Tieu ban Sinh hgc vd ngudn lgi sinh vgt Biin
PHAN L^P VA SANG LQC VI KHUAN TU* BUN CUA NHA M A Y SAN XUAT FUCOIDAN CO KHA NANG SINH ENZYME BE NGAN
M A C H FUCOIDAN
Cao Thi Thiiy H^ng\ Biii Minh L^', Phan Thi Hoai Trinh', Pham Dire Th!nh\
Huynh Hoang Nhir Khanh', va Tran Dinh Man^
' ViSn NghiSn cim va LTng dung Cong ngh? Nha Trang, VAST 02 - Hung Vuang - Nha Trang - Khanh Hoa
^Vien Cong ngh? sinh hoc, VAST.
18 - Hoang Qu6c Viet - Chi Giiy - Ha Noi Email: [email protected] Tom tat:
Fucoidan Id mgt polysaccharide dua tren nin a-L-fucose duac chiet xudt tir rong Nau. Fucoidan cd hogt tinh sinh hgc rdt phong phii vd thii vi. Tuy nhien, cdc cdng bd vi enzym be ngan mgch fucoidan cdn rdt it. O day chiing tdi cdng bd ve viec phdn lap vd sdng Igc vi khudn cd khd nang sinh enzyme be ngdn mgch fucoidan^ ciia rong Nau. Biin lay tir nhd mdy sdn xudt fuoidan duac sir dung 1dm ngudn phdn lap vi khudn. Cdc ca chat duac diing di sir dung de tim kiim enzyme fuoicoidanase la fucoidan tir Sargassum mcclurei, Sargassum polycystum vd Padina. Chiing tdi dd phdn lap duac 30 chiing vi khudn, trong do 5 chiing dir^yc phdt hien cd khd ndng sinh enzyme be ngdn mach fucoidan (SW9, SWI4. SW21, SW22. SW28). Chiing vi khudn SW21 duac tim thdy cd hogt tinh enzyme cao nhdt trin ca chat fucoidan cita cdc lodi rong Sargassum mcclurei F2 vd Sargassum polycystum Fl vd Padina.
ISOLATION AND SCREENING BACTERIA FROM MUD OF FUCOIDAN MANUFACTORY PRODUCING FUCOIDANASE Abstract:
Fucoidans are matrix polysaccharides from marine brown algae, consisting of an a-L fucose backbone. In spite of their interest as biologically active compounds, a little convenient source with fucoidanase activity are available for the degradation of the fucalean algae. We here report on the isolation of
bacterial strain capable of degradation various brown algae fucoidans. Muds from fucoidan manufacture were used as source to isolate bacteria. Fucoidans from Sargassum mcclurei, Sargassum polycystum and Padina of Vietnam sea were used as substrates for testing fucoidanase activity. From thirty isolated marine bacteria, five strains were found to produce-intracellular fucoidanases (SW9. SW14, SW21. SW22. SW28). SW2I was found to have the highest fucoianase activity on fucoidan from Sargassum mcclurei F2, Sargassum polycystum Fl and Padina.
Hgi nghi Khoa hgc vd Cdng ngh? bien todn qudc Idn thir V
1. MO DAU
Fucoidan, mpt sulphat polysacarit sinh hpc duac chiet tii rong Nau da vi dang thu hut s\c quan tam nghien cuu trong nhirng nam gan day nha sa huu nhiing hoat tinh dugc ly tuyet vai nhu: khang dong, khang khuan va khang virut, khang khoi huyet, khang u, khang viem nhi6m, khang tang sinh, khang thu tinh. Chung con dugc su; dung rat thanh cong trong viec phong ngua va diSu tri cac benh pho bien nhu cao huyet ap, viem khcrp, di ung va beo phi [1]. Tuy nhien moi lien quan giira hoat tinh sinh hgc va cau true ciia chung vIn chua dugc giai thich ro rang do cau tnic phiic tap va khong theo qui luat ciia fucoidan tir cac nguon goc khac nhau [4].
Cau tnic ciia fucoidan phuc tgp va phu thugc'vao nguon goc loai rong ma no dupe chi^t ra. Trong m^ch phan tii ciia chiing chiia chii y6u cac goc fucose dupe sulphat hoa 6 cac vi tri khac nhau va Hen ket vai nhau thong qua mpt so Uen ket nhu (1-3), (1-4), (1-2) it theo qui lu?t. Ngoai ra trong phan tit ciia chiing con c6 m|t mpt so cac duang dan khac nhau nhu galactose, mannose, xylose, glucose va cac m^ch nhanh c6 chiia cac goc fucosyl, galatosy[2].
Chinh vi vay vi?c nghien ciiu anh huang ciia tirng yeu t6 cau tnic (trpng lugng phan hi, nhom the, mach nhanh, kieu lien ket) len ho^t tinh sinh hpc cua fucoidan la mgt thach thiic khong nho doi vai cac nha khoa hpc tren the giai [5]. Dieu nay chi dugc giai quyet hi?u qua khi sii dung cac loai enzym c6 kha nang be gay mach fiicoidan nhu la mgt cong cu de nghien ciiu cau tnic cung nhu tuang quan giua cau tnic va hoat tinh sinh hpc ciia fucoidan[5].
Cho den nay, cac nha khoa hgc da tim ra 02 loal enzyme 0-glycosyl hydrolase c6 hoat tinh vdi fucoidan do la fucoidanase va a-L-fucosidase[3][4]. Hoat tinh ciia cdc a-L- fucosidase dan gian chi la su giai phong flicose ra khoi doan dau cu6i khong khu ciia mOt phan tli polysacarit trong khi hoat tinh ciia fucodanase dupe giai thich bai 2 kiSu be gay lien ket glycosics, hoac la su be gay lien ket ben trong phan tu fucoidan lam giam nhanh trpng lugng phan tu (npi fucoidanase) hoac la be gSy cac hen kit ngoai cimg ciia phan tir fucoidan liy di cac oligosacarit dan den trgng lugng phan tii giam tii tir (ngoai fucoidanase)[3][4]. Hai loai enzyme nay chii yiu dupe tim thiy trong cac dpng v^t than mem, cac loai vi khuan biin va doi khi dugc tim thay tren cac sinh vat khac[7][l 1].
Trong thap ky tra lai day c6 nhilu nghien cihi thii vi vS enzyme phan lap tir sinh v^t bien dugc cong b6. Sakai va cgng s\r da phan lap tir bien Nh|t Ban mgt vi khuin bien (Flavobateriacea) c6 the c5t nhieu loai fucoidan khac nhau dugc chiet tir cac loai rong Laminariales, kiellmaniella crassifolia, Undaria pinatifida, va Lessonia nigrescens, nodosum[9][lO].
Vi?t Nam co t6ng chi6u dai ba biin ban 3.600 km vdi khi h|u thay d6i tir can nhi?t ddi d phan phia Bac den nhiet ddi d phin phia Nam cua ddt nude, rat thuan lgi cho nhilu loai rong phat trien. Trong da dang rong bien nay, cac loai rong nau thugc chi rong mo (Sargassum) la nguon lgi ron^ biin tu nhien Idi nhSt ciia nude ta, cho den nay da phat hi?n dugc han 60 loai phan bo ph6 biin va rgng khlp ven biin va cac dab vdi san lugng khai
Tieu ban Sinh hgc vd ngudn lai sinh vgt Biin
thac hang nam udc tinh vao khoang 10.000 tan khd. Tuy nhien, viec khai thac va sii dimg rong ma d Viet Nam con chua phii hgp va nam dudi tilm nang ciia ngudn nguyen U?u nay.
Trong bai bao nay chiing toi tiln hanh phan lap va sang Igc cac vi sinh vat theo dinh hudng sinh enzyme be ng^n mach fiicoidan tir bim cua nha may san xu3t fiicoidan theo nguyen tac nai nao cd nguon ca chat, nai dd cd vi sinh v£it sinh sdng.
2. NGUYEN LlEU VA PHU'ONG PHAP 2.1. Thu mSu
Mau biin dugc thu tai cong ciia nha may san xuit fucoidan. Mhi dugc thu trong cac binh vd trimg, gdm nude va bim rong da phan hiiy sau do dem vl phong thi nghi?m dl phan l|p.
2.2. Phuffng phap phan lap
Vi sinh vat dugc phan l$p tren mdi trudng chi chua ngudn carbon duy nhSt la fiicoidan (2g/100ml nude biin) va moi trudng dugc ddng r4n b5ng agar (15g/L). U d 30°C cho din khi thay xuat hien khuan lac vi sinh vat thi.tien hanh lam thuSn tren moi trudng cd thanh phSn nude bien 750ml,^ nude may 250ml, K2HPO4 0,lg, MgS04 0,lg, saccharose 5g, pepton lOg, agar 20g, dieu chinh pH 7-7,2. Day ciing chinh la mdi trudng thach nghieng dl giu vi sinh vat cho cac thir nghi?m tiep theo. Cac chiing sach dugc giii trong mdi trudng ban ran vdi thanh phan dinh dudng tuang tu nhu tren nhung agar chi cd 3g/L [9].
2.3. Phu-tfng phap sang loc
Cac chiing vi khuan thuan chiing sau khi phan lap dugc nudi tang sinh trong moi trudng long vdi thanh phan dinh dudng nhu sau nude bien 250ml, nude may 750ml, K2HPO4 0,lg, MgS04 0,lg, pepton lOg, hicoidan tinh Ig. Cac vi khuan nay dugc nuoi cay lac d 30**C, 120rpm, 24 gid, ly tam d 4*'C, thu sinh khdi te bao va phan dich mdi trudng (I).
Pha vd sinh khdi te bao bing nitrogen Idng, hoa tan vao dem acetate pH 5,2, ly tam thu djch chilt (II). Tiln hanh khao sat hoat tinh enzym fucoidanase phan (I) va (II) theo phuang phap Nelson [8]. Ham lugng protein dugc xac dinh theo phuang phap Lowry [6],
3. KET QUA VA THAO LUAN
3.1. Phan lap vi sinh vat cd kha nang sinh enzyme be ngdn mach Fucoidan
Biin ciia nha may san xu5t fiicoidan chinh la ba thai rong Nau, trong qua trinh chilt fucoidan, ca ch3t nay van con sdt I^i trong ba thai. Ba thai rong N2u de lau ngay, la mpt ngudn dinh dudng dl vi sinh vat sinh trudng, phat trien. De sinh trudng va phat trien, chiing phai tilt ra enzyme dl chuyen hda cac ca chat co trong ba thai rong NSu, trong dd cd fucoidan. Vi thi, cd thi hy vpng day la ngudn tim kiem vi sinh vat cd kha nang sinh enzyme be ngSn mach fuoidan cao. Ket qua phan lap dugc the hien d bang 1:
Hgi nghi Khoa hoc vd Cdng ngh? bien todn qudc Idn thu V
Bdng
sh
TT 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30/ : Ddc diem khudn lac cdc chiing vi sinh vgt phdn lap dugrc Kihieu
chiing SWl SW2 SW3 SW4 SW5 SW6 . SW7 SW8 SW9 SWIO SWll SW12 SW13 SW14 SWl 5 SW16 ' SW17
SW18 SW19 SW20 SW21 SW22 SW23 SW24 SW25 SW26 SW27 SW28 SW29 SW30
Dac diim khuan lac Trdn, dep
Tron, Idi, bdng, mau vang sang_
Dep, vien rang cua, khdng mau Trdn, bdng, kich thudc nhd, mau vang Trdn, bong, Idi, mau trang due Nhd, li ti, mau trang Mau vang Dep, vien rang cua Trdn, dep, trang Man vang
Trdn, be mat nhan nheo, mau vang Trdn, dep, mau vang
Dep, vien rang cua, mau cam Bdng loi, mau trang siia
Trdn, mau tr5ng v D?p, vien rang cua ••':
Nhay, idi, man trang sira Vien gd ghe, dep, mau vang Loi, be mat nhan nheo, mau tring Trdn, dep, bdng
Bdng, Idi, trang sQa Dep, mau vang
Dep, be mat nhan nheo, mau trang Bong, loi, mau vang
Mali vang chanh Mau vang sang Khdng mau Mau cam Dep, vien rang cua Dep, mau vang
Tvr cac mlu bun va nude thai thu nhan dirgrc, chung tdi da phan lap duoc 30 chung yi khuan. Cac chiing vi khudn nay cd dac didm khudn lac khac nhau (hinh dang, mau sac khuan lac), nen so bd chiing tdi nhan djnh day la cac chiing khac nhau. Cac chiing vi khuan nay dugc lam thuan tren moi trudng dia trudng thach va kiem tra dp thuan chiing cua vi sinh vat tren kinh hien vi. Cac chiing thudn duac sir dyng &h sang* loc kha nang sinh enzyme be ngan mach fiicoidan.
Tieu ban Sinh hgc vd ngudn lgi sinh vgt BiJn
3.2. Sang Igc vi sinh v^t pbSn lap dugrc theo dinh hirdng sinh enzyme fucoidanase Chiing vi khuan thuan chimg dugc len men va khao sat kha nang sinh enzyme fiicoidanase tren 3 ca chit fucoidan chiet tii 3 loai rong khac nhau. Dich chilt ngo^ bao va npi bao deu dugc sii dimg dl thii nghiSm hoat tinh. Tuy nhien, kit qua thu nhan dugc chi cd djch chiet enzyme ngi bao cd hoat tinh va kit qua sang Ipc dugc thi hien d bang 2:
Bdng 2: Kit qud sdng Igc vi khuan theo dinh hudms fiicoidanase
si
TT 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Kihifu chiing SWl SW2 SW3 SW4 SW5 SW6 SW7 SW8 SW9 SWIO SWll SW12 SW13 SW14 SW15 SW16 SW17 SW18 SW19 SW20 SW21 SW22 SW23 SW24 SW25 SW26 SW27 SW28 SW29 SW30
Ho^t tinh enzyme fucoidanase U/mg protein Fucoidan F2
S. mcclurei 0 0,226
0 0 0,270
0 0 0 0,602 0,274 0,377 0 0 0,308
0 0 0 0 0 0 1,263
0 0 0 0 0 0 0,248
0 0
Fucoidan Padina sp.
0 0,198
0 0 0,156
0 0,555
0 0,963 0,456 0,589 0 0 1,051
0- 0 0 0 0 0 1,418 1,188 0 0,104
0 0 0 1,347
0 0
Fucoidan Fl S. polycystum
0 0 0 0 0 0 0 0 0,342
0 0 0 0 0 0 0 0 0 0 0 0,684
0 0 0 0 0 0 0 0 0
Hoi nghi Khoa hgc vd Cdng nghe bien todn qudc lan thu V
Nhin vao bang 2, chiing tdi nhan thay trong 30 chiing vi khuan dugc sang loc thi cd 8 chiing vi khuan sinh enzyme be ngan iriach fucoidan cda rong S. mcclurei, 11 chiing vi khuan sinh enzyme be ngan mach fucoidan ciia rong Padiana sp. va 2 chiing vi khuan sinh enzyme be ngSn mach fucoidan cua rong S. polycystum. Trong do^ 5 chung (SW9, SW14, SW21, SW22, SW28) la nhimg chiing sinh enzyme cd hoat tinh cao nhlt. Enzyme ciia cac chung vi khuan nay tac dpng khac nhau tren 3 loai ca chdt fucoidan vi fucoidan chilt tir 3 loai rong khac nhau se cd cau tnic va thanh phan khac nhau. Enzyme cua chiing SW21 tac dgng len ca 3 ca chat fiicoidan va deu cd hoat tinh cao nhat, do do chiing SW21 dugc coi la chiing cd tiem nang va dugc lua chpn de thuc hien cac nghien cuu tiep theo.
4. KET LU^LN
Chiing tdi da phan lap dugc 30 chiing vi khuan tir biin diai trong qua trinh san xuSt fucoidan cua nha may san xuat Fucoidan. Trong dd, 5 chiing (SW9, SW14, SW21, SW22, SW28) cd kha nang nang sinh enzyme be ngan mach fucoidan tren 3 loEii ca chSt fucoidan dugc chiet tir 3 loai rong Nau S. mcclurei, S. polycystum, va Padina sp. vai hoat tinh cao.
Chung SW21 la chiing vi khuan cd hoat tinh enzyme cao nhlt tren ca 3 loai fiicoidan.
Enzyme cd hoat tinh be ng3n mach fucoidan deu la enzyme npi bao. Nhu vSy, bun til ba thai ciia nha may san xuat fucoidan la mpt ngudn tim kiem vi sinh vat sinh enzyme be ngSn msich fiicoidan cd hieu qua.
LOflCAMOfN
Tap the tac gia xin chan thanh cam an lanh d?o va cac thanh vien phong Hda phan tich va trien khai cdng nghe, lanh d^o Vien Nghien cuu va Ung dimg Cdng nghe Nha Trang ^ hd trp ve mat kinh phi va tao dieu kien giup dd chiing tdi hoan thanh dk tai nay.
TAI LIEU THAM KHAO
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[2] Chevolot L, MuUoy B, Ratiskol J, FoucauU A, Colliec-Jouault S (2001) A disaccharide repeat unit is the major structure in fucoidans firom two species of brown algae. Carbohydr Res 330, 529-535.
[3] Daniel R, Berteau O, Jozefonvicz J, Goasdoue N (1999) Degradation of algal (Ascophyllum nodosum) fucoidan by an enzymatic activity contained in digestive glands of the marine mollusc Pecten maximus. Carbohydr Res 322,291-297.
[4] 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 Gakkaishi58, 1499-1503
[5] Kitamura K, Matsuo M, Yasui T (1992) Enzymic degradation of fucoidan hy fucoidanase fi-om the hepatopancreas of Patinopecten yessoensis. Biosci Biotechnol Biochem 56, 490-494.
Tieu ban Sinh hgc vd nguon lgi sinh vgt Bien.
[6] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folm phenol reagent. J. Biol. Chem. 193: 265-275
[7] Morigana T, Araki T, Ito M, Kitamikado M (1981) A search for flicoidan-degrading bacteria in coastal sea envirorunents of Japan. Bull Jpn Soc Sci Fish 47,621-625.
[8] Nelson TE (1944), "A photometric adaptation of the Somogy method for the determination of glucose", J. Biol. Chem. 153: 375-381.
[9] Sakai T, Ishizuka K, Kato I (2003b) Isolation and characterization of a fiicoidan- degrading marine bacterium. Mar Biotechnol 5,409-416
[10] Sakai T, Kimura H, Kato I (2002) A marine strain of Flavobacteriaceae utilizes brown seaweed fucoidan. Mar Biotechnol 4, 399-405.
[11] Thanassi NM, Nakada HI (1967) Enzymic degradation of fucoidan by enzymes from the hepatopancreas of abalone, Haliotis species. Arch Biochem Biophys 118, 172-177.
