TINH SACH VA K H A O S A T D A C D I ^ M C O A ENZYME PHYTASE TIJ SPERGILLUS NIGER PE1
Phan Thj Bich Trim Trvdng Dg/ Hgc Cin Tha
T 6 M TAT
Mflt enzyme ngo^i bio phytase (myo-inositol hcxakisphosphate 3-phosphohydroiase, EC 3.1.3.8) tijr Aspergillus niger PEl dirge liiil s?ch c6 hoat tinh cao g ^ 14,7 lin va hi?u suat thu hSi ho^t tinh khoSng 37,4% so vdi djch trich phytase Uio. T i ^ trinh tinh syl dugc tien hinh qua cic giai d o ^ hia ammonium sulfate nflng dfl 80-90% ket hgp vdi sac ky trao d6i ion duong tr&i gel SP Streamline, sic ki mong tac ky oirdc trSn gel Phenyl Sepharose vl sk ky Igc gel tren gel sephadex G75. Di?n di SDS-PAGE vi s&
ky loc gel cho thay phytase tinh s?ch la mot chufli polypeptide voi trpng luyng phin tii k b o ^ 87 kDa. Phytase tit Aspergillus nipi PE I hoat dflng tflt nhat fl pH 3,5 va nhiet d$ 65 "C. (5 nong dg 1 mM va SmM Ca * c6 tac dyng boat hoa hojt tinh phytase nhung Ifj b|
lie chS bdi mgt so ion Fe^*, Mg^*, Mn^', Na', K* and Zn^*
MdeAu
Phytate, muoi cua acid phyttc Id mpt hinh thijc liru trip phosphate du^i dgng myo-inositol chiem trin 80% ting sii phospho trong ngu d c vd cdy hp dgu. Cdc d$ng v$t nhai Igi cd thi tieu h6a phytate thSng qua hogt dOng ciia enzyme phytase dinTc tgo ra bai cdc logi nam vd vi khuin ky khf hi^n dign bong d^ cd cua chung. SI,F thCiy phdn phytate bdi enzyme phytase gidi phdng phosphate vd ca vd myo-inosilol tt;' do Id rit cin thilt cung d p lu^grng phosphate ctio co ttii dOng v$L Tuy nhidn, khi khOng du^e thiJy phdn phytate lgi Id thdnh phan khdng dinh du^ng tnsng thifc dn c6 nguln gAc tir thi,rc vdt do chOng kit \vyp cdc khodng chit thilt ylu nhu* canxi, magiS, sdt vd kem tgo phi>c phytate-khodng khOng hda tan trong du-dng rupt ngdn d n si,r hip thu khodng Ddc bi^t doi vdi cdc lodi d$ng vgt dg ddy m$t ti^l nhu' \gn, gia d m vd d sir dgng phospho ti> phytate rit kdm do thilu enzyme phytase trong dirdng tidu hda. Do dd, phosphate vd ccr cin di/(rc b l sung vdo th(>c in cOa chOng de ddp i>ng ydu d u phosphate ddm bdo tang tn/dng vd lu'p'ng phytate i\i thCra bj ddo thai gdy d nhiim mdi trudng .
v l n d l d t h i dugc gidi quylt bang d c h si> dung enzyme phytase tru-c tilp hogc tCr cdc nguln vi sinh vdt bo sung vdo thi>c an gia sOc (Simell ef al.. 1989) nhim gidm thip lugng phospho phi thdi Id mdt Ig^i the cOa d c ca sd chan null vi nd Id mdt chit gdy d nhiem. Hidn nay, mdt vdi nghidn cuu v l phytase tCr nam Aspergillus niger hoang dgi (Sariyska el al., 2005), Aspergillus niger 11T53A9 (Greiner ef at.. 2009), Rhizopus oligosporus ATCC 22959 (Casey vd Walsh, 2004) hogc tLF vi khuan Bacillus sp. DSII (Young-Ok Kim ef al., 1998) d i du'Q'c thM'C hi#n nhung con khd mdi me d Vl^t nam..
Tgi Vidn nghidn cuu vd phdt trien Cdng ngh# Sinh hpc Dgi hgc Can Tha da phan tdp duoc chiing Aspergillus nigwPB d kha ndng sinh phytase rat cao, trong phgm vi bdi bdo ndy chOng tdi binh bdy d c ket qud v l khd ndng tdch chilt, Bnh sgch vd khdo sdt mOt vdi ddc dilm y l enzyme phytase nhim su dung higu qud nguln enzyme nay gdp phin cho s\f phdt triln ngdnh chdn nu^ d ddng bdng sdng Cuu long.
VAT LI^U VA PHlfONG P H A P V | t ll$u
Chiing mdc Aspergillus niger PEl h> Phdng Cdng Nghd Enzyme, toro-ng Dgi Hpc Can Tha.
Hda chit
Albumin huyet thanh bd, Coomassie Blue G250, ethanol, acid phosphoric, glycine, Tris-HCI (Merck), sodium phytate (Sigma), gel trao doi ion SP-sb^aline, gel phenyl sepharose, Sephadex G75, hod chit cho SDS-PAGE (BioRad), protein
chuin (Fermentas)... , Phuang phdp ' + Hdm li/(mg protein dugc xdc dinh theo phu'ang phdp Bradford.
+ Hogt tfnh phytase dui^c xdc djnh trong 1 ml t h i tich enzyme d nhiet dd 55°C trong 30 phOt vdi dung djch d§m 0,2 M glycin-HCl pH 3,5 chua 1 mM natriphytate. Lugng ortho-phosphate sinh ra dugc dinh lugno theo phuang phfip
HeinonenvdLahti(1981). ' 1 Don vj hogf dS riing cda enzyme bieu hi$n qua U/mg protein. Mdt don vj hoat tinh cua phytase (U) dui?c xic dinli M
/u^ng enzyme phytase cin thiit thdy phin natriphytate, tuvmg duxmg 1 pmol phosphate vd ca giai phdng trong mdt phiit Cmg vtri cic didu kiin thi nghiim.
Dign di SOS-PAGE theo Hames (1998) vdi gel polyacrylamide 12%. Xac dinh khoi luang phdn tu' cua protein dgatrfin thang protein chuan cua Fermentas (14,4-116,0 kDa).
Thu nh|ln phytase thd
Nudi d y Aspergillus niger PEl trong mdi trudng bdn ran theo Nguyln Thj Xudn Dung vd dfing tac gid (2012). Sau 5 ngay nudi d y tiln hdnh ly trfch phytase bang dung djch d§m 0,2 M glycine-HCI pH 3,5 thu dugc dich phytase thd.
> " , ; 0 N G NGHE SINH HOC TOAN QUOC 2013 Tinh sgch s c b$ phytase b i n g phuwng phdp tiia
I u f e t e ' 3 n ^ l n ? v ^ K ? ° i r ' w ^ " ° ' "^ ^'=®'°" t^'^) * "^^ *" '^ ''•''• '••2' ^-^- ^•^' ""-^ "^ •"•6- Tua phan doan ammonium
«Anh k i t r,i • ^ 7?° ^^"^ ' ^ ^ ^ " f i ^^"^ ^"^Q protein va &) hogt tinh phytase tren ca chit natriphytate. So phytase ^^ ^^ ^*'^^"°' ^^ "=^9" phuang phap tua so bd t i t nhlt. Hip tyc tien hdnh chgy sic ky tinh sgch Tinh sgch phytase bang ky t h u i t sac k^
S i S . S "^u'-fc ' t " ' ^ ' ^ ^ ' ®^3/n//rie; Sau khi tinh sgch bang tiJa sa bd, phytase duac hoa tan bong ddm 0,2 M
^u^hX" D f • u ^ ^J]° ""9 '^•'^^ ''"^ "=91 sac ky bao doi km tr6n cfit SP- Streamline (1,5 x 40 cm) vdi toe do ddng 0,8 ml/phut. Protein hap thy difgc rii-a gidi bdi gradient nfing dfi 0-0,5M NaCl, thu khoang 30 phan doan.
^i'^^h^^'-'^ ^ n£«Jc (ren cgt phenyl sepharose: Phdn dogn protein d hoat tinh phytase ttiu dugc sau khi qua cdt
^A n fl i/°h °.' o " y^^ *• " ^^ '^^° °'^^ "^^ ^"^"9 *^^ ky " ' ^ c P^ieiy" Sepharose vdi kich thudc d t (1,5 x 30 cm), tdc ap U.0 mi/pnut. bip dung dung djch d#m 0,2 M glyeine-HCl pH 3,5 chua 30% ammonium sulfate (AS) bao hda. Tiln hanh rua giai protein bam b-en cpt bang gradient ndng dp giam din tu 30%-0% AS bao hda, thu khdng 45 phdn dogn.
Sic ky' Igc gel trdn cgt Sephadex: Phan doan protein c6 hoat tinh phytase sau khi qua cot Phenyl Sepharose dugc thim tich logi muoi trong dgm 20 mM Tris-HCI pH 7,5 + 0,15 M NaCl. Sau dd cho cfit loc gel Sephadex G75 vdi cOng dung d|cn dgm. toe dO ddng chay 0,5 ml/phut. Phytase tinh sgch dugc dfing khS bing thilt bi VirTis (USA).
Cdc phdn doan protein sau khi n>a giai d cdc cdt sac ki duoc thu nhdn bin hanh xdc anh ham luang prertein, do hogt tmh phytase. Dlin di SDS-PAGE kilm tra dp tinh sach cua enzyme. '
Khao sdt cac y l u to anh hir6ng den hogt tinh phytase «nh sach Xac dinh pH toi tm
Enzyme tinh sach dugc hda tan vdi ndng dp 1 mg/ml d cac gid tri. pH khac nhau vdi cdc dung djch ddm glyeine-HCl (pH 2,5; 3,5); natri acetate (pH 4,5; 5,5); maleate (pH 6,5) vd Tris-HCI (pH 7,5) Enzjmie dugc 0 d cde pH ndi tren trong 30 phOt va tifin hdnh do hogt tinh phytase d nhigt dd d 55''C frSn ca chit nabiphytate vd xdc dinh hdm lugng phospho sinh ra theo phuang phdp Heinonen vd Lahti.
Xic dinh nhiit dd toi uv
Phytase,tinh sach dugc hda tan trong dgm 0,2 M glycine-HCI pH 3,5 vdi nfing dd 1 mg/ml va Ci d d c nhiet do khdc nhau 35 0, 45 C, 55 C, 65 C, 75 C, 85 C trong 10 phiit. Tiln hdnh do hogt tlnh phytase tgi cdc nhidt dd tren vdi ca d a t mudi natriphytate vd xdc dinh ham lugng phospho sinh ra theo phuang phdp Heinonen vd Lahti.
Khao sit anh hwdng cua cac ion kim loai den hoat tinh phytase
B l sung cde mudi Ca^*, Mg^*, Mn^*, Na*, K* vd Zn^* d nong dp 1 mM vd 5 mM vdo dung dieh phyatse sau khi tlnh sgch.
u d 4=^: trong 30 phiit. Sau dd tiln hanh do hogt tinh phytase. Hogt tinh phytase cdn lgi (%) dugc so sdnh vdi hoat ttnh phytase mlu d l i chung khdng bd sung ion kim loai.
K ^ QUA VA T H A O LUAN Thu nhan phytase thfi
Nudi cay A. niger PEl d mfii trudng bdn ran trong 5 binh tam giac thu dugc 335ml dich trich phytase thd d hogt tinh rieng 1,65 U/mg protein, mgc du cd hoat tinh rieng thip han phytase thd tu A. niger 11T53A9 Id 2,6 U/mg protein (Greiner ef al., 2009) nhung ddy la nguln enzyme c6 hoat tinh kha cao va d l dang thu nhgn nen rat thudn tifin cho vide tinh sgch vd khao sat d c dac diem cua phytase.
Tinh sach so> bg phytase bang phirang phdp tiJa
Djch frloh phytase thd dugc tOa phdn dogn ammonium sulfete (AS) bao hda dugc ghi nhgn hoat tfnh neng cua phytase cao d hai phSn doan tiia 80% den 90%. Hoat tinh neng frung binh c^a hai phan dogn ndy khoang 6,24 U/mg, gap 3,8 lln so vdi hogt dp rieng d mlu thfi Id 1,65 U/mg vdi hieu suat thu hot hogt tinh phytase cua hai phdn dogn protein nay dgt 67,8%. So vdi tOa djch trfch phytase thd vdi aceton vd ethanol mac du cho hieu suit thu hoi cao nhung fi'ogt tinh rieng phytase thip han khoang 3 lan cd t h i do phytase bj biln tfnh mdt phan bdi cdc dung mdi huu ca. Nhu vdy, neu tOa djeh trich phytase thd bing phdn dogn AS bao hda tCr 80%-90% se loai duac kha nhilu protein tgp. Dieu ndy r i t Ehudn Igi cho cac budc tmh sgch ke tiep - tinh sach bdng phuang phap sac ky' Kit qua kha phu hgp vdi nghidn ci>u tinh sgch phytase ngogi baotij'A n/ger11T53A9 cua Greiner va ddng tde gia (2009) dat hoat tinh ridng cao nhlt khituadjd phytase thd vdi AS bao hda trong khfiang rfing han ICF 0-90%.
Tinh sgch bang cdc ky thudt sac ky
Qua khao sat so bp, dilm dang dign cCia phytase tu A. niger trong khoang 4,5-5,0 va phytase ben trong mdi tnfdng pH thip, VI vdy, gel trao dli ion duang SP-Streamline Id budc tnh sach phytase dau tien dugc Uiuc hign. Djch trich phytase thd sau khi qua cfit SP-Streamline cht thu dugc mpt tfnh protein cd hoat tinh rifing phytase la 8,56 U/mg cao gip 5.2 lln hoat tfnh rifing d a miu thd la 1,65 U/mg vd gip 1,4 fan hogt tfnh rieng cua mau tua AS bdo hda. Thu nhdn phytase thim tfch logi mudi NaCl, bo sung muoi AS 30% bdo hda va cho qua cpt_ Phenyl Sepharose thu nhdn tfnn protein d hogt Hnh cua phytase 17,41 U/mg rao gip 10,6 lan hogt linh rifing cda mau thd Id 1,65 U/mg. Cufii cung thu nhgn phytase cho qua cfit fpc gel sephadex G75 de logi bd d c protein tap thu mpt dinh protein duy nhlt d hoat tfnh rifing phytase cao gip 14,7 lln so vdi hoat tinh phytase mlu thd ban dau. Ket qua the hien d bang 1:
HOI NGH! KHOA HQC C O N G NGHf SINH H O C '
Bdng 1: Bdng ting kit cac bude tinh s^ich phytase tCr A. niger PEl
Cac budc tinh sgch
MiuthO TOa AS SP-streamlira Phenyl sepharore Sephadex G75
T6ng(mg) 33 5,89 3,97 1,24 0,84
Protdn Hi9u s u i t (%)
100 17,85 12,03 3,76 2,55
Hogt tinh Tfing(U)
54,28 36,73 34,11 21,66 20,33
enzyme H i d u s u l t (%)
100 67,67 62,84 39,90 37,45
Ho^t tlnh ddc hieu (U/mg) 1,65 6,24 8,56 17,41 24.20
sgch 1 3,78 5,19 10,55 14,72 Di|n di SDS-PAGE (Hinh 1) vdi s^ hidn dign cOa p-
mercaptoethanol cho thiy d i d n n ^ bdng protein duy nhlt d trpng lugng phdn ti> khodng 87 kDa. Nhu vdy, phytase dugc tinh sgch tO A. niger PEl d d u tnic dan phdn ddc trrnig cho [diytase cua nim, phu hpp vdi d c k i t qud d a Greiner vd dong tdc gid (2009) nghidn ci>u trdn phytase ngogi bdo ti> A.niger 11T53A9 cd trgng lugng phdn to* Id 85 kDa, Pandey va ding tde gia (2001) nghidn ci>u phytase mOt d chiing Aspergillus sp d trpng lugng phdn b> tCr 40 din 100 kDa. Tuy nhifin, nghien ci>u ndy khde vdi kit qui tnrdc day d a Sariyska vd dfing tac gid (2005) trin phytase ngogi bao tinh sgch tir A. niger hoang dgi d frpng lirgng phdn ti> Id 39 kDa.
Khdo sdt d c y i u td anh hudng den hoat tinh phytase tinh sgch
Phytase sau khi qua d c budc tlnh s g d sa bp vdi 80%-
90% AS bdo hda vd d c budc s i c kj?. Tiln hdnh thu nh$n, ^ , „ , ^ ^ . ^ „ , . „ „ ^„„ ^ « . ^ „ ™ „ „ . y„„y .. ^,„^, thdm tfch logi mufii, ddng khfi vd khdo sdt mOt s l d§c p/ieny/sep/ianMe, g/^ng6:p/iy(aseqt/asep/?a(/exG75 dilm.
Hlnh 1: Oi$n di SDS-PAGE phytase Asp. Niger PEL Giing 1:
pmtein chuin. giing 2- phytase thd, giing 3: phytase sau tOaAS.
giing 4: phytase qua cdt SP-streallne, giing 5: phytase qua c^
pH t6l uv cho ho^t d^ng ciia phytase
pH td mdt trong cde y l u t l quan trpng dnh hudng len hogt tlnh d a enzyme. Kit qud * Hlnh 2 eho thiy phytase tir nim mdc A. niger PEl hogt dfing mgnh d mdi tnrdng acid trong khodng pH 2,5 - 4,5 vd dgt gid trj tdi uu tgi pH 3 5 Ddc dilm ndy phO hgp vdi dilu kign tifiu hda trong dg ddy (pH 2.0 - 4,0) ctia dfing vgt, vl v$y d t h i dCing bo sung trong thi>c in gia sOc vdi hidu qud cao (Casey, Walsh.. 2004).
Kit qui cOng phii hgp vdi hlu h i t d c nghidn ci>u khi xdc (^nh phytase d A. niger hogt dfing d pH trong vOng add theo Gargova vd Sariyska (2003) phytase ti> chiing nam mic A. niger 307 d pH hogt dfing trong khoang 2 5 - 5 0 Tiong ktu dfi, phytase tCr vi khuin ed pH tfii uu cao hon (pH 6,5 - 7,5), * Bacillus pH tfii uu d a phytase Id 7 0 (Kim et al 1998}
rifing d Enferoftacfer.sp phytase cd pH tfii uu la 7,0 - 7,5 (Yoon etal., 1996).
Hlnh 2: Anh hufrng cua pH ldn hogt tlnh phytase "'"'* ^'- ^*^ hudng cOa nhif t d$ ldn hogt tlnh phytase |
Nhi^td^tdltfu i
S ' t ! i ? ™ L ^ T n - * ^ ' " ^ " AT ' ' " ^ " ^"^ "^"9 ™' P"- ^^'^* ^^ '^ y^" ^ ''"^^ t ^ " 9 Snh hudng Idn din hogt tfnh xDcnhi£^!dZT^hlJZ ^nl^ ' J l T T l f ^ ^^f " ! " 9 "^^"i "^'^'' P"^^" ' ^ '^'^ « " ^ " i e khdng bin dudi tdc dyng cfia nhidt dO cao. phdn tu enzyme bi biln tfnh thay dfii v l ni§t clu tnic, din din lam mit hogt tlnh ;^c tdc.
65*C " N h ^ d t ^ S f l ^ t ^ r n n t ^ & f ^ ^ . ^ r ' ^ " " ^^^ ^°^^ ''^"S ' " ^ " ^ * "f^'^» ^^ kh^ <^° ^ SS-^S^C, dgt tdi uu tgi gid t.1 65 C. Nhifit dfi ndy cung nim trong khodng hoat dpng ciia cdc phytase khac nhau t£r 40 din 77°C (Kim ef al.. 1998)! Tiiy nhidn nhilt iJfi tfii uu eua phytase tinh sach cao han so vdi k i t qud d a Sariyska vd dfing tdc gid (2005) ly trich phytase ngog, bdo tu lodi Aspe,B,//us niger hoang dgi, Greiner vd dfing tac gia (2009) tir Aspergillus niger 11T53A9 vdi nhi^l dO
; ( J U N L J N ( 3 M t S I N H M V U I O A N Q U O C 2 0 1 3
K+ zn2+
Hlnh 4: Anh h i r d t i g cua Ion ldm logi l i n ho^t tinh phytase Wi i r u Id 5 5 ° C tgi p H 2 , 6 2 v d 5 8 ° C t a i p H 5.5 t u a n g u n g . & nhifit dp t h i p trong k h o a n g 3 5 - 4 5 ° C phytase hogt d f i n g y l u . T g i n h i ^ t d d 8 5 ° C p h y t a s e m i t h o d n t d n hogt t l n h .
Anh hirimg cOa cic ion kim lo^i den hoait tinh phytase
N h i m i m g d u n g p h y t a s e trong thiJc d n gia sue v i v d y v t ^ c k h a o s d t d n h h u d n g c i i a m d t d ion kim l o g ! p h a b i l n t r o n g mfii t r u d n g Id d n t h i l L D i / a v d o k i t q u d H i n h 4 eho t h i y . p h y t a s e b> A niger P E l s a u khi tinh s g c h d hai nfing d p ion C a 1 m M v d 5 m M d i u l a m t d n g hogt t i n h d a p h y t a s e t i r 1 0 3 - 1 0 8 % s o v d i d l i c h u n g . H i e u q u d i>c c h l p h y t a s e t d n g khi t a n g n d n g d d b d s u n g d c ion k i m loai t i r 1 m M d i n 5 m M v d bj u c c h l m g n h n h l t Id i o n 2 n * d n f i n g dd 5 m M Idm g i d m k h o a n g 5 8 % hogt t i n h s o v d i ddi chi>ng. T u y nhidn mire d p t d n g h i f i u q u d i>e c h e ndy d mfii ion kim logi lgi k h d c n h a u . D f i i v d i M g ^ * vd K* chSnh l$ch hi$u q u i ire c h e k h d n g c a o d hai nfing d f i 1 m M v d 5 m M . T r o n g khi d d , ion Fe^*, M n ^ v d ZTT m u c c h e n h Ifieh ndy d i n 1 3 - 1 5 % higu q u d d c c h e . R i e n g d f i i v d i N a * d 1 m M hogt t i n h phytase g i d m 7 % n h u n g 6' n f i n g d f i 5 m M hogt t l n h g i a m d e n 4 0 % .
C d c k i t q u a trdn c u n g p h i i h g p v d i n g h i d n ci>u eua Sariyska v a d d n g tac gid (2005) ttnh s g c h phytase n g o g i b a o ti>
Asper^llus niger hoang^dgi c u n g xac djnh ion C a ^ * hogt hda phytase n h u n g d nfing d f i t h a p h a n 0,1 m M v d tang Ifin k h o i n g 1 0 7 , 8 % , ion F e , N a * , K * v d M n ^ * u c c h l h o g t dOng phytase v d i hogt tinh d n Igi Id 59,26 % , 9 4 , 5 9 % , 9 5 , 0 8 % vd 9 6 , 3 8 % t u a n g i>ng. T h e o Helsinki (2000) c d c ion kim logi a n h h u d n g Idm g i d m hogt t l n h ciia phytase d t h i d o d c Ion ndy t g o p h u c h d a t a n k d m v d i c a c h i t phytate ldm g i d m nfing d p h o l e lifin k i t v d i e n z y m e ldm thay dfii d u t n i c d i n d i n g i a m hogt t l n h . N h u v g y , d l trdnh ldm g i a m hogt tfnh phytase khi bfi s u n g c h i i n g v d o t h d c dn d n chu y n h i l u d i n h d m l u g n g d c ion kim Iogi.
K £ T L U A N
N g h i i n ci>u d a t h u n h d n d u g c e n z y m e phytase vfin d f i Bnh s g c h 14,7 l l n s o v d i phytase thd trich ly tir sinh khfii n i m mfic A. niger P E l khi q u a cde b t r d c t u a p h d n d o g n a m m o n i u m sulfate bdo hda tir 8 0 % - 9 0 % , s i c ky trao doi ion d u o n g trfin gel S P - S U e a m l i n e , s i c k^ t u a n g tdc k y n u d e tren gel Phenyl-Sepharose vd cfit Ipc gel sephadex G 7 5 . P h y t a s e tinh sgch thu d u g c d c l u t n i c d o n p h d n v d i trpng l u g n g p h d n t i i k h d n g 8 7 k D a d u g c xdc dinh bdng dign dt S D S - P A G E . pH vd nhifit d p tfii u u c u a p h y t a s e l l n l u g t la 3,5 vd 6 5 ° C , d u g c hogt hda k h d m g n h b d i ion C a ' * d 5 m M vd d i u bj i r c c h l b d i mfit s f i ion kim logi Fe^*' Mg^*, M n ^ ' , N a * , K* v d Z n d d hai nfing d f i 1 m M v d 5 m M
T A I LIEU T H A M K H A O
Nguyln Thi Xuan Dung, N g u y l n V i f t Khoa, Nguyln Vdn Tlnh, TrSn Nguyin Nti$t Khoa vd Ldm Thj Kim Chung (2012). Toi uu hda mdi tnrdng nu6i c l y Aspergillus Niger dh tang h i f u suat sdn sinh phytase. Tap chi Khoa hoc Tn/c/ng Bai hpc C3n Tha 2 4 ; 222-232.
Greiner R, Lucineia G, Sonia C (2009). Punfication and charactensabon of an extracellular phytase from Aspergillus niger 11T53A9.
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Sariyska MV, Gargova SA, Koleva LA, Angelov Al (2005), Aspergillus niger F^iytase: purification and charactenzation Sotechnol S/o/ecftEq 19(3): 98-105.
Simell M, Turunen M, Piiranen J . Vaara T (1989). Feed and food applications of phytase. Lecture at 3rd Meet Industrial Applications ol Enzymes, Barcelona. Spam.
] Purifk:atkxi and properties of a thermostable phytase from Bacillus sp. DSI.
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Yoon SJ. Choi YJ, Min HK, Oio K K, Kim JW, Lee SC, Jung YH (1996). Isolation and identificafion of phytase-producing bactutun. ]k Enterobacter sp. 4, and enzymatic properties of phytase enzyme. Enz Microbial Technol 18: 449-454.
PURIFICATION AND CHARACTERIZATION OF PHYTASE ENZYME FROM ^
ASPERGILLUS NIGER PE1 w
Phan Thj Bich Tram
College of Agriculture and Applied Biology - Can Tho University ^"
An extracellular phytase (myo-ioositol hexakisphosphate 3-phosphohydrolase, EC 3.1.3.8) bom Asper^llus niger V^\ was purified „'L about 14.7-fold to ^parent liomogeneity with activity recoveiy of 37.4% referred to the phytase activity in die crude extract. ';
PuiificatioD was actiieved by 80-90% ammonium sulphate precipitation m combination with diromatography on ion SP-streamline ' exchange cohnnn, Phenyl-Sepharose hydrophobic cohimn and sephadex G75 gelfiltiation column. The molecular weight of purified ',.
phytase estim^ed by SDS-PAGE in combin^on with gel filtration was a monomeric protein of about 87 kDa. The optimum pH and '^
temperature for phytase activity were at pH 3.5 and SSX, respectively. The enzyme was activated by Ca^ * and inhibited by a number '
of ion including Fe^*, Mg^, Mn^*, Na*, K* and Zn^'' at 1 mM and 5 niM concentrations. iil.
Key words: phytase, SP-Streamline, Phenyl Sepharose, Sephadex 075, Aspergillus niger PEl, phytate degradation.