i hc Khoa hc T nhin_ HQGHN ----------

BI TIU LUNng dng enzym keratinaza trong phn hy lng gia cm lm thc n chn nui Ging vin : Nguyn Th HNhm 7 K57 CNKTMTDanh sch thnh vin:1. Phm Th Liu2. inh Th Diu Linh3. Hunh Th Phng Linh

Ni DungI. Cht thi lng gia cm1.1 Thc trng cht thi lng gia cm1.2 Keratin1.2.1 Ngun gc Keratin 1.2.2 Cu trc Keratin1.2.3 Tnh cht keratinII. Enzym Keratinaza2.1 c tnh enzyme Keratinaza2.2 Ngun gc enzyme keratinazaIII. ng dng enzyme keratinaza trong phn hy lng gia cm3.1 C ch phn hy keratin3.2 Cc yu t nh hng n hiu sut phn hy3.3 Sn xut thc n chn nuiIV. Kt lunV. Ti liu tham kho

I.Cht Thi Lng Gia Cm1.1 Thc trng rc thi lng gia cm Pht trin ngnh chn nui l mt phn quan trng trong gii quyt vn lng thc, thc phm trn th gii. Qu trnh ny ng thi to ra lng ln rc thi lng v. Mi nm trn th gii c vi triu tn lng v c to ra t ngnh ch bin gia cm v t l tng hng nm l khong 4,5%. y l cht thi rt kh x l do thnh phn chnh l keratin - mt loi protein c kh nng chng li cc tc nhn phn hy cao, thi gian lng v t phn hy hon ton trong t nhin l t 5 7 nm. Ph phm lng gia sc gia cm thng c x l bng phng php chn lp hoc t chy, thm ch thi trc tip ra mi trng, gy nhim nghim trng cho mi trng t, nc, khng kh ( hnh 1) Hnh 1. Cht thi lng gia cm khng c thu gom x lX l lng v khng ng quy nh gy nh hng nghim trng n sc khe con ngi v mi trng ( hnh 2 ) Hnh 2 .X l, ch bin lng v khng ng quy nh Do bn cht ca lng gia cm l protein nn nu c x l ph hp th c th tr thnh ngun protein b sung hiu qu vo thc n chn nui, em li hiu qu cao v kinh t v c th gii quyt cc vn lin quan n mi trng. Tuy nhin hin nay cc c s sn xut thng p dng cng ngh hin i sn xut bt lng thng mi. Qu trnh ny tn rt nhiu nng lng v cng khng thy phn hon ton c lng v. Nhng phng php ny ph hy amino acid lm gim cht lng v t l tiu ha Protein.Bng 1: Thnh phn t l ca protein v acid amin trong lng v

( ngun latshaw et al . 1994) Vic s dng enzyme thy phn l phng n hiu qu, khng tn nng lng m hiu sut thy phn gn nh hon ton. Trong nhng nm gn y, cc nh khoa hc trn th gii c nhiu nghin cu v cc vi sinh vt c hot tnh keratinaza, v c im v ng dng ca chng, bc u phc v cho vic phn hy ph phm lng dng lm thc n chn nui v gii quyt c nhiu vn mi trng.

1.2 Keratin1.2.2 Ngun gc Keratin t nhin c to thnh t cc t bo sng ca c th. Trong t nhin keratin l thnh phn chnh cu to nn cc cu trc nh lng, tc, mng, guc, sng, da, biu m ca t bo ng vt c xng sng Ty thuc vo loi ging v tui ca ng vt m t l lng trn trng lng c th c s khc nhau. i vi g trng thnh lng v chim 5 7 % khi lng c th. i vi gia sc nh ln th t l ny l 0,5 0,8 % khi lng c th. Keratin chim n 90% khi lng lng v nn y c xem nh l ngun protein tim nng thay th cho mt s ngun protein khc b sung vo thnh phn thc n chn nui. 1.2.3 Cu trc ca keratin Keratin l nhm cc protein c cu trc si c trong t bo biu m ca ng vt c xng sng v l thnh phn ch yu cu to nn cc thnh phn ph nh mng, tc, lng, Keratin cc loi ng vt c xng sng khc nhau c trnh t acid amin v cu trc c bn tng t nhau. Trnh t acid amin quyt nh n cc lin kt trong phn t v tnh cht ca nhng cu trc bc cao hn, cng nh nh hng n cc lin kt trong phn t keratin. Cc chui acid amin ny c cun cht, dng xon ( keratin) hoc dng phin ( keratin ) v gp np to thnh cu trc ba chiu. ( hnh 3) Hnh 3 : cu to hnh hc ca keratin v keratin Trong s cc acid min to nn cc phn t keratin th cyscein l ph bin nht (c th chim ti 24%).Trong cu trc ca acid amin cyscein c cha lu hunh c kh nng to s lin kt cho cht ch nh vo nhng cu ni disunfua. Theo hm lng lu hunh Keratin c phn thnh hai nhm chnh da theo . Keratin cng: hin din nhiu tc, mng, guc, vi hm lng cu ni disunfua cao. keratin mm: cc cu ni disunfua t hn v ch yu da, m. 1.2.4 Tnh cht ca keratin Keratin l protein rt bn, rt kh ha tan v b phn hy bi cc proteaza thng thng nh : trypsin, papain, pepsin. Keratin c th ha tan hon ton trong cc dung dch c cha cht bin tnh nh ur. Keratin trong dung dch c th lp rp li si trung gian. Hu ht cc t bo cha nhiu keratin m chng ta gp l t bo cht v c th c rng ra hoc b cc t bo mi y ln. Nu cc t bo cht ny c lu gi trong tnh trng tt, chng s c tc dng nh mt lp cch in, cch nhit, khng thm nc, bo v cc t bo pha di.II. Enzym Keratinaza2.1 c Tnh Ca Keratinaza Keratinaza c tn IUB l EC 3.4.99. Trng lng phn t 33kDa. Keratinaza t vi sinh vt c pH ti u nm trong khong t trung tnh n kim (6-9) v l loi enzyme a nhit, nhit ti u t 30oC n 80oC, c khi t ti 100oC. Keratinaza l loi enzyme c kh nng phn hy c cht cha Keratin trong t nhin. Keratinaza ch yu c xp vo nhm proteaza serin do c n 97% cu trc ging vi cu trc ca proteaza serin v cng b c ch bi cc cht c ch proteaza serin. Hnh 4 : enzyme keratinaza Tuy Keratin l mt dng protein kh b phn hy, nhng thc t ngoi t nhin cc dng c cht cha Keratin nh lng, mng, sngvn b phn hy. iu ny c c l do hot ng ca cc vi sinh vt (vi khun, x khun, nm mc) c kh nng sinh ra enzym Keratinaza. Keratinase ch yu tn cng vo cc lin kt disulfide (S-S), lm suy gim cc protein dng si fibrin, elastin, collagen v si protein khc.Tuy nhin kh nng to ra enzym ny ca vi sinh vt ph thuc vo nhiu yu t nh chng vi sinh vt, ngun c cht v c iu kin ca mi trng. y c xem nh ngun enzym quan trng c kh nng ng dng vo nhiu lnh vc khc nhau nh cng nghip da, phn bn, thc n chn nui, y dc

2.2 Ngun Gc Keratinaza l mt dng protease rt ph bin trong th gii vi sinh vt. Vi sinh vt tng hp Keratinaza c phn lp t nhiu v tr a l khc nhau, t t Nam cc n sui nc nng, k c iu kin mi trng k kh v hiu kh. n nay Keratinaza c nghin cu ch yu c ngun gc t vi khun, c bit l t chi Bacillus. ( hnh 5 ) Vi khun Bacillus phn b rng trong t nhin, nht l trong t. Chng tham gia tch cc vo s phn hy vt cht hu c nh kh nng sinh nhiu loi enzyme ngoi bo. y l nhng vi khun hnh que, Gram dng, sinh trng hiu kh hoc k kh khng bt buc v hnh thnh ni bo t Hnh 5: vi khun bacillus Hnh 6: nm Chrysosporium Trong s cc loi thuc chi Bacillus th B.licheniformis v B.subtilis c nghin cu l c kh nng thy phn Keratin tt. B.licheniformis l vi khun Gram dng, c tm thy nhiu trong t, nhit tng trng ti u khong 30oC, sinh enzyme ti a 37oC v c kh nng sinh bo t mi trng khc nghit. B.subtilis l trc khun Gram dng, hiu kh ty tin, sinh ni bo t Ngoi vi khun th cn mt s nhm vi sinh vt c kh nng sinh ra enzyme Keratinaza. Nhm vi khun: vi khun Gram dng c kh nng phn hy Keratin bao gm Lysobacter, Kocurica, Nesternokia v mt vi dng Gram m nh Vibrio, Xanthomonas, Stenotrophomonas Nhm x khun ch yu l nhm Streptomyces. Nhm nm: Chrysosporium ( hnh 6) , Fusarium, Geomyces, Monodictys, UrocladiumIII. ng Dng Enzym Keratinaza Trong Phn Hy Lng Gia Cm3.1. C Ch Phn Hy Keratinaza c trong t bo vi khun s b gy cc cu ni disulfide trong cu trc ca keratin, to thnh cc chui protein c cu trc n gin hn, h tr cho qu trnh phn hy ca enzyme ngoi bo. Sn phm cui cng ca qu trnh phn hy bao gm peptide mch ngn c kh nng ha tan v cc axit amin. Nh vy qu trnh phn hy keratin bao gm hai bc l ph hy cc cu ni disulfide v phn hy chui protein Disulphide bonds NH2 NH2 CH2 - S S - CH2 CH2 S- S- - CH2NH CO NH CO CO -----------------HN CO NHR hydrogen bonds R t0, H+/OH R R NH CO + E NH + COOC NH CO NH NH3+ ------ -OOC NH3+ -OOCNH ion-ion interactions CO NH CO CO HN CO NHR R R R t0,enzym ,H+ +H2O NH2-CH-COOH + NH2-CH-COOH + . + peptide mch ngn R CH- SH 3.2 Cc Yu T nh Hng n Qu Trnh Phn Hy Lng3.2.1 nh hng ca nhit v pH Nhit v PH l 2 yu t nh hng rt ln n s tn ti v pht trin ca vi sinh vt. Nhit qu thp hoc qu cao s lm c ch qu trnh trao i cht, tiu dit t bo vi sinh vt.( Hnh 8). Nng H+ c trong mi trng lm thay i tnh thm ca t bo, nh hng n h enzyme trn thnh t bo cng nh cc enzyme ngoi bo t, lm nh hng n kh nng trao i cht v pht trin ca t bo vsv. ( Ngun: G.-T. Park, H.-J. Son et al.2007) Hnh 8. nh hng ca nhit n mt v hot ng ca enzyme kerstinza

( Ngun: G.-T. Park, H.-J. Son et al.2007)Hnh 9. nh hng ca pH n mt v hot ng ca enzyme kerstinza Bng 2 : nh hng ca nhit v pH n hiu sut phn hy keratin ca enzyme keratinas (Ngun: Dng Trng Tn. i Hc Cn Th .2013) T kt qu ca hnh 8, hnh 9 v bng 2 cho thy kh nng phn hy bt lng gia cm t hiu sut cao nht l 49,06% 370C v pH 7,5. Khi phn tch cng mt nhit : hiu sut phn hy cao nht pH 7,5 , gim dn cc iu kin pH thp hoc cao hn 7,5 v thp nht pH 6. Khi phn tch cng mt pH : hiu sut phn hy cao nht 370C v gim nhanh khi nhit t 450C tr ln. C th kt lun ti iu kin 370C v pH 7,5 cho hiu sut phn hy bt lng cao nht ( Mc nhit v pH ny s c s dng cho cc th nghim v s nh hng tip theo)

3.2.2 nh hng ca ngun dinh dng Cacbon

(Ngun: Dng Trng Tn. i Hc Cn Th .2013) Hnh 10 .nh hng ca ngun dinh dng cacbon n kh nng phn hy keratin ca enzym Keratinaza. T Hnh 10 ta thy, s c mt ca glucose hay sucrose trong mi trng s lm gim kh nng phn hy keratin do: glucose v sucrose lm c ch kh nng sinh ra enzyme keratinaza t vi khun, vi khun khng th phn hy keratin c na dn n thiu ht ngun dinh dng v gim s sinh trng, pht trin. R ng cng l mt tc nhn gy c ch qu trnh sinh ra keratinase t vi khun, n lm gim kh nng phn hy keratin mc d thnh phn chnh ca n l carbohydrate cng vitamin v khong cht, mt ngun dinh dng rt tt cho s pht trin tng sinh khi ca vi khun. Trong mi trng c b sung glucose, sucrose hay r ng u lm gim kh nng phn hy bt lng gia cm. Kh nng phn hy c cht bt lng t c cao nht khi trong mi trng ch cha lng l ngun dinh dng duy nht.

3.2.3 nh hng ca ngun dinh dng Nit

(Ngun: Dng Trng Tn. i Hc Cn Th .2013Hnh 11. nh hng ca ngun dinh dng nit n kh nng phn hy keratin ca enzymT kt qu ca hnh 11 cho thy, vic b sung thm cc ngun dinh dng cha nit khc nhau lm cho kh nng phn hy keratin b gim. T l lng b phn hy cao nht khi khng b sung. Nh vy kh nng phn hy lng ca vi khun ph thuc nhiu vo thnh phn dinh dng ca mi trng. Khi mi trng ch cha c cht keratin l ngun dinh dng duy nht, vi khun s b kch thch tit ra nhiu enzyme keratinaza phn hy c cht c trong mi trng hp thu vo t bo. Khi mi trng xut hin ngun dinh dng khc d tiu ha hn nh Yeast extract v bt u nnh, vi khun s u tin s dng ngun dinh dng ny trc, sau khi ngun dinh dng ny cn kit mi chuyn sang s dng ngun keratin trong mi trng. y l l do khin kh nng phn hy lng b gim.

3.3 Ch Bin Thc n Chn Nui3.3.1 Bt lng v thy phn theo phng php hin i ( bt lng v thng mi Ti cc c s ch bin hin i, lng v c nu di p sut cao, s dng hi nc trc tip thy phn mt phn protein, ph hy cc cu ni bn trong cu trc keratin. Sau bt lng v thy phn s c x l bng men pepsin to ra sn phm thc n c tnh ngon ming cao. Hoc lng v c th phn hu theo qui trnh: kh bi Na2S2O5 ri thu phn bng NaOH, sau c trung ho bi HCL. Dch sau thu phn trn vi cm (t l 2/1) c mu vng sng, hi kht v c hm lng protein c th tiu ha c l 50%. ( inh Vn Ci, Vin Khoa hc Nng nghip min Nam) Tuy nhin qu trnh ny i hi rt nhiu nng lng v chi ph u t sn xut ln. Gi 1kg bt lng v thng mi t 11.000 13.000 ( Thc n chn nui Bo Minh). Qu trnh gia nhit v x l ha hc cng lm ph hy mt s axit amin nhy cm vi nhit c trong thnh phn lng, gy gim st gi tr dinh dng ca sn phm. 3.3.2 Bt lng v thy phn bng enzyme keratinase Phng php ch bin Lng ct nh c 5 ngy vi keratinase, thu phn v phi kh n khi lng gy ra . u im ca sn phm Phn hy cht thi lng nh enzyme keratinaza tit ra t vi khun to ra mt ngun protein c t l dinh dng cn bng hn, ci thin gi tr dinh dng ca sn phm, gi li c cc axit amin thit yu nh Methionine, Lysine, trypophan. Hm lng protein c th tiu ha c cao chim ti 70% trong khu phn. Th nghim trn g, ngi ta nhn thy rng, Kh nng tiu ho ca bt lng thy phn vi Keratinase g trng thnh l 82% cao hn so vi bt lng thy phn thng mi (60%). Khi cho g tht n thc n hn hp c bt lng v ny, ngi ta thy mu sc ca da vng hn, tht c v m , khng c mi l ca bt lng v.Chi ph sn xut thp => Gi thnh thp hnNhc im : mi hi ca lng Tuy phng php thy phn bng enzyme keratinaza cho ra sn phm thc n c nhiu u im vt tri hn. nhng do phng php ny cha c m rng v iu kin sn xut nguyn liu t vi khun v nm cha p ng nn phn ln cc cng ty sn xut loi thc n ny vn p dng phng php thy phn hin i.

IV. Kt Lun Enzym Keratinaza c phn lp t vi khun Bacillus c nghin cu l c kh nng thy phn Keratin tt nht. Keratinase ch yu tn cng vo cc lin kt disulfide(S-S), lm suy gim cc protein dng si fibrin, elastin, collagen v si protein khc . 370 C v pH 7,5 vi khun pht trin tt nht v kh nng phn hy bt lng cao nht. Cc ngun dinh dng cha cacbon v nit khc nhau c nh hng n hot ng ca vi khun. gy c ch v lm gim kh nng phn hy keratin. Kh nng phn hy bt lng cao nht khi trong mi trng ch c c cht keratin l ngun dinh dng duy nht. Bin php phn hy lng sinh hc khc phc c nhc im trc . To ra sn phm bt lng v thy phn cht lng u vit hn, chi ph sn xut thp, gi thnh sn phm gim. S dng vi sinh vt phn hy ngun cht thi ny l hung i mi trong vic gii quyt nhim mi trng v ng dng trong thc n chn nui.

V.Ti Liu Tham Kho- Ashis K. Mukherjee, Sudhir K. Rai, Naba K. Bordoloi (2011), Biodegradation of waste chicken-feathers by an alkaline -keratinase (Mukartinase) purified from a mutant Brevibacillus sp. strain AS-S10-II, International Biodeterioration & Biodegradation 65, 1229-1237.- Taha I. Zaghloul, Amira M. Embaby, Ahmed R. Elmahdy (2011), Biodegradation of chicken feathers waste directed by Bacillus subtilis recombinant cells: Scaling up in a laboratory scale fermentor, Bioresource Technology 102, 23872393.- Geun-Tae Park, Hong-Joo Son (2009), Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium, Microbiological Research164, 478- 485.- Salon 129:Wool Dyeing History, with Focus on Dyeing of Rugs by Pierre Galafassi, http://www.turkotek.com/salon_00129/salon.html- Bad Hair Days? Chemistry to the Rescue by Marlene M. Gutierrez http://www.teachers.yale.edu/curriculum/viewer/initiative_11.05.04_u- L Th Thu Huyn, Phn lp vi khun c hot tnh keratinaza v mt s c tnh ca enzim (2012)- Dng Trng tn, Kho st cc yu t nh hng n s pht trin v kh nng phn hy lng gia cm cu vi khun Bacillus Cereus K13 ( 2013)