Tạo đột biến nâng cao hoạt tính sinh tổng hợp Polyβ Hydroxybutyrate của vi khuẩn Alcaligenes Latus VN1

Top chi Cong nghe Sinh hoc 6(4): 489-496, 2008

TAO DOT BIEN NANG CAO HOAT TINH SINH TONG HOP POL Y-13HYDROXYBUTYRATE CEA VI KHUAN ALCALIGENES LATUS VN1
Pham Thanh

Train Dinh Man', Yutaka Tokiwa2

1 Vien

Ging nghe sinh hoc, Vien Khoa hoc va Cong nghe Viet Nam
for Biological Resources and Functions, National Institute of Advanced Industrial Science and
Technology (AIST), Tsukuba, Japan

2Institute

TOM TAT
Poly-p-hydroxybutyrate (PHB) la mit loci plastic chiu nhigt co kha nang bi phan hily sinh hoc. PHB
dang duqc quan tam nghien tiro nhieu vi no co the giai quy6t &roc van
nhiem moi throng do chAt
thai nhaa CO nguOn goc ter dAu moi gay ra. Ngay nay, hau hat rththig nghien ciru trong filth vac nay tap
ming vao vigc tach vi sinh vat sin sinh PHB tir nhting ngu6n khac nhau va cai thign kha nang cita chimg.
Trong nghien ciru nay, tia UV ducic sir clang nhu tic nhan vat 1.S, anh hiring len chimg Alcaligenes latus
VN1 da thu tilting dit bien cO hoat tinh sinh tong hop PHB cao. Sau khi chiau tia UV di thu &roc 40
tren tong so 198 chimg co hoat tinh sinh PHB cao. Nang suit tich lily PHB cita cac chung thu &roc cao
han hen chung dai mit cach coy nein. Nhung chimg khic cho nang suit tich lily PHB bang hoac thap
han chimg be mg. Phan ion cac chung dit bien sinh PHB cao thu ducic tai thiri diem 30 phut chign tia
UV. Tai thai diem do hau het vi Ichufin bi diet. Trong thing dit bien throe chin loc, chung dit bien A.
latus VN1-20 la tit nhAt, no co kha nang tich lay tai 55,6% PHB/trong luong kho to bao. Nang suet sinh
PHB 6 chimg dit bien da cai thign ducrc khoang 40% so veil chimg dai. San pham PHB thu duce khi len
men chimg dit bien A. latus VN1-20 tren mei truing co se/ sau 72 h da dtroc tech chit bang chloroform
cho di tinh sach cao va chit luting tot dap Ung yen cau img dung ding nghigp.

Tie khoa: Alcaligenes latus, dOt ban, PHB, poly- fl-hydroxybutyrate, polymer phOn hr y sinh hoc
MO DAU
Nhaa c6 nguin goc tit dial moi dang dtroc dung
ring rai trong cuic sing hang ngay cue chung ta.
Tuy nhien, chung gay o nhiem moi truing nghiem
trong boi trong tv nhien cac sin pham nhoa n6i
chung can den hang ngan narri mai phan tiny duoc.
Them vao, do str can kigt nguin dan m6 cang thitc
day vigc can thiet phai tim ra cac nguon vat lieu mai
de thay the. Cac loaf polymer phan hily sinh hoc sinh
ra to cac vi,sinh vat la giai phap tit nhat.
Poly-(3inydroxybutyrate (PHB) thugc Whom
poly-hydrayalkanoates (PHAs) la cac polyester c6
tinh chat ttrcmg to nhua tong hop. PHB dang doge
quan tam nghien cau nhieu vi chimg co dac tinh deo
din hei via tinh chju nhigt cao. Dic bigt, PHB CO kha
nang bj phan hay hoin toan boi vi sinh vat, khing co
deic to va co the duqc sin xuit to nhing nguon c6
the tai tao. PHB duqc tong hqp to nhien trong to bao
chat dm vi khuan nhu the du tra carbon va nang
!Pun trong spot pha sinh truing tinh khi cac to bao
bj giai han mit hoic nhieu nhan to can thiet cite mai

triremg nhu N, P, S, Mg, K, Fe hoac 02 nhung lai du
thira nguOn C (Anderson, Dawes, 1990).
Trong horn 250 loll vi khan dirge th8ng bao co
kha nang tich lily PHB, Alcaligenes laws la vi Idmin
co kha !tang tich lily PHB cao trong suit pha sinh
truing Idling dai hOi so giai han dinh cluing (Wang,
Lee, 1997).

Daniel va doing tic gia (2003) gay at biin vii
muc dich nang cao kha nang tich lily PHB tren
Azospirillum brasilense bang sic nhigt, say Icho khong
khi va bang chign tia UV. Hikmet va ding tic gia
(2003) nghien ciru tao dit bien tren B. megatherium
Y6, B. subtilis K8, B. sphaericus X3 va B. firmus G2
bang chit acriflavin va 5-bromourasil. Nhieu nghien
tiro khic cling thong bao su sinh PHB thay dei trong
nhimg chiinp dit bien (Ojumu et al., 2004). Do do, tic
nhan dit bien ducx xem nhu co tie'm nang trong vigc
cai thign nang suit PHB o vi
Trong nghien dm nay chting fel da dung tic
nhan dit Wen vat ly la tia UV de nang cao kha nang
tich lily PHB cite cang vi khan A. laws VN I.
489


Tap chi Cong nghe Sinh hpc 6(4): 489-496, 2008
VAT LIEU VA PHIJONG PHAP
Vi sinh vet
Chung vi khan A. latus VN1 phan lap tar dat
nhiem au Viet Nam c6 kha nang tich luy PHB
lchoang 40% tong lucmg kho dm 4 brio.
Nuoi cay
Cac nuoi cay tren thach dia, nuoi cay lac, nhan
giong va len men duqc den hanh tren mei truang co
ser (/1): Glucose 30 g; Peptone 10 g; Cao men 5 g;
Na2Mo04.2H20 0,5 mg; Na2W04.2H20 0,5 mg;
MnSO4 0,5 mg; Fe SO4.7H20 10 mg; CaC12.2H20
20 mg; NaC1 0,1 g; MgSO4.7H20 0,2 g;

(N114)2SO4 1 g; KH2PO4 0,2 g; K2HPO4 1,6 g;
pH = 7 (Lee, Chang, 1995).
Nuoi city len men: Qua trinh len men duqc thuc
hien trong binh len men 5 1 (Bioflo 3000, New
Brunswick Scientific Co., Edison, NJ) chira 2,51 mei
truerng. Nhiet de duqc duy trl 6 30°C, toe de khuay
300 vonf/phirt, clang thoi Ichi 1,0 wm. pH = 7,0 ± 0,1
duqc dieu khien tu dung trong suet qua trinh len
men. Sau 20, 24, 28, 44, 48, 52, 68 va 72 h len men:
xac djnh OD, tong luqng kho to bio, ham lucmg
PHB, Chiang khir.
Phtrang phip phin tich
Sinh truing caa vi khuaAn duqc xac djnh bang
do met de quang hoc (OD) djch nuoi 6 7,. = 620 nm.
Sinh khzii thu &Km tar ly tam 20 ml djch nuoi
(10000 yang/phut, 4°C, 10 phut) dupe say kho
105°C den trong ltryng killing dai va can (can phan
tich dien to AB 204, Mettler Toledo, They st).
pH cuoi cua djch nuoi dirge xac djnh bang may
do pH.

Chpn loc dQt biEn throng tinh biing dinh tinh
Dot bien throng firth duqc tech tren moi truang
thach c6 bo sung 1% thutic nhuem Nile blue A
(Sigma). Nuoi nhung dia cay in 30°C. Sau 5 ngay nuoi
ay, quan sat cac khuiti lac dueri anh sang cue tim
(235 nm). Nhang Idiutin lac duong firth PHB c6 mau
yang huynh quang chuyen sang da cam, nhung Ichuen
lac am tinh c6 mau trang (Hilcmet et al., 2003).
Chpn !pc thit Min throng tinh bring djnh lsrpng

Cac got bien duang tinh chop IQc tar thach dia
duqc nuoi tren mei truing djch the co se/ trong 48,h
in 30°C, lac 200 yang/phut. Cuoi giai doan nuoi cay
tian hanh do OD.
xac djnh ham luqng PHB, cac mau duqc ly
tam (15 phut, 6000 vong/phat). Sinh khoi duqc dra 2
lin vii nuerc khir ion vi duqc say kho chlin kitting.
Te bao kho duqc thiry phan trong NaOH 5N in 95°C
trong 2 h va duqc trung haa bin HC1 (Belma et al.,
2002). Ham lucmg PHB trong to bao duqc phan tich
bang sac ki long cao ip HPLC (high-performance
liquid chromatography) so yen duerng chuan PHB.
Chili rat va tinh sach san pluim
Mqt gto bao kho duqc then yeti 10 ml
(acetone:con 90° viii tSr 1:1), lac trong 20 phut. Sau
d6 hoa vio 100 ml chloroform va &Km chuyen sang
he thong soxhlet, chiet rut 6 65°C trong 16 h loci
lipit va nhimg phan tar Ichong phai PHB. Loc, thu
PHB hem tan trong chloroform. Sau da chloroform
duqc cho bay hoi. PHB tinh sach thu &Km bang each
kit taa vai con (Belma et al., 2002).
Xdc djnh ciiu frac viz thIc aim If h6a es& min pluint
Cau trac cua san phim tech chiet dirge xac djnh
thong qua phe hong ngoai (IR).

Ham hem glucose trong min dirge phan tich
bang kit Glucose C2 ten may so rnau quang hoc 6
= 505 nm.

trung binh (Me) can san

, Kited luqng phan
pham duqc xac djnh bang sic IcS, loc gel GPC (Gel
permeation chromatography) so von 0,5% PHB
chuan (Sigma).

Tgo apt ban

KET QUA VA THAO LUAN

Hai mum ml to bao chimg dai nuoi qua dem
(met de' Idioang 5.108 4 bhol ml) duqc cho vao dia
petri 0 90 mm va det chiuig duai tia UV sang ngan
(245 nm) tar 1 don VL-6LC UV Wilber-Lourmat,
Cedex, Phap) trong 60 phut. Vi khuan song sat duqc
xac dinh car sau 10 phut, (let bien duqc thu 6 tS, le
gay chit 99,99% (Hikmet et al., 2003).

PHB la met vet lieu polymer ten tai duai dang
hat nhu the du tit carbon va nang luqng trong to
bao vi khan (Reddy et al., 2003). Trong thuc to
ngotti cac chang tu nhien, chang tai to hqp, lived
to can san xuat PHB tar cac chung dot bien (Gang et
al., 2004).

490


Tap chi Cong nghe Sinh hoc 6(4): 489-496, 2008
A. latus &Qv Iva chqn de nfhien cfru vi no co
the san sinh PHB de clang chiet rut yeti nang suit

cao (Wang, Lee, 1997).

a

Tao dat ban

Anh sang UV (245 nm khoang cach 20 cm)
chieu len huyen phit cua 20 ml chi:mg dai duqc nu8i
cay qua dem (mat de khoang 5.108 to bao/m1).

Hinh 1. KhAn lac tren thach dia

sau 10 phut dueri anh sang UV, xic djnh sv sting sot
cua vi khuan tren mai trirong thach dia co se.
Nhang ket qua quan sat duqc (Hinh 1, 2, Bang
1) cho thay duai tic gong cua anh sang UV, so linyng
vi khuan trong (Lich nuoi gian nhanh cheng theo theri
gian. Sau 20 phin dueri anti huOngcua tia UV,
99,94% vi khuan da bi diet. TO theri diem nay nhting
khan lac don song sot ten thach dia duqc tach de
Iciem tra hog firth so veri chi:mg dai.

ca sb sau thbi gian chi& tia UV. (Theri gian chi& UV x de pha loang dich vi

khuan).

Thad gian (phut)
0

khan sau thbi gian

Tit 14 song sot (%)

100

Wit 0 tobao (CFUlm')

109

Bang 1. TS', la song sot cua vi
chit' anh hied/ng cua tia UV.

107
105

103

10

2, 765

20

0,0061

30

0,000508

0 10 20 30 AO 50 00

40

0,000114

Th.:Asian chau UV (Omit)

50

0,000048

60

0,000008

101

Hinh 2. Mat dg to bao vi khan sau theri gian

chit, anh huerng cua tia UV.
491


Tv chi Cong nghe Sinh hqc 6(4): 489-496, 2008
Sabi) chip lQc dOt bien co the bing dinh tinh PHB
Vi khuin c6 kha &trg song sot dtrai,so tic d'Ong
dat tia UV duqc chuyen sang nuoi cay teen m8i
truing thoch ca sa c6 bo sung 1% Nile blue A. Sau
khi quan sat dirai anh sang cut tim (245 nm), nhang
khuan lac co matt yang huYnh quang chuyen sang da

cam duqc Lich, bb qua nhung khan lac mau trong.
T6ng s6 198 khuin lac co hoat tinh sinh PHB da
dirqc thu nhan va tiep tic chop lac bang dinh lugmg.
Chan loc dOt bien au the hen chung dai bing
djnh luvng PHB
Cac chung dr boat tinh ainh tong hqp PHB duqc
chan Jac va tiep tic nuoi cay tren mai truing djch
the de xac djnh kha nang tich lay PHB nqi bao. Ket
qua phan tich sau khi thay phan to bao bang NaOH
va do tren HPLC cho thay chi co 40 trong so 198
chung sinh PHB thu duqc trong nghien dm chi ra so

sinh twang va kha nang tich liiy PHB cao hen chiing
dai mit tech CO y nghia. Nhang chimg khic the
, hien
nang suet tich lay bang hoac thap hen chung b8 mg.
Phan 16n cac chung dOt bien sinh PHB yeti rang
suit cao hen chung dai da thu dugc tai thii diem 30
phut chieu tia UV. 0 thai diem nay hau het vi khuan
da chet.
Lach va ding tic gia (1990), ding thong bao sr
sinh PHB tang 6 chung det bien Bacillus (Reddy et
al., 2003). Trong nghien ciru dm ha, 2 chimg dijt
bien B. subtilis B8 va B. firmus D7 thu duqc c6 nang
suit tang gap doi so vai chimg dai. Trong so cac
chung d8t bien duang tinh duqc ching toi chin lac
dila tren nang suit PHB cao
chung dai, chung
dOt bien A. latus VN1-20 la tot nit& (Hinh 3). N6 co
kha nang tich lily tai 55,6% PHB/trang lugmg kho to

bao. Chung nay duqc lila chan de len men thu hOi
PHB. Trong khi chung dai chi tich lay duqc 39,3%
PHB/trong lacing kho to bao.

0.8

0.6 1
0

00
tt

04

0.2

0

dai

13 20 39 55 120 122 139 142 195
Chung

Hinh 3. Sinh tnrerng VA kha nang tich by PHB cua 9 chung dOt bien &rang tinh cao nhat so v6i chOng
Nu6i cay len men
Nuoi cay len men co dieu khien la phuong phap
chung nhat duqc dimg de nghien dal dOng hac sinh
truang to bao va su too Minh sin pham; moi quan he
gift sinh truong to bao va dieu kien mOi tivang
(Wang, Lee, 1997).

Chang toi da tien hanh nuoi cay len men chimg
dOt bien A. latus VN1-20 (Hinh 4) de, nghien thu
dOng hac ctia qui trinh sinh PHB c6 dieu khien pH
trong dieu kien tucmg to vai chin dai. Sinh trtrong
cua vi khuan, so tich lily PHB, so tieu thu glucose
492

duqc xac dinh de tau duqc sr thay deri trong suet
qua trinh len men.
Deng huc qui trinh len men cita chiing dOt bien A.
latus VN1-20 va chung dai duqc the hien 6 (Hinh 6).
Nhung ket qua thu duqc cho thiy c6 sr tang sinh
truing cua chimg dOt bien so veri chinig dai. Trong
truing hqp chung dOt bien, sinh truing nhanh da
duqc quan sat, to bao dot tin pha tinh sau 28 h. Sinh
truing 6 chimg dai la cham to lac bit diu len men
cho tai 28 h. Sau do, sinh throng tang va dot tai pha
tinh 6 44 h. Sinh twang toi da cua chung (tat bien


Tgp chi Cling nghe Sinh hgc 6(4): 489-496, 2008

dat 15,42 g/I a thai diem 28 h, trong khi veri chfing
dai la 10,14 g/1 sau 48 h.

Sinh twang cua chting dai cham va kern han. Sau
72 h len men ham luang glucose con lai trong mei
tatting van cao. Dieu nay dr nghia la nhu cau del hal
co chat cho len men dm chimg dai la khong cao.

Viec tang sinh khoi cita vi sinh vat hien ty le
nghich veri su giam uguon carbon trong mei true:111g.
Doi veri chung dOt bien it han 1 g/I glucose con lai
cuoi caa qua trinh len men. Khi glucose trong mei
traang can kiet sir len men phai dimg lai ngay, neu
tiep.tuc thi sin pham PHB se bj phan hit), trer thanh
nguon carbon va nang lugng cho sinh truang cua vi
khuan. Vi vay trong truang hap chting drat bien, qua
trinh len men nen dung lai sau khoing 30 h nuoi cay.

Sin pham PHB a chung dai dat tkda 0,415 g/g
sau 52 h nuoi cay. Vii chimg got bien sin phim
PHB tang len va dot raj da 0,583 g/g sau 28 h nuoi
cay. Ket qui nay cho thay ca the sinh tong hap PHB
dadtrac thiet lap thanh cong a ching dOt bien. Nang
suit sinh PHB a thing (Vat bien da dirge cii thien
khoing 40% so veri chung dai.

Hinh 4. Qua trinh len men thu nhan PHB cua chOng
dert bier' VN1-20.

VN1-20.

Hinh 5. San phArn PHB tinh sach ta, chung dOt Wan

Chung dai

Chung adt hien
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libidsilan by mom 0$
-6- PH8

(9f9)

--•-- Glucose du (rngld1)

Hinh 6. Eking hoc qua trinh len men dm chUng dai va chung dOt bien VN1-20.
493


Tgp chi Cong nghe Sinh hpc 6(4): 489-496, 2008
dinh hap thu 1725,42 tuang Ong viii nhem chitc
cho phep khang (firth san pham thu duqc la PHB.

Ket qui tich chiet PHB
Him luqng PHB tinh sach thu duqc sau len men
chimg dOt hien A. latus VNI-20 (Hinh 5) so vai tong
sinh kh6i kho dem chitt dat khoing 53%. So sanh
voiket,qui xac dinh ham lung PHB trong sinh kh6i
bang sac Id long cao ap HPLC (58,3%) cho thy co
su chenh 16ch. Sai so nay li do stn hao but san pham
trong qui trinh tach chiet.
TO bio chung vi Ichuan d6t hien VN1-20 tich luy
cac hat PHB duqc quan sat ro dual kinh hi6n vi din
tin (Hinh 7)
Xic dinh cAu true vi dic diem ISr boa ctia san
pham

Hinh 7. Anh kinh hi6n vi Men tin to bao chung dOt
lat qua pho hling ngoai (Hinh 8) cho ditty c6 Wen VN1-20 china tint PHB (X 25000 x).

0 se

x x ;,,xxsrxes.,,,,..„.

22

3

Hinh 8. Phi IR cOa san pham

?Non

2000
Mr...nun-41w cm-1

3000

1000

1500

1000

500

PHB.

ChUng dgt hien

Chung dai
hal
loo.00

100.00Col

50.00

50.00

0.00

20.00

10.00

0.00

lain)
Mw 4,46679 x 105 Da

Hinh 9. Khoi luvng phan tin trung binh cua san pham PHB.
494

Mw: 3,99455 x 105 Da


Tap chi Cong nghe Sinh hoc 6(4): 489-496, 2008
Sau khi chiet rat can tach PHB has tan trong
chloroform !way
bao ye, polymer khoi bj phan
hay. San pham phai c6
luong phan tir trung
binh (Mw) > 250000 Da mai &roc chap nhin cho

img dtmg, cong nghiep. Tinh chit IS, hoc
PHB
tan! d6 ket tinh, dO deo, deo dan h6i phu thu6c vao
kh6i luong phan tir. Kh6i lucmg phan tit cua sin
pham thi chat anh huang bed chung vi sinh vat &roc
sir dung, dieu kien nuoi cay va dO tinh sach cua mitt
thu duqc. PHB co khoi luong phan tir cao la hau ich
cho ang dung ding nghiep (Chen et al., 2001).
Khei luong phan ta trung binh caa san phim
PHB tinh sach thu dtroc sau qua trinh len men cfrang
dai va chung dOt bien khoang 4,47.105 va 3,99.105
Da, tuong img (Hinh, 9). So sanh vii kit& Wong
phan fir caa PHB chuan (Sigma) la 5,3.105 Da, khoi
Wong phan tir caa san phim PHB ma chung !el thu
duoc tit Chung dOt bien kha cao dap ang yeu eau ang
dung cong nghiep.
}CET LUAN
Dung tic nhan vat ly tia UV chieu len to bao
chung vi khuin A. latus VN1 da thu nhin 40 chung
d6t bien co hoot firth sinh PHB cao han chimg dai.
Da sa cac chung dOt hien sinh PHB cao deu thu
dtroc tai Uteri diem 30 phut chi& tia UV.
Chang dOt bien A. latus VN1-20 la tit nhit, n6 c6
Icha nang rich lay tai 55,6% PHB, tong khi chung dai
chi tich Iuy duvc 39,3% PHB/ tong Ming kho to bap.
Sinh truing ciut chung dot bien dot tai da 15,42
g/I sau 28 h len men, trong khi vii chung dai la
10,14 g/1 sau 48 h.
Nang suit sinh PHB a chung dOt bien da cai
thien duqc Ichoing 40% so vii chung dai.

Buerc ditu chimg tea da Lich chi& thanh cong sin
phim PHB c6 d6 tinh sach cao va chit Wong tot dap
ling yen au ang dung cong nghiep.
Lai cAm an: Cong !rink claw Mac hien veil sa ha
try kinh phi caa De tai cap Vin Cong nghesinh
hpc 2007 va chitcmg trinh hop tic quac to
"Research on Biomass Technology 2006" ta chac
bed JICA, Nhat Ban.

TAI LIEU THAM KHAO
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495


Tgp chi Cong nghe Sinh hoc 6(4): 489-496, 2008

IMPROVEMENT OF POLY-B-HYDROXYBUTYRATE PRODUCTION OF
ALCALIGENES LATUS VN1 STRAIN BY MUTAGENESIS SELECTION
Pham Thanh

Tran Dinh Man', Yutaka Tokiwa2

'Institute of Biotechnology

21nstitute for Biological Resources and Functions, National Institute of Advanced Industrial Science and
Technology (AIST), Tsukuba, Japan

SUMMARY
Poly-p-hydroxybutyrate (PHB) is a biodegradable plastic that has attracted much attention due to
solution to the existing grave problems of plastic waste. Today, most research efforts in this field
concentrate on the isolation of PHB producing microorganisms from different sources and improvement
of PHB production abilities of microorganisms. This study was under taken by applying UV light
mutagen on Alcaligenes latus VN1 to attempt to obtain mutant strains with higher PHB percentage yield.
Forty of the 198 obtained putative mutant strains showed significantly higher PHB yield, while other
strains showed PHB yield equal to or less than the wild type strain. Most of putative mutant strains have
been selected at around 30 minutes of UV light treatment. A PHB yield of 55.6% of total dried weight
could be achieved from one mutant strain (A. latus VN1-20). This strain has increased yield to about 40%
in compare to the wild type strain. The results after extracting of PHB product by chloroform showed
that this bacterium was able to accumulate intracellular PHB with high molecular weight in basal
medium after 72 hours of fermentation. PHB product could be developed for industrial applications.
Keywords: Alcaligenes latus, biodegradable plastic, mutation, PHB, poly-fi-hydroxybutyrate

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