B CNG THNG TRNG I HC CNG NGHIP THC PHM TP.

HCM KHOA CNG NGH THC PHM B MN: HA SINH HC THC PHM

TI:

TM HIU V NG DNG CA NHM ENZYME CELLULASE

GVHD: NGUYN TH THU SANG SVTH: NGUYN HONG PHC HUNH TN T PHM QUC HUY NGUYN TN PHC V MINH TR 2005100031 2005100054 2005100171 2005100040 2008100088

NHM 02_LP 01DHTP1_SNG TH 2_TIT 1,2

TP.H CH MINH - 2012

B CNG THNG TRNG I HC CNG NGHIP THC PHM TP.HCM KHOA CNG NGH THC PHM B MN: HA SINH HC THC PHM

TI:

TM HIU V NG DNG CA NHM ENZYME CELLULASE

GVHD: NGUYN TH THU SANG SVTH: NGUYN HONG PHC HUNH TN T PHM QUC HUY NGUYN TN PHC V MINH TR 2005100031 2005100054 2005100171 2005100040 2008100088

NHM 02_LP 01DHTP1_SNG TH 2_TIT 1,2

TP.H CH MINH - 2012

MC LC LI NI U DANH SCH CC HNH..................................................................................... 1 M U ................................................................................................................. 2 1. Yu cu thc t v l do chn ti .................................................................... 2 2. Mc ch nghin cu ............................................................................................ 2 3. i tng v phng php nghin cu ................................................................ 3 4. Kt cu ca bi tiu lun ...................................................................................... 3 CHNG 1. TNG QUAN V ENZYME CELLULASE ............................... 4 1.1. Gii thiu chung ......................................................................................... 4 1.2. Cu trc ca enzyme cellulase ................................................................... 4 1.3. Tnh cht ca enzyme cellulase .................................................................. 5 1.4. Ngun gc ca enzyme cellulase ............................................................... 6 1.5. C cht ca enzyme cellulase ..................................................................... 6 1.6. Phn loi enzyme cellulase ......................................................................... 6 1.7. C ch tc dng ca enzyme cellulase ....................................................... 7 1.7.1. C ch 1,4- -D-glucan cellobiohydrolase (EC 3.2.1.91) ............... 7 1.7.2. C ch 1,4- -D-glucan 4-glucanohydrolase (EC 3.2.1.4) .............. 8 1.7.3. C ch -D-glucoside glucohydrolase (EC 3.2.1.21) ..................... 9 1.8. Hot lc ca enzyme cellulase ................................................................... 10 1.9. Cc yu t nh hng n hot lc ca enzyme cellulase ......................... 11 1.9.1. nh hng ca nhit .................................................................... 11 1.9.2. nh hng ca pH ............................................................................ 11

1.9.3. nh hng ca ion kim loi ............................................................. 12 1.9.4. nh hng ca nng enzyme ...................................................... 12 1.9.5. nh hng ca nng c cht [S] ................................................. 13 CHNG 2. CC NG DNG CA NHM ENZYME CELLULASE ....... 17 2.1. S lc v ng dng ca nhm enzyme cellulase ..................................... 17 2.2. Cc ng dng ca nhm enzyme cellulase ................................................. 18 2.2.1. Trong cng nghip giy .................................................................... 18 2.2.2. Trong cng nghip dt ...................................................................... 19 2.2.3. Trong x l mi trng..................................................................... 20 2.2.4. Trong sn xut thc n gia sc.......................................................... 20 2.2.5. Trong k thut di truyn ................................................................... 21 2.2.6. Trong cng nghip ch bin thc phm ............................................ 21 2.2.6.1. Trong nghip sn xut bia ....................................................... 21 2.2.6.2. Trong cng nghip sn xut c ph ......................................... 21 2.2.6.3. Trong sn xut cc loi nc qu ............................................ 22 2.2.7. Trong sn xut cn ............................................................................ 23 2.2.8. Trong sn xut bt git v cht ty ra ............................................. 24 2.2.9. Trong sn xut nhin liu sinh hc ................................................... 25 2.2.10. Trong sn xut dung mi hu c .................................................... 25 2.2.11. Trong nui cy t bo v ti t hp gene ........................................ 25 2.2.12. Trong cng nghip x l rc thi v sn xut phn bn vi sinh ..... 25 2.2.13. Trong sn xut agar-agar ................................................................ 26 TI LIU THAM KHO ..................................................................................... 27

LI NI U S pht trin nhanh chng ca cng ngh thc phm trong hai thp nin gn y i hi s hiu bit ton din v kh nng phn tch cc cht trong thc phm v phng php sn xut thc phm. m bo yu cu ny, cn c kin thc ton din v ha sinh thc phm, vi sinh thc phm, cc quy trnh ch bin thc phm,... Trong bi tiu lun ny, nhm chng ti tp trung nghin cu v cc ng dng ca nhm enzyme cellulase trong tt c cc lnh vc ni chung v nhm ngnh cng ngh thc phm ni ring. Enzyme cellulase l mt enzyme rt ph bin trong cng nghip, ch ng sau amylase v protease nn cc ng dng ca chng i vi cng nghip l rt nhiu. Qua bi tiu lun ny, mc tiu m nhm chng ti hng ti l xy dng mt cch tng i hon chnh v cc ng dng ca nhm enzyme cellulase trong cng nghip, qua c th cung cp cc kin thc hu ch v y hn cho mi ngi v nhm enzyme ny. Bi tiu lun c chia lm hai chng: Chng 1: Tng quan v enyme cellulase Chng 2: Cc ng dng ca nhm enzyme cellulase D c gng rt nhiu nhng trong qu trnh thc hin cng khng th trnh khi nhng sai st, rt mong nhn c nhng kin ng gp ca C nhng bi tiu lun v sau s y v chnh xc hn. TP TH NHM

DANH SCH CC HNH Hnh 1.1. Enzyme cellulase ..................................................................................... 5 Hnh 1.2. C ch hot ng ca exoglucanase ....................................................... 8 Hnh 1.3. C ch hot ng ca endoglucanase ..................................................... 8 Hnh 1.4. C ch hot ng ca -glucosidase .................................................... 9 Hnh 1.5. Qu trnh phn gii cellulose ca cellulase ............................................ 9 Hnh 1.6. S ph thuc ca vn tc phn ng vo [E] ........................................... 13 Hnh 1.7. Bin thin vn tc phn ng theo nng c cht ................................. 15 Hnh 1.8. S ph thuc ca tc phn ng vo nng c cht ........................ 16 Hnh 2.1. Si giy c ti ch ............................................................................... 19 Hnh 2.2. C ch thy phn phn t cellulose(A) v phc h cellulose(B) ca cc Enzyme thuc h phc cellulase .............................................................................. 23

Ha sinh thc phm

M u

M U 1. Yu cu thc t v l do chn ti Enzyme l loi protein, xc tc cho mi phn ng sinh hc trong mi t bo ca sinh vt. Enzyme rt quan trng vi c th con ngi, nu khng c enzyme th mi s chuyn ha trong c th con ngi b nh tr v con ngi khng th sng c. Ngoi ra n cn ng vai tr rt quan trng trong cng nghip ch bin thc phm, trong y hc, trong k thut phn tch, cng ngh gene v bo v mi trng. Nghin cu, sn xut v ng dng enzyme c pht trin rt mnh t u th k XX n nay. Cng ngh sn xut enzyme em li li nhun rt ln cho nhiu nc. Vit Nam l mt trong nhng nc c nhiu nghin cu v ng dng enzyme. Tuy nhin, cng ngh enzyme ca ta cha thc s pht trin do y l mt ngnh i hi cng ngh cao. Trong tng lai, ha hn y s l mt ngnh cng nghip c u t pht trin rng bi nhng ng dng v tnh tin li ca n trong nhiu lnh vc v i sng. Enzyme cellulase l mt trong nhng enzyme c vai tr quan trng trong chuyn ha cht hu c c trong thin nhin, v c ngha rt ln trong cng nghip thc phm (ru, bia,...), bo v mi trng... Do c nhiu ng dng trong thc t song vic ng dng chng vo thc tin khng phi l iu d dng nn l l do v sao vn Tm hiu v ng dng ca nhm enzyme cellulase c chng ti chn l ti tiu lun. 2. Mc ch nghin cu Mc tiu ca ti l kho st cc c tnh c bn ca enzyme cellulase v hng ng dng rng ri ca enzyme ny trong cng nghip ni chung v cng nghip thc phm ni ring. Hng i chnh ca ti m chng ti hng n l kh nng ng dng thc tin cao ca loi enzyme ny. 2

Ha sinh thc phm

M u

3. i tng v phng php nghin cu i tng nghin cu ca ti l enzyme cellulase. Phng php nghin cu trong bi tiu lun ny c dng ch yu l phng php nghin cu l thuyt, phn tch, tng hp nhm lm r vn c nghin cu v gip ngi c c ci nhn tng qut hn v loi enzyme ny. 4. Kt cu ca bi tiu lun Tiu lun vi ti: TM HIU V NG DNG CA NHM ENZYME CELLULASE c thc hin vi hai chng chnh: Chng 1: Tng quan v enzyme cellulase Chng 2: Cc ng dng ca nhm enzyme cellulase

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

CHNG 1. TNG QUAN V ENZYME CELLULASE 1.1. Gii thiu chung Cellulose l thnh phn c bn ca t bo thc vt, v vy n c mt trong mi loi rau qu cng nh trong cc nguyn liu, ph liu ca cc ngnh trng trt v lm nghip. Tuy nhin, ngi v ng vt khng c kh nng phn gii cellulose. N ch c gi tr lm tng tiu ha, nhng vi lng ln n tr nn v ch hay cn tr tiu ha. Cellulase l mt phc h enzyme c tc dng thy phn cellulose thng qua vic thy phn lin kt -1,4-glucoside trong cellulose. Cellulase c kh nng thy phn cellulose thnh ng. Con ngi v ng vt khng c kh nng phn gii cellulose, n c vai tr cht n. Phng php tm ch phm cellulase cn hn ch. Ch phm cellulase thng dng : Tng cht lng thc phm v thc n gia sc. Tng hiu sut trch ly cc cht t nguyn liu thc vt.

Enzyme cellulase c nghin cu t rt lu trn th gii. y l enzyme c ng dng rt rng ri, ch ng sau protease v amylase. 1.2. Cu trc ca enzyme cellulase Cellulase c bn cht l protein c cu to t cc n v l axit amin, cc axit amin c ni vi nhau bi lien kt peptid CO-NH- . Ngoi ra, trong cu trc cn c nhng phn ph khc, Cu trc hon chnh ca cc loi enzyme nhm endoglucanase (EG) v exoglucanase (CBH) ging nhau trong h cellulase ca nm si, gm mt trung tm xc tc v mt ui tn cng, phn ui ny xut pht t trung tm xc tc v c gn them vng glycosil ha, cui ui l vng gn kt vi cellulose (CBD: cellulose binding domain). Vng ny c vai tr to lin kt vi cellulose tinh th. Trong qu trnh phn hy cellulose c s tng quan mnh gia kh nng xc tc phn 4

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

gii cellulose ca cc enzyme v i lc ca enzyme ny i vi cellulose. Hn na, hot tnh ca cellulase da vo tinh th cellulose v kh nng kt hp ca CBD vi cellulose. iu ny chng t CBD lm gia tng hot tnh cellulase i vi tinh th cellulose. S c mt ca CBD s h tr cho enzyme cellulase thc hin vic ct t nhiu lin kt trong cellulose tinh th. Vng gn kt vi cellulose c cu to khc vi lin kt thng thng ca protein v vic thay i chiu di ca vng glycosil ha c nh hng n hot tnh xc tc ca enzyme.

Hnh 1.1. Enzyme cellulase 1.3. Tnh cht ca enzyme cellulase Cellulase thy phn cellulose t nhin v cc dn xut nh carboxymethyl cellulose (CMC) hoc hydroxyethyl cellulose (HEC). Cellulase ct lin kt -1,4glucosid trong cellulose, lichenin v cc -D-glucan ca ng cc. bn nhit v tnh c hiu c cht c th khc nhau. Cellulase hot ng pH t 3-7, nhng pH ti thch trong khong 4-5. Nhit ti u t 40-50 C. Hot tnh cellulose b ph hy hon ton 80 C trong 10 n 15 pht.

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

Cellulase b c ch bi cc sn phm phn ng ca n nh glucose, cellobiose v b c ch hon ton bi Hg. Ngoi ra, cellulose cn b c ch bi cc ion kim loi khc nh Mn, Ag, Zn nhng mc nh. Trng lng ca enzyme cellulose thay i t 30 -110 KDa (Begunin, 1990; Gilkes v cng s, 1991). 1.4. Ngun gc ca enzyme cellulase c thu nhn t cc ngun khc nhau: ng vt: dch tit d dy b, cc nhm thn mm Thc vt: trong ht ng cc ny mm nh i mch, yn mch, la m mch en Vi sinh vt: cc loi x khun, vi khun, nm si, nm men Trong thc t ngi ta thng thu nhn enzyme cellulase t vi sinh vt. Cc chng vi sinh vt thng s dng: Nm mc: Aspergillus niger, Aspergillus oryzae, Aspergillus candidus X khun: Actinomyces griseus, Streptomyces reticuli Vi khun: Acetobacter xylinum, Bacillus subtilis, Bacillus pumilis 1.5. C cht ca enzyme cellulase Cellulose l c cht ca enzyme cellulase. Cellulose l mt polysaccharide phong ph nht trong t nhin. Vic phn hy sinh hc cellulose bi vi sinh vt l mt trong nhng bc chnh ca chu trnh carbon trn tri t. 1.6. Phn loi enzyme cellulase

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

V phng din ha hc, cellulose l mt polyme c cu to t cc n v

-glucose ni vi nhau bng lin kt -1,4-glucoside. Cellulose l thnh phn

polysaccharide ch yu ca vch t bo thc vt. Da vo c im ca c cht v c ch phn ct, enzyme cellulase c chia thnh ba loi: 1,4- -D-glucan cellobiohydrolase (EC 3.2.1.91) 1,4- -D-glucan 4-glucanohydrolase (EC 3.2.1.4)

-D-glucoside glucohydrolase (EC 3.2.1.21)

Cc enzyme ny c tm thy trong vi khun sng trong d dy c b v mi v trong mt s nm nh Trichoderma, Aspergillus,... 1.7. C ch tc dng ca enzyme cellulase 1.7.1. C ch 1,4- -D-glucan cellobiohydrolase (EC 3.2.1.91) Enzyme ny cn c tn gi khc nh: exoglucanase, exo- -1,4-glucanase, cellobiohydrolase, exo cellobiohydrolase, exo- -1,4-glucan cellobiohydrolase, 1,4-

-D-glucan cellobiosidase, cellobiosidase, CBH 1, C1 cellulase, avicelase.

Enzyme ny thy phn lin kt 1,4- -D-glucoside t u khng kh ca chui cellulose to thnh celllobiose.

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

Hnh 1.2. C ch hot ng ca exoglucanase 1.7.2. C ch 1,4- -D-glucan 4-glucanohydrolase (EC 3.2.1.4) Enzyme ny cn c tn gi khc nh: Endoglucanase, Endo-1,4- -Dglucanase, Endo-1,4 glucanase, -1,4-endoglucan hydrolase, Carboxymethyl cellulase, Celludextrinase, Cellulase A, Cellulosin AP, Alkali Cellulase, Cellulase A3, 9,5 Cellulase, Avicelase, Pancellase SS. Enzyme ny thy phn ngu nhin lin kt 1,4- -D-glucoside gia mch ca chui cellulose, lichenin v cc -D-glucan ca ng cc.

Hnh 1.3. C ch hot ng ca endoglucanase 8

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

1.7.3. C ch -D-glucoside glucohydrolase (EC 3.2.1.21) Enzyme ny c tn gi khc nh l: glucosidase, D glucosidase,

1,6 glucosidase, glucoside glucohydrolase, p-nitrophenyl glucosidase,

aryl- glucosidase, gentiobiase, cellobiase, emulsin, elaterase, arbutinase,

amygdalinase, primeverosidase, amygdalase, limarase, salicilinase. Enzyme ny thy phn gc -D-glucoside khng kh u tn cng phng thch ra -D-glucose.

Hnh 1.4. C ch hot ng ca -glucosidase

Hnh 1.5. Qu trnh phn gii cellulose ca cellulase

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

1.8. Hot lc ca enzyme cellulase (c ch thy phn cellulose) Thy phn cellulose phi c s tham gia ca c ba loi enzyme cellulose nh endoglucanase, exoglucanase v -glucosidase. Thiu mt trong ba loi enzyme trn th khng th thy phn phn t cellulose n cng. T nhng nghin cu ring l i vi tng loi enzyme n nghin cu tc ng tng hp ca c ba loi enzyme cellulose, nhiu nh khoa hc u a ra kt lun chung l cc loi enzyme cellulose s thay phin nhau phn hy cellulose to thnh sn phm cui cng l glucose. C nhiu cch gii thch khc nhau v c ch tc ng ca cellulose, trong cch gii thch do Erikson a ra c nhiu ngi cng nhn hn c. Theo Erikson v cng tc vin (1980), c ch tc ng hip ng ca 3 loi cellulose nh sau: u tin endoglucanase tc ng vo vng v nh hnh trn b mt cellulose, ct lin kt -1,4-glucosid v to ra cc u mch t do. Tip exoglucanase tn cng ct ra tng on cellobiose t u mch c to thnh. Kt qu tc ng ca endoglucanase v exoglucanase to ra cc celloligosaccharit mch ngn, cellobiose, glucose. -glucosidase thy phn tip v to thnh glucose. Cc loi vi sinh vt c kh nng sinh tng hp cellulose trong iu kin t nhin thng b nh hng bi tc ng nhiu mt ca cc yu t ngoi cnh nn c loi pht trin rt mnh, c loi li pht trin yu. Chnh v th, vic phn hy cellulose trong t nhin c tin hnh khng ng b, xy ra rt chm. Trong iu kin phng th nghim hay iu kin cng nghip, vic phn hy cellulose bng enzyme, ngoi cc yu t k thut nh nhit m pH, nng c cht, lng enzyme, mt yu t ht sc quan trng l tnh ng b ca h enzyme cellulose t nhiu ngun vi sinh vt khc nhau. Qu trnh thy phn cellulose ch c th c tin hnh n sn phm cui cng khi s dng ng b ba loi enzyme cellulose.

10

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

Mi loi vi sinh vt ch c kh nng sinh tng hp u vit mt loi enzyme. Chnh v th cn phi khai thc enzyme cellulose t nhiu ngun vi sinh vt. 1.9. Cc yu t nh hng n hot lc ca enzyme cellulase 1.9.1. nh hng ca nhit Nhit nh hng n s pht trin, kh nng sinh tng hp enzyme ca vi sinh vt cng nh tnh cht ca enzyme c tng hp. Sinh trng v sinh tng hp enzyme thng b km hm nhanh chng nhit cao hn nhit thch hp. Vn tc phn ng do enzyme xc tc ch tng ln khi tng nhit trong mt gii hn nht nh, cha nh hng n cu trc ca enzyme. Hot tnh enzyme t cc i nhit thch hp, khong nhit thch hp ca nhiu enzyme vo khong 40 500C. nhit cao, enzyme b bin tnh lm hot tnh gim mnh hoc mt hot tnh, cn nhit thp di 00C, hot tnh enzyme b gim nhiu nhng li c th phc hi khi a v nhit thch hp. Cellulase t A. niger NRRL 363 hot ng mnh nht 500C, trong khi cellulase t A. niger Z10 l 400C, cn hot tnh xc tc ca endoglucanase III t Trichoderma reesei t ti a 550C. Theo kt qu nghin cu ca Kiamoto v cs (1996), cc endoglucanase t chng A. oryzae KBN616 hot ng tt nht torng di nhit 45 550C. 1.9.2. nh hng ca pH Kh nng hot ng ca enzyme cn ph thuc vo pH mi trng phn ng. Ty thuc vo bn cht ca enzyme m pH thch hp enzyme hot ng c th trung tnh, kim hoc acid. Theo nghin cu trc y cho thy, pH ti thch cho hot ng ca cellulase t A. niger NRRL 363 l 5,5; ca cellulase t A. niger Z10 l 4,5 v 7,5; ca endoglucanase III t Trichoderma reesei l 4,0 5,0; ca endoglucanase t A. oryzae KBN616 l 4,0 5,0. Khi nghin cu nh hng ca pH ln hot tnh 11

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

endoglucanase t chng nm a acid A. terreus M11, Gao v cng s (2008) cho rng enzyme ny c kh nng hot ng trong di pH 2 5, trong pH 2 l tt nht. 1.9.3. nh hng ca ion kim loi Cc ion kim loi c th km hm hoc hot ha s hot ng ca cc enzyme. Cc ion kim loi nng nng nht nh c th gy bin tnh v km hm khng thun nghch enzyme. Sharma v cs (1995) nhn thy, ion Ca2+ lm tng hot tnh ca enzyme cellulase ca Baccillus sp. D04 ln 40% so vi i chng, cn Mg2+ lm gim nh (hot tnh cn li 92%) v Zn2+ c ch mnh hot tnh ca enzyme ny (hot tnh cn li bng 37% so vi i chng). Theo nghin cu ca Gao v cng s (2008), endoglucanase t A. terreus M11 b gim 77% hot tnh khi vi Hg2+ (2mM), 59% khi vi Cu2+ (2mM). Ngoi ra, cc dung mi hu c, cc cht ty ra cng nh hng mnh m n hot tnh ca enzyme. Ty thuc vo bn cht ca cc cht trn cng nh bn cht ca enzyme m tnh cht v mc nh hng ti hot ng ca enzyme l khc nhau. Nghin cu ca Trnh nh Kh (2006) trn chng Penicillium sp. DTQ HK1 cho thy, cc dung mi hu c methnol, ethanol, isopropanol v aceton u c ch hot ng ca cellulase c bit l n butanol c ch mnh nht, hot tnh cellulase ch cn 33 63%. Cc cht ty ra tween 20, tween 80, SDS v triton X 100 u lm gim hot tnh cellulase mc khc nhau, trong SDS lm gim mnh hot tnh cellulase ch cn 18 34%. 1.9.4. nh hng ca nng enzyme Trong iu kin d tha c cht, ngha l [S] >>[E] th tc phn ng ph thuc vo [E], v= K[E] c dng y=ax. Nh ngi ta o [E] bng cch o vn tc phn ng do enzyme xc tc. C nhiu trng hp trong mi trng c cha cht km hm hay hot ha th vn tc phn ng do enzyme xc tc khng ph thuc tuyn tnh vi [E] . 12

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

Hnh 1.6. S ph thuc ca vn tc phn ng vo [E] 1.9.5. nh hng ca nng c cht [S] Ta kho st trng hp n gin nht: ch mt c cht

Gi v1 l vn tc ca phn ng to thnh phc cht ES. Gi v-1 l vn tc ca phn ng to phn ly phc cht ES to thnh E v S. Gi v2 l vn tc ca phn ng to thnh E v P (sn phm).
v1 k1[ E ][ S ] v1 k1[ ES ] v2 k2 [ ES ]

Khi h thng t trng thi cn bng ta c:

k1[ ES ] k2 [ ES ] k1[ E ][S ]

(k-1+k2)[ES] = k+1[E][S]

(2) 13

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

Gi E0 l nng ban u: [E0]=[E]+[ES]=>[E]=[E0]-[ES] Thay tr s [E] t (3) vo (2) ta c: (k-1+k2)[ES] = k1([E0]-[ES]) [S]
[ ES ] k1[ E0 ][ S ] k1 k2 k1[ S ]

(3)

Nu t K m

k1 k2 k`

(Km: gi l hng s Michalis Menten)

Ta c: [ ES ]

[ E0 ][ S ] km [ S ]

Mt khc vn tc phn ng to thnh sn phm P l:

V k2 [ ES ]

Thay [ES] bng gi tr trn ta thu c:

v k2 [ E0 ][ S ] Km [S ]

(4)

Qua y ta thy nng enzyme cng cao th vn tc phn ng enzyme cng ln. Vn tc t cc i khi ton b enzyme lin kt vi c cht, ngha l:

Vmax= k2[E0]
Thay vo phng trnh (4) ta c:
v Vmax [S ] Km [S ]

(5)

Phng trnh (5) gi l phng trnh Michelis Menten Km gi l hng s Michelis Menten c trng cho mi enzyme. Km c trng cho i lc ca enzyme vi c cht, Km c tr s cng nh th i lc ca enzyme vi c cht cng ln, ngha l vn tc ca phn ng do enzyme xc tc cng ln. 14

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

ngha thc tin ca hng s Michelis l ch n chnh l gi tr ca nng c cht khi tc phn ng bng tc ti a. Thay V v v bng cc con s tng ng 1 v 0,5 vo phng trnh trn, ta s thy r iu . Nh vy, K m c o bng n v nng , tc l mol/l. Hng s Michaelis l mt hng s rt quan trng. N xc nh i lc ca enzyme vi c cht. Km cng nh th i lc ny cng ln, tc phn ng cng cao v tc ti a V t gi tr nng c cht cng thp. Trn c s phng trnh Michaelis-Menten, bng cch xy dng ng biu din s ph thuc ca v vo [S] v bng th xc nh tc ti a V ta c th tm thy gi tr ca [S], v = V/2, tc gi tr ca Km (hnh 2.7).

Hnh 1.7. Bin thin vn tc phn ng theo nng c cht Khi tng [S] th v phn ng tng, tng [S] n mt gi tr no th v t n gi tr vmax v s khng tng na nu ta vn tip tc tng [S]. Khi Km [S ] th v0 Vmax Nm 1934. Lineweaver v Burk, trn c s ca phng trnh (5) nghch o bin thnh dng ng thng y = ax+b, n c ngha ln i vi vic nghin cu km hm enzyme. 15
1 2

Ha sinh thc phm

Chng 1.Tng quan v enzyme cellulase

Hnh 1.8. S ph thuc ca tc phn ng vo nng c cht Trong nhiu phn ng do enzyme xc tc c 2 hay nhiu c cht, v d hexokinase xc tc phn ng:

ATP + glucose

hexokinase

ADP + glucose 6 phosphate

16

Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

CHNG 2. CC NG DNG CA NHM ENZYME CELLULASE 2.1. S lc v ng dng ca nhm enzyme cellulase ng dng trc tin ca cellulase i vi ch bin thc phm l dng n tng hp th, nng cao phm cht v v v lm mm nhiu loi thc phm thc vt. c bit l i vi thc n cho tr con v ni chung cht lng thc phm c tng ln. Mt s nc dng cellulase x l cc loi rau qu nh bp ci, hnh, c rt, khoai ty, to v lng thc go. Ngi ta cn x l c ch, cc loi to bin. Trong sn xut bnh m t la mch en, vic s dng enzyme ny s lm tng cht lng bnh, nh vo vic thy phn mt phn pentosan c trong bt. Vic ngm trong dung dch enzyme s thc y qu trnh ha tan cc thnh phn ca thc vt. Hn hp sau thng c s dng: exo- v endo-cellulase, v mannosidase v pectolytic enzyme. V d ng dng trong sn xut qu nghin, hoc rau nghin (puree), qu trnh phn gii l ch, qu trnh sn xut bt khoai ty,... Glycosidase (cellulase v amylase t Aspergillus Niger) khi s dng chung vi proteinase s loi b c v t tm. V tm c lm lng v tut ra d dng di vi nc. Cellulase thng c sn xut bng phng php ln men cc loi nm mc hoc vi khun. Ti chu u, c t nht nm loi cellulase c sn xut t cc vi sinh vt bin i gene nh Aspergillus, Bacillus, Trichoderma, nhng ch c cellulase sn xut Trichoderma l c s dng trong cng nghip thc phm. Bn cnh enzyme cellulase cn c hemicellulase, enzyme ny thng c s dng chung vi amylase, glucanase, cellulase, xylanase. Thng ng dng trong cng nghip sn xut bnh, sn xut cn, sn xut ru vang, sn xut nc gii kht t qu. Hemicellulase thng c sn xut bng phng php ln men cc loi nm mc hoc vi khun. Ti chu u ch c mt loi hemicellulase c sn xut t 17

Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

Bacillus bin i gene. s dng trong cng nghip thc phm, ngi ta thng dng hemicellulase sn xut t nm mc nh Aspergillus Niger. 2.2. Cc ng dng ca nhm enzyme cellulase 2.2.1. Trong cng nghip giy Trong cng nghip sn xut bt giy v giy, b sung cc loi enzyme trong khu nghin bt, ty trng v xeo giy c vai tr rt quan trng. Nguyn liu ban u cha hm lng cao cc cht kh tan l lignin v mt phn hemicellulose, nn trong qu trnh nghin tch ring cc si g thnh bt mn gp nhiu kh khn. Trong cng on nghin bt giy, b sung endoglucanase s lm thay i nh cu hnh ca si cellulose, tng kh nng nghin v tit kim khong 20% nng lng cho qu trnh nghin c hc. Trc khi nghin ha hc, g c x l vi endoglucanase v hn hp cc enzyme hemicellulase, pectinase s lm tng kh nng khuch tn ha cht vo pha trong g v hiu qu kh lignin. Trong cng ngh ti ch giy, cc loi giy thi cn c ty mc trc khi sn xut cc loi giy in, giy vit. Endoglucanase v hemicellulase c dng ty trng mc in trn giy.

Enzyme cellulase c b sung vo nhiu giai on khc nhau trong qu trnh sn xut giy: Trong giai on nghin bt giy c hc: Ngi ta b sung enzyme cellobiohydrolase nhm lm thay i nh cu hnh ca si cellulose, t lm tng kh nng nghin v tit kim 20% nng lng. Trong giai on nghin bt giy ha hc: Ngoi enzyme cellulase, ngi ta cn b sung thm enzyme hemicellulase v peptinase nhm ph v lp v ngoi ca g, gip tng cng kh nng khuych tn ca ha cht vo bn trong g, nhm lm tng hiu qu kh lignin. 18

Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

Trong giai on ty trng giy: y l giai on rt quan trng trong sn xut giy. Trc y, ngi ta thng s dng acid HCl ty trng giy; tuy nhin, HCl thi ra trong qu trnh sn xut lm nhim mi trng v gy hi i vi sc khe con ngi. Ngy nay, ngi ta thng s dng enzyme cellulase trong giai on ty trng giy cng nh trong qu trnh ti ch giy. u im ca phng php ny l gi cho si giy khng b n mn nhiu v khng nh hng n mi trng.

Hnh 2.1. Si giy c ti ch 2.2.2. Trong cng nghip dt Trong cng nghip dt, ngi ta s dng enzyme cellulase gi mu vi sng, bn v khng b sn c. i vi vi jean, cellulase c dng lm mm vi jean v to ra cc vt stone washed. Trc y cc vt stone washed c lm th cng bng cch dng bt ch ln vi jean, lm mt lp kim trn b mt vi v to ra nhng si ch trng. Hin nay ngi ta s dng enzyme cellulase trong giai on git vi jean thay cho vic s dng bt. Enzyme cellulase ch phn hy theo cc vt kim trn vi jean nhum mu to ra cc vt stone washed. Cc vt stone washed c to ra bng 19

Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

phng php ny bn hn bng cch dng bt. Ngoi ra, ngi ta c th tng m nht ca cc vt ny bng cch tng hay gim hm lng cellulase s dng trong giai on git. 2.2.3. Trong x l mi trng Cc cht thi hu c chim mt khi lng rt ln trong tng s cht thi hu c hin nay cc th v cc khu cng nghip. Trong cc cht thi hu c c ngun gc thc vt, cellulose chim khong 50%. Cc cht thi hu c cha cellulose thng l nhng cht rt kh phn hy trong iu kin t nhin. Nu cc cht hu c phn hy trong iu kin t nhin th thi gian phn hy rt lu (khong hn tm thng iu kin kh hu nhit i); tuy nhin nu b sung vi sinh vt giu cellulase th thi gian phn hy s rt ngn ch cn khong mt thng. iu ny rt c ngha trong vic bo v mi trng (hn ch s nhim nc, khng kh v t) ng thi thc y qu trnh chuyn ha vt cht trong t nhin. Ngoi vic b sung trc tip vi sinh vt vo b x l rc thi th vic to ra cc ch phm vi sinh c cha cc vi sinh vt sinh ra cellulase c nghin cu v sn xut. Phc h cellulase c s dng x l ngun nc thi do cc nh my giy thi ra. Nguyn liu lm giy l g (sinh khi ca thc vt bc cao). Sinh khi ny cha rt nhiu loi polysaccharide, trong cc polysaccharide quan trng quyt nh ti cht lng, s lng giy l cellulose. V vy, nc thi ca cc nh my giy, cc c s ch bin g, cc xng mc khi b sung cc ch phm cha phc h cellulase em li hiu qu cao. 2.2.4. Trong sn xut thc n gia sc Ngun ph liu giu cellulose trong t nhin rt a dng, phong ph nh: rm r, b ma, ci bp,...Tuy nhin, phn ln cc ch phm ny b vt b, hoc c lm phn bn, hoc c s dng lm cht t,...Trong khi , ngun nguyn liu r tin 20

Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

dng trong ch bin thc n gia sc li thiu. V vy, vic ng dng enzyme cellulase phn hy ngun ph phm cha cellulose va dng hoc lm ngun thc n cho gia sc, va gp phn bo v mi trng l mt vn rt ng quan tm. Hin nay ngi ta dng vi sinh vt ln men b ma ca nh my ng La Ng b sung vo thc n gia sc. 2.2.5. Trong k thut di truyn Ch phm enzyme cellulase tinh khit c ng dng trong k thut di truyn v enzyme cellulase ph v t bo thc vt m khng lm tn thng cc c quan bn trong t bo, m bo s nguyn vn ca cc nhn t di truyn. Do , phng php ny nhanh chng thay th cc phng php c hc v ha hc. 2.2.6. Trong cng nghip ch bin thc phm 2.2.6.1. Trong cng nghip sn xut bia Trong cng ngh sn xut bia, cc ch phm enzyme amylase, protease v glucanase c s dng ngn chn s to thnh cc diacetyl, do gim lng diacetyl c to thnh, rt ngn thi gian cn thit bia. Trong dch ln men c cha mt lng -glucan, cht ny nh hng ti kh nng lc v gy c cho bia. 2.2.6.2. Trong cng nghip sn xut c ph Trong qu trnh sn xut c ph Vit Nam, c ph ch yu c sn xut bng phng php kh, phng php ny cho cht lng c ph khng cao. tin hnh nng cao cht lng c ph, phng php ln men c p dng. l qu trnh s dng phc h enzyme cellulase v pectinase x l bc v c ph v lm tng kh nng ly trch dch qu. Trong khu bc v, cellulose gy hin tng thm mu, lm gim cht lng sau khi sy, ng thi cn tr cho vic bc v. Khi s dng ch phm 21

Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

A. niger c tn thng mi l Biovina-09 c hot tnh pectinase v cellulase cho thy s lng c ph c bc v tng, ht c ph c bc v bng ch phm khng cn nht nh ht khng s dng ch phm enzyme v hiu sut bc v kh cao. Trong qu trnh ly trch dch qu, cellulose v pectin cn tr s thot cc cht ha tan trong t bo ra ngoi t bo. Khi s dng ch phm Biotin-09 hiu sut trch ly cao hn mu khng s dng l 46%. 2.2.6.3. Trong sn xut cc loi nc qu Trong sn xut nc hoa qu khng cn ta, cellulase c thm vo giai on dch ha, hemicellulase s em li hiu qu ca ch phm, lm cho ng ha ca nc qu c tht tt hn. Dch qu sau khi p chit thng cha cc thnh phn t bo tht qu v cc cht x c bn cht polysaccharide lm cho dch c nht cao v c mu c. Glucanase thng c s dng ph v thnh t bo, thy phn cc polysaccharide lm gim nht ca dch qu to thun li cho qu trnh tch chit v lm trong. Glucanase kt hp vi cc hemicellulase, pectinase trong ch phm enzyme
Viscozyme 120L c s dng ch yu ph v mng t bo u tng.

Mi dng enzyme trong phc cellulase tham gia thy phn phn t c cht theo mt c ch ring. Tuy nhin, nhng enzyme ny thng phi hp thy phn hon ton phn t c cht thnh sn phm n gin l glucose. Dch ny thng cha cc thnh phn t bo tht qu v cc thnh phn ca polysaccharide lm cho dch qu c nht cao. tng hiu sut trch li dch qu, gim bt nht, tng mc cm quan nc qu v gim bt mt s cng on, vic b sung endoglucanase rt quan trng. Enzyme ny l im mu cht ci thin hiu sut dch ha. S kt hp ca glucanase v pectinase s ph hy hon ton mng t bo. Trong qu trnh sn xut, giai on dch ha b sung hn hp cc enzyme cellulase, hemicellulase s em li hiu qu ca ch phm, lm cho ng th ca nc qu c tht s tt hn. 22

Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

Endo--1,4-glucanase tham gia thy phn cc lin kt -1,4 glucoside bn trong cc phn t cellulose v mt s loi polysaccaride tng t khc. Sn phm phn cch l cc oligosaccaride. Exoglucanase thy phn cc lin kt u kh v u khng kh ca phn t c cht, gii phng cc oligosccaride, cellobiose v glucose. glucosidase thy phn cc phn t cellodextrin v cellobiose v glucose. -Glucosidase thy phn cc phn t cellodextrin v cellobiose to thnh cc phn t glucose.

Hnh 2.2. C ch thy phn phn t cellulose(A) v phc h cellulose(B) ca cc Enzyme thuc h phc cellulase 2.2.7. Trong sn xut cn Cc hp cht hu c c cha cellulose c nghin nh v trn vi dch enzyme cellulase. Hn hp c khuy trn u phn hy cellulose. Dch ng c to ra l hn hp ca nhiu loi ng nh: xylose, glucose, galactose, mannose, arabinose, acid glucuronic, acid galacturonic,...Ln men hn hp ng ny bng nm men Saccharomyces cerevisiae to ethanol.

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Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

Ngun ph liu nng nghip v lm nghip c bn cht l lignocellulose ang c nghin cu sn xut cn sinh hc. l mt ngun nguyn liu di do, khng nhng gip hn ch c s cnh tranh ngun t dng cho sn xut thc phm m cn gip cho vic ti s dng cc ngun ph liu mt cch hiu qu nht. Vic sn xut ethanol t ngun ny em li nhiu ngun li nhng s pht trin ca n ang b hn ch bi nhng kh khn v mt li nhun kinh t v k thut cha ti u. Thng thng ngi ta trn cht hu c c cha cellulose vi mt hn hp cc enzyme nh: cellulase v hemicellulase, ligninase,... c c t qu trnh nui cy cc chng nm mc Aspergillus niger,Trichoderma reesei,Aspergillus terreus.

2.2.8. Trong sn xut bt git v cht ty ra Enzyme cellulase c ng dng trong bt git v cht ty ra ch yu nhm mc ch lm mm vi cotton. Ngoi ra, khi b sung kt hp cellulase v mt s enzyme khc nh protease, lipase, amylase,....s lm cho bt git v cc cht ty ra c th ty sch mt s cc vt bn kh git nh vt mc, vt bn c ngun gc protein, cc vt loang du m,... 24

Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

2.2.9. Trong sn xut nhin liu sinh hc Nhin liu sinh hc (biofuel) l loi nhin liu c th phc hi c, c to ra t sn phm ca ngnh nng nghip, thc phm hay qu trnh ti ch cc sn phm du n v du thc vt. Nhin liu sinh hc c th c s dng trong cc thit b thng thng hay ng c diesel. Nhin liu sinh hc s dng n gin, khng c v khng cha lu hunh hay cc hp cht thm. Ngoi ra, nhin liu sinh hc c th pha trn vi xng du to ra hn hp cha nhin liu sinh hc. Bioethanol v biodiesel l hai loi nhin liu sinh hc c s dng rng ri nht cho ngnh giao thng vn ti; trong , bioethanol c sn xut nh enzyme cellulase thy phn sinh khi cellulose cn biodiesel l mt ester ca acid bo v ankyl, c to ra t du m thc vt, m ng vt hay m ti ch. 2.2.10. Trong cng nghip sn xut dung mi hu c Trong giai on ng ha ca qu trnh sn xut ethanol, amylase l thnh phn chnh trong qu trnh thy phn. Tuy nhin, vic b sung mt s enzym nh cellulase,hemicelluase s ph hy t bo,gip tng lng ng to thnh v y nhanh tc tip xc ca tinh bt vi amylase,dn ti hiu sut thu hi ru tng n 1,5%. 2.2.11. ng dng trong nui cy t bo v ti t hp gen S dng cellulase ph v thnh t bo to t bo trn (protoplast), c ngha rt ln trong vic tin hnh cc k thut chuyn np gen: dung hp t bo trn to t bo lai mang c im ca c t bo b m. 2.2.12. Trong cng nghip x l rc thi v sn xut phn bn vi sinh Rc thi l ngun chnh gy nn nhim mi trng dn ti mt cn bng sinh thi v ph hy mi trng sng, e da ti sc khe v cuc sng con ngi. Thnh 25

Ha sinh thc phm

Chng 2.ng dng ca enzyme cellulase

phn hu c chnh trong rc thi l cellulose, nn vic s dng cng ngh vi sinh trong x l rc thi ci thin mi trng rt c hiu qu. Enzyme ny c kh nng thy phn cht thi cha cellulose, chuyn ho cc hp cht kiu lignocellulose v cellulose trong rc thi to nn ngun nng lng thng qua cc sn phm ng, ethanol, kh sinh hc hay cc cc sn phm giu nng lng khc. Ngoi vic b sung trc tip vi sinh vt vo b x l rc thi th vic to ra cc ch phm vi sinh c cha cc vi sinh vt sinh ra cellulase c nghin cu v sn xut. 2.2.13. Trong sn xut agar-agar Trong sn xut agar-agar, tc dng ca ch phm cellulase s lm tng cht lng agar-agar hn so vi phng php dng acid ph v thnh t bo. c bit l vic s dng ch phm cellulase tn thu cc ph liu thc vt em thy phn, dng lm thc n gia sc v cng ngh len men. Nhng ng dng ca cellulase trong cng nghip thc phm c kt qu rt tt. Tuy nhin hn ch ln nht l rt kh thu c ch phm enzyme cellulase c hot cao.

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TI LIU THAM KHO Ti liu Ting Vit [1]. Nguyn c Lng v mt s tc gi, Cng ngh enzyme, NXB i hc quc gia TP.HCM, 2004 [2]. [3]. GS.TS Mai Xun Lng, Gio Trnh Enzyme, NXB i hc Lt, 2005 Nguyn Th Thu Sang, Bi ging Ha Sinh hc thc phm, i hc Cng Nghip Thc Phm TP.HCM (lu hnh ni b), 2011 [4]. Nguyn Vn Tun, Lun vn Thc s khoa hc sinh hc, Tuyn chn, nui cy chng Aspergillus Awamori sinh tng hp Endo- -1,4-Glucanase v nh gi tnh cht l ha ca Endo- -1,4-Glucanase, NXB i hc Thi Nguyn, 2009 Ti liu Internet [5]. L Th Hng Nga, Lun vn Thc s khoa hc, http://www.google.com.vn/url?sa=t&rct=j&q=&esrc=s&frm=1&source=web &cd=2&cts=1331624206975&ved=0CCYQFjAB&url=http%3A%2F%2Fwww. nsl.hcmus.edu.vn%2Fgreenstone%2Fcollect%2Fthesiskh%2Farchives%2FHAS H011d.dir%2F5.PDF&ei=6vheT4DfBu6diAeo5dHJBw&usg=AFQjCNF4wGNd VbS4b5QMJ5xvXY2E-YKTQQ

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