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.

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;

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

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


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

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).

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
3C0


--•-- 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.,,,,..„.

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

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
Anderson AJ, Dawes EA (1990) Occurrence,
metabolism, metabolic role and industrial uses of
bacterial polyhydroxyalkanoates. Microbial Rev 54:
450-472.
Belma Aslim, Zehra Nur Yuksekdag, Yavuz Beyatli
(2002) Determination of PHB growth quantities of
certain Bacillus species isolated from soil. Turk
Electron J Biotechnol special issue: 24-30.
Chen Guogiang, Wu Qiong, Chen Jinchun (2001)
Biosynthesis of polyhydroxyalkanoates. Tsinghua Sci
Technol 6(3): 193-199.
Daniel Kadouri, Edouard Jurkevitch, Yaacov Okon
(2003) Involvement of the Reserve Material Poly-fthydroxybutyrate in Azospirillum brasilense Stress
Endurance and Root Colonization. Appl Environ
Microbial 69(6): 3244-3250.
Gang Guk Choi, Hyung Woo Kim, Young Ha Rhee
(2004) Enzymatic & Non-enzymatic degradation of
PHB-PHV. Copolyesters Produced by Alcaligenes sp.
MT16. J Microbial 42(4): 346-352.
Hikmet Katircioglu, Belma Aslim, Zehra Nur

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

• Author for correspondence: Tel: 84-4-37567103;E-mail: ptha04Avahoo.com
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