I O N G N G H f SINH HOC T O A N Q U O C 2013
TINH SACH VA XAC OINH BAC DlfeM HOAT DONG CCiA ENZYME PROTEASE l/A NHiET Tl> C H O N G XA K H U A N STREPTOMYCES
PHAEOLUTEICHROMATOGENS X598
Trin Thj L# QuySn, Ddo Thj Linmg, Trinh Thanh Trung. Trinh Van Anh, Diwng Van Hg-p van Vi sinh vit vi C6ng ngh$ Sinh hqc, B^i hgc Qu6c gia Ha Noi
TOMTAT
Protease ia m5t nhom c i c enzyme c6 Uia nang phin o i l c i c Uen ket pq>Ude tiong pMn hi pnitein t?w i h ^
cic phan tir ammo acid. Protease dupe flm t h ^ & nhi&i vi sinh vjt k U c nhau g6m c i nh6m nhin chuin va nhan sa nhu nim moc, vi khuAn, x» khuin.
d H )daan, d | c bi^l l i streptomyces. dugc biit den Wri kha nang t i ^ nhieu loai protease vio mSi tniong nuoi cly. M{lt protease chiu ohi^t diti;« tinh sach t£r chung x? khuin Streplon^cespkaeoluteichromatogaies X598 v i xic dinh cac die d i ^ hojt dpng. Enzyme d u ^ tinh sach qua c^t sic k j trao d6i ion CM dgt mile d J tinh s?ch 16 lin vdi tJ 1$ ihu h6i I i 13%, Qua c§t sic ky loc gel protease tinh sgch dugrc thu 6 phan dofin 26-30 c6 Wch thudc 26 kDa tren bin d i ^ di SDS-PAGE. Protease tinh s ^ cfi nhi?t d$ host d ^ tfiiini l i 50°C v i pH t6i uu la 8. Ho?t tlnh protease tang nh? khi c6 mjt cic ion K , Ca , Na*, Fe" * n6ng do tii 0,5 d&i 5 mM, trong khi d6 cic ion Mg"*, Mn**, EDTA v i SDS lam giim hofi tlnh protease. Protease cua chitag X598 ben n h i ^ 6 6 0 ^ 6 thoi gian xii ly l i 30 phiit ho9t tinh cua e o q m e diqr tri 82% so v6i d6i chiing. Khi xii ly nhi?t a 8 0 ^ ho^t tinh sau 30 phut
Ti khoa. protease, tinh s?ch, ho?t tinh, Streptomycespkaeoluieichrumatogenes X598, hogt d ^ g t6i uu
Md'DAU
Protease la nh6m enzyme cong nghi§p quan trpng nh4t, chilm gan 60% thj phin e n z ^ e cflng nghi?p tren th6 gi6i.
Protease dtfgc sir dyng trong cong nghiep tiy ri>a, thuijc da, khai khoang. do mgc dich y hgc. chl biln thyc phim. c h l biln thi>c an chSn nufli cung nhi/ xi> t;^ chit thSi. Cdc vi sinh v^t \i ngu6n sinh pnstease dir?c quan tdm nhat bin sif da dgng v l mSt h6a sinh vi tiem ning iing dgng cOa chung trong cflng ngh$ sinh hoc {Lazim e( al.. 2009). Protease vi sinh v|it chilm khoSng 40% tdng so enzyme diriTC ban trfin toin t h i giai. Gan 6iy. cic pnatease thu-cng phim chii ylu \i dc sSn phim tO nim, vi khuan va xg khuin (Lazim ef ai., 2009). Xg khuan. dgc bi§t la streptomycetes, du'Q'c xem Id nhflm vi sinh vdt tilt da enzyme protease vdo mfli triFcrng nufli d y , Trong s6 cdc protease ndy cfl serine protease cua Streptomyces giiseus vd Streptomyces fradiae da du'pc xdc djnh cdc dgc dilm enzyme vd ciu tnic. Ngodl ra cflng cfl nhilu cflng b6 v l d$c dilm hogt dflng vd tinh sach cua cdc protease kilm duvc phdn Idp tir cdc iodi khdc thuflc chi Streptomyces (Bressoilier ef a/.,ig99).
Trong cflng b l ndy, chung tfli trinh bdy ddc ^ I m tinh sach va mflt so dac dilm hogt d ^ g cfla enzyme protease va nhi#t tir chung xg khuin Streptomyces phaeoluteichromalogenes X598.
NGUVeN ueu VA PHU'aNG P H A P
ChOng gidng vd mfli tnro>ng nuoi cly: Chflng x^ khuin Streptomyces phaeduteichromatogenes X598 dug'C cung d p b6i VTCC (Bdo tdng giong chuan Vi sinh vgt- Vign Vi sinh vgt vd Cflng ngh§ sinh hoc). Mfli tm-flrng YG (g/l:cao nam men-10. g1ucose-10) di/g^ si> dgng cho Ifln men djch t h i thu protease.
Tlnh sgch. Djch nli sau Ifln men dug^ cfl dgc bang phvcmg phdp Igc tilp tuyin qua mdng 300 kDa va 10 kDa. sau dfl tOa ammonium sunfat 30-80% bdo hfla, cdn tua 6irac hda tan trong MQ vd tra Ifln cdt CM sepharose si> dung d$m Tris- HCI 0,02 M, pH8,0. Phdn doan c6 hogt tlnh protease dug'C tinh sach bang sac ky Igc gel Superdex 75 Hiload 16/60 da du-g-c cdn bang vfl'i dgm 20 mM photphat pH 8, difln di SDS-PAGE phdn dogn co hogt tfnh protease (Bressoilier et a/., 1999).
Xdc dfnh dgc d i l m enzyme. Cdc d$c dilm hogt dgng cua protease diung X598 gom nhifit dfl vd pH hogt dgng t l i ini.
bin nhigt, anh hifflng cOa ion kim logi dirg'c nghien ci>u theo Bressoilier vd ding tac gia (1999).
11 Xdc djnh hoat tinh protease. Hogt tlnh protease du^c xdc djnh tiieo Lazim vd dong tac gid (2009).
i! K £ T Q U A V A T H A O LUAN Tlnh sgch
Djch nufli d y cfla chflng X598 dirg'c tinh sgch vd cfl dgc lln lu^t qua mdng 300 kDa vd mdng 10 kDa SLF dyng mdy Igc tiep tuyin, dich cfl ddc 6iriyc tfla bdng ammonium sulMe phan doan 30-80%, cgn tua dug'C hfla tan vdo ntfdc MQ roi tra Ifin c0t sac ky trao d l i ion CM sepharose. Kit qua trfin hinh 1 cho thay lirgng protein tiifli ra 6 birirc n>a cgt \an trong khi iiogt tfnh protease hlu nhu khong cfl, cfln hogt tinh protease cao d hiri/c thfli cgt, dieu ndy chi>ng tfl enzyme protease ciJa chflng X598 bdm cflt CM sepharose. D l kilm tra enzyme sau khi tiln lidnh sac ky trao dli ion fli tinh sgch chua.
d|ch enzyme & cdc phdn dogn cfl hogt tfnh dugc tiln hdnh chgy dign di SDS-PAGE.
H O i N G H j K H O A H O G C O N U N u n i ; : ^
CM 598
Mnh 1. S i c ky d i thih »?ch protease chiing X598 theo phifong p h i p s i c ky trao d i i ion CM-sefrfiarose
7 10 13 1« 1 * 22 2S 2B 31 34 37 40 43 4S 49 51 55 sa
Hlnh 3. S i c ky dA tlnh s f c h protease chijng X598 theo phuwng Hinh 2. Di$n di SDS-PAGE cQa cdc phSn dO^n bdm i i |
" " " ' ''"'' ' sau khi ch^y sac liy trao d d i ion CM-sepharose. CMSIt protease thu d i r o c t i r sSc ky trao d l l ion CM-sephmM p h ^ s i c ky l9c gel
Marker CM598 G598
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60^-*-70^-»-8ffc|
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protease m u d i J ^ l t , s i c k y V S ' ^ " ' ' ' ° ^ ^ ^ ' <B) cila protease chfing X598. ' ^ j
Hlnh dnh difin di SDS-PAGE (Hinh 21 chn i h i i , H U I , ^
" o n cpi sac ky Igc gel. T u hmh 3, cho thSy p n j t e a s e c h l au(»c thol ra ft phan dcnn"
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DNG NGHE SINH HQC TOAN QUOC 2013
26 den 30. De xdc nhdn t^i dg tinh sgch cua protease, cdc phan dogn cfl hogt tinh protease du'ac frfln lai voi nhau, cfl dac Oang phuwng phdp dflng kho vd tiln hdnh kilm tra bdng phuwng phdp difin di SDS- PAGE.
^?i ^ S ^'^ '^^" **' ^'^^•'^**^^ (f^'"'^ 4), chflng toi ttily ch! xuat hifin mflt bang protein duy nhat sau qud trinh sdc ky Igc hi t,' '^^"'^9 to rdng protease thu duvc sau qua trinh sac ky Igc gel Id protease tinh sgch. Dya vdo kich thu'6c pnan ftj protem maker rfiflng toi xdc djnh protease sinh ra tir chung X598 cfl kich thu'fl'c phan tfl- khoang 26 kDa. Tinh 2f ttf"^^'^ ^^ *^° ' ° " ' Pi^^^se tfl- djch nufli d y chung X598 da du'O'c tinh sach 16 lan so vd'i ban dau va higu sudt mu hfli hoat tfnh dgt 12,3% v6i hogt dfl rieng Id 29,6 U/pg. Tren doi tuwng xg ktiuan tac gia Singh (2011) da cflng 00 protease tu chung Streptomyces sp. A6 du-crc tnh sgch 34,56 lln qua sac ky Igc gel co khli lu'ang phdn tfl' 20 kDa (Singh yaChhatpar, 2011); hay protease ti> Streptomyces sp. AB1 cfl khfli lutyng phdn ti> 29.8 kDa 6irac cong bo hai Jaouadi vd dflng tdc gid (2010); cfln Bressoilier va ding tac gia (1999).ad tinh sach vd xac djnh kfch thu'dc phan tu' protease tU' Streptomyces albidoflavus Id 18 kDa.
D$c diem cfla protease chflng X598
Anh huvmg cua nhiit dd den ho^t ddng cua protease X598
Phan LPng enzyme ca chat duryc tifin hdnh d cac nhigt dfl 20. 30, 40, 50, 60 vd 70°G. Oo thi hlnh 5A cho thiy protease cua Chung X598 hoat tot d nhifit dg tfl' 30-60°C, trong dfl nhiet dfl t l i u-u Id 50°C. Difiu ndy churg to protease cua chung on 1 on "/^'^*-J'®" ^ ^ " ^ '*"' '* enzyme protease ir cdc nhigt dg khdc nhau tCr 60 den 80°C trong khoang thoi gian 10.
20 vd 30 phflt roi Idm phdn u'ng fl' nhifit dO toi u-u doi v6i enzyme (SO^C). Du-a vao d l thj hinh 5B ta thay khi xfl' 1^ 6 nhiet dg 60°C protease cfla chung X598 bi gidm hoat tinh >ai6ng con 82% sau 30 phut xi> ly nhifit. Q nhifit dfl xfl- \^ cao hon, hoat dfl protease bj gtam rat manh, chi con 18% sau khi xfl' 1^ cr nhifit do 80°C sau 30 phflt xfl- IJ.
Hlnh 5. Nhi^t dg hogt dpng t i l u'u (A) vi khd n3ng b^n nhiet (B) cua protease chung X598
Anh hu^g cOa pH d4n hoat ding cua protease.
Kit qud fl' hinh 6 cho thay protease cfla chflng X598 hoat dflng tot 6' pH tie 6 din 9. Trong dfl pH toi u'u protease cfla chflng X598 la pH 8.
Anh hirdng cua Ion ldm loai vi chit tiy rua din hoat ddng ciJa protease.
Ket qud hlnh 7 cho thiy d c ion K*, Ca^*, Na*, Fe^* diu cfl tac dyng Idm tang hogt tinh protease fl' cac nong dg tu' 0,5 den 5 mM. Cdc ion l\^g^*, Mn^*, Zn^*, Ba^*, Cu^*, EDTA va SDS deu Idm giam hoat tlnh protease.
-I
Hlnh 7. Anh hu^ng cOa ion kim lOfil len ho^it tinh protease cua chflng X598
Nghifin cfl-u v l ddc tinh cfla protease xg khuan, Singh va Chhatpar (2011) cflng bo protease tu- chflng Streptomyces sp.
A6 cfl hogt tfnh cao nhlt fl' pH gan kiem 7-9 va 6' nhiet dfl 55°C; Bajai vd Sharma {2011) cflng bo chflng Streptomyces sp. DP2 sinh protease hogt dflng t i l nhat fl' pH 8 vd Itha nang bin nhigt tfl'i 90 °C trong 1 giir, va bj giam hoat tfnh bo-i EDTA (25%), SDS (16%), va PMSF (6%); hay Jaoaudi vd dflng tdc (2010) cflng bo mflt loai protease qu;/ phan l|p tLr Streptomyces sp. strain ABI hogt dgng t l i yu 6' pH 11.5, nhigt dfl 75''C, ion Mg^* 6' nong dfl 5 mM lam tang hoat tfnh cfla enzyme ndy
HOI NGHI K H O A H Q U U U " " "
Z ^ ^ ^ « « ^ S^^-^y^^ , . a e o « / c « ™ W e - X598 06 kich « ^ ph.n . * khoing 26 kDa. ^
" ° ' ° ° ^ " „„„l>„l,^a50»CvipH8.,enzyitieduytrl82%hostltnhkliii4|i N h * « v i pH * « . cho hoi«ajrr™= r S j a ^ o ^ y cL?c6n 18% host tlnh. Ion K", Ca". Na*! Fa'* S
nh«l 6(rc trong 30 phOt «*i 8<«: «ong 30 ph^ Z ^ ^ EDTA v i SDS lam giSm host tlnh protease. ^ hoat tlhh protease, con cic ton Mg . Mn ,Zn . Ba , ou , c
T A I U 6 U THAIII K H A O
Baw BK s w ™ P (20,1) An a t e l H h a n n C o i . ™ . e«lra»llula, p r o l . a s , from a n a * eclated Sl^cntycs sp. w . , B « e c « i o i a < e ) : 7 2 M 2 . . ... ^ „ , . « . • _ d BrassoKf P. U l o u n ^ a u F. U,>to M..Va™uil B (1999) P r t c a n o n and oha^Manjabon of a keratnolytk: senna pnaaas. i j
StfBptomycesBltiidoflavus'.AEM.p-2570-2576 1
« .- j a r. ir™...^!. C7 Rcirik H 7atal N Bslar S (2010) Purification and charactenzation of a thermoslath
s^'^ns^'S^.^?^^"^"™ ^'^' - *" '"*'" °"»""= ^'""''- «*^"' ^^
101(21):8361-9.
Uiam H Mankal H Slama N Baftalia. I. Umam F (2009) PfoducSon and opBmlzalion of Ihanmophilic alkaline pjoleass in solidiJm fenfenle'llon l>y Sriplanyc^ sf. CN902. J Ind MJjoDWBWecftnol. 36:531-537
Singh AK. Chhatpar HS (2011) Purficatton. charactenzation and Ihennodynamics of antifungal protease from Sfmplom/ces sp. At.J Basic Ukmbiol 51(4):424-32.
PURIFICATION AND CHARATERIZATION OF THERMOPHILIC PROTEASE FROM STREPTOMYCES PHAEOLUTEICHROMATOGENS X598
Tran TW Le Quyen, Dao TW Luong, Trinh Thanh Tmng, Duong Van Hop Insitute of Micmtlology and Biotechnology. VNU
SUMMARY I Aclinomycetcs. puliculariy strqrtomycetcs, are known to secrete multiple proteases into the culture medium. Some of that
proteases, the serine piMeases of Str^tomyces griseus and Streptomyces fradiae, have been characterized structurally and cnzymatically. There also have been many desoiplioDS of isolation and partial characterization of alkahne protease activities fiwn Other memben of the genus Str^tomyces. In these prokaiyotic microorganisms, extracellular proteases are involved mainly in hydrolysis of large polypeptide substrates into smaller molecular entities which can subsequently be absorbed by the cells. Hie physiological role of extracellular proteases in di^entiation of some Streptomyces species has been demonstrated previously, hi (ha study, a 26 kDa alkaline protease fiom Streptomyces phaeoluteichromalogenes X598 was purified to 16 folds by gel chromatognplij' and the final yield was 13?i. The final product had a specific activity of about 29.6 ^U/mg. The enzyme exhibited highest activity U pH 8 and 50°C. The protease activity also increased (108-135%) with increasing concentration of ion K"^, Ca^^ Na% Fe^* (0.5-j mM) Enzyme activity was reduced by Zn^* (65%). Mn^* (64%), Uf* (49%), EDTA (49%), Cu^* (31%), SDS (28%), Ba^* (17%). TTie enzyme was stable at 60*C, more than 80% of the maximal activity was conserved at this temperature value after 30 min. ProMase X598 was not stable at 80^, it was only 18% residual activity for 30 min.
Key words: protease, punfication, activity, Streptomyces phaeoluteichromalogenes X598, optimal activity.