Tgp chi Cdng ngh4 Sinh hgc 11(2): 293-302, 2013
NGHlfeN ClTU QUY TRINH THU NHAN CEPHALOSPORIN C TlT DICH LEN MEN CHUNG NAM ACREMONIUM CHRYSOGENUM 6963-4
Nguyen Thi Hong L i e n \ H o T u y e n \ Ha Huy K i ^ Le Gia Hy^
'Vien Cdng nghe sinh hgc, Vien Hdn ldm Khoa hoc vd Cdng nghi Viit Nam
^Trung tdm Hda sinh, Vien Khoa hgc sdn xudl cdng nghi mdi vd Bdo ve mdi tnrdng
TOM T A T
Cephalosporin C (CPC), khang sinh thuoc nhom P-lactam, co hoat tinh khang khuan khong cao nhimg la nguyen li|u ban diu dS san xuit cac cephalosporin ban t6ng hop, dong khang sinh duoc sii dung rong rai trong dieu tri cac benh nhiem khuan, chiem vi tri quan trpng tren thi trucmg khang smh CPC duoc len men san xuat nhd cac chung nam moc Acremonium chrysogenum. Trong nghien ciiu nay, chiing loi lien hanh lach chiet CPC tir djch len men ciia chiing A chrysogenum 6963-4 Sinh khoi nam moc va cac thanh phan khong tan co trong dich len men dupc loai bo bang each loc, sii dung diatonute litm chat tro loc CPC c6 Uong dich len men khong ben nhi?l, boat tinh khang smh suy giam manh khi 6 nhiet do cao, CPC ben nhat a pH 7, hoat tinh chi giam 13% 6 60°C Uong 40 phiit nhung a pH 10 boat tinh khang sinh bi mat hoan toan. Qua trinh tach chiet dupc nen hiinh 6 nhift do 5-10''C, pH 3-7 CPC co trong dich loc duoc hap phu hoan toan vao than boat tinh. CPC-Zn dupc ket Ilia nhieu nhat trong dung moi aceton 6 pH 3 (3,56 mg/m!). Qua trinh tach chi^t va tinh sach CPC tir djch l§n men dat hi^u suat 61% Che pham cephalosponn C nhan dupc co kha n3ng iic che vi khuan Alcaligenes faecalis. Phan tich sac ky ban mong, pho tti ngoai, pho hong ngoai, pho khoi va sSc ky long cao ap cho ihay, chl phSm CPC nhan dupc tuong tu voi CPC chuan
Tir khda: Cephalosporin C, dfch len men, hdp phu, tdch chiel, than hoat tinh, trao doi icm
MO DAU
Cephalosporin C (CPC), khang sinh tu nhien thuoc nhom p-lactam, duoc sinh tong hgp tii nam Acremonium chrysogenum. Tuy CPC c6 boat linh khang khuan khong cao, nhung no la tien chat ciia 7- aminocephalosporanic acid (7-ACA), tii do san xual ra cac cephalosporin ban long hpp co boat tinh khang stnh manh, pho khang khuan rpng, it dpc, khong bi nhcm thuoc, ma con rat ben viing voi enzym p-lactamase Su thanh cong cua mot qua trinh san xudt CPC phu thupc vao boat tinh ciia chung giong, moi truong, dieu kien len men va dac biet la each thuc tach chi^l, thu hoi san pham. Theo Wildfeuer (1985) m6i khang sinh (KS) d^u co cac ky thuat tinh sach rieng, tuy nhien viec tach CPC tir dich len men co th§ ti^n hanh bang nhieu phuong phap nhu, hap phy carbon; hap phu polyme phi ion, frao d6i ion; chiSl xudt b^ng dung moi; ket liia tao muoi; kel tua t?o din xudl ciia acid; chiet dong soi va CO dich len men, Cac phucmg phap nay da duac img dyng thanh cong 66 san xual CPC a quy mo l»Jn trong do ky thu|t hjp phu de tach chiet va linh sach CPC dugc su dyng rpng r3i hon ca la nho vao chuoi ben ua nu6c a-aminoadipyl ciia CPC Than boat tinh c6 tha hdp phy CPC tir dich len men rdt hieu qua va
nha hap phu bang hon hop dung moi va nuoc Mpt trong nhiing khac biet ciia phuang phap nay do la than phai dugc thay men hoac duac boat hoa de tai su dung. CPC CO the tiep tuc duac tinh sach neu cho hap phu qua nhua trao doi ion. Kha nang hap phy CPC vao than boat tinh va cac vat lieu trao doi ion cao hon cac chat hap phy polyme phi ion cijng nhu cac dung nnfli hun co (Mishra et ai, 2007). CPC la KS ngoai bao, da phan tich luy trong dich ten men.
Trong dung dich, CPC a trang thai khong phan cue nen kho ket tinh. Do do can chuyen CPC thanh dang muoi natn hay muoi kem thi moi co the ket tinh dugc (Neto ^f a/., 2011)
Bai bao nay trinh bay ket qua nghien ciru cac yeu to anh huong toi qua trinh tach chiet va xay dung quy Irinh lach chiet CPC lu chiing nam A. chrysogenum 6963-4 (AC 6963-4)
NGUYEN LIEU VA PHU'ONG PHAP Nguyen lieu
Chiing AC 6963-4 sinh long hgp CPC va chiing VI khuan kiem dinh Alcaligenes faecalis NCAIM B02076 (AF76) nhan dupc tir bg suu tap gifing ciia phong Cong nghe len men, Vi?n Cong nghe sinh 293
Nguyen Thj Hong Lien et ai hoc. Chiing AC 6963-4 dugc bao quan, hoal hoa va
nhan gi6ng tren moi truong YPS (Vialla el ai, 1997) Cac hoa chdt: CPC (CeHi^N^OsS.Zn) cua hang Sigma (CHLB Dire); cao nam men, DL- methionme ciia Nhal Ban; saccharose, glucose ciia Viet Nam.
Moi triroTig len men (g/1)
Saccharose 36; glucose 27; bgt dau tuang 24;
cao nam men 20; DL-Melhionin 4; CaS04 7,5, CaCOj 10; (NH4)2S04 8; pH 6,4 (Vialla et ai. 1997).
Phu'ong p h a p
Len men sinh tong hffp CPC:
Qua trinh len men dugc thuc hien tren thiet hi BioflollO (Brunswick Scientific) 6 2 8 ^ , tdc do khudy 500 v/p, ty le Hep giong la 5% vA', ly le cap khi la 1 Ih khi/1 lit moi truong/I phiil ( I w m ) . Sau 96 h, dich len men dugc thu hoi khi ham lugng CPC dal cao nhat.
Xdc dinh hogt tinh khdng sinh (HTKS).
Theo phuang phap dye 16 thach, khoanh giay lgc, dugc the hien qua duang kinh vong vo khuan (D^vk. iTiui) l^^t hgp hieu chinh theo phuang phap Dmitrieva (Egorov, 1976) c6 so sanh voi CPC.Zn chuan da biel nong dp va the hien bang mg/ml.
Xdc dinh khd nang ben nhiet vd pH cua CPC trong dich len men.
Dich len men sau khi ly tam loai sinh khoi dugc chinh ve cac pH' 3, 7, 10 va dugc gift o c a c nhiet do:
40, 60, 80, 100°C keo dai 40 phut, sau do dua ve nhiel dp phong de xac dmh HTK.S
Cdc phirffng phdp phdn tich
Phan tich sac ky lop mong de danh gia dp tinh sach ciia CPC, duac thuc hien tren ban sac ky 25 TLC aluminium Sillicagel 60 F254 trang san co do day 0,2 mm, Sau khi chay sac ky, ban mong dupc lam kho 6 nhiet dp 30°C, hien mau bang ninhydrin
Pho tu ngoai (UV) danh gia do tinh sach va nhan biet CPC qua kha nang hap thy hue xa dien tir, dugc ghi tren may GBC-Instrument 2855 tai Vien Ky thual Khoa hgc hinh su. Bo Cong an
Pho hong ngoai dugc diing de nghien ciru dao dpng ciia nguyen tu hay nhom nguyen Hi Irong phan Hi ciia CPC, Pho hong ngoai dugc ghi duoi dang vien nen KBr tren may FT-IR Impact 410. Ph6 dugc do tai Vien Hoa hoc, Vien Khoa hoc va Cong nghe Viet Nam
Kh6i ph6 dugc do fren may HPLC-MS: ESIMS, tai Vien Hoa hgc cac hgp chat thien nhien, Vien Khoa hgc va Cong nghe Viet Nam de xac dinh kh6i lupng phan tu cua mSu CPC tach chiet dugc.
s i c ky long cao ap (HPLC) danh gia dp tmh sach ciia mSu CPC lach chief dugc, sit dung col Bondapak C I S (5(im, 250x4,6 mm) lai Trung lam Ky thuat Tieu chuan Do luong chat lugng 1, Tong cyc Tieu chudn do luong chat lugng.
KET QUA VA THAO LUAN
Khao sat phirong p h a p thu hoi djch sau I6n men Ket thiic len men, thong thuang djch len men c6 ih§ duoc thu h6i bang hai each: ly tam hoac lpc. Ly tam chi ap dung dugc cho cac me len men nho mot vai lit djch, khi Ien men lugng Ion trong san xuat lon thi can phai lpc. Dich sau khi len men co dp nhat rat cao, luong sinh khoi nam nhieu (khoang 30 mg/ml), dang sgi, ben quyen vao nhau gay bi be mat lpc nen can phai c6 chat trg lpc (diatomite) de tao chan loc.
Do dialomite thupc dang chat tro, deu tao thanh cac khoang rong nho ben trong nen de dang cho phan dich trong di qua, giir lai sgi nam va cac chat huySn phii, Vi vay diatomite duoc su dung lam chal trp lpc dich sau len men de thu hoi CPC
Chung loi da lien hanh thir nghiem thu hoi dich len men theo 3 phuong phap: (1) ly tam lOOOO vong/phut trong 10 phut, (2) lpc co sir dyng hot frg loc diatomite ciia Nhat, (3) lgc co sii dyng bpt frg lgc diatomile cua Viet Nam. Phuang phap 2 va 3 ciing ioai dugc hoan loan sinh khoi nam moc va cac thanh phan khong tan khac co trong dich sau len men nhu phuang phap 1, hon nua, HTKS cua cac man thu theo ca 3 phuong phap deu giong nhau (Hinh I). Sii dung 0,3% bot tra lpc diatomite cua Viet Nam de lpc moi truong sau len men da dam bao dp trong ciia dich loc, toe dp lpc nhanh va giam dugc chi phi cho qua trinh thu h6i CPC.
Xac dinh tinh bhn nhiet ciia C P C & cac p H khac n h a u
Khang sinh rat nhay cam voi sir thay doi ciia nhiet dp va pH. Trong dieu kien nhiet dg va pH khong phii hgp, hoat tinh khang khuan ciia khang sinh bj suy giam manh Kha nSng ben nhiet va ben pH se anh huang den qua trinh tach chief va bao quan ve sau. Do do, can nghifin cim 4nh huong ciia chung den boat tinh CPC co trong djch len men.
294
Tgp chi Cdng nghe Sinh hoc 11(2): 293-302, 2013 Djch lgc sau khi len men co pH 7,7 dugc dieu chinh v^ cac pH 3, 7, 10 va xac dinh boat tinh CPC ban dau (Dwio mm). Ket qua: voi pH dich nguyen 7,7 thi Dwk= 32 mm, 6 pH 3, 7, 10 thi D„vk tucmg img se la 22,1 mm, 32 mra va 18,5 mm. Sau d6, djch len men dugc gia nhiet ten 40, 60, 80 va I OO^C, giu a cac nhiet do do 40 phiit, xac dinh boat tinh CPC con lai trong dich.
Hinh 1, Hogt tinh khSng smh cua djch len men t i i chiing AC 6963-4 v&\ chung kiem dinh AF76: thu hoi bang ly tam (1), lpc c6 bOt trg- lpc Nh$l (2) va bgt tro lpc Vi^t Nam (3).
Gianhift40|diut - ^ p H = 3 -3-pH=7 -*-pHd|ch-ft-pH=10i
Nhetdpro I Hinh 2. Anh hudng ciia pH vd nhi$f dO d^n hogt tmh CPC
Kel qua nghien ciru dugc the hien tren hinh 2 cho thdy, CPC co frong djch len men khong ben nhi^t v^ chju anh huong rdt ldn boi gia trj pH 0 tat ci cic pH, HTKS deu giam ddn khi nhi?t dp iSng.
pH 7 da gitip Iam ISng kha nSng ben nhi?t ciia CPC
CO frong dich len men. 6 pH 7, HTKS frong djch len men ben nhat va kha nang chiu nhiet cao hem ca, tuy nhien, khi giu o 60°C trong 40 phiil, HTKS giam 13%. CPC trong djch len men khong ben trong moi truong kiem, hi mat hoan loan boat linh sau 40 phiit 6 6 0 X . Vi vay, a cac buac nghien cim liep theo djch sau len men dugc chinh ve pH 7 va cac giai doan ciia qua trinh tach chiet deu dugc tien hanh trong dieu kien lanh (nhiet dp 4 - l O X ) va pH 3 - 7 sao cho boat tinh CPC it bi suy giam nhat.
XSy d u n g quy trinh tach chiet CPC tir djch sau len men (Hinh 4)
De tach chiet duoc CPC tu dich len men, dung dich CPC dupc hap phu vao than boat tinh sau do thu than boat tinh va tien hanh nha hap phy de thu djch CO chira CPC, tiep theo CPC duoc chuyen qua dang muoi kem va muoi natn de linh sach va ket linh Ket qua cac buoc tach chiet CPC lu dich sau len men thu dupc nhu sau:
1. Hap phy khang sinh vao than hoal tinh- Ghosh va dong tac gia da chi ra rang co the diing mpt so dung moi nhu n-butanol, bulyl acetate .. de chiet CPC nhung thuc te lugng CPC di vao dung moi chi dat khoang 50 - 60% va con giam tiep trong qua trinh tinh sach va kel linh sau nay (Ghosh et ai, 1997). Trong nghien cuu nay, de hap phu hieu qua CPC chung toi da diing than boat tinh. Than boat tinh dugc bo sung vao dich lpc (pH 7) de hap phu CPC, dSng then chinh ve pH 3 bang HCl 5% nham muc dich CPC dupc hap phy hoan toan vao than, giu 6 4"C trong 24 h, CPC dugc lach ra khoi than boat linh bang hon hpp aceton. nuac theo ty ie 3.2, chinh vh pH 5 bang NH4OH 5% Lpc, loai than, hon hop aceton co chiia CPC dugc co duoi ap sual giam (10 mm Hg), Than va dich dung moi sau c6 chan khong khong con co HTKS.
2. K6t tua CPC-Zn: Bfl sung Zn-acetal 20% vao dich sau CO chan khong de dua CPC ve dang muoi Zn. Chinh v^ cac pH 3, 7, 10, khudy o S^C trong 30 phiit va bo sung them cac dung moi khac nhau de thir kha nang k^l liia ciia CPC-Zn. GiiJ a nhi?i do 5- WC, sau 24 h lgc, rira tiia bdng chinh dung moi dang thii nghiem theo ty le dung moi nuac (4:1) da lam lanh 5-I0''C, lam kho trong chan khong va thu dugc san phdm CPC tho Luang CPC co trong dich len men ban ddu la 5,088 mg/ml, lupng CPC-Zn kel tua a cac pH, dung moi khac nhau the hien a bang 1
Trong cac dung moi thir nghiem, CPC-Zn deu ket nia nhi6u nhdt 6 pH 3 nhung o pH 10 thi khang smh khong thi tiia dugc Dang chii y hon ca la aceton.
Nguyen Thj Hong Lien el ai frong dung moi nay, lugng CPC-Zn thu dupc la 3,56 hop do CPC-Zn it lan frong aceton (CPC-Zn tan frong mg/ml, dat hieu suat tiia 70% so voi lugng CPC ban nuoc gap 30 Ian frong aceton). Nhu vay co thi sir dau CO frong dich len men, Ket qua nay hoan toan phu dung aceton de ket tua muoi CPC-Zn.
Bang 1. L u g n g CPC-Zn kel tiia trong cac pH va dung moi khac nhau.
D u n g moi n-butanol I so-propyl alcotiol A c e t o n Methanol Ethanol Chloroform Iso-amyl alcohol
Luo'ng CPC-Zn tiia duo<c (mg/ml) p H 3
2,26 2,54 3,56 1,77 1,68 2,23 1,78
p H 7 1,58 2.37 3,02 1,29 1,31 2,08 1,14
3 Chuyen CPC-Zn thanh CPC-Na qua trao doi ion: Hoa tan lugng CPC-Zn co dugc o tren trong dung dich NaCI 0,5 M, sau do chinh v l pH 3 o 5"C bang HCl 5%, Cho dung dich vao cot nhua trao d6i ion Amberlile IR-120- polystyrol -SO3H, ChiSl CPC-Na thu dugc bang dung dich methanol : nuac theo ty le (4,1), vai l6c dp 0,4 ml/phiit, sau do giam toe do chi&t 0,15 ml/phiit. Dung dich thu dugc duac CO duai ap suat giam (10 mm Hg).
4. Tinh sach CPC-Na: Su dyng aceton d l kk tiia khang sinh co trong dung dich sau khi co duai ap sudt giam. Lpc, nia tiia bing aceton lanh S X . Tua dupc hoa tan lai trong nuac cdt va chinh vk pH 3 bang HCl 5% Lpc loai lap chdt, chinh dung dich vk pH 7 bang NaOH 5% va b6 sung aceton, CPC-Na ket tua, gift trong 12 h a 5 - I O T Sau do loc, lam kho trong chan khong thu dupc 3,103 mg/ml CPC-Na, dat hieu suat thu hfli 6 1 % so vai lupng CPC ban dau co trong dich len men.
Dp linh sach ciia khang sinh trong qua trinh tach chiel dupc kiem tra bang sdc ky ban mong.
Ket qua cho thay do tinh sach cua CPC duoc tarig len theo qua Irinh tach chiet. Sau khi co duoi ap suat giam, dich nha hdp phy co 6 vk chung to con nhieu vet tap chdl c6 phan ung duong tinh voi thu6c thu ninhydrin (vach 1) va con 3 vk sau khi tach qua cot trao doi ion (vach 2), san pham cu6i cung, CPC-Na chi con 1 \k (vach 3), ca 3 mdu d i u co l' vet dam the hien la CPC, Urong ung vai CPC chudn o vach so 4 CO In s6 Rf = 0,3.
_ Vel chay cua dung
Hinb 3. Sac ky lop mong (TLC) cua CPC qua cac giai dogn tach chiet 1 Dung djch nha hap phu d u g c co duo-i Sp suit giam. co 6 vet, 2 Dung dich sau khi t^ch qua cot trao d6i cation, CO 3 vet, 3 CPC-Na sau khi lam tinh khi§t: co 1 vel (Rf = 0 3}, 4 CPC chuan c6 1 vet (Rf - 0 3)
Djnh tinh san phdm CPC thu dugc Khd ndng khdng khudn
Kiem tra kha nang khang khudn cua mdu CPC lach chieltir dich len men chimg AC 6963-4 va mau CPC chuan, kit qua tren (Hinh 5) cho thdy, mdu CPC tho Ihu dugc cung co hoat tinh khang khuan giong nhu CPC chudn.
Tgp chi Cdng nghe Sinh hgc 11(2)- 293-302, 2013 Dgc tinh hda ly
Tren sdc ky ban mong (Hinh 3) thi hien Rf ciia CPC chuan va che pham CPC tho luang duong nhau (Rf = 0,33). Ngoai sdc ky ban mong, CPC c h l phdm
dugc so sanh voi CPC chuan thong qua p h i tu ngoai pho hong ngoai, sac ky long cao ap va khii p h i . P h i hi rigoai (UV) cua CPC chl pham luong ur CPC chuan, chung deu hdp thu cue dai a buoc song X - 258 nm.
Hinh 4. So d i tSch chi^l CPC tii- d|ch I6n men chimg A chrysogenum 6963-4 CPC chiet xuat tu moi truang len men duac cho ch^y pho hong ngoai (IR) cimg voi CPC chuan de so sanh. Kel qua ch^y pho hong ngoai tren (Hinh 6) cho thdy, cac dinh (peak) ciia CPC chuan va CPC chill xudl tir dich len men triing nhau, the hi?n san pham len men ciia chiing AC
6963-4 la CPC Dac biel, cac peak tren hmh dac trung cho cac nhom chuc nhu' peak 2873,52 cm"
d^c trung cho nhom -C-S-C-, peak 1362.06 cm"' dac trung cho nhom acetat -0-CO-CHi va peak 1648,75 c m ' dac trung cho nhom amid bac hai - CO-NH-.
297
Nguyen Thi H6ng Lien et ai Khoi pho ciia CPC chiet xuat tu moi truang
(Hinh 7) cho thdy, trgng lugng phan tu ciia mau CPC chilt xudt dugc la 415, do da mdt di I nguyen lii H nen con 414, luong duong voi pic 413,7 (pic a); sau mdt them I phan tu CO:, nen con (414 - 44 = 370), tuong duang voi pic 369,8 (pic b), tuong tu nhu the vai cac peak c, d, e, f va g Nhu vay, CPC chiet xuat
dugc CO frgng lugng phan tii bang frpng lirgng phan Hi cua CPC chudn (MW = 415).
P h i sdc ky long cao ap ciia CPC chuan va CPC chill xudt dugc the hien tren (Hmh 8) cho thay, ihcri gian luu cua CPC chiet xuat dupc triing voi CPC chudn, khoang 5,5 phiil
Hinh 5 HTKS cua CPC tho va CPC chuan uc che chiing vi khuan A faecalis 76 So 1 CPC-Na c h u i n (100 jig/ml). So 2, 3, 4 CPC-Na che pham t u dich len men bign chiing AC 6963-4 (120 ng/ml).
4000 3500 3000 2500 2000 Wavenumbera (cm-1) Dale: Wed Apr 01 02:52 51 2009
Hinh 6. Pho h6ng ngogi cua CPC A CPC chuan, B CPC che pham.
Tgp chi Cong nghf Sinh hoi 11(2) 293-302,2013
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; : H { C H , I , C » ( - Y S - , Mol W I . 415/12 CO.H g i - N Y ^ - O v l c H .
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[ M - H ) =415-] =414 (pic = 4 1 3 , 7 ) . a [ M - H ] - C O , = 4 1 4 - 4 4 = 370 (pic = 369.a) b
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[ M - H ] - - C O , = 4 1 4 - 4 4 = 370 (pic = 369-5) c I M - H ] -CO2 -CH^ = 4 1 4 - - 4 4 - -16 = 354 ( pic 353.5 ) . t [ M - H ] -CO; -CH^ - C O , = 3 5 4 ^ = 3 1 0 ( p i c = 309,4)
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Nguyin Thi Hong Lien et ai
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(M+Hr-4i54-] = 4i6 (pic=415.S) :r [M+J^ar-41S + 23 = 43e(pic=437^) :g
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Hinh 7, Pho khoi cua CPC chiet xuSt tif moi truang
Tap chi Cdng nghi Sinh hgc 11(2): 293-302, 2013
HPIX: cua CPC chiet xudi tiT moi tru&ng Hinh 8. Pho HPLC cua CPC chuan va CPC chiet xuSt ti> moi truong
KET LUAN
Dich len men chung AC 6963-4 da duoc thu hSi bang each lpc c6 su dung diatomite cua Viet Nam lam chat tro loc, CPC tu moi truang duoc tach chiet bang each dieu chinh pH, nhiet do t6i uu va k^t tiia trong dung moi aceton 6 pH 3 Chiing toi da xay dirng dupc quy trinh tach chi^t CPC tii dich Icn men VOI hieu suat dat 6 1 % Cha phdm CPC thu duoc co cac dac tinh hoa ly giong vai CPC chuan.
Loi cam on: COng Irinh dugc thuc hien Inn kmh phi cua ai ldi cdp nhd niroc CNHD.DT.004/08-11 thuoc Chuong trinh khoa hgc vd cdng nghi trong diim Quoc gia phdl tnen cdng nghi hda dirac din nam 2020 CO sir dung trang thiit bi ciia phong Thi nghiem Irgng diim cdng nghp gen.
TAI LIEU THAM KHAO
Egorov NX (1976) Thuc tdp vi sinh vdt hoc (Nguyen Lan Dung dich) Nha .\uat ban Khoa hpc va Ky ihuat. Ha Noi.
Ghosh AC, Maihur RK, Dutta NN (1997) Extraction and purilicalion of cephalosporin antibioUcs, Biotreatment.
Downstream Processing and Modelling Adv Bioehcm Eng/Biaiet lino! 56 111-145
Mishra P. Srivastava P. Mishr.i PK. Kundu S (2007) Recovery of cephalosporin C from aqueous solution usiny polymeric adsorbent hid J Che Technol \A 592-596 Neto AB. Bustamante MC, Oliveira JH, Granato AC, Bellao f, Junm AC, BaiboAi M, Hokka CO 12011) Preliminary Studies for Cephamycm C Punncalinn Technique Appl Biochem Biaiei linol
Vialla A. Catani CF. Junior RB. Azevedo JL (1997) Cephalosponn C production and genetic improvemenl ot the fungus Atreniomum chryuigenum base on morphological mutant isolation BruJGenlO 165-170 Wildfeuer ME (1985) Approaches lo L-ephalosponn C purification from fermentation broth, in Punfication ol Fermenlalion Products, LeRoiih D. Schiloach J, Leaby TJ, eds, ACS Symposium Series, Amer Chem Son.
Washington, DC: 155-174
Nguyen Thi Hong Lien etal.
ESTABLISHMENT OF A PROCEDURE FOR RECOVERY OF CEPHALOSPORIN C FROM FERMENTATION BROTH OF FUNGUS ACREMONIUM CHRYSOGENNUM 6963-4
Nguyen Thi Hong Lien', Ho Tuyen', Ha Huy K e \ Le Gia B y ' ' 'institute of Biotechnology. Vietnam Academy of Science and Technology.
'Centre of Biochemistry, Institute of New Technology and Environmental Protection
SUMMARY
Cephalosponn C is a P-lactam antibiotic with not very high activity, but which is the starting material for the production of vanous semi-synthetic cephalosporins widely used in the treatment of bacterial diseases and occupy an important position m the pharmaceuncal market. Cephalosporin C is produced by specific high yield strains of Acremonium chrysogenum, a filamentous strictly aerobic fungus This study concentrated on extracUon of the cephalosporin C from fermentation broth of A chrysogenum Biomass and insoluble substances of fermentation broth were removed by a filter with the help of diatomite. CPC from fermentation broih was thermal unstable, the activity of antibiotics was decreased at high temperatures. CPC was most stable at pH 7, the acuvity decreased about 13% at 60°C for 40 minutes bul at pH 10, the activity of antibiotics was completely lost. The extraction process was earned out at 5-10°C, pH 3-7. Cephalosporin C in the culUire filtrate was full adsorbed on the activated carbon. Cephalosponn C was precipitated in acetone at pH 3. The extraction of CPC from the fermentation broth was 61 % efficiency Preparations obtained CPC was able to inhibit ofthe bacterial strain Alcaligenes faecalis. Analysis from the thin-layer chromatogram, ultraviolet and infrared specfrophotometry, mass spectrometry and high-pressure liquid chromatogram showed that the extracted CPC was similar lo the standard reference CPC .
Keywords Cephalosponn C, active carbon, adsorption, extraction, fermenlauon broth, ion exchange
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