B khoa hc v cng ngh Vin Cng ngh thc phm 301 Nguyn Tri, Thanh Xun, H Ni BO CO TNG KTKHOA HC VA KY THUT ti cp nh nc NGHIN CU NG DUNG CNG NGH ENZYMTRONG CH BIN MT S NNG SN THUC PHM M s :KC04 07 Ch nhim ti cp nh nc :PGS. TS. Ng Tin Hin ti nhnh cp nh nc Nghin cu cng ngh sn xut mt s loi du bo bng lipazaCh nhim ti nhnh cp nh nc : PGS. TS. ng th Thu H Ni,10 2004 Bn tho vit xong thng 09 2004 Ti liu ny c chun b trn c s kt qu thc hin ti cp nh nc, m s :KC04 - 07. Danh sch ngi thc Hin STTH v tnC quan cng tcMc 1ng Th Thui Hc Bch Khoa HN 2Quyn nh ThiVinCNSHTTKHTNv CNQG 3Phng Th Thui Hc Bch Khoa HN 4Hong Lani Hc Bch Khoa HN 5 Bin Cngi Hc Bch Khoa HN 6Thiu Linh Thui Hc Bch Khoa HN 7Nguyn Th Snhi Hc Bch Khoa HN Tm tt ni dung Lipase (EC 3.1.1.3) l enzym c ng dng trong nhiu ngnh nh cng nghip thc phm, cng nghip ho hc, cng nghip m phm, cng nghipda,trongydcvccngnhcngnghipkhcdockhnng xc tc thu phn triglyxerit thnh di, mono glyxerit hoc glyxerol v cc axit bo nh hot ng trn b mt phn pha du nc. Trong cng nghip duvchtbo,vicsdnglipasertphbin.Ckhonghn100 loi lipase khc nhau c dng chuyn i lipit thnh cc cht khc Hinnay,VitNamsdngmtlnglnlipasetrongngnh cngnghipthcphm,cngnghiptyra,hohc,yhcsongcc chphmlipaseangsdngphnlnlnhpkhu.Dovy,mcch ca ti tp trung nghin cu thu nhn v s dng hiu qu lipase t hai i tng l nm men Candida rugosa v Bacillus spPhng php nghin cu-Visinhvtcnuicytheophngphpnuichmc2chng Candida rugosa v Bacillus sp trn my lc lc n nhit Shellab v trn thit b ln men 2 lt.-Xc nh hot lipase bng phng php chun . -C nh lipase trn nylon 6 bng lin kt ng ho tr. -TchvtinhchlipasebngetanolvscktraoiionDEAE cellulose.-Bngphngphptonhcthcnghimtiumitrngnuicy theo m hnh bc 1 ca Box Wilson. -So snh sn phm phomat c ch bin c b sung lipase v khng b sung lipase bng phng php cm quan thc phm. ti thu c nhng kt qu chnh sau: -Tm c iu kin nui cy ti u cho sinh tng hp lipase t Candida rugosa cho mt lt l:Mitrngnuigm:KH2PO4:15g;K2HPO4:5,5g; (NH4)2SO4:4g;MgSO4:1g;FeCl3:10mg;inositon:0,004mg;biotin: 0,008mg;Thiamine.HCl:0,2mg;Duoliu:1%;axitpalmitic:1.2g; H2O: 1000ml;S t bo nm men cy ban u: 2,6.106 tb/ml canh trng;Thi gian nui: 40 gi -Tm c iu kin nui cy ti u cho sinh tng lp lipase t Bacillus sp l: mi trng BMGY vi thnh phn: (1% (w/v) cao nm men, 2% (w/v)bacto-peptone,100mMkaliphosphate,pH7.0,4x10-5%(w/v) biotin v 1% (v/v) methanol) 30C lc 220 vng/pht -Nng cn thch hp cho kt ta thu lipase l 90%. Lipase sau kt ta cn cho chy sc k trn sc k cho thy lipase sau khi qua ct trao i ion DEAE - cellulose xut hin 3 pick. Tin hnh xc nh hot lipase cc phn on. Kt qu cho thy ch c pick 1 v pick 3 c hot tnh lipase (69UI/ml v 77UI/ml) v Lip1 v Lip3 c khi lng phn t l58v62kDa.Lipasesauccbclmsch:kttaetanol,sck traoiionDEAECellulosethucchphmchotring 55,16U/mgprotein,mctinhschl12,5ln;hiusutthuhil 37,06%.- kho st c cc iu kin ti u c nh lipase trn Nylon-6 l: nng HCl cht mang l 3,5N; glutaraldehit l 10%, nng lipase em c nh l 1mg/ml (550U/ml); s dng dung dch m phosphat pH 7,7. - xc nh mt s c tnh ca lipase c nh trn Nylon-6 Nhittiul70C,bn vng nhit 40 - 60C, sau 18 gi hot cn li l 52,4 66,7%. pH ti u l 7,0; bn vng pH 7 - 7,5; sau 18 gi hot cn 50,6 - 57%. - xc nh mt s c tnh ca lipase tan t Candida rugosa Nhit ti u l 60C, bn vng nhit 40 - 50C, sau 18 gi hot cn li l 50,2 - 59%. pHtiul8,0;bnvngpH7,5-8,0,sau18gihotcn 53,9 - 62%. im ng in ca lipase l pI = 7,5 nh hng ca cc ion kim loi n hot tnh ca lipase:- Ion Pb2+ c ch mnh (lm gim hot ti 33,26%) - Cc ion Cu2+, Fe3+, Zn2+ c ch yu (lm gim hot tnh 13%)- Cc ion Na+, K+ khng nh hng.-Cc ion Mg2+, Ca2+ c kh nng hot ho lipase. -Mt s c tnh lipase t Bacillus sp Tnh c hiu c cht i vi tributyrin l cao nht: 101 U/ml. Nhit ti u l 70C (91,1 U/ml). Lipase c bn nhit, sau 24 gi hot tnh vn cn 63%. pH 9,5 l thch hp nht cho phn ng, hot tnh t 108,1 U/ml. pH9,0mTris-HClvpH10,0mglycinekhngnhhngti bn ca lipase, hot tnh cn li vn t 117%. Ca2+ tng hot tnh lipase ln 10-12%, ngc li K1+, Mg2+, Fe3+ gim hot tnh lipase t 11-42%. Cc dung mi hu c u gim hot tnh lipase. Acetone, 2-propanol v ethanol gim mt na, methanol gim mt phn t. Ccchttynhhnglnhottnhlipase.TritonX-100tnghot tnh ln 63%. Tween 20 gi nguyn hot tnh (107%). Tween 80 gim mt na. Cn SDS km hm hot tnh lipase 3%. -khostkhnngthuphnduutngcalipasecnhthu c du c ch s axit t 80,5. -Sdnglipasetrongsnxutphomatktquchothyphomatcb sunglipaselmtngccchtiuchtlngcmquan(musc, hng thm,...)so vi sn phm phomat khng b sung lipase MC LC M u.......................................................................................... TNG QUAN TI LIU ................... 1.1. Cc ngun thu lipase .................................................... 1.2. cc yu t nh hng n kh nng sinh tng hp lipase............................................................................................................ 1.2.1. nh hng ca thnh phn mi trng dinh dng......... 1.2.2. nh hng ca iu kin nui............................................. 1.3. Cu to ca lipase: ............. 1.3.1. Khi lng phn t.. 1.3.2. Trnh t axit amin, cu trc khng gian.. 1.4.Mt vi c tnh ca lipase . 1.4.1.Cht hot ho v cht km hm 1.4.2. Tnh c hiu c cht 1.4.3. C ch ng hc xc tc ca lipase 1.4.4. Nhit v pH hot ng ca lipase 1.5. Tch tinh ch lipase 1.6. Cc phng php c nh lipase . 1.6.1. Phng php gn enzym bng lin kt ng ho tr 1.6.2. Phng php gi enzym trong khun gel 1.6.3.Phngphphpphvtltrnccchtmangc cha hoc khng cha in tch. 1.7. ng dng ca lipase 1.7.1. Trong cng nghip sn xut cc cht ty ra....... 1.7.2. Trong cng nghip thc phm. 1.7.3. Trong cng nghip dc v ho cht....................... 1.7.4. Trong cng nghip thuc da 1.7.5 Trong cng nghip m phm. NGUYN LIU V PHNG PHP ................. 2.1. Nguyn liu... 1 2 2 3 3 5 5 5 6 8 8 9 11 12 12 14 14 15 16 16 17 17 18 19 19 20 20 2.1.1. Chng vi sinh vt.. 2.1.2. Cht mang Nylon-6.. 2.1.3. Ho cht 2.1.4. Thit b s dng. 2.1.5. Mi trng nghin cu. 2.2. Phng php nghin cu 2.2.1. Phng php vi sinh vt ... 2.2.2. Phng php ho sinh.. KT QU V THO LUN................. 3.1. Tm iu kin nui cy thch hp thu lipase 3.1.1. Tm iu kin nui cy thch hp thu lipase t Candida rugosa............................................................................................ 3.1.1.1. Kho st nh hng ca thi gian nui ti sinh tng hp lipaza................................................................................................. 3.1.1.2. Kho st nh hng ca tc lc n qu trnh sinh tng hp lipaza................................................................................ 3.1.1.3.Khostnhhngcanguncacbonnsinhtng hp lipaza..................................................................................... 3.1.1.4.Khostnhhngcanngaxitpalmiticn sinh tng hp lipaza..................................................................... 3.1.1.5. Ti u ho iu kin nui chm Candida rugosa 3.1.2.Tm iu kin nui thu lipase t Bacillus sp 3.2. Tch tinh ch lipase t Candida rugosa 3.2.1. Kho st nng etanol thch hp kt ta lipase 3.2.2. Tinh sch lipase bng phng php sc k trao i ion trn h thng FPLC ...................................................... 3.3. Kho st cc iu kin ti u c nh lipase t Candida rugosa trn Nylon-6. 3.3.1. nh hng ca nng HCl x l ht n kh nng c nh enzym lipase.. 20 20 20 20 21 22 22 22 25 25 25 25 25 26 26 27 31 32 32 34 35 36 3.3.2.nhhngcanngglutaraldehitnkhnng c nh enzym.. 3.3.3. nh hng ca nng enzym n kh nng c nh enzym... 3.3.4. nh hng ca dung dch m n kh nng c nh enzym lipase.......... 3.4. Xc nh mt s c tnh ca Lipase .................................... 3.4.1. Mt s c tnh ca lipase t do v c nh t Candida rugosa............................................................................................ 3.3.1.1. nh hng ca nhit ti hot lipase t Candida rugosa........................................................................................ 3.4.1.2. nh hng ca nhit ti tnh bn lipase t do v c nh............................................................................................... 3.4.1.3. nh hng ca pH ti hot lipase t do v c nh 3.4.1.4. nh hng ca pH ti tnh bn lipase t do v c nh 3.4.1.5. nh hng ca ion kim loi ti hot lipase t do 3.4.1.6. Xc nh im ng in ca lipase ............................... 3.4.2. Mt s c tnh ca lipase Bacillus-sp.............................. 3.5.KHOSTKHNNGNGDNGLIPASEt Candida rugosa............................................................................... 3.5.1. Kho st kh nng thu phn du vi lipase c nh 3.5.2.Khostkhnngsdnglipasetantrongsnxut phomat............................................................................................ KT LUN....................................................................... TI LIU THAM KHO............................................... 36 37 38 40 40 40 41 43 45 47 49 50 55 55 56 59 62 M u Lipaselenzymxctcthuphntriglyxeritthnhdi,mono glyxerit hoc glyxerol v cc axit bo nh hot ng trn b mt phn pha dunc.Lipase(EC3.1.1.3)lenzymlinhhotcthsdngxctc nhiu loi phn ng khc nhau, c kh nng chu kim, chu nhit. Chnh v th, lipase c ng dng rng ri trong cng nghip nh cng nghip thc phm, cng nghip ho hc, cng nghip m phm, cng nghip da, trong y dc v cc ngnh cng nghip khc[2]. Lipasecphnbcngvt,thcvtvvisinhvt,song trong cng nghip ngun thu lipase ln nht l t vi sinh vt. Trong c nm mc, nm men, vi khun v x khun u c kh nng tng hp lipase.Vo nhng nm cui th k XX, tng sn lng cc loi enzym ton thgiivokhong1300tn/nm[3].Thtrngthgiivenzymcng nghiptrongnm1994ctnhkhong1tlaM,tronglipase chim7%(70triu) [sheldon, 1996]. Trong cng nghip du v cht bo, vicsdnglipasertphbin.Ckhonghn100loilipasekhc nhau c dng chuyn i lipit thnh cc cht khc[13]. Hin nay, Vit Nam s dng rt ph bin lipase trong ngnh cng nghip thc phm, cng nghip ty ra, ho hc, y hc v cn mt lng ln lipase, song cc ch phm lipase ang s dng phn ln l nhp khu. Do vy, ti tp trung nghin cu thu nhn v s dng hiu qu lipase t hai i tng l nm men Candida rugosa v Bacillus sp Tng quan 1.1. CC NGUN THU LIPASE (EC.3.1.1.3) Lipase c phn b rng trong cc loi vi sinh vt, ng vt v c thc vt. ngi v ng vt c xng sng, nhiu lipase kim sot s thu phn, s hp th, s to thnh cht bo v chuyn ho lipoprotein. Lipase ngvtchialm3nhmdatrnvtrvhotngcachng:lipase thc phm, lipase m v lipase sa. thcvt,lipasectmthymdtrcahtdu,htng cc trong qu trnh ny mm ca ht. Hu ht cc lipase khng hot ng trong thi k ng ng, ngoi tr lipase tm thy ht u caston[16]. Lng ln lipase trong cng nghip c sn xut t cc loi vi sinh vt: vi khun, nm mc, nm men[10].Vi khun: Lipase ngoi bo ca Streptomyces rimous R6554 W c tch t dch lc canh trng bng sc k ct. Streptomyces l vi khun t Gram (+) th hin kh nng c bit i vi s tng hp cc cht trao i th cp v s dng lipase thu phn ngoi bo ph hu nguyn liu hu c trong mi trng sng t nhin. Tuy nhin cho n nay, ch c mt vi lipaseStreptomycescxcnhhottnh[10].Ngoira,ngita cngtmthylipasectrongmtsloivikhunkhcnh Pseudomonascepaciachottnhngaynhitcao,trongmitrng kim v k nc[5]. Nmmc:Lipasectmthymtsloinh:Aspergillus, Rhizopus(tchtquda),Rhizopusoryzae(phnlptduda), Pythiumnltimum (trong dung dch hu c, khng c nh tng), v Mucor sp humicola lamuginosa[6].Nmmen:NmmenCandidarugosalmtngunsnxutlipase quan trng. Cc c tnh xc tc cu trc ho sinh ca lipase trong loi nm menCandidarugosaccngbtrnnhiutiliu[7].Ctnht7 ging lipase t Candida rugosa trong c 4 ging c xc nh c tnh v 3 ging c dng sn xut enzym thng phm. 1.2. cc yu t nh hng n kh nng sinh tng hp lipase Cnhiuyutnhhngnsinhtnghpenzym.Nhngnhngc imvtnhcht,sinhl,sinhhocavisinhvtcnghaquytnh hnc.Vvyvictuynchnchngchotlctngitrongt nhinsautngcngkhnngsinhtnghpcaocachngbng phng php gy t bin lm thay i ngun gen trung tm nh cc yu t ho hc, vt l,c mt ngha v cng quan trng. Khngphittcmivisinhvtuckhnngsinhtnghpenzym nhnhau,ngaycnhngchngcngchi,cngloicngcthrtkhc nhauvlngenzymdochngsnsinhra.Vthvictuynchnccvi sinhvtckhnngtonhiuenzymcnphilunkthpchtchvi vic kim tra hot lc ca hng chc, hng trm chng vi sinh vt tm ra nhng chng c kh nng sinh tng hp enzym cao nht. Ngoivictunchnchngvisinhvtckhnngsinhtnghpcch enzymchotcaocnphitinhnhnuicychngtrongcciu kintiunhmmbokhnngsinhtnghpvduytrhotlc enzym ca chng. 1.2.1. nh hng ca thnh phn mi trng dinh dng Thnhphnmitrnglnhntctcngquantrngnhotng cng nh qu trnh tng hp enzym ca vi sinh vtj, mun sinh trng, pht trin c cn phi c cc hot cht cha C, N, H v O y . Ngoi ra vi sinh vt cn c cung cp cc cht khong Mg, Ca, S, P, Fe, K v mt s cht cm ng c trng ca tng enzym. nh hng ca ngun cacbon Cc ngun cacbon khc nhau s nh hng khc nhau ln s pht trin v sn sinh enzymca mi sinh vt. E. Dalmau v cng s (2000) nghin cusnhhngcanguncacbonnsinhtnghplipasecachng Candidarugosabngphngphpnuibnlintcchothycc hydratcacbon v cc axit khng lin quan n cht bo th khng lm tng ssnxutlipase.Hiusutthucenzymcaonhtviccngun cacbon l lipit hoc axit bo. S kt hp 2 loi c cht hydratcacbon v axit bo khng ci thin s sinh tng hp lipase. Glucose km hm s sinh tng hp lipase, ngc li, tween 80 kch thch s tng hp lipase. nh hng ca ngun nit Nit l yu t nh hng ln n s sinh tng hp ca nhiu enzym, v nit lnguncungcpvtliu(nguyntnit)choqutrnhsinhtnghp protein enzym trong qu trnh pht trin.Silbel F. v cng s (2002) nghin cunhhngcanguncacbonvnitnssinhtnghplipaseca chng Candida rugosa cho thy trong nhng ngun cacbon v nit nghin cu th hot lipase ca canh trng nui t c cao nht (5,58 U/ml) khimitrngnuichangunnitlcaonmmenvproteose-pepton vi ngun cacbon l du oliu. V hot t thp nht khi mi trng nui cy cha ngun nit l trypton v ngun cacbon l lactose. Tuy nhin nh hngcangunnitnstnghplipasecaccchngkhcnhaul khc nhau, do vy trong nghin cu ca Melisa E. L. Gutara v cng s cho thykhilnmenbmtchngPenicilliumsimplicissimumthnhhng ca ngun nit n sinh tng hp lipase l khng ng k. nh hng ca ngun khongCc nguyn t a lng v vi lng c nh hng ln ti s sinh trng v sinhtnghplipasecavisinhvt.TheocngbcaRohitSharmav cngs(2001)chothyMgcnhhngnbnnhitcaenzym, nuthiuMgtrongmitrngnuithssinhtnghplipasebgim xung.Phosphonhhngtrctiptisinhsncanmmcvccvi sinh vt. V vy mun tng cng s tng hp lipase cn cung cp y lng phospho y lng cn thit ca nguyn t ny. Lu hunh kch thch s to thnh lipase nm men, ngun lu hunh dng l (NH4)2SO4.Khichnmitrngdinhdngnuivisinhvtcnchnthnh phn,chtlngvstngquanvkhilnggiacccut.Cc nghincutrcychothyckhnngtngcngsinhtnghp enzymlnhngchclnvhnnabngcchthayithnhphnmi trng mt cch chun xc, T l ti u gia cc cu t chnh, tuyn chn v cho thm cc cht cm ng c hiu lc, b sung lng cn thit cc cht khong cng nh cc yu t kch thch sinh trng. 1.2.2. nh hng ca iu kin nui iu kin nui c nh hng ln n qu trnh sinh trng v pht trin ca vi sinh vt, iu kin nui y bao gm cc yu t m, pH, thng kh, tc lc, nhit mi trng pH mi trng pHbanucamicngnhhngkhngnhtisphttrinvkh nngtoenzym.Jau-YannWu(2000)nghincussinhtnghplipase cho thy hot lipase c tng hp cao nht pH 8,5. thng kh Trongqutrnhsinhtrngvisinhvttiuth25-35%chtdinhdng ca mi trng v thi ra mt lng ln nhit v CO2. V vy phi h nhit bng thng gi vi khng kh v trng c m tng i gn 100%, ch thngkhvtrngcthlintchocgimonhocthngkht nhin. Trong mi trng nui cy chm phi m bo sao cho oxy ho tan khng thp hn 20% bo ho (F. Valero, 1998). Mt t bo S lng t bo vi sinh vt cng c nh hng ti sinh tng hp lipase ca nmmenCandidarugosa.Trongcngmtmitrngnui,nuslng t bo qu t s lm ko di pha n nh v pha logarit v nh hng n s sinh tngr hp lipase, ngc li khi s lng t bo ban u qu nhiu pha logit cha hon thnh th mi trng cn kit ngun dinh dng v vy lipase cng c tng hp t hn. Do vy ta cn phi nghin cu tm mt t bo ban u thch hp cho kh nng sinh tng hp lipase cao. 1.3. CU TO CA LIPASE 1.3.1. Khi lng phn t Cngnhccenzymkhc,lipasecththutm,cquanng, thcvtvvisinhvt.Cclipaseucnhngtnhchtchung,song chng cng khc nhau v mt s tnh cht nh: tnh c hiu c cht, mc phn gii c cht, khi lng phn t, thnh phn axit amin v iu kin hot ng. S khc nhau ny khng nhng gia cc loi m ngay c gia cc chng trong cng mt loi[7]. Khi lng phn t ca lipase dao ng kh nhiu gia cc chng vi sinhvt:Candidarugosac2ngphnlLipA(58kDa)vLipB (62kDa);Geotrichumcandidumkhong60.000Da[34],Aspergillusniger 97.000 Da, Penicillium crustosum 29.300 Da, C.Paralipolytica 55.900 Da. Awai v cng s cng tch c t P.cyclopum hai lipase c khi lng phn t 27 kDa v 30 kDa. Lipase c sn xut bi P.camemberti l mt gluco-protein c khi lng phn t l 38 kDa.1.3.2. Trnh t axit amin Thnh phn axit amin ca lipase t cc ngun khc nhau cng khc nhau, nhng nhiu trnh t axit amin c suy ra t trnh t ca lipase tch dng,tchothycnhngimtngnggianhngcutrc khnggiancalipase.Chnghnnhtrnhtca20axitaminuca lipase t ng vt c v ging vi vng N- ca lipase t P.camemberti v gingmtphnvilipasetHumicolalanuginosavAspergillusoryzae nhng khc nhau so vi lipase t P.Cyclopium lipase 1. So snh tip u N tiptheocalipase1tP.expansiumvP.soliumchothyngoi16ca 20axitaminulkhcnhaucnlilnhnhau.Nhngaxitamincui c15trongs20axitaminlngcnldolipaseP.expansiumv P.solium[11]. ivilipasetCandidarugosathsspxptrnhtNca nhng protein th hin s tng ng vi lipase 2 v 3 nhng khng tng ng vi lipase 1[13]. S quyt nh trnh t N- trn 19 axit amin ch l s tng ng cao vi nhng lipase tng t .Nichungcrttnhngtrnhtgingnhauhonton,ngoitr nhngtrnhtquanhvngtrungtmhotng.Khisosnhtrnht axit amin vng quanh trung tm hot ng ca nhm gm 3 enzym l protease, lipase,cutinasevienzymesterrase,ngitathystnticatrnht bo th GxGxG v tr serin. Ngoi l duy nht bit vi trnh t bo th trn l s thay th ca alanin cho glyxin th 2 trong subtilisin. Khng mt trnh t bo th no c tm thy quanh vng gc histidin hoc a.aspastic (glutamic)catrungtmhotngtrtrnghpcaccenzymtng ng t cc loi h hng rt gn. MELALALSLI ASVAAAPTAT LANGDTITGL NAIINEAFLG40 IPFAEPPVGN LRFKDPVPYS GSLDGQKFTS YGPSCMQQNP80EGTYEENLPK AALDLVMQSK VFEAVSPSSE DCLTINVVRP 120 PGTKAGANLP VMLWIFGGGF EVGGTSTFPP AQMITKSIAM160 GKPIIHVSVN YRVSSWGFLA GDEIKAEGSA NAGLKDQRLG200 MQWVADNIAA FGGDPTKVTI FGESAGSMSV MCHILWNDGD240 NTYKGKPLFR AGIMQSGAMV PSDAVDGIYG NEIFDLLASN 280 AGCGSASDKL ACLRGVSSDT LEDATNNTPG FLAYSSLRLS320 YLPRPDGVNI TDDMYALVRE GKYANIPVII GDQNDEGTFF360 GTSSLNVTTD AQAREYFKQS FVHASDAEID TLMTAYPGDI400 TQGSPFDTGI LNALTPQFKR ISAVLGDLGF TLARRYFLNH 440 YTGGTKYSFL SKQLSGLPVL GTFHSNDIVF QDYLLGSGSL 480 IYNNAFIAFA TDLDPNTAGL LVKWPEYTSS SQSGNNLMMI 520 NALGLYTGKD NFRTAGYDAL FSNPPSFFV 549 Hnh 1. Trnh t axit amin ca lipase t Candida rugosa 1.3.3. Cu trc khng gian crtnhiuthnghimctrinkhainhmtmhiucutrc ca lipase, trong cu trc ba chiu ca lipase c coi l c s hiu c phn ng ng hc, c hiu c cht, tin on nhng c im sinh hovhotngcalipasetibmtphnphadunc.Lipaset Rhizomun miehei l enzym thu phn u tin c cu trc bc 3 c xc nh bi tinh th hc tia X[16]. Dng phng php tia X pht hin cu trc /. Lin kt song songlmcunivicutrcxonchnhthnhcutrcphctp. Cccudisunfuactmthytrongphntlipaseclnhmlmn nh cu trc gp np ca enzym. Trong cu trc ny protein hnh cu cha mtnhnknctonnmtchuikhngcccnhcnghaquan trngchosnnhphnt.Bncnhcncvinhmcccv nhngphntclinkt.Scmtcanhngphntnclm tng lc lin kt ca Vander Wall v lc phn tn trong nhn, v vy s n nh v cu trc khng gian ca phn t enzym c tng ln[16]. Hnh 2. Cu trc khng gian ca lipase t Candida rugosa [21] Nhmquantrngnhttothnhtrungtmhotngcalipasel b ba AspHisSer. Lipase tham gia vo v tr xc tc viproteinase v esterase. i lp vi proteinase, trung tm xc tc ca n khng phn b bmtngoimnmdichuixonbmt.Vtrhotngcami lipase u c Ser, His v Asp (hoc Glu) v hon ton b che khut bi mt on np cu to bi mt hoc hai chui xon. B mt mang Trypsin hon tonkhngccvtcngqualiviukncbaoquanhtrungtm hotng.Tronghuhtcutrclipase, userinhot ng trong chui pentapeptit c trnh t Gly-X1-Ser-X2-Gly cu trc cng c tm thy protease serin (Boel v cng s, 1998). 1.4. MT VI C TNH CA LIPASE1.4.1. Cht hot ho v cht km hm Phnngdoenzymxctcphthucvonhiuyut,trong chthothovchtkmhmcngnhhngtivntcphnngv hot enzym trong phn ng. Hot enzym c th b thay i di tc dng ca mt s cht c bn cht ho hc khc nhau.* Cht hot ho enzym Chtnylmtng hot xc tc ca enzym. Cc cht ny c bn cht ho hc khc nhau, c th l cc anion, cc ion kim loi hoc cc cht hu c c cu to phc tp hn.Cc cht hot ng cation nh cc mui amon c a vo lm tng kh nng thu phn du oliu ca lipase t Mucor [Shamkant Patkar v Fredrick Bjorkling, 1992]. DidierRotticcivcngschngminhrnglipasetCandida antaractica c th c hot ho bi cc cht hot ng b mt. Trong mi trngcchtlaxetatp-nitrophenylcchachthotngionSDS (Sodiumdodecylsulfate)thtcphnngtngln.ChngtSDSc tc dng trong vic tng cng tc phn ng ca lipase[9].Tuy nhin tc dng kch hot ch gii hn nhng nng xc nh, vt qu gii hn ny c th lm gim hot ca enzym. * Cht km hm enzym Cc cht lm gim hot ca enzym gi l cht km hm (c ch-Inhibitor), k hiu l I. Cc cht ny c th l nhng ion, cc phn t v c, huc,kcccprotein.Cchpchtkmhmhottnhlipasecth bng cch thay i cu trc lipase hoc tc ng vo tnh cht b mt.C chhot ng km hm lipase bi protein v polypeptit vn cha cnghincurrng.gii thch hot ng ca s km hm ny c hai gi thuyt c a ra: (1) cht km hm protein c hp ph ln b mt v gy ra s thay i cc tnh cht ca b mt ; (2) S tc ng qua li trc tip ca protein vi lipase v cnh tranh vi c cht.Ngi ta lm nghincuvkmhmlipasettutngngavlipasetRhizopus arrhizus,R.delemarvimtlotccproteinphbingm:myoglobin, serumalbumin,ovalbumin,meltin.Ttcccproteinnykmhmlipase t tu tng nga khi khng c mt colipase v mui mt. Mt iu th v l lipase t R.delmarb km hm bi cc protein ny trong khi lipase t R. Arrhizus khng h b nh hng bi cc protein [24]. 1.4.2. Tnh c hiu c cht * c im ca ngun c cht Lipase (EC.3.1.1.3) l enzym xc tc trong qu trnh thu phncc triglyceridekhngtrnncmtphncchncvlipit.Ccesteca axit cacboxinic mch ngn d tan trong nc v c thu phn t t vi th tch nc ln v c th thu phn hon ton. Cc cht to thnh ca qu trnhthuphnl1,2-hoc2,3-diglyxerit;2-monoglyxerit;1-hoc3- monoglyxerit v glyxerol. Da vo cc iu kin ca phn ng thu phn tcncnbngvccthnhphntrunggianchuithuphn c tch ra nh s kt tinh, chng ct hoc sc k[16]. Lipaselchtxctcthuphnchuitriglyxeritdihoccceste metyl alcohol ca mt chui di cc axit bo lipase Triglyxerit +H2O ------>iglyxerit + axit bo * Thu phn cc lin kt este gia glyxerol v cc axit bo Lipaselmtchtxctcsinhhccsdngvonhiumc chkhcnhau.Trnthgiipdngthnhcnglipasetccngun khcnhauxctcchosthuphn,stnghpchtbovstraoi nhmcanhngchteste[13].Tnhchtcthkhphbinvittc cc lipase l phn ng ca chng trong mi trng khng ng nht, trong lipasehunhchthhinchcnngringmtphnphadu nc[16]. Lipase c s dng rt nhiu trong tng hp ho hu c vi vai tr l mt nhm enzym c kh nng phn gii hydro t triacylglyxerol, trn b mt phn pha du nc[7]. Gn y ngi ta pht hin c lipase t cy ci du c tnh cht xc tc s thu phn triolein nhanh hn khong 70 ln so vi s thu phn ca tri-linolein di nhng iu kin khng c nc, lipase ny xc tc s este ho axit oleic bng butanol[14]. Lipasexctcphnngthuphndukhngtanthnhccsn phm ho tan; phn ng ny cng c th xy ra theo chiu ngc li trong lipase xc tc to thnh acyl glyxerol t glyxerol v axit bo. 1.4.3. C ch ng hc xc tc ca lipase Theo cc nghin cu lipase c nhm serin, histidin v axit aspastic trungtmhotngcan.Vicccchtkhngtantrongnc,hot tnh ca lipase t c cc i ch khi n c phn tn vo gia b mt phnphadunc.Qutrnhcgilqutrnhhothophn pha[24] (hnh 3) S Eis*Es*Es*S -------------------------------------------------------------------Mt phn pha

Sw E E*E*Sw Nc Pw Hnh3.Mhnhhotngcalipasetrncchthotanvkhngtan trong nc. S thay i hnh th ca lipase trong qu trnh hot ng xc tc. M hnh ny do Verger xut (1980). Trong : E: Lipase ho tan khng hot ng E*: Lipase ho tan dng hot ng Es*: Lipase hot ng c hp phEis*: Lipase khng hot ng c hp ph Sw: C cht tan trong nc S: C cht khng tan trong nc E*Sw v Es*S: Phc hp lipase- c cht Khikhngcmtnchockhicncvilngnh,phnng este ho v phn ng este c u tin. Trong trng hp ny, nhng c tnhnhtcxctcvtnhchiuphthucnhiuvoiukin phn ng v bn cht c cht [Paul Woolley & Steffen B. Petersen, 1992]. 1.4.4. Nhit v pH hot ng ca lipase Nhit v pH l hai yu t nh hng rt nhiu n hot ng cng nh tnh bn ca lipase. Lipase thu t cc ngun khc nhau chu nh hng bi nhit v pH khc nhau. TheoMarijaAblamicvIwanalescie,lipasetnghptchng StreptomycesriromusR6554Whotngmnhnhtdinhit50-60C, pH ti u l 9,5. iu kin pH v nhit khng thch hp, lipase ubkmhmhotngvgimtnhbn.enzympH9,5;nhit 23C sau 24 gi hot gim 5%, pH 10-11 hot gim 55%. nhit 30Chotgimcn25%,90Chotcn30%sovihot lipase ti 55C[17]. Lipase t chng S.riromus bn cng nhit 20-50C v mt hot tnh nhanh vng nhit cao hn 70C[17]. LipasetchngPenicilliumcyclopiumcpHtiutrongkhong 4,5-7,0[10]. Lipase t nm Pythium ultium c pH hot ng iu kin ti u l 7,5-8,5. Cn i vi lipase t chng Rhizopus oryae c pH ti u l 7,5[6]. aslipasehotngnhit50-60C,tuynhincmtvi chnghotngnhitthphn.ChngPenicilliumcyclopium, Pythium ultium v Rhizopus oryae chu nhit km, s b c ch khi nhit ln ti 50C[6].Lipase t Candida rugosa n nh trong mi trng kim pH t 7-10 v c hot mnh nht pH 8,5 [Sharon v cng s, 1998]. 1.5.TCH TINH CH LIPASE Tu vo mc ch s dng m ngi ta tinh sch enzym cc mc khc nhau. c c enzym thun khit hn, ngi ta tin hnh tinh chenzymtchphmenzymth.Cnhiuphngphptinhch: Phng php kt ta da trn tnh ho tan, cc phng php sc k trao i iondatrntchincaenzym;phngphplcgeldavokhi lng phn t, kch thc enzym; cc phng php sc k i lc da vo v tr lin kt c hiu ca enzym. Hin nay, vic tch tinh ch enzym c tinhnhdatrnskthpcanhiuphngphp.Ccphngphp sckthngcsdnghncvnchophpthunhiuchphm enzym c thun khit cao. Vn tch enzym thun khit vn cn gp phimtskhkhndolngenzymctrongtbotnhiuclntp cht v lun c ng thi vi cc protein khc cng c cc tnh cht l ho gingnhau,enzymlikhngbnvdbmtkhnngxctcdotc ng ca cc yu t bn ngoi[4]. i vi cc enzym ni bo do cc phn t enzym khng c kh nng i qua mng t bo do cn phi ph v cu trc ca t bo bng cc bin phpchc(nghinvibtthutinhhocnghobngthitbng ho) hoc ph v t bo bng tc dng ca cc dung mi hu c trong sng siu m Sau khi ph v t bo enzym c chit ri loi tp cht ri mi c tch tinh ch bng cc phng php khc nhau. Lipasecnhiunhkhoahctrnthgiinghincunhm lmtinhschhnvthngcthchinquanhiuct.M. KambourovavcngstinhnhtinhschlipasetBacillus stearothermophilusMC7qualcgelttinhschl1,56vhiusut thu hi l 75%; qua ct Sephadex G-20 t tinh sch l 16,47, hiu sut thuhil60%;quactDEAE-xenlulothtinhschl19,25vhiu sut thu hi l 10,2%. Qua kim tra tinh sch bng chy in di protein bintnhtrnSDS-PAGExcnhckhilngphntl66kDa (2003)[20]. Lipase t Pseudomonas mendocina PK-12CS c tinh sch qua ct scktraoianionDE-52tctinhschl240,99vhiusut thuhil14,78%.Quakimtratinhschbngchyindiprotein bintnhtrnSDS-PAGExcnhckhilngphntl80kDa (2003)[33]. R.K.SaxenavcngstchvtinhschlipasetAspergillus carneus qua ct tng tc k nc Octyl Sepharose t c mc tinh sch l 24,1 v hiu sut thu hi l 38,4% (2003)[25].ivilipasetCandidarugosa,M.A.Pernasvcngstin hnh tinh sch qua ct DEAE- Sephacel t tinh sch l 2,3 v hiu sut thu hi l 4,9%. Qua kim tra tinh sch bng chy in di protein bin tnhtrnSDS-PAGExcnhckhilngphntl60kDa (2000)[15]. 1.6.CC PHNG PHP C NH LIPASEEnzym c nh l nhng enzym c gn ln trn nhng cht mang khnghotanhocccenzymclinktlivinhautoiphnt enzymkhngtan.Vicsdngenzymcnhcnghatolnnhc th s dng nhiu ln mi lng enzym em li hiu qu kinh t cao. Dng enzym c nh ln vo trong sn phm sau ngi ta c th tch ra khi sn phm d dng nn khng lm nh hng n mu sc, mi v v cht lngthcphm.Sdngenzymcnhcthlmngngphnng nhanh chng bng cch ly ch phm ra khi phn ng khi cn thit. Chnh v vy m enzym c nh c s dng rng ri v ngi ta cng tm ra c rt nhiu phng php c nh enzym. 1.5.1. Phng php gn enzym bng lin kt ng ho tr Cnhenzymbnglinktnghotrviccpolymec hotholmttrongnhngphngphpcnhenzymphbinnht, mbolinktvngchccaenzymvichtmang.Bnchtca phngphpnylenzymcgnvichtmangthngquacuni. Cu ni ny c kch thc khng ln v c hai u, mt u ni vi cht mang polyme, u cn li ni vi enzym. V d glutaraldehit cng hayc s dng lm cu ni gn enzym v n cha hai nhm aldehit hai u ca phn t. Hai nhm ny gi tr pH trung tnh s phn ng vi cc nhmNH2tdo.Nhvy,mtucaglutaraldehitcgnvocht mang cn u kia s c gn vo enzym[4]. Chtmangthngcsdnglccpolymekhcnhaunh: celluloza, agaroza, alginic axit, chitin, collagen, keratin v cc dn xut ca chng;hocsdngccpolymetnghpnhpolymecaacrylicaxit, polyurethan, cc dn xut N-vinylpyrolidon v cc loi polyme khc[4]. Datrncslthuyt,Carneiro-da-Cunhavcngsc nhlipasetCandidarugosalnmngxenlulozavdnxutca celluloza, s dng tc nhn hot ho l cacboiimit (HN=C=NH) thu c ktqucnhtrnmngcellulozaaxetat hot ng l 0(mols-1m-2); trn Textile cotton tissue l 1002(mols-1m-2) (1999)[18]. S dng phng php ny, theo P. Padmini c th c nh lipase trn htpolyamid(Nylon-6).Nylon-6clhngnhcsdnglmcht mangchoviccnhlipase.Trongqutrnhcnhsdng glutaraldehit10%,nngHCl3,5Nxlhtvnngenzyml 1mg/ml trong m photphat 7,7[23]. 1.6.2. Phng php gi enzym trong khun gel Nguyn tc ca phng php ny l gi enzym vo trong khun gel bngcchtrnghphogelkhicmtngthigelvenzym.Saukhi ktthcqutrnhtrnghpcagelthenzymcgitrongcckhun gel . Gelthngsdnglgelpolyacrylamithocagaroza,Na-alginat. Cnhiuphngphpgienzymvokhungel:gididnght (vin); gi enzym trong cc l nh ca gel dng si; gi enzym trong bao vi th (Microcapsul); phng php tin polyme. cnhlipasetCandidarugosatheophngphpny,ngi tagnenzymvotrongkhungeldidnght.Monomecdng y l Na alginat. Alginat t lu c dng to mng gel c nh t bo v enzym xc tc cc phn ng trong cc dung mi ho tan. Mng gel Ca alginat c tc dng lm cho enzym n nh hn, bn nhit hn khi n bc trongcckhungel.Lipasesaukhi cc nh trong cc ht alginat c thcxctcphnnglintchoccthtisdngnhiulncho hiuqukinhtcao[28].OConnellvJ.Varleydatrnphngphp nycnhlipasetCandidarugosalnmngkhCGAs(Colloidal Gas Aphrons) thu c hiu sut c nh l 80% (2001)[22]. 1.6.3. Phngphphpphvtltrnccchtmangcchahoc khng cha in tch Phng php c nh enzym c s dng ph bin nht l phng php hp ph vt l khng thng qua lin kt ho tr. Qu trnh thc hin hp ph kh n gin: cht hp ph v enzym c trn ln vi nhau trong mtkhongthigianchophpshpphxyranhtngtccacc linktion,knc,hydrogenvlcVanderWals.Tuynhinphng phpnycnhcimlqutrnhgiihpphenzymddngxyra bi s thay i pH, nhit v thnh phn ion[4]. Nuchtmangkhngclxp,enzymchpphtrnbmt cht mang. Nu cht mang c l xp, enzym s c hp ph nh lin kt ion. Cc cht mang v c thng c s dng l nylon, cc loi chitin ca vtm,maicua,btsanh,thanhottnh,thanci,gch,,si,ct Cht mang hu c thng s dng l dn xut celluloza. Lipase c th c c nh trn ht Amberlite-XAD16 bng phng php hp ph vt l ln b mt ht[35].Theo phng php ny, lipase t Candida rugosa cng c th c cnhtrndungmihucmetanolchohiusutcnhl12,6%; etanolhiusutt15,2%vaxetonhiusutl16,3%(SolMonterov cng s)[29]. Cngdatrnphngphphpphvtllnchtmang,Tien- Chieh Hung v cng s tin hnh c nh lipase t Candida rugosa trn Chitosan s dngglutaraldehit lm tc nhn hot ho (2003)[31]. 1.7. NG DNG CA LIPASE Lipase c ng dng rng ri trong cng nghip sn xut bt git, tng hp cc cht hu c, trong cng nghip ch bin sa v pho mt, cng nghip sn xut dc phm, ho cng, m phm v cc cng nghip khc. Ngoiralipasecsdngrngritrongcchotngbinisinh hctrongqutrnhthuphncngnhqutrnhtnghpvkimsot c cc bc trong cng nghip giy, bt giy. 1.7.1. Trong cng nghip sn xut cc cht ty ra Hinnaythtrnglnnhtchoccloilipaselvicsdngn nhnhngchtcuthnhtrongccloibtgitcngnghgitkh. Ngy nay lipase c dng trong bt git thu phn cc vt du m m conngititraquno,chnmtrongiukinnhitthpvpH caovinhngchthohcchottnhbmt[Gormen&Malmos, 1991].Mtslipasedngthngphmcsdngtrongngnh thuctyl:LipasehumicolacaNovoNordisk,lipolasehoclipase P.glumacecaUnilever[Estellvcngs,1985]csdngtrong ngnhcngnghiptyra.Nhngnhnglipasednglmbtgitcho nhnghgianhginnhviccproteasevhotngchiuqu trong iu kin kim. 1.7.2. Trong cng nghip thc phm Trongcngnghipthcphmngitasdnglipasecibin cctriacyglycerol,nngcaochtlngccloiduthcvtrtin,cht lngthpbngcchthaythccaxitbonobiccaxitbokhngno, trong dung mi him nc. Vd:Lipozyme(Novo)cdngsnxutbCocoatng ng t du c v cc axit stearic s dng trong cng nghip ch bin hp [Bjorkling, 1991]. ivithcphmcchbintsa,lipasecsdngrng ri lm tng mi v, lm tng chn ca phomat, hoc ch bin nhng snphmnhphomatvphngiilipit[Bech,1992].Nhngaxitbot domchngn(chyulC4,C6)cthuphnradotnglipasevo sa bo dn n s tng hng v c trng cho sa. Trong khi nu cc axit bo mch trung bnh (C12, C14) c to ra s c xu hng to ra mi v ta nh x phng cho sn phm. Thm vo nhng axit bo t do ny c th tham gia vo cc phn ng ho hc n gin vi vi sinh vt dn n victnghpnhnggiavcmithmkhcnhau.Nhngngunlipase dngchovictnghngvphomattrnmcanhngloingvt nhai li (d non, cu, b). Mt s ch phm lipase c s dng lm tng mivvlmchnphomatcthutmtschngnh:M.miehei, A.niger hay A. oryzae. Lipasecsdnglmtngnhngchtbonhvicchuyn este ho ti mt triglycerit c gi tr cao, b cocoa, c mt hn hp 1,3-2-oleoyl-glycerolchano(palmitic,streraicvaxitoleic)cthcch xut t loi du r tin chit xut tng hp thn cy c (1,3-dipalmitoy 1-2-oleoyl-glycerol)viaxittritearrinhayaxitsteariclnltl1,3s lipasectrng[Mastuaovcngs,1981],trongkhiphngphp chuyn i este ho ho hc di nhng iu kin kim c th dn n s tothnhsnphmmangtnhngunhinvtothnhsnphmph khngmongmun.Bngvicsdngphngphpchunbgingkhc nhau(axitpalmitichunhkhngctronghaivtrvi1,3-dioleoyl- 2palmitoyl-glycerol)cthcthaythbngnhngchtthaythsa bocchitxutphnnhiutcyc,dupalmitoylglyceridesvi nhng axit bo khng no [King & Padly, 1990]. Triglycerides cng vi axit octanoic v axit decanoic v tr 1,3- v mt axit bo cn thit v tr 2- c th c sn xut theo cch ny dnh cho nhng bnh nhn khng dch tu hoc km hp th.1.7.3. Trong cng nghip dc v ho cht Trong nhng nm gn y enzym ngy cng c s dng nhiu v rngritrongyhc,snxutmtsthucvdngtrctipcha bnh.nhiunctrnth gii, c bit Nht Bn hng nm ngi ta sn xut mt lng ln ch phm enzym vi sinh vt c tinh khit cao trong c mt ca lipase. Trong dc phm lipase c ng dng rng ri trong qu trnh sn xut thuc cha bnh cho ngi v cc loi ho cht bo v thc vt (thuc tr su, tr c)[19]. Gn y hn, trong lnh vc ho hc tinh khit, ngi tadnglipasetoccchttrunggiantnghpquaqutrnhthu phn lp th chn lc, ng dng trong sn xut thuc tr su Pyretinoidic. Ngoi ra trong phng th nghim cc lipase cng c nh ho hc hu c dnglmcngctnghpmtschtkhcnhphngiiRaxemic epoxiestenhlipasecaRhizopusarhisus[J.ALaitte(Php)].Vimong muncthsnxutmtloithuclmginngmchvnh,ngita da vo tnh cht chn lc thu phn ca lipase trong mi trng dung mi hu c. Cclipasecthxctcesteho,chuyniestehovesteho tng tc trong cc dung mi hu c vi lng nc thp [Kazlaus kas v Bornscheuer,1998;Godbergvcngs,1989],ngdngchnhcacc lipase trong ho hu c l s phn gii cc hn hp enantiomeric. C mt vi nhm phn t lin quan n s truyn tnh trng du hiu lm gia tng nhng cn bnh d ng, vim l nhng lipit hay lipit aqnalogues. S truyn tn hiu gia v trong ni bo sinh vt lin quan gn gi vi nhng loi lipit n l nh phosphatidylinositol, lipase cng nh photpholipase u c th c s dng tng hp trn phm vi ln.1.7.4. Trong cng nghip thuc da Qutrnhthucdayucuphiblpchtbodida.Sdng phi hp lipase vi cc enzym thu phn khc nh protease m ra con ngmichocngnghipthucda.Mtqutrnhenzymmichosn xut t da sng n thuc da bao gm vic git, r lng v ra sch trong bnccpHkhong8-13chamtenzymakim.Rrngcns dng phi hp lipase vi cc protease kim hay trung tnh v mt cht hot ng b mt khc. Ti Nga c bng sng ch s dng ch phm enzym thuc da cu vi cc c tnh u vit hn nh bn, co gin tng v gim cng[36].1.7.5. Trong cng nghip m phm Lipase c ng dng nhiu trong cng nghip m phm sn xut ccchthotngbmtvcchpchtthm.Nhngthnhphnch yutrongmphmvnchoaciuchbngphnngesterho glycerolcxctcbilipase.Vicchuyn ester ho ca 3,7- dimethyl 4,7- octadien- 1- ol bi lipase t nhiu ngun vi sinh vt khc nhau iuchoxidehoahnglmthngliuquantrngtrongsnxutnc hoa[36].NGUYN LIU V PHNG PHP NGHIN CU 2.1. NGUYN LIU 2.1.1. Chng vi sinh vt Candida rugosa nhn t Vin bo tng ging vi sinh vt Vin Cng ngh sinh hc v Cng ngh Thc phm- i hc Bch Khoa- H Ni. ChngBacillusspnhntPhngCngnghGenengvt-Vin Cng ngh Sinh hc 2.1.2. Cht mang Nylon-6: ca hng Sigma- M (d= 1.084) Nylon-6 l cht nha k thut ni ting l c cng cao v sc khng cao i vi cht ho hc v du m. Mc d vy, Nylon-6 c nhit chuyn bin nha thp v nhit bin dng thp. l iu cn thit trnlnNylon-6viccpolymekhctothnhccchtckhnng chu nhit cao(1996)[26]. Nylon-6clhngnhcsdnglmchtmangchovicc nhlipase.Ncungcpccnhmaminhotngmnhcuiphnt polyme, c th to phn ng vi nhm lin kt glutaraldehit [8]. H2-N-(CH2)5-CO-[-NH-(CH2)5-CO-]n-NH-(CH2)5-COOH Nylon-6 2.1.3. Ho cht Duoliu,caonmmen,pepton,glucoza,dchchitmalt,KH2PO4, K2HPO4, (NH4)2SO4, MgSO4.7H2O, FeCl3, CaCl2, NaOH, Inositon, Biotin, thiamine.HCl,Axitpalmitic,cn99%,glutaraldehit,htNylon-6,vmt s ho cht cn dng khc. 2.1.4. Thit b s dng - T cy vi sinh vt (clearn benchop) - Ni hp tit trng - My o mu quang in UV-Vis - My o pH 900 Prescisa : Nht : Nht : Thu in : Thu S - My khuy t gia nhit (Magnetic stirrer) - My lc n nhit Shellab - My lc ht chn khng - My ly tm lnh allegra 64R - H thng sc k ct FPLC V mt s thit b thng thng khc. : c : M : c : M : Thu in 2.1.5. Mi trng nghin cu:* Mi trng hot ho chng Candida rugosa (g/l) - Dch chit malt - Cao nm men - Pepton - Glucoza : 3g : 3g : 5g : 10g Bsungncctntngthtchl1000ml.Khtrnghnhp iu kin 1 atm, 110C trong 30 pht. * Mi trng gi ging Candida rugosa: thnh phn ging mi trng hot ho * Mi trng sinh tng hp lipase t Candida rugosa (g/l) - KH2PO4

- K2HPO4 - (NH4)2SO4 - MgSO4.7H2O - FeCl3 - Inositon - Biotin - Thiamine.HCl - Axit palmitic - C cht du oliu 1% :15g : 5,5g : 4g : 1g : 10mg : 0,004mg : 0,008mg : 0,2mg : 1,2g Bsungncctntngthtchl1000ml.Khtrnghnhp iu kin 1 atm, 110C trong 45 pht. MitrngnuicyBacillus(BMGY):(1%(w/v)caonmmen,2% (w/v)bacto-peptone,100mMkaliphosphate,pH7.0,4x10-5%(w/v) biotin v 1% (v/v) methanol) 30C lc 220 vng/pht. 2.2. PHNG PHP NGHIN CU 2.2.1. Phng php vi sinh vtHothochngnmmenCandidarugosatrongmitrnghot ho nhit 30C, tc lc 200 vng/pht trong 24 gi.Sautinhnhnuicysinhtnghplipasebngphngphp nuicychmtrnmitrngsinhtnghpnhit30C,tclc 200 vng/pht trong 40 gi. 2.2.2. Phng php ho sinh Phng php c nh lipase trn Nylon-6 [23] * Nguyn l S dng phng php gn enzym ln cht mang bng lin kt ng hotr.Datrnhotenzymtrcvsaukhicnh,xcnhc hiu sut c nh enzym. * Tin hnh Ly30ghtNylon-6clctrong600mldungdchHCl3,5N 45C trong 15 pht (tc lc 200 vng/pht). Sau nhng ht ny c tch ra bng my lc chn khng v ra nhiu ln bng nc ct. Sau c b sung 150ml glutaraldehit 10% v lc vi tc 200 vng/pht 30C trong 30 pht. Nhng ht ny li c tch ra bng my lc chn khng. Saukhihotho,nhnghtnyctrnvi100mldungdch enzym(1mg/ml)trongmphosphatpH=7,7;rilc45Ctrong120 pht (tc lc 200 vng/pht).Sauhnhpc4Cquam.Ricranhiuln bng nc ct, mi ln 20ml loi nhng enzym khng c gn kt.Phng php xc nh hot lipase bng chun [32] * Nguyn l Da trn kh nng xc tc thu phn lipit ca lipase, xc nh lng axit bo to ra trong 15 pht bng lng NaOH cn b sung duy tr pH cnh.HiusgialngNaOHsauvtrckhibsungenzymc trng cho hot tnh xc tc ca lipase. * Tin hnh: Ly10mldun+200mlncct+1%Gumarabic->nhtan bng my xay sinh t trong 5 pht, to huyn ph. Ly20mldungdchhuynphchovoccthutinhtlnmy khuy t, iu chnh nhit ti 65C. Thm 470 l dung dch CaCl2 o pH v iu chnh pH ti 8,3 (chun bngNaOH10mM).SaukhinnhpHvnhit,otthuphn ca dung dch c cht trong 15 pht gi pH lun lun bng 8,3 (lng NaOH tiu tn l a ml). Sauthm20ldungdchlipasevohnhpcchtvchun bngNaOH10mMtpH=8,3vbtutnhlngtiuhaoNaOH trong 15 pht gi pH lun lun = 8,3 (lng NaOH tiu tn l b ml). * n v hot lipase Mt n v hot lipase (U) c xc nh nh l lng enzym c kh nng chuyn ho to 1 micromol axt bo trong thi gian 1 pht. Hot lipase trong 1ml dch ni c tnh theo cng thc: (b-a)*0,01 Hot lipase = -------------- U/ml t*v Vit: thi gian o t thu phn (pht) v: Th tch enzym (ml) a: Lng NaOH 10mM tiu tn khi cha c enzym (l) b: Lng NaOH 10mM tiu tn khi c enzym (l) 0,01: h s chuyn i nng Phng php tch tinh ch lipase bng sc k trao i ion DEAE cellulose [15] * Nguyn l:Datrnphngphpscktraoiion,chnhlstraoiion caproteintchinviioncanhatraoiion.Trongnhmtch in(+)lchtmangtraoiviccion(-)vdo chngcgil cc cht trao i anion v ngc li chng c gi l cht trao i cation. * Tin hnh: Dchenzymsaukttaphnonbngetanolnng60%c choquactDEAEcelluloseviiukintchphnonnhsau:ct c cn bng trong m Tris-HCl 25mM, pH 7,5; tc chy 1 ml/pht vi gradien nng 0-0,25M NaCl, mi phn on thu mu l 1ml/ng. mi phn on o hm lng protein, xc nh hot lipase bit c phn on tch lipase tt nht. KT QU V THO LUN 3.1. Tm iu kin nui cy thch hp thu lipase 3.1.1. Tm iu kin nui cy thch hp thu lipase t Candida rugosa 3.1.1.1. Kho st nh hng ca thi gian nui ti sinh tng hp lipaza Chng nm men c nui cy trn mi trng MT2, vi cc iu kin: nhit nui l 300C, tc lc 150 vng/pht. Kho st vi 6 thi giannuikhcnhau:24,30,36,42,48,54gi.Saumimcthigian trn dch nui cy c ly ra v xc nh hot . Kt qu c th hin bng 1. Bng 1: nh hng ca thi gian nui n sinh tng hp lipaza Thi gian (gi)Hot lipaza (U/ml) 242 305.2 367.5 428.33 486.9 543.9 Ktqunghincuchothy,chngnghincuckhnngsinh tng hp lipaza ngay t nhng gi u nui cy v t cc i sau 42 gi nui, cho hot lc 8.33 U/ml. 3.1.1.2.Khostnhhngcatclcnqutrnhsinhtng hp lipaza Thnghimctinhnh4tclckhcnhau:100,150, 200, 250 vng/pht trn mi trng sinh tng hp lipaza nhit 30oC, thi gian nui 42h. Kt qu th hin bng 2 Bng 2. nh hng ca tc lc n qu trnh sinh tng hp lipaza Tc lc (v/p) Hot lipaza 100 5150 8,33200 4250 3Ktqubngtrnchothyqu trnh sinh tng hp lipaza t cc i tc lc 150 vng/pht. 3.1.1.3.Khostnhhngcanguncacbonnsinhtnghp lipaza Chngtitinhnhkhostvi5nguncacbonkhcnhautrn mitrngsinhtnghplipazanhit30oC,tclc150 vng/pht,ccht1%duoliu,thigiannui42h.Ktquthhin bng 3 Bng 3. nh hng ca ngun cacbon n sinh tng hp lipaza Ngun cacbon ( 2g/l)Hot lipaza (U/ml) Glucoza8,67 Galactoza12,30 Tween 806,67 Axit Palmitic18,00 Glyxerol12,67 Qua bng trn cho thy hot lipaza t c cao nht trn ngun cacbon l axit palmitic, cc ngun cacbon khc nh glyxerol v galactoza cngchohottngicao.Vynguncacbonthchhpnhtcho sinhtnghplipazacachngnghinculaxitpalmitic. Kt qu ny cngphhpviktqunghincucatcgiE.Dalmau(2000)khi nghincunhhngcaccnguncacbonkhcnhaunsinhtng hp lipaza cu chng candida rugosa. 3.1.1.4. Kho st nh hng ca nng axit palmitic n sinh tng hp lipaza Tin hnh nui cy chng nm men trn mi trng sinh tng hp lipaza vi cc iu kin thch hp tm c trn, thay i hm lng axitpalmitictrongmitrng(g/l):0,5;1;1,5;2;2,5;3.Sau42hnui cy xc nh hot trong dch canh trng thu c kt qu trong bng 4 sau y. Bng 4. nh hng ca nng axit palmitic Nng axit palmitic (g/l)Hot lipaza (U/ml) 0.512.3 115.83 1.517.67 213.67 2.59.33 35.67 T kt qu trn ta thy nng axit palmitic cng nh hng nhiu ti kh nng sinh tng hp lipaza. Nng axit palmitic 1,5 g/l cho hot cao nht l 17.67 (UI/ml). 3.1.1.5. Ti u ho iu kin nui chm Candida rugosa Tcciukinkhostctrnchngtitinhnhtiu ho bc 1 iu kin nui chng Candida rugosa theo 3 yu t sau: X1: axit palmitic (g/l) X2: S t bo nm men (tb/ ml ) X3: thi gian (gi) Khi y hm mc tiu l Y: hot lipaza (U/ml) Cc mc th nghim Mc th nghimX1X2X3 Mc gc1.5442 Khong bin i0.521 Mc trn2646 Mc di1238 Lp ma trn th nghim v kt qu Sj2 0.62 2.74 1.13 0.15 0.29 0.36 0.11 0.65

=6.05Ytb 10.12 15.5 12.62 15.03 15.25 15.21 17.44 18.1

=119.27Y2 10.67 14.33 11.87 14.76 14.87 15.63 17.67 17.53 Y1 9.56 16.67 13.37 15.3 15.63 14.78 17..2 18.67 Thc34 34 34 34 42 42 42 42 X3 M - - - - + + + + Thc 2 2 6 6 2 2 6 6 X2 M - - + + - - + + Thc 0.05 0.15 0.05 0.15 0.05 0.15 0.05 0.15 X1 M - + - + - + - + STN 1 2 3 4 5 6 7 8 X l thng k cc kt qu thc nghim thu c Kim tra ng nht ca ma trn theo chun Cochran Nu Gb > Gtt th ma trn ng nht Gtt = max Sj2= 0.45 Sj2 Gb tra bng = 0.6798 Gb>Gttnnccsliucocngmtchnhxcnh nhau v c th chp nhn c Xc nh cc h s ca m hnh theo cng thc Nyb= b0=14.91b1=1.05 b2=0.88b3=1.59 Phng trnh hi quy c dng : y = 14.91 + 1.05x1 + 0.88x2 + 1.59x3 Kim tra s c ngha ca cc h s |bi| t.Sb = 2.12*0.31 = 0.6572vi t l chun student tra bng (p=0,05; f=16) = 2.12 ta thy cc h s |bo|,|b1|,|b2|,|b3| u 0.6572 Tt c cc h s trn u c ngha Vy phng trnh hi qui cui cng l:y = 14.91 + 1.05x1 + 0.88x2 + 1.59x3 Kimtrasthchngcaphngtrnhhiquytheochun Fisher STTM hnhYttYtn(Ytt-Ytn)2 114.91 - 1.05 - 0.88 - 1.5911.3910.121.61 214.91 + 1.05 - 0.88 - 1.59 13.4915.54.04 314.91 - 1.05 + 0.88 - 1.59 13.1512.620.28 414.91 + 1.05 + 0.88 - 1.59 15.2515.030.05 514.91 - 1.05 - 0.88 + 1.59 14.5715.250.46 614.91 + 1.05 - 0.88 + 1.59 16.6715.212.31 714.91 - 1.05 + 0.88 + 1.59 16.3317.441.23 814.91 + 1.05 + 0.88 + 1.59 18.4318.10.11 3.93 B - N ytn) - (ytt S= t) St , y min(S) St , y (S max Ftt= St = 3.93 / 4 = 0.98Ftt=0.98/0.38 = 2.58 Fb = 3.01 tra bng vif1= N - B = 4 f2= N ( k -1) = 8 Trong N: s th nghim B: s h s c ngha k: s ln lp li Ftt FbM hnh trn l thch ngTi u ho theo Box - Wilson i = X0 - cn di = cn trn - X0 b11= 1,05*0,5 = 0.525 b22= 0,92*2 = 1.84 b33= 1,61*4 = 6.44 t gi tr ln nht nn ta chn X3 lm Xcs Chn bc nhy ca X3 l 3 = 2 1 = (b11/ b33). 3 =0,2 2 = (b22/ b33). 3 = 0.6 Kt qu ti u theo Box - Wilson STTX1 X2 X3 Hot 1123816.17 21.22.64018.67 31.43.24211.67 41.63.8449.33 51.84.4466.67

Tktqutrnchothyqutrnhsinhtnghpenzimlipazaca chng nm menCandida rugosa t hiu qu tt nht th nghim th 2 vi iu kin:Hm lng axit palmitic: 1.2 g/l S t bo nm men : 2,6.106 tb/ml canh trng Thi gian nui: 40 gi 3.1.2.Tm iu kin nui thu lipase t Bacillus sp Chng ti nui cy chng vi sinh trong 6 mi trng khc nhau v thnhphn(bng5)vxcnhhottnhlipasetrongdchnisau120 gi nui cy. Kt qu cho thy hot enzym t cao nht khi nui cy trongmitrngBMMY(67U/mlcanhtrng).Mitrngnys c chn cho cc nghin cu tip theo. Bng 5. Hot tnh lipase Bacillus- sp nui cy 6 mi trng khc nhau Mi trng(50 ml) Thnh phnHot sau 120 gi nui cy (U/ml) ABt ng: 0,8g. Bt u tng: 1,6 g. Nc da: 50 ml. Methanol 1%. 3,2 BBt ng: 0,8g. Bt u tng: 1,6 g. Nc da: 50 ml. Methanol: 1%. Biotin: 5,1 CBt ng: 0,8g. Bt u tng: 1,6 g. Nc my: 50 ml. Methanol: 1%. 3,4 DBt ng: 0,8g. Bt u tng: 1,6 g. m K-phosphate: 50 ml. Methanol: 1%. 10,2 EBt ng: 0,8g. Bt u tng: 1,6 g. m K-phosphate: 50 ml. Methanol: 1%. Biotin 8,3 BMMY67,0 Th nghim tip sau y nhm kho st nh hng camt s c cht cmngtikhnngsinhtnghplipase.MitrngcbnBMMY cb sung mt s loi du hoc gum arabic (bng 2). Tin hnh nui vi 6 loi mi trng khc nhau, c 24 gi ly mu xcnhhottnhvtcphttrincavikhun(OD).Ktqucho thy cc c cht ny khng lm tng kh nng sinh tng hp enzym ca chng nghin cu. Bng 6. nh hng ca du v gum arabic ti kh nng sinh tng hp lipase Bacillus-sp Hot (U/ml)TT Cc cht b sung vo mi trng BMMY 24 h 48 h 72 h 96 h 120 h 144 h 1i chng (BMMY) 16,0 37,2 42,0 48,4 67,054,021% Gum arabic16,0 16,5 17,7 22,0 32,241,031% du Marvela21,0 23,0 32,0 37,0 44,044,041% du Marvela + 1% Gum arabic23,3 24,2 36,0 37,0 45,050,051% du m19,0 21,2 38,0 42,0 42,042,861% du m + 1% Gum arabic 30,0 36,0 46,0 52,0 56,062,0 3.2. Tch v tinh ch lipase t Candida rugosa 3.2.1.KhostnngetanolthchhpkttalipazatCandida rugosa Tin hnh th nghim kt ta lipaza vi cc nng khc nhau: 60, 70, 80, 90%, ly tm lnh, thu kt ta cho bc hi dung mi ta thu c lipaza th. Kt qu xc nh hot c th hin bng 7 Bng 7. Kt ta lipaza cc nng etanol khc nhau Nng etanol (%v/v) Hot lipaza trc khi kt ta (UI/ml) Hot lipaza sau khikt ta cn (UI/ml) Hiu sut thu lipaza (%) 5716.7300 6716.7300 7616.731.337.95 8116.735.6721.94 8616.739.8358.76 9016.7312.3373.7 9116.733.3319.9 9316.7300 0246810121457 67 76 81 86 90 91 93nng cn( cn)hot lipaza (U/ml)Hnh 1: kt ta enzim bng etanol T kt qu thu c trn ta nhn thy cc nng cn trong khong 0 -67% c cho ta kt ta nhng khng c hot enzim, ch khi nng cn t 67% tr ln l c hot v nng cn l 90% th cho ta hot caonht.Quathnghimnychothynngcnt67%tr xungchcproteintpbkttavvychngtacthkttaphn on loi b bt protein tp. 3.1.2. Tinh sch lipase bng phng php sc k trao i ion-DEAE-cellulose trn thit b FPLCDchenzymsautabngetanolcly15mlchovoct ccnbngtrongmTris-HCl25mMpH7,5;tcchyl 1,5ml/pht. Dung dch y lipase l NaCl vi gradien nng 0-0,25M; mi phn on thu1ml/ng.Kt qu c biu din trn sc k hnh 2 010203040506070801 81624324048566472808896104112120Phn on (1ml)A/280nm00.20.40.60.811.21.41.61.8Hot (U/ml) 0,251NaCHnh 2. Sc k tinh sch lipase Sckhnh2chothylipasesaukhiquacttraoiion DEAE-cellulosexuthin3pick.Tinhnhxcnhhotlipase cc phn on thuc cc pick. Kt qu cho thy ch c pick 1 v pick 3 c hot tnh lipase (69UI/ml v 77UI/ml). iu ny ph hp vi nghin cu caM.A.Pernas(2000)[15]cngbLip1vLip3ckhilng phn t l 58 v 62 kDa. Kt qu cc bc tch v lm sch c th hin bng 8 Bng 8. Kt qu tch lm sch lipase Hot lipase Cc bc lm sch Tng th tch (ml) Protein tng(mg)Tng (U) Ring (U/mgPr) Mc sch (ln) Hiu sut (%) Dch th20034015004,41(1)(100) Sau kt ta etanol 2066,8124518,644,2283,00DEAE - Cellulose810,0855655,1612,5037,06Ktqusckhnh2vbng8chothylipasesauccbc lmschlintip:kttaetanol,scktraoiionDEAECellulose thucchphmchotring55,16U/mgprotein,mctinh sch ca lipase l 12,5 ln; hiu sut thu hi l 37,06%.3.3. KHO ST CC IU KIN TI U C NH LIPASE t Candida rugosa trn nylon 63.3.1.nhhngcanngHClhothoNylon-6nkhnng c nh lipase Nylon-6lmt polyamit tch in (+). Lipase l enzym kim tnh chanhm(OH-)vvysdngHClhothochtmang,lmtng in tch (+) l cn thit. Do vy tin hnh th nghim bng cch s dng lipasesaukttuetanolcnhtrnNylon-6ccnngHClkhc nhau vi cc iu kin n nh: Tng lng ht Nylon-6 em x l: 3g Nng glutaraldehit : 10% m kali phosphat: pH 7,7 Tc lc : 200 vng/phtKt qu biu din bng 9 Bng 9. nh hng ca nng HCl hot ho Nylon-6n kh nng c nh lipase Nng HCl (N) Tng th tch nc ra (ml) Hot cn li trong nc ra (U) Hiu sut c nh enzym (%) i chng (lipase tan) -550100 333,5276,449,75 3,533,5226,158,90 433,5245,355,40 4,533,5259,652,80 Bng9chothy,vinngHCl3,5Nhiusutcnht 58,9% l cao nht. C th vi nng HCl thp cha kh nng hot ho cht mang, cn vi nng cao lm thay i pH, nh hng n kh nnglinktlipasevochtmang.Ktqunyphhpvitiliu cng b trc y[23]. 3.3.2.nhhngcanngglutaraldehitnkhnngcnh lipase Tinhnhcnhlipaseviccnngglutaraldehitkhcnhau (8%, 10%, 12%) v cc iu kin: Tng lng ht Nylon-6 em x l: 3g Nng HCl: 3,5N m kali phosphat : pH 7.7 Tc lc: 200 vng/pht Kt qu kho st th hin bng 10 Bng 10. nh hng ca nng glutaraldehit n kh nng c nh lipase Nng glutaraldehit (%) Tng th tch nc ra (ml) Hot cn li trong nc ra (U) Hiu sut c nh enzym (%) i chng (lipase tan) -550100 833,5293,146,7 1033,5209,461,9 1233,5376,831,5 Quaktqukhostchothynngglutaraldehit10%cho hiu sut c nh lipase cao nht (61,9%). Nh vt nng glutaraldehit 10% l nng thch hp lin kt enzym li vi nhau v tng lin kt ln cht mang Nylon-6. 3.3.3. nh hng ca nng lipase n kh nng c nhTin hnh c nh lipase cc nng enzym khc nhau (0,5; 1; 1,5; 2 mg/ml) v c nh cc iu kin:Tng lng ht Nylon-6 em x l: 3gNng HCl : 3,5N m kali phosphat : pH 7.7 Tc lc: 200 vng/pht Kt qu c th hin bng 11 Bng 11. nh hng ca nng enzym n kh nng c nh lipase Nng enzym (mg/ml) Tng th tch nc ra (ml) Hot cn li trong nc ra (U) Hiu sut c nh (%) 0,5 (270U)33,5160,8040,45 1 (550U)33,5224,4559,20 1,5 (820U)33,5375,2054,25 2 (1100U)33,5753,7531,50 KhostkhnngcnhlipasetrnNylon-6viccnng enzymkhcnhautathyvinngenzyml1mg/ml,hiusutc nhcaonht(59,2%).Ktqukhostnyphhpviktqu nghin cu trc y[23]. 3.3.4. nh hng ca dung dch m n kh nng c nh lipase Tinhnhkhost3dungdchmkhcnhau:mphotphat, m borat, m a nng vi cc iu kin c nh: Tng lng ht Nylon-6 em x l: 3g Nng HCl: 3,5N Nng glutaraldehit: 10% Tc lc: 200 vng/phtKt qu kho st th hin bng 12. Bng 12. nh hng ca dung dch m n kh nng c nh lipase Cc dung dch m Tng th tch nc ra (ml) Hot cn li trong nc ra (U) Hiu sut c nh (%) i chng (lipase tan) -550100 m phosphat33,5251,2554,30 m borat33,5335,0039,10 m a nng33,5418,7523,86 Quaktqukhostviccloimchothy hiu sut c nh vi m phosphat l cao nht (54,3%). Nh vy m thch hp cho vic c nh lipase trn Nylon-6 l m phosphat pH 7,7. Ph hp vi kt qu nghin cu trc y[23]. 3.4. c tnh ca lipase 3.4.1. Mt s c tnh ca lipase t do v c nh t Candida rugosa 3.3.1.1. nh hng ca nhit ti hot lipase t Candida rugosa Nhitlyuttcngmnhmtihotcaenzym.Khi nhit tng th vn tc phn ng cng tng nhng n mt gii hn no th vn tc t cc i, nu tip tc tng th vn tc enzym s gim v nhit cao s lm bin tnh protein gy bt hot enzym. Nhit m ti enzym hot ng mnh nht gi l nhit ti u. Mi enzym c mt vng hot ng khc nhau. xc nh nhit ti u ca lipase ta tin hnh xc nh hot cc nhit 55, 60, 65, 70, 75C bng phng php chun theo m t trong phn phng php nghin cu. Kt qu th hin bng 9 v hnh 3 Bng 13. nh hng ca nhit ti hot lipase t do v c nh Nhit (C)Hot lipase t do (U/g) Hot lipase c nh (U/g) 4019,50,5 5527,02,5 6029,75,5 6527,510,0 7020,515,0 7516,511,5 8012,57,5 0510152025303535 40 45 50 55 60 65 70 75 80Nhit ( C)Hot (U/g)Lipaza tanLipaza co dinh Hnh 3. nh hng ca nhit ti hot lipase t do v c nh Theohnh3chothykhinhittngt60Ctrlnhot lipasecnhtngdnvtcciti70C(15U/g).ivilipase tan, nhit 60C hot lipase t cc i (29,7U/g) v gim dn khi nhit tng ln.Nh vy nhit thch hp cho lipase t do v c nh hot ng l khc nhau. Nhit ti u ca lipase c nh cao hn ca lipase t do v lipase c nh c lp cht mang bo v nn c kh nng chu nhit. 3.4.1.2. nh hng ca nhit ti tnh bn lipase t do v c nh Tinhnhthnghimvilipasetdovcnhccnhit khc nhau: 40, 50, 60, 70oC vi thi gian xc nh trong 18 gi, c sau 6 gi xc nh hot mt ln.Kt qu xc nh tnh bn nhit ca lipase t do v c nh c th hin bng 14 v hnh 4. Bng 14. nh hng ca nhit ti tnh bn lipase t do Thi gian (gi) 061218 Nhit (0C) Hot U/g%U/g%U/g%U/g% 4029,510023,680,020,268,517,459,0 5029,510021,773,517,659,614,850,2 6029,510018,964,014,248,212,943,7 7029,510015,452,311,739,59,632,5 01020304050607080901000 5 10 15 20Thi gian (gi)Hot (%)40 C50 C60 C70 C Hnh 4. nh hng ca nhit ti tnh bn ca lipase t do Kt qu cho thy, i vi enzym t do vng bn nhit 40-50C, sau 18 gi hot lipase cn 50,2 - 59% . 70C hot gim mnh sau 18 gi ch cn 32,5%. Bng 15. nh hng ca nhit ti tnh bn ca lipase c nh Thi gian (gi) 061218 Nhit (0C)Hot U/g%U/g%U/g%U/g% 402110017,885,516,076,214,066,7 502110015,975,513,966,512,459,0 602110014,066,712,057,111,052,4 702110013,061,910,248,58,339,3 01020304050607080901000 5 10 15 20Thi gian (gi)Hot (%)40 C50 C60 C70 C Hnh 5. nh hng ca nhit ti tnh bn lipase c nh Kt qu cho thy lipase c nh bn vng nhit 40 - 60C (sau 18 gi hot cn 52,4 66,7% so vi hot ban u). Sau 18 gi, 70oC hot lipase c nh cn 39,3%. Nh vy mi nc nhit , hot lipase c nh tng khong 10% so vi lipase t do. 3.4.1.3. nh hng ca pH ti hot lipase t do v c nh pHcnhhngkhlnnvntcphnngcaenzym,mi enzym hot ng mnh trong 1 di pH khc nhau. gi tr pH m ti enzym hot ng mnh nht gi l pH ti u. Tin hnh xc nh hot lipase t do v c nh vi cc gi tr pH thay i t 7,0 - 8,3 vi cng c cht du u nnh 5%, nhit 65C.Kt qu th nghim c th hin bng 16 v biu din trn hnh 6 Bng 16. nh hng ca pH ti hot lipasePHHot lipase t do (U/g) Hot lipase c nh (U/g) 6,517,511,5 721,515,0 7,525,512,5 828,57,5 8,526,52,5 921,51,5 0510152025306 6.5 7 7.5 8 8.5 9pHHot (U/g)Lipaza tanLipaza co dinhHnh 6. nh hng ca pH ti hot lipaseQua bng 16 v hnh 6 cho thy hot lipase c nhhot ng mnh nht pH 7,0 (15 U/g). Khi tng pH ln trn 9,0 th hot enzym gim ch cn 1,5 U/g.i vi lipase t do, hot ng mnh pH 8,0 (28,5U/g). iu ny cng ph hp vi l thuyt, lipase c c nh ln cht mang polyamit tch in dng nn pH ti u chuyn v vng axit. 3.4.1.4. nh hng ca pH ti tnh bn lipase t do v c nh xc nh nh hng pH ti tnh bn lipase t do v c nh, tin hnh th nghim cc gi tr pH khc nhau: 6,5; 7,0; 7,5; 8,0 vi nhit 30Ctrongthigian18gi.Csau2gixcnhhotlipase.Kt qu th hin bng 17 v hnh 7Bng 17. nh hng ca pH ti tnh bn lipase tan Thi gian (gi) 061218pH Hot U/g%U/g%U/g%U/g% 6,529,510018,362,013,345,09,632,5 729,510021,272,016,255,012,843,5 7,529,510023,680,019,265,015,953,9 829,510025,185,021,272,018,362,0 01020304050607080901000 5 10 15 20Thi gian (gi)Hot (%)pH 6,5pH 7pH 7,5pH 8Hnh 7. nh hng ca pH ti tnh bn lipase tan Kt qu cho thy lipase t do bn vng pH 7,5 8,0 sau 18 gi vn cn 53,9 - 62 %. Bng 18. nh hng ca pH ti tnh bn lipase c nh Thi gian (gi) 061218pH Hot U/g%U/g%U/g%U/g% 6.52110014,066,79,947,08,239,0 72110016,779,513,865,712,057,0 7.52110016,076,212,258,210,650,6 82110012,057,18,239,16,129,0 01020304050607080901000 5 10 15 20Thi gian (gi)Hot (%)pH6,5pH 7pH 7,5pH 8 Hnh 8. nh hng ca pH ti tnh bn lipase c nh Kt qu bng 18 v hnh 8 cho thy lipase c nh bn vngpH 7 - 7,5 sau 18 gi hot cn 50,6 57%. Nh vy lipase c nh vng axit hn so vi lipase t do. 3.4.1.5. nh hng ca ion kim loi ti hot lipase t do xc nh nh hng ca cc ion kim loi ln kh nng xc tc ca enzym tin hnh nghin cu i vi cc ion kim loi: Ca2+, Na+, K+, Cu2+, Zn2+, Fe3+, Mg2+, Pb2+. Tin hnh enzym vi dung dch m cha cc ion kim loi trn vi nng 10mM trong 30 pht, iu kin nhit phng(30C)emxcnhhottheophngphpchunso vi mu i chng khng ion kim loi bit c mc nh hng ca chng vi enzym lipase.Kt qu th hin bng 19 v hnh 9 Bng 19. nh hng ca ion kim loi n hot lipase tdo Cc ion kim loi (10-3M) Hot lipase (U/ml)% hot Mu i chng54,0100 Ca2+ 58,0107,40 Mg2+ 57,5106,48 Na+ 53,599,07 K+ 53,098,15 Cu2+ 47,087,03 Fe3+ 46,586,11 Zn2+ 46,586,11 Pb2+ 35,567,74 0102030405060701 2 3 4 5 6 7 8 9Ion kim loiHoat do (U/ml) Hnh 9. nh hng ca ion kim loi n hot lipase t do Kt qu nghin cu cho thy, cc ion kim loi Na+, Ca2+, Mg2+, K+ khng nh hng nhiu n hot lipase, trong Ca2+ v Mg2+ cn c chiu hng hot ho lm tng hot lipase. Cn cc ion kim loi nng Fe3+,Cu2+,Zn2+cchyulnhotlipasekhong13-14%.Pb2+c ch lipase mnh nht, n lm gim hot khong 33%.3.4.1.6. Xc nh im ng in ca lipase t do im ng in (pI) l pH m ti phn t protein trung ho v in. Ti im ng in, phn t protein enzym hot ng km v d b ktta.imngincaproteinenzymkhngnhngcnghal thuytmcncnghathctintrongsnxutvvycnphixc nhimngincaenzymbngcchoccadungdch enzym khi kt ta vi cn theo Droxov (1970) Tinhnhly5mldchenzymcha5-8mgproteinenzym,10ml mboratccpHkhcnhaut6,6n9,0.Sauchokttabng cn96vinngcnl60%.lnhtrong30pht,lyraohm lngproteinbcsng=620nm.pHnodchenzymsautac ccaonht,tclccaonht,limnginpIca enzym nghin cu. Kt qu th hin bng 20.Bng 20. Kt qu xc nh im ng in ca lipase t do PHOD620 6,60,577 7,00,631 7,50,704 8,00,688 8,40,615 8,80,566 9,10,454 Kt qu thu c bng 20 cho thy ti pH 7,5 dch enzym ta c c cao nht. Vy pI ca lipase l 7,5 3.4.2. Mt s c tnh ca lipase Bacillus-sp c hiu c cht Lipase Bacillus c hot tnh c hiu i vi tng c cht v du n rt khc nhau. Hot tnh i vi du olive thp nht ch t 26 U/ml, i vi ccloidukhcnhdumtinhluyn,dunnh,Neptune,Meizan, Nakydaco t 53-63 U/ml, i vi du Tng an t 79 U/ml v i vi tributyrin t 101 U/ml (hnh 10). Hnh10.HotlipaseBacillus chiucatngccht.1:Du olive;2:Dum(tinhluyn);3: Dunnh(tinhluyn);4:Du neptune(duc+duhtci);5: DuMeizan(duolein+duht ci+dunnh);6:DuNakydaco (duoleinda+dunnh);7:DuTngan(duolein+dunnh + du canola); 8: Tributyrin (1%). 0204060801001201 2 3 4 5 6 7 8Cc c chtHot tnh (U/ml)nh hng ca nhit Nhit ti u ca lipase chng Bacillus chu nhit ny c xc nh qua vic o hot tnh dch ni nhit phn ng khc nhau t 30n75Cvicchtlduntnghpnng5%vpH8,0. Khi nhit tng t 30 n 75C th hot tnh lipase cng tng theo vi tc u t 31 n 91 n v/ml (hnh 11). Hot tnh t ti u nhit 70C l 91 n v/ml (hnh 11). Sau nhit 70C, hot tnh lipase c gim cht t (89 U/ml nhit 75C). Vi biu nhit ti u ny, lipaseBacillusthucvomttrongcclipaseanhitcnghin cu trn th gii. 02040608010030 35 40 45 50 55 60 65 70 75Nhit (C)Hot tnh (U/ml)Hnh 11. nh hng ca nhit n hot lipase. nh hng ca nhit n bn ca lipase Di tc ng ca nhit , hot tnh lipase t nhiu u b nh hng. Mcnhhngtythucvotngloilipase khc nhau v ty theo khongthigianenzymcnhit.Trongqutrnhxl nhit,cthtrctinmtslinkthydrotrongmnglilinkt hydro tham gia vo vic gi vng cu trc ca lipase b t gy dn n cu trc ca enzyme b bin i, lm cho c cht khng lin kt c vi enzymevkhngbchuynhaditcdngcaenzyme.Nuxl enzymemtnhitqucao,cclinkttrongphntenzymes hon ton b ph hy lm enzyme b mt hot tnh v lc ny khi a v nhit bnh thng th hot tnh ca n khng th hi phc li c.Thigianlipasecngluvnhitcngcaothhottnhcnli cng thp. nhit 30C, hot tnh lipase gim nh cho ti 8 gi (86%) v sau 24 gi hot tnh cn li l 63% (hnh 12). nhit 40 v 50C, th hot tnh - nhit tng t nhau, chng gim mnh u cho ti 6 gi(32v34%),sauhottnhgimnhchoti24gi(29v 22%). i vi nhit 60 v 70C, th hot tnh - nhit cng tng tnhau,chnggimrtnhanhchsau1gi(31v20%)vsau gim dn cho ti zero (70C) v 17% (60C). Ktqunychothy bn ca lipase ph thuc vo nhit v thi gian . 0204060801001200 1 3 6 8Thi gian (gi)Hot tnh tng i (%) 24Hnh 12. nh hng ca nhit n bn lipase. Nhit 30C (X); 40C (Q); 50C (V); 60C (X); 70C (O). nh hng ca pH n hot tnh pH hn hp phn ng c nh hng r rt n hot tnh enzyme v n linquannmcionhacchtvbncalipase.tmra khongpHtiuchohottnhlipase,xutphttmtsthngtinv pH ti u ca mt s chng Bacillus, chng ti bt u d tm pH t 7,0 v i dn v pha acid v kim. Hnh 13. nh hng ca pH n hot tnh lipase. Hot tnh lipase t l thun vi tng pH hn hp phn ng trong mt khong pH gii hn 5,0-9,5. pH 5,0, hot tnhlipaselzerocnghallipasenykhnghotngtrongmi trngacid.HottnhtngnhanhukhipHtngln7,5(76U/ml). HottnhbtutngchmkhipHit7,5(76U/ml)ln9,5(108 U/ml) v t cc i ti pH 9,5. Hot tnh gim nh pH 10,0 (98 U/ml). DipHcnthitcholipasehotngl7,5-9,5,mtloilipasea kim. c tnh ny cng vi kh nng thy phn tt cc loi du n khc nhauchothyylmtloienzymertthchhpchongnhcng nghip sn xut cc cht ty ra hot ng mt mi trng c tnh kim cao. 0204060801001205 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10pH hn hp phn ngHot tnh (U/ml)nh hng ca pH n bn ca lipase Ging nh nhit , pH cng nh hng r rt n bn lipase. Khi pH4,hottnhlipasechcn12%,vkhipH5-7hottnhcnli mt na (48-58%) (hinh 14). Lipase gi nguyn hot tnh (92-117%) khi mitrngpH9,0-10(hnh14), nhngkhimitrngqukim(pH 11)hottnhcnlich23%.Cng mtpH,nhngtrongccmkhc nhau, hot tnh cn li cng khc nhau (hnh14).iunychothyccion cngnhhngtibnlipase.Nhvy lipase c bn cao mi trng kim v thp mi trng acid. 0204060801001201404 5 6 7 8 9 10 1pH hn hp lipaseHot tnh tng i (%)1Hnh14.nhhngcapHnbnlipase.macetate(X);m phosphate (Q); m Tris-HCl (V); m glycine (X). nh hng ca ion kim loi n hot tnh Theonhiutiliunghincuvcutrccalipasechothylipase khngphilmtenzymekimloi.Tcngcakimloinlipase trongqutrnhxctcphnngcththeonhiucchkhcnhau:(a) Thamgiavovicgivngnnhcudngkhnggiancnthitca phn t lipase m bo vic thc hin cc phn ng enzyme. (b) C tc dng lm tng tc phn ng thng qua vic to thnh phc vi c cht. (c)Thamgialmcunikthpcchtvitrungtmhottnhca enzyme. KtquchothyccionkimloihatrI,IIvIIIunhhngn hot tnh ca lipase. K+, Mg2+, Fe3+ lm gim ng k hot tnh ca lipase (80-89%ivi1mMv57-83%ivi10mM)trongkhiCa2+li c nh hng tch cc n hot tnh ca lipase (tng 10-12%) (hnh 15). Hnh15.nh hngcaion kimloinhottnh lipase.Ctgchngang:KCl;ctcar:MgCl2;ctxygch:CaCl2;ct thang: FeCl3. 0204060801001200 1 10 1 10 1 10 1 10Nng ion (mM)Hot tnh tng i (%)nh hng ca dung mi hu c Scmtcadungmihuc1%lmgimhottnhlipase. Acetone,2-propanolvethanolgimhottnhlipasemtna,cn methanol gim mt phn tnh hng ca cht ty ra MtschttyrachottnhbmtnhSDS,Tween20,Tween80, Triton X-100 nh hng n hot tnh lipase. Triton X-100 tng hot tnh lipaseln63%vTween20ginguynchottnhlipase(107%), trong khi Tween 80 gim mt na, cn SDS th c ch lipase hot tnh cn khong 3% (hnh 17). 0204060801001201401601801 2 3 4 5Cc cht ty raHot tnh tng i (%) Hnh 17. nh hng ca cht ty ra n bn ca lipase. 1: i chng. 2: SDS. 3: Tween 20. 4: Tween 80; 5: Triton X-100. 3.5.KHOSTKHNNGNGDNGLIPASEtCandida rugosa 3.5.1. Kho st kh nng thu phn du vi lipase c nh Trongcngnghipdcngitathngsnxutvinnangc chachtbovichsaxit80100.Cngnhtrongkhuphn thc n king ca nhng bnh nhn nhim m trong mu nu s dng cc loi du n thc vt c thu phn s b ch s axit tng n 80 100 th slmgimhmlngmtrongmu.Dovychngtinghincu kho st kh nng thu phn du u nnh ca lipase c nh.Dolipasechckhnngthuphndutrnbmtlinphav vy chng ti tin hnh kho st s thu phn du n bng cch cho hn hp du: nc vi cc t l khc nhau 1:1, 2:1, 1:2 chy qua ct c cha enzym c nh 2 ln vi tc chy 15 git/pht nhit phng v c b sung CaCl2 5mM 2%. Sau xc nh ch s axit ca du sau khi thc hin phn ng thu phn. Kt qu kho st th hin bng 21 Bng 21. nh hng t l du: nc n kh nng thu phn du ca lipase Ch s axitT l du: nc Ln 1Ln 2 1:140,3260,14 2:161,3082,50 1:250,4970,14 Kt qu bng 21 cho thy vi t l du: nc l 2:1 kh nng thu phn calipaselttnhtchochsaxitl82,5.Nnchngtissdng iu kin ny cho thu phn vi lng du ln hn sau ny.3.5.2. Kho st kh nng s dng lipase tan trong sn xut phomat Trongqutrnhsn xut phomat t sa ti, giai on ln men ng t phomat ngi ta s dng renin v mt s proteaza axit tnh hoc kimtnhlmtcnhnngtsa.Tronggiaionchncthb sung lipase nhm lm tng hng v c trng cho phomat.khostkhnngsdnglipasetrongqutrnhsnxut phomat, tin hnh th nghim trn 2 mu. Mu 1: sn xut phomat c b sung 50% renin + 50% proteaza ng t sa. Mu 2: sn xut phomat b sung 50% renin + 50% proteaza + 0,001% lipase. C2mucchnnhit410oCtrong6tun.Sau nh gi cht lng bng phng php cm quan. nhgibngphiuchoim(thangim10)theoccchtiu cht lng phomat. Kt qu biu din bng 22 Bng 22:Kt qu nh gi cht lng phomat bng phng php cm quan imCh tiu Mu 1(Khng c lipase)Mu 2 (c lipase)Mu sc1,142,21 Mi kem4,645,92 Mi chua5,854,57 Mi i3,281,71 Mi b3,645,00 Mi lactic4,005,50 Mi l1,000,35 V ngt4,075,78 V chua5,575,35 V ng2,141,42 V mn7,786,85 chc4,005,92 tan5,854,21 mn5,785,28 dnh5,285,42 Cm gic bo5,006,28 Cht lng tng hp6,427,85 Ktqunhgicmquanccchtiucasnphmphomatcv khng b sung lipase cho thy cc ch tiu ca sn phm phomat c s dng lipase u tt hn c v mu sc v hng v so vi phomat khng b sung lipase. KT LUN -iu kin nui cy ti u cho sinh tng hp lipase t Candida rugosa cho mt lt l:Mi trng nui gm: KH2PO4: 15 g; K2HPO4:5,5 g; (NH4)2SO4:4g; MgSO4:1g;FeCl3:10mg;inositon:0,004mg;biotin:0,008mg; Thiamine.HCl:0,2mg;Duoliu:1%;axitpalmitic:1.2g;H2O: 1000ml;S t bo nm men cy ban u: 2,6.106 tb/ml canh trng;Thi gian nui: 40 gi -iu kin nui cy ti u cho sinh tng lp lipase t Bacillus sp l: mi trng BMGY vi thnh phn: (1% (w/v) cao nm men, 2% (w/v) bacto-peptone,100mMkaliphosphate,pH7.0, 4 x 10-5% (w/v) biotin v 1% (v/v) methanol) 30C lc 220 vng/pht -Nng cn thch hp cho kt ta thu lipase l 90%. Lipase sau kt ta cnchochyscktrnsckchothylipasesaukhiquacttrao iionDEAE-cellulosexuthin3pick.Tinhnhxcnhhot lipase cc phn on. Kt qu cho thy ch c pick 1 v pick 3 c hot tnh lipase (69UI/ml v 77UI/ml) v Lip1 v Lip3 c khi lng phn t l 58 v 62 kDa. Lipase sau cc bc lm sch: kt ta etanol, sc k trao iionDEAECellulosethucchphmchotring55,16 U/mg protein, mc tinh sch l 12,5 ln; hiu sut thu hi l 37,06%.-iu kin ti u c nh lipase trn Nylon-6 l: nng HCl thch hp cho hot ho cht mang l 3,5N; glutaraldehit l 10%, nng lipase em c nh l 1mg/ml (550U/ml); s dng dung dch m phosphat pH 7,7. -Mt s c tnh ca lipase c nh trn Nylon-6 Nhittiul70C,bnvngnhit40-60C,sau18gi hot cn li l 52,4 66,7%. pH ti u l 7,0; bn vng pH 7 - 7,5; sau 18 gi hot cn 50,6 - 57%. -Mt s c tnh ca lipase tan t Candida rugosa Nhittiul60C,bnvngnhit40-50C,sau18gi hot cn li l 50,2 - 59%. pH ti u l 8,0; bn vng pH 7,5 - 8,0, sau 18 gi hot cn 53,9 - 62%. im ng in ca lipase l pI = 7,5 nh hng ca cc ion kim loi n hot tnh ca lipase:- Ion Pb2+ c ch mnh (lm gim hot ti 33,26%) - Cc ion Cu2+, Fe3+, Zn2+ c ch yu (lm gim hot tnh 13%)- Cc ion Na+, K+ khng nh hng.- Cc ion Mg2+, Ca2+ c kh nng hot ho lipase. -Mt s c tnh lipase t Bacillus sp Tnh c hiu c cht i vi tributyrin l cao nht: 101 U/ml. Nhit ti u l 70C (91,1 U/ml). Lipase c bn nhit, sau 24 gi hot tnh vn cn 63%. pH 9,5 l thch hp nht cho phn ng, hot tnh t 108,1 U/ml. pH 9,0 m Tris-HCl v pH 10,0 m glycine khng nh hng ti bn ca lipase, hot tnh cn li vn t 117%. Ca2+tnghottnhlipaseln10-12%,ngcliK1+, Mg2+, Fe3+ gim hot tnh lipase t 11-42%. Ccdungmihucugimhottnhlipase.Acetone,2-propanol v ethanol gim mt na, methanol gim mt phn t. Cc cht ty nh hng ln hot tnh lipase. Triton X-100 tng hot tnh ln63%.Tween20ginguynhottnh(107%).Tween80gimmt na. Cn SDS km hm hot tnh lipase 3%. - kho st kh nng ng dng ca lipase trong cng nghip dc, thc phm: Tlduncl2:1,tcchyl15git/pht,nnglipase 10U/mldu,nhitphng,thucduunnhcchsaxit 80,5. Vihmlnglipasel0,001%bsungvoqutrnhsnxut phomatthucccchtiuchtlngsnphmtthnsnphm phomat khng b sung lipase c mu sc v hng v.