Tgp chi Cong nghe Sinh hoc 10(2): 353-364. 201
S A N G L Q C V A D A N H G I A K H A N A N G PH.AN G I A I P H Y T A T F C U A M Q T S O C H I J N G PSEUDOMONAS STVTZERI P H A N L A P O D O N G B A N G S O N G C I J U L O N G
Cao NgQC D i ? p ' . Nguyen Thj Ngge Anh^
' Vien Nghien ciru vd Phdl trien Cong nghi s'lnh hpc. Dpi hpc Can Tho
^Tnrdng Pho thong Tnmg hpc Thgnh An. Iniyin Vinh Thgnh. Thdnh pho C 'dn Tiny
TOM T A T
Ngu coc. hat d(iu va hgi lay dau la nliOng hoa mau lu^ng tren 90% dien tich dat tr6ng iren Irii dat, cung cSp nguon dinh duong cho nguai \ri gia sue Tuy nhien nhOng hoa mau lai chira nhicu acid phytic (dang muoi la phytale) la nguon phospho chinh (ch e n hon 80 ) ng ngu coc v.i h.il dau. Phytase la enzyme c6 the thuy giai acid phytic tliimh dgng lan de lieu cl 1 u u nl u I o. gij cL^m va ca. vi?c bo sung lan vo cho khiu phan cua \at nuoi Uen da khong dugc 1 ^ a\.\ pi o b Iiai ra ngoai niui truong va gay 6 nhilm nguon nuoc Gop phAn nang cao klia ning hap I u I tytis, pho pi oru hCtu dyng tir nguon phytale \a muoi phytin co nhieu trong ngu coc \ a dau, cho e n tro a g nu dl b e la dong vat co dg day don ngSn, nghien cim nay tap trung vao \-i?c nhgn dien gen ma h i pi yti e o / lo o is sintzei i va img dyng phytase de cai ihien chat lugng phan hCiu co \,i ihirc iSn gia cam Kei qua cho thay co 3S/147 chung P .\iiiizeii tao halo tren moi trucmg bo sung acid phytic Chin trong .^S chung /' stutreri duoc xac d|nh mang gen ma hoa phytase bang PCR. Gen phyi cua 5 chung duoc chon co nntc dg tuong dong nucleotide \ 6i gen phyt cua P. siinzeri A1501 la 99%, Cay pha he di truyen cua 5 chung din^c giai Irinh ty (2a, LN2a, N7a, 8s, 16s) \oi P stutzeri AI50I chuan cho thay cac chung \i kliuan nay eo moi quan he di truyen ral gan nhau. Voi hai co chat la hon hop cam - dau nanh va phan hihi CO, t5t ca 9 chung P sturzen (2&, LN2a, N7a. 8s. i6s, B9C1, LNlc. 13f2, B5a)dk co kha nang hoa tan lan huu co tot Chung Pseudomonas siutzeri BT2a cho ham luijng lan hoa lan toi nhat tren ca hai ca chat co the lam ca so cho san xuai che pham sinh hoc phuc s'u cho san xuat phan hiiu co chat liigmg cao va bo sung thiic an gia sue va thuy san
Tir khoa: gen ma hoa pin lose, hoa tan idn, phytase, pin late. Pseudomonas stiilzei i
MODAU
Phosphaus la nguyen to co vai tro guan trong trong moi co the song, tham gia cau tao te bao va la thanh phan quan trcmg trong cau trite cua phan tu di truyen deo\\Tibonucleoic acid (DNA) va ribonucleic acid (RNA). D6ng then phosphorus con la thanh phan cua mot so enzyme quan trpng tham gia ehuyen hoa protein, lipid, carbohydrate, ho hap te bao, kich hoat vitamin nhom B va tham gia van ehuyen nang luong thong qua cac hop chat cao nang nhu adenine triphosphate (ATP), adenine diphosphate (ADP).
Neu thigu phosphorus a thyc vat thi qua trinh t6ng h(?p protein bi ngimg tre, la cay nho lai, ban la hep, cay sinh trucmg cham, qua trinh chin ciing bj keo dai.
Thi^u phosphorus o dpng vat gay mem xuong, thit nhao, chan an, r6i logn tam than, giam san lugng sira, giam ti le thu diai, sinh truang chgm,Tuy nhien.
phosphorus trong tu nhien phan Ion ton tai a dgng kh6 tieu sinh vat khong hap thu duoc, chii ySu la dgng mu6i vo ca kho tan co trong dat, cac quang thien nhien nhu apatit. phosphate s4t, phosphate nhom va dang phosphorus huu ca co trong dat, xac
ba dong thyc vat, Dac biet phytale la chat du trii phosphorus hiiu co chinh trong ngii coc nhu dau tuong la 60%, ngo la 72%, liia mi la 77% (Pham Thj Tran Chau, Phan Tuin NghTa, 2007). Day la ngu6n thuc an pho bien cua vat nuoi do do kliong nhiing lam lang phi thiic an ma con gop phan gay 6 nhiem moi truong.
Ngay nay co rat nhieu nghien cim nhSm lam tang lugng phosphorus hiru dyng eho cay trong va vat nuoi, trong do nghien ciiu ve ung dyng cac enzyme tir vi sinh vat dang rat duoc quan tam, Va enzyme rit hieu qua trong xiic tac thiiy phan fihytate nham giai phong phosphorus de tieu va cac chat dinh dirong kh^c la phytase. Co rat nhieu vi sinh vat san xuat phytase nhu Pseudomonas spp. (Richardson, Hadobas. 1997), Aspergillus spp,. Bacillus subtilis (Shimizu, 1992), Escherichia coli (Greiner el ai, 1993), Milsiiokelta spp. (Lan et ai. 2002),... cho nen viec nghien cuu cac nhom vi sinh vat, dac biet la vi khudn, CO kha nang san xuat phytase va ling dyng vi khudn nay d^ cai thi?n lugng phosphorus hihi dung trong phin bon va thuc an gia siic, gia cim. Phytase con duac dimg trong ch^ bien thiic an thuy san de 353
Cao Ngoc l)iC'p& Nguyen Thi Ngyc Anh Iiim guim liri,mg phosphoius thai la moi iitiVriig niu're.
giani ngti> co g.iy hiC'ii luitiig plui iliri>iig do o nliiem phosphorus v.i gi.im chi phi bo ,sung phospli.ile \ o l o vao thirc iin (Ki-rovno 211(111), 1 liC'ii nay. ticn llie r.i.ri, d i e pham ph>l,ise duoi s.iii .viial \,i lluiong mgi hoa iihiini bo sung lliiie iln gi.i s m . gin cam nhir HiolU-d'"
(hilng No\o Nouliskk \leii/vme plui.ise (lulng Alltech), I iiuse (lulng M k o e o ) , O Vicl n.iin. en nlunig nghien ciru va img dyng pliM.isi' nhtmg chn yen lien M n.im Ispeigillns nigei (1 e Minh Ihien -•/
at. 2111)1, Dmh Minh lliqi . / .;/. ^HH'*), nam Triehotlcima sp (Ngincn l h | U\On "Ih,i.i d ,il.
2004) nhimg iliii.i en ngliicn cim co ban \ e cii/yiiie piiM.ise lif \i khiiiiii iihiil la vi khiiiin I'seuilmnonas slulzeii Ngoiii Ciie irng diing Iren, pluliise dir(,re dimg de t.ii Ihiijn iliiil Uri,mg d;ii. vl co nlii>ng nm aeid ph>lie \.i t.ic d;iii MI.I1 cuii iin clueiii den .''<)""
tong lu^mg pliosphouis hiVu t o limig dat. Vii;e hn sung pli.Ma-se \.io d,il l.iui tilng liaiii lii(,mg plinspli.ile
\ o lO giup i,i\ t.uig trirong nhanh h»m \.i giam ihi phi dimg phiin him phospliniu,s ilio iilu) ming (Vohni, Satyanara>iiii. 2003)
N(iUYEN l.ll-U VA PIIU()N<i PHAP
\ ill li^u
Cac chung \i khuan Pscudinnimas siutzeri diryc phan lap tir ao nuoi ca tra o cac tmh An (iiang. hen Tre. Can This, Dtxig Thiip. Kien (iiang, Tien (iiang va Vinh Long trong cac de tai lot nghiep luan viin ihgc si khoa ht,K nganh Cong nghi; smh h(.)C \ii Smh thai hpc cua Le Thi Ngoc Mii (2()(W). Ngineii Th|
Thiiy Tnnh (2009), Phgm My C.iiii (200S). Do Thi Kim Chi (2009). Phiing Ngpc Bich (2009) \ a Dtnmg Thi Bich (2009)
Phan h&u co hi?u Fofcr 333 siin xuat tgi Cniig ly Trach nhiem huu ban Phiin bon hoa smh VAC. My Tho. Tien Giang; phan huu co san xual tir cii, than biin boat hoa. banh dau. voi thanh phiin dmh diriViig: 3"o N, 3 % P.O,, 3 % K:0, 1% CaO. 2% humat, ciie vi luong Cu, Zn, Mn, Bo, ,, c:ic amino acid v;i viliimiii thiet y^u. Cam trich li va bpt diiu nanh sir dyng lam thirc kn gia site ciia Cong ly Trach nhiC'ni hOii hgn Thuc an Gia sue Kien Thanh, Sa Dec, D6ng Thiip, Phuong phdp nghien ciru
Khao sdt khd ndng hiia lan lan huu nf ctia vi khudn P. siutzeri
Ckc chiing P siutzeri dupe xac djnh kha nSng t6ng hop phytase bang each nuoi vi khuSn tren moi
Iririmg bo ,suiig aeid plivlic (Hill el al. 2007), mni (llii pelri Ciiy S diing \.\ ^ kin kip Igi Sau 24 giiniuoi liiy. quan s,il niiui, do duimg kinh vimg quang (halo) v,i iluiiiii; kinh kluiiiii Igc, Chtmg vi khuan niio igu (lir(,rc liiilii Ircn moi liiiong chirng U) vi khuiin do co khii nilng hnii Inn liin hiiu m hay chimg co khii niing limg hop |ih>liisc <'hi sti him lan phosphale cua cae eliiiiig M kliiLiii throe \.ic diiili dya theo Liiiig ihiic uiii i:di ('/ ai (1996) lii: cln so hda liin T.iiig dmmg kinh (liiiln I khtiiiii lgL)/din'mg kinh khuan Igc. Dong vi khiiiin iiiiii i 6 clu sii liiiii liin cimg ciio chimg lii c6 khii Iliing giiii phdng phosphale ciing iiiiinh Khuech d{U vd giai Irinh ttr do(in md hda phylase d r. siutzeri biing PCR
Vi khiiiin di[i;c nhiin nudi Irong dug nglii^in chira indi Iruimg LB (long) de thu sinh khdi. tach chiel DNA \\ kluiiin theo quy Irinh cua Neumann el al (l'>'>2)
De nhgn di^m gen phytase cua vi khuan P.
sliilzcri dya Ircn Irinh Ur cua / ' .siutzeri A1501 do
<pS TS Rene De Mni (Dai hpc Leuvcn, Bi) goi lang nhu siiii,
Mdi Midi I'sssi 5' - CTA CCiT CAA UGA ( . A A C ( i - 3 ' ,
Mdi ngupc PI 321R 5' - AC A K i t ACAGGC C(ri A ( i A T A - 3 '
I'h.iii img PCR- '>^••(^'3'. 40 chu k> (')5"C'3ir.
5(."C/4(r, 72-C/l'). 72'XV5'.
Su dyng doi.m mdi \udi trong phan img PCR de nhgn dnj-n vi khuiin iidi sinh dil md lii o phan tren. San phiim PCR dupe imh S.RII Iheo kit Invitrogen va giai trinh ty bfing hC' lining m.i> gi." trinh ly lu dpng ABI 3130, Sir dyng chinmg tnnh BLAST N Ak so sanh uinh ly DNA ciia ciic chung \i khuiin chpn loc [codp hiia tan Lin cao] vm trinh ty DNA cua bp gen o cac loin VI khuiin trong GenBank \a \ii> A\m% cay pha HQ gen giua cac ditng vi khuiin duyc giai trinh lu bang ph.in mem MEGA 4 I \.i phan mem BioEdil de phan tich liic trinh ly amino acid cua prolein vi khuan.
Oiinh g i i kha niing hoa lan lan hihi co' Ddi voi CO cUk la phan hOii co; Thi nghifm dupe bo tri theo the thirc hoan loan ngau nhien cr nhi?t dp phong trong 6 ngay gom co 2 nghiem thitc [m6i nghiem thirc Igp Igi 3 liin]: chiing 50 ml dich vi khuin va 50 ml nuoc cit tiet trimg cho doi chimg trong 200 g phan hChi co trong mdi bpc nylon.
D6i voi CO chit 1^ thiic 3n cho gia cim (cam ggo
Tgp chi Cong nghe Sinh hoc \t)(2)- 3 5 3 - 3 6 4 , 2 0 1 2
+ bpt d a u i i i i n h t i lc 1:1), T h i nghiC'm du'pc I h y c hiC-n n h i i i r e n i r o n g 15 i i g i i y , g d m ed 2 n g h i e m ihirc | m o i n g h i e m thirc lap Igi 3 l i ) n | v i k l n i i i n : c i i i i n g 100 m l dich v i k h u i i n vii n u d e ciit c h o d d i c l u i n g \ .in ciic boe n y l o n chira 2 0 0 g CO chiit Ciim \ d i i u n i i i i h .
T h e o d d i cac c h i l i e u ; p l l , p h o s p h a t e dc l i e u ( P O / h a \ I ' - O , ) . d p i i m , n i i i l sd \ i k l u i i i n b i i n g p h u o n g p h i i p d e m s d n g n h o g i n l ( l l o h c n . Somasaragaii. 1982).
Excel d u p e so d y n g di} p h i i n t i c h i l i d n g ke so sanh s y khiic h i e l r i t i . i ciie n g h i e m t l u i e hu s u n g \ i khuan va n g h i e m l l i u e i l d i c l u i n g k h d n g co \ i k h i i . i n ,
K E T Q U A V \ l l [ \ t ) | | i \ N
K h a n a n g l u i a l a n k i n lii'ru eo c u a e:ie c h u n g P.
siutzeri
T i r 147 c h u n g / ' stuizcii d i l d u p e phan lap tir ao nudi Cii tra o 7 t m h . \ n ( i i a n g (21 c h u n g ) . Ben T i e
(53 c h u n g ) . C i i n 1 h o ( 1 6 c h u n g ) , D o n g T h i i p (18 c l u i n g ) , K i e n Ciiiing (7 d u i n g ) . I iim ( i n i n g ( 1 6 c l u i n g ) V.I V i n h L o n g ( 1 6 c h u n g ) ed 38 c h u n g /*
:(77 l g n d u p e h a l o ( v i m g q u i i i i g I r o n g ) ( k h d n g h i n h ) l i e n n i d i t i u i m g bo s u n g a c i d p h y t i c . (.)tia kh.in Sill ed I 8 / 3 S ( 4 7 " . ) i h i m g /> .siutzeri i l i i sd hda 1.111 phosphiile 2",,. 1 7 ^ 8 ( 4 ^ " , , ) m g VI k l u i i i n i n ehi sd hda Ian lir 2 3 vii V ( 8 i " " l c h u n g i o i h i sti hda l i m eao 3 ( B a n g I ) . i i n d i f ' n g e n m i l h d a i i l i y t a s e o / ' . stntrcn h i l n f ; R
d i i n
Siiii p h i i m I'CR v d i cap m d i dflc hieu thiOl ki eho gen iiiii hdii pliyta.se. d u o c t h i ; lugn l i e n p h d d i e n di v o l ') 18 e h u n g P sinlzcn ( 2 i i . 1312, 8s. Ids ( B e n h e l . B5a ( V i n h L o n g ) , L N 2 a , l . N l c , M9C1 ( A n ( n a n g ) , N 7 a ( T i e n ( i i a n g ) ch(» b i i n g o vj tri k h o i l n g 7 3 0 b p sn v o i t h a n g chuan ( l i i n h 1). p h i i htrp v d i b i l n g cua v i k h u i i n d d i c h i m g d u o n g /* slnizen
\ 1 5 0 l N l u r v."i\ ') c h u n g P .stuizcri d u p e xiic d n i h m a n g gen m i l hd.i p l n l . i s c
Bang 1. Kha nang hoa tan lan huu co (phytale) cua c^c chung P stulzen
Ky hieu
Cho' M O I , An Giang ChSu Thanh, An Giang
Ky hi^u c h i i n g
Giong Trom, Bgn Tre Cho Lach, BSnTre
LN1( Chau Thanh, An Giang Giong Trom, Ben Tre
LN2a LN3a LNIa LN3d MN1b MNIc PB1d2 l i e
Chau Thanh, An Giang Chau Thanh, An Giang Chau Thanh, An Giang Phu Tan, An Giang Long Xuyen. An Giang Long Xuyen, An Giang Chau ThSoh, An Giang Binh Dgi, B^n Tre Binh Dai, B i n Tre Binh Dai, B^n Tre Binh Dgi, Ben Tre Binh Oai, B i n Tre Bmh Oai, B i n Tre C h a L a c h , Ben Tre Cho' Lach, B i n Tre Binh Dai, B i n Tre
4c 4s 8s D7m D8c D9a Dge B3d N7a D7a D2b B5a B9a N i a N6a NGc
Cho L ^ h , Ben Tre Cho L i c h , Ben Tre Cho Lach, Ben Tre Lai Vung, Dong Thap Lap Vo, Dong Thap Lai Vung, D6ngThap Lai Vung, DfingThap CSi Bfe, Tien Giang Cai Be, Tien Giang Cai Be, Tien Giang Cai Bg. T i l n Giang Long H 6 , Vfnh Long MSng Thit, VTnh Long Long Ho, VTnh Long Mang Thit, VTnh Long Mang Thil, VTnh Long (+)• Chi so ti> 1 - <2, (++) chi so lip 2 - <3, { +++) chi s6 > 3
Ciin Ngpc l)ii;p& Nguyen Thj Ngpc Anh
i'hiin lich Triiili l^r sen phyt tun i.ie cliiinf> / ' slulwri
Niim lining /' slulzeit (N7ii, 1:\. I N2ii, 8s va 16s) itupi nhgn divn hiing PCK iiiiing gen mil luia phyliisc v.) cd cln sd linii liin pluLspliiile klui iiio (King 1) hiiih ly ^c\\ mil lidii plul.ise eua e.i "i chung iiii\ den m niiii dd luong dniig ')')"., v.ri iiinli ly gen phvtiisc tir / ' siuizeii AI501 irong Genliiink (NC OD'M'H))
D I M Villi liiiili ly gen plivl.ise iii;i 0 chung / ' sinizt'i-i \\^i)\. 2.1, I N : ; I . N7ii. 8s. 16,s. sir dyng chinmg litiih Ml d \ 4,1. mdi quan he gcii pluiiisc cuii i,ic cluing VI kluiiin niiv diiia- i.n phii he llie liivii Hong hinh 2.
(ien phvt.isc o /' siutzeri 16s g;in voi / ' stulzen A 1.501 nli.il so vol 4 ihiiiig con l.n. ke den lii diiiiig 8s \a N7,i. edn 2 chung l.\ \,i 1 N2,i \i\ lum Nhu v,i\
5 tilling /' ,v7/(/j(';'; iiiiy luy phiin bddc^cdia phuong kliiiL iiliiiii (biing 1) nhinig cd mdi ipian h^ gen gan gin ehimg li) cac chiing vi khuiin niiy ddng hinh vk kii'ii gen Kill phiin Ikh moi quan hi^* ciic ammo acid Irimg piolein ciia 5 tliiing vi khuiin so siinli vdi cac .iiiiint) iiml uiii ihiiiig I'seiulonionas siutzeri AI501 tkU\ny. cliuiin |icleieiice strain)) b^ng phan mem Binl dn elm ili.iy (\ciii li.iiig phy chmmg Hmh 7a va 7b I phiin Imstingl) Irong sn638 iiinmo acid.
Cliimg/'. siutzeri Ids c6 nurc do iirtiiig ddng vdi (king chtiiin tiin iihiil (38.71%), ke den la 2 chung:
iliiiii]; Ks (28.21"n). chung N7ii [24,72"„i trong khi dd 2 lining chung 2.i (mirc ilnni: hinh la 9,41%) va iliiiii).' I N2.1 (niifc ilnii[j hinh l.i 7,')')",,) xa nhat so Mri ill 111)! iluiiin A l "^01 Nhir vay, kel qua so sanh lir prolein iiiiig t n ket ttiiii tutmg ty nhu voi so sanh lir 1)N\ eua 5 chung vi khuan vni dnng chuan P.
stulzen A\50\.
50(1 b p
Hinh 1 Ph6 di$n di cua s^n p h i m PCR d i r p c nhfln I6n ti> DNA cua cAc dbng vi khu^n Pseudomonas stulzen (1): Thang chuan lOOpb. (2) DOng A1501, (3) Ddng 2a; (4) D6ng LN2a, (5) D6ng 8s, (6) D6ng N7a. (7) Dong 16s, (8): Dong 69C1;
(9) Dong L N l c , (10) D d n g B 5 a , ( 1 1 ) 13f2
LN:a
<
liDt
Hinh 2. Cay pha h? gen phytase cua cic chung P siutzeri so vCri chung P. sfoizen A l 501
Tgp chi Cong nghe Smh hpc 10(2)- 353-364, 20\2
Kha nang h6a tan Ian huu co- eiia ciic chung P. Bang 2. KhA nang hda tan l^n h&u co cua cSc chung P.
001 vdi CO' chat Id cam + dgu ndtdi
Tat ca 9 chiing P. stuizcii [1312. B5a. N7ii, 16s, 8s. LNlc, 2a. LN2a. B9CI] dupe khao sat dim en kha nang hoa tan lan hOn co vdi kk qua nhu sau;
Chiing vi khuan LN2a va 1312 giai phdng lupng PO4' cao hon cac chung \ i khuAn khiic sau 3 ngay phdl tron. Vao ngay 6, chiing LN2a hoa lan P lang len 184.5 mg'lOO g va chiing B9C1 cung co k^t qua I d l ( t i r 9 0 , 7 m g / I 0 0 g ( i i g a y 3 ) l e n 183.7 mg/l00 g), Vao ngay 9. chiing LN2a vii chiing B9CI liC-p lyc lang lupng lan hda lan. cic chiing 1312, B5a. N7a, t6s. Ss. L N l c tang chgm nhimg chung 2a Igi l.ing manh (tir 176.8 mg 100 g (ngav 6) len 271.7 mg 100 g), khac bi^t nil co \ nghTa thong ke (P.Ol = 1.5) so vcri cac chung con igi.
Vao ngay 12 \ a 15 lupng phosphorus hoa tan ctia cac chiing \i kliuan tiep tyc tang va tang manh nhat la chiing 2a (Hinh 3).
Chiing P. stutzeri
D 6 I chirng Chung 2a Chung LN2a Chung 8s Chiing 1312 Chiing B9C1 Chiing B5a Chung N7a Chung L N l c Chung 16s
LSD 01 C V
Ham lu'9'ng lan hipu co- cua c i c c h i m g chon 19c Cam + d | u nanh m g ( m g PO/ZIOOg)
83,5 239.1 225,0 150,8 168,5 209,7 133,9 134,7 135,9 117,8 1,5 1,08%
Phan hffu co- (mgP,O5/100g) 151.8 233,1 224,1 208.3 203,4 203,6 233,8 220,3 231,9 223,9 4,4 1,66%
800
- -D - DC —•—2a
700 — * - 8s — 0 - 13f2—G - B5a - • - N7a 600 16s
0 3 6 . 9 12
ngay
Hinh 3, HiguquS cua9chQngP, sfuteeridln lirpng P hoa tan trong hfln hpp cam va d$u n^nh
15
) Ngpc I)ii;p& Nguyen Thj Ngpc Anh Nhu\iiy.';iiu 15 ngii\ iheo ddi lirpng lim lioii urn.
9 chung /' stiitzeii duoc kli.iu s.il Iren h(')ii hpp C<T ch.^t Ciim \;i di^ii luinh tlcii co kli.i ii.'ing lidu lan lan hfm CO lot Hiim liimig P o / nnng hinh die lining hda lan dupe dim lilng kli,ii hn-l liil ed y ngliiii llidiig ke (P.Ol l.'i) so \ni chung diu eliimg \ii Ling Ciin nhat lii cluing 2.1 hda till! dupe 2i').l m g i ' d / iimig 100 g t o tluil. ke do 1,1 chung I N : . I hnii tiin dupe 225,0 mg/IOO g, chung B9( 1 lioii t:m dupe :(I'L1
mg/IOl) g (Biing 2) v,i ihiip nliiil la chiing I6schih6a tan liung hinh dirpc I 17,8 ing/IOOg,
Dini Viio Hinh 4 cho Ihiiy mgt sd cac dong vi kluii'm deu lang viio ngiiy 1 va ngiiy 6 ciia qua trinh Ihi nghiem. Vim ngiiv 9 niiii sd deu giam. sang ngay 12 mgt .SI) vi kluiiin o hiiu hii cac dimg deu liep lyc giani till cd ddng I,N2ii. 2,i. H5a lii duy In dupe mgl sd Liio den ngiiy 15,
y?^€r-^
; < K ; = . ^ ; ^--B^ - - - •
- - a - - DC — A
— O - B 9 C 1 —m
2a — i — LN2a - -o B5a — n — N7a — •
- • - - 1 3 f 2 16s
-p-
6
Hinh 4. Sy bi^n dftng m^l s6 vi khuAn (log,oCFU/g) trong mSu cSm - d$u nanh
Doi vf'ri cir chat Id phdn hiru cir
Vao ngay thir 3. chung 2a vii LNlc htia tan dupe lupng P2O5 lan lupt la 275 mg/lOO g va 276 mg/lOO g vupl tryi so vdi cac chung con lai. Nhimg vao ngiiy 6, hai chimg vi khuan nay hda lan chgm lai trong khi cliung B5a hda tan lan hiru co tang manh tu 233 mg/lOO g (ngay 3) len 331 mg/IOO g va cac chiing 1312. 8s, 16s, LN2a, B9C1, N7a cung tang kha cao (Hinh 5).
Nhu vay sau 6 ngay theo doi. tat ca 9 chimg P.
siutzeri na, 8s, 16s, B5a, LN2a, LNlc, B9CI, N7a, 2a d^u cd kha nang hda tan lan hitu ca. Lupng PjiO^
trung binh cac chung vi khuan hda tan dupe d^u 358
khiic bi^'l CO y nghia thong ke (LSD.Ol - 4.35) so voi cluing ddi chimg va kha nang hda tan cao nhat la chung 2.1 (233.1 mg 100 g), chung B5a (233.8 mg/IOO g) \ a chung L N l c (230.9 mg/IOO g) (Bang 2) d mau phan htru co. mat sd ciia cac chiing vi khuan tang mgnh vao ngay 3 nhung lai giam vao ng>i> 6, cd the do ngudn dmh dudng ffong phan hihi CO dii giam nen mat so vi khuan bat dau giam theo v^o thdi diem nay (Hinh 6).
Hien nay tren the gidi cQng nhu d Viet nam, cac nghien ciJu va img dyng phytase de bd sung thiic an gia siic, gia cam vai cac san pham thuong mgi rao ban tren thj trudfng hay chi san xuat phytase txta co
Tgp chi Cong nghi Sinh hpc 10(2): 353-364, 20\2
chit ban ran nhimg chu ylu chi tren vi ndm (Lc Minh Thien d at.. 2003, TrSn Thj T u y a et al, 2004;
Dinh Minh Hi^p et id., 2009) vi cdng ngh? ly trich va bao quan che ph5m phytase vkn la mdt cau hdi khd cho cac nha khoa hpc Vi?t nam. Ket qua ciia chimg tdi fren vi khuin (dang don bao), cd nhOng thugn lai nhat dinh so vdi vi nam, dupe giiii tliich ve ca ch^ va budc dau chimg minh sy hi?u qua phytase
eiia VI khuan Iren ca hai ca chat (Ihitc an chan nudi va phiin hiru ca),
Vdi hai CO chdi la thitc an gia cimi (hon hpp cam- dau nanh) vili phan hiru co, qua trinh phat triSn ciia vi khuan da lam tang ham lupng phosphorus hihi dyng trong co chat va chiing cd kha nang hoa tan lan hfl-u CO nhal la /'. siutzeri 2a.
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Cao Ngpc Di^p & Nguyen Thj Nggc Anh
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Chung LN2a co 58 aminQ acid triing v6i 58 vj tri trong t6ng s6 638 ammo acid cua chiing c h u i n P. stutzen A1501 vcri i luwng dong 7,99%
( an N g g c Di?p & Nguyen Thi N g p c Anh
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Chimg N7ac6 170 ammo acid triing vdri 170 v| Iri trong lAng s i 63B ammo acid cua chung chuin P. s(uIzer)A1501 voi muc tirong d6ng 24,72%
Hinh 7b. So sanh tu'ng cdp giOa cic amino acid cua tirng chiing vi khuin vi^i chung chuin P stulzen A1501 bing phin mim BioEdit cho kit quci mirc dO ti/crng ding
KET LUAN
Co 3K 147chiJng/' i7u/reri tao duac halo (vung quang trong) tren moi tnrang bo sung acid phytic chijng to cac chiing nay co kha n^ing hoa tan lan hihi ccr va 9/38 chiing P stulzen cho san pham PCR co kich thuac phii hgp vai gen mk hoa phytase tir vi khuan doi chimg duong/* siutzeri A150I
Trinh tu DNA hi 5/9 chiing P. stulzen 2a, LN2a, 8s, 16s va N7a co dp tuang dong 99% v6i gen phytase cua P siutzeri Ps\50\. C a 9 chiing/', siutzeri 2a, LN2a, 8s, 16s, LNlc, B9C1, 13f2, N7a, B5a d^u CO kha nang hoa lan lan h&u ca a cac miic dp khac nhau trong do chung 2a hoa lan t6t nh^t tren ca hai ca chat cam - dau nanh va phan hiru co.
L i i cam on Chimg lot xin chan thdnh cdm an GS TS Rene De Mot (Dat hqc Leuven. Vircmg qudc Bi}
da lang chiing P. siutzeri AI SOI.
TAI LIEU THAM KHAO
Dirong Thi Bich (2009) Phan l|p vi khu^n Pseudomonas stulzen va vi khuan oxy hoa ammonia (AOB) lir nude, biin day ao nuoi tom va ca tra tai tinh Kien Giang, Lugn vdn
that .sTkhoahot ngiinh Smh Thai hgc. Dgi hgc Can iha.
Dmh Miiih RiC-p. Huynh Kim Mai, Ngo Dai N ^ i ? p (2009) T90 che pham phytase lit nuoi cay han ran vi nam Aspergillus mi^cr A2 Tuyen Igp hgi nghj cong nghe sinh hgc loan qiux luim :i)flV, Thai Nguyen: 594-597.
Do Thj Kim Chi (2009) Phan l^p vi khuin khu dam Pseudomonas stulzen lir JO nudi ca tra d Vinh Long va Dong Thap. Lugn VH/I thm si khoa hoc nganh Sinh thai hgc. Dgi hgc Can Thir.
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trudng Dai hoc Can Tho
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thdng III yill-l 243
\'ohia \. Saljanarayana T (2003) Phyiase Microbial Souices, Pioduclion, Purification, and polenlial Mioieihnological Applications Department of Mierobiotogi Univeisity of Delhi, India. 21 29-60
S C R E E N I N G A N D C H A R A { T E R I Z A T I O N O F S E \ E R A L P H Y T A T E D E G R A D I N G PSEi DO\IO.\.4S STi TZERI S T R A I N S F R O M C l U L O N G D E L T A
Cao Ngoc Diep'*, Ngujen Thi Ngoc Anh'
Bioiechnologi-Research andDeyelopmeni Institute. Can Tho Lnnersit}
-Thanh An High-Schooi Vinh Thanh District. Can Tho SUMMXR'i
Cereals, legumes and oilseed crops are grown in over 90% f the world's harvested area. These crops serve as a major source of nutrients for humans and animals An imponani constituent in these crops is phytic acid (nivo-inositol hexaphosphate). The salt form, phyiate. is the major storage form phosphorus and accounts for more than 80% of the total phosphorus in cereals and legumes Phytases are enzymes capable of hydrolyzing phytic acid lo less-phosphorylaied myo-inositol derivates and monogastric animal, such as pig, poultry and fish, are not able lo metabolize phytic acid and therefore inorganic phosphate isaddedtotheir diets to salify the phosphorus requiremeni This consequently contributes lo phosphorus pollution problems in areas of mlensive livestock production. In order to improve phosphorus utilization from phytale and phytin salts in grains and legume seeds for crop fertilizer and animal ieed, cspeiially for monogastric animals, a study was conducted to identify phyiase gene from Pseudomonas siutzeri and lo apply phyiate-degiading P^eKi/omowoi siutzeri strains in improvement of quality compost and poultry feeding The results showed that thirty-eiglit of one-hundred and forty-seven Pseudomonas siutzeri strains having a "halo" on the phytale-medium and nine of thirty-eight Pseudomonas siutzeri strains were determined positive in phytase gene PCR. The phytase gene from five selected strains were sequenced and compared with bacterial phytase gene available Genbank by BLAST N software and revealed 99% idenUfies with Pseudomonas stutzer'i AI501. The phylogenetic tree of five
* Author for correspondence: Tel: +84-913833792; Fax' +84-710-3835892; E-mail' cndiep&.ctu edii.v
Cao Ngpc Diep & Nguyen Thi Ngijc Anh .sequenced strains (2a. LN2a, N7a, 8s. 16s) wilh lelcienec I'seiidomoiias sliizeri A1501 strain showed that five slrainshiiveaiclalioiishipiloselv. All of nine sliains(2,i, l,N2a, N7a, 8s, I6.s, U9Cl,Nlc, 13(2, B5a) had good phosphaic-sulubling .ihilily m boih ol eomposl and poultry lued.Tlic Pseudomonas slulzeii BT2a slrain was a best one and will be usedas.supplemeniaiv ldi hi^ili quidiiy compost production and animal li^eds Keyivoids: jihosphoriissotnhlizaiion. pliyUise, pliyla.seeiuiuliiig iieiie. phviuie. Psendomonas.s'Inlzeri
