0 CONG NGH£ SINH HOC T O A N Q U O C 2013
SCr DUNG PECTATE LYASE TAI T 6 H Q P LOAI PECTIN TR^N VAl BONG THO
LA Tr^ng Tdi, D 5 Thj Thu Hang, Vd Hodi Bic, Le Van Truvng Viin Cdng ngh^ Sinh hQc, Vi^n Hin lim Khoa hgc vi Cdng ngh$ Vi$t Nam
T6M TAT
Pectate lyase \& enzyme phSn cit nglu nhien lign ket a-1,4 glycosidic cua polygalacturonate trong thinh te biko tiiuc v$t tb6ng qua pbin ling B-elimination 190 ra cac oligogalacturonate co lien ikt ddi giua C4 vi CS d diu duong khong khi^. Nhifaig niim gin dSy, pectate lyase dl dugc img dvmg to ldn cua chung trong cong n ^ ^ d ^ cong nghi^ sin xuit giiy vi bpt giiy, chilt diu thuc vEit, cong ngbi^ 16n men ca phe va leo men che. Pectate Ijasc t&i to hpp (rPel) tir Bacillus, subtilis dugc sii dyng dk logi pectin trfin vii bfing Ui6. K^ qui cho thiy rPel co kh& nang phan hily hifiu qui pectin Q-fin vii bdng. Sit dung 2% d)cb enzyme rPel trong d ^ phin ling dio hifiu qui phan hiiy pectin trfin vii b ^ g , niong diiong voi sii dyng 1% en^me thuang m^i Bioprq) 3000L (Novozymes) d Cling difiu ki$n xijr ly. Xur ly pectin trong vai bong bSng d>el 6 nhi^ dd t^i uu 42°C can it nhit 1 gib de dat d u ^ hieu qui t6i da.
Ti khoa • Bacillus subtilis. pectin, pectate lyase, polygalacturonate, pectate lyase tai to hop, vai bdng.
MdoAu
Sgi bdng t^ nhiSn dipgc ciiu tgo bfl-i lop ngoSi m6ng gpi Id vdch te bao so d p (primary cell wall) vi ldp d&y bSn trong \i vdch tk bdo ttii> d p (secondary cell wall). Hdm lu'ang cellulose trong si^i bflng chilm t6i 95%, chi 5% c6n lgi Id chdit khfing ptiai cellulose (non-cellulose). Cdc chit non-cellulose ndy c^fi ylu Id pectin, ngodi ra cfin co chit dj cellulose (hemicetlulose), protein vd chat sdp dinh (waxe) (Bafra, 1985; Seagull et al.. 2000). Chat non-cellulose chilm ph^n ldn trong vdch t l bdo sa d p vl vgy qud trlnh xir t^ sgi bdng chO y l u Id loai bfi vdch t l bdo sa cap. Pectin trong sgi b6ng chilm khodng 0,4-1,2%, chOng cfi val tro nhu chit kit dinh lien kit cellulose vd chit non-cellulose. Vi v§y vi§c logi bfi pectin s@ Idm cho vi§c lo^i bfi cdc chit non-cellulose khdc trong sgri bdng d§ ddng hon.
Trong cfing nghiep d$t,vdi bong sau khi d^t cfin chi>a nhilu chit non-cellulose Id nguySn nhdn gdy cdn trd thu6c nhufim thdm nhgp vdo vdi, dnh hu^ng din chat lu'ang vai nhufim. Thfing thiremg d l logi bfi cdc chit ndy, vdi bfing phdt qua cfing do^n nlu kilm (alkaline scouring) de tay logi cdc chit ndy. Bdy la phuvng phdp don gidn nhirng dnh hirfi'ng den chit lu'gng cOa sgi bfing vd gdy fi nhilm mfii tnj^ng. D l gidi quylt vdn d§ ndy, nhOng ndm gan ddy cdc nhd cfing ngh$
trdn till gifii da sO dgng enzyme pectate lyase, mfit logi enzyme pectinase kilm d l phdn hOy pectin trong sgi bfing, thay till cho hda chit kilm dfic hgi, ldm tdng khd ndng tham n u ^ cOa vai tiOng, do dfi mang l^i hi^u qud cao cho qud trlnh tiy tring (bleaching) vdnhufim mdu, Idm cho chit lu^ng vdl tot han (Buchert ef a/., 2000; Liu ef a/., 2000).
Pectate lyase (EC 4.2.2.2} Id mfit pectinase kilm phdn hiiy pofygalac^ronate tgo ra cdc oligogalacturonate. Enzyme ndy phdn cat nglu nhien lidn k i t a-1,4 glycosidic cOa polygalacturonate trong thdnh t l bdo tiii/c v(lt ttifing qua phdn i>ng (!>- elimination tgo ra cdc oligogalacturonate cfi lifin kit dfii giija C4 vd 05 a diu diro'ng khfing khO (non reducing end) (Birch et ai. 1981). Pectate lyase ti> Bacillus, subtilis dd dif^c nghidn ecru tO Idu, tuy nhiln hi$n chua cd d n g b l ndo nghidn dru irng dgng enzyme ndy ti-ong logi pectin tiong vdi bfing d l thay t h i chit kilm ddc hgi bong khdu nlu luem. v1 vdy vi$c nghidn cO'U khd ndng logi pectin b^n vai bfing cOa pectate lyase b> B. subtilis Id d n thilt, Pectate lyase tCr B.
subtilis xiic tdc phan O-ng phan huy pectin d nhiet dg gan vdi nhiet dd thuong (42 °C) do dd se gitip tilt kigm ndng lugng hon so vdi cdc che phim pectate lyase khdc hi^n dang du'g'c ban tren thj trudng nhu BioPrep 3000L (t6i uu d 55 - 60
°C).Trong bdi bdo tirrdc, chiing Idi dd cdng b l kit qud nghifin ct>u tfnh chit cua enzyme, trinh tu gen nay tCr B. subtilis phdn ldp ti> Vl$t Nam (Ol Thj Thu Hing et al., 2012). Trong bai bdo ndy chOng tdi cdng b l kit qud nghifin ci>u kha nang logi pectin trong vai t>dng bang pectate lyase ^ i t l hgp ti> chiing B. subtilis ndy.
VAT U^U vA PHlfONG P H A P V$t lifu
Miu vai: Vdi 100% sai bfing, chi s6 sg'i Ne 100/2, kilu d$t vdn dilm (Vign D^t may cung d p ) . Phinmg phdp
Thu nhin pectate lyase tii to hgp
Pectate lyase tdi t l hap (rPel) dv/gc thu nh^n tii' chOng B. subtilis tdi to hg'p da copy BSM, 6irgc tgo tO' chiDng chu B.
subfi/js 168 mang gen pe/tdi to hg'p cOa chung VBS11. Chung B. subbf/s BSM duac b^ hfia bgn mfii trutrng LB Ifing 20 MQ/ml kanamycine 6- 37°C, 200 vfing/phut qua dfim, sau dfi d y chuyin 1% sang mfii tiiffi'ng khodng chit Belistity (Stuike ef ai, 1993) cd b6 sung 20 pg/ml kanamycine. Sau 26 gid nufii d y d cCing dilu k[$n, dich enzyme ngogi bdo dug'C thu bing ly tdm 10.000 vfing/phiit trong 10 frfiiit d 4''C de logi t l bdo, djch noi chiia enzyme ngogi bdo duvc thu nhdn vd bdo qudn d -20°C cho cac thi nghifim tilp theo.
Xdc djnh ho^t tfnh pectinase bang phifomg phip dtrimg khv
Hogt tinh pectinase duac xdc djnh bang phuang phdp dudng khi> nhu mfi ta cua Bemfeld (1955). Bfi sung 50 pi djch enzyme ngogi bdo vao 450 pi dung djch p^dn ung chua 0,2% pectin tu citrus (Sgma) frong d ^ Tris 50 mM, NaCI 20 mM, CaCl2 0,1 mMpH 10. Phdn i>ng duac thyc hi$n d 42"^ tiong 1 gid. Phan dng enzyme duac kit thiic bang vific b l sung 500 pi 3,5 dinitrosaiinciiic add (DNSA), dun sfii 10 i^OI, sau dfi dung dich phan ung duvc Idm ngugi din nhigt Sg phdng. Li tdm 10.000 vdng^hut trong 5 phiit tiufi'c khi do d budc sfing 530 nm tr§n mdy do quang phi. Mfit dan vj hogt tinh enzyme (unit) duiTc djnh nghTa Id luvng enzyme pectinase phdn d t ca chit pectin tao ra 1 pM glucose trong 1 phut.
nuong phap loai pectin trin vii bong
Phuong phdp Logi pectin trdn vdi tifing duvc d i tiln dya theo phuong phap cua Morozova vd ding tdc gid (2006) Ar^
torn tat nhu sau: mlu vdi duvc d t nhd Uieo kfch thudc 3x3 cm, mdu vai duoc xu ly logi tinh bfit trudc bang'amyiesB.
Cho 1 milng vdi dd xO- ty amylase vao Ing falcon co chua 10 ml dem phdn Crng (100 mM Na3C03; pH 9,0) vd 100 ui pectate lyase. Mau doi chiing duvc ldm bjong t\r nhung thay enzyme bdng dgm. Cac mlu vai duvc xO" ly d 42°C lie 100 vong/fSiut trong 1 gid de enzyme phdn dng viri pectin tong vdi. Lly vdi ra bing panh, cho mil mieng vdi vao mfi binh tam gidc cfi chda 50 ml nude d t , rua bang each lie nhg khodng 1 phut Lay vdi ra, iogi nude bing cdch ii tdn 3.000 vdng/phut, 5 phiit Lap lai budc n>a 3 lan. Mdu thi nghifim duac chukn bj 3 miu song song, kit qud Irung blnh ciia 3 mlu thf nghifim dug^ si> dung de ddnh gid kit qud.
Nhu^ ruthenium red
Be dinh tfnh pectin cdn lgi bong vdi, mlu vdi sau khi xO-1^ duoc nhufirn vdi rhutfienium red 0,05% trang 30 phiiL D|tfi nhufim du di/grc logi bang li tdm nhu 3.000 vdng/phiit, 5 phiit, rua vdi bdng nuoe d t nhu mfi ta d phin trfin.
Oinh gii kha nang loai pectin tnmg vii bing phirang phip cin
Vai trudc va sau xi> h^ vdi pectinase duvc danh gid mdc dfi gidm ti'gng lugng vdi theo phuang phdp ciia Tzanov vi ddng tdc gia (2001). Cdc birerc duac tfim tat nhu sau: vai taroc khi d n duoc lam khfi d 105°C, 1 gid trong tiJ sly cCffg vdi hgp d^mg miu. Trong suit qud tiinh cdn, miu duoc dung tiong hgp kin de ti^nh hai I m trong khfing khi bi hiit trd 1^
lam mit tfnh chinh xde cua ttif nghiem. Vige danh gid su gidm trgng luong eua vdi eon duoc qui dfli ra tigng lirgng tuong d& theo %. Trgng luong gidm tucmg dli duvc djnh nghTa Id tivng lifg'ng vdi bj giam so vdi mlu vdi gidm Idn nhat (100%) tinh theo don vj %. Gid tii tiung binh eua 3 mlu thf nghifim Igp Igi duoc sd dyng d l ddnh gid kit qud.
K ^ QUA VA THAO LUAN Thu nh$n pectate lyase tai t6 hgp
Chiing B. subb'lis tdi to hop BSM duoc nudi d y trfin mfii bufing BMM d l sinh pectate lyase sau dd enzyme duvc thu nh$n nhu mfi ta tix)ng phin phuang phdp. Hogt igc enzyme thu duvc duvc ddnh gid bdng phuong phdp dudng khO, Kit qud thu duvc cho tiily hogt luc pectate lyase d a chung tdi t l hvp thu duoc bang 30% chl phim enzyme thifong mgi Bioprep 3000L(Novozymes) (Bdng 1).
Bang 1. Hoat l | ^ cua enzyme sO dgng
Khi ning phin hOy pectin tin vii b6ng cua rPel
D I danh gid kha ndng phdn buy pectin trfin vdi bfing, djch enzyme rPei diroc su dgng nhu md ta trong phan phirong phdp vdi tt lfi enzyme/dfim id 1 %. Pectate lyase Bloprep 3O00L vd nude d t duac sd dgng nhu Id mlu ddi chdng dmrn vd dli Chung dm. Khd ndng kigi bfi pectin tifin vdi bfing ttid duvc ddnh gid qua su giim trong lugng vai sau xu 1:^. m qud cho thiy tiipng lugng vdi sau xd IJ vdi rPel gidm ddng k l (0,86%) so vdi tnrdc xu \^, gin bing vdi mlu enzyme ttiuong mgi BioPrep 3000L (0.89%) trong khi mlu doi chung am giam khfing ddng k l (Bdng 2). Dilu nay chdng tfi iPd cfi khd ndng logi pectin cd trong vdi bong.
Bdng 2. Sg glim trpng lu^ng cQa vii bong sau khi xi> ly pectin bang rPel Mau thi nghi#m
rPel BioPrep 3000L DC am (nude cdt)
Trpnq luvng v i i (ma) Trudc x d ly
370,6 379,0 365,3
Sau XLP1^
33B,6 345.0 363.2
Trpng luvng vdi gidm
32.0 34.0 2,1
{%) 0.86 0,89 0,06
Vai -khdng
xvtl;/v6i rPel
Vai dagcxxt ly voi rPel
Hinh 1. Vii da dup-c xu> ly vdi enzyme rPel va nhuom ruttienium red 0,05%
Kit qua thi nghifim kilm tra pectin ttfin vai bfing bang nhufim vdi vdi 0,05% nrthenium red cho thay mlu dli chCmg fl»
bit mlu vdi ttiule nhufim r i t dgm ttong khi miu xu 1^ bang rPel till bdt mdu r i t nhgt. dilu ndy cd t h i gidi ttifch lfl * phin Idn pectin tren vai bfing tixing miu thf nghifim da bi enzyme phan huy do dfi mit khd nang bat mdu v6^ nhuOmvS njthenium red (Hinh 1).
-~ - t c CONG NGHE SINH HQC TOAN QUOC 2013
Khio sat n6ng d ^ enzyme rPel thich hgp de phin hOy pectin trin vai b6ng
o l bilt duvc kfvng enzyme rPel thfch hvp ph&i huy pectin bfin vai^ bfing, rPei duvc b l sung vdo mlu xu ly vdi eae ning dfi khdc nhau. Mlu enzyme thucmg mgi BioPrep 3000L duvc bo sung 1% duvc si> dgng nhu doi chdng duong, u 1 gid d 45°C. K i t qua cho tiily lugng enzyme rPel l>i sung 2% t h i b'ch dfim eho khd nang thiiy phdn pectin ti^n vai bfing cao nhlt vd tuong duvng m l u bo sung 1% enzyme BioPrep 3000L (Bang 3). Nhu vdy vdi mdt luvng nhfi rPei da cd thi logi bd duoc pectin t l i da trdn vai bdng.
Bdng 3. Khdo sat nftng d^ enzyme rPel thfch hp-p d i phan hAy pectin tren vai bdng
Trpng lupng vai (mg) Trpng lupng val giam
3 2 1 0.5 0,2
E)C duong 1 % (BnPrep 3000L) DC dm (nude ( ^ )
Trudc xd ly 378,5 371,4 370,7 347,2 357,7 379,0 367,3
S a u x u l y 344,8 338,4 338.6 323,8 346.5 345.3 364.8
(mg) 33,7 33,0 32,1 23.4 11,2 33.7 2,5
(%)
0.8S 0,89 0,86 0.67 0.31 0.89 0.06 l^io sit tiibf gian thich hgp phan hdy pectin trin val b6ng bing rPel
Bk ddnh gid khd ndng phdn buy pectin trfin vai bfing theo thfii gian. luang rPel dirac sd dung trong ttif nghifim Id 2%.
Phuang phdp cdn vdi eOng duvc sd di,ing d l ddnh gid. Ket qud hlnh 2 cho thiy sau 60 phiit luang pectin trang vdi bfing ttifi bj ptian hOy dgt t l i da (100%). Thdi gian u llu hon eung khong ldm gidm trgng luvng d a vdi hon do dd thdi gian u 80 phOt la thdi gian thieh hop d l rPel phdn hiiy pectin ttong vdi t>dng dgt hifiu qud cao nhat (Hlnh 2).
T^ittrnweTttp,
UUi
15 30 OD 90 12Q Ihiitsa&iiit Hlnh 2. Thdi gian thich hp-p ph3n huy pectin trin vii bong bing rPeiSu dvng pectinase d l logi bo pectin trong vdi bfing da duoc cde nhd khoa hgc quan tdm nhung ndm gin ddy. Calafell vd ding tdc gia (2005) dd nghifin cdu so sdnh sd dgng Bioprep 3000L (Id pectate lyase) vd pectinase 62L (Id mfit endo polraalacbjronase) d l logi pectin trfin vdi bfing eho thiy Bioprop 3000L cd khd ndng logi pectin t i t han vd vdi sau khi xd ti bdng Bioprep 3000L bit mdu thulc nhufim t i t han pectinase 62L. Klug-Santner vd ding tdc gid (2006} da chl ra petite lyase ti> Bacillus pumilus BK2 d khd nang logi 80% pectin d a svi bdng. Ket qud nghifin cdu cOa chiing tdi cho thiy pectate lyase nguln glc ti> B. subWis cd khd ndng logi pectin ttfin vdi bfing hifiu qud, sd dgng 2% djch enzyme rPel sau 1 gid xd i<^ d nhifit dfi t l i uu 42°C pectin hau nhu da duac logi bd toi da. Enzyme rPel duac thu tivc tilp tu djdi Ifin men chiing BSM nhung eho hogt igc enzyme bang 30% wizyme tiiuang mgi Bioprep 3000L cho thiy khd ndng toi uu dilu kifin bilu hifin chung ndy d l ndng hogt tfnh enzyme tiKmg duong vdi enzyme thuong mgi Id hodn todn cd ttie. Mdt khdc, rPel xu ly pectin trong vdi bdng d nhifit dfi t l i uu 42 °C sfi tiet kifim ndng luvng hon khi sd dgng enzyme thuang mgi BioPrep 3000L (nhifit dg t l i uu d 55 - 60 °C).
K ^ L U A N
Enzyme rPel efi khd nang phdn huy hifiu qud pectin trdn vdi tifing thd. Cj nong dfi 2%, thdi gian 1 gid, rPel phan huy pectin dgt hifiu qud eao nhlt. Kit qud ndy md ra triln vpng d t h i dng dyng enzyme pectate lyase tdi t l hgp tir B.
subWis d l logi pectin trong vdi bfing ttid thay ttil xdt trang khdu nlu kiem trong cdng nghfi xu 1^ vai bfing. giiip gidm thilu fi nhilm mfii trudng.
TAI LI^U THAM KHAO
Batra SK (1985) Handbook of Rber Science and Technology. Lewin M. and Pearce EM, Editor 727-807.
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Biichert J. and Pere J. Puolakka A, Nousiainei P (2000) Scourmg of cotton with pectinases. proteases and lipases. Textile Chemist and Cokxist i Amencan Dyesluff Reporter 31 (5). 48-52.
H Q I N G H ! K H O A H O C C O N G N G H E S I N H j
Calafell M, Klug-Sanfrier B. Guel«tz G, and Garriga P (2005) Dyeing behaviour of cotton fabnc bioscoured wilh pectate • / « « ; «
polygalacturonase. Cotoratkm Technt^ogy 121:291-297. ^ 0 5 Thj Thu HiiSg. Vo Hoai B i c , Le Vdn Trudng (2012) SSng Igc va ntian ddng gen ma h6a pectate lyase tir Baallus subtilis c6 ngu^
g l c vm Nwn. Tap chl Sirdi hoc 34(4):485-492.
Klu^^antner BG. Schnitzhofer W, Vrsanska M. Weber J, Agrawal PB, Nierstrasz VA. Guebitz GM (2006) Puriffeatfan an ctiaracterization of a new bnscouring pectate lyase from Bacillus pumilus BK2. JSibfec/inof 121: p. 390-401.
Uu J, B Condon, and HL Showmaker III (1999) Single-bath biopreparation and dyeing of textiles Novo Nomisk BioChem NoHh Amenca. inc (Franldinton, NC): US.
Morozova W , Semenova MV, Salanovich TN. Okunev ON, Koshelev AV. Bubnova TV, KrkiievsldT GE, Timatkov AG, Bary^nn NV, Sinitsyn AP (2006) Apfrifcabon of neutral-alkaline pectate lyase lo cotton fabric boil off. AppI Biochem Microbiol 42:603-608 Seagull RW. Oliven V. Murphy K, Binder A, and Kottiari S (2000) Cotton fiber growth and devdopment 2. Changes in cell diameter and wall birefringence JCoflartSc("4:97-104.
StulkeJ.HanschkeR,HeckerM(1993)Tempora/ac(/vatfon of H-glucanase synthesis in Bucillus sutriilis is mediated by the GTPpooi.
J Gen Microbiol 139{9):2041-2045.
Tzanov T, Costa SA, GObitz GM, Cavaco-Paulo A (2002) Hydrogen peroxide generation witti immobilized glucose oxidase (or texlile bleaching. J B/o(ec/ino( 93(1): 87-94.
USING RECOMBINANT PECTATE LYASE FOR REMOVAL OF PECTIN FROM RAW COTTON FABRIC
Le T r o n g Tai, D o Thi T h u H a n g , V o Hoai Bac and L e V a n T r u o n g ' Instate of Bmtechnc^ogy, VAST
SUMMARY
Pectate lyase cleaves a-1,4 glycosidic linkages of polygalacturonate m plant cell wall by fl-eliminalion product oligogalacturonates, fonning unsaturated linkages between C4 and C5 in reducing sugar. Recently, pectate lyase have been used in several industry such as textile processing, paper and pulp industry, oil extraction, coffee and tea feimentations. Recombinant pectate lyase of BaciH\e subtilis (rPel) have used to remove pectin from raw cotton fabnc. The resulting shown that pectin from raw cotton fabric could be removed well by rPel. Usmg 2% of rPel in buffer lead to decomposition ef^cient of pectin from raw cotton &bric, equivalent to IK of commercial enzyme BioPrq) 3000L (Novozymes) in the same processing conditions. The treatment of pectin from raw cottos fabric by rPel in temperature 42°C requires at least one hour incubation to achieve maximum efficiency.
Keywords: Bacillus subtilis, cotton fabric, pectin, pectate lyase, polygalacturonate, recombinant pectate lyase
' Author for correspondence- Tel. 84-4-37652880. Fax' 84-4-37560339; E-mail: [email protected]