Hdi nghj Khoa hgc vd Cdng ngh? bien todn qudc Idn thir V
PHAN LAP VA SANG LQC VI KHUAN BIEN SINH ENZYM BE NGAN MACH POLYSACCARIDE TlT R O N G NAU Cao Thi Thiiv H5ng\ Biii Minh LyS Phan Thi Hoai Trinh', Pham Due Thjnh',
Huynh Hoang Nhir Khanh\ va Le Mai Hmmg ^ 1. Vien Nghien cuu va U'ng dung Cdng ngh? Nha Trarig, VAST
2. Vien Hda hpc cac Hgp chit thien nhien, VAST.
Email: caohang.nitra(S).gmail.com Tom tat:
Dd phdn lap duac 30 chung vi khudn tir rong bien, 15 chimg vl khudn tir d^ng vgt khdng xuang sdng bien vd 6 chung vi khudn tir nuac bien cua vung Bien Bdng Viet Nam. Cdc ca chat duac diing di sdng Igc vi sinh vgt bien sinh enzym be ngdn mgch polysaccarit Id fucoidan chiet tir rong Sargassum mcclurei, Sargassum polycystum cua Viet Nam vd Fucus evanescens cua L.B.Nga, laminaran tir rong Laminaria cichorioides. polymanuronic axit tir rong Fucus evanescens cda L.B.Nga. Kit qud sdng Igc da phdt hi^n 8 chiing vi khudn sinh enzym be ngdn mgch fucoidan, 12 chiing vi khudn sinh enzym be ngdn mgch polymanuronic axit vd 30 chiing sinh enzym be ngdn mgch laminaran trong tdng sd 51 chung vi khudn phdn lap duac.
ISOLATION AND SCREENING MARINE BACTERID PRODUCING ENZYMES DEGRADE POLYSACCHARITE FROM BROWN SEAWEEDS
Thirty bacteria strains were isolated from seaweeds, 15 bacteria strains were isolated from marine invertebrates and 6 bacteria strains were isolated from seawater in the East Sea of Vietnam. The substrate were used to screen marine microorganisms produce enzymes that degrade polysaccharlte are Fucoidan from Sargassum mcclurei, Sargassum polycystum of Vietnam and Fucus
evanescens of Russia, laminaran from Laminaria cichorioides seaweed and polymanuronic acid from fucus evanescens of Russia. Eight bacteria strains were found to contain fiicoidanase, 12 bacteria strains produced enzyme that degrade polymanuronic and 30 bacteria strains contained laminarinase in total 51 isolated strains.
1. MO DAU
Nude ta cd bien Ddng bao hpc toan bd phia Ddng va phia Nam vdi di?n tich mat nude bien rong hon 1.000.000 km^, dugc thien nhien ban tang cho ngudn tai nguyen rong bien rat da dang va phong phu, trong dd rong Ma (Sargassum), da dugc-xac djnh dugc tren 60 loai vdi san lugng khai thac udc tinh len tdi 10.000 tin khd/nam. Trong rong Nau, ham lugng polysaccaride chilm khoang tir 40-80% khdi luang rong khd, la nhiing chit cd gia
Tieu ban Sinh hgc vd nguon lgi sinh vgt Biin
tri nhat [7]. Cac polysaccharide tir rong Nau cu the la Laminaran, Fucoidan, Uronan tan trong nude va acid alginic tan trong kilm da thu hiit su chii y nhilu cua cac nha khoa hpc do boat tinh sinh hgc va cau tnic dac thii ciia chiing [10].
Laminaran dugc biet den nhu la chat khang u , tia bao ve nang lugng va tia biic xa, i'Chat kich hoEit su mien dich ciia ca [4]. Polyuronan cd hoat tinh khang u nao [10], Dac biet, facoidan la mpt sulfated hetero-polysaccharide, dugc tim thiy trong vach tl bao rong nau hoat tinh sinh hgc vd cimg phong phii va thii vi: HoEit tinh chdng dong mau va chdng ddng tu, hoat tinh khang u, tang cudng tinh miln dich, hoat tinh khang vi nit, tri benh da day, benh ve da [3].
Nhd cac tinh chat nay ma fucoidan, laminaran va polyuronan cd rat nhieu trien vpng dugc sii dung cho lam thudc chtia benh, nude hoa, che bien thiic an, nuoi trdng thiiy san va vi sinh vat hgc [3]. Tuy nhien, nhieu boat tinh sinh hpc cua polysaccaride phu thupc vao cau tnic hda hgc cua chiing. Cau tnic hda hgc ciia polysaccaride chilt tir rong nau rit da dan^. Dac tinh cau tnic va thanh phan hda hgc ciia cac polysaccarit nay phu thudc vao ngudn thu, miia thu hoach, tudi ciia rong va cac nhan td khac. Them vao dd nhiing nghien ciiu g3n dSy cho thay boat tinh sinh hgc ciia cac polysaccaride rong nau manh hem gap nhilu lln khi trgng lugng ph3n tii ciia chiing nhd han lOKda [6]. Vi vay, dl giiip cho viec nghien ciiu ca che tac dung cua cac polysaccaride rong nau len cac tl bao sinh vSt va tiln tdi sir dung chiing d6 lam dugc lieu, thi viec xac djnh can tnic hda hgc ciia chiing la dieu ki?n tien quyet va dang thu hiit sir chii ;^ cua cac nha khoa hgc tren thi gidi.
Cac phuang phap sir dung ly hoa trudc khi phan tich dugc sii dung hien tai (thiiy phan de-sulfat hda, de actyl hda) thudng ddi hdi dieu ki?n acid hoEic kiem manh d nhigt do cao, do dd cd the lam thay ddi cau tnic ciia polysaccaride va lam mat boat tinh sinh hpc quy cua chiing. Vi v^y, enzyme la cdng c\i hiiu hi$u nhat de de thuc hi?n cac phSn tich nay [2].
6 Vi?t Nam, cdc nghien cihi ve vi sinh vat va enzym chuyen hda cac polysaccaride tren dat lien dS va dang dugc trien khai kha manh tai cdc vien nghien cuu, trudng dai hgc. Tuy nhien, cac nghien cuu vl vi sinh vat bien d nude ta cdn rat it, dac biet la cac nghien ciiu ve vi sinh vdt biin sinh enzyme phan giai cac polysaccaride rong biin dl su dyng lam dugc Ii?u. Vi v$y day la mpt hudng mdi, cdn dupe md ra de nghien ciru. Trong bai bdo nay chiing tdi trinh bay sa bg qua trinh phan lap va sang loc vi sinh vat bien sinh enzyme cd kha nang be ngan m^ch polysacharide.-
2. NGUYEN LIEU VA PHU'ONG PHAP 2.L Thu mSu
Cac mlu nude biin va rong biin va ddng vat than mem bien dupe thu tai viing bien Nha Trang va cdc vimg biin Viet Nam trong chuyen tau khao sat bien ciia con tau
"Akadamik Oparin" thdng 5/2010.
2.2. Phuang phap phan lap vi sinh vat
Vi sinh vat bien dugc phan lap tren mdi trudng thach vdi thanh phan gdm 750ml nude biin, nude may 250nil,K2HPO4 0,Ig,MgSO4 0,lg, glucose 3g, pepton 2g, dich chiet nam men Ig va agar 18-20g, pH 7-7,2. U d 30°C, khi thay xult hien khuin lac vi khuIn thi tiln hanh lam
Hgi nghi Khoa hgc vd Cdng ngh? bien todn quoc lan thuV
thuin tren mdi trudng tuang tu mdi trudng phan lap. Cac chiing thuan dugc giii trong moi trudng ban rdn vdi thanh phIn dinh dudng tuang tu nhu ten va agar chi cd 3g/l [5].
2.3. Phucmg phap sang loc enzyme be ng^n m^ch fucoidan tu vi sinh vat
Cac chung vi khuin thuIn chiing dugc nudi tang sinh trong mdi trudng long vdi thanh phIn tuang tu mdi trudng phan lap. Cac chiing vi khuin dugc nudi cly lac d SO^C, 120rpm, 24 gid. Dich chiet ndi bao va ngoai bao dugc sir dung de thii nghiem ho?it tinh enzym[l]. Tiln hanh khao sat boat tinh enzym be ngdn m^ch polysaccarit theo phuang phap Nelson [9]. Ham lugng protein dugc xac dinh theo phuong phap Lowry [8].
3. KET QUA VA THAO L U i ^ 3.1. PhSn lap vi sinh vat bien
Vi khudn biin dugc phan lap tir cac ngudn rong Nau vi day la ngudn ca chat polysaccarit ddi dao, do dd, kha nang tim kiem vi sinh vat sinh etizym be ngan mach polysaccaride cao nhlt. Ddng vat than mem bien (hai sam, sen bien, cau gai) la rJiiing loai an rong bien nen chiing cdn phai tlet ra enzyme de tieu hda polysaccarride, rong Ndu sdng trong mdi trudng nude biin, khi chit di se bi vi sinh vat ky sinh va he vi sinh vat ngoai sinh tin cdng phan buy, mdi trudng nude bien xung quanh cay rong tan lui sinh song ciing la mgt ngudn phan lap vi sinh vat cd hieu qua. Dua tren ca sd dd, chiing tdi tien hanh phan lap vi sinh vat bien va ket qua thu dugc the hien d bang 1:
STT 1 2
3 •
4 5 6 7 8 9 10 11 12 13 14 15 16 17
Bdng 1: Ddc diem khudn lac vi Nguon phan
lap Rong nau (30
chung)
Ky hifu Chung 38-14-4-1 38-14-4-2 38-I4-5-I 38-14-5-2 38-14-5-3 38-14-5-4 38-14-12-1 38-14-12-2 Fl F2 F3 F4 F5 FI4 FI5 F16 F22
khudn bien phdn lap dicac DSc diem khuan lac MQC lan, mau trang Mau trang
Khuan lac nho, khong mau Mau be
Mautiang Mau be Mau be Mau trang
Khuan lac trang, nhay, bong Khuan lac trang, tron, co nhan Khuan lac tron, mau vang, bong Khuan lac tring sua, nhay bong Khuan lac tring, tron, loi Tr5ng sQa, tron, bong, tron Tron, I6i, vang nhat, co vien sang D?t, bdng, tiing, bmh bSu.dyc Nd
Tieu ban Sinh hgc vd ngudn lgi sinh vgt Bien
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Ndikh
Hai sara (3 chiing)
Sen bien (5 chiing)
C i u gai (7 chiing)
Nucfc bien (6 chiing)
jngxdc dinh F23 F24 NITRA-108 NITRA-I09 NITRA-IIO NITRA-111 NITRA-112 NITRA-113 NITRA-114 N I T R A - I I 5 NITRA-132 NITRA-133 N n R A - 1 3 4 038-4-3-2 0 3 8 - 4 - 3 - 7 038-5-4-1 038-5-3-1 038-5-3-3.1 038 -5-3-3.2 038-m-l-2 038J18-2-1 038-1-3-5 038-1-3-3.1 038-1-3-7.1 038-1-3-7.2 038-1-3-11.1 038-1-3-11.2 0 3 8 - 7 ^ - 2 NlTRA-26 NITRA-27 NlTRA-131 NITRA-77 NITRA-78 NITRA-79
Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Mau kem Mau vang Mau vang Vang cam sang BS mat nhan nheo. vang sang V ^ g sang
Mau be H6ng sang Mau vang Mau kem, nhSn nheo Nd
Nd
Khuan lac nau vang Khuan lac nau vang Nd
Nd Nd Nd Nd Nd Nd
Bang 1 cho thay, da phan ISp dugc 51 chung vi khuin biSn. Trong do nguon rong nau CO s6 lugng vi khuin phan lap dugc cao nhjt (30 chung), hai sam (Schung), sen bi6n (5 chung), cku gai (7 chiing) va nuac biSn (6 chung). Nhin so bo ve dac diSm khuin lac, cac chung vi khuin nay co hinh dang, kich thuoc khac nhau nen co the chung la nhung chiing vi khuin khac nhau. Cac chiing vi khuin nay dugc lam thuin tren inoi tnicmg dia thach va kiem tra dp thuin chiing qua kinh hiSn vi dien tu. Cac chting vi khuin thuin chiing dugc su ' dung di sang Igc hogt tinh sinh enzym be ngan mach polysaccaride.
Hdi nghi Khoa hgc vd Cdng ngh? bien todn quoc idn thit V
3.2. Sang loc vi sinh vat bien theo dinh hirdng sinh en^one be n g i a mach polysaccaride Bdng 2: Kit qud sdng Ipc vi sinh vgt bien sinh enzyme be ngdn mgch polysaccaride ST
T 1 2 3 4 i 6 8
;
9 10 11 l i 13 14 15 16 1/
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ii 34 35- 36 il
m
3940 41 42 43 44 45 46 47 48 49 50 51
C h u n g vi sinh vat 38-14-4-1 38-14-4-2 38-14-5-1 38-14-5-2 38-14-5-3 38-14-5-4 38-14-12-1 38-14-12-2 Fl F2 F3 F4 F5 F14 FI5 F16 F22 F23 F24 NITRA-108 NITRA-109 NITRA-IIO NITRA-111 NITRA-112 NITRA-113 NITRA-I14 NiTRA-115 NlTRA-132 NlTRA-133 NITRA-134 0 3 8 - 4 - 3 - 7 038-5-4-1 038-4-3-2 038-5-3-1 038-5-3-3.1 038 -5-3-3.2 038-m-l-2 038-48-2-1 038-1-3-5 03S-I-3-II.1 038-1-3-11.2 038-1-3-7.1 0 3 8 - I - 3 - 7 a 038-1-3-3.1 038-7-4-2 NITRA-26 NITRA-27 NlTRA-131 NITRA-77 NITRA-78 NlTRA-79
Hoat tinh enzyme U/mg prvtein Fucoidanase
S.mcc 0 0 0 0 0 0 0 0 0 0 0 0
"
0.487 0 0 0 0 0.312
0 0 0 0 0 0 0 0 0,432 0.562 0.321 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
S.po 0 0 0 0 0 0 0 0 0 0 0 0 0 0,512
0 0 0 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 0 0,837
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Fu.ev 0 0 0 0 0 0 0 0 0,210
0 0 0 0 0,538
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.262 0,404 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
a
Polymanuronic acid lyase Fii.ev 0,012 0 0 0 0 2 3 1 0,432 0 0 0.911
0 0 0 0 1,950
0 0.054
0 0 0,453
0 0 0.432
0 0 0,129
0 0.673
0 0 0 0,021
0 0 0 0 0 0 0 0 0 0 0 0 0 0.056
0 0 0 0 0 0
Laminaranasp L.dc ^ 4.131 ^ 0 J 7 8 H
0 0 - <
0.431 0.765 0 0 H 2,534
0 1,794
0 5.892
0 0,798
0 1.374
0 0.497 3.412 0.983 0.560 0.450 0,430 0.123 0 0.765 3.980 _^
4.905 4,321 0.760 7.903 10.985 0 0 0 0.210
0 0 0 OjOl
0 0 0 27.68
0 0 0 J 2 6 0.431 0.120 0 J 3 4 S.mcc: Sargassum mcclurei. S.po- Sargassum polycystum, Fu.ev: Fucus evanaescens, Lcic: Laminaria cichorioides
Tieu ban Sinh hgc vd nguon lgi sinh vgt Bien
Ket qua d Bang 2 cho thay, trong 51 chiing vi khuan dugc sang Ipe thi cd 8 chiing cd kha nang sinh enzyme be ngan mach fucoidan. Trong dd, sd chung chiing sinh enzyme be ngan mach fucoidan cua rong S. mcclurei, S. polycystum va Fucus evdnnescens theo thii tu la 5,2 va 4. Dieu nay la do 3 loai ca chat fucoidan dugc chilt tir 3 loai rong khdc nhau nen chiing cd cau tnic khac nhau, vi v$y ma enzyme tdc dgng len 3 ca chat nay khdc nhau.
Trong 8 chiing vi khuan cd boat tinh fucoidanase thi 6 chiing cd ngudn gdc tir rong bien va 2 chiing cd ngudn gdc tit dgng vat than mem bien. Chiing F14 va 038-4-3-2 la nhung chiing cd boat tinh cao. Nhu vay, rong ndu Id ngudn phan l^p vi sinh vdt sinh enzyme fucoidanase cd hlSu qua.
Nhin vao Bang 2, ta ciing nh?n thay cd 12 chiing vi khudn sinh enzyme polymannuronic acid lyase trong dd chiing .F14 sinh enzym cd boat tinh cao nhlt vd cd 30 chimg sinh enzyme laminarianase trong dd chung 038-7-4-2 cd boat tinh cao nhat. Nhan thdy rang ti le vi khuan sinh enzym laminarianase cao nhat (30/51) va boat tinh enzyme cung cao han nhiSu so vdi cac nhdm enzyme khdc vi laminaran dugc hinh thanh tir cac gdc D-glucans la loai dudng chii yeu vi sinh v^t sir dyng trong qua trinh chuyen hda, vi vay, chiing uu ti£n sd dung nguon dinh dudng thuan lgi nhdt. Cdc chiing vi khuan phan lap tir nude bien khdng nh|tn thay khd nang sinh enzyme be ngan mach fucoidan va polymannuronic acid, cdn hoat tinh laminarianase thi thap so vdi vi khuan phan lap tu cdc nguon khac.
4. K E T L U ^
Tir cac ngudn rong ndu, dgng v^t than mim bien va nude bien thudc viing bien Dong Vi?t Nam, chiing tdi da phan lap dugc 51 chiing vi khudn thuin chiing. Trong dd cd 8 chiing sinh enzyme fiicoidanase (chiing F14 va 038-4-3-2 cd boat tinh cao nhlt), 12 chiing smh enzyme polymanuronic acid lyase (chiing F14 cd boat tinh cao nhlt) va 30 chiing sinh enzyme laminarianase (chiing 038-7-4-2 cd boat tinh cao nhlt). Nguin rong nau la ngudn cd quin thi vi khuin phong phii nhdt va kha nang sinh enzyme ciia nhdm vi khuin ndy ciing cao han so vdi cdc nhdm khac. Cac chiing vi khuan bien nay la mpt ngudn tiem nang de tim kiem cac chat cd hoat tinh sinh hpc.
LOfI CAM ON
Tap the tac gia xin chan thanh cam an PGS.TS. Pham" Qudc Long, chu nhiem tilu dv an sd 1/19/47 "Hgp tac Viet Nam - CHLB Nga vl dilu tra khao sat ngudn hoat chit va da dang sinh hgc biin Vi^t Nam" da tao dilu kien thuan lgi cho chiing tdi tham gia lam vi?c ciing cdc nha khoa hgc LB.Nga tren con tdu "Akademik Oparin" va lam vi6c tai Vien Hda sinh Hiiu ca Thai Binh Duang - LB.Nga. Chiing tdi ciing xin cdm an cac thanh vien phdng Hda phan tich va triln khai cdng nghe, lanh dao Vien Nghien ciiu va Ung dyng Cdng nghe Nha Trang cung tap the cdc nha khoa hpc thudc Vien Hda sinh hiru ca Thdi Binh Duang da tao dilu kien giiip da chiing tdi hoan thanh ket qua nay.
Hdi nghj Khoa hgc vd Cdng ngh? bien todn qudc Idn thu V
TAI IA%\3 THAM KHAO
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[5] Descamps el al. (2006), Isolation and cuture of a marine bacterium degrading the sulfated fucans firom marine brown algae. Mar Biotechnol 8:27-39.
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[7] Kuznetsova T. A., Zaporozhets T. C, Besednova N. N. et al., (2003), Immunostimuliruyuschaya i antikuagulyantnaya aktivnost fucoidana iz buroi vodorosli Okhotskovo morya Fucus evanescens// Antibiotiki i khimioterapiya.,T.
48. N4, 11-13,
[8] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folm phenol reagent. J. Biol. Chem. 193: 265-275.
[9] Nelson TE (1944), "A photometric adaptation of the Somogy method for the determination of glucose", J. Biol. Chem. 153: 375-381.
[10] Zvyagintseva, Shevchenko N.M, Nazarenko E.L, et al., (2005), Water-soluble polysaccharides of some brown algae of the Russian Far-East. Structure and biological action of low-molecular mass polyuronans. Journal of Experiment Marine Biology and Ecology 320 123-131.