1 11 10BILIZA TION AND CHARACTERIZATION OF RECO IBINED LEVAlVSUCRASE FROI Escllericllia coli BL21 (D£3) pLysS-lsbl-bkl IN LEVAN AND POLY(R) -3- HYDROXI'BL'TJRATE BIONANOPARTICLES SYST£ 1

• Main Author: Cellyana, Jessica
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/38564
• Institution: Institut Teknologi Bandung

Le1•amucrav! I\ an en;ymt? !hat cata r:::e\ the \./lerose l rdro nir.eaction and fructose po men:::atwnmto lem11. l.emn1' afmc:tose po rmer that 1s composed of fructofuratlmyl re\!dW!'\ uInch are lmked by fi-0.6) g rcosidu.: 111 the main cham and fJ- C.l) g(rcu\. ultc 111 the branch cham. Levan 1. produced by se1•eral tJ7NS qf plant\, fungi, and most b_\ bacterw. I e l'anli luch Hproduced by bacteria lun h1gher degree of po men:::at1011 compared to otha otgamwm. !hi\ hwpo(rmer hm been applied w a111iox1dallf. bwnanopart1clt! drug delm!ry sy.\tem. and an altemat11'e for art1{icta/ \weetenen. In thl\\•tudy, lemm11c.nl\e wm expre\wd by f j.••,chenchia coli BL21 (D/ 3) pLysS­ l\bl-hk I by I PIG mduct1o11. I he lemnsucrase gene in the clone was obtamedjrom the haloplu!tc Bactlfu, l1chemfornm BK I bacterw uluc.•h had been iwJiatedfrom the sa/me rnud crater (?( Bledug Kuwu. Purwodadt, Central Jam. Indonesia. lmlatwn and c/onmg of the le1•am11crme gene from tlu. bactena has been done before. Inth/\ study, the recombmantlemmucrase obtwned had optimum pH of 6. optunum temperature of -10 ( •. meltmg po1111 of -13 -( •. and 1/. half-l1fe qf decay actii'I(J.'"' "'.8 hour.\. Jhese results are 111 accordance wllh the opllmum cond111ons (?f leransucrase from Ractllu\ !tchemfortnl\, the murce qf the lemmucrase gene. I emn \ylllhe\.1\ 111 l'ltro u.\lllf( rl!combmwJI le1•amukrthe and 1:!% (b \/lerose substrate solutiOn produced 3.ON wam\ of lemn -5 mi. of culture. l.emn obtained mtheform ojyellou1\h-11lute m/uA Ana n'' (f lewm \. lmcture., using the Founer lra/1\form infrared ,\/Jec/roswpy (!- TIR) shmn:d mmlantle\ Hllh \landard /el'(.t/1 from D-winia herb1cola. General(r. the l rdro l'-"" reactwn cata r:::ed by lemnsucrase follows the Aflchaeli\­ Afemen k1net1n, for example le1•an.mcra\e from Ractlhl\• whttll.\. Howe1•er, 111 th1., \ltuly lem11suc.:rase had allustenc characlert.\tlc.\ that followed H11f kme11cs. The kinetin parameten obtamed were the dlssocta/1011 con\lan/ (K J ) wluch 11as 30.-1 m\1, the max1m11m react/Oil rate o•," ) 1tluch u m 2:!.9-1 m.\1 .\ . and the H11! constant (h) 2.2-1. !he H1// cons/all/ mlue .\hou s that three lemnsucrase domams, C'-termmal. N-terminal. and transll/011\ hare the cooperati1•1v charactert\Ctlc 111 Iv cata nl\ reac:tiom. J.e,•an\ucra\1! with mmlar clwracten\IIC\ 1\ found 111 /emmucra.\e from l.actohac:lllu\ rl!uteri. flu, \llu r ua\ alll!mpl to tmmoh1l1=e recomhmalll !l!l'atlsucn.m! 111 the hwnallopar/1(:/e \T\Iem that earned 0111 hy m \'tiro /emn mu.f por (R)-3- hydroxyhuzrrate lt uh elllrapmenl method at 25°C wuh llrnllg 'peed rf 500 1pm. PHR t.\ a po rl rdroxyalkanoale dertl'(f/11'1! po rmer produced hy \l!l'erai!Jpes of hactata. PHR has mwr hydroxyl.functiOIIal group.\ \llch m /el'(ln, thentl was also uwd to lfnmobt!t=e /emmHcraw m thr\ .\tudy. Lffictellc.)' of tmmohilt=allon and aclll'tly of immobilt=ed en=_yme.\ohtamed wm about 50°/o and 96 11'tJ (f lllllial protem cmtcelllratwn and free lenlll\llc.'!'a\e ac/JI'tty. lmmohiit=ed lel'aii\UCI'a\1! nllllOfJllrttcle\ uas 1111.!Cl\llred to he :! 18.5 13.nm H11h Zeta polenttal of -63..,m J • Hherea.\ tmmohtiJ=ed l'HR-/enm,ucrme ua\ meamred 10 he -2x 90.8 n111 with poleIIIia/ Zeta-6-1.-1m I: 1he 1111!£1.\111'1!1111!111 remit\of the Zeta potentwlllldtc:ale that the /H o nanoparttc:/e \ han! Ju h \tahl/tzr. .Ve a/11•e m/ue \ mdtcate that /emmukrase 1\ \till ad\Orhed on the \lo:face (!(the nanopamcle\. I 1!\'all\llcra'ie tmmohilt=atum can q{[l!cl thl! dec'{\' 111111! (!f en=_rme pe '(ormance. Immohtli=ed /e1•amucraw ac.lll'lt\' hm half-l1fe of R.J hour\ wlule m1moh1ft=ed PHR-Ienmwkrme 1.\ -. .,hour.\. I emn cw1 mam/am the structure and actll'iZr r?f lemmucrase well so w to mcre(l\e the decay tune qf en=yme ac:ll\'lty. !tJmwhilt=ation rllemmuLnrse '!(feet\ the kmetic parameten of the en=_rme. Immoh1ft=ed fe,•an-lemnsukrml! IIWWparttcle.\ k111etlc parameten are K 1 2f. (i mAl. JlnlLX" 2-1.--1 m\1' . h l.fl uherem tmmohtlt=ed PHB-Iemnsukrase 11anoparttde. kmet1cpcmuneten are A. :!8.6 m\1, r, . 23.3-1 mAts '. h 2.21. /Jecremmg mlue of H1ll coll\lanl (h) mdtcatn that the JWOCI!\\ of lmmohllt=atiOII ca11 ' fleet the naturl! of cooperatiOn betuee11 lel'atl\llcrase domatm. l•ree recombmanl le,•amltcra\e tmmohJ!t=atton also mcreme' the 111mm•a n11mher of en=_rme 1-ree recomhmanl lemmucraw ha.\ k, :!2-1.9 ' , lmmohtlt=ed /e,•an-/el'a/1\llcra\e na11oparttde\ hm k 506 3 \.-J_ and tmmoht!t=ed PH R-lemno,;ukrase ha.\ k, -1-19, I s . /he mctwmng \'Uflle of k ,, \hou morl! prodltcl\that can he prod11ced hy one en=_rme 11111t111 a il'<'ll tune. Ihus. lfmnohlft=atlo/1 of lemmucrase mnanoparllde \')'\l<'m\ IIS/11 lenm and PHR i\ a hwcompatthle 'Y'lem.