Tp ch Cng ngh Sinh hc 7(4): 493-500, 2009

1

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

Bi Hng Qun
1, 2
, Nguyn c Lng
3
1
Vin Cng ngh sinh hc v Thc phm, i hc Cng nghip Thnh ph H Ch Minh
2
Trng Cao ng Kinh t K thut Bnh Dng
3
i hc Bch khoa Thnh ph H Ch Minh
TM TT
Nm men Pichia anomala VTCC Y7087 c ti u ha qu trnh nui cy nhm thu c sn lng
lipase cc i. P. anomala VTCC Y7087 c kh nng sinh tng hp lipase pH ti u 9,0; iu ny ha hn
lipase c kh nng hot ng pH kim (pH > 9) v c th ng dng trong cng nghip cht ty ra. 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 , dch chit nm men, t l ging v du n l ba yu t c tc ng mnh
nht. 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 chit nm men (3,0%), t l ging (2,37%; 10
8
t bo/ml)
v du n (4,19%) cho sn lng lipase cc i theo m hnh 18,8078 (U/ml). M hnh c p dng vo
thc t vi kt qu 100 ml nui cy lc l 18,95 (U/ml) v 3 l trong ni ln men t ng l 22,52 (U/ml).

T kha: Lipase, ni ln men, Pichia anomala VTCC Y0787, Plackett -Burman, RSM-CCD
M DU
Lipase (EC 3.1.1.3) l mt nhm enzyme thy
phn cc triacylglycerol. Mi quan tm cng nh
vic s dng lipase trong cc ngnh cng nghip
gia tng trong nhng nm gn y. Vic ng dng
lipase gia tng ng thi vi nhng pht hin ng
dng lipase trong cc lnh vc mi lm gia tng nhu
cu lipase. Th phn lipase trn th trng th gii
ngy cng tng. Trong , lipase t cc vi sinh vt c
mt ngha v cng quan trng trong cc ngnh
cng nghip cng ngh sinh hc hin nay. 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).
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).
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),
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). Tuy nhin,
ti u sinh tng hp lipase theo ma trn Plackett-
Burman v phng php p ng b mt - phng
n cu trc c tm cha c p dng Vit Nam v
vic tm kim ngun lipase mi c kh nng chu
kim, chu acid, chu nhit vn l mt ch thu ht
s ch ca cc nh khoa hc.
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
Bi Hng Qun & Nguyn c Lng
2
cng nghip. Bt c mt qu trnh ln men no cng
b nh hng bi cc yu t vt l cng nh sinh ha
hc khc nhau. Bc u tin ti u ha l sng
lc cc yu t quan trng. Mt cch n gin v
thun tin l ti u tng yu t trong khi gi nguyn
cc yu t khc. Cch thc hin ny tn thi gian v
khng xc nh c s tc ng qua li ca cc yu
t. 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.
Nm men P. anomala c nghin cu su
v sinh tng hp ethyl acetate, c t git, phytase,
kh nng khng nm v iu khin sinh hc trong
qu trnh bo qun ng cc (Fredlund et al., 2002).
Lipase t chng P. anomala hot ng pH 5,5 v
7,5 (Hou, 1994). Lipase P. anomala cha c
nghin cu nhiu nh cc lipase t nm men khc
nh C. rugosa. Vit Nam, P. anomala c
phn lp t bnh men trong mt s nghin cu gn
y (Dung et al., 2007; Vu Nguyen Thanh et al.,
2008).
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 thu c sn
lng lipase cc i t dch nui cy nm men P.
anomala VTCC Y0787.
VT LIU V PHNG PHP
Mi trng nui cy v gi ging
Nm men P. anomala VTCC Y0787 do Vin
vi sinh vt v Cng ngh sinh hc (IBMT-VNU)
cung cp, c gi trn mi trng thch nghing
Sabouraund 2% glucose 13
o
C v trong glycerol
20/80
o
C. Mi trng c bn gm (g/l): 1 g
K
2
HPO
4
; 0,5g MgSO
4
c dng kim tra nh
hng ca ngun N v ngun 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 9. 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
Hot tnh lipase c nh lng theo phng
php chun lin tc vi 10 ml nh tng du -
nc bng 0,1 N NaOH trn my Metrohm 702 SM
Titrino (Metrohm Ltd. Switzerland) vi in cc pH
(Vario pH). 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 9,0 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 nm men P.
anomala VTCC Y0787, 11 yu t c chn l mt r
ng, glucose, NaNO
3
, (NH
4
)
2
SO
4
, (NH
4
)
2
HPO
4
,

pH, nhit , chit nm men, tui ging, t l ging,
du n 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 7(4): 493-500, 2009
3
Bng 1. Cc bin trong ma trn Plackett-Burman v nh hng ca chng.

Yu t Mc Mc nh hng
K hiu Tn yu t Thp (-1) Cao (+1) nh hng Prob > F
X1 R ng (%) 0,0 10,0 -1,92
a
0,0184
X2 Glucose (%) 0,0 2,5 0,75
b

X3 NaNO3 (%) 0,2 1,2 0,25
b

X4 (NH4)2SO4 (%) 0,0 0,9 0,25
b

X5 (NH4)2HPO4 (%) 0,1 0,8 -2,22
a
0,0106
X6 Chit nm men (%) 0,5 1,0 7,35
a
< 0,0001
X7 Nhit (
o
C) 25,0 37,0 0,75
b

X8 pH 5,0 10,0 -1,82
a
0,0224
X9 Tui ging (h) 12,0 24,0 -0,55
b

X10 T l ging (%) 1,0 3,0 3,58
a
0,0014
X11 T l du n (%) 2,0 5,0 3,28
a
0,002
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) (105 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 3,2 3,48
2 +1 +1 -1 +1 -1 -1 -1 +1 +1 +1 -1 3,5 3,78
3 -1 +1 +1 -1 +1 -1 -1 -1 +1 +1 +1 9,0 8,58
4 +1 -1 +1 +1 -1 +1 -1 -1 -1 +1 +1 16,0 16,23
5 +1 +1 -1 +1 +1 -1 +1 -1 -1 -1 +1 4,1 3,08
6 +1 +1 +1 -1 +1 +1 -1 +1 -1 -1 -1 5,6 5,33
7 -1 +1 +1 +1 -1 +1 +1 -1 +1 -1 -1 12,0 11,28
8 -1 -1 +1 +1 +1 -1 +1 +1 -1 +1 -1 4,0 3,48
9 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 +1 9,5 10,53
10 +1 -1 -1 -1 +1 +1 +1 -1 +1 +1 -1 10,2 10,73
11 -1 +1 -1 -1 -1 +1 +1 +1 -1 +1 +1 17,1 16,33
12 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 3,2 3,93


Bng 3. Nng ba yu t dng trong RSM-CCD.

Yu t Tn Phm vi nghin cu Mc
- -1 0 +1 +
x1 Chit nm men (%w/v) 0,32 - 3,68 0,32 1,0 2,0 3,0 3,68
x2 T l ging (%v/v) 0,82 - 4,18 0,82 1,5 2,5 3,5 4,18
x3 Du n (%v/v) 0,98 - 6,02 0,98 2,0 3,5 5,0 6,02

Bi Hng Qun & Nguyn c Lng
4
Bng 4. K hoch thc nghim theo RSM-CCD ti u ha sn lng lipase.

Th nghim Mi trng c bn Lipase (U/ml) - 105 h
x1 x2 x3 Thc nghim Suy t m hnh
1 -1 -1 -1 9,23 9,53
2 +1 -1 -1 16,81 16,11
3 -1 +1 -1 13,45 13,50
4 +1 +1 -1 17,61 17,17
5 -1 -1 +1 13,52 13,72
6 +1 -1 +1 18,41 18,12
7 -1 +1 +1 15,12 15,58
8 +1 +1 +1 17,61 17,08
9 - 0 0 12,51 11,79
10 + 0 0 17,53 18,58
11 0 - 0 13,45 13,63
12 0 + 0 15,93 16,09
13 0 0 - 13,61 13,96
14 0 0 + 17,42 17,40
15 0 0 0 17,25 17,52
16 0 0 0 18,01 17,52
17 0 0 0 17,52 17,52
18 0 0 0 17,13 17,52
19 0 0 0 18,22 17,52
20 0 0 0 17,02 17,52

nh gi m hnh thc nghim
Ln men th nghim m hnh ti u c thc
hin vi quy m 100 ml nui cy lc v 3 l trn ni
ln men Bioflo 110 Fermentor/Bioreactor 5 l (New
Brunswick Scientific, Edison, New Jersey USA).
Ni ln men c iu khin t ng pH 9,0; nhit
25C; 300 rpm v tc kh 1 vvm. Mu c
ly v kim tra thi im thch hp.
KT QU
P. anomala VTCC Y0787 c th pht trin pH
kh rng t 5 - 12. iu ny ph hp vi nghin cu
trc y v nm men ny (Fredlund et al., 2003).
Gi tr pH thch hp nht sinh tng hp lipase l
9,0; iu ny ha hn lipase P. anomala VTCC
Y0787 c kh nng hot ng pH kim (pH > 9)
v c th ng dng trong cng nghip ty ra.
Nhit pht trin ca nm men t 13 - 37C.
nhit cao hn 37C nm men pht trin chm v
yu. Nhit di 13C khng c thc hin.
Nhit thch hp cho sinh tng hp lipase khong
25C.
Thi gian sinh tng hp lipase mnh nht sau 96
h v gim xung sau 168 h.
Khi c mt c cht, P. anomala VTCC Y0787
nui trong mi trng glucose sinh tng hp lipase
yu. Mi trng ch c du n nh l ngun carbon
duy nht, P. anomala VTCC Y0787 sinh tng hp
lipase mnh. kim tra nh hng ca cc ngun
nitrogen khc nhau ln sn lng lipase, cc ngun
nitrogen c b sung mc 0,2% nitrogen vo mi
trng c bn vi 3,0% du n nh l ngun c cht
cm ng v ngun C duy nht. Trong s cc ngun
nitrogen, dch chit nm men (YE), (NH
4
)
2
HPO
4
,
NaNO
3
, (NH
4
)
2
SO
4
c nh hng n kh nng sinh
tng hp lipase ca P. anomala VTCC Y0787.
Sng lc cc yu t nh hng quan trng n
sn lng lipase P. anomala VTCC Y0787
Ma trn Plackett-Burman thu c sn lng
lipase t 3,08 - 16,33 U/ml dch nui cy (Bng 2).
Gi tr nh hng ca tng yu t ln sn lng
Tp ch Cng ngh Sinh hc 7(4): 493-500, 2009
5
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 P. anomala VTCC Y0787. Dch chit nm
men, du n v t l ging nh hng mnh nht n
sn lng lipase vi mc ngha = 0,05. Do ,
dch chit nm men, du n v t l ging c chn
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 nh hng chnh 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 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 m hnh sau:
Y = 17,52 + 2,02x
1
+ 0,73x
2
+ 1,02x
3
- 0,73x
1
x
2
-
0,54x
1
x
3
- 0,53x
2
x
3
- 0,82x
1
2
- 0,94x
2
2
- 0,65x
3
2

Trong , Y l sn lng lipase (U/ml); x
1
, x
2
, x
3

ln lt l t l chit nm men, t l ging v du n
(%). H s hi quy (R
2
) tnh c l 0,9616. iu
ny th hin rng c 96,16% 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. Gi tr R
2
tin on l 0,7714 ph hp
vi R
2
iu chnh l 0,9270 ( lch 0,1556 < 0,2).
T l tn hiu so vi nhiu l 19,301 > 4 ch ra rng
tn hiu y .
Biu p ng b mt (Hnh 1) 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 (18,8078 U/ml) gi tr
cc yu t: chit nm men (3,0%), t l ging
(2,37%; 10
8
t bo/ml), t l du n (4,19%). T l
chit nm men cng tng th sn lng lipase cng
tng, nhng ngc li t l du n v t l ging ch
tng n mt mc no y th bt u gim.
Th nghim m hnh 100 ml v 3 l
Chng ti th nghim m hnh 100 ml nui
cy lc 200 rpm v 3 l trn ni ln men t ng 5 l.
Th nghim lp li 3 ln 100 ml; 1 ln 3 l v c
trnh by trong bng 5. Kt qu cho thy, ch
nui cy lc t c sn lng lipase (18,95
1,5 U/ml) tng ng vi m hnh d on (18,8078
U/ml). ch nui trn ni ln men nhit
25
o
C 3; pH 9,0 0,2; lu lng kh 1 vvm; tc
cnh khuy tuabin 300 rpm, sn lng t c
22,52 U/ml.
















T l ging (%)
Hnh 1. Mt p ng sn lng lipase theo t l ging v
du n.

1.50 2.00 2.50 3.00 3.50
2.00
2.75
3.50
4.25
5.00
Lipase
17.1071
17.6794
17.6794
18.2517
18.7665
Predict 18.8085
X1 2.37
X2 4.19
D

n

(
%
)


Lipase (U/ml)
Chit nm men (%)
Hnh 2. Mt p ng sn lng lipase theo t l ging v
chit nm men.

1.00 1.63 2.25 2.88 3.50
1.50
2.00
2.50
3.00
3.50
Lipase
14.8177
15.9624
17.1071
17.6794
17.6794
18.2517
18.7665
Predict 18.8085
X1 3.00
X2 2.37
T

g
i

n
g

(
%
)


Lipase (U/ml)

Bi Hng Qun & Nguyn c Lng
6
Bng 5. Sinh tng hp lipase t P. anomala VTCC Y0787
ghi nhn c t 100 ml, 3 l mi trng ti u.

Th tch mi
trng
Ch nui
cy
Lipase (U/ml)
100 ml Lc 200 rpm 18,95 1,5 (105 h)
3 l Ni ln men t
ng
22,52 (96 h)

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.
Mc d chit nm men c th cho sn lng
lipase cc i nhng chi ph ca chit nm men kh
cao so vi cc ngun nitrogen khc. iu ny cng
l mt cn tr khi tin hnh gia tng quy m sn
xut. Do iu kin c hn, chng ti ch chn ngun
c cht l du lc (u phng). Theo nh nhng
nghin cu trc y, lipase c tnh c hiu c cht
rt cao, do cn phi tin hnh nghin cu trn cc
loi du khc nhau.
Lipase ca P. anomala c khong pH hot ng
rng

(Hou, 1994) v ha hn l mt enzyme chu
kim tt nhng iu ng tic l chng ti cha tm
thy cng b no v sn lng ca lipase t P.
anomala tin hnh so snh. Tuy nhin, vi sn
lng thu c thp nh kt qu nghin cu trnh
by th s rt kh khn p dng vo sn xut cng
nghip quy m ln hn. Do , chng P. anomala
VTCC Y0787 ch nn dng trong nghin cu c bn
cc c im, tnh cht ca lipase v dng lm ngun
to th vin b gen m ha lipase.
Hin nay, cc chng vi sinh vt dng tng
hp lipase cho sn lng cao thng l cc chng ti
t hp (Pignde et al., 2000; Zhao et al., 2008). Sn
lng lipase t cc chng hoang di thng khng
cao. Chng ti cng gy t bin chng hoang di
bng cc tc nhn vt l v/hoc ha hc (kt qu
khng cng b) nhng do s lng sng lc ln,
khng c du chun kh d c c s khoa hc vng
chc (ch da vo vng phn gii trn mi trng c
cht du) dn n kt qu khng r rng. Do , vi
s pht trin ca cng ngh gen nh hin nay, cn
thit phi phn lp cc gen m ha cho lipase c c
im qu (chu kim, chu acid, chu nhit, chu
dung mi hu c) t nhiu ngun khc nhau
(prokaryote v eukaryote) v to dng bng cc h
thng biu hin (vector v t bo ch) thch hp
tng sn lng.
KT LUN
xc nh P. anomala VTCC Y0787 pht trin
pH t 5 - 12, nhit t 13 - 37
o
C.
xc nh P. anomala VTCC Y0787 sinh tng
hp lipase ti u pH 9,0; nhit 25
o
C; 96 h.
T 11 yu t ban u sng lc v chn c 3
yu t l dch chit nm men (3%), t l ging
(2,37%; 10
8
t bo/ml) v du n (4,19%) cho sn
lng lipase t chng P. anomala VTCC Y0787 cc
i theo m hnh l 18,8078 (U/ml).
ln men th nghim m hnh ti u vi quy
m 100 ml nui cy lc v 3 l trn ni ln men
Bioflo 110 Fermentor/Bioreactor 5 l (New
Brunswick Scientific, Edison, New Jersey USA) thu
c sn lng lipase P. anomala VTCC Y0787 ln
lt l 18,95 (U/ml) v 22,52 (U/ml).
Li cm n: Chng ti xin gi li cm n n Ban
Ch nhim b mn CNSH H Bch khoa Tp. HCM
to iu kin cho chng ti thc hin ti ny.
Chng ti xin cm n TS. Quyn nh Thi c
nhng gp chng ti hon thin ti ny.

TI LIU THAM KHO
Castillo E Del (2007) Process Optimization A Statistical
Approach. Springer Science. New York, USA: 118-122.
de A Vieira AP, da Silva MAP, Langone MAP (2006)
Biodiesel production via esterification reactions catalyzed
by lipase. Latin Am Appl Res 36: 283-288.
Dennis KJL (1995) Screening properties of certain two-
level designs. Metrika 42: 99-118.
Dung NTP, Rombouts FM, Nout MJR (2007).
Characteristics of some traditional Vietnamese starch-
based rice wine fermentation starters (men). LWT 40: 130-
135.
Tp ch Cng ngh Sinh hc 7(4): 493-500, 2009
7
Fredlund E, Druvefors U, Boysen ME, Lingsten KJ,
Schnurer J (2002) Physiological characteristics of the
biocontrol yeast Pichia anomala J121. FEMS Yeast Res 2:
395-402.
Hasan F, Shah AA, Hameed A (2006) Industrial
application of microbial lipases. Enzyme Microb Technol
39: 235-251.
Hou TC (1994) pH dependence and thermostability of
lipases from cultures from the ARS Culture Collection. J
Ind Microbiol 13: 242-248.
Jeger EK, Ransac S, Dijkstra B, Colson C, Heuvel M,
Misset O (1994). Bacterial lipases. FEMS Microbiol Rev
15: 29-63.
Li Y, Liu Z, Cui F, Liu Z, Zhao H (2007) Application of
Plackett-Burman experimental design and Doehlert design
to evaluate nutritional requirements for xylanase
production by Alternaria mali ND-16. Appl Microbiol
Biotechnol 77: 285-291.
Liu C, Sun ZT, Du JH, Wang J (2008) Response surface
optimization of fermentation conditions for producing
xylanase by Aspergillus niger SL-05. J Ind Microbiol
Biotechnol 35: 703-711.
Nguyn S L Thanh, Quyn nh Thi, Trng Th Bch
Hu (2006) Ti u mt s iu kin nui cy chng nm
Geotrichum sp. DTQ-26.3 sinh tng hp lipase. Tp ch
Cng ngh Sinh hc 4: 455-462.
Nguyn S L Thanh, Quyn nh Thi (2007) Mt s tnh
cht ha l ca lipase ngoi bo chng Geotrichum sp.
DTQ-26.3. Tp ch Cng ngh Sinh hc 5: 31-40.
Pignde G, Wang HJ, Fudale F, Seman M, Gaillardin C,
Nicaud JM (2000). Autocloning and amplification of LIP2
in Yarrowia lipolytica. Appl Environ Microbiol 66(8):
3283-3289.
Plackett RL, Burman JP (1946) The design of optimum
multifactorial experiments. Biometrika 37: 305-325.
Quyn nh Thi, Nguyn Th By, Mai Th Thanh,
Nguyn Th Tho, L Th Thu Giang, Nguyn Th Tuyt
Nhung, Nguyn Ngc Dng (2003) Phn lp chng vi
khun sinh tng hp lipase t nc thi v kho st hot
tnh lipase ca 102 chng Pseudomonas. Tp ch Di truyn
& ng dng 4: 37-41.
Quyn nh Thi, L Th Thu Giang, Nguyn Th Tho
(2004) Biu hin cao lipase kim v chu nhit ca chng
Ralstonia sp. M1 E. coli. Tp ch Di truyn & ng dng
4: 38-42.
Quyen DT, Nguyen TT, Le TTG, Kim HK, Oh TK, Lee
JK (2004) A novel lipase/chaperone pair from Ralstonia
sp. M1: Analysis of the folding interaction and evidence
for gene loss in Ralstonia solanacearum. Mol Gen
Genomics 272: 538-554.
Quyen DT, Le TTG, Nguyen TT, Oh TK, Lee JK (2005)
High-level heterologous expression and properties of a
novel lipase from Ralstonia sp. M1. Prot Expr Purif 39:
97-106.
Quyen DT, Nguyen SLT, Dao TT (2007) A novel esterase
from Ralstonia sp. M1: Gene cloning, sequencing, high-
level expression and characterization. Prot Expr Purif 51:
133-140.
Quyen DT, Le TTG, Nguyen TT, Oh TK, Lee JK (2007)
Production and properties of an extracellular alkaline,
thermostable, highly organic-solvent-resistant and
detergent-inducible lipase from Ralstonia sp. M1. ASEAN
J Sci Technol Dev 24: 237-251.
Starace CA (1983) Detergent enzymes-past, present and
future. J Am Oil Chem Soc 60(5): 1203-1207.
Vu Nguyen Thanh, Le Thuy Mai, Duong Anh Tuan (2008)
Microbial diversity of traditional Vietnamese alcohol
fermentation starters (banh men) as determined by PCR-
mediated DGGE. Int J Food Microbiol 128(2): 268-273.
Vakhlu J, Kour A (2006) Yeast lipases: enzyme
purification, biochemical properties and gene cloning.
Electronic J Biotechnol 9(1): 69-85.
Zhao W, Wang J, Deng R, Wang Y (2008) Scale up
fermentation of recombinant Candida rugosa lipase
expressed in Pichia pastoris using GAP promoter. J Ind
Microbiol Technol 35: 189-195.

OPTIMIZATION LIPASE PRODUCTION BY PICHIA ANOMALA VTCC Y0787 USING
DESIGN OF PLACKETTBURMAN MATRIX AND CENTRAL COMPOSITE DESIGNS-
RESPONSE SURFACE METHODOLOGY

Bui Hong Quan
1, 2
, Nguyen Duc Luong
3, *1
1
Institute of Biotechnology and Food Technology, Ho Chi Minh University of Industry

2
Binh Duong Economic and Technology College

3
University of Technology, Vietnam National University, Ho Chi Minh City

* Author for correspondence: Tel: 84-8-38607273; E-mail: buihongquan@hui.edu.vn
Bi Hng Qun & Nguyn c Lng
8
SUMMARY
The yeast Pichia anomala VTCC Y0787 was optimized for maximum lipase production. The pH optimum
for this secretion was 9.0; this lipase was active in an alkaline pH range (pH > 9) and had a potential
application in detergent industry. 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, yeast (%),
inoculums size (%) and vegetable oil (%) were identified as significant factors. After screening, these factors
were subsequently optimized using the response surface methodology (RSM) - Central Composite Designs
(CCD). These optimal levels were found out yeast extract (3.0%), inoculums size (2.37%; 10
8
cells/ml) and
vegetable oil (4.19%) in which the lipase yield was the highest. The regression equations (model) obtained and
predicted maximum lipase yield 18.8078 (U/ml). We also verified model in shaking flasks (100 ml) as well as
automatic fermentor (3 litres). Lipase production for 100 ml and 3 litres recorded from supernatant was 18.95
(U/ml) and 22.52 (U/ml), respectively.
Keywords: fermentor, lipase, Plackett-Burman, Pichia anomala VTCC Y0787, Response Surface Methodology
(RSM)-Central composite designs (CCD)