TifP cm maa hoc va Cong nghe 54 13) (2016) 323-331 DOI- 10.,5625/0866-708)054/3/7,44
^f^^n,^Mn.l^^ ^ ° * = ^'^*= ™ ™ G X A K H U A N T H U O C C f f l MICROMONOSPORA C O H O A T T I N H S I N H H O C TlT T R A M T I C H
V I N H H A L O N G - C A T B A '
Pha" l s n " r , ; L ' ^ . ' t ' - ' ^^.™ '5"'''"'' ^ ' " ' ^ ° Mai Anh', Pham Viet Ctrfrng'•^
Phan, Van Ctrcmg , Doan Thj Mai Htrong', Brian T. Murphy^ dhSu Van Mini'
^ Vien Hoa slnl, hiin. Vien Han lun, KHCNVN. IS Hoang Quic Viel. du Gidv. m Nol '''^'"••"•''"ofl^edlclnalaeniislry und Phamiacognosv. College of Phannacy
University of niinois al Chicago
'Vien Nghlm cm Khoa hpe Miin Trung. Vlen Hin ldm KHCNVN. TP Hui Email; ihmlnhbk@gmali.com
Bin Toa soan: 23/9/2015; ChSp nhan dang; 28/4/2016
TOM TAT
d.ng::::ti*^c^t*stct.^rL^'?^ i;^^''^ ="• '^^^^p ^^ ^^ -^ ^^
dang va mai la Trong nghitacii^ nav ehunV^rt, \ ^ ° . * ' " ^ '" ''" " ' " "^= ""^ ""= <>"' trSm lich b,anthu thip I Wnh Ha Ung ^ a t n ? C K" ' T " I " ' " " ^ "" '^"^" '* ' "'«"
Al, dich len men duoc c h i f s lln vTethyl acetate h u h " ' ' ' ' ? """ """'' ™ ' '"'*"8 khang khuSn. Tit kit qua sang loc da chon d^o!. f I ^ • f " ' " '""^ ™ ".*' <•'"'' h"?' tinh hieu la. G016, G017 va 1^019 Ca 3 ehoL d' ."""^ ' ° ^ ° ' """ '='''"8 '<'"'*'' •=»<> "^31 ky ATCC292I2 Jo, M l c d = 64 u l l MK TjS " / " f M ? ; ^ " * ' " ^ £«./-»™CC»/„ec„fa
l " m . ' ^ ' " ' " ° ' " — ' " - • ^oat tinh khang vi s,nh vat, MlC, trinh tu ,6S ARN
1. MO BAU
mai ^£:^Si^i^th i^itz""' T' T"""'""' '"""^ "^""" v4
chat tu nhten do v, s,nh vS'inh radl duoc °6n^ b"' ™H " ' " ' " " ^ "'" ^'^' ^'""l^ '^ "'' ""P sinh ra nr x, khuin, 38 •/. tt, ndm , ? 17% ° vl toul S f ^ 1 ' ° " f ^ ' ' ? '"' ' ' °''° """^
nguon quan trpng trong vtec san xiiSi ,h ' 1 . 1 , , ' ' ' " " " ' " '"'"' ^ ^ c danh gia la hien tu''chung,'vil J^i^L^Zl^Zotr!^^^ "" '' ''" ""'"' '•"' ""' ^"^ P"*'
L§ Thi HOnq Minn va >v/vR
Cac loai thugc chi Micromonospora la xa khuan chiem uu the, co the dupc phan lap tii moi micmg song dual nuoc nhu suoi. ho biin, tram tich song, cat bai bien, bpt bien va tram tich bien [4. 5]. Micromonospora ciing \a\ chi Streptomyces dupc biet den nhieu nhat de tong hgp khang sinh, dac biet la khang sinh nhom aminoglycoside, enediyne, va khang sinh oligosaccharide [6].
Khang smh pho bien trong linh vuc y te nhu gentamicin va netamicin thupc nhom khang sinh aminoglycoside thu dugc tu Micromonospora.
Ngoai ra, Micromonospora con la nguon san xuat thuoc khang sinh chong khoi u. Dang chii y - Micromonospora lupini sp. Nov san xuat cac anthraquinon, lupinacidins A va B khang u [7]. Cac phan tir hoat tinh sinh hpc khac, nhu vitamin B12 va cac hgp chat khang nam cung dugc tong hpp rii cac loai Micromonospora [8, 9]. Dac biet, cac loai Micromonospora bien gan day da noi len nhu la nguon cua che pham sinh hgc [10]. Cac alkaloid, diazepinomicin khang khuan dugc xac dinh tir rapt loai Micromonospora bien cho thay tiem nang ciia no nhu la mfit ung cu vien thuoc tuong lai do chat chong oxy hoa va ch6ng protease boat dpng [11]. Hign nhien la xa khuan nhu la mpt nguon day tiem nang va phong phii ciia cac chat co hoat tinh sinh hpc
Trong khuon kho bai bao nay, chiing toi trinh bay ket qua ve nuoi ciy, sang lgc hoat tinh khang vi sinh vat kiem dinh va dinh danh bang sinh hpc phan tu ciia mpt s6 chiing vi sinh vat co hoat tinh cao dugc phan laptirtram tich bien Ha Long, Cat Ba.
2. VAT LIEU VA PHI/ONG PHAP NGHIEN ClTU
Cac hoa chat: Casitone, malt extract, yeast extract. Peptone, Instant ocean, CaCOj, NZSG, Tinh bpt tan, Agar,cac khang sinh cycloheximide. Streptomycin. . dugc mua ciia hang Himedia (An Do), Sigma (Due), dung de pha cac loai moi trudng A I, Ml, SWA, A+, SCA, NZSG va ISPI, ISP2 sir dung trong phan lap, nuoi cay, dinh danh va thii hoat tinh.
Kit tach DNA tong s6 ciia hang Madison, My, master mix cho PCR cua hang Bioneer, Han Quoc, bg BigDyeR Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) sii dung cho phan ung dgc trinh tu DNA; chi thi DNA chuSn (hivitrogen), Taq DNA polymeraza Cac CM moi de khuech dai gen 16s rDN A (16sF, 16sR)
Cac Chung vi sinh vat ki8m dinh g6m: 3 chiing vi khuSn Gram am (-): Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, Salmonella enterica ATCC13076; 3 Chung Vl khuan Gram duong (-i-): Enlerococcus faecalis ATCC29212, Stapphylococus aureus ATCC25923, Bacillus cereus ATCC 13245; 1 chimg nkm men Candida albicans ATCC]023\
2.2. Phinmg phap nghien cun - Thu thgp mdu
Cac mau tram tich bien duoc liy tai cac dia didm va dp sau khac nhau tai day bi6n viing H?
Long - Cat Ba dugc luu giu trong cac 6ng Eppendorf vo triing, dugc bao quan lanh trong thoi gian van chuyen ve phong thi nghiem va dugc t i k hanh phan lap ngay.
- Phan lap cdc chung xei khudn
Nghien c&u phan tap vi sang loc cac chuna xa khuin thuac chi Micramanaspara ...
I den c6n"kh;^,^-°"^"'h*'' ""^ ' ™ l " ' ' " ' ° ' " * " * ' ™ " ^ 8 . <iung thanh inox cho qua ngon hra - dOng mat^vo^ i ^ " ^ ' ' ' ••°' '*'"' ™° *^° *™ '^'"' '^' '^^ ^ "^i*' I* "» °C tong 8 phut,
7 3 ? n 2 1 ™ r t " , T , , ^ T " ' "''"^° -"'• ^ ' ' -^'^ •""": '"'«. wng tu am 6 28 - 30 "C ttt , 7 30ngay,luachoncackhuanlaccaychuyenlamsachsangm6,tmcmgAl[I2,13].
' -Tfo Cfn chiit tho tir dich nuoi ciy
'^ men f g c S S t S m a l t ^ l T " " ' I - ' " ' ° ™ ' •"*"« '" * ™ " «" 81"=°^=, 4 g/l nim
> 7 S v fu6i c l Tch , r f ""'"l.l'.en.dnstant ocean), pH 7.0, tat 200 mm va 30 "C. Sau
I «->^-^t"i"ss^^^:ig^^:rsr;h^
- Phirtmgpha xdc dinh hoal Hnh lihmg khudn
dinh vf nam nhim d T r T g S e ^ a ^ k h t i ' g L u t " " ' "h " ' " " ' ' ° - ' " ° - '**"« " ^"* ^«' •"«•"
sung 50 m dung d|ch vi khuan va nim >i n6ng do 5 10'CFU/ml li o 37 ' c ^ -,1 T ^- " "
i ^ e S r ^ r a t t t ^ l ^ ^ * - ^"^?°'^>-"^P"^^"'^
- Phtrong phip Oinh danh cac chung xa khuin
hot ^^7^!,^h^uT&'™'?'' "^*^"'" °^ '^" •'^°' '™s ""•^1' --." •."h
model JSM-54KILV,E<?Lmu d i e h f'^" "'.'""^'.SEM (banning electron microscopy) .hai.cua Chung ! ^ Z : 2 ^ t ^ ^ ^ ^ ^ ^ S ^ l ! : * " t ' " 1 ^ °^= ^•^•" ^^"^
khuan tl khi smh. khuin ti cochit, hinh t h r k l ^ l T c tl7) "' '"'° ^ ° " ' ' " ' " ^*= ™»
phim la khoang 1500 bp Sa„ ph£, PCR d n / ^ h™ '^uj^"^ ^ ^^ """ ''"^ '<''='' '^""^ ^^^
Giai trinh ttt gen I6S rl^A da duoc .huctsn h ^ '"^ ' " J " * ' ^ * ="" " " ^ '™"»gen cua hang Bioscenec Trinh tu geHuac L H b ^ T^ '^"'-"l"^ " ' " ''«"S ^Bl PRISM 3 IOO du lieu ngan hang gen eua NBcT S y phai i h c h u 7 f '"". """"-"V" " - ™ » ^""' ^ ^ MEGA version 4.1 [19). P ^™"° '"'" ''''"= My dung bang chuong tinh
Le Thi Hdng Minh va NNK
3. KET QUA VA THAO LUAN 3.1. Kk qua phan lap va xac dinh hoat tinh khang k h u i n
9 miu trim tich bien thu thap dugc a Vinh Ha Long - Cat Ba, dugc xii li theo phuong phdp da neu, cay chai len cac moi trudng phan lap v6i cac nong do khac nhau sau do nuoi 6 28 "C tu 7 - 30 ngay (Hinh 1 va Hinh 2).
Hinh I Mot so hinh anh thu thap mau a Ha Long - Cat E
Hinh 2 Hinh thanh khuan lac tren cac moi trudng phan lap sau 14-30 ngay nuoi.
Sau khi tien hanh lua chpn cac khuan lac rieng re d§c trung cho cac m l u , cSy ria cac khiifc,?
lac do ra dia moi trudng de thuan khifet va phuc vu cho cac chiing nghien ciju tiep theo. 39 chung^
CO hinh thai va mau sac khuan lac khac nhau, cac chiing nay dupc nuoi c i y trong moi trucmg ffi'' neu 6 phan phuong phap, dich len men duac chi6t xufit voi ethyl acetate (5 lin) thu can tho, xic ' djnh ho^t tinh khang vi sinh vat kiem djnh vai can tho. KSt qua da s i n g lpc dugc 3 chiing c6 hoal tinh khang vi sinh vat cao la G016, G017, G019 (Bang 1).
s T T
T e n chiing
N o n g d p
!
2 3 K
G 0 1 6 G 0 1 7 G 0 1 9 Strep C y c
Bdng /, G i a tn MIC ciia can chiet E t O A c c u a 3
G r a m dircmg (+) E.faecalis
ATCC29212 MIC(jig/mi)
64 128 64 256
Strep; S t S aureus ATCC25923 M[C(;ig/ml)
256
256
r e p t o m y c i n B cereiLS ATCC13245 MiC(ng/m))
128 128 128 128
K S
c h u n g CO h o g t t i n h c a o . G r a m Sm (-) Ecoli
ATCC25922 MIC(|ig/ml)
32
K h a n g sinh P.aeragmosa ATCC27853 MlC()ig/m])
256
-
C y c - C y c ATCC1307
6 I^IC(,ig/ml)
128
o h e x i m i d e Nim C.albicam ATCC 10231 MIC(iig/m]}
2 8 ^ 32 %
- 1
• •
Ket qua xac dinh hoat tinh khang vi sinh vat kiem dmh cita can chiet tho cho t h i v ca 3 Chung deu co kha nang (rc c h j cac chung Gram duong E. faecalis ATCC 13124 vm MIC = 64 pg/ml, M l C o o i , - 128 pg/ml, MlCoai, - 64 pg/ml vkB.cereus ATCC 13245 vai MIC =.°°128
1 ctn/ phin lip va sang loc CRC chung xa khuin thnnn nhi Micromonospora .
jig'ml, chi co^ Chung G016 c6 kha nang uc chl S aureus ATCC25923 vtM MIC™. = 256 ug/ml Afcc;023f V 7 M " "' T / ' , t!?' "'"' " '"' "" '"^ "^^ ZcZdldaaittfns
«7 kJ ng s L ^ ' ^ f ^™' - ' e.g""!. M l C c , . 8 pg/ml. Giai thich nguyen nhan stt nhay earn h M thai c t SIC Jon" d " ' : ; ^ T ° " " '"• ™ ° " " " """"^ c6 thj do stt khac biet vi
3.2. Ket qua djnh danh
tt.n S i ^ S L l t h a c t a : e f n ° t : r b * 6 t ' ; s C A f ' o ° " ' ' ' ' i '"'' """' ''' """^ '' "^^^ * 3° "^
svi khuin tl CO chTnhat S L ,t, ? • ' ? ' ^u?" " " '''"*' "^"^ ''='' "^ ^'^^ ^^ SEM cho thiy thi rit yl Biat XcsTiliir """ ' " , * " T " " " " ^ S . "hung soi khuin t, khi s,nh va min;.n bl°mat va I h C ctryiS Z ^ ' n Z T '^""^ " ' " ' '"" ' ^ ° >* " " « "^'
(A) (3)
Hinh 3 Bao ti CUa chung G016(A), G017(B), G019(C) duoi kinh hiSn vi dien liquet (SEM).
w (Sx Qr^;s^ttStii;t)"G^r" ™'' -^^ -^-^ --- "-^
G017 va nau den sau kht hinh L n h ba t* 6 hifng GoTo m „ h " l ^ C a c T ' d ™ " '""' K""""' vcri dac diem phan loai eua chi Mlcromonosporapl y , ' ' * " " ' " " " > • P ^ ^ W
khuan n CO cha, cua 3 Chung G016{.^), GOI 7(B), G0I9(C,
L§ Thi Hong Minh va NNK
Xac djnh ninh tu gen 16S ARN riboxom cita 3 chiing G016(A), G017(B), 0 0 1 9 ( C ) . Cac trinh tu thu ducrc dugc phan tich bed phan mem BtoEdit va so sanh vtji c a sa dir lieu tren GenBank bang phin m i m Blast. Ket qua cho thay trinh ttt gen 16S ARN riboxom cita cac chting nghien cmi co do tuang dong cao (hon 99 % ) so vtri cac chung tren ngan hang Gene Quoc te (Hinh 5).
(3016 Ha tong Cat Ba VN (3019 Ha Long Cai BaVN KNM56224 Mcrom,nospora sp (30,7 Ha Long Cai Ba VN KM456225 Mcfomonosfrara sp Kl^39505 Mcrorronospora sp.
B^544,49 Mcrannnpspora sp,
£1,7,4258 Mcrormnospora sp HF674981 Mcromonospora sp EU214935Streploiivcessp St
M,07 hde S7-SC3 niai ,73709 Oiina R, Msxica S12 SFAtfJ
H'mh 5. cay phal s,nh loai dua [ten trinh hf gen 168 rRNA cho thiy mil quan he giita cac chiing nghien cuu vij, cac thanh vien da, d,en cua cac chi lyticromonospora sp.
Nghien cim quan he ciia cac chung tten cay phat sinh loai (Hinh 5) cho thiy, chimg G0I6, GO 17, GO 19 CO moi quan he gan gill vtji cac chiing thugc chi Micromonospora ., Trong do' Chung GO 16 va GO 19 CO do luong dong cao (han 99 % ) so vai Micromonospora M60 Co ma si tren GenBank la KM 456226; chiing G 0 I 7 nrong d6ng (hon 99 % ) vol chiing Micromonospora M95 mang ma so KM 456224 duijc phan lap tit vimg ngap mjn a An Do
Tit mpt so ket qua nghien cuu cac chung iiflcromomspora d l san xuit cac hgp chit co hoat ttnh s,nh hoc da dugc de cap. Dang chit y, mot chiing ua man Micromonospora lomalvlllensis d(igc phan lap ttr Polysyncralon lllhoslrolum. Chung nay tao ra hai lomaiviticins A va B gay dgc te bao khoi u manh, cau trite cua lomaiviticins gin tuang t u viji khang sinh kanamycin...Cac thiocoraline khang u dugc tach chilt tir Micromonospora phan lap tir mgt Mo IM 251" ^ " " " " ^ ""^ ^""^ ""^ ''""'^ •"* "'*" ™ ' ' 1^-96212, CO tinh chit gay dgc t l Nghien Cliu cac hgp chit tni, c i p ti, ngu6n vi sinh vat biln a Vict Nam mtjt chi dugc bit dau CO rat it cac nghien cuu da cong bo, mac dii ngu6n da dang vi sinh vat biln ctia nutjc ta la rat Ion nho v, tii dia It tiep giap vcri bten. Do vay, viec tiln hanh nghien ciru cac hgp chit thii cip cua nguon vi smh vat bien Viet Nam nham phat hien cac chit co hoat tinh sinh hgc cao co gta t J mig dung trong hnh vuc y dugc la hu(jng nghien cliu cin thilt, hua hen nh,lu triln vgng.
4. K E T LUAN
k h u a J ' t ^ . T ' ; ' ™ ° ' - " ' ' ' ' i " ' " u " ' ' P ' * ' ' ' ' ° ^ " ' ' ' " ? '•""'E- Cat Ba, da phan lap dugc 39 chung X, khuan. Tu ket qua sang loc boat tinh khang khuBn da lua chgn duoc 3 chung co hoat tinh cao
Nghien ciru phan lap va sang lgc cac chiing ya khuin thuoc chl Mlcramanosaara
^ S ARN nboxom Ket qua cho thay chimg G016 va G019 tuang di^ng cao (hon 99 % ) so v(ii W T v r Z : M'""'" '' ™ ^* " ' " * ° ^ ^ '^ ™ 4 5 6 2 2 6 f c h 4 G017 tumtg d^n^ h i ng?p man g An Dp. Cac ket qua ban dau thu dugc mti ra nien vong nghten cim vl nhom J ^ r i T n h " r ™ D u ^ d- "r-r""'"-'-' •* • " - » * " « ^'^-' >* -J- t,lm nang quan t n g trong nnh VIIC Y Dugc de chong lat cac tac nhan gay benh.
TAI LIEU THAM KHAO
Fenical W, Jensen P. R. -Developing a new resource for drug dtscovetr- marine actinomycete bacteria, Nat. Chem. Biology 2 (2006) 666-673 "'scoveiy. marine '• p"o°9S'5':i6.' " ' ' " ° '• ^ " ' " * " "^^ '"'°y'^- '"y-^"^ Pf P'-^g^'"^- •>• Antibiotics 62
^ !l"ve? *!• " " ^ - ''""r* ^" ^"""^ ^" ^"' S' -«'c™™™o«/,<,™ r,/<,„,^a,„„ sp nov a novel actinomycetes from mangrove sediments. Int. J. Sysf Evol. Mictobiol. !s (2008) ' "chrty";. Meduer. Ectl'Tpoo",'"-"'""'"^'"'^ '" "= ""''' ^"= - " ' " ' " ^ - i f - B "
' M ? c ' r r o : s o % r r e \ n X : . c h a ' l ; t a t s " f t ^ h' ^ " ^ . P ^ ™ - "^ ' - ° - i v c (2008)243-261 "*""= " " " '" Australia, Mar Drugs 6
"' MZ^hl'^!;^2oZ:l2T' ™ ° " " ' " " ' ^ - ' " — c t , n o m y c e t e s , C u . . O p , „
actinomycete / l ^ t c ^ ^ o L ^ L , Z J N o v T o ™ ' c I T " d ° ° " f cT "" ^'^''''^''^
17(2007)3702-3705. '^ " P '^V''*«'"'8»n.c & Medicinal Chemistry Letters o Wagman G. H., Gannon R.D., Weinstein M I D j .-
Micromonospora, Apphed Microb,„loj;; ,7 (19W) 648 649 " "'""""'" ' " ' "^
' Lotd"„n!'l9'88 p ^ l , 12-2141°'"°° " ' " " ™ " ^ "^ -tinomycetes. Academic Press,
'"•^^td^:iUdr:;L.:ic:i-^j;^-:;--»>^.^^^^^^^^
. . Usama R. A., Matthias S , Eman M O Tania S 1„.l - n ,7
;;• ^jiez^i^ft-^s-Soos;^^^^
I J. ""..."tts b. r, Ctoss T.-Actinomycetes In-MethnHc , \^
(London) 4 (I97I) 295-334. Methods in Microbiology, Academic Press 14. C<!dricO., SkylarC, BtndiyaK, MashalMA Hs, , .
Cuong Pham, Cathenne L S Brian T u L P "^ "-' '^'^'' °- Q"'" S , Va enne L.b., Bnan T. Mutphy and Alexandet S M, Tool fo Tool for
Le I til HOna Minh va /V/VK
characterizing bactenal protein synthesis inhibitors, Antimierob. Agents Chemother. 57 (2013)5994-6002.
15. Hadacek F., Greger H. - Test of antifimgal natural products methodolagies, comparability of resuh and assay choise. Phytochem. Anal. 90 (2000) 137-147.
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168-173.
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Microbiology 11 (1963) 335-338.
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Isolation and Identification of Actinomycetes Isoptericola variabilis From Cauvery River Soil Sample, bit.J.Curr. Microbiology 2 (2013)236-245.
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ABSTRACT
ISOLATION AND SCREENING OF ANTIMICROBIAL COMPOUNDS OF MICROMONOSPORA STRAINS ISOLATED FROM HA LONG - CAT BA BAY'S
SEDIMENTS
Le Thi Hong Minh'', Vu Thi Quyen', Nguyen Mai Anh', Pham Viet Cuong''^
Pham Van Cuong , Doan Thi Mai Huong', Brian T. Murph/, Chau Van Minh' 'Institute of Marine Biochemitiy. VAST, 18 Hoang Quoc Viet, Cau Giay Hanoi
'Department of Medicinal Chemistry and Pharmacognosy College of Pharmacy University of Illinois at Chicago
Mientnmg Institute of Scientific Reseach. VAST Hue city Email: lhminhbk@gmail.com
iJ
Nghien c&u phan l^p ,>n sang (oc cac chimg xa khuin thugc chl Mlcramonasaorn
M,croorganisms are especially tntciested due to the ability to produce secondarv ompounds with htgh value application. The bioactive substances c o l give us new and™ r e^
wTh ethv^t ' " ^ " " ' ^ ^ / ™ c - t e d m AI medimn, ferinentation brodts were extracted 5 ttmes rlrence bac.ena%t°° t'h " " ? " ' f ''"""' "="• ""'"='" '° ' « ' ""= ""'"'>' ^ S " - ' som rererence bactena From the results of screening, 3 strains of actinomycetes that have the highest
I b T o n TZ" ^?'^''' f}'- ° " " ' ° " ' " " ' - ^*"=<'- "Th^c sLins have the qui e tmng C r r e r A T C C 1 3 ° 2 4 ? T . ' ' S ; r "'"'""• " ^ ' > ' — " / - * ATCC292I2 and iJiiciuui cereu, AICCI3245 wnh MIC values less than or equal to the MIC value nf ,he
momhobgiealanHnhv^o"' '. ''^'"-'' ' ^ " = " " ° * »'^'"'' ^'''' ^""'"^ "ere subjected to r S s t d l a t h S , n f c m « ' c „ ' f - ; ^ ' ' ' r ""='' °" "^ "^^ Bene sequences Tlie obtained
indicate that snams O0I6, GOI 7, and G0I9 belong to Genus Micromonospora.
s:ZZt ''"'"'"P>"^"=^' '^•"'•'""'"•ospoi-u, antimictobial activity, MIC, I6S rRNA gene
