Tgp chi Cong ngh? Sinh hoc 10(2): 335-343, 2012
N G H I E N C U t J S A N X U A T K E R A T I N A S E K I E M TI) C H I I N G VI K H U A N DS23 P H A N L A P 6 H A I P H O N G
Dang Thj Thiiy DiroTig', Nguyen Vftn Hieu'. P h a m T h a n h Huyen', Khuat HSku T h a n h S Phi Quyet Tien', Le Gia Hy'*
^Vien Cong nghe smh hpc. Vi^ii khoa hpc vd Cong nghi' Vi^l Nam
^Vien Cong nghe sinh hpc vd Cong ngli4 tlnrc pham. Dgi hpc Bach khoa Ha A'p/
TOM TAT
Tir cac nuu dat va nude Igi Do Son • Hai Phong, cluing toi d3 phan l^ip va luyen chgn dugc chiing co ki hieu DS23 c6 kha ning sinh keralinase kiem cao, on djnh, didi gian sinh trudng nhanh, cd the phdl trien tot Uen nhieu mdi truimg. Kci qua phan logi bing bg kit API 50CHB cho thay chimg DS23 thugc chi Bacillus, ket hgp vdi phan tich trinh tg gen l6SrDNA thu dugc chiing DS23 thugc loSi Bacillus liclnmfoi mis vdi dg tuong ddng la 99%, trinh tg da dang ki trong GenBank dudi ni3 so GU004539 (Bacillus hcheniformis strain DS23). Chiing B licheniformis DS2.' sinh long hgp keralinase kiem lot nliat trong dieu kien: pH 8,0; nhiet dp 37''C; the tich moi trucmg 20 ml tren th^ tich binh 251) ml. thanh phan moi trucmg (g/l): glucose 10,0; peptone 10,0; K2HPO4 1,0; MgSOj 7H:0 0.2. Na.-CO; 3.0; ndng dg chat cam img keratin 0,3% v^ ty I? liep giong 2%, Hogt tinh keratinase cue dgi sau 72 h len men, dgt 63,8 U/iiil, Djch len men chiing B licheniformis DS23 ihiiy phan hoan t e n Idng ga sau 72-96 h ii.
Ttr khoa keratinase kiem . sinh long bap. Bacillus licheniformis. thuy phdn long ga, ISSrDNA.
MODAU
Keratmase [EC 3 4.21/24/99 11] thugc nhdm protease, mgt sd nghien cuu trudc day cho rSng keratinase thugc ho serin protease do chiing cd dp hiong ddng Ien tdi 9 7 % vdi protease kiem va cd the bi lie che bdi cac chat tic che Wong tu nhu hg enzyme nay (Lin etal., 1995; Bressolier el al, 1999;
Gupta, Ramnani, 2006).
Keratinase dugc sinh tdng hpp manh trong moi trudng chiia keratin, loai protem khdng tan nay thudng tim thSy trong long, tdc, simg, vay, mong vudt (keratm mach dai) va lop te bao bieu bi ngoai cimg (keratin mach ngin) ciia dpng vat. Keratin cimg vdi collagen va elastin dugc phan loai vao nhdm protein cau tnic sgi, ma 4^c tnmg ciia chiing la chiia rit nhi^u lien kk disulfiia (S-S), do do chiing rk b^n vdi cac tac nhan Ii, hda va sinh hgc (Korkmaz et al, 2004), Keratinase cd kha nang phan cdt lien k6t disulfiia cua phan tu keratin, do do thuy phan keratin thanh cdc protein hoa tan, peptide mach ngan va axit amin. Ngoai keratin, keratinase co kha nang xiic tac thuy phfin khdng d|ic hi§u tren collagen, elastin va mgt sd protein khdng mang cSu tnic spi nhu casein, bovine serum albumin, gelatine... (Lm et ai, 1995;
Letoumeau el fl/.,I998; BressoUier et a/., 1999). Do vay, keratinase dugc nghien ciiu ling dung trong cac qua Irinh thiiy phan Idng d l tao thinh cac hgp chat
cd the bd sung vao thiic an gia siic, phan bdn hihi co, chit tky rua, w...(Gupta, Ramnani, 2006;
Deivasigamani, Alagajipan, 2008). Mpt sd tac gia da phan lap cdc chiing nam, xa khuan va vi khuan cd kha nang sinh tdng hgp keratinase nhu: Alternaria tenuissima, Aspergillus nidulans (Saber et al, 2010), Chryseobaderium sp. (Brandelli, Riffel, 2005);
Streptomyces spp (Letoumeau et ai. 1998; Tapia, Simoes, 2008; Kansoh et ai, 2009), Bacillus sp.
(Giongo d al, 2007; Xu d ai, 2009), Bacillus licheniformis (Kokrmaz et at., 2004; Paul et ai, 2007) v a S subtillis sp (Matikeviciene eM/., 2009).
Trong bai bao nay, chiing tdi trinh bay ket qua nghien ciiu tuyin chgn, dinh ten, cac dieu kien len men sinh tong hgp keratinase kiem cao va kha nang phan giai long ga ciia chimg vi khuan phan lap d Viet Nam.
VAT LIEU VA PHUONG PHAP Vat li^u
Cac m l u dSt va nude dugc thu thap tai Dd Son - Hai Phdng dugc sii dgng di phan I^p cac chung vi sinh vat co kha nang phan buy keratin cao.
PhSn loai vi khuSn hSng kit sinh hda API 50 CHB
Sau khi dugc xac dinh la vi khuan Gram duong, 335
l^ng Thj Thuy Duong etai 190 biio lir. eluing vi khuiin dugc Iga chpn duge phiin
lojji b^ng bp kit API 511C1IB (HmMericux. Phap) LiW 1.0 ml djch huyen phu vi khuan bii sung \;ii>
10 ml mdi truimg klioiiiii:. sau dd djch lir i^ng nidi truimg khoAng cd chira \i kliiuin bd dugc sung viui cae gieng iht'r ciia bg kit. Dimg d;iii paralln phu kin len he null cae iiieng l).i\ bg kit \ii nudi it nhigt dp 37 C. Dgc ket qua siui 24 \a 4S h. So sanh kel qua thu dupe \di bang pluin logi API cliiiiui dc d|nli liin, (iiai (rinh l y g e n mil hda IdS rHN \
DN.A idiiLi so cua vi khuan duge tiich ihco phumig pli.ip liuoi md la btn Sainbrook ct al, 1989, Gen I6S iDN \ LU.I \ I khiiiin IIIHK kliiicdi d^i hflng PCR sti dgiii: ciip moi (.1 I 5'- 1 \ \ t Ai A l ( . ( A U r K GA.U d - l ' vA (iRl 5'- GGlG"l(jAl.G(jGC<i(i!(iK>l A-V, San pham cua phan img dugc kiem tra iii-ii gel agarose T o . tmh sach bang bg kit PureLmk' '-DNA Purification (Invitrogen) \.i 1:1.11 trinh tg Iren inily iniiy dgc trinh tg tg dgng \HI PRISM'3I0()-Avanl Genetic Analyzer ^Applied IJiosv stems, Foster City. CA.
USA) tai Vign Cdng nghe smh hgc. ViC'ii Khoa hpc
\a Cdna nghe Vicl ^.^lll Trinh tg nucleotide dugc
\ u h' bang phan mem DNAstar de xac dinh h? sd tuong ddng \ a dung ca\ phan loai,
Mdi trirfrng nuoi c:i>
M61 trudng tuyen chpn so bp cac chiing sinh keratinase MP (g I) Cao thit 5; peptone 10; NaCI 5.
H j O b d s u n g d u 1 hi. pH 8.6 Mdi trucmg AMI (g/l):
Glucose 10. Cao nam men 5: peptone 5, K^HPOj I, MgS04,7H20 0.2 ; Na:C (). 1.5; \ a nude bd sung du I lit. Mdi trudng ihay Ihi-(g/l). Ri dudng 10; Bgl dgu tucmg 5; K:HP0j I; MgSOj 7H:(K).2 . Na.-CO; 1.5;
va nude bd sung du I In Xirly CO* chat
Long ga sau khi thu nhgn \c dugc rira 3 l^n bang nude may de loai bo tgp chat, sau do dugc rua vdi nude cat, say khd a 60''C trong dioi gian 4K h \ a lam ngupi ve nhigt dg phdng. Long ga khd dupe bao quan a S^C den khi su dgng
Keratm de hda tan dugc dieu ch^ tir long ga theo phuong phap ciia Wawrzkiewicz el al, 1987, Bd sung vao 10 g Idng ga sach 500 ml dimethylsulphoxide (DMSO) va dun each thuy hdn hgp d lOO^C trong 2 h. Sau do bd sung vao h6n hgp 1000 ml acetone -70°C, giQ- d -70°C trong 2 h. ly tam 10000 vdn^phut trong 10 phiit. PhSn kk tua dugc nia lai 2 lan bang nude cat va s l y khd trong may hut chan khdng. Keratin thu dupe b3ng phuong 336
phiip iren dugc hd sung vao mdi trudng nhflm tuygn chpn Ciic chimg cd hogt linh keratinase cao va sij dyng trong cdc pli;ui img thir hogt tinh enzyme.
\iK' djnh hoal linh keratinase
Trong xdc djnh hogt linh keratinase ca ch3t azocasein cd the dugc sir dgng thay cho co chat a/okeratin. De xac djnh keralinase, chung tdi sir dgng ca clnit a7ncascin dga theo phuong phap ciia Jahan et al. 2010. Tien h^nh bd sung 0.2 ml dung d k h azocasein 1% trong dgm NaOH-Glycine 0.05M pll 10 vao 0.4 ml d|ch enzyme va ii hon hgp a "iTC iinii): 30 phul Phan img dupe dimg bang each b6 sung 1.4 ml dung d|ch tricloacelic acid (TCA) 10%, il 4"C trong i 5 phiil. sau dd li tam hon hgp d tdc dg lOllOO vong/phin trong 10 phtit. Do dp hap th\i ciia dung djch siiu li liim tt buoc .sdng 440 nm. Mau doi chimg dugc lien hanh tuong tg nhu mau thi nghiem nhimg b<) sung 1.4 ml dung djch TCA UCoxaohon hgp CO chal - enzyme tai thdi diem bit dau phan img.
Mill dcni \ 1 boat dp cua keratinase dupe djnh nghia la lut,mg enzyme cd the ehuyen hda co chat lam tSng 0,01 don vj dg hap thy anh sang o budc sdng 440 nm cua mau ddi chung so \oi mau thi nghiem sau 30 phiil phan img trong dieu Id^n thi nghiem.
\ : i c djnh a n h huung cua dieu ki^n audi cay den kha ndng sinh tdng h g p keratinase
Danh gia anh huong cua thanh phan mdi trudng
\ a dieu ki?n nudi cay den kha nSng sinh tnrong \a tdng hgp keratinase cua ehimg B. licheniformis DS23 bang each tha\ doi lan lugt timg yeu td trong khi co dinh Ciic yeu td edn lai cua mdi irudngco so .AMI.
•\nh huong ngudn nitrogen, carbon va chat cam img se dugc xac djnh bang each thay the cac ngudn carbon va nitrogen ctia mdi truong .AM I bing mgt so ngudn nitrogen va carbon khac vcri lugng tuong duong vdi cac dieu kien nudi cay khong thay ddi: toe dg lie 200 vdng/phiit, nhi?t dg nudi 37°C va thdi gian thu hdi la 72 h,
Anh huong cua dieu kien nudi cay se dugc nghien cim bang each thay ddi dieu kien muon khao sat trong khi cd djnh cac yeu td khac nhu: nhiet do nudi ciy (25"C; 2S-X\ 30°C, 37°C; 40°C; 45°C;
50 C va 55'C). pH mdi trudng ban diu (6,0; 7,0, 8,0; 9,0; 10.0; 11.0 va 12.0). ih^ tich djch len men trong binh tam gidc 250 ml (20, 35, 50 va 65 ml); ty 1^ tiep gidng (0,5, 1, 2. 4, 8 va 10%). Kha nang sinh tdng hgp keratinase theo thdi gian dugc xat; djnh b i n g each liy mau 9 h/1 lin. Tat ca dugc t i ^ h a n h nghien ctru trong binh tam giac 250 ml voi toe dglSc on dinh d 200 vdng/phiit va sii dyng mdi tnrdng dS
Tgp chi Cong nghi Sinh hoc 10(2)' 335-343,2012 xac djnh thSnh phan carbon, nitrogen va ndng dg chit cam ling thich hgp
D^ng g i i k h a ndng thiiy phSn long gk cua chung B. licheniformis DS23
Chimg B. licheniformis DS23 dugc Ien men tren mdi trudng da thay the co bd sung Idng ga va ki^m tra hogt tinh keratinase sau 24; 48; 72; 96 va 120 h nudi, dong thdi quan sat mirc dg Ihiiy phan theo thdi gian len men.
KET QUA VA THAO LUAN
khuan ed kha ndng phan Tu\en chgn c&c chung \
huy keratin cao
Tir 20 mau dat \ii nude thu thgp tai vimg bien Dd Son - Hai Phdng, chimg tdi da phan lap dugc 140 chiing vi khuan sinh protease cao tren mdi trudng MP thgch cd bd sung 1% casein. Tir ciic chiing co hoat tinh protease, tien hanh kiem tra kha nang sinh keratinase bang phuong phdp c,i\ chain diem tren dia thach MP cd bo sung 0 . 1 % keratin (Hinh I), cac
Hinh 1. Kha nang sinh enzyme ngoai bdo tren moi trifdng MP b6 sung 0 , 1 % keratin cua chiing DS23. HT23 vS HT24,
chung sinh keralmiise cao tren dla Uigch dugc nudi tren mdi trudng AMI, dich keratinase ngogi bao dugc thu Igi va kiem tra hogt linh tren dTa thgch chira 0.05" n keratin (Hinh 2). Trong sd 140 chimg ban diiu, chimg DS23 cd hoat tinh keratinase cao, 6n djnh, CO the phat trien tdt tren nhieu mdi trudng da dugc chgn de ngliien ciru,
Phan loai chiing vi khu^n DS23
Chung DS23 cd kha nang sinh bao tii tren moi trudng MPA cd bd sung MnSOj Ket hgp dac di^m hinh thiii, klia nSng sinh b^o tii va sir dgng kit phan loai API 50CHB cho thay. chiing DS23 thugc chi Bacillus (Bang I), Ket qua phan tich trinh tu gen 16S rDNA chi ra rang chimg DS23 thugc loai Bacillus licheniformis vdi dp tuong dong la 99% so vdi trinh tg ciia cac chimg Bacillus licheniformis trong GenBank, Trinh tg nucleotide ciia gen 16S rDNA cua chiing DS23 da dugc dang ki uong GenBank dudi ma sd la GU004539 (Bacillus licheniformis strain DS23), da so sanh uinh tu 16S rDNA ciia chimg Bacillus licheniformis suain DS23 vdi cac trinh tu tren GenBank (Hinh 3),
#
Hinh 2. Vong hoat tinh keratinase ngoai bao tren moi th^ch chira 0,05% keratin cua chung DS23 voi th^ tich dich l§n men (pi).
Wnh 3. So sanh trtnh V i>09n 16S lON cua Chung vl i<huin DS23 M cSc Unh V gan cua cSc chung vi khuSn khSc t i n GenBai*
Tnmg 06 HQ143567 iBaallus Ithenlomis PG9),HQ143565 (Sacifcs tOimilomis PG5),HQ143568 (Saci/us tchmtforms PG10 , eCl»l539 (BBC/to Scheomrms DS23)i HM753e25 (BaoJl/s Iktientoms WSE-KSU301 )i HO7093e2 (Baci/bt khenihmis BL1);
HQ703897 (Baagus Ichen/amb NBM60);HQ703943 (Sacl/us khenlomie NBM42) v4 HQ641338 (Bacijus Ic/ienfomiis BGt 1)-
l ) i ) n g T l i | T h u y D u i m g C/K/.
Bang 1
STT 0 1 2 3 4 5 6 7 8 g 10 11 12 13 14 15 16 17 IB 19 20 21 22 23 24
D^-ic di^m-.iiih lio.iM
C o ' c h ^ t Dill billing Glyrerol Erythiilol D Ai.ibinoso L-Arnbinoso Ribose D Xylose L- Xylose Ad on! tot l l - M e t y i n r.iii, o/ii Golaclose Glucose Fructose Manose S O I bos e R h „ u n t r . , Duixiloi Inozilol Mannitol Sorbitol ii-Melyl-D. Manozit u-Melyl-D- Glucozit N • A xyl-G luc OS a mi n Amygdalin Arbutm
Co' c h i t Eaculin Snlixln XolobloBO M.inlosc Laclose MeltbioHO Saccoiose Trehalose Inulin Melezilose Rafinose Tmh bW Glycogen Xylilol Gentiobiose D-Tufanose D-Lyxose D-Tagalose D-Fucose L-Focose D-Arabitol L-Arab«lol Gluconat 2-kelogluconat 5-ketogluconal Ghi c h i i : (+) d u o n g Unh, ( - ) i m tinh, ( i ) chua i.
A n h h i r i i n g c i i a n g u o n c i i r h o n
K h a n ^ n g t d n g h o p keratinase eao nhat ciui c h u n g B Tichentfoimis I ) S 2 3 k h i n g u d n c a r b i m c d t r o n g m d i t r u i m g la gluco.se (3X,5 U / m l ) , tiep dOn la r i d u d n g ( 1 4 , 7 U / m l ) . Cac n g u d n c a r b o n k h a c n h u saccarose, lactose, maltose, m a n i t o l . t i n h bpt c h o k h a n a n g sinh I d n g h g p keralina.se thiip. c h i dgt k h o a n g 6 - 2 0 % so v d i g l u c o s e ( H i n h 4 ) . K e l q u a n a y k h d n g t u o n g d d n g v d i m g t so n g h i e n c i m k h a n a n g sinh keralinase c h i i n g 5 . licheniformis M K Z - 3 . B. licheniformis P W D - I c h o r i n g , s t i d g n g g l u c o s e n h u n g u d n carbon c h i n h l a m g i a m h i ? u q u i i s i n h l o n g h a p e n z y m e ciia te bao d o g l u c o s e ire c h ^ q u a t r i n h d d n g h d a ( B r a n d e l l i et ai, 2 0 0 5 ; G u p t a et al., 2 0 0 6 , H o s s a i n d al, 2 0 0 7 , K a i n o o r , N a i k , 2 0 1 0 ) . K h i n g h i e n c i m k e r a t i n a s e tir c h i i n g B. licheniformis L Y 4 , L i n va Y i n , 2 0 1 0 c u n g d u a ra k i t l u g n t u o n g 3 3 8
Hinh 4 Anh h u d n g cOa ngu6n carbon khd nSng smh t4ng hgp keratinase cua chung DS23. •
Tap chi Cong nghe Sinh hpc 10(2) V.5-U3. 21112 lu, lion iiiia.juii liic gia tien eho ning, su iKiii!; vo Iriiii iihu nguon ciirbon chinh liini Uiiig dting kc lioiii linh keialinasc Xiuii phiil tii viec sii dung cliiiii;;
gidng \ii mdi Iruitng co ban tioiig Ciic nghien m i i l.i khac nhau. dan diin kha nang sinh long hor|i eii/\iue Ihu dugc la khdng gidng nhau. hon ml,!, kha lu'iiig sinh keriiliiiase khdng iihiTng phg lluide vim iigiioii carbon iiiii cim phu lluide vao nit. ihiinh pli;in khiic.
dac biet la ngudn nitrogen.
Anh hudii<>ciia ii^iidn nilro^ieii
rhii\ ihe bdn hgp peplone \ii i..io ii.iiii men irong mdi tiuinig A M I lan luifl bang cao ihii. eao nam men. peptone, tr\plone. ure. (Nl l | ) M ) , \a NIIjCl cho thay ngudn iiitiogon co anh limmg k'm den kha II.IIIL; sinh kei'iiliiuise eiia cluing li lieheniforinis l)s2 5 iluip nliiii hi ure chi dgt kluxing 2"o (1.87 Uinl) so \ d i ngudn nitrogen cd trniig mdi tuiiniii CO ban Trong so cac ngudn nitrogen nghien cim, peptone eho hieu qua tdng hop eii/\iiie cm nh.il dat 40.15 U m l . tidp dd dCm cuo llijl. ii\puiiic \ii NHjCl \oi boat tinh tuong inig 30.2 U m l ; 22.'-l5l' ml \a 22.5 I ' ml (Hinh 5), Kel qua iiaj timg tuong ddiiL: \oi nghien cim cua llossam et at., 201)7 tren chiing B. Iichcni/mnns M/K-3 cho raiitj iigiidii nitrogen hiru co nhu peplone. li\'plone. cao niiiii men linn tiing hogt tinh keratinase nhimg khdng cao biing ngudn nitrogen \ d ca la N l l j t l , Ddi \oi chung Bacillus sp. JB99, KauKXir \a N.nk. 2010 dil chi ra ngudn nitrogen la cao nam men. peptone \a casein kich thich su tdng hpp keralinase. dgc biet lii cao nam men d nhieu ndng do. tJupta \a Ramnam (2006) da chi ra MCC SU dung co chal keratin nhu ngudn nitrogen \a carbon duy nhat trong indi trudng cho hieu qua cao Uong tdng hgp keralinaie
\ i i l i hiioii;^ ciiii nong do co elial emu ling
^ 120 1 - ^ 100
r "
b 60
•S -10 f 20 •
X °^
w .
Nguon nito-
ffinh 5. Anh hudng cOa nguon nitrogen len kha nSng smh long hgp keratinase cOa chiing DS23.
I hen I la chill ea
u cua f a i MI /licng, 2(111), keiali 111 img smh long hup kcriilinasc neu t d iii.ii Uong mdi Iruong len men MU ndng dd nho. hen hiinh bd sung ketiUin dii \ u li \ao mdi iririrng Icn iiieii eiia cluing H tirlieni/oniiis l)S2t vdi nongdg di 11.1 I " c h i nhi' Ihi' ;iiig cluing smh
\iiie lot iihiil a ndng dttchiil cai
(SV: I ' m l \,i 54.1 11 mil. l.ing h<m 10"» so vui mdi liiiinig khdng bd sung, I.iigng kenilinase smh ra i;i,iiii Midiig IS. V'„ khi ndng i.\(i keralm bd sung (iiiig len 1"« (Hinh 6), Tuirng lu nhu Iren, (iupla vii Kaiiiiiiii. 2006 eho i.ing keialin kich ihich phan img siiiheii/\me
«^
tin Hoat 120 100 ao
60 40 20 0
• •
N..
J 02 04 06 08 1 Nong do Keratin (%)
1 2
Hinh 6 Anh hu'ong cua n6ng do keralm len kha nSng smh long hop keratinase cua chimg DS23
Anh huong cua nhiet do
DO \aL dmh nhiet do nao tdt nhat cho qua trinh smh keratinase ciia chung B lieheniforinis DS23.
chiing ldi da thay ddi nhiet dd len men: 25, 30, 37.
40. 45, 50 va SS^C. K^t qua cho thiy. chiing phat ll icii va smh hoat linh cao trong khoang nhict dg 30- 45"C (Hinh 7), Nhict do thich hgp nhat cho quii trinh sinh tdng hpp keratinase la 37°t\ hoat tinh dat 55U/ml. Khi so siinli nhiet do phat Uien va sinh keratinase ciia chiing B licheniformis pS23 vdi cac chiing B. licheniformis khac trong mpt sd nghien cim trudc day, nhan th^y rang phan Idn cac chiing B licheniformis ddu sinh trudng va sinh enzyme d dieu kien nhiet dp tir 30 den 5 0 ^ (Gupta, Ramnani, 2006), Nhiet do tdi uu cho sinh keratinase ciia chiing B. licheniformis HK-I (Korkmaz d al, 2004) la 50°C. trong khi chung B- licheniformis MZK-3 sinh enzyme tdt d 40''C (Hossain et al. 2007)
339
D$ng Th i Th uy Duang et al
a ID 3S 40 IS so SS N h i v l d O l " ' »
Hinh 7, Anh hudng cii.i nhi0l dft nuOi cdy 6kn khfi nflng smh l i n g hgrp keralinase ki^m ciia chimg DS23
\ n h hirdnt: eiia p l l nidi li-ir<Vn[> han dnu Ngoai nhivl dg. pH cd lac ddng hiii den qua trinh sinh trudng, phdl uien s.\ MIIII idng hgn cn/yine cua indi chung vi sinh vgl Kel qua cho thay, cluing fl, tuheniformis l)S2l sinh keratinase lot nhal o pi 17
\ a pH8, vol hogt tinh Ian lugt dgt 50 va 55 I' ml (Hinh 8) O pH ' 7 hogt tinh chi edn 54''o boat tinh cgc dai, trong mdi trudng cd pH Idn hon X hogt linh giam mgnh: pH 9 giam xuong 3S"n va pH 12 xudng 2% so vdi viing pH cho boat tinh cgc dai. Ket qua tren tuong ddng voi mpt sd nghien ciru cho rJing, hau het cac chimg B. licheniformis sinh tdng hgp keratinase tdt nhat trong mdi truong kiem nh^
(Korkmark et ai, 2004, Gupta, Ramnani. 2006;
Hossain era/., 2007).
pHinoi nuoiiieltiiii dHii Hinh 8. Anh h u d n g cua pH mOi tru£rng ban d i u akn khS nang smh t6ng hgp keratmase ciia chung S, lichonilormis DS23
Anh hirong ciia the tich djch nuoi ban dau vk ty 1$
tiep giong
Chiing S, licheniformis DS23 sau khi dugc nhan gidng trong thai gian 18 h dugc bd sung vao cdc moi
truimg len men vdi cSc ty I^ thay doi: 0,5; I; 2; 4; 8 vii 10%. Ket qua nhgn dugc cho thay, sg sinh tnrdng, phiil Irien vii long hgp keratinase ciia chiing B.
lieheiiifhrmis DS23 chju knh hudng Idn cua the tich giong ban d^u, vdi lugng giong cay \ko lir 0,5, 1 vk 2"n tilling Miili iiutnig vii smh keralinase thap hon nhieu so vdi khi ciiy giong > 2 % dgt 35.23; 48,02 vh 82.04% so vdi 4. K v;i 10% (Hinh 9), ket qua nay ciing phii hgp vdi nghien cinj ve chiing Bacillus siiblilis KD-N2 (Cai. Zheng, 2009).
II illlJ
Hinh 9. Anh h u d n g cua ty l< t i i p giAng (%v/v) d i n kh^ nSng smh lAng hop keratmase cu.j chung 6 Icheniormis DS23.
The tich dich nudi ban dau cd anh huong Idn den qua trinh smh iruong va sinh enzyme cua chiing B licheniformis DS23, tien hanh thay ddi the tich djch nuoi cay ban dau 20. 35, 50 va 65 ml trong binh tam giac 250 ml thu dugc ket qua la chiing sinh truong tdt nhat va sinh keratinase trong binh chira 20 ml/250 ml dgt 55.18 \J ml. hogt tinh keratinase giam xudng 8 3 % vdi the lich 35 ml. 45" n voi 50 ml va 29% vdi 65 ml (Hinh 10). Nhu vgy, chung B.
licheniformis DS23 cd nhu cau o\y cao, ket qua nay ciing phii hgp \di nghien cuu ciia Cai va Zheng, 2009 khi danh gia kha nang sinh keratinase ciia chimg Bacillus siihtdis KD-N2.
Ikx
2 0 35 65Tkrltch d|tb nncllml) Hinh 10. Anh h u d n g ciia th4 t i d i djch nu6i ban a i u a i n khi nang smh keratinase ciia chOng DS23,
Tgp chi Cong nghe Sinh hpc 10(2): 335-343. 2012 Sinh tong h g p keratinase cua chung B.
licheniformis DS23 theo thoi gian
Dpng thai len men phu thupc vao t^t cii cAc y^u td lien quan: chung giong. moi Uudng len men (thanh phan carbon, nitrogen, cac nguyen id vi lugng...). dieu kien duy tri len men (nhigl dg, pH, oxy hda tan,..). De nghien ciru anh hudng thin gian den su sinh keratinase cua chiing B. licheniformis DS23 trong binh tam giac. chiing tdi sir dyng mdi truong va dieu kien nudi cay Iga chpn trong phan ket qua phia tren. Ket qua cho tliay chiing xuat hign hogl tinh keratinase sau '•> h len men. tir dd tang nhanh den 27 h, sau 27 h hogt linh enzyin co \ u hirtVng King chain theo thai gian \ii dat cue dgi sau 72 h len men vdi hogt tinh dat 63.8 U^nl (Hinh 11). Hogl tinh giam dpt nggt sau SI h. sau dd tang tro Igi va giam lai tgi thdi diem 99 h len men Nhihig nghien cuu trudc day cho thay. thdi gian cho sinh tdng hgp keratinase kiem cue dai thay ddi tir 30 - 96 h len men tii\ timg chung vi sinh vgt (Gupta, Ramnani.
2006: Xu el al. 200'»1 Nhu vay cd thi thky. chiing fi licheniformis DS23 sinh keratinase tuong ddi cham,
Kha nang phan huy long ga cua ch ung u . licheniformis DS23
Keratinase co the dugc sir dung de phan hiiy long ga thanh cac hgp chat cd khdi lugng phan tu nhd, cd gia tri dinh dudng cao. cd the bd sung lam thuc an cho gia siic, gia cam va nudi trdng thiiy san.
Quan sat Idng ga ngam trong cac binh chira dich nudi c5y chimg B licheniformis DS23 cho thay. cac sgi
long ga bat dau bi thiiy phan thanh cac manh vyn va lang xudng day binh tam giac sau 24 h, Cac sgi long ga dupe cat thanh manh nhd ngay cang nhi^u trongmdi trudng len men trong thdi gian len men 48, 72 va 96 h (Hinh 12 A, B). Sau thdi gian len men 120 h, phan sgi Idng ga bi phan hiiy nhanh va ho^n loan, trong khi phan ong long bj phan hiiy cham hon va phan cui'ing Idng khdng bi anh hudng (Hinh I2C) Tac dung thiiy phan Idng ga ciia keratmase ciia chung DS23 cd mgt trong dich len men hoan loan phii hgp vdi ket lugn ve kha nSng phan cat Idng g^
thanh ciie cau phan prolein ho?ic dudi protem cd kich thudc nho hon ciia cac chung vi khuan B licheniformis (Lin el ai, 1995, Letoumeau d (//„1998. Bressolliet d at..\999. Korkmaz d
«/..2004)
5 i " 36 4.*^ '•A 6 3 ' Tlioigiiiit (2I01 Hinh 11. Anh huong cua thm gian nuoi cay din kha nang smh keratinase cua chimg DS23
Hinh 12. Qua trinh len men thuy phSn Idng ga ciia chOng B. licheniformis DS23 A 60, Thoi diem ban d h B l Long ga i> thoi ai^m ban 3^0.2: Long ga sau 96 hl&n men trong binh tam giac
D(lngTlij Thuy Duong tVu/,
Tir cac man diii vii nude i,ii I )d Sim - I kii I'hdiig, dii phiin Igp dugc 140 cluing vi khiiiin id hogl linh piolciise kiem, Trong do cluing l)S2l id klii\ iiiing sinh keralinase kiciii eao. dn d|iili. Ihdi gian sinh Huong nhanh, cd ihe ph.it iiicii idi Iren nhieu mdi truimg. Cluing l),S2l l o kli.i ii.iii[! sinh bao tu uen mni Ituimg M l ' \ i.'> bd sung M n s n , Kel hgp dgc diein hinh thiit v,i su dgng kit ph.in hui API ^IK III) chn ihi'iy chung l ) S : t lliiigc chi Bticitltis Kin phiin lich Hinh ig gen 16S iRNA iihriii dugc d m lli,i>
chung h.s^l thugc lo.ii liacillus liehcinforniis \(ri do liioiij: ddnj; lii ')9%. ninh tg miilcolule cua gen I6S I KNA CU.I cluing I S21 dii dirge liriii); ki Irong (Icnliimk dudi mil so l.i (>lHllltsig {tUicittiis tuheniUnmis strain h s : 3 ) C h u n g / * tulicnilnnnis l)S2 ' smh tdng hgp kei.iiin.ise kiem mgnh nhat imiig dicit kii-ii pll mdi iruinig ban diiu K,l). nliivl dg 17 C.
the Itch dicli len men iioiig binh uiiii gi.ii 25()ml |;i 20ml \iTi nidi tiuimg len men lliitli hgp (i; 1) (ilucose 10.0; pepmiie 10.0. K.III'O, 1.0.
MySO, 7H..() (1.2 Na-CO; VO m bd stmg 0,3%
keralm \.i t> Ig liep gidng -1"„ S.iii 72 h Ien men hogt linh keratmase cua cluing dat ctre dgi 63.8 I )/ml, .Su dung dich nudi de phiin huy long ga cho ilii'iy. suu 24 h Idng ga hat dau bi phiin huy va ihuy phiin hoan loan phan Idng to \ii dug Idng siiu 120 h l-iii cam on: Cdng tnnh ndy dirpc thin hn'ii hoi kinh phi de Idi idp Mui nuoc DTUS US (W'.SHCB l\gtiien dm Cling nghe sun \iidt eiizini protcuzti \,i ciiziin lipaza lir chimg yi smh vtil ldi to hop tit' img dung trong cong iighicp ihiidi da yd son \ ndi t hat lay i ira) ihui'ic De dn phdl h icn vd irng thing cdng nghi '•'I'b hoc tnmg Imh vin ii'nig lu^hiip thi bien den ndm 2020
ir.inL'\it'ai'ellubr sp growing on i.iv\
TAI LIEU THAM KHAO Brandelli A, RilTel A (2(I(1S| Prodiii keralinase Irom Chiwinb.ii leiiuiu lealhers, lileeU on J limteehnol S( I), 35-42, Uressollie P, LctourniMii I", llrdaci M. Verneuil h (IW)) Puriliealion and Characteri/iiiion ol';! Ki-i.itinnluii. Serine Proteinase from Sireptoiiiyi e.s athUloflavu Appl Invnon Microbiol 65(6): 2570-2571).
Cai C, Zheng X (2ll()»n Medium oplinii/;ilion lor keralinase production in hair subslriite by a new Bacillus siihiihs KD-N2 using response siirliice methodology, J Ind Microbiol Biotechnol 3U{7). 875-883
Deivasigamani li Alagappan KM (2(l()S) Industrial 342
:i|ipIii';ilion ol ki'iiiliiiiise mid soluhle proteins I'rom Icathi^r kei ;ilins,,//';/nvm/i/(w/2')((i)'<) 11-9.16
tiiongo JN. I iKJis IS, Casiiiiii F. Ileeb P. Brandelli A (2007) Kcialinolylie pioliiises ii\'Baciltiis species isolated I'rom llie Aina/,oii hiisin showing lemmkable de-hairing iielivily, il.nhl I \h, mhi,,! Bioiechnol 23: 375-3H2.
(Iiiplii K. R;iiiiii;iiii P (2(l(lfi) Mitrobinl keralm.Lscs iind Iheir prosperive iipplii.ilinns ;ii] overview Appl Muivhiol liioteihnotH) 2l-,n
llnss.im MS, A/iid AK. Abu Sayem S\l. Moslafa G.
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k;imooi I'S, Niiik (iR (20111) Production and eluMilciiii/alion ol lealher dci;r.iiling kiT.ilinasc ffdm l!,llillu.^•,\^ j n w hidJBloh;hnd9-3M-Vm Kiinsoh Al.. Mossiny RN. I"l-Ihimeed I.KA (2009) Keiminase Pi.Kkkti.'ii ITOI.I I LIIIKIS U.isk-s Using Sonic I.K.il Sncplomu.s k o U i s Utyl J Biisu .ippl Sti 3{2]
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77-sn
l.n IIM. Yin IJ (2(110) I e.iilier meal and rice husk enhaneed ki.r,ilin,isi.s prixluelion by BacilliLS licheniformis YJ4 .intl vh.ii.iikis olproduied keraliiuisL-s J .Mtiriih' Sa Teih IS(3),-IsS-lh.S
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.\u B. Zhong (.). I.iiig X, 'I'any Y. Huang Z (2009) Isolation and characlerization ol" a new keralinolylie bacterium thai exhibits ignifieaiii leather-degrading capnbiln\ Afi J Biotechnol 8( 18), 4590-4596 S T U D Y O N P R O D I C T I O N O F A L K A I IM-
DS23 I S O L A T E D IN HAI P H ( ) \ ( ;
K E R A I I N A . S E B Y B A C T E R I A L S T R A I N
Dang Thi Thu> Duong'. N<;ii>cn Nan Hieu'. Pham Thanh I h n e n ' , Khuat Huu Thanh^ Phi Quyet Tien'. Le Gia Hy'"
'institute of Biotechnology. I idnam .-tcailenn of Science and Technology
^Institute of Biotechnology and Food Technologi. Hanoi i nn ei sin- of Technology SUMM \ R \
From various soil and water samples collected from Do Son - Hai Phong ihe bacteria producing keratinase were isolated and screened, .\inong them, the besi keratmase pioducer designated as DS23 showed a great growth rate, a strong kei atinase acii\ity and be able io develop in a grealnumbers of gi owing media The strain DS23 was first idemified ,is Bacillus branch using the API 50CHB kit whereas its 16s rRNA gene sequence similaniies v\ere 99°o with B hchenifoi mis. The optimum conditions for keratinase activily of this train DS23 were pHS. 3"' C. in the medium coniaimnu per litre: glucose 10.0. peptone 10,0. K:HP04 1,0. MgSOj 7H;0 0.2. Na;C tX 3.0 with 11..'"n keratin solube as keralinase inductor and the initial bacteria concentration of 2%.
The best keralinase actiMi\ of 63.8 U/ml was obtained after 72 hours of incubation The crude enzyme from culture medium incubaied was able lo bydroKze the native feather.
Keywords: Bacillus licheniformis. alkaline protease, kerralinase. feather degradalion
Author for correspondence: E-mail: legiahvl
