HOI NGHI KHOA HOU uuiNu. .>
r,^,roAr,icuiDFRnXIDASE(HRP)TR£N VAT Lieu KHUNG HOU ,
^ g r L O A l T e f o ' ^ M O F r S ^ ^ B6, NH6M CHCTC A M I N
Ding HSl Long, Nguyen Van V ^ f ^ ' f ^ Z s i B h Thu Huong, Nguyin T h t Anh, HU Duy Trinh, Nguyl.
Quyin. Luong Van Son, H6 Th| Oanh, oo irung oy, DlnhTuytn*
«#n Hda hoc. Viin Han lam Khoa hgc va Cong nghe w a Nam
... ,. i - J , j . K-„. „„ iHn, loai rBiO-MOFs) chl tao xuc tic sinh hpc v6i hlfu suit c6 djnh V * c6 di.4 cic CB,™ ! » vK u a . hybnd khcg h™ ^'TJ'^ISCTAhgc i i v S „ d j q t m l « , . g vSmStkho.h«cT, pmtein CO. hos tIri, . i do b e . o ^ ^ S n ^ l S r S y tTtog « S b i , cic k*r,»i .6og h w vi bifef ttoh Mll-101 X g ci!
nhu dioh hmSng iing di«g cong ngh.w Trong 1 * ^ ^ ^ 2 I H B P 1 vi dinh gii tfnh chfi xiic tic dia enzyme c4 drnh Mn, phinltagphinhi5,AlmmrcdS<AM)XScd»;«mJioaiyc^^^ phSn tfch nhiSt TGA, hSpphvftihihSp phu Nito (BET) Ci.
S t ' ^ S t d S ; ^ t o ^ b T M ™ S : d " : : ^ " r n ^ S ^ h ; „ ^ " i o < - N H O c d h i ^ J c « d W . c n z ^ ^ b ^ nhi^ cao hon Mil-101.
r,>ittrf«: horseradish peroxidase CHRP), c6d,nh enzyme, BiO-MOFs, chirc nang hoa nh6m chuc amin, NUI-101-NH,
MtiroAu
Paroxidase tCr cO cfii ngva (Horseradish pert>»dase - HRP, EC 1.11.1.7) IS rnOtgly^proteinci khfii Ii/7n9pharit&
2 7 * 3 9 ^ a L l/mcJ pdypeptida ciia 308 amino acid (Welinder, 1976), di.grc bilt d i n vfi'i nhilu ..ng dgng trong oOng m h V h i sSTl^RP <S ^ K g me t l bSo thy^ v|t vS dOng vHt xuc tdc cho phSn Crng oxy h6a khC hydrogen peiDjddB S mfit sfi hpp chit nhl.: ascoiLla, ferrocyanide. cytochrome C, mOt s l thulc nhuOm thuOc nhfim anthraquinol. Clu S^=Sa enzl^m peroxydase gim hai trung tam kim loai, mot nhom ion sat hoa trj 111, hai nguySn tir canx. (Veitch vi cs.
2004). Duvc thi hi§n frfin Hlnh 1.
Hlnh 1. cJlu tniic hda hpc v i khong gian cua Horseradish peroxidase (Veitch vi cs 2004)
Xilc tac enzyme HRP thi/dng dug'C c l djnh tr6n cdc ch4t mang vo co va cdc polyme hOu co'. cdc hp'p chit polyme ci ngufin glc tu-nhiSn, cic lo?! silica mao qudn tmng binh nhu: MCM-41, SBA-15, cdc polymer ting hp'p, polysaccharide tg nhifin v.v.,. Yfiu clu dfii v6i cdc chdt mang cin co b l mgt rifing i6n, kich thuoc hgt v^t li§u nhl, c l u trCic mao quin vd d$c dilm phfin ci/c b l m|t tuong thich voi kich Uiu6c phSn thu enzyme, nham ktiuylch tdn, hip phy tio$c t^io ll*n kit hfia hpc giD^ enzyme vi>i chit mang ran. Ok cfi thi tao cdc clu noi li&n kit giOa enzyme vd b l m$t chit mang, ngufii ta biln tlnh be m$t b l m$t ho|c hogt hfia cfic nhfim chuc cfi san tren chit mang bdi cdc nhfim chirc amin, hydnsxyl. cacboxyl (NHj, COOH. OH...) (Veitch, 2004; Welinder, 1993).
Vdt liOu khung hOru co kim kigi (Metal Organic Frameworks - MOFs) v6i su kit hiTp nhung uu dilm cua d$c tlnh v 6 ^ vd h&u CO dang du?c nghifin cuu, phdt triln nhO'ng kha nang i>ng dgng rit dpc ddo cua no trong phdn tdch tiln hop 1 ^ CO/CO2 nhim gidm thilu hi§u ung nha kinh, tdng trd Hydrogen Idm nhifin lifiu sgcti, xilc tdc chgn lpc c l u hlnh trong tm phdn irng hfia duvc, ldm chat mang cho cac loai thulc ch&ng ung thu...( Nguyin vd cs, 2012; Ligia ef al., 201^
Yuanbiao et ai, 2012). Dgc bifit hifin nay trfin thi gio'i co mfit hufing nghien ciiu rit mfi'i Id phdt tnIn cdc vit li§u MOF^
sinh hoc (Bio-MOFs) bing each biln tinh b l mat vdt liSu MOFs d l cfi djnh cdc enzyme tao nfin cdc xuc tdc sinh hijc trong ting hgrp hfia duvc, chl tgo nhien lifiu smh hpc vd xu ly moi trufi'ng (Bio-Catalyst) (Ligia et al.. 2012). Ddy Id c*
v$t lifiu mfi'i thdn thifin vfii mfii tru&ng, cfi nhung dac tinh rat uu vifit: b l mdt rifing Ifin, co cdu tnic mao qudn dgng I&ng (cage), clu tnic dgng kSnh (chanel), vd clu true Idp the (chiral) Yuanbiao et ai, 2012; Chen et ai. 2008).
Tgi \fifit Nam cung nhu hfin ttil gioi da cfi^ cdc nghifin cuu d dinh enzyme su dung cac vdt lifiu vfi co vd hiju co trfin ^ sfi' silica, dioxittitan hogc polyme tfi' nguon polysaccharide, chitin, chitosan... nhung chua cfi cfing b l ndo v l d di®
enzyme peoxidase (HRP) voi kich thufi'C phan tu Ifin (-44.000 Dalton) frfin vdt lifiu khung hOu co kim iogi {MOFJT Phfing Xuc tdc I/ng dgng - Vifin Hfia hpc - Vifin Hdn Idm KH&CN Vifit Nam da tiln hanh ktido sdt trfin mOt sfi vdt liO"
vd dd cfi nhung kit qud kt^ quan, qua dfi cfi the lam rfi thfim vfi co chl cfi ^nh enzyme ding th&i bing h i p phg vfitlj vd sg- tgo thdnh cdc clu nfii lifin ket giOa nhfim amin cua enzyme va ntifim carboxyl ton tai trfin cdc c l u nfii (ligand hiW CO) cfi sin trfin vdt lifiu MOFs duoc hogt hfia.
Hlnh 2. Anh SEM tinh thi cOa Mll-101 (Ting hifp tgi Ph6ng Xue tdc l>ng dgng - Vi?n Hoa hpc) THXJK NGHIDM
Hfia c h i t vd t h i l t bt
Chnsmium (111) nitrat: Cr(N03)3.9HaO (Trung Qulc), axit 1,4-benzen dicartjoxylic: HzBDC (Merck), Cetyl frimettiylammonium bromid: CTABr (Merclt), axit flohydric: HF (Trung Quoc), 3-aminopropyl friethoxysilan: ; ^ S (Merck), toluen (Taing Qufic), etanol (Trung Qulc), nude cit: HzO; Dung djch dfim axetat nahi pH = 4,7 nong dp 0,1 N; Alizarin red S (ARS) (cfi vai trfi nhu chit khi>. ban diu cfi mdu vdng, khi bj oxy hfia khfing mau) hogc indigocarmin (Ban diu cfi mdu xanh, khi bj oxy hfia khfing mdu); 60 pi HzOz 0.2 N; Peroxidase nguon glc tu cay cai ngga dirge chilt tdch vd tinh sgch tgi Phfing Hogt tinh sinh hpc Vifin Hfia hpc - Vifin Hdn ldm lOioa hpc vd Cfing Nghfi Vifit Nam; Mdy ly tdm HERMLE Z 400 K (DO'c); Mdy quang phi hip thy UV-Vis Lamda 35 (My).
Cdc mSu xOc tdc duoc khdo*5dt cdc ddc dilm hfia 1;^ bdng cdc phuong phdp: Phi nhieu xg tia Ronghen (XRD): Mdy D8 Advance-Bruker; Pho hing ngogi FT-IR: trfin mdy THERMO NICOLET; Phuong phdp hien vi difin tu quet (SEM) duoc ghi tr«n mdy NIHE S-4800.
Phircmg phdp t i n g hp'p v | t li$u Mll-101
Mil-101 dup'c ting hp'p bing phuong phdp thfiy nhifit vfi'i ngufin kim logi Id Cr(N03)3.9H20, c l u noi hO'u co Id HzBDC, HF. Hin hvp dup'c khuly trOn ding nhlt trong 1h. Sau dfi hon hop duoc dua vdo lp Teflon co dp ddy 0,6 cm, dgt trong autoclave, tiln hinh thu;^ nhifit d 220°C trong 9h. Sdn phim thu duprc dem lpc bang giay lpc sfi 2 lly dung djch cho lpc bing gily Ipc sfi 5 rfii sly d 10O^C trong 3 gift' -* sdn phim ran A. Sdn phim A + etanol (95%) + ngam trong autoclave d 100 C trong 22 gifi'. Sau dfi dem Ipc, n>a bang etanol nfing, sly kho thu duvc sdn pham dgng bpt mdu xanh ngd -^ MIL- 101. Cdc mSu vdt lifiu dup'c do XRD. FTIR vd BET.
Chi>c ndng hfia nhdm amin ldn v$t li$u MIL-101
Cdc mlu Mil-101 dup'c tiln hdnh g i n nhfim amine NH2- nhu sau: 0,5 g Mil-101 dup'c hoat hoa 6 423°K trong 12 gifir, sau dfi duvc phdn tdn trong 30ml toluene. Tilp tgc cho vdo hfin hgrp frfin 0,75 mmol 3-aminopropyltrimethoxysilane (APS). Hon hp'p duoc khuly hfii luu trang 12 gifi'. San phim dup'c loc n>a v6i nirdc cit / etanol sau dfi sly khfi. K^ hifiu mlu Id Mil-101-NH2. Cdc mlu v$t ll$u duvc do XRD, FTIR, BET.
Co djnh enzyme horseradish peroxidase len v$t ll^u MOFs
St> dgng 100 mg v$t lifiu MIL-101-NHz da tfing hp'p cho mil thi nghifim, vgt lifiu dup'c n>a bang 5 ml dung dich dfim, frfin diu tn>ng 10 phut bing mdy Voltex, sidu I m trong 5 phiit, sau dfi iy tdm fi' toe dO 9000 vfing/phut trong 10 phOt, thu Igi phin r l n . Tiln hdnh gdn enzyme bing phuong phdp tlm: 3 ml dung djch dfim duoc bo sung, cho 0,2 ml HRP dfing c6 djnh. djnh mi>c bing dfim din 5 ml, de 2 gifi-, moi 5 phut trpn 1 lln fi' dieu kifin nhifit dfi phfing (26°C). Phan xiic tdc ran thu dup'c bing mdy ly tdm tic dO 5000 vfing/phut vd rua bdng dung d[ch dfim 4 lan^sau dfi thuc hifin phdn ung phdn huy Alizarin red S trfin xuc tdc enzyme c6 djnh. Phan Crng tren cung thu'C hifin trfin mdu dfii chung khfing cfi enzyme vd mfit trufi'ng phdn img dirge thay bing dung djch dfim. Phdn ung enzyme thuc hifin d dieu kifin: 2 ml dfim; 0,5 ml;
Alizarin red S 0,001 N; 1 ml H208 0,2 N, khuly trpn frfin may khuly ti> lifin tuc d nhifit dfi phfing (30°C). Cdc mau dung djch hon hgp phdn Ong duvc lly ra theo thfii gian phan ung 0, 10, 20, 30, 40 va 60 phut, tdch phin long dem xdc djnh nfing dp Alizarin red S U^n mdy quang phfi hip thg UV-Vis (Lamda 35) tgi bud'c sfing 525 nm.
Hogt dfing cua enzyme dup'c ddnh gid Id kha ndng tgo bpt khi khi enzyme »jc tac cho phdn irng khu HzOz tgo ra Oz.
Oxy m6i sinh ndy oxy hfia (Alizarin red S) Idm mit mdu chat nay. Hoat dfi cua enzyme co ^ 1 duoc xdc djnh bang phuang phdp do m§t dfi hip thg quang hpc (OD) d bud'c song 405 nm trfin may Tecan Genios, so sanh vcri dudng chuin bidu diin tuong quan giO'a nong dfi Wizarin red S vd OD.
K^T QUA VA T H A O LUAN
D|c d i l m hfia ly v$t ligu MIM01 vd Mil-IOI-NHz
D l Khdo sdt cdc thfing s l cua vdt lifiu cQng nhu khang djnh sg cfi mgt cua nhfim ehuc -NHz da duvc biln tinh trfin Mil- 101, cdc m i u Mil-101 vd MII-IOI-NHz duvc ddc truong bang cac phucmg phap pho, tCr do da co ntiung ehi>ng tfi d§ cfi
HOI NGHj K H O A H O C C O N ^ J N o r w j
Hlnh 3 Phi XRD cO. MIL-101 (.). «IL.101-NH.(b) (ghi M 0,1 h,c KHTN - 0 , 1 h,c OGHN)
P h i h i p (h(* hdng ngo?/ FT-/R ciia cic v^t liiu Mil-101 vi Mil-iOI-NH,
4000 3500 30OO 2500 2000 ISOO Wavenumbers {cm-1>
hllnh 4. Phi IR cua MIL-101 tnr6c vk sau khi bi4n h'nh NHt(do tgi Qai hpc Bdch Khoa Hd Npi)
Kit qud phfi FT-IR thu duvc trinh bdy frfin hlnh 4. Trfin hlnh ta thiy d mlu Mil -101 ton tgi cdc dtnh dac tning cho cfc nhfim, lifin kit trong vdt lifiu Mil-101. Dinh fi ddl tan s l 1623 cm'MdehTJ'ng cho dao dfing bat doi xung cua lien kit C=^
Dinh d vung tin sfi 1300 -1800 cm'^ d|c trung cho dao dfing cua nhfim carboxylic trong axit terephthalic. Dinh d tfln f 1166 cm'' vfi'i cufimg dfi nhfi d$e tnmg cho dao dfing cOa lifin kit Cr-O. Ddc bifit fi-sau khi chiic nang hfia nhfim-NS Ifin MtL-101 xuit hifin ddi phfi fif tin s l 1544 cm"' dgc trung cho dao dfing cua nhom amino. Chipng tfi da bien tfnh nMB amin t h ^ h cfing.
Dl^n dch mao quin vi kich thirdc mao quin cUa v$t ll^u
c l u friie mao qudn cua cdc vgt lifiu cung dup'c khing dtnh qua duong ding nhifit hdp phy / nhd hip phy Nz. Kit qu*
trlnh bdy frfin Hinh 5 vd Hinh 6 cho thiy, trdn dud'ng ding nhifit hap phg / nhd hip phy Nz cua cdc v^t lifiu MIL-101 vi MlL-101-NHz diu xuit hifin vfing t r i dgng iV (theo phdn logi cua iUPAC) d$c fr^ng cho sy cfi mdt cua logi mao qu*
trung blnh. ~^
Sau khi chCrc ndng hfia nhfim amin -NHz Ifin MIL-101, difin tich b l mdt rifing SBET va the tich mao qudn cOa . NHi da gidm di so vfii MIL-101 (bdng 1), dilu do dup'c gidi thich do cac nhfim amin so d p dd tin tgi trfin cdc maa, ldm che phu mfit phin b l mgt cua hgt tinh the Mll-101,
Mllfl
naag
• m
•mn
«nn nnn .nn
•m
i m n
/ ' /
' ^*
>"
•111-1 (
n - K Jill i i . i
1 -A( Mipe l-NH m i l
-AdiirplD
' J
1
1
j !
j
'
1
012 010 ooe
O M
-on?
fe -
fe . ( • 1 , 1
i ^ ! . ^ i •
•e ,-e-Mil-101
t . 1 — MiMOI-NHJ , 1
! : ' i
Pore Diaineter (nm)
™'' !.!lim^jS"l''!;'H,''f?''''S'""? ''^? I""" "''™ ""• HI"!' «• a"*w PM" "» w-^h »"*= ™» qiS" oia "lii-'oi »»
Bdng 1. Cdc thfing s l d u tnic cfia cac vit li$u
V$t li$u SBET(ni'/g) Vpo„(cm'/g) Duong kinh mao qudn (nm)
MIL-101 MIL-IOI-NH2
2964 1697
0.19 0.17 Khi ning c6 ^nh Enzyme HRP cOa Mil-IOI-NHi
Hlnh 7. Phi UV-Vis cda hin ho-p phan Cmg phin hu;^ Alizarin red S bing xdc tdc enzyme HRP c£ dinh tren Mil-101-NH2 Cdc mlu v|lt 1i$u sau khi c l djnh enzyme duvc bao qudn bong thoi gian 7 ngay fi' nhifit dp phfing (26°C), sau dfi cho tham gia ptidn i>ng v6i Alizarin red S d l kilm tra dfi bin cua enzyme. Phdn O'ng enzyme bao gim: 10 mg chit mang (vdt lifiu Mil-IOI-NHz) + e n z ^ e HRP; 13,5 ml dfim RB; 0,5 ml Alizarin red S 0,003 N; 6 ml HzOz 2%, d l phdn ung xay ra 6'nhi^tdO phfing.
Hlnh 8. Hoat tlnh xilc tac cua enzyme HRP t^r do vk enzyme HRP co d|nh trin Mil-101-NH2
Hinh 8 t h i hi$n hogt tinh xiic tdc cOa enzyme HRP a cdc dilu kifin Wide nhau. & dgng tu do, HRP gin nhu khfing cfi hogt tinh, Vnk hi§n qua ning dO Alizarin red S duiyc giu nguyfin. Khi dup'c c6 djnh trfin Mil-101-NH2, HRP da bat diu cfi hogt tinh nhung khd nang xiic tdc v l n cfin rit thip (ehuyen hfia duvc 5% Alizarin red S). Thir trong dllu kifin b l sung 5 ml HzOz, enzyme HRP co djnh trfin Mil-IOI-NHz t h i hifin rit ro rfit khd nang xdc tdc phan ung, ngodi thoi gian h i p phg.
chl trong 20 phiit, logi xiic tdc ndy da xuc tdc chuyin hfia dup'c 35% so vfi'i ning dfi Alizarin red S ban dau. De khao sat
H O I N G H I K H O A H O C C O N G N G H b a i i N n n y L . i ^ .. ,
d f i ben ntMfit. enzyme NKh* co o n n i r e n i w i i - i u i - i i n z A u i , «-v " " • ^ - , = ~ j - • ' . - . . . - . J . T T J ' ' . : — " " ' " j w i t t j AJizann t S s ^ ^ { t 7 % ) n h u n g chi>ng tfi enzyme HRP/Mil-101-NHz b i n v o i nhifit r i t n h i l u s o v o i e n z y m e H R P ^ ^ , HzOz Id d l l u kifin r i t quan frpng d l i v f i i khd nang xuc tac cua H R P .
K f T L U A N
Cdc k i t q u d nghiSn et>u c 6 djnh enzyme horseradish peoxydase H R P (eu cdi n g y a ) b * n vdt lifiu k h u n g h u u c o kim lo, M i l l O l v d vgt lifiu b i l n Knh M i H O I - N H z cho t h i y c v c h l g d n e n z y m e H P R trfin h f i frifing m a o q u d n vdt lifiu cfi th§ bao g i m diing t h f i i s i / h i p p h u vgt ly v d s u tgo thdnh cdc c l u n l i lifin k i t gi&a n h o m a m m eua e n z y m e v d nhfim caiboxyi ( t i n tgi frfin cdc ( 5 u noi (ligand h u u c a ) cfi sdn frfin vgt lifiu M O F s d u v c hoat h o a .
Hogt tlnh enzyme c l djnh HPR/Mil-101-NHz chO'ng minh vdt lifiu Mil-101 c f i t i i m n d n g l a m c h i t m a n g d ^ n h enzyme e4 t r i l n vpng frong c h l tao xfic tdc sinh hpc (BiO-MOFs).
T A I L I ^ U T H A M K H A O
Vertch. Nigel C. (2004). Horseradish peroxWase' a modem view of a classic enzyme. Phytochemlstry 65:249-259
Welinder K.G., Gajhede M. (1993). Stiucture and evolution of peroxidases. Plant Peroxidases- Biochemistry and Phycology. UnivenHf of Geneva: 35-42,
NguySn Olnh Tuyfen, Nguyln Th4 Anh. NguySn Ngpc Tilng. Dfi Tnjng Sy. VQ Anh T u I n , T r i n Thi Nhu Mai, D l Thu Huong, NguyJn DuyTrinh, Hendrick Kosslick (2012). Tong hpp, dgc tm-ng.bi^n llnh vat lieu khung hOu CO kim logi Mtl-101 vd Hnh chat quang xuc Be tmng phdn i>ng phin huy dur>g d|ch 4-fiitrophenol. Tap chi Hoa hoc 49 {5fiS): 181-185.
Ugia Fnjnza, Nteoleta Gheorghe. Constantin Paul Ganea, Reinhard Eckelt& Hendrik Kosslick (2012). Oxidation activity of horsetadish
^oxidase tiosted In molecular sieves, spectroscopic investigations show hindenng of the enzyme activity. Reaction Kinetics flfec/»n/Sfm and Cata'KS's 105(1): 195-205.
Yuanbiao Huang. Songjuan L u , Zujin Un, Weijin Li, Xlnfa Li, Rong Cao (2012). Facile synthesis of palladium nanopartidei encapsulated In amine-functionalized mesoporaus metal-organic Irameworks and catalytic for dehalogenallon of aryl chlorides J Ca(a/ys2fl2:111-117.
Chen YZ, Yang CT, Ching CB. Xu R (2008) Immobilization of lipases on hydrophobilized zirconia nanoparticles: highly enantioselecUv and reusable bkx:atalysts. Langmuir 24 (16): 8877-6684.
Lindsay JP, Womnsbecher RF, Dordick JS (2003). Surface hydrophobicity of silica supports effects activity of immobilized enzymesi organic reactions The 225(f>>4C5A/s(rana/iUeef/ng'. 23-27.
IMMOBILIZATION OF HORSERADISH PEROXIDASE (HRP) ON METAL ORGANIC FRAMEWORK (BIO-MOF) FUNCTIONALIZED BY AMINE GROUPS
D a n g H a l Lxino, N g u y e n V a n T u y e n , B a T h i C h a m , N g u y e n T h i P h u o n g D u n g , N g u y e n N g o c T u n g , Nguyen Tli Quyen^, L u o n g V a n S o n . H o T h i O a n h , D o T r u n g Sy, D o T h u H u o n g , N g u y e n T h e A n h , H a D u y T r i n h , N g u y e n Dinh
institute of Chemistry. Vietnam Academy of Science and Technology <
SUMIWARY
Iminobiiizai™ of tnzynes on hybnd solids mttai organic fameworks material. (BiO-MOFs) as biocalalysB with higli pro J oadoig ytdd, lugh «aivity, aabilily and reusabie which are interested io both scieoce and iodnslry fields. In this report the hyW M.I-IOI inateriaU were synlhesaed via hydrothemial treatment and innetlonalizcd wilh S-aminopropyllrielhoxysilane (APIBS).
post-synthesis grafting and weni then used to immobilize Hor,e»dish peroxydase (HRP). Tlie onino-flmctionafeed materials « ehmetenzed by vanons teehni^ies; XRD, SEM, IR and N2 adsoipdon-desoiplion (BET). From characterization results, il indiod that these matenals so I mamlained their stmcture after fimetionalization. The dau of IR and TOA-DTA analysis demonstmted I.
Zr ^^l^T? . . T l ' i . ^ ' " ^ " ^ ° ' " " " " " • " * " »f AfTES-fiincdonafad sample,. Hie HRP immobilized.
fcnetionaiized matenals eriubited higher catalytic activity and stability for conversion of Alizarin red S (ARS).
X ^ ^ r r t Horseradish peroxydase (HRP), enzyme immobilization, BiO-MOFs, hmctionalized amino group, Mil-lOI-NH,
•Author lor eor.ssBood»«e:T«:.84-913223900, Email: loyen„guyend,nh2003Syahoo.com