309 ext. 890 E-mail: buihongquan@hui.edu.vn
Hunh Th Thanh Hin et al.
818
(3.53%), under which the highest lipase yield was obtained. The predicted maximum yield of lipase attained to
21.459 (U/ml) after 72h culture.
Keywords: Lipase, Plackett-Burman, Bacillus licheniformis GBDTY1, Response Surface Methodology (RSM)-
Central composite designs (CCD)