Tgp chi Cdng ngh? Sinh hgc 11(2)' 265-274, 2013
NGHIEN c u t ! PHAN LAP VA ANH HlTONG CUA MOT SO DIEU KIEN NUOl CAY LEN KHA NANG SINH TONG HOP LACCASE BOI CHUNG NAM THU THAP TlT ROM MUC NINH BINH
T r a n Thi T h u H i l n , Hoang Thi Nhung, Nguyen Hai Van, N g u y i n Thi L a n Anh, Dinh Thi T h u Hiing, Dang Thi CSm Ha
Vi^n Cdng nghe sinh hgc, Vien Hdn ldm Khoa hgc vd Cdng nghe Viet Nam
TOM TAT
Laccase la nhom enzyme o:,y hoa co chua phan tir dong, duac phan bo rgng rai a vi sinh vat nhu nam dam, nam soi, xa khuan va vi khuan. Nam dam va nam sgi la cac vi sinh vat co kha nang sinh long hgp laccase duac nghien ciru nhi6u hon ca D6 tim hieu kha nang sinh laccase cua nSm soi phan lap tir cac moi truang smh thai kh^c nhau trong do co nguon rom ra muc, nghien ciru da dugc thuc hien. Tir rom muc dugc thu thap oNinh Binh, chiing nam sgi FNBLal da duoc phan lap tren moi tmdng PDA vai hoat tinh ban dau la 200 U/i Bang phuang phap phan loai tmyen thong ket hgp voi xac djnh mgl phan trinh tu gen ma hoa I8S rRNA, chiing nam FNBLal duac xep vao nganh nam nang Ascomycetes, chi Myroihecium va dugc dat ten la Myroihecium sp FNBLal Chiing FNBLal co kha nang sinh laccase cao trong dieu kien pH 6,5; chat cam (mg la CUSO4 (0,1 mM), nguon carbon la glucose (12 g/1), nguon nitrogen la hon hgp casein I (g/1) va NaNOj (3 g/l). Tren moi trudng thich hgp, chiing FNBLal co hoat tinh cao nhat trong binh tam giac la 30016 U/1 sau 96 h. Hoat tinh laccase cao nhat khi len men trong bioreactor co dung lich 15 lit la 10,410 U/l sau 57 h.
Tirkhoa: Chdt cdm img, laccase, len men. Myroihecium. nnn ra
MO DAU
Enzyme ngoai bao ciia vi sinh vat la nguon vat li?u quan trpng cho cac nghien ciru lay doc chat hO'u CO kho phan hiiy (POP) nhu cac loai thuoc nhuom, dal nhiem chat diet co. Laccase la enzyme co tiem nang cao trong cac irng dung khac nhu cong nghiep thuc pham, d u a c pham v V Dac biet, laccase tham gia phan irng xuc tac OM hoa khi co mat cua oxy doi vdi hang loat cac CO chat la chat 6 nhiem khac nhau nhu cac hgp chat diphenol, polyphenol, cac dan xuat ciia phenol, cac hop chat diamin, amin vong thom, benzenthiol, iham chi la cac hap chat vo c o nhu iot (Nyanhongo et ai, 2002). Enzyme ngoai bao laccase thuoc nhom oxygen oxidoreductase, enzyme nay chua bon nguyen tir dong d vi tri xuc tac. Laccase xuc tac qua trinh oxy hoa khu nhieu hpp chat phenolic khac nhau cung nhu cac diamine va amin tham, dong thdi khir oxy phan tu thanh nudc. Laccase la mpt trong cac loai enzyme dup'c ung dung kha pho bien trong cac nganh cong nghiep do chiing co pho c a chat rpng va su d^^g ch5t nhan dien tir cudi cimg la oxy phan tir chii khong phai can cac chat nhan dien tu khac nhu NAD(P)* (Ruggaber^r ai, 2006).
Laccase dupc sir dung trong mpl so nganh cong nghiep del nhuom, cong nghiep hoa my pham, hoa thuc pham, dugc pham ,v,v, (Rubia et ai, 2002). Laccase co tiem nang cao trong cong nghe nano sinh hpc de tao cac biosensor c6 dp nhay cao. Ngoai ra, laccase con dugc su dung trong chuyen boa (lien xu ly) cac hop chat lignocellulose va xii ly cac chat 6 nhicm kho phan hiiy, Tir lignocellulose da qua qua trinh tien xu ly va sau do ten men se tao thanh dudng, Tir day thong qua cac cong nghe tinh che smh hpc se lao ra 12 cum hoa chat kien tao gom 1,4- diacid (succinic acid, fumaric acid, malic acid), 2,5- furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, ievulinic acid, 3- hydroxybutyrolactone, glycerol, sorbitol, xylitol.
Cac vat lieu ban dau nay se la ca sd de san xuat cac san pham hang hoa dua tren nen tang smh hpc thay the nguyen lieu hoa thach
Trong nghien cim nay, chimg toi lien hanh phan lap chiing nam sinh enzyme ngoai bao laccase tu rom ra muc ihu thap d Nmh Binh va nghien ciru mpt so yeu to moi trudng nham nang cao kha nang smh long hgp laccase ciia chiing.
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Trau Thi Thu Hien et a VAT LIEU VA PHU'ONG PHAP
Vat lieu
Bon mau rom muc tir Ninh Binh da duoc thu thap de phan lap va sang lpc cac chung nam co kha nang sinh long hpp laccase.
P h u o n g p h a p nghien ciru Mdi trudng nuoi cdy
Cac moi trudng dugc sir dung trong nghien ciru g6m moi trudng PDA (potato, dextrose, agar) (the U.S Food and Drug Administration, 2011), moi trudng PG (dich chiet khoai tay 200 g/1, glucose 10 g/1) va moi trudng gom dich chiel khoai tay 200 g/1, casein 1 g/l, NaNOj 3 g/1, cam 5 g/l. glucose 12 g/l.
Moi trudng dugc khu triing d II5"C trong 30 phut. Hon hop vitamin, hon hgp vi lugng da dugc bo sung vao moi trudng trudc khi su dung (Nguyen Nguyen Quang, Dang Thi C i m Ha, 2010).
Phdn lap vd sdng lgc cdc chiing ndm cd khd ndng sinh laccase
Phdn lap cdc chiing ndm
Ram muc duac cat nho vdi kich thudc 100 - 200 (im sau do dal len dia ihach co chua san moi trudng PDA bo sung guaiacol de lam chat chi thi va cac dTa thach nay duoc nuoi d 30"C trong ihdi gian til 3-5 ngay
Sdng Igc cdc chiing n CO khd ndng sinh laccase Cac chimg nam sau khi duac tach sach duoc nuoi lac tren moi trudng PG vdi t6c dp lac 200 vong/phiil d SCC. Hoat tinh laccase ciia cac chung nam dugc xac dinh sau 5 ngay nuoi cay (Nguyen Nguyen Quang, Dang Thi Ckm Ha. 2010).
Phuffiigphdp xdc dinh hogt linh laccase Hoal iinh laccase dugc xac dinh dua tren su oxy hoa ABTS {2.T-azmo-his (3- ethylbenzthiazoline-6-sulfonic acid) cua laccase lao thanh hap chat hap thu d budc song 420 nm (X.I20 = 36,000 M"',cm'') d dieu kien thi nghiem (1 ggerte/(;/., 1996) Hon hop phan ung (I ml) g6m 600 |il dem natri acetate pH 3 (20 mM), 200 ul ABTS (2,5 mM) va 200 ^] dich enzyme. Mot don Vl hoat do laccase la lugng enzyme c^n thiSt d l lao ihanh 1 jaM san pham tir ABTS Irong thdi gian I phlil. d dicu kien thi nghiem.
Khdo sdt cdc Miu kien mdi trucfng dnh hudng den khd ndng sinh trudng vd sinh laccase cda ching FNBLal
Moi Irudng co kha nang sinh laccase cao nhat dugc chgn lpc theo cac yeu to sau:
Anh hucmg cda pH moi truang nudi cdy Dai pH 4-8 da dugc sir dung de danh gia kha nang sinh Irudng va sinh laccase tren moi trudng PG cua chiing nam FNBLal, Danh gia su thay doi hoal tinh enzyme theo thdi gian tir sau mpt ngay nuoi cky cho den khi boat tinh enzyme giam da dugc tien hanh,
Anh hir&ng ciia nguon glucose, cdm vd nguon nitrogen
Ba nguon cacbon va nitrogen da dugc chpn de khao sat la: glucose co nong dp la 6, 8, 10, 12 va 14 g/1; cam la 1, 3, 5, 7 va 9 g/1 va nguon nitrogen la NaNOj, KNO3, NH4CI, NH4NO3, pepton. tripton va casein d moi ngudn nitrogen thi nghiem dong then duoc Uen hanh vdi ndng dp lan lucrt la I, 2, 3, 4 va 5
Anh hir&ng ciia cdc chdt cdm img
Cac hop chat 4-hydroxybenzoic acid, feruhc acid, vanilin, veratryl alcohol, guaiacol, CUSO4 da dupc sir dung nhu la cac chat cam img de danh gia su anh hudng den sinh long hop laccase cua FNBLal,
Phdn logi chiing nam FNBLal
Chung nam FNBLal duac phan loai theo hinh thai va xac dinh trinh tu gen ma hda 18S rRNA va so sanh vdl trinh tu tuong irng cua cac chung nam da dugc cong b6 tren GenBank (NCBI). DNA l6ng s6 dugc tach chiel theo phuang phap da dugc mo ta trudc day (Dao Thi Nggc Anh, Dang Thi Cam Hii 2009), Cap m6i EF4f: 5' GGAAGGG(G/A)TGTATTTATTAG-3' v Fung5R: 5-GTAAAGTCCTGGTTCCC-3' dugc siJ dung de khuech dai doan gen ma hoa 18S rRNA tu DNA tong so cua chiing nam nghien ciru, San pham DNA sau khi dugc khuech dai bang ky thuat PCR dugc lam sach bang kit QIAGen San pham PCR sau khi tmh sach dugc xac dmh trinh tu true liep bang may doc trinh ty tu dong Automated Genetic Analyzer ciia hang Applied Biosystems (My). Su
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Tgp chi Cdng nghi Sinh hgc 11(2): 265-274, 2013
dung cac phSn m6m Clustal X, Mega5 da xay dimg cay phat sinh chung loai cua chiing nkm nghien cuu.
'• long trong bioreactor Phutmg phdp len me
Mdi trucmg len men
Sau khi lira chgn dugc cac dieu kien nuoi c i y ihich hgp, chimg nSm FNBLal da dugc Ien men frong bioreactor vdi the tich 15 lit. Moi trudng len men gom 1,5 mM CUSO4; 12 g/1 glucose; 3 g/1 NaNOi; 1 g/l casein; 5 g/l cam; dich chifit khoai tay 2 0 0 g / l v a p H 6 , 5 .
Diiu ki4n len men
Cac chting vi sinh vat dupc nhan gi6ng trong cac binh tam giac 250 ml dk thu dii lugng giOng cung cap cho len men trong bioreactor, Len men duac lien hanh Irong bioreactor vdi th^ tich 15 lit moi Irudng giong nhu mdi trudng da nhan giong, Lugng giong bo sung tu 3-5% thS lich djch len men Len men d dieu kien nhiel do phong, toe dp khuay 200 vong/phiit, khi d u p c sue lien luc bang may nen khi vdi luu lugng khi 80 lit/phiu. Trudc
khi vao thiing len men, khong khi d u a c di qua mgt mang loc khi vd trung dugc gan tren ong dan khi. Dich len men dugc lay mau dinh ky sau 2-4 h va xac dinh boat tinh laccase theo thdi gian lay mau tren,
KET QUA VA THAO LUAN
Sang I9C chung n a m cd kha nSng sinh tong hgp laccase
Tir 4 mau ram ihu thap lu Ninh Binh da phan lap dugc 7 chiing nam co ten la FNBLal, FNBLa2, FNBLa3, FNBLa4, FNBLaS, FNBLa6, FNBLa7, Trong 7 chiing nay chi co ! chiing nam FNBLai co kha nang tao mau nau do tren moi trudng PDA c6 chua guaiacol sau 6 ngay nuoi cay (Hinh I), Ket qua nay chirng to chiing co kha nang sinh long hgp cac enzyme ngoai bao thupc nhom peroxidase (MnP, LiP) hoac oxidoreductase (laccase). Chiing FNBLal da dugc chgn cho cac nghien cim Hep theo nham thu laccase co boat tinh cao nhal.
Hinh 1 Smh trudng cua chiing nam FNBLal tren moi Infong PDA chifa guaiacol la chat chi thi
Phdn loai chting n a m F N B L a l
Chting FNBLalco khuan lac mgc lan rpng.
khuan ly khi sinh dang sgi mau trSng, bao lu mau den. Dudi kinh hien vi quang hpc, sgi nam c6 dang spi phan nhanh, bao tu dinh. Dua vao khoa phan loai cua Samson RA (1984), chiing FNBLal co the dugc xep viio nganh nam nang Ascomycetes, chi Myroihecium.
Dfi \km rd cac k^t qua phan loai truyen thong, mpt phan trinh tu gen ma hda 18S rRNA cua chung FNBLal da dugc xac dinh vdi kich thudc 523 nucleotit va cd muc dp tuong dong 9 9 % vdi cac chiing Myroihecium sp. 7C50, Myroihecium verrucaria strain Z16. Trinh ty gen ma hoa 18S
rRNA ciia chiing FNBLal da dugc dSng ky tren GenBank vdi ma so JX514175, M61 quan he phat sinh chiing loai ciia chiing FNBLal vdi mgt sd chung vi nam da dugc xay dyng va trinh bay d hinh 2.
Tir hinh 2 ta thay chiing FNBLal rat gan gui vdl loai nam Myroihecium verrucaria. Trong do, dai dien la chiing nam Myroihecium verrucaria NF- 05 va Myroihecium verrucaria 24G-4. Chung NF- 05 dugc phan lap tir Khu Bao ton thien nhien Liangshui ciia Trung Quoc co boat tinh laccase cao nhal la 40,2 U/ml sau 13 ngay len men (Zhao et al..2012). Chiing 24G-4 sinh long hpp laccase tho CO hoat tinh laccase cao nhat dat 10 U/l d pH 10 (Sulistyaningdyah el ai. 2003) Theo mgt so nghien
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ciru, chi Myroihecium gdm nhieu loai cd kha nang sinh long hgp enzyme tham gia phan huy cellulose (Moreira et ai, 2005; Okunowo et ai. 2010). Cho den nay cung da co nhieu cong bo ve kha nang sinh tong hgp laccase cua chi nay (Wang et ai.2009).
Loai Myroihecium melanosporum cung da dugc Wang va dong tac gia (2009) chimg minh c6 kha
Tran Thi Thu Hien etai nang sinh tdng hgp laccase.. Cac chiing nam con lai chua dugc cong bo ve kha nang sinh long hgp laccase. N h u vay, dua vao mgt sd dac diem hinh thai va mgt phan trinh lu gen m3 hoa 18S rRNA, chimg F N B L a l cd the dugc xep vao cht Myroihecium va dugc dat ten la Myroihecium sp, F N B L a l .
—Didymostilbe echinofibrosa s
—Elaphocordycer>s ophi
AR2824 strain NBRC -Paecilomyces canadensis
•Ph/locorclyceps ninctiukispora Ascomycete %Q HV_19C strain LMSA1 08 027 /iscomycofa sp SAUFC1-5
—Tolypocladium mflatum
—Cordyceps bifusispora strain ARS 5690
—Elapnocordyceps subsessilis strain OSC 71235
— MyrottKcium sp. FNBLal
_ fJyiothoc.am verrucaria
—Hyrottiecium sp 7C50
—Hyrothecium verrucaria strain Z16
—Necliia carta strain UMB 39 01
—Uncultured fungus clone ST2F5
—Uncultured Pezizomycotina clone ST3C2
—Cosmospora coccinea strain AR2741 Hinh 2, Cay phal sinh chung loai ciJa chung n
Anh hudng cua mot s6 di£u kien nuoi c5y d i n kha nang sinh tong h o p laccase cua chung Myroihecium sp. FNBLal
Anh hudng ctia pH mdi trudng
pH ban dau la mgl trong cac yeu t6 anh hudng den su sinh trudng cung nhu kha nang sinh enzyme cua vi sinh vat. Sau 7 ngay nuoi ciy. chiing Myroihecium &p FNBLalsmh trudng t 6 t d p H 6 - 7 va laccase c6 boat tinh cao nhSt la 1.391 U/t d pH 6.5. C) pH 4 boat tinh enzyme tk thip dat 34 U/l (Hinh 3). Nhu vay, pH 6,5 phii hpp cho su sinh trudng Vii sinh laccase cao nhit d chiing Myroihecium sp FNBLal
Theo nhieu tai lieu da cong b6 trong va ngoai nudc thi nam thudng sinh trudng t6t d pH hoi axit.
Cac chiing nam sai Aspergillus ochraceus NCIM- 1146 (Parshetti el ai, 2007), Pleurotiis oslreatus Dl (Pozdnyakova el ai, 2010), Pemcillium
ochrochloron MTCC 517 (Shedbalkav el ai, 2008) sinh irudng toi uu theo thir ty tai 5,4; 6 va 6.5, Chiing Aspergillus sp FNAl sinh trudng va sinh enzyme tdt d pH 5 (Dao Thi Nggc Anh, Dang Thj Cam Ha, 2009). Tuy nhien. mot so ket qua nghien cuu khac cho thay laccase v^n duoc sinh ra 6 pH ban dau kiem nhu d chiing Montospora sp. vdi pH ban diu la 8,5 (Wang et ai.lOOC) Dac linh nay phy thugc rat nhieu vao nguon goc cua chung.
Anh huffng cua nong do glucose din kha nang sinh laccase ciia chiing Myrothecium sp. FNBLal
Nguon glucose trong moi trudng PG co nongd?
la 10 g/i, trong nghien cim nay nong dp glucose lan lucrt la 6, 8, 10, 12 va 14 g/1 da dugc khao sat miic dp anh hudng len kha nang sinh long hgp laccase cita chimg FNBLal. Sau 5 ngay nuoi cay. hoat tinh enzyme tao ra bdi chung Myrothecium sp FNBLal da dugc xac djnh (Hinh 4).
Tgp chi Cdng nghi Sinh hgc 11(2) 265-274,2013
Tir ket qua trinh bay d hinh 4 cho thay chiing enzyme cua chung FNBLal dat cao nhit la 2.633 U/l Myrothecium sp. FNBLal co kha nang sinh trudng tren moi trugng co n6ng dp glucose la 12 g/1. Do tot tren ca 5 nong dp glucose neu tren. Tuy nhien, vay, glucose vdF nfing do 12 g/I dugc lya chgn cho anh hudng cua nong do glucose len kha nang sinh cac nghien cud tiap theo
t6ng hgp laccase khong gi6ng nhau. Hoat linh
Hinh 3. Anh hudng ciia pH len kha nang sinh laccase d chung Myrothec
B24h a4Sh a72h B9Gh H120h
Nong dp glucose (g/l)
Hinh 4. Anh hirdng cua nong do glucose den kha nang sinh laccase a chung Myrothecium sp FNBLal
Anh hudng cdc nguon nitrogen den khd ndng sinh laccase cda chung Myrothecium sp. FNBLal
De nghien ciiu anh hudng ciia nguon nitrogen len kha nang sinh laccase bdi chung Myrothecium sp.
FNBLal cac nguon nitrogen huu co va vo ca khdc nhau da dupc bo sung vao moi Irudng PG.
Hoat tinh enzyme trong dich nuoi cay cua chiing FNBLal dugc xac dmh sau 24, 48. 72, 84 va 96 h (Hinh 5).
Tir hinh 5 ta thiy, chiing nam sgi Myrothecium sp, FNBLal sinh laccase cao nhal sau 96 h nuoi cay vdl ngu6n nitrogen la NaNO., (3 g/l) va boat tinh
enzyme cao nhat la 3.650 U/l tiep do la KNO3 vdi hoat tinh enzyme la 3.417 U/l. Dot vdi nguon nitrogen huu co, hoat tinh laccase cao nhat tren moi trudng CO chira casein (1 g/1) dat 2 545 U/l. Trong khi boat tinh laccase ciia chiing Myrothecium sp.
FNBLal tren nguon nitrogen la pepton (3 g/l) va tripton (4 g/l) chi dal lin lugt la :,093 U/l va 1 048 U/l (Hinh 5) Nhu vay NaNOi la nguon nitrogen thich hop cho su sinh long hgp laccase bdi chiing Myrothecium sp, FNBLal, Dieu nay kha khac biel so vdi cac nghien ciru khac tren the gidi Cac kel qua nghien ciru gan day cong bo su tao thanh laccase tren nguon nitrogen hiru co cao ban so vdi nitrogen vd CO Nhieu chung nam sinh laccase tren moi trudng chua nguon nitrogen hiiu co nhu Pleurolus oslreatus
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Tran Thi Thu Hien et al.
32 (Hou et ai. 2004), Trameles pubescens MB89 va Penicillium simplicissimum H5 (Zeng et ai, 2006) thich hgp vdi pepton con Coriolus versicolor NCIM 996 thich hop vdi cao nam men v.v. nhung ciing cd mgt so chiing toi uu vdi nguon nitrogen vd c a nhu (NH4)2S04 ciia loai Debaryomyces polymophus (Yang era/,, 2005).
Do hoat tinh laccase ciia chung Myrothecium sp.
FNBLal cao vdi ngudn nitrogen vo c a la NaNOs va KNO3 va hoat tinh cao nhat vdi nguon nitrogen h5u ca la casein va pepton nen moi trudng chiia h6n hpp ciia cac ngudn nitrogen tren da dugc nghien curu. Sau 5 ngay nudi cay, hoat tinh laccase ciia Myrothecium sp. FNBLal dugc trinh bay d hinh 6.
Hinh 5. Anh hudng ciia nguon nitrogen len su smh long hap laccase d chung Myrottiecium sp FNBLal
Nguon nitrogen
Hinh 6, Anh hudng cua nguon nitrogen len kha nang sinh long ho^p laccase d chung Myrothecium sp FNBLal
Tren hinh 6 la thiy boat tinh laccase cao nhll 2 lin so vdi khi khdng kk hgp hai ngu6n nitrogen (7.676 U/l) khi bo sung vao moi trudng PG ngu6n nay. Do vay hfin hgp casein va NaNO] da dugc chpn de bo sung vao mdi trudng PG de cho cac nghien niyogen k^l hgp casein 1 g/l va NaNOj 3 g/l cao g
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Tgp chi Cdng nghi Sinh hgc l l i 2 i 265-2"4. 2013
cuu tbu laccase tiep theo.
Anh hudng cua nong do cdm ggo din khd ndng smh laccase cua Myrothecium sp. FNBLal
Cam gao cd lugng dinh dudng rat cao MJI ham lugng chat beo chira bao hda. vitamin nhdm E, nhdm B. phylate, kera. can xi. kali deu r i t cao. Theo cac nghien ciru, 65° o chat dinh dudng ciia gao tap trung
d cam, tbanh thin cua cam co nhieu loai vitamin \3 chat beo toL Vi va> cam gao dugc dua vao nghien ciru trong cdng trinh nay
Ket q u i thu dugc cho thiy vdi nong do cam £20 la 5 g l thi hoai tinh laccase dat esc nhit idi 12 ^69 U 1 tang 1,7 lin so vdi mau doi chiing (Hinh 7). Do
\ay. 5 g l cam da dugc bd sung vao moi midng PG trong nhiing nghien cuu tiep theo.
:-:oc:
c^ 12000 b
^ 10000 0 8000
;; 6000 Z -iOCC
^ 2000
0 - ' — ' -• -
3 5 7 9 f & ching N6ng(}ocamgao(g/I)
Hinh 7. Anh hudng cua ndng (36 cam gao den kha nar>g sinh laccase d chung Myrothecium sp FNBLal
y< - i . ; ;
Anh hudng cua cdc chat cdm irng den khd ndng smh laccase cda Myrothecium sp. FNBLal
Cac nghien cim tang kha nang long hgp laccase cua \ SN' bing cac chat cam img da %a dang ird thanh mdt trong cac bien phap mang lai hieu qua kha l>' thii. Cac ngudn na\ bao gdm cac ammo acid, cac hgp chat thanu cac chat chiel tir thuc \ a t \ a Cu"*
(Cavallazzi et ai. 20051 Trong nghien ciiu nay. cac chat cam irng khac nhau dang dugc su dung d nhieu nghien cim gan day bdi cac nha khoa hpc tren the gioi da dugc chgn de bd sung vao mdi trudng. Anh huong ciia chiing len kha nang sinh tons hgp laccase bdi Myroihecium sp FNBLal da dugc khao sat %a kel qua duoc trinh bay d hinh S
Tir hinh S ta thiy hoat tinh laccase ciia chung Myrothecium sp FNBLal da tang rat cao dat 29 5~5 U/l trong moi trudng chua chat cam img CuSOj. tiep do la ferrulic acid 21 681 U l \ a \eran>- alcohol la P 259 U/l sau 5 ngay nuoi ca\ Cac chat cam ung con Ifi cd boat tinh xap xi hoac iSng khdng dang ke
so vdl mau ddi chimg.
Rubia \ a dong tac 21a 12( i' <Z 1 da nhan iha\ hieu suat sinh long hgp laccase cao nhai 1 t^^'^ L" 1 doi \ d i loai nam dam Phanerochaete fla\ io-alba khi vanillin dugc sir dung lam chat cam ung. Tuy nhien, khong phai cac chai cam img deu lam tang hoat unh cua laccase (Cavallazd et at.. 2005J. \ I du nhu chung Lentinula endodes L F \ ' 5 2 hoan toan bi uc che smh tdng hap laccase khi bd sung gallic acid ha\
catechol. Chung nay sinh laccase cao nhat tren ammonium tanrate \ d i hoat tinh 251 L" ml sau 30 ngay (Ca\ allazzi et ai. 20051 Cung a chime nay ihi p-hydroxyben20ic acid \ a vanillm lam giam boat tinh laccase so vdi doi chung Trudng hop na> ciing gidng nhu o chimg Myroihecium sp. FNBLal co hoat tinh thap hon so \ d i doi chimg khi bo sung vanillin. Nhu \ a \ . chat cam ung la mot trong nhirng yeu to lam tang hoat tinh laccase nhung moi chung lai chi phii hop \ di mot sd chat va nong do nhat dmh.
Ddi vdl chung .'ilyrothecium sp. FN'BLal, chli cam ling lot nhit la CuSO*.
Tran Thi Thu Hien eta.
Anh huffng cua ndng do CuSO^ den khd ndng smh laccase Myrothecium sp. FNBLal
Do bo sung CuSO^ co vai tro cam ung sinh laccase cao nhat d chiing Myrothecium sp. FNBLal nen nong dp CUSO4 khac nhau da dupc chpn de nghien cim tim ra nong dp CUSO4 toi uu (Hinh 9).
Tir hinh 9 la thay khi bo sung CUSO4 d nong do 0,06-0,17 mM. boat linh laccase deu tang cao so vdi
dot chiing. Hoat tinh laccase cao nhal la 30.016 U/m d ndng do CUSO4 1,5 mM, Theo nghien cuu cuj Xiao va dong tac gia (2007). chiing nan Trichoderma harzianum WLI co boat linh laccasi dal 4,36 U/ml khi bo sung I mM CUSO4 vao moi trudng nuoi cay, Trong khi chiing FBHl 1 lai co kha nang smh enzyme rat tot tren chal cam img 4- hydroxybenzoic acid (6,940 U/l) (Nguyen Nguyen Quang. Dang Thi d m Ha, 2010).
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Chal cam iJng
Hinh 8. Ho^t tinh laccase cua chiing Myrothecium sp FNBLal tren cac ch§t cam upng khac nhau
0 06 0,08 0,1 0 12 0.15 0.17 Nong dp CuS04 (nrW)
Hinh 9. Anh hudng ciia ndng 6Q CUSO4 d^n kha nang smh laccase d chung Myrothecium sp. FNBLal 272
Tgp chi Cdng nghi Sinh hgc 1I{2)- 265-274, 2013
Len men chung Myrothecium sp. FNBLal v&i quy md 15 lit
Vdi thanh phan moi trudng da chpn lpc, nghien cuu len men d quy md 15 lit da dugc thye hien nham thu enzyme tho cho nhiSu muc dich nghien ciru khac. Enzyme tho dugc sir dung dh nghien ciru kha nang loai mau thu6c nhugm. PAH, phenol -V.v Cac enzyme tho thu dugc duac tmh
che de nghien ciiu cac dac tinh. Ngoai ra, enzyme thu dugc se cung cap cho cac nghien cuu bao quan, co dinh nham xu ly chat 6 nhilm d cac qui mo khac nhau. Qua trinh len men chiing Myrothecium sp. FNBLal vdi the lich 15 lit co toe dp sue khi 0,8 m^/h va khuay 150 vong/phiit, lay mau 2 h mgt lan da duac thye hien. Kel qua qua trinh len men dugc trinh bay d hinh 10.
i J.5000 i 3 1 | o o o o
i 5000 X
0 1
1
i
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30 33 36 39 42 45 48 51 54 57 60 53 66 70
Th6igian(h) [ Hinh 10. Hoat tinh laccase cua chung Myroihecium sp FNBLal trong qua trinh len men
Tir hinh 10 ta thay hoat tinh laccase trong 24 h dau tien lang rat cham. Tai thdi diem 24 h. hoal tinh chi dat 50 U/l, nhung sau 3 h hoat tinh da tang len den 400 U/l, Sau 30 h, boat tinh lang rat tuyen tinh va dat cue dai la 10 410 U/l tai 57 h. Hoat tinh nay dat dugc chi sau hon 2 ngay len men, nhanh hon nhieu so vdi khi nghien ciru d binh tam giac. Nguyen nhan co the do dieu kien sue khi va khuay manh nen lugng oxy hoa lan cao dan den nam phal Irien manh.
Tir 60 d^n 70 h, boat tinh laccase duy tri on dinh d mirc 9.509 U/l nen thi nghiem len men da dugc dirng tai thdi diem nay. Tuy nhien, chiing Myrothecium sp.
FNBLal sinh long hop laccase thap hon gan 3 lan so vdi klii khao sat cac dieu kien nuoi cay Irong binh tam giac, Nguyen nhan co the do len men trong binh len men ldn chua kiem soat duoc tat ca cac yeu to nhu: nhi?l dp (len men d nhi^t do phong), sue khi, khuay dao bang canh khuay (cac binh thi nghiem su dyng may lie) v.v Khong giong nhu chiing Myroihecium sp. FNBLal trong nghien ciru nay, chiing FBH11 phan lap tir dat di?t co/ dioxin l^i san bay Da Ning co boat tinh laccase cao nhal trong binh tam giac la 1,469 U/l nhung khi len men d quy mo 15 lit thi hoat tinh lai tSng len rit nhieu 6 073 U/l (Nguyin Nguyen Quang. Dang Thi Cam Ha, 2010) Djch enzyme thu va ly tam sau khi len men chung Myrothecium sp. FNBLal duoc bao quan d 4''C cho c^c nghi&n cuu khac.
KET LUAN
Tir 4 mau rom muc thugc tinh Ninh Binh, chiing nam FNBLal dugc phan lap co kha nang sinh long hgp laccase vdi boat tinh ban dau la 200 U/l Bang phuong phap phan loai truyen thong ket hgp vdi xac dinh mpt phan trinh ty gen ma hoa 18S rRNA. chiing nam soi FNBLal dugc xep vao chi Myrothecium vdi ten la Myroihecium sp FNBLal Chimg nay co kha nang sinh tong hgp laccase cao d pH 6,5 Irong moi trudng CO 1,5 mM CuSOj, 12 g/1 glucose, 3 g/1 NaNOj; I g I casein; 5 g/1 cam, dich chiet khoai lay 200 g/1 Khao sat d dieu kien nuoi cay trong binh tam giac, chiing nam sgi FNBLal c6 hoat tinh cao nhat la 30,016 U/l sau 96 h, Hoat tinh laccase cao nhit khi len men chim Irong bioreactor co dung tich 15 lit la 10 410 U/l sau 57 h
TAI LIEU THAM KHAO
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STUDY ON ISOLATION AND CULTURE CONDITIONS ON LACCASE PRODUCTION OF FUNGI ISOLATED FROM ROTTEN RICE STRAW IN NINHBINH PROVINCE
Tran Thi Thu Hien. Hoang Thi Nhung, Nguyen Hai Van, Nguyen Thi Lan Anh, Dinh Thi Thu Hang, Dang Thi Cam Ha*
Institute of Biolechnology, Vietnam Academy of Science and Technology
SUMMARY
Laccases are copper-containing oxidase enzymes, widely distributed in microorganisms such as basidomycetes, filamentous fungi, aclinomycetes and bacteria. Basidomycetes and filamentous fungi have been more studied on capacity of laccase Synthesis than the others The study was conducted m order to know capacity of laccase production from Ascomycetes isolated from different enviroments such as rotlen nee straw.
From four samples of rotten rice straw collected in Ninh Binh, FNBLal suain was isolaied on PDA medium with initial activity of 200 U/i, Based on traditional method and sequence of 18S the partial rRNA coding gene, FNBLal sUain belonged to Myroihecium genus and was named Myrothecium sp, FNBLal. FNBLal was capable of producing laccase with high activity at pH 6,5; 0,1 mM CUSO4 as an inducer, 12 g/1 glucose as a carbon source and a mixture of 1 g/1 casein and 3 g/1 NaNOj as nitrogen sources. On suitable medium, FNBLal strain produced laccase with the highest activity of 30.016 U/l and 10,410 U/l in 250 ml-flasks after 96 h and 15 hter bioreactor after 57 h, respectively.
Keywords: Fermentation, inducer, laccase, Myrothecium, rice straw
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