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Bai Giang Cong Nghe Enzyme PDF
Bai Giang Cong Nghe Enzyme PDF
HIAST
C NG NG H E NZ YM E
(Ti liu lu hnh ni b)
TPHCM, 03/2013
CNG NGH ENZYME i
MC LC
1. M U ................................................................................................................................. 1
1.1. Lch s nghin cu v pht trin cng ngh enzyme........................................................ 1
1.2. Khi nim v enzyme........................................................................................................ 2
1.2.1. Bn cht sinh hc ca enzyme ................................................................................... 3
1.2.2. Bn cht ha hc ca enzyme.................................................................................... 4
1.2.3. Cu trc ca enzyme.................................................................................................. 5
1.3. C ch tc dng ca enzyme............................................................................................. 7
1.3.1. C ch xc tc ca enzyme ....................................................................................... 7
1.3.2. Nng lng xc tc .................................................................................................... 8
1.3.3. S to thnh phc hp enzyme c cht .................................................................. 8
1.3.4. ng hc ca phn ng enzyme................................................................................ 9
1.3.5. Cc yu t nh hng n phn ng enzyme .......................................................... 10
1.3.5.1. Nhit ............................................................................................................ 10
1.3.5.2. pH ..................................................................................................................... 11
1.3.5.3. Cht km hm ................................................................................................... 11
1.3.5.4. Cht hot ha .................................................................................................... 12
1.3.6. Tnh cht c hiu ca enzyme................................................................................ 13
1.4. Phn loi enzyme ............................................................................................................ 13
1.4.1. Danh php quc t ca enzyme ............................................................................... 13
1.4.2. Phn loi enzyme ..................................................................................................... 14
1.4.2.1. Oxydoreductase ................................................................................................ 14
1.4.2.2. Transpherase..................................................................................................... 14
1.4.2.3. Hydrolase ......................................................................................................... 15
1.4.2.4. Lipase ............................................................................................................... 15
1.4.2.5. Isomerase .......................................................................................................... 15
1.4.2.6. Ligase ............................................................................................................... 15
1.5. Phng php tch v lm sch enzyme .......................................................................... 15
1.5.1. Cc phng php ph v t bo .............................................................................. 15
1.5.2. Phng php gy bin tnh chn lc ....................................................................... 16
1.6. Phng php xc nh kh nng xc tc ca enzyme..................................................... 17
1.6.1. Cc nhm phng php xc nh kh nng xc tc ca E ...................................... 17
1.6.2. n v hot ......................................................................................................... 17
2. K THUT C BN TRONG CNG NGH ENZYME .................................................. 18
2.1. ngha ca k thut tinh sch trong cng ngh enzyme ............................................... 18
CNG NGH ENZYME ii
2.2. K thut c bn ............................................................................................................... 19
2.2.1. Cc phng php c hc tch enzyme .................................................................... 19
2.2.2. Cc phng php ph v t bo sinh vt................................................................. 19
2.2.2.1. Ph v t bo bng phng php c hc: ........................................................ 20
2.2.2.2. Ph v t bo bng phng php khng phi c hc: ..................................... 20
2.2.2.3. Cc phng php c c .................................................................................. 21
2.2.2.4. Cc phng php tinh sch enzyme................................................................. 21
2.2.2.5. To sn phm enzyme ...................................................................................... 21
3. ENZYME C NH ............................................................................................................. 22
3.1. Chuyn ha sinh hc....................................................................................................... 22
3.1.1. Chuyn ha vt cht do enzyme t do hay enzyme ha tan.................................... 22
3.1.2. Chuyn ha vt cht bng enzyme khng ha tan .................................................. 22
3.1.3. Chuyn ha vt cht do cc qu trnh ln men ........................................................ 23
3.1.4. Chuyn ha vt cht do t bo c nh .................................................................... 23
3.2. c im ca enzyme c nh ........................................................................................ 23
3.3. Phng php to enzyme c nh ................................................................................... 23
3.3.1. Cc yu t nh hng n enzyme c nh ............................................................. 23
3.3.2. Phng php c nh enzyme ................................................................................. 25
3.3.2.1. Cht mang dng c nh enzyme ................................................................ 25
3.3.2.2. Phng php c nh enzyme .......................................................................... 25
3.4. ng dng ca enzyme c nh ....................................................................................... 26
3.4.1. Sn xut fructose nh enzyme glucose isomerase .................................................. 27
3.4.2. Sn xut L-amino acid nh enzyme aminoacylase c nh .................................... 28
4. THU NHN ENZYME......................................................................................................... 28
4.1. Chn ngun nguyn liu ................................................................................................. 28
4.2. Thu nhn enzyme ............................................................................................................ 31
5. NG DNG CA ENZYME .............................................................................................. 32
5.1. Tnh hnh ng dng enzyme trong cng nghip trn th gii ........................................ 32
5.2. ng dng trong y hc ..................................................................................................... 34
5.3. ng dng trong ha hc ................................................................................................. 34
5.4. ng dng trong cng nghip .......................................................................................... 34
5.4.1. ng dng trong cng nghip thc phm ................................................................. 34
5.4.2. ng dng trong cng nghip dt ............................................................................. 36
5.4.3. ng dng trong cng nghip thuc da .................................................................... 36
5.5. ng dng trong nng nghip .......................................................................................... 36
TI LIU THAM KHO ............................................................................................................. 36
CNG NGH ENZYME 1
1. M U
1.1. Lch s nghin cu v pht trin cng ngh enzyme
Enzyme hc l mn hc nghin cu v vt cht phn t c hot tnh sinh hc. Mn hc ny pht trin rt
mnh nh nhng thnh tu ca mn ha sinh hc, vi sinh vt hc, di truyn hc v c nhng tin b ca
ngnh ha hc v vt l hc.
Bng 1.1. Nhng ct mc quan trng trong nghin cu v pht trin mn enzyme hc[2]
Tanabec co xy dng quy trnh cng nghip sn xut amino acid. S dng
12 1969
glucose isomerase trong sn xut dch ng giu fructose
CNG NGH ENZYME 2
pht hin hng trm loi enzyme khc nhau, a vo sn xut cng nghip v
16 1984 - nay ng dng rng ri enzyme trong sn xut v i sng. k thut enzyme c nh, t
bo c nh a cng ngh enzyme t c nhiu kt qu cao
Diastase: l enzyme xc tc cho phn ng thy phn tinh bt thnh maltose. Diastase l enzyme u tin
c pht hin ra. N c chit tch t malt vo nm 1833 bi Anselme Payen v Jean-Francois Persoz
nh Ha hc ti nh my ng ca Php. Tn diastase xut pht t ting Hy Lp (diastasis), trong
dch nha nng, cc enzyme ny thy phn tinh bt trong ht i mch thnh cc ng ha tan v v th
c th tch phn b malt vi phn cn li ca ht. Ngy nay, diastase l , hoc -amylase.
Rennet l enzyme c sn xut trong d dy ca ng vt c v khi cn trong giai on b sa m v
thng c s dng lm phomai. Rennet l mt hn hp enzyme, bao gm: cc enzyme thy phn
protein (protease) gy ng t sa, c th tch phn sa ng (phn cht rn) v phn huyt thanh sa
(phn cht lng). Nhng enzyme hot ng trong d dy b c gi l chymosin hoc rennin. Ngoi ra,
cng c nhng enzyme quan trng khc nh: pepsin v lipase.
Protease: (cn gi l peptidase hoc proteinase) l enzyme thy phn protein
-amylase l enzyme thy phn lin kt alpha ca phn t polysaccharide nh tinh bt v glycogen thnh
glucose v maltose. Alpha amylase c tm thy ch yu ngi v ng vt c v khc, ngoi ra,
chng cn c mt trong cc loi ht c cha tinh bt v c tit ra bi mt s loi nm.
Urease: l enzyme xc tc phn ng thy phn ure thnh carbon dioxide (CO2) v amoniac (NH3), hin
din trong vi khun ng rut. Urease c tm thy trong vi khun, nm men v thc vt.
1.2. Khi nim v enzyme
Sinh vt c phn ra 2 nhm da vo cu to c th ca chng: sinh vt n bo v sinh vt a bo. Mc
d chng c s khc bit rt ln v cu to c th v nhng c im sinh l khc nhau nhng chng u
ging nhau v trao i cht vi mi trng bn ngoi v mt s c im v bin d di truyn.
Sinh vt c xem nh l mt h thng m c lin quan cht ch n qu trnh trao i cht ca c th
trong t bo v gia t bo vi mi trng ngoi. Qu trnh trao i cht bn trong t bo v gia t bo
vi mi trng bn ngoi l biu hin sinh ng nht ca s sng. S khc nhau gia t bo sng v vt
cht khng phi s sng chnh l kh nng trao i cht ny. Khi c th khng cn kh nng trao i cht
th c th s cht. Do mi quan h gi c th vi bn ngoi l mi quan h hu c.
Qu trnh trao i cht bao gm qu trnh d ha v qu trnh ng ha. Qu trnh d ha l qu trnh phn
gii vt cht cung cp cho t bo nng lng v vt liu xy dng t bo, qu trnh ny c th xy ra
trong t bo hoc ngoi t bo.
Qu trnh di ha xy ra trong t bo l qu trnh cung cp nng lng, vt cht cho qu trnh tng hp vt
cht to ra sinh khi nhm lm i mi vt cht t bo.
Qu trnh d ha ngoi t bo phn ln ch p ng nh cu v vt cht, gip cho t bo tng hp cc
cht trong t bo. Nng lng to ra t qu trnh d ha ngoi t bo thng c gii phng dng nhit
nng.
CNG NGH ENZYME 3
Qu trnh tng hp vt chtt cho tt bo ch xy ra trong t bo. Qu trnh ny th
ng phi thu nhn nng
lng t cc phn ngng do qu trnh dd ha. Nh vy, qu trnh trao i cht cc xem nh h
h thng m
ca sinh vt i vi mi trng
ng xung quanh.
2 Polyphenoloxydase ng (Cu)
E+S ES E+P
Hnh 1.9. Cht km hm cnh tranh Hnh 1.10. Cht km hm khng cnh tranh
1.3.5.4. Cht hot ha
Cc cht c tc dng lm tng hot tnh ca enzyme gi l cc cht hot ha enzyme. Cc cht hot ha
enzyme c bn cht ha hc rt khc nhau. Chng c th l nhng anion, cc ion kim loi t th 11 n
th 55 trong bng h thng tun hon, cc cht hu c c cu trc phc tp. Tuy nhin, cc cht hot
ha ch c tc dng mt nng nht nh. Vt qu nng ny, chng s gy c ch hot ng ca
enzyme
CNG NGH ENZYME 13
nng hot ha, cc cht hot ha thng lm nhim v chuyn n nhm hydrogen hotho nhng cht c
kh nng ph v mt s lin ktt trong phn t tin enzyme hoc cc chtt c tc dng
d phc hi cc nhm
chc nng trong trung tm hott ng ca enzyme
Thi gian
1 Lng c cht v sn phm
Nng enzyme
Thi gian
3 Nng enzyme
Lng c cht hay sn phm
1.6.2. n v hot
Kh nng xc tc ca enzyme c xc nh thng qua hot hot ng ca enzyme. Hot hot ng
ca enzyme c xc nh thng qua n v hot . Ngi ta biu din n v hot qua nhng n v
sau:
a - n v hot quc t (UI): n v hot quc t l lng enzyme c kh nng xc tc lm chuyn
ha 1 micromol c cht trong 1 pht iu kin tiu chun
b - katal (kat): n v kat l lng enzyme c kh nng xc tc lm chuyn ha c 1mol c cht sau 1
giy iu kin tiu chun
1UI = 16,67nano kat (nkat)
CNG NGH ENZYME 18
c - hot ring: hot ring ca mt ch phm enzyme l s n v UI (hoc mt n v katal) ng
vi 1 ml dung dch (nu l dung dch) hoc 1mg protein (nu l bt kh) ca ch phm enzyme. Khi bit
c khi lng phn t ca enzyme ta hon ton c th tnh c hot ring ca phn t.
d - hot ring ca phn t: hot ring ca phn t enzyme l s phn t c cht c chuyn ha
bi mt phn t enzyme trong mt n v thi gian.
Vic xc nh hot ca enzyme thng c cc nh khoa hc a ra nhng phng php rt c th
cho tng loi enzyme, ng vi tng loi c cht. Tuy nhin, khi tin hnh xc nh hot ca enzyme
cn lu nhng im c bn sau:
Khi tin hnh xc nh hot ca enzyme cn phi cho lng c cht trong mt gii hn thch hp
tha bo ha enzyme. Lng c cht cho vo khng c qu nhiu. Nu lng c cht qu
nhiu s gy c ch hot ng ca enzyme
i vi nhng enzyme d mt hot tnh cn phi c cht hot ha hoc lm bn enzyme, th cn phi
cho nhng cht ny vo trc khi cho c cht vo thc hin phn ng.
Khi xc nh hot enzyme cn ch phi thc hin trong iu kin pH c nh v thch hp vi
hot ng ti u ca enzyme nghin cu. pH thng thay i ty thuc vo c cht v dung dch m
Khi xc nh hot enzyme cn lu n nhit . Cc nh khoa hc cho rng khng nn xc nh
hot enzyme nhit ti u ca enzyme m nhit thp hn nhit ti u mt t
trnh kh nng lm bin tnh enzyme khi c s bin ng v nhit
Thi gian xc nh hot enzyme ch nn ko di khong 5 - 10 pht, khng nn ko di qu, d
gy gim hot ca enzyme. Tuy nhin, cng c nhng trng hp ko di n 24 gi i vi
nhng enzyme c kh nng hot ng yu. Trong trng hp phi ko di thi gian, cn thit phi
cho cht c ch hot ng hoc tiu dit vi sinh vt
Ngoi ra, hin nay trn th gii c rt nhiu cch biu th n v hot ca enzyme nh:
MWU n v biu th lng enzyme cn thit chuyn ha mt mg tinh bt ha tan n dextrin trong 30
pht iu kin th nghim;
SKB l lng enzyme cn thit dextrin ha 1g beta-dextrin gii hn kch thc xc nh sau 1 gi
iu kin th nghim
2. K THUT C BN TRONG CNG NGH ENZYME
2.1. ngha ca k thut tinh sch trong cng ngh enzyme
Cng ngh enzyme c bt u t giai on sn xut ch phm enzyme th v kt thc giai on to
thnh ch phm tinh khit.
Bt u t ch phm enzyme th, ngi ta tin hnh tinh sch enzyme bng nhiu k thut khc nhau.
Cc k thut tinh sch thng a n nhng kt qu quan trng nh sau:
Khi lng ch phm enzyme s c gim dn qua tng bc tinh sch. Ch phm enzyme th thng
cha nhng thnh phn c bn sau:
Nc chim khi lng ln nht
Protein khng c hot tnh sinh hc
Cc tp cht t t bo (nu l ngun ng vt v thc vt), mi trng nui cy (nu l ngun t vsv)
Enzyme (hay protein c hot tnh sinh hc)
Nh vy, qu trnh tinh sch l qu trnh loi b ba phn trn, ch nhn sn phm cui l protein c hot
tnh sinh hc. Vic loi b thnh phn u khng th thc hin trong mt giai on m phi thc hin qua
nhiu giai on khc nhau, mi giai on ta s loi b c mt phn khng phi l enzyme ra khi hn
hp. Khi cc thnh phn khng phi l enzyme c loi dn ra, khi lng ch phm s gim theo.
Nh vy, nu enzyme c tinh sch theo nhng k thut khc nhau s thu nhn nhng gi tr quan trng
sau:
Gim khi lng. Tr s ny c ngha cho vic ng bao b, vn chuyn, s dng v bo qun.
Tng c hot tnh enzyme c ngha rt ln trong qu trnh s dng sau ny.
Tng gi tr kinh t thng qua gi thnh sn phm
gii quyt c trong nhng mc tiu trn, ta thc hin nhng k thut c bn sau:
CNG NGH ENZYME 19
2.2. K thut c bn
2.2.1. Cc phng php c hc tch enzyme
Cc phng php c hc c ng dng nhiu tch enzyme khi t bo v cc thnh phn khc gm 2
phng php:
Phng php ly tm: ly tm l tch vt cht rn ra khi dung dch. Trong cng ngh enzyme, phng
php ly tm thng c ng dng kh rng ri thu nhn dung dch enzyme, dung dch ny cha
cc enzyme ngoi bo. Enzyme ni bo nm trong t bo sinh vt mun thu nhn enzyme ni bo ta
cn phi tin hnh ph v t bo.
Phn ln enzyme ngoi bo l enzyme ha tan. Nh vy khi tin hnh ly tm, dung dch c tch khi
cc thnh phn rn l dung idjch enzyme th. Dung dch enzyme th ny cn cha thnh phn sau:
Protein c hot tnh sinh hc
Cc cht ha tan khc
Nc
Phng php ly tm ch tch c thnh phn rn c t trng ln hn dung dch. Dch thu c cha phi
l ch phm enzyme tinh khit m l ch phm enzyme th, v cn cha protein khng hot ng, nc
v cc cht ha tan khc.
trnh hin tng bin tnh ca enzyme, trong nhng mu nghin cu, ngi ta thng tin hnh ly
tm lnh, cn trong sn xut theo quy m cng nghip, ngi ta thng tin hnh trong iu kin nhit
thp.
Trong quy m cng nghip, ngi ta thng tin hnh ly tm thu nhn dung dch enzyme bng my ly
tm lin tc. Phng php ly tm lin tc c nhng u im l thi gian thu nhn sn phm nhanh, lin
tc v khng gy nh hng ln n hot tnh enzyme.
Phng php lc: trong cng ngh enzyme, sau khi ph v thnh t bo sinh vt hay sau khi ln men,
ngi ta thng s dng qu trnh lc thu nhn dung dch enzyme ni bo, enzyme ngoi bo ha
tan.
Lc cng l phng php tch thnh phn rn ra khi dung dch. Lc l mt phng php khi thc hin
thng gp nhiu kh khn nh kch thc vt cht di t bo thng rt nh v nht ca dung dch
thng rt cao. C hai yu t ny u lm cn tr qu trnh lc. Tc lc ph thuc rt ln vo: din
tch b mt vt liu lc, p sut khi lc, nht dch lc
Trong cng nghip sn xut enzyme, ngi ta thng s dng cc kiu sau:
Lc p: thng c s dng trong trng hp dung dch cn lc c khi lng nh. Phng
php ny s dng lc enzyme rt c hiu qu.
Lc chn khng: l phng php c ng dng nhiu nht trong nghin cu v trong sn xut
cc sn phm sinh hc c hot tnh. Trong lc chn khng quay c s dng nhiu hn c.
Lc theo dng chy ct ngang: trong nhng nm gn y, nhiu nh my sn xut enzyme s
dng phng php lc theo dng chy ct ngang. Theo , dng dung dch lc s chy song song
b mt nguyn liu lc. Phn lc s c thot qua vt liu lc v i xung pha di. Phng
php ny c u im l lm gim sc khng qu trnh lc ca vt liu rn.
Lc thng thng: phng php lc thng thng c t rt lu, hin nay, mt s nh my
vn cn s dng. Phng php ny dn c thay bng phng php khc nhanh hn, c hiu
qu hn.
2.2.2. Cc phng php ph v t bo sinh vt
Mc ch ca vic ph v t bo l gii phng ton b cc cht c trong t bo. Cng vi cc cht c
trong t bo c gii phng ra ngoi l enzyme ni bo.
i vi t bo ng vt v t bo thc vt ta phi tin hnh nghin nt trc khi thc hin cc qu trnh
trch ly, lc, c c, lm sch v sy
i vi t bo vsv phi tin hnh tch bng phng php ly tm hoc bng phng php lc. Phn dch
lc thu c ca mt trong hai phng php ny l dch ch phm enzyme ngoi bo. Phn sinh khi vsv
sau khi ly tm hoc lc cha enzyme ni bo. Vic ph v t bo ng vt v thc vt khng my kh
CNG NGH ENZYME 20
khn nhng ph v t bo vsv rt phc tp v t bo vsv va c kch thc nh, va c thnh t bo c
cu trc kh c trng. Do , ph v t bo vsv i hi phi c nhng phng php c bit.
2.2.2.1. Ph v t bo bng phng php c hc:
Mc ch ca ph v t bo l gii phng cc cht c trong t bo v m bo c hot tnh ca enzyme
ni bo.
i vi t bo ng vt (thng s dng ton b c quan hay m bo) c cha enzyme v cn phi
c tr nhit thp trnh mt hot tnh enzyme. Trc khi tr lnh cn loi b m hot cc
thnh phn khc bm theo m bo , m cn c x l nhanh v phi c thu nhn enzyme trong
thi gian khng qu 4 gi k t khi git m.
i vi t bo v m thc vt cng cn phi c lm sch v c tr lnh nu cha tin hnh thu
nhn enzyme ngay.
T bo ng vt v thc vt thng rt d ph v bng cc phng php c hc. Ring i vi t bo
vsv, vic ph v t bo gp phi kh khn nht nh:
Th nht: t bo vsv c kch thc qu nh, vic ph v t bo bng phng php nghin nu
khng c cht tr nghin s khng c hiu qu.
Th hai: vsv l c th n bo, khi pht trin trong mi trng (b l mt lng) c nhiu c cht
thy phn, chng s to ra nhiu enzyme ngoi bo tng ng. Cc enzyme ngoi bo ny ha
han trong mt v d dng tch chng ra khi dung dch nui cy. Do trong cng nghip, tr
trng hp phi thu nhn enzyme ni bo ngi ta mi tin hnh nghin t bo vsv
Phng php ng ha p lc cao: l phng php c ng dng rng ri nht ph v t bo vsv.
Huyn ph tb vsv s c nn vi mt p sut cao, chng va chm rt mnh vo vnh ng. Tb b ph v
bi lc ct v sc nn.
Phng php nghin m: l phng php c s dng rng ri trong cng nghip sn xut enzyme.
Trong qu trnh nghin, ngi ta s dng nhng vin bi thy tinh c kch thc 0,2 1mm tng qu
trnh ph v t bo. Trong nhiu trng hp, ngi ta khng dng vin bi thy tinh m ngi ta dng ht
thy tinh v ht thy tinh c ma st cao hn
2.2.2.2. Ph v t bo bng phng php khng phi c hc:
Ngoi 2 phng php c hc trn, ngi ta cn ph v t bo bng phng php sau:
Phng php ha hc: da vo kh nng to ra p sut thm thu mnh, hoc kh nng oxy ha mnh
ca cc cht ha hc ph v thnh t bo. Phng php ny khng i hi p sut cao, nn t chi ph v
d thc hin. Tuy nhin, v trong qu trnh thc hin, ngi ta s dng ha cht nn cc ha cht ny
thng ln vo trong hn hp, i hi qu trnh tch rt phc tp. Mt trong nhng ha cht s dng
nhiu l acetone
Phng php vt l: l s dng siu m, thng c s dng trong quy m ptn cha s dng trong cng
nghip. Phng php vt l l to shock nhit hoc shock thm thu. Nguyn tc ca phng php ny l
khi a nhit ca huyn ph tb xung nhit rt thp, ngay lp tc nng nhit ln 40oC khi thnh
t bo s b ph v v ngi ta thu c huyn ph t bo v. Phng php ny thng d thc hin, m
bo c hot tnh ca enzyme nhng hiu sut ph v t bo khng cao.
Phng php sinh hc: c 2 phng php hin ang c s dng rng ri l phng php t phn v
phng php thy phn bng enzyme a t bn ngoi vo.
Phng php t phn l to iu kin ti u cho mt s enzyme c kh nng phn gii mt s thnh phn
ca thnh t bo, cc enzyme ny phi l nhng enzyme c trong tb v l ca tb . Bnh thng cc
enzyme ny khng hot hot ng mnh, nhng nu iu kin nhit , pH, nc bn ngoi tb m trng
vi mc hot ti u ca chng, th chng s hot ng mnh v thy phn thnh t bo, lm cht t
bo.
T phn l qu trnh c p dng nhiu trong ph v thnh t bo cc loi nm men. Ngi ta thng
tin hnh t phn huyn ph t bo nm men nhit 48 52oC trong khong thi gian 6 24 gi.
Phng php ny c nhiu nhc im v trong qu trnh thy phn, cc enzyme c trong t bo khng
ch thy phn cc cht thnh t bo m c nhng cht c trong t bo, thm ch enzyme cng b ph
hy. Phng php ny hin khng c s dng nhiu trong cng nghip.
CNG NGH ENZYME 21
2.2.2.3. Cc phng php c c
Dung dch enzyme th thng cha lng enzyme khng nhiu. x l mt khi ln dung dch enzyme
phi tn t nhiu cng sc chi ph cho x l ny rt cao. Mt khc lng enzyme c trong dch enzyme
th qu t, hot tnh enzyme khng cao. Chnh v th phi c c dung dch enzyme nhm mc ch:
Lm tng hot tnh ca enzyme
Lm gim chi ph v cng sc cho vic x l sau ny
Tng kh nng bo qun enzyme
Gim chi ph vn chuyn, bo qun
Tng hiu sut tc ng ca enzyme i vi c cht
t c mc ch , ngi ta tin hnh c c enzyme bng phng php sau:
Phng php nhit: s dng nhit c c enzyme thng kh khn v enzyme l protein rt nhy cm
vi nhit . Qu trnh c c nhit l qu trnh lm bc hi nc, lm cho lng nc trong dung dch
gim i, hm lng enzyme trong dung dch s tng ln, hot tnh enzyme cng tng cao. Tuy nhin phi
ch n iu kin nhit khng ch n trong mt khong cho php th hot tnh enzyme mi c
bo ton.
Phng php kt ta: enzyme l mt phc hp protein c kh nng kt ta vi mt s dung mi
Kt ta bng mui: mui nng cao c th tc dng vi phn t nc bao quanh protein enzyme v
lm chuyn ha in tch, lm thay i tnh ha tan ca enzyme. Trong cng nghip thng s dng
mui ammonium sulfate. Nhit tin hnh kt ta: 35 40oC, nng mun: 20 80%
Kt ta bng dung mi hu c: dung mi hu c c nh hng rt ln n kh nng ha tan ca
enzyme, nh hng solvat ha ca phn t nc xung quanh phn t enzyme b thay i, tng tc
gia cc phn t enzyme s tng ln. Trong cng nghip sn xut enzyme ngi ta thng s dng
ethanol v acetone kt ta enzyme. Nhit kt ta: 3 10oC, t l v nng cc dung mi hu
c dng kt ta enzyme c xc nh bng thc nghim cho tng loi enzyme v tng nng
enzyme c trong dung dch enzyme.
Kt ta enzyme im ng in: protein l cht lng cc. Mc ha tan ca enzyme ph thuc
vo pH. Kt ta enzyme im ng in thng thc hin quy m nh. Thi gian to im ng
in v thi gian lu to kt ta bn vng thng lm thay i hot tnh enzyme.
Phng php siu lc: l phng php s dng mng lc c l lc siu nh. c s dng rng ri
thu nhn cc cht c khi lng phn t trong khong 1000 300.000Da. Ngi ta thng s dng
cellulose acetate v cc polymer hu c nh: poly sulfone, polyvinylidene v polypropylene lm nguyn
liu mng lc.
2.2.2.4. Cc phng php tinh sch enzyme
C 3 phng php tinh sch enzyme chnh:
Phng php kt tinh
Phng php in di
Phng php sc k
Sc k lc gel
Sc k trao i ion
Sc k i lc
2.2.2.5. To sn phm enzyme
Sau khi c lm sch, ngi ta tin hnh thc hin giai on cui cng l to sn phm, ng bao. Ch
phm enzyme c th c to thnh cc loi sn phm sau: enzyme dng dung dch, enzyme dng huyn
ph, enzyme dng bt kh, enzyme dng vin nh
Enzyme dng dung dch hay dng huyn ph: c nhc im l rt kh bo qun. do ngi ta
thng a chng vo dung dch cc cht bo qun. mt khc khi s dng ngi ta cng phi gi n
trong iu kin nhit nht nh, khng c bo qun nhit cao
Enzyme dng bt kh hoc dng vin nh: thng d dng bo qun v kh nng bo qun rt lu.
ngoi ra, cc ch phm enzyme ny thng d vn chuyn.
CNG NGH ENZYME 22
3. ENZYME C NH
3.1. Chuyn ha sinh hc
Qu trnh chuyn ha sinh hc l qu trnh vt cht trong t nhin c sinh vt chuyn t dng ny sang
dng khc thng qua hot ng sng ca t bo. Hot ng sng ca t bo l qu trnh trao i cht gia
bn ngoi v bn trong t bo thng qua hot ng ca enzyme v ca t bo.
Nh hot ng xc tc ca enzyme to cho t bo sinh vt v c th sinh vt nh mt h thng m.
nng lng cha trong vt cht lun lun c bin i, lun lun c trao i gia bn trong v bn
ngoi c th.
Ton b chuyn ha sinh hc c thc hin bi 4 qu trnh sau:
Qu trnh hot ng ca enzyme ngoi bo (enzyme t do)
Qu trnh hot ng ca cc vi sinh vt trong qu trnh ln men.
Qu trnh hot ng ca t bo c nh
Qu trnh chuyn ha ca enzyme c nh
3.1.1. Chuyn ha vt cht do enzyme t do hay enzyme ha tan
Enzyme t do hay enzyme c tch khi t bo, chng c kh nng ha tan trong mi trng nc v
thc hin cc phn ng ngoi t bo sinh vt. Nhng enzyme ny cn c gi l enzyme ngoi bo. Cc
enzyme ny ng vai tr rt quan trng trong s chuyn ha vt cht ngoi thin nhin v c trong sn
xut v i sng. Tuy nhin, khi s dng cc enzyme ny, cc nh khoa hc cng cho thy chng c
nhng nhc im sau:
Trong qu trnh tham gia phn ng, cc loi enzyme ny thng ln vo sn phm. Vic tch sn
phm cui ra khi enzyme ha tan l mt cng vic rt kh khn v i hi chi ph khng nh. Nhiu
sn phm ca qu trnh thy phn i hi khng c cha tp cht v cc thnh phn khc nh
enzyme. V d nh trong sn xut glucose s dng trong y hc phi tuyt i sch c v phng din
ha hc v v sinh hc.
Sau phn ng, nu tch c enzyme ra khi phn ng thc hin cc phn ng tip theo, enzyme
s khng gi c hot tnh nh hot tnh ban u, nh vy vic ti s dng enzyme s khng c hiu
qu, trong nhiu trng hp enzyme ny khng s dng c na.
Cc enzyme ha tan thng km bn nhit, acid, kim, dung mi hu c hay mt s ion kim loi nng.
Do , vic s dng cc bin php bo v chng trong phn ng l iu rt cn thit
3.1.2. Chuyn ha vt cht bng enzyme khng ha tan
Enzyme khng ha tan hay enzyme c nh c hiu theo c ngha hp v ngha rng
Theo ngha hp: enzyme khng ha tan l nhng enzyme c a vo nhng pha ring r, pha ny
c tch ring vi pha dung dch t do. Pha enzyme khng ha tan trong nc v c gn vi nhng
polimer a nc c trng lng phn t ln
Theo ngha rng: cc cht xc tc c nh l cc enzyme, t bo trng thi sng trng thi cho php
s dng li. Nh vy, theo ngha rng enzyme khng ha tan bao gm c enzyme c c nh vo mt
cht mang, bao gm c enzyme c trong c th sng c c nh trong cc bnh phn ng sinh hc c
gn kt mt cht mang cho php ta s dng nhiu ln.
Enzyme khng ha tan hay cn gi l enzyme c nh thng l nhng enzyme ha tan c gn vo mt
cht mang bng nhiu k thut khc nhau. Nh qu trnh ny m enzyme t trng thi ha tan chuyn
sang dng khng ha tan. khi chuyn t trng thi ha tan sang trng thi khng ha tan, enzyme khng
ha tan c nhng u im sau:
Enzyme khng ha tan c th c s dng nhiu ln, hot tnh ca enzyme khng ha tan t b thay
i trong nhng ln ti s dng. c im ny ca enzyme khng ha tan c ngha rt ln trong k
thut, nh ta c th ti s dng nhiu ln v s lm gim chi ph cho vic sn xut enzyme. y l
u im ln nht ca vic thu nhn v ng dng enzyme khng ha tan.
Enzyme khng ha tan khng ln vo sn phm cui ca phn ng enzyme, do chng ta khng
phi chi ph cho vic tch enzyme ra khi sn phm. Sn phm cui thu c s coi nh sn phm
tng i sch.
CNG NGH ENZYME 23
T 2 c im trn cho thy s dng enzyme khng ha tan c ngha kinh t hn s dng enzyme ha
tan nhiu ln.
3.1.3. Chuyn ha vt cht do cc qu trnh ln men
Qu trnh ln men l qu trnh chuyn ha sinh hc c thc hin bi cc t bo vi sinh vt sng t do.
Ln men l qu trnh chuyn ha vt cht c trng cho ring vi sinh vt. Cc qu trnh ln men l tp
hp rt nhiu cc qu trnh enzyme. , ton b cc phn ng xy ra trong t bo theo mt trt t c
quyt nh bi h thng gen c trong nucleic acid.
Khc vi enzyme ngoi t bo, qu trnh ln men thng xy ra phc tp hn, cc c cht ngoi t bo
phi thm thu c vo trong t bo. Ti y s to ra cc sn phm bc mt (sinh khi), sn phm bc
hai (sn phm trao i cht). Qu trnh to ra cc sn phm trn ch c thc hin trong t bo sng. Nu
t bo cht th cc qu trnh chuyn ha li mang ngha khc. l qu trnh thy phn bi enzyme
ngoi bo.
Qu trnh chuyn ha sinh hc ny xy ra thng xuyn, lin tc trong thin nhin v c loi ngi
ng dng vo sn xut hng ngn nm nay nh sn xut ru, bia, nc gii kht, phomai, tng, chao,
nc mm v pht trin mnh trong cng ngh ln men hin i nh amino acid, khng sinh, cc cht
kch thch tng trng.
3.1.4. Chuyn ha vt cht do t bo c nh
T bo c nh l t bo vi sinh vt c gn vo mt cht mang. Cc cht mang v vi sinh vt gn vo
c a vo bnh phn ng sinh hc (bioreactor). C cht c a vo t nh ca bnh phn ng
sinh hc v sn phm c ly ra y bnh phn ng sinh hc. Qu trnh ny c thc hin lin tc.
T bo c nh c ng dng rng ri, em li hiu qu kinh t rt r rt
3.2. c im ca enzyme c nh
Khi gn enzyme ha tan vo mt s cht mang, enzyme ny c nhng c im khc hn enzyme ha tan.
Hot tnh ca enzyme khng ha tan thng nh hn hot tnh ring ca enzyme ha tan cng loi.
S d c s thay i v hot tnh ring ca enzyme khng ha tan l do nhng nguyn nhn sau:
a) Do nh hng in tch ca cht mang. Khi gn enzyme vo cht mang c in tch khc vi in
tch ca enzyme, cu trc khng gian ca enzyme c s thay i mc nht nh. V s thay
i cu trc khng gian ca enzyme nn s tng tc ca enzyme v c cht chm li, phn ng
s xy ra yu hn. Mc gim hot tnh ca enzyme ph thuc vo mc thay i cu trc
khng gian ca enzyme khi ta gn chng vo cht mang.
b) Do enzyme b nht vo mt gel. Khi ta nht mt enzyme vo mt gel no , gel s bao ly phn
t enzyme to thnh mt vt cn i vi c cht ca enzyme. Do , s tip xc gia trung tm
hot ng ca enzyme vi c cht gp kh khn hn so vi s tip xc gia c cht vi enzyme
t do
Enzyme khng ha tan hon ton tun theo nh lut Michaelis menten. Tuy nhin, trong phn ng
gia c cht vi enzyme khong ha tan c nhng sai khc nht nh:
a) C th xy ra hin tng cnh tranh c cht vi enzyme v cht mang
b) Hin tng cn tr s khuch tn c cht v cc sn phm ca phn ng lm gim gim tc
phn ng
Enzyme khng ha tan c tnh bn nhit hn enzyme ha tan cng loi v chng c bo v bi cht
mang
Enzyme khng ha tan c xu hng chuyn dch pH ti u sang kim hoc acid so vi pH ti u ca
enzyme ha tan ch khng trng vi pH ti u ca enzyme ha tan cng loi
Enzyme khng ha tan c kh nng bo qun tt hn enzyme ha tan cng loi
Ta c th ti s dng enzyme khng ha tan nhiu ln. Trong khi enzyme ha tan ch c th s
dng mt ln. c im ny rt c ngha v kinh t khi ta tin hnh cc phn ng theo quy m cng
nghip
3.3. Phng php to enzyme c nh
3.3.1. Cc yu t nh hng n enzyme c nh
Khi gn ln cht mang, enzyme b gii hn trong mt phm vi mi trng xc nh, cu to khng gian
ca phn t c th b thay i, do lm bin i mt s tnh cht ca enzyme ban u (enzyme ha tan).
CNG NGH ENZYME 24
Enzyme c th thay i v pH, nhit hot ng, cng nh cc gi tr ca hng s Michaelis menten
v tnh cht c hiu, tuy nhin nhng thay i ny cn ph thuc nhiu vo bn cht ha hc ca cht
mang. Ni chung enzyme c nh thng bn vi cc tc nhn bin tnh hn, nhng hot ring thng
thp hn enzyme ha tan.
Mt trong nhng nguyn nhn lm gim hot tnh ca enzyme c nh l tng tc protein protein gia
cc phn t enzyme c lin kt. Trong mi trng hp u thy hot ring ca ch phm b gim
i cng vi s tng lng enzyme c kt hp vo trn mt n v trng lng ca cht mang.
Nhn chung, cc enzyme c nh vn gi c tnh c hiu ca mnh. Thm vo , nh lin kt vi cht
mang m chng c bn vng ln i vi tc ng khc nhau.
a) Hot tnh ca enzyme khng tan ph thuc vo bn cht v tnh cht ha hc ca cht mang
Cc tnh cht l hc ca cht mang nh tnh ha tan, tnh bn vng c hc, trng, in tch, tnh ho
nc v k nc u c nh hng nht nh n lng enzyme c lin kt, tnh bn v hot tnh
sinh hc ca dn xut enzyme c nh. Bn cht ha hc ca cht mang cng c nh hng ng k n
kh nng to thnh dn xut enzyme, v ti kh nng cht mang hp ph khng c trng cc cht t mi
trng phn ng.
Dn xut enzyme khng tan thng c tnh bn nhit cao so vi enzyme tan do kt qu ca s nh v
enzyme to nn. Cht mang cng c tc dng hn ch s bin tnh ca enzyme trong cc dung mi c kh
nng lm t mi lin kt hydrogen v lin kt k nc.
Khi enzyme c gii hn trong phm vi mi trng xc nh, s xy ra nhiu kiu tng tc khc nhau
ca cht mang polymer ln mi trng vi m bao xung quanh phn t enzyme c nh.
Th nht: polymer nh vo nhng tnh cht l ha hc c trng s ko ti b mt ca enzyme (hoc
y khi enzyme) c cht, sn phm ca phn ng v cc phn t khc, iu ny lm tng (hay gim)
nng ca nhng cht ny trong mi trng vi m st enzyme.
Th hai: bn thn cht mang polymer ngn cn s khuch tn t do ca cc phn t theo hng ti
enzyme cng nh i khi enzyme, t nh hng n hiu qu xc tc ca enzyme.
b) Hot tnh ca enzyme khng tan ph thuc vo s khuch tn ca c cht, sn phm v cc
phn t khc
Tc khuch tn ca c cht, sn phm v cc cht khc ph thuc vo cc yu t:
Kch thc l gel ca cht mang polymer
Trng lng phn t ca c cht
S chnh lch nng gia vng mi trng vi m xung quanh enzyme v dung dch t do
Nhng hn ch v mt khuch tn cng c ngha ng k trong qu trnh s dng h thng enzyme c
nh. ng knh l ca cht mang polymer v trng lng phn t ca c cht ng vai tr hng u.
Song nhng hn ch khuch tn khng ch n gin nh vy bi v cn c s khc bit nng gia
vng mi trng xung quanh enzyme v dung dch t do. Hot tnh xc tc ca enzyme dn ti s thay
i nng ca c cht v sn phm phn ng vng mi trng vi m xung enzyme. iu ny lm
xut hin gradient nng ca c cht v sn phm ca dung dch v cht mang cha enzyme c nh.
S xut hin ca gradient nng ni trn c gii thch bi tc khuch tn thp cc phn t trong
gel v hot tnh xc tc cao ca enzyme
Nhng gii hn khuch tn c th c th hin hai dng: hng ro khuch tn bn ngoi v bn trong.
Ro khuch tn bn ngoi xut hin l do c s tn ti ca lp mng dung mi khng pha trn bao xung
quanh ht polymer. Cc cht khuch tn vo lp ny nh c s kt hp ca khuch tn phn t th ng
v s i lu. dy ca lp ny ph thuc vo tc khuy trn ca dung mi xung quanh cc ht
cha enzyme c nh. Vic gia tng tc pha trn s lm gim ro khuch tn bn ngoi, v vy trong
bt k quy trnh cng ngh no, tc khuy o ng vai tr ht sc quan trng.
Ro khuch tn bn trong l s gii hn khuch tn t do bn trong cht mang polymer do chnh cht
mang gy ra. Trong gii hn ca ht polymer ch c s khuch tn phn t th ng khng b nh hng
bi tc pha trn. Ro khuch tn bn trong s biu hin r rng hn nu nh enzyme c c nh
bng cch a n vo trong cht mang polymer ch khng phi gn n vo b mt ca cht mang.
CNG NGH ENZYME 25
c) nh hng ca pH ln enzyme khng ha tan
in tch ca cht mang c nh hng ng k ln pH ti u ca enzyme c nh. pH ti u ca enzyme
c nh c th dch chuyn v pha pH kim hay pH acid so vi pH ti u ca enzyme ha tan. S dch
chuyn pH ti u l do nh hng ca trng tnh in ca cht mang to nn. S c mt ca trng tnh
in mnh dn ti nng H+ gn cht mang v trong dung dch khc nhau. Khi lc ion thp th nng
H+ gn cht mang in tch m cao hn, cn gn cht mang in tch dng thp hn trong dung dch.
Kt qu l pH ti u ca enzyme lin kt ng ha tr vi cht mang c in tch m s chuyn dch sang
pha pH kim so vi enzyme ha tan, ngc li nu enzyme lin kt ng ha tr vi cht mang mang
in tch dng th pH ti u s chuyn dch sang pha pH acid. Cn khi lc ion ln in tch ca cht
mang c lm trung ha bi cc ion tri du v pH ti u ca enzyme c nh gn ging nh ca
enzyme ha tan.
3.3.2. Phng php c nh enzyme
3.3.2.1. Cht mang dng c nh enzyme
Mc ch cui cng ca vic c nh enzyme l gn c enzyme vo mt cht mang no ta c th
thc hin phn ng enzyme nhiu ln. Cng vic ny cn thc hin nhng bc sau:
Chn cht mang ph hp vi enzyme cn gn vo n
Hot ha cht mang cho kh nng gn enzyme tt hn
Tin hnh cc k thut ph hp gn enzyme vo cht mang
Yu cu ca mt cht mang l tng:
mt cht mang l tng s dng trong c nh enzyme iu trc ht l cn phi r. iu ny c lin
quan n hiu qu kinh t ca quy trnh cng ngh, c bit c ngha khi quy trnh ng dng
quy m cng nghip
Cht mang phi c tnh cht c l bn vng, n nh. Nh m cht mang mi chu c cc iu
kin ca mi trng nh khuy trn, p lc trong cc quy trnh sn xut
V mt ha hc, cht mang phi bn vng, khng tan trong mi trng phn ng. Cht mang khng
c lm mt hot tnh enzyme. Cht mang khng gy ra nhng phn ng hp ph khng c hiu.
Cht mang phi c tnh khng khun cao, bn vng vi s tn cng ca vi sinh vt.
Cht mang phi c trng tt, c din tch b mt tip xc ln. Tnh cht ny ca cht mang va
lm tng kh nng c nh enzyme va tng kh nng tip xc ca c cht vi enzyme, nh lm
tng hot tnh ca enzyme c nh v s ln ti s dng
Cht mang c th c cu trc l xp, siu l, c th dng ht, dng mng, dng phim mng
Tt c cc cht mang dng trong c nh enzyme c chia lm hai nhm: cht mang polymer hu c,
cht mang v c
Cht mang l polymer hu c: polymer tng hp (polyacrylamide, polyester, polyvinylalcohol) v
polymer t nhin (polysaccharide: cellulose, agarose, dextran, sephadex v cc dn xut ca chng,
alginate, carrageenan, chitin, chitosan; protein: gelatin, keratin, albumin)
Cht mang v c: si bng thy tinh, silicum oxide, alluminium oxide, mangesium oxide
3.3.2.2. Phng php c nh enzyme
Trong t bo sinh vt tn ti sn c enzyme dng ha tan v c enzyme khng ha tan. Mun thc hin
c cc phng php to enzyme khng ha tan ta phi tch v thu nhn c enzyme t t bo vsv.
Phng php nht enzyme: c 2 phng php:
Nht enzyme trong gel: nht enzyme trong gel l phng php da trn c s to ra mt mng bc
hay mt polymer. Cc cht tham gia phn ng v sn phm ca phn ng c th thm thu vo trong
hoc ra ngoi thng qua mang bao bc ny v enzyem s c gi nguyn trong khun gel .
Cch thc hin: enzyme c trn vo trong mt polymer. Sau tin hnh cc qu trnh trng hp vi
s c mt ca mt hay nhiu tc nhn khu mch, to ra phc chelate. Khi enzyme s c bao bc
trong khong khng ca gel mi to thnh. Polymer c s dng y l acrylamide
Nht enzyme trong h si: Phng php nht enzyme trong h si c kh nng xc tc phn ng tt
hn phng php nht enzyme trong gel. Cc si s dng nht enzyme thng l nhng si nhn
CNG NGH ENZYME 26
to. Cc loi si ny c bn vi acid, kim, cc loi ion v cc dung mi hu c ha tan. Cellulose
acetate thng c s dng nhiu hn c
Phng php to vi nang nht enzyme: cc vi nang c to ra nht enzyme thng c kch thc
1 100m. Cc vi nang c tnh cht l cho cc c cht v sn phm phn ng qua li t do.
Phng php ny c u im l enzyme c nht trong kh bn vi mi tc ng bn ngoi v s
dng c nhiu ln.
Phng php to lin kt enzyme vi cht mang:
y l nhng phng php khng phi nht enzyme vo mt cht mang m gn enzyme vo cht mang.
Nh vy, khng phi tt c cc vt liu u c th s dng lm cht mang m chng phi c tnh cht c
bit mi gn enzyme c vo cht mang. Tnh cht c bit ny:
Cht mang phi bn vi acid, kim
Cht mang pahri gn c vi enzyme theo nhng c ch nht nh
Cht mang khng c tham gia phn ng vi c cht
Ty theo tnh cht ca cht mang, ngi ta thc hin gn enzyme vo cht mang theo nhng c ch sau:
Phng php hp ph: nh kh nng hp ph ca nguyn liu lm cht mang, enzyme s c gn
vo b mt ca cht mang. Kh nng hp ph ca cht mang cng cao, enzyme cng c gn nhiu
vo cht mang. Do , kh nng ti s dng enzyme c nh cng cao. Trong thc t ngi ta thng
s dng cc cht c kh nng hp ph cao nh: cellulose, agarose, chitin, polyacrylamide, nylon,
nha trao i ion, silicagel, thy tinh.
Phng php to lin kt ion: da vo kh nng to lin kt ion gia cht mang v enzyme ngi ta
gn enzyme vo cht mang. Lin kt ion thng khng bn bng s hp ph ca enzyme i vi cht
mang. Cht mang thng s dng: DEAE-sephadex
Phng php to lin kt kim loi: ngi ta lm tng hot tnh b mt ca cht mang bng nhng hp
cht kim loi. T cc enzyme s gn cht hn vo cc cht mang. Nhng cht mang thng c
hot ha b mt bng kim loi: cellulose, gelatin, bng thy tinh, nylon, nhm, silicagel
Phng php to lin kt ng ha tr: da trn nguyn tc ca s tng tc ng ha tr gia phn
t enzyme v cht mang. Cc cht mang ny phi l nhng cht khng c tan trong nc. Phng
php ny c ng dng rt rng trong quy m cng nghip.
Hnh 3.1. S sn xut fructose siro t tinh bt bp s dng enzyme glucose isomerase c nh
C nhiu cng on tch lc v x l gia qu trnh ng ha v ng phn ha phi tha mn cc iu
kin c bn ca enzyme hc: ti u ha n nh nhit ca -amylase c s dng trong giai on
ha lng (nhit ca giai on ny vo khong 105oC) th cn s dng ion canxi. Mt khc, nhng ion
ny c ch glucose isomerase, v chng c loi b nh qu trnh trao i ion trc khi dung dch
ng dextrose c cho vo thit b thc hin phn ng isomer ha.
CNG NGH ENZYME 28
3.4.2. Sn xut L-amino acid nh enzyme aminoacylase c nh
Nhu cu s dng L-amino acid trong thc phm v y hc gia tng nhanh chng. L-amino acid c
tin hnh nghin cu sn xut t cng ngh vi sinh vt hay bng phng php tng hp ha hc. Cc
amino acid c tng hp bng phng php ha hc thng gp mt s bt li do to ra c dng ng
phn D-amino acid, dng ng phn ny khng c gi tr dinh dng. Do , trong qu trnh sn xut
ngi ta mong mun ch thu c dng ng phn L trong sn phm cui.
Nguyn l chung ca phn ng chuyn ha Dl-amino acid thnh dng L-amino acid di s xc tc bi
enzyme aminoacylase:
Phn ng xy ra trong mt thit b dng ct c cha enzyme aminoacylase c nh. L-amino acid mong
mun c tch ra khi dng acyl D-amino acid da trn s khc bit v ha tan. Sau D-acylamino
acid c racemic ha thnh dng DL-amino acid tip tc quay tr li l phn ng c cha enzyme
aminoacylase c nh.
Hnh 3.2. S sn xut L-amino acid s dng amino acylase ca cng ty Taabe Seiyaku, Nht Bn
Hin nay, loi aminoacylase gn vi DEAE-Sephadex bng lin kt ion c chn s dng bi v n c
hot tnh cao, d dng chun b, c kh nng ti sinh v n nh.
4. THU NHN ENZYME
4.1. Chn ngun nguyn liu
Enzyme l nhng cht xc tc sinh hc, c nhiu trong c th sng. Vic iu ch chng bng phng
php ha hc vi s lng ln l vic lm rt kh khn v y tn km nu khng mun ni l iu
khng tng, nn ngi ta thng thu nhn chng t cc ngun sinh hc. Mc d enzyme c trong tt c
cc c quan, m ca ng vt thc vt cng nh trong t bo vi sinh vt, song vic tch enzyme p ng
yu cu v mt kinh t ch c th tin hnh khi nguyn liu c cha mt lng ln enzyme cng nh cho
php thu c enzyme vi hiu sut cao v d dng tinh ch chng. Vic phn b ca enzyme trong t
bo cng khng ng u, trong mt loi t bo cng c th c nhiu enzyme ny song khng c enzyme
khc. Lng enzyme li thay i ty theo giai on sinh trng pht trin ca sinh vt v ty theo loi
nn chng ta phi chn ngun nguyn liu thch hp cho vic chit rt v tinh ch enzyme. C ba ngun
nguyn liu sinh hc c bn:
CNG NGH ENZYME 29
Cc m v c quan ng vt
M v c quan thc vt
T bo vi sinh vt.
Cc m v c quan ng vt
Trong tt c cc nguyn liu c ngun gc ng vt th tuyn tu, mng nhy d dy, tim... dng tch
enzyme rt thun li. Dch tu tng c cha amylase, lipase, protease, ribonuclease v mt s enzyme
khc.
T ngn t ca d dy b ngh ngi ta c th thu nhn ch phm renin lm ng sa trong sn xut
fomat. Ngi ta cng sn xut pepsin t d dy ng vt. Nhng khc vi pepsin, renin c kh nng
ng t sa cao m khng thy phn su sc casein. Renin l ch phm enzyme c gi tr ln trong cng
nghip.
M v c quan thc vt
thc vt: thng thng enzyme hay c mt cc c quan d tr nh ht, c, qu. C quan d tr giu
cht g th nhiu enzyme chuyn ha cht y. V d trong ht cy thu du c nhiu lipase, trong ht u
tng c nhiu enzyme urease.
Thc ny mm cha nhiu - amylase, c khoai lang li c nhiu - amylase. Ngi ta thu c
mt s ch phm enzyme thy phn nh papain, bromelain, fixin t thc vt bc cao.
Papain thu c t mu nha u xanh, bromelain thu c t cc b phn (l, thn, qu) cy da, cn
fixin c tch t dch p thn v l cy Ficus.
Qua cc ngun nguyn liu ng, thc vt chnh c th t chit xut cc ch phm enzyme, chng ta
thy rng hai ngun nguyn liu ny khng th dng sn xut cc ch phm enzyme vi quy m ln
bi cc nhc im sau y:
Chu k sinh trng ca chng di
Ngun nguyn liu ny khng ci to c.
Nhiu nguyn liu dng lm thc phm (dng n) khng th dng lm nguyn liu sn xut vi
quy m ln cc ch phm enzyme nhm tho mn cc nhu cu ca nn kinh t quc dn.
Dng vi sinh vt lm ngun nguyn liu sn xut cc ch phm enzyme c nhiu u im ni bt v c
tnh cht c o vt xa so vi ngun nguyn liu t ng vt, thc vt, cng nh s khc phc c
mi kh khn v hn ch trn.
T bo vi sinh vt
Trc ht vi sinh vt l ngun nguyn liu v tn sn xut enzyme vi s lng ln. y cng l
ngun nguyn liu m con ngi ch ng to ra c. Chu k sinh trng ca vi sinh vt ngn (t 16 -
100 gi) v vy c th nui cy hng trm ln trong nm.
Enzyme vi sinh vt c hot tnh rt mnh, vt xa cc sinh vt khc. V vy ch cn mt lng nh
enzyme c th chuyn ha mt lng ln c cht. S liu tnh ton cho bit, trong vng 24 gi, vi sinh
vt c kh nng chuyn ha mt lng thc n gp 30 - 40 ln so vi trng lng c th chng. Trong khi
, h enzyme ca con ln trn 50 kg ch c th chuyn ha c vi kg thc n trong ngy.
H enzyme vi sinh vt v cng phong ph. Vi sinh vt c kh nng tng hp nhiu loi enzyme khc
nhau, trong c nhng enzyme ng, thc vt khng tng hp c. V d cellulase, raxemase...Phn
ln cc thc n nui vi sinh vt li d kim v gi r. Nhiu vi sinh vt cho enzyme thng c kh
nng pht trin trn cc mi trng n gin, gi r, d kim nh cc ph liu ca cc ngnh sn xut.
Hn na, c th dng nhng nguyn liu khng phi thc phm, nhng dung dch mui v c nui vi
sinh vt. V vy dng vi sinh vt lm ngun thu enzyme s mang li gi thnh r, thi gian nhanh v hiu
qu kinh t cao.
Vi sinh vt sinh sn pht trin vi tc cc k nhanh chng, khi lng li nh, kch thc b, nhng t
l enzyme trong t bo tng i ln nn quy trnh sn xut ch phm enzyme kh d dng, hiu sut thu
hi cao. Lng enzyme c th c sn xut ra trong mt thi gian ngn. i vi mt s trng hp c
th dng 100% sinh khi vi sinh vt lm ngun enzyme.
CNG NGH ENZYME 30
Vi sinh vt rt nhy cm i vi tc ng ca mi trng, thnh phn dinh dng nui chng cng nh
mt s tc nhn l ha, c hc khc. Do c th thay i nhng iu kin nui cy chn ging to
nhng chng t bin cho ta hm lng enzyme ng k vi hot tnh xc tc cao. C th ni rng, nh
ngun enzyme vi sinh vt, ngi ta c th iu khin s tng hp enzyme d dng hn cc ngun nguyn
liu khc tng lng enzyme c tng hp hoc tng hp nh hng enzyme.
Tuy vy trong qu trnh chn ngun nguyn liu t vi sinh vt, cn lu mt s vi sinh vt c kh nng
sinh c t c bin php x l thch hp. Ni chung cc vi sinh vt mun c s dng lm ngun
nguyn liu tch enzyme cn phi tho mn cc iu kin sau:
Kh nng tng hp enzyme mnh trong mt thi gian ngn.
D tch enzyme v khng sinh c t.
C mt iu l th l: trong iu kin bnh thng, vi sinh vt ch tng hp ra mt lng enzyme va
cho hot ng sinh l c th ca chng (thng c gi l s tng hp enzyme "bn th"). Nu khi tng
hm lng mt s cht hoc thm mt s cht mi vo mi trng nui cy, c bit l c cht ca
enzyme, th s tng hp enzyme tng ng tng ln mt cch ng k, khc thng c khi cn tng hp
enzyme mi: hin tng trn gi l s cm ng sinh tng hp enzyme. Cht gy nn s cm ng sinh
tng hp gi l cht cm ng. S tng hp mt lng ng k enzyme gi l siu tng hp enzyme.
thu c ngun enzyme di do t vi sinh vt, cn phi nui cy chng. C hai phng php nui
cy vi sinh vt thu enzyme: phng php nui cy b mt v phng php nui cy b su hay l
phng php ni v phng php chm.
Trong phng php nui cy b mt, ngi ta cho vi sinh vt pht trin v bao ph trn b mt cc hot
cht dinh dng rn, c lm m, dng lm mi trng (cm go, cm np, cm m, bp xay nh...).
mi trng xp ngi ta trn thm mt lng nh mt ca... Sau khi nui thi gian vi sinh vt
tng hp enzyme mi trng c sy nh, nghin nh. Ch phm thu c dng rn - th. Mun c
ch phm tinh khit phi qua giai on tch v tinh ch enzyme.
Khc vi phng php nui cy b mt, trong phng php nui cy b su ngi ta cho vi sinh vt pht
trin trong mi trng lng. Nguyn liu chnh v ph bin l dch ng glucose, fructose, maltose,
saccharose... dch thy phn cellulose, tinh bt... Ngun nit hu c thng dng l nc chit
bp, chit malt, dch t phn nm men. Cn chn pH ph hp vi chng vi sinh vt v s tng hp
enzyme theo mong mun. Sau khi nui, ta thu c canh trng lng - dng th.
lm tng lng enzyme vi sinh vt chng ta cn ch tuyn la v chn ging cc chng vi sinh vt
c hot tnh enzyme cao, tng hp c enzyme cn thit v vi s lng nhiu. Cc chng c phn
lp theo phng php thng thng ch tng hp mt lng nh enzyme (enzyme bn th), do cn tin
hnh gy t bin bng cc phng php sinh hc, l, ha hc... to chng c kh nng siu tng hp
enzyme. Vi sinh vt sau khi c tuyn chn, cn c nhn ging v nui trong iu kin ti u
chng sinh trng tt, tng hp nhiu enzyme.
Ngoi ra cn phi chn mi trng v thnh phn mi trng dinh dng c nh hng trc tip n s
sinh trng v tng hp enzyme ca vi sinh vt. Trong thnh phn mi trng phi c cc cht m
bo c s sinh trng bnh thng ca vi sinh vt v tng hp enzyme.
c bit lu l tng s tng hp enzyme ngi ta thng da vo hin tng cm ng. V nu nh
trong thnh phn mi trng c cc cht cm ng th cht hay sn phm phn gii ca n s km hm
hoc lm yu tc dng km to ca cht km hm nhm bo m kh nng sinh tng hp enzyme cho
khng b cn tr. Cht cm ng tng hp enzyme cho thm vo mi trng nui thng l c cht tng
ng ca enzyme cn tng hp.
V d: Mun tch - amylase nm mc (Asp.Oryzae), ngi ta cho vo mi trng nui cy tinh bt,
maltose, isomaltose, oligosaccharid... c cha lin kt - 1,6 glucozid. Mun tch pectinase Asp.Niger,
ngi ta cho thm vo mi trng pectin. i vi hemicellulase th cht cm ng l hemicellulose; cn
i vi proteinase cht cm ng c hiu lc l protein, bt u nnh, lng, sng nghin nh (
Actinomyces fradiae). Cht cm ng cng c th l nhng cht ging c cht v nhng sn phm thy
phn ca chng. V d: thay cho protein th peptid v thay cho tinh bt th erithrodextrin u c tc dng
cm ng.
C nhiu yu t nh hng i vi mi trng nui cy. Nhit nui cy thng thng t 25 30oC.
Tr s pH ban u ca mi trng (ch yu mi trng nc) cng c th gy nh hng no n s
CNG NGH ENZYME 31
too thnh enzyme, nhng khi ccng cn tnh n kh nng bin ii nhanh chng ch ch s bi vi sinh
vt. Thng thng i vi - amylase, pH tti u cho s sinh tng hp p (pH = 7 - 8) khc vi pH ti u
cho hot ng ca n (pH = 4,7 - 4,9). Cc enzyme ng ha khc ca nm m c nh glucoamylase th
pH ti u cho s sinh tng hpp v cho ho hot ng l chung nhau (4,5 - 5,0). thng kh cng rt cn
thit cho vic sinh tng hpp enzyme. V vvy mi trng b mt ngi ta thngng thm cht
ch xp nh tru
vo, cn mi trng b su (mi tr
trng dch th) , th ngi ta thng lc (nu
u enzyme cn
c lc th vic
ny cc k quan trng). m m ccng rt quan trng (ch c tc dng nui cy y b
b mt), ph thuc vo
thnh phn mi trng b mt.
Mt iu cn ni thm naa l enzyme th thng cha cc t bo sinh vt gii l cc
c enzyme trong t bo
(intracellular), nhng n cng
ng c th
th c cc sinh vt tit ra mi trng sng.
ng. l cc enzyme ngoi t
t
bo (extracellular). Enzyme vi sinh vvt thng chit l enzyme ngoi bo.
4.2. Thu nhn enzyme
Quy trnh thu nhn enzyme t ngu
ngun thc vt v ng vt
Nghin
Trch ly
Lc
Kt ta
Canh trng ln
men vi sinh vt
Phn ly lng/rn
Canh trng
Lc
Kt ta