Li Cam ne hoan thanh khoa luan nay ngoai s no lc cua ban than trc tien toi xin to long biet n sau sac en co giao TS. Nguyen Hien Trang, co giao TS. o Th Bch Thuy a tan tnh hng dan, truyen at cho toi nhng kien thc bo ch va can thiet trong thi gian hoc tap, thc hien e tai va hoan thanh khoa luan tot nghiep. Tiep en toi xin chan thanh cam n cac anh ch cong tac tai phong th nghiem thuoc vien Tai nguyen, Moi trng va Cong nghe sinh hoc, ai hoc Hue a tan tnh hng dan, giup , tao ieu kien cho toi hoan thanh khoa luan tot nghiep nay. ong thi toi xin gi li cam n en Ban giam hieu;

Phong ao tao; Khoa C kh - cong nghe cung quy thay co trng ai hoc Nong Lam Hue a trang b hanh trang cho toi trong suot 4 nam hoc va qua. Cuoi cung, toi xin cam n gia nh va ban be a tao ieu kien thuan li ca ve mat vat chat lan tinh than e toi co the hoan thanh c khoa luan tot nghiep nay. Mot lan na toi xin chan thanh cam n. Hue, thang 5 nam 2010 Sinh vien thc hien Hoang Nguyen Nhat Minh

DANH MC T VIT TT MTCB: Mi trng c bn MTTCB: Mi trng ti u c bn Glu: Glucose Sac: Saccharose Lac: Lactose TB: Tinh bt Cas: Casein CN: Cao nm N: NH4Cl C: i chng

DANH MC BNG BIU, HNH NH Bng 3.1 Bng phn tch phng sai.......................................................23 Hnh 4.1. nh hng ca ngun Carbon n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9..................26 Hnh 4.2. nh hng ca ngun Nitrogen n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9..................27 Hnh 4.3. nh hng ca ngun phi hp n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9..................28 Hnh 4.4. nh hng ca hm lng tinh bt khc nhau n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9........29 Hnh 4.5. nh hng ca pH ban u n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9..................30 Hnh 4.6. nh hng ca nhit n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9................................32 Hnh 4.7. nh hng ca thi gian n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9................................33

MC LC

PHN 1 T VN Ngy nay, cng vi s pht trin mnh m ca Cng ngh sinh hc, cc ch phm enzym c sn xut ngy cng nhiu v c s dng trong hu ht cc lnh vc nh: ch bin thc phm, nng nghip, chn nui, y t S lng cc enzym c sn xut hng nm trn th gii, ch yu l cc nc ang pht trin nh chu u, M, Nht Bn khong trn 300000 tn vi gi tr trn 500 triu USD. Trong c khong 600 tn protease tinh khit c sn xut t vi sinh vt bao gm khong 500 tn t vi khun v 100 tn t nm mc [1]. Protease l mt trong nhng enzym c ng dng rng ri trong mi lnh vc, c bit l trong cng nghip ch bin thc phm (ng t sa lm pho mt, lm mm tht, b sung lm tng cht lng sn phm trong sn xut bia, x l ph ph phm trong ch bin thc phm). Hin nay trn th gii c nhiu nghin cu, a vo ng dng cc ch phm protease trong cng nghip ch bin thc phm v thng mi ho ch phm protease. Vit Nam cng c nhiu cng trnh cng b v vic nghin cu s dng protease, tuy nhin cc cng trnh nghin cu cn nhiu hn ch. Ngi ta c th thu nhn protease t nhiu ngun khc nhau: ng vt, thc vt v vi sinh vt. Vi sinh vt chnh l i tng c th sn xut enzym ni chung v protease ni ring vi s lng nhiu v gi thnh r. Nhng kt qu t c trong lnh vc nghin cu v protease vi sinh vt ch yu tp trung vo i tng Bacillus sp [17], [18], [19], [24], [25], [27], [28], [29], [34], [36], [37], [38] Chng Bacillus amyloliquefaciens c kh nng sinh protease, tuy nhin c rt t cc cng trnh trong nc v trn th gii nghin cu v iu kin nui cy chng ny sinh tng hp protease ngoi bo. Xut pht t nhng vn trn chng ti thc hin ti: Nghin cu mt s yu t nh hng ln kh nng sinh tng hp protease ngoi bo ca Bacillus amyloliquefaciens T9.

PHN 2 TNG QUAN TI LIU 2.1. Tng quan v enzym protease [2] 2.1.1. Khi nim v phn loi Protease l cc enzym xc tc s thy phn lin kt peptit (CO-NH) trong phn t protein v cc c cht tng t.-CH - C - N -CH R O H R - CH - C - OH R O HN - CH H R

+

H2O

+

Nhiu protease c kh nng lin kt este v vn chuyn acid amin. Hip hi Ha sinh v Sinh hc phn t quc t (1984) ngh s dng thut ng peptidase ch cc enzym thy phn mi lin kt peptide (thuc phn nhm E.C 3.4) [10]. Cc peptidase bao gm hai nhm enzym : - Endopeptidase: Nhm enzym xc tc thy phn cc mi lin kt peptid ni mch. Thuc nhm ny c cc enzym sau: papain, bromelain, ficin, trypsin, renin, rennin, chymotrypsin [10]. - Exopeptidase: Nhm enzym xc tc thy phn cc mi lin kt peptide u mch, c chia thnh 2 phn nhm: + Cacboxypeptidase: xc tc s thy phn lin kt peptit u C ca mch polypeptit v gii phng ra mt amino acid hoc mt dipeptide. + Aminopeptidase: xc tc s thy phn lin kt peptit u N t do ca mch polypeptit gii phng ra mt amino acid, mt dipeptide hoc mt tripeptide. Da vo s c mt ca cc nhm chc nng trung tm hot ng, cc protease c chia lm 4 nhm sau: serine protease, cysteine protease, aspartic protease v metallo protease [10].

- Serine protease: L nhng protease c nhm -OH ca gc serine trong trung tm hot ng, c vai tr c bit quan trng i vi hot ng xc tc ca enzym. Nhm ny bao gm hai nhm nh: chymotrypsin v subtilisin. Nhm chymotrypsin bao gm cc enzym ng vt nh chymotrypsin, trypsin, elastase. Nhm subtilisin bao gm hai loi enzym ca vi khun nh subtilisin Carlsberg, subtilisin BPN. Cc serine protease thng hot ng mnh vng kim tnh v th hin tnh c hiu c cht tng i rng. - Cysteine protease: Cc protease thuc nhm ny c nhm -SH trong trung tm hot ng. Cysteine protease bao gm cc protease thc vt nh papain, actinidin, bromelain, mt vi protease ng vt v protease k sinh trng. Cc cysteine protease thng hot ng mnh pH trung tnh, c tnh c hiu rng v ch hot ng c khi nhm -SH trong trung tm hot ng ca n khng b bao vy. - Aspartic protease: Hu ht cc aspartic protease thuc nhm pepsin. Nhm pepsin bao gm cc enzym tiu ha nh pepsin, chymosin, cathepsin, renin, cc protease ca nm mc. Cc aspartic protease c cha nhm carboxyl trong trung tm hot ng v thng hot ng mnh pH trung tnh. - Metallo protease: y l nhm protease c tm thy vi khun, nm mc cng nh cc sinh vt bc cao. Cc metallo protease thng hot ng mnh nht vng pH trung tnh v hot gim mnh di tc dng ca EDTA. Ngoi ra, protease c phn loi mt cch n gin hn thnh 3 nhm: - Protease acid: pH < 6 - Protease trung tnh: pH = 6-8 - Protease kim: pH = 8-11 Protease cn thit cho cc sinh vt sng, rt a dng v chc nng t mc t bo, c quan n c th nn c phn b rt rng ri trn nhiu i tng t vi sinh vt (vi khun, nm v virus) n thc vt (u , da...) v ng vt (gan, d dy b...). Trong c th, cc protease m nhim nhiu chc nng sinh l nh: hot ha zymogen, ng mu v phn hy si fibrin ca cc mu ng, gii phng hormon v cc peptid c hot tnh sinh hc t cc tin

cht, vn chuyn protein qua mng...[7]. Ngoi ra, cc protease c th hot ng nh cc yu t pht trin ca c t bo c tnh v t bo bnh thng l tng s phn chia t bo, sinh tng hp ADN...[2]. 2.1.2. Ngun thu nhn protease Protease c th c thu nhn t thc vt, ng vt v vi sinh vt [42], [12]. Tuy nhin ngun enzym vi sinh vt phong ph nht. 2.1.2.1. Ngun ng vt - Ty tng: y l ngun enzym sm nht, lu di nht v c cha nhiu enzym nht. - D dy b: trong ngn th t ca d dy b c tn ti enzym thuc nhm protease tn l rennin. Enzym ny t lu c s dng ph bin trong cng ngh phomat. Rennin lm bin i casein thnh paracasein c kh nng kt ta trong mi trng sa c nng Ca2+. y l qu trnh ng t sa rt in hnh, c nghin cu v ng dng y nht. Trong thc t nhiu ch phm rennin b nhim pepsin (trong trng hp thu ch phm rennin b qu th, khi d dy b pht trin y c kh nng tit ra pepsin) th kh nng ng t sa km i. 2.1.2.2. Ngun thc vt C 3 loi protease thc vt nh Bromelain, Papain v Ficin. Papain thu c t nha ca l, thn, qu u (Carica papaya), Bromelain thu t qu, chi da, v da (Pineapple plant). Cc enzym ny c s dng chng li hin tng kt ta trng ca bia khi lm lnh (chilling proofing) do kt ta protein. Nhng ng dng khc ca protease thc vt ny l trong cng ngh lm mm tht v trong mc tiu tiu ho. Ficin thu c t nha cy h Ficus (Ficus carica). Enzym ny c s dng thu phn protein t nhin. 2.1.2.3. Ngun vi sinh vt Protease vi sinh vt c th c thu nhn t vi khun, nm [10], [34] v x khun. So vi protease ng vt v thc vt, protease vi sinh vt c nhng c im khc bit, h protease vi sinh vt l mt h thng rt phc tp bao gm nhiu enzym rt ging nhau v cu trc, khi lng v hnh dng phn t nn rt kh tch ra di dng tinh th ng nht. Cng do l phc h gm

nhiu enzym khc nhau nn protease vi sinh vt thng c tnh c hiu rng ri cho sn phm thy phn trit v a dng [13]. * Vi khun Lng protease sn xut t vi khun c c tnh vo khong 500 tn, chim 59% lng enzym c s dng [9]. Protease ca ng vt hay thc vt ch cha mt trong hai loi endopeptidase hoc exopeptidase, ring vi khun c kh nng sinh ra c hai loi trn, do protease ca vi khun c tnh c hiu c cht cao. Chng c kh nng phn hy ti 80% cc lin kt peptide trong phn t protein [9]. Trong cc chng vi khun c kh nng tng hp mnh protease l Bacillus subtilis, B. mesentericus, B. thermorpoteoliticus v mt s ging thuc chi Clostridium. Trong , B. subtilis c kh nng tng hp protease mnh nht [2]. Cc vi khun thng tng hp cc protease hoat ng thch hp vng pH trung tnh v kim yu. Cc protease trung tnh ca vi khun hot ng khong pH hp (pH = 5-8) v c kh nng chu nhit thp. Cc protease trung tnh to ra dch thy phn protein thc phm t ng hn so vi protease ng vt v tng gi tr dinh dng. Cc protease trung tnh c kh nng i lc cao i vi cc amino acid a bo v thm. Chng c sinh ra nhiu bi B. subtilis, B. mesentericus, B. thermorpoteoliticus v mt s ging thuc chi Clostridium. Protease kim tnh ca vi khun c kh nng hot ng v n nh pH kim. Protease kim tnh chim 25% tng s enzym thng mi, s lng ln protease ny rt quan trng cho vic nghin cu v l thuyt v ng dng trong cng nghip. Protease kim tnh c sn xut bi vi khun v bit n nhiu nht l subtilisin Carlsberg, subtilisin BPN [22]. Protease ca Bacillus a kim c im ng in bng 11, khi lng phn t t 20.000-30.000. n nh trong khong pH = 6-12 v hot ng trong khong pH rng t 7-12 . * Nm Nm to ra nhiu loi enzym hn so vi vi khun. Nhiu loi nm mc c kh nng tng hp mt lng ln protease c ng dng trong cng nghip thc phm l cc chng: Aspergillus oryzae, A. terricola, A. fumigatus,

A. saitoi, Penicillium chysogenum Cc loi nm mc ny c kh nng tng hp c ba loi protease: acid, kim v trung tnh. Cc protease ca nm hot ng trong vng pH kh rng (pH = 4-11) v th hin tnh c hiu c cht cao. Tuy nhin chng c tc phn ng thp hn v chu nhit km hn so vi cc enzym ca vi khun. Cc protease acid tnh ca nm c pH thch hp t 4-4,5 v n nh pH t 2,5-6. Cc protease trung tnh ca nm l nhng metallo protease c pH thch hp bng 7 v b bt hot bi cc hp cht cng cua [10]. Protease kim tnh ca nm, c bit h Aspergillus mang n li ch trong cng nghip thc phm v y hc. Trong s cc protease kim tnh thuc h Aspergillus, protease kim tnh (Alp) t Aspergillus oryzae (EC. 3.4.21.63) c nghin cu c bit chi tit [22]. Mt s nm mc khc nh: A. candidatus, P. cameberti, P. roqueforti cng c kh nng tng hp protease c kh nng ng t sa s dng trong sn xut pho mat. * X khun V phng din tng hp protease, x khun c nghin cu t hn vi khun v nm mc. Tuy nhin, ngi ta cng tm c mt s chng c kh nng tng hp protease cao nh: Streptomyces grieus, S. fradiae, S. erythreus... [23]. Cc ch phm protease t x khun c bit nhiu l pronase (Nht) c tch chit t S. grieus, enzym ny c c tnh c hiu rng, c kh nng thy phn ti 90% lin kt peptide ca nhiu protein thnh amino acid. Lin X (c), ngi ta cng tch c ch phm tng t t S. grieus c tn l protelin. T S. fradiae cng c th tch chit c keratinase thy phn karetin. M, ch phm c sn xut c tn l M-Zim dng trong sn xut da. Protease t S. fradiae cng c hoat tnh elastase cao, do chng c dng trong cng nghip ch bin tht. Nh vy, cc ch phm enzym c sn xut t vi sinh vt rt a dng, phong ph v chng loi v tnh cht. C th ni vi sinh vt l ngun nguyn liu thch hp nht sn xut enzym quy m ln dng trong cng nghip v i sng [7]. Enzym protease t ngun ng vt v thc vt c loi ngi khai thc v ng dng hng ngn nm sn xut v phc v trong

cuc sng. Enzym thu nhn t cc ngun ny thng rt kh v hiu qu kinh t khng cao. Ngun enzym t vi sinh vt dn dn thay th enzym t ng vt v thc vt do hng lot nhng u im v sinh l vi sinh vt v v k thut sn xut nh sau [9]: + Trong t bo vi sinh vt c cha nhiu enzym, h enzym ca vi sinh vt v cng phong ph. T vi sinh vt c th thu mt lng ln cc enzym khc nhau. C nhng enzym ch c th khai thc t vi sinh vt nh: Cenlulose, raxemase, nitrogenase + Cc enzym ca vi sinh vt c hot tnh rt mnh vt xa cc enzym ca cc sinh vt khc. + Vi sinh vt rt nhy cm vi tc ng mi trng nn khi thay i iu kin sng hoc tc ng thch hp bng cc yu t khc nhau c th lm thay i d dng h enzym v hot tnh enzym ca chng. iu cho php thu c cc enzym theo mun vi hot tnh cao. V d nh i vi Aspergillus niger mun thu ch phm protease th cho vo mi trng cc c cht c cha nhiu protein nh kh lc, kh u tng, nc chit nm men cn mun thu c pectinase th thay cc c cht bng bt c rt, ng, c ci, cc mui v c l nhng loi c cha nhiu cht pectin. + Vi sinh vt sinh sn cc k nhanh chng, c th thu c mt khi lng t bo ln trong khong thi gian rt ngn. T bo vi sinh vt c kch thc b nhng t l enzym trong t bo tng i ln nn quy trnh sn xut d dng, thao tc thun li, hiu sut cao. Trong mt thi gian ngn vi mt phm vi nh, ta c th sn xut ra mt lng ln cc ch phm enzym mong mun. + Phn ln thc n nui cy vi sinh vt l d kim v r tin. C th dng cc ph liu, ph phm ca cc ngnh sn xut cng nghip khc, hoc nguyn liu t nhin nh du m, kh t v hu nh bt c cht no trong thin nhin t cc cht v c n gin n cc cht hu c phc tp u c vi sinh vt ng ho ht. + C th d dng iu khin cc iu kin ti u trong qu trnh nui cy c th thu c hiu sut cao trong sn xut.

Tm li, tuy vi sinh vt v cng nh b nhng v phng din khai thc enzym n vt xa cc ng vt v thc vt khc. Tuy nhin trong mi trng hp cn lu kh nng sinh c t (gy c, gy bnh) c bin php phng nga, x l thch hp. sn xut ch phm enzym, ngi ta c th phn lp cc ging vi sinh vt c trong t nhin hoc cc ging t bin c la chn theo hng c li nht, ch tng hp u th mt loi enzym nht nh cn thit no . 2.1.3. Cc yu t nh hng n qu trnh tng hp protease vi sinh vt Qu trnh tng hp enzym ni chung cng nh tng hp protease vi sinh vt chu nh hng ca nhiu yu t khc nhau nh m, nhit , pH, thng kh, thnh phn mi trng... Nm vng c quy lut nh hng ca cc yu t ny chng ta c th iu khin hot ng tng hp enzym vi sinh vt theo mun. * nh hng ca nhit nui cy Nhit nh hng n s pht trin, kh nng sinh tng hp enzym ca vi sinh vt cng nh tnh cht ca enzym c tng hp. Ty tng loi vi sinh vt khc nhau m c ngng nhit thch hp khc nhau, v d nh cc loi nm mc pht trin thch hp nhit 22-33C, cn nhit thch hp cho cc loi vi khun thng thng khong 35-55C, c bit c loi vi khun nh Bacillus istaticus... pht trin mnh 60-65C. Nhn chung a s cc loi enzym c tng hp t vi sinh vt u khng bn vi nhit v b km hm nhanh chng nhit cao hn nhit thch hp. nh hng ca nhit nui cy ln hot tnh enzym mt s loi nm mc khc cng vy, nh khi tng nhit nui cy Aspergillus candidus hay Mucor sp. renninus n 36C, s lm mt hon ton hoc lm gim kh nng lm ng t sa ca dch mi trng [14]. * nh hng ca pH mi trng Trong phng php nui cy b mt, pH mi trng t nh hng n qu trnh sinh tng hp enzym ca vi sinh vt. Hn na, pH mi trng hu nh khng thay i trong qu trnh pht trin ca vi sinh vt. i vi cc loi nm mc, pH thch hp cho qu trnh sinh tng hp protease vo khong 6-

6,5. Trong khi , hu nh cc loi vi khun u pht trin tt v to ra lng enzym ln pH gn trung tnh (6,6-7,4) [14]. Ngc li, i vi phng php nui cy chm, pH mi trng c nh hng rt ln, nhiu khi ng vai tr quyt nh i vi s tch ly protease trong mi trng. iu ny th hin kh r cc loi thuc chi Bacillus. ng lu l pH mi trng thng c th thay i sau khi kh trng v trong qu trnh pht trin ca vi sinh vt. pH ban u ca mi trng thch hp cho s pht trin ca vi sinh vt khc nhau l khng ging nhau. i vi cc nm mc pH ny vo khong 3,8-5,6 cn i vi vi khun l 6,2-7,4. Trong qu trnh nui cy, ty theo thnh phn mi trng v cc sn phm ca qu trnh trao i cht vi sinh vt, pH mi trng chuyn dn v pha acid hoc kim [14]. i vi cc loi thuc chi Bacillus, pH mi trng nui cy thng c xu hng chuyn v kim tnh. Do c th cn c vo gi tr pH mi trng sau khi nui cy d on lng protease c tch ly trong mi trng. Theo mt s tc gi, pH mi trng c nh hng ln n t l cc protease c tng hp. V vy nu gi gi tr mt ngng nht nh trong sut qu trnh nui cy, th vi sinh vt s tng hp mt dng protease xc nh v chim u th trong mi trng [14]. * nh hng ca thnh phn mi trng Thnh phn mi trng c nh hng rt ln n kh nng sinh tng hp enzym ca cc loi vi sinh vt. Thnh phn mi trng thch hp cho mt chng vi sinh vt sinh protease trc ht phi tho mn s tng trng ca chng . Tuy nhin, khng phi lc no s pht trin tt ca vi sinh vt cng m bo hiu sut sinh enzym tt. Thng thng, hiu sut sinh enzym t c tt nht trn mi trng c kh nng cung cp cho s pht trin mt cch va phi ca vi sinh vt [10]. tng hm lng enzym trong mi trng nui cy cn la chn ngun carbon, nitrogen, mui khong thch hp, chn t l thch hp gia cc thnh phn ny v c bit l cn s dng cc cht cm ng. Thnh phn mi trng thng bao gm hn hp ca ngun carbon (tinh bt, glucose, r ng, xiro bp, cm m), cc ngun nit (protein thy phn,

gelatin, casein, cc mui amoni), cc cht kch thch sinh trng (cao nm, nc ng...), v mui khong [10]. i vi mt vi sinh vt nht nh, iu kin thch hp cho qu trnh sinh tng hp cc enzym khc nhau l khng ging nhau. Nm vng cc quy lut nh hng ca cc yu t ny c th iu khin vi sinh vt tng hp nh hng mt enzym xc nh [14]. - nh hng ca ngun Carbon Ngun carbon thng dng nui cy vi sinh vt l cc glucid, bao gm momo-di v c polysaccharide (tinh bt). Cc cht ny c nh hng khc nhau n s tng hp enzym ni chung cng nh tng hp protease vi sinh vt ni ring. c tnh tc dng ca ngun carbon ty thuc vo bn cht ha hc ca chng v c tnh sinh l ca vi sinh vt. Ngun carbon v nitrogen t nhin thng dng trong mi trng nui cy l bt m, bt u tng, cm v nc chit ca n. C khi mi trng ch cn nc chit ng li tng hp protease tt nht [14]. Trong s cc loi monosaccharide, glucose c nh hng rt khc nhau n qu trnh tng hp enzym ni chung cng nh qu trnh sinh tng hp protease ni ring [36], [39]. Trong nhiu trng hp n lm gim r rt lng enzym c tng hp [19]. Mt s tc gi nghin cu nh hng ca 11 loi mono v disaccharide khc nhau n kh nng sinh tng hp protease acid cc chng Aspergillus flavus 74, Aspergillus awamorii 200, Aspergillus oryzae 79 v cho thy ngun carbon tt nht i vi cc chng ny l fructose. Sau l maltose hoc saccharose (i vi Asp. oryzae 79). Kh nng tng hp protease ca c 3 chng trn u rt thp khi s dng lactose lm ngun carbon. Mt khc, nng glucid thch hp nht cho qu trnh sinh tng hp protease cng thay i ty loi vi sinh vt. V d nh i vi Aspergillus awamorii 200 v Aspergillus flavus 74, nng glucose trong mi trng t 5-6% l thch hp nht nhng i vi Aspergillus flavus 116 li l 3%.

Polysaccharide tinh bt cng c nh hng n kh nng sinh tng hp protease mt s loi nm mc. Tuy nhin, nh hng ca n cn ty thuc vo loi tinh bt v loi nm mc nui cy. Ngoi glucid c th s dng hydrocarbua lm ngun carbon duy nht trong mi trng dinh dng cho mt s vi sinh vt tng hp protease nh Pseudomonas acruginosa 3454, Pseudomonas aeruginos IFO 3455 [14]. - nh hng ca ngun Nitrogen Ngun nitrogen dng nui cy vi sinh vt c th l cc hp cht hu c v v c. Cc hp cht hu c c th l cao ng, bt u tng, kh lc, casein, pepton..., cn ngun v c thng l NH4NO3, NH4Cl... [10]. Ngun nitrogen hu c thng c nh hng tt n qu trnh tng hp protease v chng c vai tr nh cht cm ng ca qu trnh ny [36]. Cc ngun nitrogen hu c thng dng l cc loi tinh bt khc nhau, nc chit ca cm, nm men t phn, pepton, protein... Cc nguyn liu ny u c hm lng acid amin t do tng i thp [14]. i vi cc loi Aspergillus (Aspergillus oryzae, Aspergillus awamorii, Aspergillus niger, Aspergillus flavus), dng m hu c c bit l protein v pepton l thnh phn quan trng nht ca mi trng i vi qu trnh tng hp protease acid. Khi thay NaNO3 trong mi trng Czapek bng casein s lm tng ng k lng protease acid trong mi trng nui cy. nh hng tch cc cng xy ra khi s dng phi hp ngun nitrogen hu c v v c [14]. Cc amino acid c nh hng khc nhau n qu trnh sinh tng hp protease vi sinh vt. Trong , mt s amino acid c vai tr kch thch tng hp protease nhng mt s khc li c tc dng km hm. Theo cc nh nghin cu th tc dng km hm ca cc acid amin c th l do khi c s hin din ca cc loi amino acid trong mi trng m t bo c th s dng c th chng khng cn thit phi tng hp protease na [14]. i vi vi khun v x khun, s dng phi hp ngun nitrogen v c v hu c cng c nh hng r rt n kh nng to protease. Ngun nitrogen tt nht cho vi khun thng l (NH4)2HPO4, trong khi mui chloride, sulfate,

nitrate ammonium thng c tc dng khng tt n qu trnh tng hp protease. Ngun nitrogen hu c c s dng ph bin l pepton, casein, albumin, bt u tng... Trong pepton nng thp thng l cht cm ng tt cho qu trnh tng hp protease [9], [28], [36]. Ngoi vic chn ngun v lng carbon, nitrogen thch hp cn cn phi chn t l thch hp gia carbon : nitrogen. Nh vy mi bo m kh nng tng hp mt lng ln protease t cc loi vi sinh vt. Theo Iulius (1969) [14], t l carbon : nitrogen thch hp Aspergillus oryzae tng hp protease acid t 8 : 1 n 2 : 1. Nu nng carbon vt qu ngng ti thch vn khng c nh hng n qu trnh sinh tng hp protease. Tuy nhin nu tng lng nitrogen trong mi trng qu nng thch hp s lm km hm qu trnh tng hp protease [14]. - nh hng ca ngun phosphate v sulphate Ngoi nitrogen v carbon, photphate v sulphate c nh hng quan trng n qu trnh tng hp protease. Ni chung phosphate c nh hng xu n qu trnh tng hp protease acid, tuy nhin KH2PO4 thng c nh hng tt n qu trnh tng hp enzym thy phn [27], l nh tnh cht m ca n. Mt s nghin cu cho thy ngun sulphate dng SO42- ha tan nng thp (10-4M) c vai tr kch thch tng hp protease acid Murcor pusillus, nhng nng cao hn c tc dng km hm r rt qu trnh ny. Tuy nhin sulphate cng c nh hng khc nhau n qu trnh tng hp cc loi enzym ni chung, cng nh protease ni bo v ngoi bo ni ring. Do n c vai tr iu ha v la chn qu trnh sinh tng hp enzym, protein trong v ngoi t bo vi sinh vt [14]. Ngoi cc ngun carbon v nitrogen, cc nguyn t khong cng c nh hng r rt n qu trnh sinh tng hp enzym. Cc nguyn t ny bao gm cc nguyn t vi lng v a lng. Cc nguyn t vi lng nh st, ng, km, coban... thng c trong nc hoc trong thnh phn hu c khc ca mi trng [10].

2.1.4. ng dng ca protease Cng ngh enzym l mt trong bn b phn c s ca cng ngh sinh hc. S hnh thnh v pht trin cng ngh enzym nhiu nc thng bt u t cc enzym thy phn, c bit l cc enzym xc tc cho phn ng thy phn protein (protease) v cc enzym phn gii polysaccharide kiu tinh bt (amylase). Lng protease c s dng trong thc t chim t l kh ln, khong 50% ton b ch phm enzym c s dng [8]. Protease c ng dng rng ri trong sn xut cng nghip nh: sn xut cht ty ra, ch bin thc phm, ho hc, dc phm, cng nghip thuc da v cht chn on...[16], [18], [21], [24], [27], [28], [38]. 2.1.4.1. ng dng trong cng nghip thuc da Qu trnh ch bin da bao gm mt s cng on nh ngm t, ty lng, lm mm da v thuc da. Thng thng cc phng php thuc da thng dng cc ha cht c hi nh natri sulfide, lm nh hng rt nghim trng n mi trng khi nc thi ca nh my ny thi ra sng. Vic s dng enzym thay th cc ha cht rt thnh cng trong vic nng cao cht lng da v lm gim nhim mi trng. Protease c s dng lm mm da nh s thy phn mt phn protein ca da, ch yu l collagen, thnh phn chnh lm cho da b cng. Kt qu loi b khi da cc cht nht v lm cho da c mm do nht nh, tnh cht c hon thin hn sau khi thuc da. Trc y, lm mm da ngi ta dng protease c phn lp t c quan tiu ha ca ng vt. Hin nay, vic a cc protease tch t vi khun (B. mesentericus, B. subtilis), nm mc (A. oryzae, A. flavus) v x khun (S. fradiae, S. griseus, S. rimosus...) vo cng nghip thuc da em li nhiu kt qu v dn dn chim mt v tr quan trng [2]. 2.1.4.2. ng dng trong cng nghip dt: Protease vi sinh vt c s dng lm sch t tm, ty t nhn to (cc si nhn to c ty bng cc dung dch casein, gelatin) si c bng, d nhum. Protease c tc dng thy phn lp protein serisin lm dnh bt cc si t t nhin, lm bong v tch ri cc loi t tm, do lm gim lng ho cht ty trng.

2.1.4.3. ng dng trong cng nghip hng liu m phm Trong cng nghip sn xut x phng, kem m phm, thuc nh rng ngi ta cho enzym vo c tc dng lm da mn, lm sch rng, ty m hi, vt bn. Vo nhng nm 60, mt na mt s loi bt git chu u, chu M u c cha protease. Vic s dng protease trong bt git chim khong 25% lng enzym bn trn th gii. Protease l mt trong nhng thnh phn khng th thiu trong tt c cc loi cht ty ra, t cht ty ra dng trong gia nh n nhng cht lm sch knh hoc rng gi v kem nh rng. Vic ng dng enzym vo cc cht ty ra nhiu nht l trong bt git. Cc protease thch hp b sung vo cht ty ra thng c tnh c hiu c cht rng d dng loi b cc vt bn do thc n, mu v cc cht do c th con ngi tit ra. Mt tiu chun quan trng khc ca cc protease dng trong cht ty ra l hot ng tt trong iu kin nhit v pH cao cng nh phi thch hp vi cc tc nhn oxy ha v cc cht km hm c trong thnh phn ca cht ty ra. 2.1.4.4. ng dng trong y hc Ch phm protease thun khit cao c ng dng trong y hc nhm loi b cc b phn hoi t, loi tr cholesterin bm trn thnh mch mu cng nh h tr tiu ho. Ngoi ra, protease cn gip hnh thnh so vt thng cng nh iu tr cc bnh tim-mch, nht l bnh vim tc ng mch [3], [15]. 2.1.4.5. ng dng trong cng nghip thc phm Vic s dng cc protease trong cng nghip thc phm c t lu i. Chng c s dng mt cch bnh thng trong nhiu mc ch khc nhau nh lm mm tht, sn xut phomat, lm bnh nng, sn xut bia, rt ngn cc quy trnh sn xut nc chm, nc mm [12], [35], [36]. * Cng nghip tht S dng cc ch phm protease lm mm tht. Ngoi papain c th s dng cc ch phm protease ca vi sinh vt thy phn mt phn no cc protein trong thnh phn ca tht. Kt qu l cht lng ca cc loi tht tng

cao, c th chuyn tht loi thp thnh tht c phm cht cao. Khng nhng th, thi gian chn ca tht c th rt xung nhiu ln. * Sn xut bia Ch phm protease c ngha quan trng trong vic lm tng bn ca bia v rt ngn thi gian lc. Protease ca A. oryzae c dng thy phn protein trong ht ng cc, to iu kin x l bia tt hn [9]. Trong qu trnh sn xut bia, cc ch phm protease thng c b sung vo hai giai on [10]: - Trong qu trnh ng ha, ngi ta thng b sung protease nhm thy phn na vi cc protein phn t ln to nn cc protein trung gian. Sn phm thy phn na vi ny s c tc dng lm bn bt bia. Cc protease c s dng rng ri nht t trc n nay l ch phm ca vi khunBacillus amyloliquefaciens.

- Sau qu trnh ln men, khi cc protein khng b loi b ht do nhit hay do kt ta lnh, cc polypeptide c th lin kt vi cc hp cht polyphenol cc giai on cui ca qu trnh sn xut bia gy c bia. Cc ch phm enzym thy phn protein nh papain, bromelain, ficin thng c b sung trong qu trnh lc bia nhm kim sot c s to thnh cc kt ta lnh v ko di thi gian bo qun bia. * Sn xut cc sn phm t u nnh Hin nay, nhng ng dng cc dch thy phn ca u rt rng ri. Chng hn nh sn xut nc gii kht v trong vic to nn thc n king, b sung cht dinh dng v lm thc n tr em, b sung vo nc chm (x du) lm tng hng thm cho sn phm. u nnh l ngun thc phm dinh dng giu protein, tuy nhin trong thnh phn ca n li cha cc cht c ch nh contitrypsin, lectin lm gim kh nng tiu ho khi s dng. V vy, hin nay ngi ta nghin cu vic b sung enzym tiu ho subtilisin ca Bacillus subtilis vo x l u nnh tng hm lng cht ho tan, gim mc c ch trypsin.

* Trong cng nghip sn xut bnh nng: Protease lm gim thi gian trn, tng do v lm nhuyn bt, to xp v n tt hn. * Cng nghip sa ng dng ch yu ca protease trong cng nghip sa l lm ng t sn xut phomat. Protease t mt s vi sinh vt nh A. candidus, P. roquerti, B. mesentericus, c dng trong sn xut pho mt [5]. Protease khng nhng c kh nng thy phn protein m cn c kh nng lm ng t na. Tnh cht ny ca renin cng nh ca pepsin t lu c ngi ta s dng vo cng nghip thc phm. Protease ca nm mc v vi khun cng th hin kh nng . * Ch bin thu sn B sung ch phm protease trong sn xut bt c c tc dng d dng tch tht khi xng, da nhm rt ngn thi gian sn xut, tng hiu qu kinh t. Ngoi ra ch phm enzym cn c ng dng trong sn xut nc mm. Khi sn xut nc mm (v mt s loi mm) th thi gian ch bin thng l di nht, hiu sut thu phn ( m) li ph thuc rt nhiu a phng, phng php gi nn, nguyn liu c. Nn hin nay quy trnh sn xut nc mm ngn ngy c hon thin trong s dng ch phm enzym thc vt (bromelain v papain) v vi sinh vt rt ngn thi gian lm v ci thin hng v ca nc mm. Tuy nhin vn cn mt s tn ti cn phi hon thin thm v cng ngh. 2.2. Tnh hnh nghin cu enzym protease 2.2.1. Tnh hnh nghin cu enzym trong nc Hu nh mi phn ng ho hc trong c th sng u cn phi c vai tr xc tc ca enzym - cht xc tc sinh hc. Chnh v vy, cc nghin cu v enzym thu ht c s quan tm ca cc cn b ho sinh, sinh hc thc nghim v nhiu nh nghin cu cc lnh vc lin quan khc. Cc nghin cu nhm theo hng tch, tinh sch enzym, to cc ch phm c sch khc nhau, nghin cu cu trc, mi lin quan gia cu trc v hot tnh sinh hc

ca enzym, kh nng ng dng enzym trong thc t [7]. Gn y nht l nhng sng to mi m, mang tnh ng dng ln v c s dng rng ri nh: + Th Bch Thu, Trn Th X (2004), Nghin cu nh hng ca mt s yu t ln kh nng sinh Protease ca Bacillus Subtilis. Nghin cu ny kho st nh hng ca ngun carbon, nit, phi hp ng thi ngun carbon v nit, nh hng ca pH ban u v nhit nui cy ln kh nng sinh protease ca Bacillus subtilis c phn lp t v tm [11].+ Trn Quc Hin v cng s (2006), Nghin cu thu nhn ch phm

Protease t rut c Basa (pangasius bocourti). Nghin cu ny kho st qu trnh trch ly v tinh sch enzym protease t rut c Basa (Pangasius bocourti). Dch chit protease kim thu c t rut c tng hot tnh cao nht l 15,79UI/gCKNT (cht kh ni tng) trong iu kin chit: t l mu/dung mi 1/1(w/w); pH 9,5; nhit 350C; thi gian chit 10 pht. Dung mi isopropanol l tc nhn thch hp nht kt ta protease trong dch chit t rut c Basa. Vi t l th tch dch chit enzym v th tch isopropanol l 15/85, mc tinh sch ch phm protease kim l 1,65 ln v hiu sut thu hi enzym t c l 90,42% [45]. + Ti u mt s iu kin nui cy chng vi sinh vt bin Acinetobacter sp. QN6 sinh tng hp Protease c thc hin bi Quyn nh Thi v cng s (2007), nghin cu ny chng minh c cc iu kin thun li nhm nui cy vi hiu sut cao nht i vi chng vi sinh vt bin Acinetobacter sp. QN6 sinh tng hp protease [43]. + Phan Th Bch Trm v cng s (2007), tin hnh Tinh sch v kho st c im ca cc Serine Protease t Trn Qu. Bc u kho st h enzym protease t trn qu (Perionyx excavatus) cho thy phn ln cc protase trong dch chit enzym th c th tinh sch s b bng ta phn on vi amonium sulfat nng trong khong 30-80%. Nhit v pH ti u cho enzym th hot ng trn c cht casein l 550C v pH trong khong kim 1012 [44]. + V Ngc Bi nghin cu ng dng protease Bacillus subtilis trong sn xut bt m thy phn t c Mi. Qua nghin cu cho thy protease B. subtilis c th thy phn mnh m c tht c Mi v hon ton

c th s dng enzym ny trong sn xut bt m thy phn. Khi b sung protease B. subtilis vi nng enzym 0,3% vo hn hp c tht c mi v thy phn 500C [40]. 2.2.2. Vn sn xut enzym protease trn th gii Trong cc protease, cc enzym ca h tiu ha c nghin cu sm hn c. Nm 1857, Corvisart tch c trypsin t dch ty, l protease u tin nhn c dng ch phm. Nm 1861, Brucke cng tch c pepsin t dch d dy ch dng tng i tinh khit. Ngoi cc enzym ca h tiu ha, ngi ta cng quan st u tin v cc protease trong mu [14]. Cc protease thc vt c pht hin mun hn. Nm 1879, Wurtz c xem l ngi u tin tch c protease thc vt. n nay ngi ta nghin cu c kh y v cu trc phn t ca nhiu protease nh: papain, trypsin, chymotrypsin, subtilisin [14]. Cc protease ca vi sinh vt mi c ch nghin cu nhiu t nm 1950, mc d t nm 1918- 1919 Waksman pht hin c kh nng phn gii protein ca x khun. Trong hn 10 nm nay s cng trnh nghin cu protease vi sinh vt tng ln ng k nhiu hn cc protease ca ng v thc vt. Nhng kt qu t c trong lnh vc ny gp phn m rng quy m sn xut enzym v ng dng enzym trong thc t [14]. Trong cng nghip thc phm, ngi ta s dng protease sn xut phomat t sa (Reddy v cng s, 2007) [35] hay ch bin cc sn phm giu protein t u tng (Gupta v cng s, 2002) [23]; trong cng nghip thuc da, protease c dng thy phn mt s thnh phn phi collagen ca da v loi b cc protein phi fibrin nh albumin, globulin (Haddar v cng s, 2009) [24]; trong cht ty ra, protease l mt trong nhng thnh phn quan trng ca tt c cc loi cht ty ra, t cc cht ty ra dng trong gia nh n nhng cht lm sch knh hoc rng gi, kem nh rng (Doddapaneni v cng s, 2007) [18]. Trong nhng nm gn y, gi tr thng mi ca cc enzym cng nghip trn ton th gii t khong 1 t USD, trong ch yu l cc enzym thy phn (75%) [29], v protease l mt trong ba nhm enzym ln nht s dng trong cng nghip (60%) (Rao v cng s, 2007) [32].

T nm 1950 tr li y trn th gii c hng lot protease ng vt, thc vt v vi sinh vt c tch chit nghin cu. Thi gian gn y cc nh khoa hc trn th gii tp trung nghin cu v protease vi sinh vt v t nhiu thnh tu to ln v lnh vc ny. Protease t vi sinh vt chim ti 40% tng doanh thu ca enzym ton th gii [24]. Hin nay, s lng cc enzym c sn xut hng nm trn th gii rt ln. Nhng nc c cng ngh sn xut v ng dng protease tin tin trn th gii l: an Mch, Nht Bn, M, Anh, Php, H Lan, Trung Quc, c, o. Cc nc ny u t thch ng cho cng tc nghin cu, sn xut v ng dng protease t vi sinh vt. Chnh v th nhp sn xut protease quy m cng nghip ti cc nc pht trin hng nm tng vo khong 5- 10%. Ngy nay ngi ta c th sn xut cc enzym c nh trn cc cht mang khng tan cho php c th ti s dng enzym nhiu ln. V vy m vic ng dng protease ngy cng gia tng [5]. 2.3. Tng quan v Bacillus amyloliquefaciens Bacillus amyloliquefaciens thuc h Bacillaceae, ging hoc chi Bacillus, vi khun Gram dng tnh, catalase dng tnh, hiu kh, hnh que v di ng. Sinh vt ny c tm thy trong mu t t nhin, l vi khun hnh thnh bo t. Bacillus amyloliquefaciens c bit n vi kh nng lm suy thoi protein ngoi bo, c tm thy l c ch [41]. Nhng nghin cu trc y cho rng Bacillus amyloliquefaciens tit ra mt yu t bn nhit c hot tnh khng sinh li vi sinh vt Gram dng v c kh nng phn gii nhanh chng nguyn sinh cht ca Bacillus amyloliquefaciens. Yu t ny l mt chui peptit-lipid, cu trc ca n ng nht c tch ra t dch huyn ph ca Bacillus subtilis [21]. Bacillus amyloliquefaciens c nghin cu rng ri trong vic sn xut cc enzym cng nghip, nh -amylase, subtilisin (protease c dng lm cht ty git v cht ty ra ng knh), barnase v cht khng sinh [33]. Yerra Koteswara Rao v cng s (2006) nghin cu v chng minh c chng Bacillus amyloliquefaciens B128 l mt tc nhn kim sot sinh hc i vi Botrylis elliptica (mm bnh mu xm mc trn hoa hu) [33].

Bacillus amyloliquefaciens cn l mt trong nhng thnh phn ca sn phm dng x l mi trng nui tm v thc n b sung cho tm. Thnh phn ca cc sn phm ny l cc vi sinh vt m trong mi loi gi mt chc nng ring (Pseudomonas fluorescens, Bacillus subtilis, Bacillus amyloliquefaciens, Lactic bacteria, Rhodoturula mucilaginosa, Rhodosporidium toruloides) v tng cc sn phm l h vi sinh vt c ch, gip cho con tm pht trin kho mnh, chng li c mt s bnh v iu kin khc nghit [46]. c bit Bacillus amyloliquefaciens c ng dng rng ri trong cng nghip thuc da. George v cng s (1995) s dng protease kim ca Bacillus amyloliquefaciens cho vic lm rng lng v da. Vic x l da bng cch s dng enzym l tt nht v n mang li nhiu li th: d dng kim sot, nhanh chng v gim c cht thi. X l bng enzym s tiu hy cc sc t khng mong mun, lm tng din tch da v do thu c da sch [23].

PHN 3 I TNG V PHNG PHP NGHIN CU 3.1. i tng nghin cu Chng Bacillus amyloliquefaciens T9, c phn lp ti phng th nghim cng ngh vi sinh_vin ti nguyn mi trng, 27 Phan nh Phng, Hu v c nh danh bi cng ty Nam Khoa, 793/58 Trn Xun Son, P.Tn Hng, Q.7, TP.H Ch Minh (Ph lc 4). 3.2. Ni dung phng php nghin cu 3.2.1. Phng php vi sinh 3.2.1.1. Phng php cy tng sinh [3] Cho t bo vi khun t ng gc vo ng nghim cha 1 ml mi trng lng (1% pepton, 0,3% cao tht, 0,5% glucose, 0,5% NaCl). Tin hnh nui cy lc vi tc 200 vng/pht trong 24 gi 350C. Thu dch canh trng thc hin cho cc th nghim tip theo. 3.2.1.2. Phng php nui cy thu nhn enzym [5] Phn phi 0,5 ml dch cy tng sinh vo bnh tam gic dung tch 100 ml c cha 20 ml mi trng dinh dng (1% pepton, 0,3% cao tht, 0,5% glucose, 0,5% NaCl). Nui cy lc vi tc 200 vng/pht trong 24 gi 350C. Kt thc thi gian nui cy, canh trng cha enzym c ly tm tch sinh khi t bo 14.000 vng/pht, nhit 40C trong 10 pht. 3.2.2. Phng php ho sinh Xc nh hot protease bng phng php Anson ci tin [4] Nguyn tc: Phng php ny da vo s thy phn protein (casein) bi enzym. Sau bt hot enzym v kt ta protein cha b thy phn bng acid tricloacetic. nh lng sn phm c to thnh trong phn ng bng phn ng mu vi thuc th Folin, kt qu phn tch da vo ng chun tyrosin. n v hot protease (HP): l lng enzym m trong mt pht 300C c kh nng phn gii protein to thnh cc sn phm ha tan trong

tricloacetic acid, cho phn ng mu vi thuc th Folin tng ng vi 1 mol tyrosine. Tin hnh: Dng 6 eppendorf, 3 ng cho mu th nghim, 3 ng cho mu kim tra. i vi mu th nghim: + Cho vo 175 l dung dch enzym, tin hnh 350C, lc 500 vng/pht ( Thermoblock) trong 10 pht. + B sung 350 l casein 2% 350C, trn nh v 350C, lc 500 vng/pht, thi gian phn ng 10 pht. + Kt thc phn ng bng cch cho 875 l dung dch acid tricloacetic 5% (TCA) vo bt hot enzym v kt ta c cht khng c thu phn. + eppendorf trn nhit phng trong 20pht. + Ly tm 14.000 vng/pht nhit 40C trong 10 pht thu dch ni cha amino acid ho tan, loi kt ta casein. + Cho 500 l dch ni mi eppendorf sang ng nghim sch, b sung 2 ml Na2CO3 v lc nh. + Cho thm 500 l thuc th Folin 0,2N vo, gi nhit phng trong 20 pht. + Tin hnh o mt quang bc sng 750 nm. i vi mu kim tra: V hot enzym bng acid tricloacetic 5%, b sung casein 2%, tip tc thc hin cc bc nh trn. Tnh kt qu: Ly s liu c trn my tnh gi tr trung bnh OD ca cc mu th nghim v kim tra sau tnh: OD = ODTN - ODKT Tnh kt qu da vo phng trnh ng chun tyrosin (Ph lc 2) Cng thc tnh hot proteolitic: HP/ml = (mol tyrosin 8)/t

Trong : HP/ml: Hot chung 8: Th tch ton b hn hp phn ng (1 enzym: 2 casein: 5 TCA) t: Thi gian enzym tc dng vi c cht (t = 10 pht) 3.2.3. Phng php vt l Xc nh pH bng my o pH in t 3.2.4. Phng php x l s liu ANOVA [6] Cc kt qu th nghim thu c trong qu trnh nghin cu u c phn tch Anova v x l Ducan xc nh s sai khc gia cc trung bnh, c ngha vi tin cy p < 0,05. Ngun bin ng (Source of variation) Nghim thc (Treatment) Sai s (Error) Tng (Total) Bng 3.1 Bng phn tch phng sai Phng php b tr th nghim theo a mc yu t tc ng khc nhau n nhn t cn phn tch, mi mc c lp li 3 ln, N l tng s cc th nghim thc hin. SSA: Tng bnh phng nghim thc (biu th s sai khc do trung bnh cc nghim thc khc nhau). N-1 SST0 Bc t do (Degree of freedom) Tng bnh phng (Sum of square) SSA SSE Trung bnh bnh phng (Mean square) MSA MSE Ftnh Fbng ( = 0,05 hoc 0,01)

a-1 N-a

MSA/MSE Tra bng

SSE: Tng bnh phng sai s ngu nhin (biu th s sai khc trong cc mu ca nghim thc). SST0: Tng bnh phng ton b. MSA=SSA/a-1 MSE=SSE/N-a Nu Ftnh > Fbng th kt lun cc s trung bnh c khc nhau mc ngha .

PHN 4 KT QU V BIN LUN 4.1. Kho st nh hng ca thnh phn mi trng nui cy n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9. Thnh phn mi trng c nh hng rt ln n kh nng sinh tng hp protease. V vy, vic la chn thnh phn mi trng nui cy thch hp l rt quan trng [14]. Trong cc th nghim thm d u tin, chng ti s tin hnh b sung vo mi trng nui cy mi cht cn nghin cu l 0,5%. 4.1.1. Kho st nh hng ca ngun Carbon Tin hnh b sung cc ngun carbon khc nhau gm: glucose, saccharose, lactose, tinh bt vi hm lng l 0,5% vo mi trng c bn (MTCB) cha 1% pepton, 0,3% cao tht, 0,5% NaCl, ng thi kho st trn mu i chng. Sau khi nui cy 24 gi 350C, 200 vng/pht, tin hnh xc nh hot protease ca dch mi trng nui cy theo phng php Anson ci tin, kt qu c trnh by hnh 4.1. T kt qu hnh 4.1 cho thy, sau khi b sung cc ngun carbon nh tinh bt ho tan, glucose, saccharose, lactose vi nng 0,5% vo MTCB, ng thi kho st trn mu i chng, chng ti nhn thy tinh bt ho tan l ngun carbon kch thch Bacillus amyloliquefaciens sinh protease tt nht, hot t c 0,637 HP/ml ln hn mu i chng (0,452 HP/ml) l 1,41 ln. Kt qu ny ph hp vi Udandi Boominadhan v cng s (2009) [39] khi nghin cu trn i tng B.licheniformis cng cho rng tinh bt l ngun carbon tt nht kch thch kh nng sinh protease.

Hot (HP/ml)

0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 Glu 0,436 c

0,637 a 0,528b 0,452 0,259 d c

Sac

Lac

TB

C

Ngun Carbon n Hnh 4.1. nh hng ca ngun Carbon (0,5%) kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9

(C: mu i chng, MTCB; Glu: Glucose; Sac: Saccharose; Lac: Lactose; TB: tinh bt) i vi cc ngun carbon nh saccharose, glucose, lactose, kt qu nghin cu cho thy rng khi c mt cc cht ny trong mi trng nui cy, hot protease gim. iu ny c th c gii thch l do s c ch n kh nng sinh protease ca chng Bacillus amyloliquefaciens khi c mt cc ngun ny trong mi trng nui cy. i vi lactose, nhiu cng b cho kt lun khc nhau. Han-Seung Joo & Chung-Soon Chang (2004) [25] chng minh lactose km hm kh nng sinh protease ca Bacillus sp. I-312. Ngc li vi kt qu ny, Endang SETYORINI v cng s (2005) [19] cng b lactose l ngun carbon ti u cho kh nng sinh protease ca B.subtilis FP-133. 4.1.2. Kho st nh hng ca ngun Nitrogen Tin hnh b sung cc ngun nitrogen nh: casein, ur, cao nm, NH4Cl vi hm lng 0,5% vo MTCB. ng thi kho st trn mu i chng l MTCB. Sau khi nui cy 24 gi 350C, 200 vng/pht, tin hnh xc nh hot protease ca dch mi trng nui cy, kt qu c trnh by hnh 4.2.

Hot (HP/ml)

0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

0,651a 0,554 b 0,547 b

0,106 c 0,001d Cas Ur CN N C

Hnh 4.2. nh hng ca ngun Nitrogen n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9 (C: mu i chng; Cas: casein; CN: cao nm; N: NH4Cl) Kt qu hnh 4.2 cho thy, khi thm cao nm vo mi trng, hot protease t c cao nht so vi vic b sung cc ngun nitrogen khc. Hot t c (0,651 HP/ml) cao hn 1,19 ln so vi mu i chng (0,547 HP/ml). Trong trng hp ny c th gii thch cao nm c vai tr l cht cm ng cho qu trnh sinh tng hp protease ngoi bo. Kt qu ny ph hp vi Endang SETYORINI v cng s (2005) [19], trong cc ngun nitrogen c th nghim, cao nm c nh hng ln nht n hot ng ca protease trong iu kin nghin cu. Ngc li vi kt qu ny, khi kho st nh hng ca ngun nitrogen ln kh nng sinh protease ca chng Bacillus pumilus SG2, R. Sangeetha v cng s (2008) [36] cng b rng cao nm khng c vai tr kch thch. Mt s cng b khc ch ra rng ngun nitrogen tt nht sinh protease l casein (R. Sangeetha v cng s, 2008 [36]; Quanfu Wang v cng s, 2007 [31]). Ngun nitrogen hu c thng c nh hng tt n qu trnh tng hp protease v chng c vai tr nh cht cm ng ca qu trnh ny. Mc d ngun nitrogen v c khng hiu qu nh ngun hu c, R.

Ngun Nitrogen (0,5%)

Sangeetha v cng s (2008) [36] chng minh rng ur c tm thy l tt hn trong s ngun nitrogen v c kch thch kh nng sinh protease. 4.1.3. Phi hp ngun Carbon v Nitrogen Da vo kt qu kho st nh hng ring r ca ngun carbon v nitrogen ln hot protease ca dch mi trng nui cy, thy rng ngun carbon tt nht l tinh bt v ngun nitrogen tt nht l cao nm. Tin hnh kho st hot protease ca chng vi khun Bacillus amyloliquefaciens trn mi trng nui cy khi phi hp cc ngun carbon v nitrogen vi nhau. Trong , chng ti chn tinh bt lm ngun carbon chnh v phi hp vi cc ngun nitrogen khc. ng thi chn cao nm l ngun nitrogen chnh v phi hp vi cc ngun carbon khc. Cc mu th nghim c nui cy 24 gi 350C, 200 vng/pht, tin hnh xc nh hot protease ca dch mi trng nui cy v kt qu c trnh by hnh 4.3.0,7 0,6 0,5 0,4 0,3 0,2 0,1 0TB +C TB +U TB T +N B CN CN CN CN CN +G +S +L +T ac ac B lu

0,671 a 0,470 c 0,544 b

0,666 a 0,537 b0,556 b 0,465 c

Hot (HP/ml)

0,682 a 0,657 a

as

r

Ngun phi hp

Hnh 4.3. nh hng ca ngun phi hp n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9 (TB: Tinh bt; CN: Cao nm; Cas: Casein; N: NH4Cl; Glu: Glucose; Sac: Sacchaose; Lac: Lactose) T kt qu hnh 4.3 cho thy, ch c s kt hp ca tinh bt + casein (0,671 HP/ml), tinh bt + cao nm (0,682 HP/ml) l c tc dng lm tng hot

protease gp 1,01 v 1,02 ln so vi mu ch c ngun tinh bt (0,666 HP/ml). Nhng sau khi x l ANOVA th s sai khc ca 3 mc ny khng c ngha. S sai khc gia cc mu cao nm + cc ngun carbon khc nhau (glucose, saccharose, lactose) l c ngha nh nhau trong vic km hm hot protease. Nh vy, qua kt qu nghin cu kt hp b sung cc ngun dinh dng vo mi trng nui cy cho thy khng lm tng hot protease ca chng Bacillus amyloliquefaciens so vi khi kho st nh hng ring r ngun carbon v nitrogen. T kt qu nghin cu trn, chng ti i n kt lun rng, khi b sung tinh bt lm ngun cung cp carbon va c tc dng lm tng hot protease, va em li hiu qu kinh t v y l ngun dinh dng r tin, d s dng v rt phong ph. Kt qu ny ph hp vi L.V.A. Reddy v cng s (2007) [35] khi nghin cu trn i tng Bacillus sp. RKY3. 4.1.4. nh hng ca hm lng tinh bt xc nh hm lng tinh bt thch hp cho kh nng sinh tng hp protease ngoi bo cao ca chng Bacillus amyloliquefaciens, chng ti tin hnh kho st hm lng tinh bt trong khong t 0,25-1,75% theo cc mc: 0,25%, 0,5%, 0,75%, 1%, 1,25%, 1,5%, 1,75%. Kt qu kho st hot protease sau khi thu dch mi trng nui cy c th hin hnh 4.4.0,69 0,689 a

Hot (HP/ml)

0,68 0,67 0,66 0,65 0,64 0,63 0,62 0,61 0,6

0,672 b 0,648 c 0,647 c

e 0,632 e 0,634

0,639 d

0,25

0,5

0,75

1

1,25

1,5

1,75

Ngun tinh bt (%)

Hnh 4.4. nh hng ca hm lng tinh bt khc nhau n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9

Kt qu hnh 4.4 cho thy, hm lng tinh bt 0,75% th hot protease trong mi trng nui cy t gi tr cao nht (0,689 HP/ml) so vi cc hm lng tinh bt cn li. Nh vy, t cc kt qu nghin cu v nh hng ca thnh phn mi trng nui cy ln kh nng sinh tng hp protease ca chng Bacillus amyloliquefaciens chng ti nhn thy: trong mi trng c cha 1% pepton, 0,3% cao tht, 0,5% NaCl, 0,75% tinh bt l mi trng ti u, cho hot protease cao nht. Chng ti gi mi trng ny l mi trng ti u c bn (MTTCB). 4.2. Kho st nh hng ca pH ban u n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9 kho st nh hng ca pH ban u n kh nng sinh tng hp enzym protease ngoi bo cao, chng ti tin hnh nui cy Bacillus amyloliquefaciens trong MTTCB c iu chnh pH ban u, pH ban u ca mi trng nui cy c thay i t 5-11. Sau khi nui cy 24 gi 350C, 200 vng/pht trong cc iu kin pH ban u khc nhau, tin hnh xc nh hot protease ca dch mi trng nui cy, kt qu c trnh by hnh 4.5.Hot (HP/ml)

0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 2 3 4 0,104i0,107h

0,694 0,674b0,667c0,666c 0,617d 0,519e 0,418 f

a

0,179

g

5

6

7

8

9

10

11

Hnh 4.5. nh hng ca pH ban u n kh nng sinh tng hp pH protease ngoi bo ca chng Bacillus amyloliquefaciens T9

Kt qu hnh 4.5 cho thy, chng Bacillus amyloliquefaciens c kh nng sinh protease cao nht khi nui cy pH ban u l 5, hot t c trong iu kin nui cy l 0,694 HP/ml. Trong khong pH 6-9, hot protease trong mi trng kh n nh, gim khng ng k. Mt s cng b khc ch ra rng kh nng sinh tng hp protease cao nht khi pH=8 (Udandi Boominadhan v cng s, 2009 [39]; Laila Manni v cng s, 2008 [27]; R. Sangeetha v cng s, 2008 [36]; A. Ganesh Kumar v cng s, 2007 [26]). N. Bhaskar v cng s (2006) [17] nghin cu trn i tng Bacillus proteolyticus CFR3001 v kt lun rng hot protease tng mnh pH bng 9. Kiran Kumar Doddapaneni v cng s (2007) [18] khi nghin cu trn Bacillus cereus cho kt qu pH ti u l 10. Trong khi Han-Seung Joo, Chung-Soon Chang bo co rng chng Bacillus sp. I-312 sinh protease thch hp pH 11 [25]. 4.3. Kho st nh hng ca nhit n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9 Nhit nh hng n s pht trin, kh nng sinh tng hp enzym ca vi sinh vt cng nh tnh cht ca enzym c tng hp. Ty tng loi vi sinh vt khc nhau m c ngng nhit thch hp khc nhau. kho st nh hng ca nhit n kh nng sinh tng hp protease ngoi bo cao, chng ti tin hnh nui cy chng Bacillus amyloliquefaciens trong MTTCB, pH ban u bng 5, ng thi iu chnh nhit ban u t 30-60oC. Sau thi gian nui cy 24 gi c lc 200 vng/pht, tin hnh xc nh hot protease trong dch nui cy, kt qu c trnh by hnh 4.6. T kt qu hnh 4.6 cho thy, protease hot ng mnh trong khong nhit 30 450C v t c hot cc i 400C (0,694 HP/ml). Trong khi khi nui cy cc mc nhit 500C, 550C, 600C chng Bacillus amyloliquefaciens hu nh sinh protease thp. iu ny c gii thch l c th cc mc nhit t 500C tr ln enzym b bt hot. Ti 600C, hot gim mnh ch cn 0.050 HP/ml so vi hot cc i. Kt qu ny tri ngc vi Kiran Kumar Doddapaneni v cng s (2007) [18] khi nghin cu trn chng Bacillus cereus, cng nh kt qu nghin cu ca Mohammad Ali Amoozegar v cng s (2006) [16] trn i tng Salinivibrio sp.AF-2004 u kt lun rng 600C l nhit ti u cho kh nng sinh protease.

Hot (HP/ml)

0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

a 0,674c 0,678b 0,694 0,674c

d 0,104

e 0,069 0,050f55 60

30

35

40

45

50

Nhit (0C)

Hnh 4.6. nh hng ca nhit n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9 Nghin cu nh hng ca nhit n kh nng sinh protease, c nhiu kt lun khc nhau. Theo Quan-Fu Wang v cng s (2007) [30], nhit ti u cho kh nng sinh protease trn chng Pseudoalteromonas sp. NJ276 l 300C. N. Bhaskar v cng s (2006) [17] th li cho rng 370C l nhit ti u lm tng hot protease trn i tng Bacillus proteolyticus CFR3001. Trong khi protease ca chng Bacillus sp. I-312 n nh nhit 550C (Han-Seung Joo, Chung-Soon Chang, 2004 [25]). 4.4. Kho st nh hng ca thi gian n kh nng sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9 Nui cy chng vi khun trong MTTCB nhit 40 0C, pH ban u bng 5, lc 200 vng/pht. Tin hnh ly mu 8 gi mt ln xc nh hot enzym trong mi trng nui cy bng phng php Anson ci tin (Hnh 4.7). Qua kt qu hnh 4.7 cho thy rng, hot protease tng sau 8 gi nui cy v t cc i 16 gi (0,699 HP/ml) v tng i n nh cho n 40 gi nui cy. Sau 40 gi hot gim nh, c th do nhiu nguyn nhn nh c ch km hm ngc bi sn phm thy phn protein ca mi trng hoc phn t protease b thy phn bi chnh phn t protease khc (hin

tng autolize) hoc do vi sinh vt tng hp nn cc cht km hm trong mi trng, do s cn kit v mi trng dinh dng cng nh s tch lu cc sn phm c ca qu trnh trao i cht.Hot (HP/ml)

0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 0 8

0,699a 0,657b 0,323i

0,630ce 0,531

0,613d 0,491 f

0,465g h 0,440

0,008j 16 24 32 40 48 56 64 72

Thi gian nui cy nng Hnh 4.7. nh hng ca thi gian n kh (gi) sinh tng hp protease ngoi bo ca chng Bacillus amyloliquefaciens T9

C nhiu kt lun khc nhau v nh hng ca thi gian n kh nng sinh protease ngoi bo. Shikha v cng s (2006) [37] khi nghin cu trn chng Bacillus pantotheneticus, cng nh kt qu nghin cu ca Fikret Uyar v cng s (2003) [20] trn i tng Bacillus sp, u kt lun rng 24 gi lm tng hot protease mnh nht. R. Sangeetha v cng s (2008) [36] th li kt lun rng hot protease t cc i ti 36 gi. Da vo kt qu kho st trn, chng ti tin hnh la chn thi gian nui cy thch hp chng Bacillus amyloliquefaciens sinh protease cao nht l 16 gi.

PHN 5 KT LUN V KIN NGH 5.1. Kt lun: Kt qu nghin cu cho php chng ti kt lun v iu kin nui cy chng Bacillus amyloliquefaciens T9 sinh tng hp protease ngoi bo cao l: - Thnh phn dinh dng ca mi trng nui cy bao gm: Pepton 1% Cao tht 0,3% NaCl 0,5% Tinh bt ho tan 0,75% Trn c s , chng ti gi mi trng ny l mi trng ti u c bn (MTTCB). - pH ban u bng 5 - Nhit nui cy l 400C - Khi nui cy trong iu kin trn, chng ta c th thu nhn enzym thi im sau 16 gi nui cy. 5.2. Kin ngh: ti c tin hnh trong mt thi gian ngn nn ch mi nghin cu bc u. V vy chng ti kin ngh tip tc nghin cu: - Xc nh tnh cht ca enzym thu c - Thit lp quy trnh thu nhn ch phm enzym v nghin cu ng dng ch phm enzym ny trong cng nghip thc phm. - Nghin cu thay th thnh phn dinh dng trong MTTCB bng cc mi trng t nhin, r tin sn xut enzym quy m ln c hiu qu kinh t cao.

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