640 Tuyen tgp Hpi nghj KHOA HQC T O A N Q U 6 C Vg SINH HQC BifeN vA P H A T T R I ^ N B £ N VCTNG
PHAN LAP VA SANG LOC VI SINH VAT BIEN PHAN CAT FUCOIDAN Tir RONG NAU
Cao Thi Thuy Hing', Biii Minh Ly', Huynh Hoang Nhu Khanh', Phan Thi Hoai Trinh', Nguyen Duy Nhut', Pham Dure Thjnh', Le Mai Huong^
' Viin Nghiin dm vd Ifng dung Cong nghi Nha Trang, Viin Khoa hpc vd Cong nghi Viit Nam
^ Viin Hod hpc cdc Hap chdt thiin nhien, Viin Khoa hpc vd Cong nghi Viit Nam
I. M O DAU
Fucoidan, mgt sulphat polysacarit sinh hgc dugc chiet tir rong Nau, dac bift quan tam nghien cim nhd tinh chit sinh hgc da dang ciia no. Nhd co nhieu boat tinh dugc ly dac bift nhu chIng d6ng ty, chIng viem nhiem, dilu tilt mien djch, ire che sy phat trien u budu, khang ung thu va khang virut kl ca vi rut HIV..., va ban nira ngudn cung cap chu yeu la rong Nau ty nhien vdi trir lugng Idn d cac vimg biln fren the gidi nen fucoidan frd thanh d6i tugng day trien vgng frong vifc su dyng chiing lam dugc lifu. Tuy nhien moi lien quan giira boat tinh sinh hgc va cau tnic ciia chiing vin chua dugc giai thich ro rang do cau triic phirc tap va khong theo qui luat cua fucoidan tir cac nguon g6c khac nhau [1].
c i u fruc ciia fucoidan phirc tap va phu thudc vao ngu6n gdc loai rong ma n6 dugc chiet ra.
Chinh vi vay vifc nghien ciru anh hudng cua tung yeu to cau triic (trgng lugng phan tu, nhom tbi, mach nhanh, kilu lien kit) len boat tinh sinh hgc ciia fucoidan la mgt thach thiic kh6ng nho dii vdi cac nha khoa hgc fren the gidi. Dieu nay chi dugc giai quyet hifu qua khi sir dyng cac loai enz>'m co kha nang be gay mach fucoidan nhu la mgt cong cy de nghien ciiu cau tnic cQng nhu tuang quan giu'a cau tnic va boat tinh sinh hgc cua fucoidan.
Cho den nay cac nha khoa hgc da tim ra 02 loai enzjm O-glycosyl hydrolase cd boat tinh vdi flicoidan do la fiicoidanase va a-L-fiicosidase. Hai loai enzym nay chii yeu dugc tim thay frong cac dgng vat than mem, cac loai vi khuan bien va d6i khi dugc tim thay fren cac sinh vat khac.
Cac nha khoa hgc Phap va Nhat Ban da c6ng b6 tim thay mgt s6 loai fucoidanase tir mgt so loai dgng vat than mem bien nhu: bao ngu Haliotis sp, dgng vat than mem Pectinidae Patinopecten yessoensis, va con difp Pecten maximus co kha nang thuy phan fucoidan mgt each rat hifu nghifm tao thanh cac oligosacarit [2,3,4]. Tuy nhien viec tim kiem enzym tir dgng vat than mem bifn chi co y nghTa ve mat nghien ciru nhung khd cd the dua vao ling dyng d quy mo Idn. Vi vay, ngudi ta dac bift quan tam den enzym be gay mach fucoidan co ngu6n vi sinh vat va fren the gidi cung da co nhieu c6ng trinh nghien ciru vf van df nay. Mgt s6 nhom cac nha khoa hgc Nhat Ban [5,6] da phan lap dugc vi khuan co kha nang be gay mach fucoidan tir fram tich bien va nude bien. Trong thap ky XX trd lai day co nhieu nghien ciru thu vj ve enzym phan lap tir sinh vat bien dugc cong bo. Sakai va cgng sy [7,8] da phan lap tir biln Nhat Ban mgt vi khuan bien (Flavobateriacea) co the cat nhieu loai fucoidan khac nhau dugc chiet tir cac loai rong Laminariales, Kiellmaniella crassifolia, Undaria pinatifida, va Lessonia nigrescens...Emr^'mnky dugc cho la thugc loai sulfated fucoglucuronomannan lyase...
Vift Nam mac dii co dudng bd bien keo dai tir Bic den Nam vdi nguIn tai nguyen bien vo ciing phong phu. Trong do co rong nau lam nguyen lifu san xuat fucoidan co trir lugng len tdi
10.000 tan/nam. Tuy nhien chi co mgt vai nghien ciru sinh vat biln sinh enzym fucoidanase va a-L-fucosidase phan giai trong rong Nau dugc cong b l nhung ciing chi tren d6i tugng la dgng
Tieu ban: HOAT C H A T SINH HQC BI^N 6 4 1
vat than mim biln [9]. Vi vay chung toi tien hanh nghien cim sang lgc cac vi sinh vat biln df san xuat enzym be ngan mach fucoidan mgt each hifu qua.
II. VAT LIEU VA PHU'ONG PHAP NGHIEN ClTU 1. Dieu che fucoidan
Cac loai rong nau: Sargassum microcystum, S. mcclurei, S. olygocystum, S. duplicatum, Turbinatia ornata, Sargassum polycystum dugc sir dung lam nguon rong de tach chift fucoidan.
Rong dugc thu d vimg bien Nha Trang. Fucoidan dugc chiet tir rong bang HCl 0,1N va tiia c6n.
Fucoidan th6 dugc lam sach tiep qua sac ky cot DEAE-cellulose. Fucoidan sach dugc d6ng khd lanh va dugc diing lam ca chat de sang lgc boat tinh thuy phan fucoidan.
2. Phan lap vi sinh vat bien be ngan mach fucoidan
• Thu mau
Cac mau nude bien, biin bifn va rong nau dugc thu tai Bai Can (12°I2'30"- vT do Bac, 109" 13'30" kinh do Ddng) thugc viing bien Nha Trang. Mau dugc thu vao binh nhya v6 triing va van chuyen ve phdng thi nghifm ngay trong ngay. Mau nude bien, bim bien va rong tuai dugc dimg de phan lap ngay. Lay mgt tan rong cho vao binh tam giac cd chiia nude bien vo trimg bo sung them pepton (5g/l), de d 20''C, cho tdi khi quan sat thay sy phan ra ciia tan rong thi dem di phan lap vi sinh vat.
• Phirong phap phan lap
Vi sinh vat bien dugc phan lap tren m6i trudng chi chiia nguon carbon duy nhat la fucoidan (22/lOOml nude bien) va moi truong dugc ddng ran bang agar (15g/L). U d 30°C cho den khi thay xuat hifn khuan lac vi sinh vat thi tien hanh lam thuan tren mdi trudng co thanh phan nude biln 750ml, nude may 250ml, K2HPO4 O.lg, MgS04 O.lg, saccharose 5g, pepton lOg, agar 20g, dilu chinh pH 7-7.2. Day cung chinh la moi trudng thach nghieng de giir vi sinh vat cho cac thir nghifm tilp theo. Cac chiing sach dugc giir frong mdi trudng ban ran vdi thanh phan dinh dudng tuong ty nhu tren nhung agar chi cd 3g/L.
• Phirong phap sang loc
Cac chung vi khuan sau khi phan lap dugc nudi tang sinh frong m6i trudng long vdi thanh phin dinh dudng nhu sau nude bifn 250ml, nude may 750ml, K2HPO4 O.lg, MgS04 O.lg, pepton lOg, fucoidan tinh Ig. Cac chiing vi khuan dugc nuoi cay lac d 30°C, 120rpm, 24 gid, ly tam d 4°C, thu sinh khoi t l bao va phan djch m6i trudng (I). Pha vd sinh kh6i tf bao bing nifrogen Idng, hda tan vao dfm acetate pH 5.2, ly tam thu djch chiet (II). Tien hanh khao sat hoat tinh enzym fucoidanase phan (I) va (II) theo phuang phap Nelson.
III. K E T QUA VA THAO LUAN 1. Dieu che fucoidan sach
Chiing tdi da dif u chf dugc cac loai fucoidan tir cac loai rong nau khac nhau va dugc ki hifu nhu sau:
SI: Fucoidan tir rong 5". microcystum S2: Fucoidan tii rong S. mcclurei S3 : Fucoidan tir rong S. denticapum S4: Fucoidan tir rong S. duplicatum
642 Tuyln t$p H$i nghj KHOA HQC T O A N Q U 6 C Vg SINH HQC BlfeN VA P H A T T R | £ N B £ N VCTNG
S5: Fucoidan tir rong Turbinatia ornata S6: Fucoidan tir rong S. polycystum
1. Phan lap cac chung vi sinh vat bien theo djnh hudng sinh enzym be ngin mach fucoidan Chiing toi sir dyng m6i tnrdng phan lap vdi nguIn carbon duy nhit la fucoidan S6 chiet tii rong S. polycystum thi thu dugc cac kit qua frinh bay frong bang 1.
Nhu vay chiing toi da phan lap dugc 24 chiing vi sinh vat biln bao gIm ca nam moc va vi khuin. Chiing toi nhan thiy tir mgt nguIn phan lap cd thi thu dugc nhieu chiing vi khuan, nhu vay cd thf co nhilu chiing cimg sing cgng sinh frong cay rong. Cac vi sinh vat bien nay dugc nuoi tren m6i trudng gilng nhau, co hinh dang khuin lac khac nhau nen co the khang djnh so bg chiing la cac chiing vi sinh vat khac nhau.
STT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Bang 1
Ngu6n phan j ^ p
Rong S. seratum
Rong S. microcystum
Nude bien
Nude bien
Rong S. oligocystum
Rong S. Polycystum
Nuoc chiet fucoidan de lau ngay
Rong tan lyi
. Cac Chung vi sinh vat phan lap dipg-c
Ki h i f u c h i m g
F l F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21 F22 F23 F24
D | c diem k h u i n l^ic Khuan lgc t r i n g , nhay, bong
Khuan lac trang, tron, co nhan Khuan lgc tron, mau vang, bong Khuan lgc t r i n g sCra, nhay bong Khuan lac t r i n g , fron, loi
Khuan lgc tron, trang sCra, vien vang Khuan lgc trang sua, dgt
Khuan lac tron, d^t, vang nhat Khuan lac vang, det, vien trang Khuan lgc t r i n g , bong, loi Khuan lac tron, trang trong, dye Khuan lac trang, nhay bong
Khuan lac trang sCi'a, tron, bong, tron, dk 1mm Khuan lgc trang su'a, tran, bong, tron, dudng kinh 2mm Khuan lgc tron, loi, vang nhgt, co vien sang xung quanh Khuan lgc dgt, bong, trang, trong, hinh bau dye Moc den
Moc vang nau Moc xanh
Moc den, vien trang Moc nau
Moc vang
Moc vang d gi&a, vien t r i n g Khuan lgc vang cam
3. Sang loc vi sinh vat bien be ngan mach fucoidan
Tir cac chiing vi sinh vat bien thu dugc, chiing toi tiln hanh sang lgc cac chung cd kha nang sinh enzym phan cat fucoidan. Kiem tra hoat tinh phin djch nudi ciy va phin djch ngi bao fren cac CO chit fucoidan SI, S2, S3, S4, S5, S6 thi thu dugc kit qua cu the frong bang 2.
Tieu ban: HOAT C H A T SINH HOC B l t N 643
Bang 2. Tdng k§t cac k i t qua da dat du'g'C
STT 1 2 3 4 5 6
Kf hifu chiing
Fl F4 F10 F12 F14 F24
Ngudn phan l^p Rong S. seratum Rong S. microcystum Rong S. microcystum Rong S. oligocystum Rong S. Polycystum Kxd, rong da suy tan
Moi trudg sang lgc sir dyng fucoidan
S2 S2 S6 S6 SI SI
Co hogt tinh tren co- chit fucoidan Ngogi bao
S2, S4 81,82,84 -
- - -
Ngi bao -
- 86 86 81 81,86
Mac dii phan lap dugc 24 chung vi sinh vat bien tir bim bien, nude bien va rong bien nhung chi CO 6 chiing ngudn goc tir rong bien la c6 sinh enzym be gay mach fiicoidan (bang 2). Theo cac cdng bd frudc day [2,3,4] tbi cac enzym be gay mach fuoidan phan lap tir ddng vat than mem chi cd tac dyng len mgt loai fucoidan. Tuy nhien, d day chiing tdi thu dugc ket qua nhu sau:
ngoai nhung chiing cd tac dyng len mgt loai fucoidan (FIO, F12, F14), enzym ciia cac chiing nay cd the la enzym dac hifu chi cd tac dung len mgt loai lien kft, con c6 nhirng chiing sinh enzym cd tac dyng len bai hoac ba loai fucoidan (Fl, F4, F6), nhu vay cac chimg vi sinh vat bifn nay sinh ra mgt hdn hgp enzym be gay mach fucoidan hoac enzym ciia cac chiing nay co thf be gay nhieu loai lien ket df chiing co the thich nghi vdi dieu kifn s6ng.
Khi nudi cay cac chimg vi sinh vat phan lap dugc fren m6i trudng sang lgc vdi fucoidan SI, chi CO chung F14 va F24 sinh enzym thiiy phan fucoidan. Ca hai chiing nay deu sinh enzym ngi bao. Chung F4 dugc phan lap tir nguon rong S.microcystum, nuoi cam img tren m6i trudng sang lgc cung chira fucoidan chilt tir rong S.microcystum, djch chiet enzym cung co boat tinh tren ca chit fucoidan chiet tir rong S.microcystum, nhu vay chiing F14 chi sinh ra mgt loai enzym be gay mach fucoidan doi vdi mgt ca chit nhat dinh, enzym nay chi co tac dyng len mgt loai lien kit trong mach fucoidan. Chiing F24 dugc phan lap tu nguon rong da suy tan, va cung c6 boat tinh fucoidanase tren ca chit fucoidan ciia rong S.microcystum yd S.polycystum. Dieu nay chirng to hai loai rong nay hoac la co cac nhom chirc, hoac co mach chinh hay cac mach nhanh tuong ty nhau. Enzym sinh ra tir hai chiing nay la cac enzym khac nhau.
Khi cac chiing vi sinh vat dugc len men trong moi trucmg long vdi fucoidan S2 thi thay co chiing Fl va F4 sinh ra enzym ngoai bao. Djch nu6i cay co boat tinh tren fiiCoidan cua rong S.
microcystum va 5. mcclurei, chiing F4 con co tac dyng len fucoidan ciia rong S.duplicatum. Hai chimg nay rit dang quan tam vi khi sinh enzym ngoai bao thi de dang thu nhan enzym hoac cd thi sir dyng chiing vi sinh nay de phan cat fucoidan. Chiing F2 va F3 lai sinh enzym ngi bao, enzym co tac dyng tren fucoidan cua rong S.duplicatum.
Khi nuoi ciy cac chiing vi khuin thu fren m6i trudng sang lgc vdi fucoidan S6 (S.polycystum) thi chiing FIO va F12 sinh enzym ngi bao trfn chinh ca chat la flicoidan S. polycystum. Dieu nay hoan toan cd thi giai thich dugc vi flicodan S6 cam ling dugc sy tiet enzym ciia chung FIO va F12 nen djch enzym ciia chung ciing co tac dyng len co chat fucoidan S6.
IV. KET LUAN
Da phan lap dugc 24 chung vi sinh vat bifn theo djnh hudng sinh efizym phan cit fucoidan.
Trong dd 6 chiing cd kha nang phan cat fucoidan. Hai chiing la Fl va F4 sinh enzym ngoai bao, cd tac dyng phan cat fucoidan tir rong S. microcystum, S. mcclurei va S. duplicatum. Con 4 chung khac la FIO, F12, F14, F24 sinh enzym ngi bao cd hoat tinh cat mach fucoidan ciia rong nau S.polycystum va rong S. microcystum.
644 Tuyen tap HQi nghj KHOA HQC TOAN QU6C v£ SINH HOC B I £ N VA P H A T T R I ^ N B £ N VCfNG
Day chi la budc sang lgc sa bg, hifn tai dang tiln hanh nghien ciiu cac dieu kifn nudi cay 6 chiing nay (nhift do, thdi gian nu6i cay, pH m6i frudng) dl chiing co boat tinh cao nhat. Sau do se tiln hanh thuy phan fucoidan va phan tich cac phan doan oligosaccharit.
V. TAI LIEU THAM KHAO
1. Chevolot L, Mulloy B, Ratiskol J, Foucault A, Colliec-Jouault S (2001) A disaccharide repeat unit is the major structure in fucoidans from two species of brown algae. Carbohydr Res 330, 529-535
2. Thanassi NM, Nakada HI (1967) Enzymic degradation of fucoidan by enzymes from the hepatopancreas of abalone, Haliotis species. Arch Biochem Biophys 118, 172-177.
3. Daniel R, Berteau O, Jozefonvicz J, Goasdoue N (1999) Degradation of algal (Ascophyllum nodosum) fucoidan by an enzymatic activity contained in digestive glands 4. of the marine mollusc Pecten maximus. Carbohydr Res 322, 291-297.
5. Kitamura K, Matsuo M, Yasui T (1992) Enzymic degradation of fucoidan by fucoidanase from the hepatopancreas of Patinopecten yessoensis. Biosci Biotechnol Biochem 56,490-494
6. Morigana T, Araki T, Ito M, Kitamikado M (1981) A search for fucoidan-degrading bacteria in coastal sea environments of Japan. Bull Jpn Soc Sci Fish 47, 621-625.
7. Furukawa S, Fujikawa T, Koga D, Ide A (1992) Production of fucoidan-degrading enzymes, fucoidanase and fucoidan sulfatase by Vibrio sp. N-5. Nippon Suisan Gakkaishi 8. 58, 1499-1503
9. Sakai T, Kimura H, Kato I (2002) A marine strain of Flavobacteriaceae utilizes brown seaweed fucoidan. Mar Biotechnol 4, 399-405.
10. Sakai T, Ishizuka K, Kato I (2003b) Isolation and characterization of a fiicoidan-degrading marine bacterium. Mar Biotechnol 5, 409-416
11. A. M Zakharenko, M. I. Kusaikin, Bui Minh Ly, et al. Catalytic Properties of Endo-l,3-P- D-Glucanase from VietNamese Edible Mussel Pema viridis (2009). Russian Journal of Bioorganic Chemistry 54-61.
ISOLATION AND SCREENING O F M A R I N E B A C T E R I A D E G R A D I N G FUCOIDAN F R O M B R O W N A L G A E
Cao Thi Thuy Hang, Bui Minh Ly, Huynh Hoang Nhu Khanh et al.
SUMMARY
Sea water, sea mud where brown seaweed species live and brown seaweeds .were collected for microbial isolation. We isolated 24 sfrains marine microorganisms. Among them, there were six sfrains producing fucoidan degrade enzymes. In six sfrains of bacteria screened, there are two sfrains Fl and F4 produced exfracellular enzym degrading fucoidan from S. microcystum, S.
mcclurei and S. duplicatum. Other sfrains (FIO, F12, F14 and F24) produced infracellular enzym that enable to degrade fucoidan from S. polycystum and S. microcystum.
