- H p c CONG NGHE SINH HOC T O A N Q U O C 2013
NGHIEN CO'U T H A N H PHAN M 6 | TRUONG LEN MEN SINH T C N G HQP ACARBOSE cy >4CT/A/OPMWES sp. KCTC- 9161
Nguyen Thj Nuxmg, Do Thi Tuyen, Lfi Thanh Hodng, Quyen Dinh Thi*
Vi$n Cdng nghe sinh hgc, Vien Han lam Khoa hoc va C^ng nghe Vidt Nam
TOM TAT
Tieu dirong la mpt trong nhung bSnh rat nguy hilm, gay biln chiing kho lucmg nhSt la cdc benh nhan khong dugc dieu tri hay diSu tri khong diing miitc. Acarbose la mpt hgp chSt gia ducmg co tac dyng uc ch6 a-glucosidase dugc tim thiy a mot sfi loai vi smh vat, dac bifit cdc lodi thuoc chi Actinoplanes. Hi$n nay acarbose dugc sit dung rgng rai trong diSu tri bSnh ddi thao duong type II, c6 thi dung mgt nunh bo|c kit hgp ciing vol m6t s6 thufic khdc nhu migUtoI, vogUbose. Trong nghien cuu nay, chung Actinoplanes sp. KCTC- 9161 dugc len men trong cdc mfii tnrong khac nhau, chi ylu sii d\mg ngufin thuc pham len men truyen thong tir dudng maltose, glucose, bfit ngfi, bgt dau hiong vi khoang vi lugng voi cac thfing sfi ky thu$t: thoi gian 168 gio, nhiet dg 28''C, tfie dg 15c 200 vong/phiit. Djch len men chiing yfcftnop/awes sp. KCTC- 9161 sau 168 gio cho nang sudt smh tfing hgp acarbose dat trgn 20 mg/L, iic chl 52,24% hoat tmh a-glucosidase & moi trucmg len men chua maltose (3%). Maltose c5n duy tiri fi nong do cao trong suot qua trinh len men de sinh tong hpp acarbose co hifiu qud. Sau khi tinh sach so bfi, sdn phdm acarbose dugc Idem tra tren sac ky lop mong (TLC) voi he dung moi: ethyl acetate: methanol = 1:1 vd 5% dung dich H^O: acid formic = 5:2 (v/v) vdi he sfi Rf= 0,28.
Tirkhoa: Acaihose, Actinoplanes sp. KCTC- 9161, dich len men, TIX, a-glucosidase.
MdoAu
Tilu du'o'ng id can bpnh nguy hilm, co t h i gay nhijng bien chirng v l mdt, cdc b§nh v l thdn vd thin kinh, bpnh mgch mdu ngoai vi, nhilm tning, b§nh tim vd dOt quy, b-ong d6 bpnh vong mac do tilu du'dng Id nguyen nhan hdng dau dan den mu 16a. Hi?n nay co nhieu hu^ang nghien ci>u vd\ muc dfdi lam giam ham lu'png du'o'ng huyet cho cac b?nh nhdn bj b§nh ddi thdo du'&ng logi II, bt§n phap nham irc che sy hlnh thdnh glucose d rugt non moi duvc phdt hipn gan day dd CO nhO'ng u'u diem noi bgt hPn va chit du^c sO dpng nhilu nhat cho hudng dilu hi ndy Id acarbose.
Acarbose Id mOt hpp chit gia du'dng thupc hp C7 N-aminocyclltol, mgt san pham tp nhien (Mahmud T, 2003) do cac chijng Actinoplanes sp. nhu Adinoplanes utahensis JIB-08196 (Sun ef a/., 2012), Actinoplanes sp. A56 (Wei ef ai, 2010), Actinoplanes sp. CK0485-16 (Choi, Shin, 2003) sinh long hpp ra. Cau tnic cua acarbose gim m6t nCra id phan tir acarvlose vd nu'a con lai Id maltose. Acarviose Id dan vj 151 ciia acarbose, vd'i chCrc nang quan trpng va chu yeu Id kim ham a-glucosidase. Cdc den vj maltosyl cau thdnh n6n phdn tir acaitiose hlu h i t bat nguln tO' phdn tiF maltose. 0 niem mgc nj$t non, acarbose tac dpng bang cdch irc c^e canh tranh a-glucosidase, lam giam qua trinh phan giai carbohydrate (di, oligo vd polysaccharide) thanh monosaccharide Id dgng co t h i hip tiip duvc. Mgt khdc acarbose kh5ng Idm tang insulin huyet, khong gay d l khdng insulin, bao ton t l bdo beta, gidm ning 66 HbAlc, triglyceride va giam cdc bien chi>ng do tieu du'dng gdy ra. Vi vgy, acarbose dirge sir dung Idm gidm nong dp du-ong huyet sau an cho nhO'ng bdnh nhan tieu Qirdng Chiing toi da tien hanh tuyen chpn mpt so chiing Actinoplanes VTCC sinh tong hpp acarbose cao (06 Thi Tuyen e( a/., 2011), cung nhu- tien hanh ndng cao kha nang sinh ting hpp acarbose bang cac phiPong phdp t l i u'u dieu kien tong hpp chat i>c chl a-glucosidase . Trong nghien ciru ndy, chOng Actinoplanes sp.
KCTC- 9161 du-pc theo d5i qud trinh tong hg-p acarbose Iheo thwi gian va lua chpn thdnh phan moi tRrdng ISn men tir maltose, glucose, b5t ngo, bpt dgu tuo'ng vd cdc khodng vi luang; myc dfch toi uu mpt so thanh phan moi tru'ong ISn men de smh ting hgp acarbose nang suit cao.
VAT Ligu VA PHU'aNG P H A P Nguyen vgt li^u
Nguyen vat Hiu: Chiing giong Adinoplanes sp. KCTC- 9161 dupc mua tir Bdo tdng Gilng chuin Vi sinh vat Han Qulc (KCTC) c6 kha nang sinh tong hpp acarbose cao
Mdi truedng. M5i tru'dng thach nghieng (g/L): 20 glucose, 5 peptone, 0,5 KCI, 1,0 K2HPO4, 0,5 MgS04, 20 agar, pH = 7. M61 truong len men (g/L): 30 glucose, 30 maltose, 10 b6t ng6, 1 sodium glutamate, 2 CaCb, 0,5 FeCIs, 1 K2HPO4, 2,5 CaCOs, pH = 7,0 ; thdi gian len men 144 gid d 2&°C, lac 200 v6ng/phCit.
Hda chat: Cdc h6a chit sir dung trong thf nghigm diu d dang tinh khiet: cao nim men, peptone, casein hydrolysate dupc cung cap bdi ICN Biochemicals (My); chat chuin acarbose Sigma; cdc dung moi (ethanol, methanol, ethyl acetate, acid formic) cua Vi§t Nam hodc Trung qulc sdn xuit. Mot s6 nguyen ii§u diing d l thay the nguon carbon va mto nhu- bdt ngd, bpt ddu tuong duoc mua d ngodi thj tru'dng (Viet Nam).
Nghien ci>u sinh t i n g ho^ acarbose
Chung Actinoplanes sp. KCTC- 9161 dugc nudi cay hxjng moi frudng l§n men (Wei et a/., 2010). Ci> sau 24 gid, lay mlu 2 ml d|ch nuoi cly, xac djnh hogt tinh uc chl a-glucosidase vd kha nang smh tong hpp acarbose bang phuong phap sac k^ bdn mdng (TLC)
Lya chpn moi truwng len men cho chiing Actinoplanes sp. KCTC- 9161
Chung Actinoplanes sp. KCTC- 9161 dupc l§n men theo me d 28°C, 200 vdng/phiit, pH 7,0; tiep 10% djch nhdn gilng vao binh tam gidc cd dung tich 500 ml chua 100 ml mdi hudng len men, sir dyng cac mdi trudng len men khdc nhau (Bdng 1). Djch sau len men dupc loai bd can te bdo bang ly tam 4000 vong/phiit, trong 10 phiit d A'C, djch trong sau ly
H y i Niji-ij ^ ^ u « M V L . o u n t j w u r i b siNH HQC
tdm diKTC tinh sgch sa bd d l kiem tra khd nang smh tong hpp acarbose vd xdc djnh hogt tinh i>c a-glucosidase. Thdnh phin mdi tnfdng len men sinh tdng hpp acarijose dirpc t h i hi^n tr&n Bdng 1:
Bdng 1. Mra chgn thdnh phin mdi trvdmg lin men sinh tdng hgp acarbose Mdi
t n r t n g MTl MT2 MT3 MT4 MTS
Glucose 30
40 30
Maltose 30 30 43 50
Sucrose
30 BN 10 10
Thanh p h i n BOT
17 17
mfii t n r t n g (g/L) Casein
0,1 Glutamate
1 1 5 5
Peptone
0,2 Glycerol
5 5
~
Khodng
^ ~
+ t 4.Ghi chu: -: Khfing tham gia, +: 06 tham gia, MT: M6I tm*ng, BN: B^t ngS, BDT: BOt d^u tuwng Xdc d[nh hogt tinh i>c che a-glucosidase
Djch Idn men chiing Actinoplanes sp. KCTC- 9161 dupc si> dyng d l xdc djnh khd nang Crc chl a-glucosidase flieo phuang phdp ciia Yamaki vd Mori (2006) bing phdn li'ng giO'a a-glucosidase (Sigma) vd 4-nib-ophenyl a-D- glucopyranoslde (4-NPG. Sigma), sd dpng khay vl t h i 96 gilng, thf nghigm (TN) difpc Igp Igi 3 lln, cd doi chCrng (DC), Djch nfii 20 pi duvc cho vdo mfii gilng cCia TN, d$m 0,5M potaslum phosphate buffer (PPB) 20 pi (pH 6,7) dupc cho Wb mfii gilng cOa DC, b l sung vdo mli gilng 50 pi emzyme a-glucosidase cd nguon glc tif rudt chu^t 25 mg/ml, 50 \ii co chit 4-NPG (3 mM), vd 120 pi d|m PPB, ii hin hpp phdn i>ng d 37°C. Sau khi Ci 45 phOt, thfim 50 pi Na2(X)3 {0,67 M vdo mfii gilng d l kit thOc phdn i>ng. Hfin hpp phdn Crng dugc do d budc sdng 415 nm tren mdy Elisa reader (Blotefc ELx800, USA). Mpt dan vj a-glucosidase dupc djnh nghTa nhif Id lu-png enzyme gidi phdng 1 pmol cua 4-NPG d diiu ki^n thi nghi#m. MCrc dfi dc chl a-glucosidase dupc tinh ^ e o cdng thirc:
Trongdd, A4isocvdA4isTNldgidbiOD do tgi birdc sdng 415 nm cOacacong DC vd TN Sic ky bdn mdng
Sic ky bdn ming (thin layer chromahjgraphy-TLC) duvc sCr dyng d l phdn tfch va xdc djnh cdc nhdm chit khdc nhau eo hx)ng thdnh phin djch Ifin men dga tr6n nguydn tic cdc chit khdc nhau cd dO phan eye khdc nhau ndn dupc tdch ra i cdc vj hi khdc nhau, aud trinh ndy thyc hi§n trdn bdn mdng silica gel Merck 60 F254, ddy 0,25 mm vdi pha tTnh Id lik Idp mdng cdc chit hip phy (silicagel), pha dgng duvc hlnh thdnh theo f} lp eth^ acetate : methanol = 11 (v/v) vd Sp dung djch H2O : add formic = 5:2 ( v ^ . Djch Un men dugc ly tam 40C0 vdng/phiit trong 10 phdt d 4''C d l loai bd cdnlii bdo, sau dd tlnh sgch so bO vdi ethanol theo ty l# 1.4 (1 djch l§n men da du-pc logi bd can t l bdo vd 4 ethanol) 0 30 phut vd ly tam 12000 vdng/phOt trong 10 phut d 4°C, thu djch trong chay TLC. Cdc phan h> dudng dupc phdt hifin' bim xfing hai H2S04 10% trong ethanol 99.9%, sau dd Idm hifin mdu d nhifit dg 110°C hong 10 phCit. '- -^ ti K^T QUA V A T H A O LUAN
Sinh tong hpTj acartwse liang Ifin men chung Actinoplanes sp. KCTC- 9161
f r i ' " i ' 1 - ' ' " ° ^ ' ^ " t * sp KCTC- 9161 ban diu dupc nudi trong mdi trydng Ifin men cda Wei vd ding tdc gid (2010) sai TJ S"r^ f ' i I '^tl^ Adinoplanes VTCC- A l 779 (Dfi Thj Tuydn et al., 2011) thu djch len men, logi bd cd
^ c a r b S e / S - n l S "t^n h^- H ° *^-^^? ?'^"3 ' ^ ^ ^^ ^ ""^^ ^'^" ^^"^ ^^"^ "^t cd cung hfi s l Rf ngang vdi ch3 acarbose (Rf - 0,28) trfin hg dung moi ethyl acetate : methanol = 1:1 vd 5% (H20 : acid fonnic = 5:2) (v/v). |
A^!^ila^ri's'&?c.^Slt^l^S^!JV^^^^^ '^'IT^A"^ -^^ctmp/anes KCTC-9161 (A): (1, 3 miu acarbose lir chfl«!
mfli tnrdng ISn men (1. MTT; ^ S T M T ? 4! MT5 5 ^ T S r ^° Adinoplanes sp. KCTC-9161 trong ck Anh hu*ng eUa thanh phin mdi trudmg Ifin men din khd nang sinh ting h^p chit 0^ chl o-gluco»ldase Z i l ^ ^ r o s ^ X m ^ S S ^ r ^ l ^ ^ ^ ^ ^ - «^- — - bit ngi, ^
HQC CONG NGH$ SINH HQC TOAN QUOC 2013
Tiln hdnh idn men chung Actinoplanes sp. KCTTC- 9161 frong 5 mfii tnrdng vdi cdc thdnh phin khac nhau. Kit qud cho thiy mdi birdng Ifin men MTl bao gom glucose, maltose vd b|t ngd da cho hoat tinh Crc c h l o-glucosidase cao nhat (52,24 %). Mdi tardng MTS c6 nguon carbon tfr maltose duprc bo sung nhilu han, glucose thip han so vdi MTl va nguon nita tCr bdt ngd duac thay bang bfit dau tircrng nhung lugng sodium glutamate dirge b l sung gip 5 lan so vdi MTl dd cho hogt tlnh Crc chl a-glucosidase hrang duang vdi djch len men trong MTl (51 %). Kit qua ndy phCi hpp vdi nghien cCru cua m$t s l tac gid khi ting nguln nita tir sodium glutamate gidp tang khd nang sinh ting hap acartwse (Wet et al.. 2010; Wang et al.. 2011).
^ m
MAI tntong ten man
Hlnh 2. Ho^t Unh dc chl o-glucosfdase trong cAc mdi tivdng lin men
Cac nghiSn cdu trudc ddy da chi ra rang, trong mdi trudng ISn men, maltose va glucose la nguln carbon thfch hop cho sytang tnrdng t l bdo vdsy stnh tong hpp acarbose (Wang e( a/., 2011). Glucose ludn dupc COI la nguon carton d l sir dyng nhlt trong thdnh phin mfii trudng Ifin men (Sun e( ai, 2012). Maltose d n dupc duy tii d nong dd cao trong mdi tnrdng trong suot qud trlnh idn men de sinh ting hgp acaibose cd hifiu qud (Choi, Shin, 2003). VI vay, mdi trudng MT3 cd nguln carbon tir maltose dupc thay bang sucnase vd b l sung thdm glycenai, nguln nikr khdng dupc lly h> bdt ngd vi bpt d$u tuang md tCr casein hydrolysate vd peptone da khdng cho hogt tfnh Crc chl a-glucosidase. MT2 Id mdi tnrdng khdng dugrc bo sung glucose vd cdc thdnh phan cdn Igi gilng MT1 da cho hogt tlnh Crc chl a-glucosidase dat 45,1 % thip hon MTl. Dilu dd cang chdng Id glucose rit cin thilt cho qud trlnh Ifin men sinh ting hg'p acarbose h> c^Ong Actinoplanes sp. KCTC- 9161. MT4 vd MT5 Id hai mfli trudng cd cCing thdnh phin, tuy nhifin trong MT4 ham lugng glucose vd maltose Id ft han b'ong MTS, vl vfiy hogt tfnh uc chl a-glucosldase trong MT4 (46,6 %) thip han ruITS.
Vific lya chpn thdnh phan mdi trudng len men cd sy dnh hudng rd rfit din hogt tfnh i>c chl a-glucosidase cua d[ch Ifin men. M$t s l tdc gid da chf ra ring, Actinopinaes. sp sinh ting hpp vd tilt acarbose ngogi bdo re mdi tardng Ifin men dgt hifiu suit cao khi si> dyng nguln dinh dudng cacbon Id maltose, glucose, glycerol, nguln nita Id sodium glutamate, bfit ngfi, b|t d$u tircng; Ifin men 168 gid, 200 rpm d 28°C, pH cCia mdi hudng tir 7,0-7,2 (Sun et ai, 2012) (Wang e( ai.
2012). ChOng Actinoplanes sp. A56 sinh ting hpp acarbose cao nhlt tif 837 din 1043 mg/L hong mdi tardng chda glucose, maltose, djch chiet ngd vd glutamate cho khd nang sinh ting hpp acarbose cao (Wei et ai. 2010). Trong mdi trudng ifin men cd chira glucose, bpt ngd, bdt ddu tuang, tinh bgt tan vd glutamate cho khd nang smh ting hap acarbose dgt tdi 5000 mg/l trong 168 gid Ifin men (Li efa/., 2012).
Lya chgn nguon carbon chd y l u td- glucose, maltose vd bpt ngd hong moi tnj-dng Ifin men MTl cho hoat tfnh uc chl a- glucosidase Id cao nhlt (52,24%). Tuy nhifin, cin phdi xdc djnh nang suit sinh tong hgp acarbose tCi- qud b'lnh Ifin men hong mdi tnrdng MTl d l cd kit ludn difnh xdc hon.
Khd nang sinh ting hgp acarbose hong cdc mdi birdng khac nhau dugc kilm tra bdng phuong phdp xdc d[nh hogt tfnh O'C chl a-glucosidase, ding thdi dugc kilm tra kha ndng sinh ting hpp acarbose bing sac ky' do TLC trfin hfi dung mfii ethyl acetate : methanol = 1:1 vd 5% (H20 : acid fonnic = 5:2) (v/v) the hifin trfin Hinh IB
Ket qud sic k;^ dd cho thiy, cdc gilng 1,4,5 tuong ung vdi djch Ifin men trong mdi tardng MTl, MT4.va MT5 (thdnh phan mfii trudng chu y l u Id glucose, maltose va bfit ngd hay bgt dgu Urang) cd xuit hifin bdng ddm nfit vdi hfi s l Rf = 0,28 tuvng O'ng vdi chuan acarbose, rifing gilng 3 (dich ifin men trong MT3 khdng cd maltose) khfing xuit hifin bdng ddm ngang chuin acarbose. Gilng 2 Id dich ldn men trong mfii tnfdng khfing cd glucose (MT2) da xuit hien bdng md cd cung Rf vdi chuan acarbose. Ket qud cho thay, lua chpn ngudn cartDon tir maltose va glucose cd dnh hudng rd rfit den khd ndng sinh tfing hgp acarbose cda chdng Actinoplanes sp. KCTC- 9161. Kiem tra khd nang sinh tfing hpp acarbose trfin ban mdng TLC cung phd hpp vdi vific xdc djnh hogt tinh dc che a-glucosidase cua djch Ifin men trong cdc mfii trudng khdc nhau.
Khd nang sinh t i n g hgp acarbose cua chung Actinoplanes sp. theo thd>l glan
Chung Actinoplanes sp. KCTC- 9161 dupc nudi hong mdi tardng MTl bao gom cdc thdnh phin: glucose, maltose, bpt ngd, sodium glutamate va khodng vi lupng. Khdo sdt kha nang sinh tdng hpp acarbose tdng din tiieo thdi gian nuoi cly (Hlnh 3). 6 thdi dilm tCr 24 din 72 gid dau, acarbose chua dupc stnh ting hgp, hogt tfnh uc che a-glucosidase d giai dogn nay hlu nhu khdng cd. Hogt chat acarbose tang dan tir thdi diem 96 gid din 216 gid, vd dgt eye dgi d 192 gid ldn men (55,2 %). Tgi thdi diem 168 gid, hoat chit acarbose dupc sinh tdng hpp ra tir ehdng Actinoplanes KCTC- 9161 irc chl 52,24 %, thip hvn d thdi dilm 192 gid. Tuy nhiSn, de gidm gia thdnh cung nhu- thdi gian nudi ely, chdng tfii chpn thdi dilm 168 gid id thfch hgp nhlt cho kha nang sinh ting hpp acarbose.
So sdnh vdi kit qua sinh tong hpp chat CPC chl a-glucosidase dung d l dilu trj bfinh tilu dudng type 2 cua mdt so tac gid cho thay djch ngogi bdo cua 5 chdng Bacillus sp. d-c chl trfin 85% hogt tfnh a-glucosidase sau 4 ngay ifin men (Quyen Dinh Thi, Vu Van Hgnh, 2011). Nhu vfiy, chung Actinoplanes sp. KCTC- 9161 cho hoat tinh Crc che a-glucosldase thap han vd thdi gian Ifin men ddi han so vdi nghifin ciru cda tdc gia trfin eae chung Baallus sp .
T1t»1ftanl*nnwn(l»l
Hlnh 3. K h i n i n g i>c ch4 a-glucosidase cua acarbose tip chung Actinoplanes sp. KCTC- 9161 ttieo thfl'i gian
K£T LUAN I Mdi tm-dng ttilch h g p c h o Ifin m e n sinh t i n g h g p acarbose tCr chCing Actinoplanes sp. K C T C - 9 1 6 1 b a o g i m (g/L);
30 maltose 30 glucose, 10 bgt n g d , 1 sodium glutamate, 2 CaClz, 0,5 F e C b , 1 KzHPO^, 2,5 C a C O a , p H = 7,0. Thdi gian ttilch h g p d i o sinh t i n g h o p acarbose c a o it 168 g i d , 28°C v d i t i e dO Idc 2 0 0 r p m . C h i i n g Actinoplanes sp. KCTC- 9181 cho hogt Hnh irc e h i a-glucosidase cao n h l t dgt 5 2 , 2 4 % trong mdi t r u d n g ldn m e n M T 1 .
Idi dm ffn: Cong trinh c6 su hi trv da Chuong trinh nghien cuu khoa hpc cong nghi trprig diem quoc gia phdi tnin cans nghiep hoa dim Ai nam 2020. de tdi: "Nghien cOu quy trinli cang ngh? dieu chi acarbose ldm nguyen liiu thuoc chOa bfnh dat Ihdo duang " Bo Cong Ihutmg 2611- 2014
T A I L I ^ U T H A M K H A O
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STUDY OF FERMENTATION MEDIUM COMPONENTS FOR ACARBOSE ,
PRODUCTION BY ACTINOPLANES sp. KCTC- 9161 I
N g u y e n T h i N u o n g , D o T h i T u y e n , L e T h a n h H o a n g , Q u y e n D i n h T h i * Institue of Bhtechnology, Viet Nam Academy of Science and Technology
SUMMARY
Diabetes is one of a serious disease. It causes disability for the diabetic patients, especially untreated or inadequately treated patienn Acarbose is a pseudo-oligosaccharide compound, a a-glucosidase competitive inhibitor found in a number of microbial species, particularly species of the genus Actinoplanes. In recent years, acarbose has widely used in the treatment of type n diabetes melliliii it can be used wittiout or with the combination of other dmgs such as some migiitol, voglibose. In this shidy, Actinoplanes if.
KCTC- 9161 fermented in different fermentation media. The fermentation mainly use traditional fermentation nutrients such at maltose, glucose, com meal, soybean meal and trace minerals with the fennentation condition 168 hours, at 1%°C, shaking at 2M rpm/min. Feraientotion of Actinoplanes sp. KCTC- 9161 after 168 hours showed acarbose production over 20mg/L above 52,24i4 inhibitor activity towards from fermeutation medium with maltose (3%). Maltose should be maintained at high level diirini fwmentaiion for acariiose production effectively. After preliminary purification, acarbose product is tested on tiiin-l^
chromatography (TLC) with a solvent system: ethyl acetate methanol = 1.1 and 5% solution of H2O: fonnic acid = 5-2 (v Iv) byi factor of Rf= 0,28
Keywords: Acarbose, Actinoplanes sp. K C T C - 9161, fermentation, T L C , a-glucosidase.
• Author for correspondence: Tel, 04 7568260; Fax: 04.8363144. E-mail: ouvenOibl.ac.vn
