O N G NGHE SINH HOC TOAN QUOC 2013
NGHIDN Cl>U C 6 DINH LACCASE THO TREN GIA TH^ DS LOAI PHENOL VA MAU THUOc NHUOM
Nguyen Nguyen Quang, Dinh Thj Thu HSng, Pham L i Anh, Nguyen Thj Lan Anh, Dang Thj Cam Ha*
Vign Cdng nghg sinh hgc. Vign Hin lam Khoa hgc va Cong nghe Viet Nam
TOM TATLaccase thfl ciia chung nam Myrothecium sp FBV2 phin lap t i rimg Quoc gia Ba Vi da dirge c5 dinh len chitosan va 6ng carbon nano (CNT). Hi?u suat co djnh enzyme dat 42,8% tren vat beu chitosan co duong kinh Idn hon 6 mm khi bfl sung cyanamide (0,01 g/i). So voi chitosan, hicu suit c6 djnh laccase tren CNT cao hon khoang 1,6 lin. Mot s6 tinh chit va kha nang phan hiiy phenol, loai mau thuflc nhupm ciia laccase c6 dinh tren CNT da dirge khao sat. Ket qua cho thSy pH thich hgp cho hoat dong cua laccase c6 dinh U pH 3 trong khi laccase bf do o pH 2. Tuy nhien, enzyme c6 dinh khong bSn hon so vcti enzyme nr do 6 60°C sau 1 gio, hoat tinh con l^i khoang 17% so v6i ban diu Sau 5 gio, laccase c6 dinh co kha nang loai bo 94,2% mau axit xanh 62 (NY3) va 98% mau axit do 266 (NY7) voi nong dp ciia moi loai mau la IOO ppm. Laccase cfi dinh co kha nang loai bo 68,7% phenol vcri nfing do 20_ppm trong 24 gio. Kha nang phan huy phenol va loai mau thuoc nhugm ciia laccase cfi dinh Uen CNT cao hon tuong ling 1,5 va 3 lin so vdi laccase tv do_. Nhimg k6t qui thu dugc mgt lln niia cho thiy txkm nang iing dung cua laccase cfi dinh trang xii ly phenol, thuflc nhuflm vi cic chat fl nhiem da vong thom khac.
Til khoa. chitosan, enzyme cfl djnh, laccase, loai mSu thufic nhugm, Myrotiiecium sp., fing carbon nano MdoAu
Laccase (oxygen oxidoreductase, E.C.I.10.3.2) thu^c nhom enzyme oxidase, cy the ia polyphenol oxidase Trong phan ti> c6 chii'a 4 nguyen tir dbng co Vhi nang oxy hoa ca ch^t sir dyng phan tiF oxy Mm chit nh^n dign tir. Khac v6i phan ldn cac enzyme kh^c, laccase c6 pho co* chat rlit rflng va da dang, bao gom diphenol, polyphenol, cac din xuSt phenol, diamme, amine thom, benzenethiol, PCS, dioxin.
Trong nhOng n§m g^n diy, laccase dgc bi§t du-p'c quan tam bdri nhOng Cpng dyng rgng rai cfla no trong nhilu nganh cong nghiep khac nhau nhLP t6ng h(?p hO'u co, thyc phim, du'g'c pham, my phim v^ XLF \</ 6 nhiim nhu' loai mau thufic nhupm, phSn huy cSc hffp chit vong thom va da v6ng thom v.v. (Imran etai, 2012). Tuy nhien, enzyme ty do co nhieu nhu'p'c diem nhu- lln vSo s^n phim, hogt tfnh giam sau moi phan iJng xflc tac, khong ben vol nhiet, axit, kiem. dung moi hOu CO V V. Trong khi 36 enzyme co dinh c6 nhieu icu dilm nhu" giam gia thanh do enzyme dyoc SCH dung lap di lap lai, che pham bin hon trong Ccic dieu kidn nhiet dp, pH, ap suat Ihim thau toi uu, bSn nhiet v.v.. Enzyme co dmh thu'ong on d|nh hon so v(r\ dgng hoa tan cua nd va c6 the tai sCe dung o dang tmh khiet, ban tinh khilt hoac dang toan te bao vi du nhu' glucoisomerase c6 djnh c6 thi SLP dyng lien tyc hon 1 000 gio Irong khoang OO-OS^C. Hon thi nij-a, san pham phan Crng khong bi lan ipn vdi enzyme, de ding tb chCrc san xuat cac san pham len men bang enzyme ngoai bao. Vi smh vat sinh enzyme c6 djnh Id nguon sinh enzyme on djnh d l tai si> dung l§u dai. Vi v$y, viec nghien cCfu co djnh enzyme va vi smh v$t sinh enzyme l§n cdc chit mang thgt sy cin thilt (Nisha et al, 2012). Cac chit mang bao g6m nhilu logi nhu' chit mang hO'u co nhu- polymer ting hgp vi tu- nhien; chat mang vo co nhu' sgi bong thuy tmh, silicum oxide, alumium oxide, mangesium oxide; chat mang polysaccharide nhu- cellulose, agarose, dextran, sephadex, tinh bot, chitin, chitosan, chit mang c6 hin chat protein nhu' gelatin, keratin, albumin (Datta ef al., 2013; Tischer va Wedekind, 1999), sgi polypropylene biln tinh; vgt li#u nano nhu ong carbon nano (Feng va Ji, 2011)
Trong thong bao ndy, chung toi trinh bay mpt so kit qua nghien CIJU c l djnh laccase tho tin chung nam Myrothecium sp.
FBV2 len chitosan vd Ing carbon nano (CNT) nhdm irng dung trong iogi phenol va mau thulc nhuom.
VAT LIEU VA PHU'aNG P H A P Vat lieu
Chung nim Myrothecium sp. FBV2 du'gc phan ldp tu' ning Qulc gia Ba Vi nam trong Tap dodn gilng ciia phong Cong ngh? sinh hpc tai tgo moi tru'dng, Vi6n C6ng nghe sinh hpc 6ng CNT da Idp du^gc cung d p ti> TS. Ngo Thi Thanh Tdm - Vien Khoa hgc vdt ligu vd chitosan dygc cung d p ti> PGS TS. Pham HLFU Ly - Vien Hoa hpc. Vien Han Idm KH&CNVN. Vdt li^u chitosan du'gc phSn iogi theo kich cd thanh ba loai 1 (du-dng kinh < 2 mm), II (2 mm < du-dng kinh <
6 mm). Ill (du'dng kinh 2 6 mm). Cac logi mau du'gc si> dung la NY7 (thupc nhom azo, / ™ , = 499 nm) va NY3 (thuoc nhom anthraquinone, Ama» = 595 nm)
Phipong phap
Chung Myrothecium sp. FBV2 duoc giii giong tren moi tnjdng Czapek thach co bo sung 1% CUSO4 co vai trd la chit cam ijng. Hogt tinh laccase du-gc xac djnh bdng phuong phap so mau dua tren su oxy hda ABTS cua laccase lao thanh hgp chat hap thy d budc song 420 nm (Kunamneni et al. 2008) Laccase duoc c6 djnh len chitosan theo quy trinh sau:
dung djch phan i>ng gom 0,5 g chitosan loai III; 5 ml laccase tho (500 U/l), 44 ml dem sodium acetate 20 mM (pH 5). 1
ml cyanamide (0.5 g/l). Laccase dugc c l dinh tren vgt lieu CNT theo quy trinh sau: dung djch phan ung gim 3 ml dung
dich huyen phu CNT (3 g/l); 5 ml laccase (500 U/l), 41 ml dem sodium acetate pH 5, 1 ml 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide (0,5 g/l). Hin hop enzyme va gid the dugc khuay deu bang^may khuay tu d nhiet do
4°C tnang 1,5 gid (Feng vd Ji, 2011) Sau phdn ung, hdn hop laccase va vat lieu c l djnh dugc lay ra rua sach bang dem
sodium acetate pH 5(20 mM), lytdm 12.000 vdng/phiit d4°C. giu khd d nhiet dp 4°C cho cac thi nghiem tilp theo Higu
suat CO d]nh laccase tren gid the = (Hogt tinh laccase co dinh/hogt tinh laccase thd ban ddu) x 100 Anh hudng cac kich
thudc khdc nhau I, II, 'II cua vdt lipu chitosan !am gid thi len hieu qua co dinh laccase da dugc khao sdt. Kha nang ben
' ^-K tr^n PMT rli prtr tac dinh bang d c h u laccase co djnh trong dem sodium acetate pH 5 6 rMkI S S ' c ' t ^ r T g i ^ H S S c ' a V n S e ' d u J f x d c dfnh tgi thdi ^ I m ban diu vd sau 10. 20, 30, 40 50. 60 p h i . ^ ' h?iSa c S ^ X h o ^ t i n h laccase c l ^nh tren CNT da dugc xac <^nh bang c j ^ u laccase c6 dinh d nhi§t dQ 30^
^ ^ m Hn KCLfDH 21 sodium acetate (pH 3-5), postasium phosphate (pH &^) va carbonate-bicarbonate (pHi t^ S ^ ? 2 4 o S t l ddnh gik k h ^ S n g loai r!.au a>.t xanh 62 (NY3) va axit do 266 (NY7). laccase thd vd lacc^^
i ? n h ^ n CNT d toat Tnh 27 3 U/l du^c cho vao hin hgp gim dem sodium acetate (pH 5. 20 mMWd 100 ppm miu K S ^ g ^ r S u ^ S . ! ^ ! ^nh theo dng thdc: Phdn tram loai mdu = (dp hip thy ban diu - dO hip thy cuHyd^Wp Su b^n diu X 100 Hiw luong phenol dugc xac djnh dua tren sy Phan ^^"9 cua phenol vdi thuoc thu Foiin-Ciocatea, S *dnh hoi dSt hip thud budc sdng 750 nm {Singleton et ai. 1999). Hon hgp phan ung dugc u d 30"C trong 24 To gdr^ 0 5^1 W e L va laccase c l djnh tren CNT ( d hogt tmh 28 U/L), 4 0 ml sodium acetate 20 mM (pH 5) vi 0 ? Sphenordl d^dugc d c ndng do culi cung ta 0, 2, 4, 6. 8 va 10 ppm. Sau do lly 2 ml hon hgp phdn dng IrOn £.
vdi 0.3 ml Na^COa 20% va 0,1 ml Folin-Ciocateau, u d 30°C trang 1 gio. | K6T QUA V A T H A O LUAN
Chung Myrotiiecium sp FBV2 tuy d hoat tinh laccase khdng cao vi chua dugc toi uu kha nang sinh ting hgp enzyme ndy nhung nhdm nghign cuu vln quyet dinh sir dung enzyme thd d l nghi&n CLFU co djnh len vgt li^u chitosan vd GMT.
c l d|nh laccase trin chitosan
Enzyme dugc d dinh tren chitosan bang cdch tao lien kit amide giua nhom -COOH tren be mdt phdn ti> laccase rti nhdm -NHz tren mgd cua phan tir chitosan. phan dng tgo lien kit amide duoc xuc tdc bdi cyanamide (ho$c bdng hip phu Oelanoy el al.. 2005). Chitosan dugc sd dyng trong nghien cuu ndy cd kich thudc ldn vdi dudng kinh £ 6 mm, gim nhilu phdn tif chitosan dan xen vdi nhau. Vi thi. d nhieu cho tring trong d u tme cua vgt ligu chitosan d l chojaccasi cd thi chui vdo. Hon nua, trong dung dich, nhom -NH2 tren mgch phdn tu chitosan d l ddng nhdn th§m ion H'tir mft trudng hinh thdnh ndn goc -NH3'. glc nay d l ddng tao li§n ket ion vdi cac ion dm^ty do tren phdn td enzyme vi th& khJ ndng hip phy dugc tang ten Hoat tfnh cua laccase c l djnh da dugc xdc dinh va kit qud dugc trlnh bdy d Bdng 1.
Bdng 1. Ho^t tinh laccase tivirc va sau khi c6 dinh tren vat l i f u chitosan Enzyme Hogt linti laccase (U/L)
Laccase cA ^ n h lr6n chitosan
Td Bdng 1 ta thiy laccase da dugc d djnh tren vdt ligu chitosan Tuy nhi§n, hoat tinh laccase c l djnh chl dgt 39% so vdl hogt tlnh ban diu Theo Delanoy vd dtg (2005), hogt tfnh laccase co djnh bj gidm sau khi gdn tr§n chitosan bdi nhiiu nguydn nhdn. Trong dd, khi lien kit amide hinh thdnh d gin hoac ngay tmng tdm hoat ddng d t h i din tdi sy cSn W khflng gian ciia phdn ung cua enzyme vdi co chat. Ngoai ra trong qud trinh phdn dng vl dy nhu laccase bj ngdm tronj mfli tofdng d pH khong tuong thich dan din cac hogt dgng vdi phuc hgp laccase - chitosan nhu rua trong d§m pHB v.v. cQng Idm gidm hogt tlnh enzyme.
Anh h u ^ g ciia kich thudc vdt lieu chitosan lam gia t h i den hi|u qud c l djnh laccase
Khi d djnh laccase ty do ldn vat lipu chitosan se cd hai kha nang xay ra. thu nhlt Id xdy ra li§n kit cflng hda trj vS Ihi hai Id hip phy v$t ly. Trong qud trlnh d djnh, kich thudc cua chitosan dnh hudng rat Idn din hi§u qud c l djnh. Vdt li^ii d kich thudc cdng Idn se cd be mat tilp xiic vdi enzyme cdng Idn do dd qud trlnh hip phy se xdy ra mgnh hun Delanoy et al.. 2005) Do dd. chitosan vdi ba kich thudc khdc nhau nhu logi I vdi dudng kinh s 2 mm. logi II vdi 2 mm<
dudng kfnh s 6 mm. logi HI vdi dudng kinh > 6 mm da dugc su dung de danh gid khd nang co djnh cua enzyme (Sanfl
Bdng 2. Hogt Knh cua laccase c6 ^ n h tren v j t lieu chitosan co kich thu^o-c khac nhau^^'"°san H09I llnti laccase co djnh (U/l) Hieu suat (%) Logi I (d s 2 mm) 0 Q Logi II (2 mm< d S 6 mm) 0.78 1 9
Logi III {6 mm < d) 21.52 4 2 3
S ^ t n i n n i ^ K « f r . , h ^ ^ ' ^ d-.nh enzymo cao n h l t tren vat Ii6u chitosan c d d u d n g k i n h > 6 m m . V d i kich thirtt
S u ? r n t ^ S ? S i ^ ? ^ f ' % "^"^ ^^' ^ ^ ^ ' " ^\^^ ^"- ^'^" ^"^ '^^ "^^"^ enzyme tren chitosan loai II thip h<m r*
Tr^n .,• nn ^ = . ^ ? ' K^ ""^J ^ " ^ ' ^ ^ ^f ''^^"Q ^° ^^^ ^^'^'^ '^^" ^^^i'o^an log, I Nhu vgy, hieu qud c l dinh enzyi«
d ^ p h V h r ^ d c mN^n"^ ' ^ ' K - ' ' ? * ' " '^ ' = " ' ° ' ^ " ' ° ^ ' ' " ^ ^ " ° ^ " ' ° ^ ' " < chitosan logi Ill.Kl. qJtht Z ^ =n J ^ . ^ ^ ^ ^ - 5 """^ ^°' "^' ''^" ''^'^°^^" °^ "^'cfi thudc ldn se cd b l mat Idn han nin khd nSns mang enzyme tren be m$t se cao hon vgt ligu kich thudc nho. Hip phy vat ly khonq ldm dnh h i S o S n clu tme cto a m T i r f h i S T . " * " ? " ^ ^ ' ° ' ' " " ' • ^^' "^" "^ '•^'^^ ^^"'^ "^6 h o n k n kl? v d T e n i e ^ O ylu b I n g S S d " N h u U ^ ^ ^ ^ t S d ^ S V S r "^i?^' ' r ^ " ' ' ^ " ' " " ^^" ^^" hogt tinh J d 3 a n h s o ' v d i t S * ^ S i g)l <^ h!|u q S ? ^ o ^ h l t T d S 4 ? 8 % ' ' ' " ' " ' ° ' ' " '"' ' " ' ' " ^ " ' " ' "^^ ^''^ « - - ^^' -^h^* ^^ ^^ ^V^"^-"*
C l djnh laccase tren CNT
C C G N G N G H E S I N H H O C T O A N Q U 6 C 2 0 1 3
S a u Wii p h d n i m g d d j n h k i t thiic, C N T d u g c n>a b i n g d e m vd ly 1am d e loai b d laccase k h o n g b d m tr§n b l m | L H o a t t i n h l a c c a s e d djnh trdn C N T d a d u g c xdc dinh v d k i t q u d d u g c trinh bay d B a n g 3.
Bang 3. Ho^t tinh laccase t n i v c v k h i c 6 djnh tren CNT
E n ^ . Laccase Cho Laccase co dinh
Hoat tinh laccase (U/l)
42,33 29,23
Ti> B d n g 3 ta t h i y laccase c u n g da d u g c cd djnh tren C N T vd hogt f i n h en2yme d dinh dat 6 9 % s o v d i b a n d a u . C a n g - R o n g v a Pastorin (2009) d a Chung minh e n z y m e d u g c mang n h i l u h o n tren C N T b a n g h i p phy vgt 1;^ s o v d l lien k i t d n g hda tn C d c tdc gid nay c u n g nhdn t h i y rang e n z y m e d u g c c 6 djnh b d n g p h y o n g phap h a p p h u vat ly d hogt t i n h c a o h o n khi c l dinh b d n g lien k i t d n g hda tn d o h i p phy vat ly e n z y m e g i u d u g c c l u tnJc v d l u g n g e n z y m e h i p p h u c u n g nhieu h o n . T u y nhien, hieu q u a d djnh c u a laccase b d n g c a hai p h u o n g p h a p d e u khong cao h o n 3 4 % . N h u vdy, hi#u s u i t c l djnh laccase dgt 6 9 % trong nghien c d u ndy td do laccase od t h e d u g c c l djnh b i n g ci lien k i t d ^ g h d a tn va h i p phy vgt ly.
C d c k i t qua n g h i e n c i r u d trdn d a c h i ra r i n g hi^u q u d d djnh laccase tren C N T c a o h o n h a n so v d i tr§n chitosan ( 6 9 % s o v d l 4 2 . 7 8 % ) . N g u y e n n h a n c d t h e Id do C N T m a n g d u g c n h i l u p h a n t u e n z y m e tren b l m d t h o n chitosan v i t i t d n g u y e n t u c a c b o n c u a o n g d e u t d p t m n g tgi b l mdt, dign t i c h b l m d t l d n va hieu i r n g b l m a t p h i i h g p cho s y h i p phy vgt \</. Ngoai ra. t i t c a c d c n h d m c h u c ciia C N T d e u n a m tren b e mgt trong khi h a u h i t g l c - N H 2 cua chitosan k h d n g n d m trdn be m a t d o d u tnJc vgt lieu n d n higu s u i t d dinh b i n g lidn k i t c d n g hda tri ciia C N T c u n g c a o h o n c h i t o s a n . O a n h g i d k h a n a n g c h j u n h i e t c u a l a c c a s e c o d j n h t r e n C N T & e o ' C
Bdn c h i t cua e n z y m e la protein d o d d c h i i n g r i t nhgy d m v d i nhigt d d . D d n h g i a tinh ben n h i f t c i i a e n z y m e Id mOt trong d c khdu q u a n t r g n g n h a m a p d u n g c h i i n g trong t h y c t l . Khd n d n g chju nhiet ciia laccase c l dinh tren C N T d eO^C da d u g c khao s a t . L a c c a s e t h d vd c l djnh d u g c u d nhigt d g 60°C trong 1 g i d , hogt tinh cua laccase d u g c xdc djnh theo t h d i gian va d u g c t r i n h b d y tren H i n h 1 .
] laccase c6 djnh (•) Thiri gian (phut)
Hinh 1. Kha nang chiu n h i ^ t cua laccase c d djnh tren Hlnh 2. Anh h u d n g cua pH t d i hoat tinh c CNT(») v i t h 6 { » ) d 6 0 ° C v i t h 6 ( 4 )
T u H i n h 1 ta t h i y l a c c a s e c 6 d j n h tren C N T vd laccase t h d d i u k h o n g b i n d 6 0 ° C . Hoat tinh laccase g i a m m g n h n g a y s a u 10 phijt d i u t i e n , c h i c d n 2 4 % so v d i b a n d a u d l i v d i e n z y m e tho v d 1 7 % d l i v d i enzyme c l d j n h . K i t qui thu d u g c c u n g c h o t h i y e n z y m e c o d j n h k e m b i n n h i | t h o n e n z y m e t h d d 60''C. Khi e n z y m e d u g c d djnh len C N T d a Idp t h i h i p phy vdt ly x a y ra c a o h o n hai lan s o v d i lien k i t cOng hda tri ( C a n g - R o n g v a P a s l o n n . 2009) nen d Uie trong nghign c i r u nay laccase d u g c d d i n h len vat ligu C N T c u n g dd xay ra hien t u o n g n h u tren. D o hien t u g n g h i p phy vgt ly trang qua trinh d d j n h , khi u v d i d e m v d r u a n h i e u l l n d t h i laccase da bi nha hap p h u v a bj loai b o b d t n e n hogt t i n h cOa laccase c o d|nh g i d m s o v d i laccase thd
A n h h t p d n g c u a p H t d i h o a t t i n h c u a l a c c a s e c o d j n h t r i n C N T
p H m d i t n j d n g d n h h u d n g rat I d n d e n t i c d o p h d n irng e n z y m e . B d i p H d n h h u d n g d i n trang thdi ion hda d c g l c R cua cac g l c axit a m m t r o n g phdn t u e n z y m e , ion hda d c n h d m c h u c trong trung tam h o a ! d d n g va ion hda c o c h i t K h o d n g p H t h i c h h o p c h o hogt d f l n g cCia laccase t h f l vd c 6 dmh tCi" c h u n g F B V 2 da d u g c xac dmh ( H l n h 2).
Tren H i n h 2 ta thay, p H t h i c h h g p c h o hogt dflng c i i a laccase thd do FBV2 sinh ra d pH 2 va ciia laccase c o d m h tren C N T li p H 3. H o a t t m h l a c c a s e c l dinh d p H 2 giam x u o n g c h i dgt 75,13 % so v d i h o a t tinh ciia nd d pH 3. T r o n g khi do, hogt tlnh l a i p H 3 c i i a l a c c a s e thfl c h l dat 49,2 % so v d i hogt tinh ciia e n z y m e c l dmh tai pH 2, N h u v d y p H thich h o p cho hoat dflng c i i a l a c c a s e t d c h u n g F B V 2 da cd s y c h u y i n djch sau khi c l dmh tren CNT K i t qua nay c u n g t u o n g t y v d i nhO'ng k i t qua m d J u n g v a dtg (2005) da cong b l .
N h u vdy. l a c c a s e t u c h u n g Myrothecium sp, FBV2 d u g c d d|nh trdn C N T cd p H thich h g p djch c h u y i n t u p H 2 d i n p H 3 c d mflt y nghia rat q u a n trgng trong c d n g n g h g x u ly m d i t a r d n g v d i n h i l u n g u l n 5 n h i l m va d i l u kien k h i c nghigt n h u p H axit
H i e u s u i t l o a i m d u t h u o c n h u o m ciJa l a c c a s e t h d v d c o d j n h t r e n C N T
Loai m a u t h u l c n h u d m b d n g d c p h u o n g phap vdt ly h o d c hda hpc t h u d n g ddi hoi ky thuat c a o , ton t h d i gian vd hieu qua t h i p T r o n g n h u n g n a m g i n day, logi mdu va p h d n huy m a u b i n g p h u o n g phap sinh hpc dd d u g c coi la m o t
HOI NGH! KHOA HOC U U I N U ii'id^^^-t^mo.
uic . Q u ~ . . - . . - " ^ - - etal.. 2009). Trong dd. laccase thfl vd laccaseo.,, S c ' s d " d u r n dr'ioa7mdu liiuic nhuflrn. Hai loai mau NY3 vd NY7 thugc hai nhdm mau t u o i ^ |
" " ^ . „ r • ' j . ^ i a i _ i , _:A L-h4 r,5nn Inai m a n CUa a c c a s e c o rlinh Iron r^MT fl-llnh -^l 9 nhuong phdp thay t h i va than thien voi moi truong
u i dann dugc sir dyng d l logi mau thuoc nhugi . - ,
a n l h S . n o U a azo da dugc su dung dk danh gid kha nang loai mau cua laccase co djnh tren CNT (Hinh 3).
0 ) 3 3 •! 5 6 Thm gian (gio-l Ttim gian Itn*)
Hinh 3. Kha nang loai mau NY3 (A) va NY7 (B) bdi laccase tho (•) va laccase co djnh (•)
Kit qua trdn Hinh 3A cho thay laccase tho \ir chung FBV2 cd kha nang logi mdu NY3 tot trong khoang flidi gian ngii Sau 1 gid, laccase d djnh da logi dugc 76,9 % mdu NY3 tuong duong vdi lugng mdu ma laccase ty do loai IJLHIC trong 5 gid. Sau 5 gid, laccase d dinh da loai bo dugc 94,2 % mau NY3 trong khi dd laccase tho chi logi dugc 78,9 X So vdl mdu NY3, higu qua loai mdu NY7 ciia laccase c l djnh cao hon (hinh 3B). Sau 1 gid, laccase c l d|nh da kqj dugc h l u nhu toan bo mdu NY7. Trong khi do, laccase tho chf togi dugc 21,3 % mau NY7 sau 5 gid phan dng. N^
thuflc nhdm mau azo Id nhdm rat kho bj pha huy bdr d c tac ddng sinh hpc. K i t qua thu dugc trong nghidn ctPU ndyo chi ra ring enzyme d djnh cd y nghTa Idn khi dng dung trong cong nghe xu ly de loai mau NY7 Theo nhilu nghignaS da dugc d n g b l cho thiy hieu qua loai mau ciia enzyme d dinh dd dugc chirng minh ludn cao hon so vdi enzyme|i do (Kunamneni etai. 2008)
Hi^u s u i t p h i n huy phenol cua laccase tho vd laccase co djnh tren CNT . Phenol Id mflt hgp chat vdng thom co nguon glc ty nhien vd tu cac hoai ddng ciia con ngudi. Phan huy smh hpc phEffl
Id kit qua hogt dflng d a mpt so lugng Idn cdc chung vi sinh vat bao gom vi khuan, n i m vd xa khuan. Ddc bi^t,^
chijng vi sinh vdt d kha ndn^ smh enzyme ngoai bao nhu laccase. Do do, kha nang loai bd phenol vdi ning dfl 20-1M ppm d a laccase lir chiing nam Myrothecium sp. FBV2 c l dinh tren CNT da duoc khao sdt sau 24 gid. K i t qud Jo^ll
phenol cua laccase thfl va laccase c l djnh trfin CNT duoc trinh bay tren Hinh 4. 1
Ndnga^ phenol ID
Hinh 4. Kha nang toat ho phenol cua laccase co djnh tren CNT (•) Td Hinh 4 la thiy hi§u suit loai phenol ciia laccase
j laccase tho (•) a cac nong dp phenol khac nhau sau 24 gtt
^o ^!"|i ^a jM' do giam khi ndng do phenol trong dung dmh tang dk ndng dO 20 ppm phenol, laccase cfl dinh da Ic^, >,
S L ' " i ? n ' ' i l ' L ' i " 3 ? - " ! _ " * ? 5 * " ? « ' P^an huy phenorcu'a"ra;cas;'c6 * ( , tren CNT cao han'laccase t k 8
„ ? . ' [ " S ° i ' ' " ™'!° .""""S ?•• S " ' '™"S ' ' ' " enzyme ttio ctil loai dui/c ttxm phS hOy cao. Nhi, v^y trong ttidi g
cao tion laccase tho. Trong ttifri glM n dang ke con laccase co djnh vgn cJuy tri dirge hi^u sgi th6. cac ket qua thu W c da'chLno mmh'!!jm"2-*^° " * ' ; ["^'."8 Phenol b, loai bo, laccase c6 dinh 'se cao ton laced dgng laccase d4 lo,i b f i S c chit , S g , h ™ " " " "° ™° '^"^ "°"<l ' " " " 8 "^ '^' ™ '^'e 1"1 «'">' «"S " * « '^
K t T LUAN
Laccase thfi cua chung n4m Mvipthenum en crjvyi .^- a « . . .
enzyme d,t 42.8% tren v,t N J c h i t S a T c T X S ^ k „h™„° ton's 1 " k P h - " " " ' " " " ' = " ' ° " ™ " ° " « " = " * ' ' ^ X dinh laccase tren ong cartion nano cao hon khoing 1 6 iSn so yd" c h t e a """" ^ ' " ' ' " ' " ' ' ' ("•"' 9 " ' " ' * " ^ " * ' ' pH thich hgp cho hoat d6no cua lacrj^w ..A rt..., - .
cS dinh tren vat K u nay khong ben h " „ so vo, e n z y m e r r t " „ T « n v ' ' ° ' ' " " " ' ^ """^ ""' ' ^ - ^ ^ ^ ^ t t o i, pH=2. Enzym d i u 3 Laccase co dmh tren ong carbon nano c6 k h i „ j , 2 '' 8'°' H™' "nh con la, khoang 17% so v« W 3 lin so vft, laccase l i , do. LacMse c6 dmhTren A„o " "h?, " " " ° "? " • ' " ' " " ^ " ' ' " ^ ==<> ^ n fLrong ung 1,5.)
.nn tren ong carbon nano co kha nang loai bo 94,2% mau NY3, 98% mau NVI
: 0 N G N G H f SINH HOC T O A N Q U O C 2013
vol nong dp d a mdi lo^i mau Id 100 ppm sau 5 gid. Laccase d dinh d kha ndng loai bd 68,7% phenol vdi nong dd 20 ppm trong 24 gid
TAI L I E U THAM K H A O
Asgher M, Shah SAH, Ali M (2006). Decolonzation of some reactive textile dyes by white rot fungi isolated in Pakistan W J Microbiol Biotechnoi 22(1). 89-93
Cang-Rong JT. Pastonn G (2009) The influence of cartxin nanotubes on enzyme activity and structure, investigation of different immobirizabon procedures through enzyme kinetics and circular dichroism studies Nanotechnology 20(25) 255202
Datta S, Chnslena LR, Rajaram YRS (2013). Enzyme immobilizabon an overview on techniques and support matenals. Biotechnology
Delanoy G. b Q, Yu J . (2005), Activity and stability of laccase in conjugation with chitosan. Inter J Biol Macromol 35. 89-95, Feng W, Ji P (2011). Enzyme immobilizied on cartwn nanotubes. Biotechn<^ Advances 29: 8 8 9 ^ 9 5 .
Imran M. Asad MJ. Hard SH, Mehmood S (2012). Production and industna! apfHIcation of laccase enzyme. J Cell Moi Biol 10(1) 1-11.
Jung SK, Chae YR, Yoon JM, Cho BW. Ryu KG (2005). Immobilization of glucose oxidase on multi-wall carbon nanotubes for biofuel cell applications. Micmbio Sotechnol 15(2) 234-238.
Kunamneni A, Ghazi I, Camarero S. Ballesteros A, P b u FJ. Alcalde M (2008) Decolorization of synthetic dyes by laccase immobilized on epoxy-activaled carriers Process Biochem A3 169-178.
Nisha S, Anjn Karthick S, Gobi N (2012), A review on methods, application and properties of immobilized enzyme. Che Sci Rev Lett 1(3)-148-'55
Singleton VL, Orthofer R, Lamueia-Raventbs RM (1999), ^^aiysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent P Lester. Editor. Academic Press. Methods m Enzymol 52-178,
Tischer W. Wedekind F (1999) Immobilized enzymes Methods and applications. Top Curr Chem Vol 200 Spnnger Veriag Bertin Heidelberg.
Lii cim irn: Cong Irinh ndy dupe hodn thdnh bai hnh phi cua de tdi Dgc Igp cap Nhd mroc: "Nghien cuu cong nghe sdn xudt eniyme ngogi hao
laccase, managanese peroxidase vd lignmperoxidase (MnP vd LiP) lu vi sink vdi phuc vu xH ly cdc chdt 6 nhiim da vdng thom " giai doan 2010- 2012.
CRUDE LACCASE IMMOBILIZATION ON CARRIER MATERIALS FOR PHENOL AND DYE REMOVAL
Nguyen N g u y e n Q u a n g , D i n h Thi T h u H a n g , P h a m Le A n h , Nguyen Thi Lan A n h , Dang T h i C a m H a ' Institute of Biotechnology, VAST
