khảo sát điều kiện cố định amylase trên silica-chitosan và silica-alginate

C nh amylase trn vt liu phi hp silica gel
SVTH: Nguyn Ngc Trm
LI CM N
u tin xin cho em gi li cm n su sc nht n cha m, cc ch v em trai ca
em, nhng ngi khng ngng ng hnh v ng vin em trn tng chng ng kh
khn, gian kh em c ng lc phn u v t thnh tu nh ngy hm nay.
Xin chn thnh cm n:
- Xin gi li tri n n PGS. TS. Nguyn c Lng, mt cn b lo thnh ca
ngnh dc ht lng, cng sc gy dng cho chng em mt chng trnh hc tp tht
logic v gip chng em c ci nhn bao qut v ngnh Cng ngh Sinh hc, cng nh to
cm hng cho chng em, nh hng cho chng em c c s la chn ng n cho
hng i sau ny ca mnh.
- Tin s Hunh Ngc Oanh B mn Cng ngh Sinh hc, i hc Bch Khoa Tp.
H Ch Minh rt nhit tnh hng dn, trao i v gip tn tnh trong sut ba thng
lm lun vn.
- Cc Thy C trong B mn Cng ngh Sinh hc tn tnh ging dy v truyn
t nhng kin thc qu bu cho chng em.
- Cc bn cng lp HC07BSH i hc Bch Khoa Tp. H Ch Minh cng em
trao i, ng vin nhau trong thi gian lm lun vn.
Do thi gian nghin cu v thc hin lun vn cn hn hp nn kh trnh khi nhiu
iu s st. Em rt mong nhn c nhng li khuyn v gp t cc Thy, C trong B
mn ti ny ngy cng hon thin hn.
Em xin chn thnh cm n!
GVHD: TS. Hunh Ngc Oanh
SVTH: Nguyn Ngc Trm
TM TT TI
Ngy nay enzyme c nh c s dng rt rng ri trong cc ngnh cng nghip
nh vo kh nng ti s dng v kh nng iu khin qu trnh sn xut bng enzyme.
Nghin cu ny kho st kh nng c nh Termamyl 120L trn silica-chitosan v silicaalginate. Enzyme c nh trn silica-chitosan c nhit v pH ti u ln lt l 60oC,
pH 8 v 60oC, pH 5,5 i vi enzyme c nh trn silica-alginate. Kh nng ti s dng
ca -amylase c nh trn silica-alginate (42 ln) cao hn kh nng ti s dng ca amylase c nh trn silica-chitosan (30 ln). S ng c nh glucoamylase v -amylase
mang li hiu qu cao v c th ti s dng ln ti 44 ln 50oC, pH 5,5. Cc phn ng
c thc hin trong 30 pht, iu kin nhit v pH thch hp.
ii
SVTH: Nguyn Ngc Trm
ABSTRACT
Nowadays the immobilized enzyme used universally in industrials thanked to
capability re-use and industrial process control capability by enzyme. In this
research, a thermostable -amylase was immobilized by covalent bond onto silicachitosan beads and entrapment in silica-alginate beads. The optimum temperature
and pH of the enzyme -amylase, immobilized by covalent bond onto silicachitosan are 60oC, pH 8, respectively. For the immobilization enzyme -amylase
entrapped in silica-alginate, the optimum temperature and pH are 50 oC and pH 5,5,
respectively. The capability re-use of the immobilized enzyme -amylase entrapped
into silica-alginate beads (42 times) was higher than the immobilized enzyme onto
silica-chitosan beads (30 times). Co-immobilization glucoamylase and -amylase in
silica-alginate beads resulted in high yields and could re-use up to 44 times at 50oC
and pH 5,5. The reactions were performed within 30 minutes, at suitable
temperature and pH value.
iii
SVTH: Nguyn Ngc Trm
MC LC
LI CM N ..........................................................................................................i
TM TT TI................................................................................................. ii
MC LC .............................................................................................................. iv
DANH MC HNH ................................................................................................ vi
DANH MC BNG ........................................................................................... viii
Chng 1:
M U .......................................................................................... 1
1.1. L do chn ti ........................................................................................ 1

1.2. Mc tiu nghin cu ................................................................................... 1
1.3. Gii hn ca ti ........................................................................................ 2
Chng 2:
TNG QUAN .................................................................................. 3
2.1. Khi nim v c nh enzyme ..................................................................... 3

2.1.1.
Lch s pht trin v tnh hnh nghin cu enzyme c nh trong v
ngoi nc ....................................................................................................... 3
2.1.2.
c im ca enzyme c nh........................................................... 4
2.1.3.
u, nhc im ca enzyme c nh ................................................ 5
2.2. Vt liu c nh .......................................................................................... 6

2.2.1.
Silica gel ........................................................................................... 8
2.2.2.
Chitosan.......................................................................................... 10
2.2.3.
Alginate .......................................................................................... 12
2.3. Hot ha vt liu bng glutaraldehyde ...................................................... 15

2.4. Enzyme amylase ....................................................................................... 16
2.4.1.
-amylase ....................................................................................... 16
2.4.2.
Glucoamylase ................................................................................. 18
2.5. iu kin phn ng khi tin hnh xc nh hot enzyme ...................... 20

2.5.1.
Cc phng php xc nh hot enzyme .................................... 20
2.5.2.
Mt s lu khi xc nh hot enzyme ...................................... 21
Chng 3:
VT LIU V PHNG PHP ................................................... 22
3.1. Nguyn vt liu ........................................................................................ 22

3.2. Thit b ..................................................................................................... 23
3.3. Cc phng php phn tch....................................................................... 23
iv
SVTH: Nguyn Ngc Trm
3.4. Phng php nghin cu .......................................................................... 23

3.4.1.
To bt silica gel............................................................................. 23
3.4.2.
Chun b ht chitosan ...................................................................... 24
3.4.3.
Chun b ht silica-chitosan ............................................................ 24
3.4.4.
Hot ha ht silica-chitosan vi glutaraldehyde .............................. 24
3.4.5.
C nh Termamyl ln cc cht mang ............................................. 24
3.4.7.
Kho st nhit v pH ti u ........................................................ 25
3.4.8.
S ti s dng ................................................................................. 26
Chng 4:
KT QU v BN LUN ............................................................. 27
4.1. Enzyme t do............................................................................................ 27

4.2. Termamyl c nh trn silica gel ............................................................... 27
4.2.1.
Hiu sut c nh Termamyl trn silica gel ..................................... 27
4.2.3.
Kho st pH ti u ca Termamyl c nh trn silica gel ................ 28
4.2.4.
Ti s dng Termamyl c nh trn silica gel.................................. 29
4.3. Termamyl c nh trn silica-chitosan v silica-alginate ........................... 30

4.3.1.
iu kin c nh ............................................................................ 30
4.3.2.
iu kin phn ng ......................................................................... 35
4.3.3.
Kho st kh nng ti s dng ca -amylase c nh ..................... 38
4.4. ng c nh glucoamylase v Termamyl trn silica-alginate................... 44

4.4.1.
Kho st nhit ti u .................................................................. 44
4.4.2.
Kho st pH ti u .......................................................................... 45
4.4.3.
Ti s dng hai enzyme ng c nh trn silica-alginate ............... 46
KT LUN V KIN NGH................................................................................ 49

TI LIU THAM KHO...................................................................................... 51
PH LC A: CC PHNG PHP PHN TCH .............................................. 54
PH LC B:
BNG TNH TON ................................................................... 57
SVTH: Nguyn Ngc Trm
DANH MC HNH
Hnh 2.1: Silica gel .................................................................................................. 8
Hnh 2.2: Tng hp ht silica gel ............................................................................. 9
Hnh 2.3: Chitosan ................................................................................................. 10
Hnh 2.4: Acid alginic ............................................................................................ 13
Hnh 2.5: Cu to ca alginate ............................................................................... 13
Hnh 2.6: Cu trc ht gel alginate ......................................................................... 14
Hnh 2.7: Glutaraldehyde hot ha chitosan ........................................................... 15
Hnh 2.8: Glucoamylase thy phn amylose .......................................................... 19
Hnh 3.1: Silica gel ................................................................................................ 23
Hnh 4.1: Kho st nh hng ca nhit ln hot tnh ca Termamyl c nh trn
silica gel ................................................................................................................ 28
Hnh 4.2: Kho st nh hng ca pH n hot tnh ca Termamyl c nh trn
silica gel ................................................................................................................ 28
Hnh 4.3: Kho st kh nng ti s dng ca Termamyl c nh trn silica gel ...... 29
Hnh 4.4: Kho st lng silica gel thm vo chitosan ........................................... 30
Hnh 4.5: Ht silica-chitosan .................................................................................. 31
Hnh 4.6: Kho st lng silica gel thm vo alginate ............................................ 32
Hnh 4.7: Ht silica-alginate .................................................................................. 33
Hnh 4.8: Kho st lng Termamyl c nh trn silica-chitosan ........................... 34
Hnh 4.9: Kho st nhit ti u ca Termamyl c nh trn silica-chitosan ....... 35
Hnh 4.10: Kho st nhit ti u ca Termamyl c nh trn silica-alginate ...... 36
Hnh 4.11: Kho st pH ti u ca Termamyl c nh trn silica-chitosan ............. 37
Hnh 4.12: Kho st pH ti u ca Termamyl c nh trn silica-alginate .............. 38
Hnh 4.13: Kh nng ti s dng ca Termamyl c nh trn silica-chitosan ......... 40
vi
SVTH: Nguyn Ngc Trm
Hnh 4.14: Kh nng ti s dng ca Termamyl c nh trn silica-alginate .......... 43

Hnh 4.15: Kho st nhit ti u ca glucoamylase v Termamyl ng c nh
trn silica-alginate .................................................................................................. 45
Hnh 4.16: Kho st pH ti u ca Termamyl v glucoamylase ng c nh trn
silica-alginate......................................................................................................... 46
Hnh 4.17: Kh nng ti s dng ca glucoamylase v Termamyl ng c nh trn
silica-alginate......................................................................................................... 47
vii
SVTH: Nguyn Ngc Trm
DANH MC BNG
Bng 4.1: Cc thng s k thut ca amylase v glucoamylase .............................. 27
Bng 4.2: Hiu sut c nh -amylase trn silica gel ............................................ 27
Bng 4.3: S ln ti s dng -amylase c nh trn silica-chitosan ....................... 39
Bng 4.4: S ln ti s dng -amylase c nh trn silica-alginate ....................... 41
Bng 4.5: S ln ti s dng enzyme ng c nh trn silica-alginate................... 48
viii
Chng 1:
1.1.
SVTH: Nguyn Ngc Trm
M U
L do chn ti
Amylase xc tc thy phn cc c cht ln, nh ti cc lin kt -1,4 glucoside
(-amylase), lin kt -1,4 v lin kt -1,6 glucoside (glucoamylase) v to sn

phm a dng nh: cc oligosaccharide c gc kh, cc di-, tri- v tetrasaccharide,
glucose v isomaltose Tuy nhin, vic s dng enzyme t do gy nhiu kh khn
trong vic ng dng ln vic ti s dng (s ra tri, b bin tnh bi nhit, pH)
enzyme. Kt qu l lm tng chi ph gi thnh sn phm. tng tnh bn v n
nh trong hot tnh ca enzyme khi nhng tc nhn l ha gip vic ng dng
enzyme c thun tin v tit kim c chi ph. T , tng c nh enzyme
trong cht mang c ra i.
Hiu qu c nh enzyme bng cch s dng mt s phng php, mt trong
s l phng php by trong gel, lin kt cng ha tr. Enzyme s c c nh
vo mng li cc vi l trong ht cht mang (alginate) hoc trn b mt cc ht
(silica gel, chitosan). Phng php by trong alginate v phng php c nh
amylase trn b mt nh lin kt cng ha tr vi chitosan, hp ph trn silica gel rt
d thc hin, chi ph thp v d s dng. Hn na, bng cch thay i iu kin to
gel, n c th d dng kim sot mt s c tnh, chng hn nh dy hoc
thm thu cc cht nn khc nhau ca ht gel.
nhng nghin cu trc y, cht mang chitosan v alginate c dng c
nh amylase c nghin cu v cho kt qu tt [3, 4, 19, 20]. Tuy nhin, ch
l vt liu hu c, khng bn di cc tc nhn l ha, trong khi , vt liu v c
(silica gel) c th khc phc c nhc im ny. T yu cu trn, chng ti
tin hnh nghin cu th nghim ti: Kho st iu kin c nh amylase trn
silica-chitosan v silica-alginate.
1.2.
Mc tiu nghin cu
- Kho st iu kin c nh Termamyl ln ht silica-chitosan bng lin kt
cng ha tr.
SVTH: Nguyn Ngc Trm
- Kho st iu kin c nh Termamyl bng phng php by, nht trong gel
silica-alginate.
- Kho st nhit v pH ti u.
- Kho st kh nng ti s dng ca enzyme c nh.
1.3. Gii hn ca ti
V thi gian c hn nn ti ch dng bc kho st kh nng ti s dng
ca enzyme c nh, cha kho st kh nng bo qun ca enzyme c nh theo thi
gian, cng nh thc hin ti u ha cc yu t nh hng n enzyme c nh (theo
phn mm ti u Design Expert 8.0).
Chng 2:
SVTH: Nguyn Ngc Trm
TNG QUAN
Khi nim v c nh enzyme
2.1.
Enzyme c nh (immobilized) hay enzyme khng ha tan (insoluble enzyme)

c gn c nh vo mt vng, mt khoang nht nh, khng b ha tan trong cc
iu kin bnh thng, vn gi c hot ng xc tc [1].
Enzyme c nh c y tnh cht ca mt enzyme v c th s dng c lin
tc v lp li nhiu ln. Cc cht dng gn hoc gi enzyme thng c gi l
cht mang hay gi th. Nh vy, nu gn enzyme xc tc theo mt dy phn ng theo
ng trt t (h thng nhiu enzyme, multi-enzyme) s nhn c h thng enzyme
khng tan xc tc cho mt dy phn ng chuyn ha t c cht n sn phm cui
cng [1].
2.1.1. Lch s pht trin v tnh hnh nghin cu enzyme c nh trong v ngoi
nc
- Enzyme c nh c nghin cu u tin vo nm 1916 bi Nelson v Griffin
khi hai ng ny quan st kh nng thy phn ng saccharose ca enzyme invertase
t nm men hp ph ln than hot tnh.
- 1956, Mizt c nh enzyme catalase trn DEAE-cellulose bng lin kt ion
(ionic binding).
- 1964, Quiciio v Richards m t phng php khu mch (cross-linking) cc
enzyme carboxyl-peptidase vi glutaraldehyde. Chang tin hnh to vi nang nht
enzyme carbonic anhydrase (microcapsule).
- 1969, Wilson xy dng thnh cng xng thc nghim sn xut glucose
bng glucoamylase c nh. Chibata l ngi u tin thc hin thnh cng cng vic
sn xut vo cng nghip.
- 1973, Chibata v cng s ln u tin c nh thnh cng t bo vi sinh vt E.
coli sn xut L-aspartate t amonium fumarate bng gel acrylamide.
- T nhng thnh tu t c trn m ra nhng ng dng to ln ca
enzyme v t bo c nh trong cng nghip. Mt trong nhng bng chng thuyt
SVTH: Nguyn Ngc Trm
phc nht l s dng glucoisomerase c nh trong sn xut fructose t glucose quy

m cng nghip.
- Trn th gii hin nay s dng rng ri cng ngh c nh enzyme vo trong
cc lnh vc cng nghip thc phm, y hc, cng nghip ha cht, bo v mi trng
v cc lnh vc phn tch.
- Vit Nam, nghin cu c nh enzyme mi ch bt u vi nm gn y v
thu c kt qu cn rt hn ch.
- 1994 1995, Vin Sinh hc Nhit i cng tc vi Trng i hc Khoa
hc T nhin Thnh ph H Ch Minh nghin cu c nh enzyme glucoisomerase
trn cc ht cylochrom B, hot ha bng glutaraldehyde. Trng i hc Khoa hc T
nhin c nhiu cng trnh nghin cu c nh cc enzyme thy phn nh protease,
amylase, urease, pectinase Hin nay, Vin Sinh hc Nhit i cng ang nghin cu
s dng ch phm enzyme glucoisomerase (Novo, an Mch) sn xut fructose
syrup, tuy nhin mi ch quy m phng th nghim.
- Nhng nm gn y, phng th nghim cng ngh bc x, Vin Ht Nhn
Lt c nhiu cng trnh nghin cu c nh enzyme v t bo, cc cht c hot tnh
sinh hc khc bng k thut bc x nh c nh enzyme glucoamylase protease vi
khun t (Vibrio cholerae), t bo nm men, vi khun x l nc thi (Pseudomonas
maltophilla) v hormon ptrogesteron trn cc gi th polymer tng hp c ch to
bng k thut bc x.
2.1.2. c im ca enzyme c nh
Enzyme c nh c nhng c im khc vi enzyme ha tan nh:
- Enzyme c nh bn nhit hn enzyme ha tan do chng c bo v bi cht
mang.
- C kh nng bo qun tt hn.
- C s di chuyn pH ti thch sang kim hay acid so vi enzyme ha tan.
- C kh nng ti s dng c nhiu ln.
SVTH: Nguyn Ngc Trm
- Enzyme c nh tun theo nh lut Michaelis-menten, tuy nhin cng c s sai

khc nh:
Xy ra hin tng cnh tranh gia c cht, enzyme v cht mang.
Hin tng cn tr s khuch tn c cht v sn phm ca phn ng dn
n vn tc phn ng gim.
-
Hot tnh ca enzyme c nh thng thp hn enzyme ha tan cng loi
do 2 nguyn nhn sau:

Do nh hng in tch ca cht mang nn lm thay i cu trc khng
gian ca enzyme.
Do enzyme b nht vo trong gel nn s tip xc gia enzyme v c cht
vi tm hot ng kh khn.
2.1.3. u, nhc im ca enzyme c nh
Enzyme c nh ngy cng c s dng rng ri trong nhiu lnh vc do nhng
u im sau:
- Hiu qu kinh t cao, do enzyme c nh l ch phm sinh hc rt t tin nu
s dng dng ha tan th ch s dng c mt ln, kh thu hi. Khi enzyme c c
nh trn gi th th s dng c nhiu ln, gi thnh gim, nng cao hiu qu kinh
t.
- Sn phm thu c sau phn ng c xc tc bng enzyme c nh kh tinh
sch do enzyme c c nh trn nhng pha ring r nn khng ln vo sn phm.
iu ny c bit quan trng trong sn xut dc phm, trong cng ngh sn xut ha
cht tinh khit v trong phn tch.
- Khi thc hin bng phn ng, enzyme c nh c th dng phn ng bt k
giai on no cn thit, ch cn tch enzyme c nh ra khi c cht. i vi nhng
trng hp yu cu sn phm ch l cc sn phm trung gian th iu ny c ngha
quan trng.
- C thi gian bo qun lu hn v bn vng hn i vi cc cht km hm cng
nh cc tc nhn gy bin tnh so vi dng ha tan.
SVTH: Nguyn Ngc Trm
- V c c nh trn gi th nn enzyme c hot tnh n nh hn so vi

enzyme t do khi c s tc ng ca cc yu t mi trng nh: nhit , pH
- Enzyme c nh c bit thch hp vi cc quy trnh cng ngh lin tc, t
ng ha. Enzyme c nh c np vo cc thit b phn ng nh ct, thp vi dng
c cht i vo lin tc v u ra l sn phm.
Tuy nhin, bn cnh nhng u im th enzyme c nh cng c nhng nhc
im sau:
- Lm hn ch kh nng tip xc gia enzyme v c cht. V vy hot tnh ring
(specfic activity) ca enzyme c nh thng thp hn so vi enzyme ha tan. c
bit trong trng hp c cht c trng lng phn t ln nh: protein, tinh bt,
chitosan
- Trng hp enzyme c nh bng phng php nht (entrapment method)
trong khun gel th ch phn enzyme nm lp ngoi ca gel l hot ng.
Enzyme c c nh bng phng php cng ha tr th mt lng ng k
enzyme b mt hot tnh do cht hot ha v do phn ng gn khng c hiu xy ra
trung tm hot ng ca enzyme.
2.2.
Vt liu c nh
Theo Trevan mt cht mang l tng cn c nhng tnh cht sau:
- Gi thnh r l yu cu u tin ca mt cht mang c s dng trong c nh
enzyme.
- Phi c tnh cht c l bn vng, n nh th mi chu ng 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 v khng gy ra nhng hp ph
khng c hiu.
- Cht mang phi c tnh khng khun cao, bn vng vi s tn cng ca vi sinh
vt..
SVTH: Nguyn Ngc Trm
- Cht mang phi c trng n tt, din tch b mt tip xc ln. Tnh cht
ny ca cht mang va tng kh nng c nh enzyme, va tng kh nng tip xc ca
c cht vi enzyme, nh lm tng hot tnh 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 phin mng
Cht mang polymer hu c: t nhin v tng hp:
Nhm cc polymer t nhin:
Polysaccharide: cellulose, agarose, dextran, sephadex v cc dn xut ca chng.

Cellulose v cc dn xut nh carboxymethyl cellulose, diethylaminoethyl
cellulose. Agarose: n nh, t tin nn s dng ch yu trong y hc. Alginate,
carragenan: to gel tt nhng khng n nh trong mi trng c photphat.
Tinh bt: phong ph, r nhng km trng n, kh nng c nh km nn thng
c ghp vi cc copolymer lm tng trng n, ci thin tnh cht c l, tng kh
nng c nh vi sinh vt.
Chitin, chitosan: c cu trc siu l, d to mng, to ht, bn c l, n nh. Tuy
nhin k nc, trng n km nn ci thin ghp copolymer vi cc vinyl
monomer a nc.
Protein: thng s dng gelatin, keratin, albumin. D to mng, to ht. Tuy
nhin km bn, d nhim khun hay gy cc phn ng min dch.
Nhm cc polymer tng hp (synthetic polymer):
Polyacrylamide, polyester, polyvinylalcohol, polyvinylacetate, polyacrylic,

polystyren, polyethylen ghp vi cc vinylmonomer.
Bn c l, bn nhit, bn vi sinh vt, tr ha hc, trng n tt, c th iu
chnh kch thc siu l. Tuy nhin, gi thnh cao, qu bn vng nn gy nhim mi
trng khi kh phn hy.
Cht mang v c:
Gm si bng thy tinh, silicium oxide SiO2, alluminium oxide Al2O3, MgO
c cu trc l, c kh nng hp ph tt.
SVTH: Nguyn Ngc Trm
Ngoi ra cn c ceramic, celite (diatomic).

Trong nghin cu ny, chng ti s dng vt liu v c l silica gel (SiO2) v vt
liu hu c l chitosan v alginate. Enzyme c dng c nh l Termamyl 120L
v AMG 300L ca hng Novozyme.
2.2.1. Silica gel
2.2.1.1. Tnh cht ca silica gel
Silica gel hay gel acid silicic l mt cht sn c trong t nhin, cng thm vi
tnh nng u vit ca n trong cc qu trnh ha hc to nn cho silica gel mt v th
ng c trn trng. Cng thc ha hc n gin ca n l SiO2.nH2O (n < 2). iu
ch bng cch cho sodium silicate tc dng vi acid sunfuric kt qu to thnh dng
sol, ri sol ng t li thnh gel, sau khi ra, sy kh v nung ta thu c silica gel.
l cht rn c l xp nh, dng cc hoc vin hnh cu tu thuc phng php to
ht khi iu ch, c loi trong sut nh thy tinh, c loi c. xp thay i trong
gii hn 20 - 60%, ng knh l xp khong 3 - 10 nm, b mt ring 200 - 800 m2/g.
Hnh 2.1: Silica gel [38]

Silica gel l mt cht v c bn, khng c, d bo qun v vn chuyn d dng.
Hin nay silica gel c vai tr rt quan trng trong cng ngh ha hc t n gin n
phc tp. Silica gel c dng rt nhiu lm xc tc trong tng hp hu c ha du,
lc nc... Ngoi nhng u im v tnh ht m, cht ph b mt Nhng nm gn
SVTH: Nguyn Ngc Trm
y, silica gel c ch n nh l mt cht c nh enzyme l tng. Vi kh nng

c th to gel hnh thnh cu trc mng tinh th vi kch thc l c th iu chnh
c nh pH v nhit .
Vi tnh cht c bit ny, chng ti tin hnh c nh enzyme trn bt silica gel
v kho st kh nng hp ph ca n, cng nh cc iu kin bn ca enzyme c
hp ph.
2.2.1.2. Tng hp silica gel
Nguyn tc tng hp:
(1) Phn tn cc ht silicium oxide
(2) Nh tng ha cc lp du 65C
(3) Lm lnh h thng nh tng n nhit phng
(4) Khong ha lp v silic oxide xung quanh cc git du
Hnh 2.2: Tng hp ht silica gel [35]

Tuy nhin phng php ny ch p dng quy m cng nghip sn xut ht
silica gel (ht m). Mc khc, quy m th nghim, silica gel c to ra s c
em i c nh enzyme, lng du bm trn ht silica gel nu khng tch trit s
nh hng n hiu sut c nh enzyme sau ny.
SVTH: Nguyn Ngc Trm
2.2.1.3. ng dng silica gel trong c nh enzyme

Silica gel c dng c nh GL-7-ACA acylase, chloroperoxidase,
lipoxygenase bng lin kt cng ha tr v hp ph [27] v c nh lipoxygenase
bng phng php by trong sol-gel [3].
2.2.2. Chitosan
Chitin ((1-4)-N-acetyl-D-glucosamine), ngun nguyn liu phong ph th hai
sau cellulose, l thnh phn ch yu trong cu trc ca ng vt khng xng sng,
thnh t bo ca nm. Sn phm tha ca cng ngh sn xut thy sn nh hi sn, v
tm, v cua, tm hm, c, s u c cha chitin. Chitosan, c thu nhn bng cch
deacetyl ha 1 phn chitin, l mt polycationic polysaccharide ca 2-acetamido-2deoxy--D-glucopyranose (GlcNAc) v 2-amino-2-deoxy--D-glucopyranose (GlcN)
ni vi nhau bng lin kt 1,4 (hnh 2.3) v c gi tr pKa trong khong 6,3 6,5.
Chitosan v chitin c tnh thng mi ha v hm lng nito cao (6,89%) so vi
cellulose thay th (1,25%). Polysaccharide ny tan trong dung dch c tnh acid (pH <
6), c tnh sinh hc v l polymer c th phn hy c. Hn na, n c kh nng bm
dnh cao, cha lnh vt thng v c tnh khng khun. Ngoi ra, chitosan c
chuyn ha bi enzyme trong c th ngi nh lysozyme, Termamyl, hyaluronidase,
v c th phn hy bi vi sinh vt. Cui cng, chitosan c rt nhiu trong t nhin, n
kh r v c tnh kinh t cao.
Hnh 2.3: Chitosan [24]

2.2.2.1.
Ngun gc ca chitosan
Chitosan l mt dng chitin b kh acetyl, nhng khng ging chitin n li tan

c trong dung dch acid. Chitin l polyme sinh hc c nhiu trong thin nhin ch
10
SVTH: Nguyn Ngc Trm
ng sau cellulose. Cu trc ha hc ca chitin gn ging vi cellulose. C chitin v

chitosan u c nhiu ng dng trong cng nghip v cuc sng, c bit l trong ch
bin v bo qun thc phm.
Chitin c gc t ch "chiton", ting Hy Lp c ngha l v gip. Chitin l thnh
phn cu trc chnh trong v (b xng ngoi) ca cc ng vt khng xng sng
trong c loi gip xc (tm, cua).
2.2.2.2. c tnh ca chitosan
- L polysacharide c m khng c hi, c khi lng phn t ln.
- L mt cht rn, xp, nh, hnh vy, c th xay nh theo cc kch c khc nhau.
- Chitosan c mu trng hay vng nht, khng mi v.
- Khng tan trong nc, dung dch kim v acid m c nhng tan trong acid
long (pH6), to dung dch keo trong, c kh nng to mng tt, nhit nng chy
309 - 311oC.
2.2.2.3. Tc dng ca chitosan
- Phn hy sinh hc d hn chitin.
- Chitosan v cc dn xut ca chng u c tnh khng khun, nh c ch hot
ng ca mt s loi vi khun nh E.Coli, dit c mt s loi nm hi du ty, c
rt, u v c tc dng tt trong bo qun cc loi rau qu c v cng bn ngoi.
- Khi dng mng chitosan, d dng iu chnh m, thong khng kh cho
thc phm (nu dng bao gi bng PE th mc cung cp oxy b hn ch, nc s b
ngng ng to mi trng cho nm mc pht trin).
- Mng chitosan cng kh dai, kh x rch, c bn tng ng vi mt s
cht do vn c dng lm bao gi.
- Mng chitosan lm chm li qu trnh b thm ca rau qu. Rau qu sau khi thu
hoch s dn dn b thm, lm gim cht lng v gi tr. Rau qu b thm l do qu
trnh ln men to ra cc sn phm polyme ha ca oquinon. Nh bao gi bng mng
chitosan m c ch c hot tnh oxy ha ca cc polyphenol, lm thnh phn ca
11
SVTH: Nguyn Ngc Trm
anthocyamin, flavonoid v tng lng cc hp cht phenol t bin i, gi cho rau qu

ti lu hn.
2.2.2.4. u im ca chitosan
- D phn hu sinh hc.
- V tm ph liu l ngun nguyn liu t nhin rt di do, r tin, c sn quanh
nm, nn rt thun tin cho vic cung cp chitin v chitosan.
- Tn dng ph thi trong ch bin thy sn bo qun thc phm nc ta.
Thnh cng ny cn gp phn rt ln trong vic gii quyt tnh trng nhim mi
trng do cc cht thi t v tm gy ra.
2.2.2.5. ng dng chitosan trong c nh enzyme
C nh lipase trn chitosan c ph lp Fe3O4 [15], c nh allinase trn chitosan
bng lin kt cng ha tr sn xut allicin [20], c nh glucose oxidase trn silicachitosan [31]
2.2.3. Alginate
2.2.3.1. Ngun gc ca alginate
Alginate l thut ng dng cho cc mui acid alginic. Alginate i khi cn c
gi bng thut ng algin.
Alginate l mt polymer sinh hc c ngun gc t to bin, c nhiu trong vch
t bo to nu (Paccophyceae) di dng mui calci, magie, sodium ca acid alginic.
Alginate c ngun gc t bin nn n rt linh ng v bn. Tuy nhin, dng mui
calci v magie alginate khng tan trong nc, phi qua qu trnh tch chit alginate
thu alginate dng bt kh, tan trong nc v loi b nhng thnh phn khc ca to
bin qua qu trnh kt ta.
2.2.3.2. Cu to ca alginate
Cng thc ca acid Alginic l (C6H7O6)n t cng thc ca sodium alginate l
(C6H7NaO6)n, calci alginate (C6H7Ca1/2O6)n.
12
SVTH: Nguyn Ngc Trm
Hnh 2.4: Acid alginic [37]

Alginate l mt polymer c cu to t hai monomer l acid -D-Mannuronic
(M) v acid -L-Gluconic (G), vi cc cu ni l lin kt 1-4 glucoside, ch khc
nhm carboxyl nm trn v di mt phng ca vng pyranoza. Chng tham gia vo
cu trc thnh v mng t bo, chng thng tn ti dng G-G-G-G; M-M-MM; M-G-M-G S phn b ca thnh phn M, G bin i nhiu theo loi, do c
nhiu loi alginate v c tnh cht khc nhau.
Hnh 2.5: Cu to ca alginate [37]
13
SVTH: Nguyn Ngc Trm
2.2.3.3. Tnh cht ca alginate

Vic s dng alginate da trn 3 tnh cht chnh:
- Khi tan trong nc, alginate to dung dch nht. Cc mui alginate (Na, K, Mg)
u ha tan trong nc, to cho dung dch c nht cao. nht alginate ca rong
thay i nhiu ty thuc vo loi, vo giai on sinh trng ca rong v k thut tch
chit alginate. nht tng khi mc polymer ha tng, nng alginate tng v
nhit gim. Bnh cnh , nht ca alginate li ph thuc rt ln vo phng
php tch chit. tng nht, trong qu trnh tch chit alginate, ta c th b sung
mt s ph gia cn thit thu c sn phm c nht mong mun.
Ngoi ra, nht alginate ph thuc vo qu trnh bo qun. Khi dng bt
thnh phm, alginate vn tip tc b ct mch, sau mt thi gian, nht ca n gim
ng k. khc phc iu ny, ta c th cho vo alginate thnh phm mt lng nh
calci sao cho cha n ngng to gel. Khi s dng kt hp alginate vi cc dng gum
khc nh pectin s lm tng ng k nht ca dung dch.
Hnh 2.6: Cu trc ht gel alginate [37]

- iu kin bnh thng, cc gc COO- ca cc phn t guluronic s hnh thnh
lin kt hydro vi ion H3O+ c trong dung dch. Khi ta a vo mi trng cc ion kim
loi nh Ca2+, Ba2+, cc cation ny s thay vo v tr ca H3O+, ni cc chui
14
SVTH: Nguyn Ngc Trm
guluronic li vi nhau, to thnh mt cu trc mng li khng tan trong nc, cu

trc ny c gi l gel.
Qu trnh to gel ca alginate c tin hnh cc iu kin bnh thng v
nhit v p sut. Cu trc gel to thnh bn nhit trong khong 0 100oC. V
th, s gia nhit hu nh t lm h cu trc gel. Tuy vy, gel c th d dng ha tan tr
li khi nhng vo dung dch cha hm lng Na+, K+ hay Mg2+ cao. Do vy, nn duy
tr t l Na+/Ca+
25:1 gip cu trc gel c n nh.
Kh nng to gel ca alginate vi cc cation l khng ging nhau, bn gel tng

dn theo th t Ca2+ < Sr2+ < Ba2+. Trong , Ca2+ l tc nhn to gel c s dng
nhiu nht do tnh ph bin v khng c hi ca n.
- Alginate to dng mng mng (films) ca sodium hoc calci alginate vi dng
si (fibres) ca calci alginate. Tnh cht ny ca alginate c ng dng trong vic to
nn cc lp ph bn ngoi.
2.2.3.4. ng dng alginate trong c nh enzyme
ng c nh dextransucrase v dextranase trn alginate sn xut
isomaltooligosaccharides t sucrose [9], urease [6], invertase, -amylase c nh trn
silica-alginate [34]
2.3.
Hot ha vt liu bng glutaraldehyde
Hnh 2.7: Glutaraldehyde hot ha chitosan [28]
15
SVTH: Nguyn Ngc Trm
Cc cht c mang nhm NH2 (polysaccharide, chitin, chitosan, cellulose) u

c th s dng phng php ny.
Glutaraldehyde l cht c cha hai nhm aldehyde (-CHO) hot ha. Mt nhm
s gn vo NH2 ca enzyme.
2.4.
Enzyme amylase
2.4.1. -amylase
-amylase c kh nng phn ct cc lin kt -1,4-glucoside nm pha trong phn
t c cht mt cch ngu nhin khng theo mt trt t no c. Di tc dng ca amylase, tinh bt c th chuyn thnh maltotetrose, maltose, glucose v dextrin phn
t thp.
2.4.1.1. c tnh
Trung tm hot ng ca -amylase c cha nhm [COOH] v [NH2]. -amylase
d tan trong nc, trong cc dung dch mui v ru long.
Protein ca -amylase c tnh acid yu v tnh cht ca globulin.
im ng in nm trong vng pH 4,2 - 5,7. Phn t lng ca -amylase t
cc ngun gc khc nhau rt khc nhau (ca nm mc 45,000 50,000, ca malt
59,000) [32].
-amylase l mt metaloenzyme (enzyme c kim). Cc -amylase u cha t 130 nguyn t gam Ca/mol. Song khng t hn 1 - 6 nguyn t gam Ca/mol. Khi tch
hon ton Calci ra khi protein ca enzyme th -amylase mt ht kh nng thy phn
c cht. V calci tham gia vo s hnh thnh v n nh cu trc bc ba ca enzyme,
duy tr cu hnh hot ng ca enzyme [32]. Calci cn c tc dng m bo cho amylase c bn cc ln i vi cc tc ng gy bin tnh v s phn hy bi cc
enzyme phn gii protein. -amylase bn nhit hn so vi cc loi amylase khc.
Ngi ta cho rng c tnh ny ca -amylase c lin quan n hm lng ca Calci
trong phn t ca n (-amylase ca cc vi khun a nhit c cha Calci nhiu hn
amylase ca nm mc 3-4 ln nn n bn nhit hn). Tt c nhng -amylase u b
km hm bi nhng kim loi nng nh: Cu2+, Li+, Mg2+, Cr3+, Mn2+, Zn2+, Co2+, Sn2+
16
SVTH: Nguyn Ngc Trm
-amylase t cc ngun gc khc nhau c thnh phn amino acid khc nhau. Mi
loi -amylase c mt t hp amino acid c hiu ring, song chng u kh giu
tyrosine v tryptophan. Cc glutamic acid v aspartic chim khong tng lng
amino acicd cu thnh phn t enzyme. Trong -amylase rt t methionine v ch c
khong 7-10 gc cysteine, tr amylase ca Bacillus licheniformis khng c cc lin kt
sulfhydryl v disulfhydryl.
- iu kin hot ng ca -amylase t cc ngun khc nhau cng khc nhau.
pH ti thch cho hot ng ca -amylase t nm si l 4,5 4,8 (c th hot ng tt
trong vng pH 4,5 4,8) ca i mch ny mm l 5,3 v ca vi khun l 5,8 6,0
(hot ng tt trong vng pH 5,5 6,8).
- bn i vi tc dng acid cng khc nhau. -amylase ca nm si bn vng
vi acid tt hn l ca malt v vi khun. pH 3,6 v 0oC -amylase ca malt b v
hot hon ton sau 15 30 pht, -amylase ca vi khun b v hot 50% trong khi
hot lc ca -amylase ca nm si hu nh khng gim bao nhiu [32].
- pH < 4,0 -amylase ca vi khun b v hot hon ton -amylase ca nm si
rt nhy vi nhit (nhit ti thch 50oC). -amylase ca thc mm v ca malt bn
nhit hn v hot ng ti thch 58 60oC, -amylase ca vi khun c bn cao
hn c.
2.4.1.2. Gii thiu v enzyme Termamyl
Termamyl l ch phm enzyme dng lng cha -amylase chu c nhit cao
v c sn xut bi chng men Bacillus licheniformis (enzyme ny l mt -amylase
thy phn lin kt 1,4 -glucoside thnh amylose v amylopectin.
Mt s ng dng ca Termamyl trong cng nghip (thng tin do hng Novo cung
cp) [38]:
- Trong k ngh tinh bt: Termamyl c dng cho vic dch ha lin tc tinh
bt trong ni hi hoc trong nhng thit b tng t hot ng nhit t 105 110oC v v vy li dng c tnh n nh nhit cao ca enzyme.
17
SVTH: Nguyn Ngc Trm
- Trong k ngh nu cn: Termamyl c dng phn tn tinh bt khi nghin

cu v chng ct. V giai on ny, cng li dng c n nh nhit ca
enzyme.
- Trong k ngh nu bia: Termamyl c dng gip cho vic dch ha c
d dng. Do n nh nhit cao ca enzyme.
- Trong k ngh ng: Termamyl c s dng ph v lng tinh bt hin
din trong ma.
- Trong k ngh dt: Termamyl c s dng tc cao, nhit cao r h
trc khi nhum. Loi enzyme k thut c p dng cho cng ngh ny.
2.4.1.3. ng dng -amylase trong c nh
-amylase c nh trn mt s vt liu nh silica-alginate [34], silica gel [23],
polyanilin [4], chitosan v Amberlite MB-150 [8]
2.4.2. Glucoamylase
Glucoamylase l mt enzyme quan trng thuc h enzyme amylase. Enzyme ny
ang thu ht s quan tm ca nhiu nh sn xut nh vo kh nng thy phn lin kt
-1,4 glycoside v -1,6 glycoside thm ch c lin kt -1,3 glycoside trong mch
amylose v amylopectin thu c sn phm trit l glucose. Nh vo c tnh
ny, glucoamylase c s dng trong cng nghip sn xut ng glucose v mt
tinh bt.
Glucoamylase (EC 3.2.1.3) c bit nh l amyloglucosidase, glucamylase,
maltase, saccharogenic amylase v -amylase [29], l nhm quan trng ca nhm
enzyme thy phn tinh bt. N c s dng rng ri trong sn xut cng nghip cc
loi ng syrup. Glucoamylase gii phng -D-glucose t u khng kh ca h tinh
bt, bng s thy phn lin kt -1,4 v thy phn lin kt -1,6 mc thp hn.
Theo l thuyt, n c th phn ct tn gc tinh bt thnh glucose, nhng s thy phn
h tinh bt l thun nghch v to ra lng ln cc sn phm khc ngoi glucose nh
di-, tri- v tetra-saccharide, hu ht l isomaltose [25].
18
SVTH: Nguyn Ngc Trm
Hnh 2.8: Glucoamylase thy phn amylose [36]

c nhiu n lc ci thin hiu sut ca glucoamylase bng cch gy t
bin v thay i protein khc nhau t c hai mc tiu chnh. Mc tiu u tin
l sn xut glucoamylase tinh khit hn. Mt vn vi cc enzyme truyn thng
c s dng l khi nng cao, mt s ng sn xut s ngng t khc nhau bi
cc sn phm nh isomaltose. H khng mong mun v ngt nh ng v khng th
tip tc x l to thnh fructose. Mc tiu th hai l ci thin tnh chu nhit cao
hn. iu ny cho php thc hin phn ng nhit cao hn v c th lm gim
thi gian sn xut v gim nguy c nhim [25].
Cc enzyme chu nhit mi s c to t cc sinh vt t nhin chu nhit v a
nhit. Cch tip cn ny hin ang c p dng rng ri v nhng glucoamyase a
nhit khc nhau c nhn bn v tnh [26].
Mt s loi chu nhit nh Thermomyces lanuginosus, c bit n nh
Humicola lanuginosa, Humicola grisea varindica, Acremoniella thermophila,
Monotospora lanuginose v Sepedonium lanuginosum, c glucoamylase tt bng amylase v -glucosidase [5]. Trong nhng nghin cu khc nhau v cc c tnh l
ha khc nhau ca hot tnh glucoamylase t nm chu nhit T. lanuginosus, enzyme
c m t v on gen m ha cho glucoamylase c phn lp v nhn dng.
19
SVTH: Nguyn Ngc Trm
Hiu qu mang li khi x l bng glucoamylase cao hn so vi -amylase. Do

vy, glucoamylase hin ang c cc nh nghin cu hng ti v kh nng thy
phn tinh bt ca chng. Mt s nghin cu c nh glucoamylase trn cc vt liu
khc nhau nh gelatin [9], ceramic [22], cellulose [21]
2.5. iu kin phn ng khi tin hnh xc nh hot enzyme
- Trong phn ng xc nh hot enzyme, cn s dng c cht v cofactor
mc d tha (nu enzyme cn cofactor) v thi gian xc nh cng ngn cng tt.
- Cn phi chn la cc iu kin sao cho c c s bin i tuyn tnh gia
nng enzyme, thi gian phn ng v mc chuyn ha c cht (trong gii hn
nng c cht la chn).
- Mt s enzyme b c ch bi c cht nng qu cao; hoc b gim hot
nhanh chng theo thi gian nhit phn tch. Do phn ng phn tch nn thc
hin trong khong thi gian nht nh v nng enzyme ln c cht thch hp.
2.5.1. Cc phng php xc nh hot enzyme
2.5.1.1. Phn tch lin tc: l phng php o c cht b bin i hay sn phm to
thnh mt cch lin tc theo thi gian. Tuy nhin yu cu i vi thit b o l phi c
b phn n nh nhit. y l mt hn ch ca phng php ny. ng thi, do phi
theo di s bin i ca cht phn ng mt cch lin tc nn kh thc hin phn tch
hot nhiu mu enzyme cng lc.
2.5.1.2. Phn tch gin on: l phng php cho enzyme tc dng vi c cht sau
mt khong thi gian nht nh th ngng phn ng enzyme bng cch thch hp v
sau o lng c cht cn li hoc sn phm to thnh. ngng phn ng c th
dung cc tc nhn lm bt hot enzyme: nhit cao, thay i pH, dung cht to phc
hay tch enzyme ra khi hn hp Phng php ny khc phc nhng hn ch ca
phng php o lin tc, phn ng c th tin hnh trong t m hay b nhit n nh,
c th tin hnh mt lc nhiu mu tuy nhin vn t ra l phi tm cch lm
ngng phn ng thch hp nht.
Trong nghin cu ny, chng ti dng phng php phn tch gin on, cho
enzyme c nh phn ng vi c cht theo m (gin on), mi m l 30 pht, sau
20
SVTH: Nguyn Ngc Trm
dng phng php ha hc xc nh hot ca enzyme c nh. C th l dng

cc phn ng ha hc nh lng c cht mt i hay lng sn phm to thnh.
Thng thng phi chn phn ng to nn cc phc cht mu c hp thu nh sng
cc i vng no t nh lng hp cht ny.
Phng php nh lng c cht cn li hay sn phm to thnh c th n gin
l s o c cht cn li hay sn phm to thnh mt cch trc tip. Tuy nhin trong
nhiu trng hp, c th o mt cch gin tip v do php o phc tp hn.
2.5.2. Mt s lu khi xc nh hot enzyme
- Khi xc nh hot enzyme cn chn iu kin pH v nhit phn tch
vng thch hp. Vi nhng enzyme ln u tin nghin cu, nn chn pH trung tnh,
300C 370C. Sau c nhng iu chnh sau nu cn thit.
- Bn cnh pH v nhit , cn lu c cht, thnh phn m, lc ion hay nng
mui, cc cht lm bn.
- Cc phn tch hot enzyme phi thc hin mt gi tr pH n nh, nn phi
dng mt loi m hay mt h thng m thch hp no . Nng m thng
dng l 20 50 nM. Tuy vy cc phn ng sinh acid, base cn phi dng m nng
cao hn trnh thay i pH trong qu trnh th nghim. Cn chn loi m thch hp
trnh lm kt ta cc yu t cn thit cho hot ng ca enzyme nh Ca2+, Zn2+.
- C cht v sn phm, m u phi t cng nhit phn tch khi tip xc vi
nhau bt u phn ng. Nu phn tch nhiu mu, thi gian bt u v kt thc
phn ng phi duy tr nh nhau.
- Lun c mu kim tra thch hp trnh sai st.
- Phi la chn phng php lm ngng phn ng thch hp, trnh lm bin i
c cht hay sn phm cn o; hay can thip qu mnh vo php nh lng sn phm
phn ng enzyme (cht lm ngng phn ng c cng bc song hp th nh sng cn
o vi cht phn ng, hay c ch phn ng to mu tip theo ca phn tch)
Vic xc nh hot ca enzyme ch cho s liu tin cy khi chn c phng
php thch hp v c cc bc tin hnh ng.
21
Chng 3:
SVTH: Nguyn Ngc Trm
VT LIU V PHNG PHP
Cc th nghim c tin hnh ti phng th nghim Cng ngh Sinh hc,

Trng i Hc Bch Khoa Thnh Ph H Ch Minh.
3.1.
Nguyn vt liu
3.1.1. Nguyn liu

Enzyme amylase c s dng trong nghin cu ny l Termamyl 120L (amylase) v AMG 300L (glucoamylase) ca hng Novozym.
3.1.2. Ha cht
3.1.2.1. Vt liu c nh
- Sodium alginate (Trung Quc)
- Chitosan (i hc Nha Trang)
- Na2SiO3.9H2O
- (NH4)2SO4 khan
3.1.2.2. Dung dch to gel
- Dung dch CaCl2 3%
- Dung dch NaOH 0,3M
3.1.2.3. Cht hot ha
- Glutaraldehyde (Merck)
3.1.2.4. Cht chun
- D-glucose
- Albumin
3.1.2.5. Ha cht nh lng
- DNS (Dinitrosalicilic acid)
- Dung dch Bradford
22
3.2.
SVTH: Nguyn Ngc Trm
Thit b
My o quang ph, cn k thut, cn phn tch.
3.3.
Cc phng php phn tch
- Phng php xc nh hm lng protein bng phng php Bradford (xem

ph lc 1a).
- Xc nh hot tnh enzyme bng phng php xc nh hm lng ng kh
DNS (xem cch tnh ti ph lc 2a).
3.4.
Phng php nghin cu
3.4.1. To bt silica gel

u tin, dung dch (NH4)2SO4 1M c cho vo cc. Tip n cho dung dch
Na2SiO3 1M vo, va cho va khuy u trong 1 gi. Sau dung dch c yn
qua m nhit phng. Silica gel c ra sch vi nc ct v sy kh. Ta thu
c silica gel th. Xem s to bt silica gel ti ph lc 3a.
Silica gel th c x l vi dung dch HNO3 5% trung ha lng base
trong gel. Sau , gel c hot ha vi glutaraldehyde 10% trong 3 gi 4oC.
Tip theo, enzyme c cho vo gel thc hin qu trnh c nh. Thi gian c
nh enzyme l 3 gi v sau enzyme c nh c em i xc nh hot tnh
(phng php xc nh hm lng ng kh bng DNS) v hm lng protein
(phng php Bradford).
Hnh 3.1: Silica gel

23
SVTH: Nguyn Ngc Trm
3.4.2. Chun b ht chitosan

Ht chitosan 3% c chun b bng vic un nh trong acic acetic 1,5% v
khuy u. Dung dch thu c c to ht vi ng knh 3mm trong dung dch
NaOH 0,3M. Ht va to c khuy lin tc 2 gi nhit phng. Sau khi n
nh kch thc, ht c thu hi bng cch lc v ra nhiu ln bng nc ct.
Cui cng, ht c em i tr lnh 4oC cho n khi s dng.
3.4.3. Chun b ht silica-chitosan
Xc nh hm lng silica gel thm vo chitosan 3% nhm to ht gel silicachitosan c cu trc bn, kh nng hot ng n nh v ti u nht. Vic kho st
c tin hnh nh sau:
Bt silica gel cho vo trong dung dch chitosan 3% ln lt vi lng l 1%,
2%, 3%, 4%, 5% (w/v). Hn hp c trn u trc khi to ht vi ng knh
3mm trong dung dch NaOH 0,3M. Ht va to c khuy lin tc 2 gi nhit
phng. Sau khi ht n nh kch thc, c thu hi bng cch lc v c ra
nhiu ln bng nc ct. Cui cng, ht c em i tr lnh 4oC cho n khi s
dng.
3.4.4. Hot ha ht silica-chitosan vi glutaraldehyde
Nhm gip silica-chitosan c kh nng c nh enzyme c tt hn,
glutaraldehyde c s dng. Theo , gc CHO ca glutaraldehyde s lin kt
vi gc NH2 ca enzyme v chitosan. Phng php c tin hnh nh sau:
Ht silica-chitosan c hot ha vi dung dch glutaraldehyde 2,5% v
4oC trong 3 gi. Sau , ht silica-chitosan c ra sch vi nc ct nhiu ln
loi b glutaraldehyde t do. Sau ht c bo qun kh 4oC.
3.4.5. C nh Termamyl ln cc cht mang
3.4.5.1. Silica gel
Cho 5ml Termamyl vo 1g silica gel v 4oC trong 3 gi. Sau tin hnh
ra vi nc ct. Nc ra thu c nh mc ln 50ml xc nh hm lng
protein khng b hp ph.
24
SVTH: Nguyn Ngc Trm
3.4.5.2. Silica-chitosan
Cho vo 1g silica-chitosan tng ng l 0,1; 0,2; 0,3; 0,4; 0,5ml Termamyl v
4oC trong 3 gi. Sau tin hnh ra vi nc ct. Nc ra thu c nh mc
ln 10ml xc nh hm lng protein khng b hp ph.
3.4.5.3.
Silica-alginate
0,2ml Termamyl c trn chung vi 2g hn hp silica gel-alginate c nng

tng ng l 0; 1,5; 3; 4,5; 6; 7,5%. Hn hp c trn u v yn trong 1
gi. Sau , nhng ht gel c nh c to bng cch nh hn hp trn vo dung
dch CaCl2 3%. Ht c ngm trong dung dch CaCl2 trong 2 gi n nh cu
trc. Cui cng, tin hnh lc v ra sch CaCl2 vi nc ct. Ht c nh c tr
4oC cho n khi s dng.
3.4.6. ng c nh Termamyl v AMG trn silica-alginate
0,2ml Termamyl:AMG (1:1) c trn chung vi 2g hn hp silica-alginate c
nng tng ng l 0; 1,5; 3; 4,5; 6; 7,5%. Hn hp c trn u v yn
trong 1 gi. Sau , nhng ht gel c nh c to bng cch nh hn hp trn vo
dung dch CaCl2 3%. Ht c ngm trong dung dch CaCl2 trong 2 gi n nh
cu trc. Cui cng, tin hnh lc v ra sch CaCl2 vi nc ct. Ht c nh c
tr 4oC cho n khi s dng.
3.4.7. Kho st nhit v pH ti u
Theo nghin cu ca Dilek Kl Apar (2004), nhit v pH nh hng ng
k n hot tnh ca enzyme c nh [18]. V vy, vic xc nh nhit v pH ti
u ca enzyme c nh trn tng loi vt liu l rt cn thit. N quyt nh n
hiu sut v kh nng ng dng ca enzyme c nh.
3.4.7.1.
Kho st nhit ti u ca enzyme c nh trn cc vt liu
a) Silica gel
- Bt silica gel sau khi c hot ha bi glutaraldehyde v c nh Termamyl
c tin hnh kho st nhit ti u. Nhit c kho st tng ng l nhit
phng, 70, 85 v 100oC.
25
SVTH: Nguyn Ngc Trm
- Sau khi xc nh c nhit ti u, enzyme c nh trn bt silica gel

c tin hnh kho st pH ti u. Nhng gi tr pH c kho st l 6,0; 6,5; 7,0;
7,5.
b) Silica-chitosan
- Ht silica-chitosan c tin hnh kho st cc nhit tng ng l 40,
50, 60, 70, 80oC.
- Tip theo, enzyme c nh c kho st pH. Nhng gi tr pH c kho
st l pH 7,0; 7,5; 8; 8,5.
c) Silica-alginate
- Ht silica-alginate sau khi c nh Termamyl c tin hnh kho st nhit
ti u. Nhng mc nhit kho st l 40, 50, 60, 70, 80oC. V tng t i vi
ng c nh Termamyl v AMG trn silica-alginate.
3.4.7.2. Kho st pH ti u ca enzyme c nh trn cc vt liu
a) Silica gel
- Sau khi xc nh c nhit ti u, enzyme c nh trn bt silica gel
c tin hnh kho st pH ti u. Nhng gi tr pH c kho st l 6,0; 6,5; 7,0;
7,5.
b) Silica-chitosan
- Tng t vi enzyme c nh trn silica gel, enzyme c nh trn silicachitosan c kho st pH. Nhng gi tr pH c kho st l pH 7,0; 7,5; 8; 8,5.
c) Silica-alginate
- Enzyme c nh trn silica-alginate c kho st cc gi tr pH l pH 5,0;
5,5; 6,0; 6,5.
3.4.8.
S ti s dng
Mi loi ht s c tin hnh kho st kh nng ti s dng ca chng iu

kin nhit v pH ti u. Phn ng c din ra lin tc (c 30 pht li thay c
cht mi v phn ng).
26
SVTH: Nguyn Ngc Trm
KT QU v BN LUN
Chng 4:
4.1. Enzyme t do
Bng 4.1: Cc thng s k thut ca amylase v glucoamylase

Cc thng s
Termamyl
glucoamylase
Hot tnh (UI)
11.657
13.663
Hm lng protein
12.829
199.489
Nhit ti u* (oC)
80-90
55-60
pH ti u*
5,5 6,8
4,5
* Theo ti liu c cung cp t hng Novozym [36].

(Chi tit xem ph lc 1b)
4.2. Termamyl c nh trn silica gel

4.2.1. Hiu sut c nh Termamyl trn silica gel
Bng 4.2: Hiu sut c nh Termamyl trn silica gel
Hiu sut c nh
- Hm lng protein
- Hot tnh
Termamyl c nh trn silica gel

66,66 %
5,12%
(Chi tit xem ph lc 2b)
Hiu sut c nh hm lng protein trn silica gel l kh cao. Tuy nhin, hot
tnh ca enzyme sau khi c nh li kh thp.
4.2.2. Kho st nhit ti u ca Termamyl c nh trn silica gel
Theo hnh 4.1, nhit cng tng th hot tnh ca enzyme c nh cng tng.
Tuy nhin, do iu kin thit b phng th nghim khng cho php nn nhit
27
SVTH: Nguyn Ngc Trm
kho st ch dng 100oC. Mt khc, enzyme c nh trn silica gel cn c th chu

c nhit cao hn m vn gi c hot tnh xc tc.
Hot tnh (UI)
1.2
1
0.8
0.6
0.4
0.2
0
nhit phng
70
85
100
Nhit (oC)
Hnh 4.1: Kho st nh hng ca nhit ln hot tnh ca Termamyl c nh trn

silica gel
4.2.3. Kho st pH ti u ca Termamyl c nh trn silica gel
Hot tnh (UI)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
5
5.5
6.5
pH
Hnh 4.2: Kho st nh hng ca pH n hot tnh ca Termamyl c nh trn

silica gel
28
SVTH: Nguyn Ngc Trm
Hnh 4.2 biu din hot tnh ca Termamyl c nh trn silica gel. Theo ,
hot tnh ca enzyme c nh t nh ti pH 5,5 v khi pH tng dn t pH 5,5 7
th hot tnh ca enzyme c nh c xu hng gim dn. iu ny chng t pH ca
enzyme c nh c nh hot ng ti pH 5,5 so vi enzyme t do c khong hot
ng pH hiu qu l pH 5,5 7.
4.2.4. Ti s dng Termamyl c nh trn silica gel
Hnh 4.3 biu din hot tnh cn li ca Termamyl c nh theo s ln ti s
dng. y, ta tin hnh so snh gia hai mu silica gel. Mt mu c hot ha
glutaraldehyde sau em i c nh enzyme v mt mu khng hot ha
glutaraldehyde ri em i c nh enzyme.
Hiu sut phn ng (%)
120
100
80
100
100
87.29
ban u
75.01
ln 1
66.84
60.48
60
ln 2
40
ln 3
22.66
23.43
20
Mu silica gel
0
khng hot ha glutaraldehyde
c hot ha glutaraldehyde
Hnh 4.3: Kho st kh nng ti s dng ca Termamyl c nh trn silica gel

Theo nghin cu ca M. Vezir Kahraman v cng s (2007), Termamyl c
c nh trn silica gel bng lin kt cng ha tr, c hot tnh l 98% sau 6 ln ti s
dng v 81,4% sau 25 ln ti s dng [23]. T , c th kt lun rng c nh
Termamyl trn silica gel bng phng php cng ha tr cho kt qu cao hn hn
phng php hp ph.
Glutaraldehyde l cht hot ha chng ti th nghim xem c hiu qu trong
vic c nh Termamyl trn silica gel c hiu qu hay khng. V kt qu l hot tnh
29
SVTH: Nguyn Ngc Trm
cn li ca c hai mu enzyme c nh trn silica gel u c kh nng ti s dng

khng tt (ch ti s dng c 2 ln), tc ra tri kh cao v khng c s khc
bit r lm trong hot tnh ca enzyme c nh trn silica gel c v khng c hot
ha vi glutaraldehyde. Qua cho thy, glutaraldehyde khng ci thin nhiu
trong vic lm tng kh nng c nh enzyme ln silica gel.
Nh ta bit, silica gel l mt cht mang v c, tng i tr vi nhit v
cc iu kin l ha khc. Nh vy, n c kh nng gip cho enzyme c nh trn
n chu c cc iu kin l ha ca mi trng sn xut cng nghip. Tuy nhin,
v lin kt trong ni b enzyme l lin kt mng tinh th, nn khng th gi c
enzyme bn trong mng ca chng. T vn , chng ti quyt nh phi
hp silica gel vi mt s cht mang hu c nhm ci thin tnh trng ra tri ny.
Cht mang hu c chng ti chn l chitosan v alginate.
4.3. Termamyl c nh trn silica-chitosan v silica-alginate
4.3.1. iu kin c nh
4.3.1.1.
Kho st lng silica gel thm vo chitosan v alginate
a) Kho st lng silica gel thm vo chitosan

Hiu sut c nh (%)
50
49
48
47
46
45
44
43
42
0
silica gel/chitosan (%)
Hnh 4.4: Kho st lng silica gel thm vo chitosan
30
SVTH: Nguyn Ngc Trm
Trong , silica/chitosan l t l khi lng silica gel thm vo trong 3g dung

dch chitosan. Vi gi tr 0% tng ng vi dung dch chitosan nguyn cht, khng
b sung silica gel.
Theo hnh 4.4, hot tnh ca Termamyl tng dn khi lng silica gel thm vo
tng t 1 4% v t nh 4%. Tuy nhin, khi t l silica gel thm vo ln n 5%
th ht chitosan khng bn, b ph v cu trc (hnh 4.5 g) v v nguyn nhn nn
hot tnh ca Termamyl c phn gim hn so vi t l 4% silica.
(a)
(b)
(c)
(d)
(e)
(g)
Hnh 4.5: Ht silica-chitosan

Trong , a, b, c, d, e, g tng ng ln lt vi t l silica/chitosan l 0%, 1%, 2%, 3%, 4%,
5%.
gi tr 0%, hot tnh ca Termamyl tng i cao, tuy nhin khi thm silica
gel vo th hot tnh ca Termamyl c phn gim i. iu ny c th gii thch rng
chnh s c mt ca silica gel trong cu trc gel ca chitosan lm tng tr lc
lm cn tr c cht i vo v sn phm i ra, lm cho hot tnh ca Termamyl b
gim i. Mt khc, s c mt ca silica gel cng lm gim tnh bn vng trong cu
trc ca chitosan (iu ny c biu hin r nht gi tr 5% silica). Tuy nhin,
nu lng silica gel cho vo ph hp, n s h tr trong cu trc mng li ca
chitosan, s gip cho ht gel silica-chitosan c tnh bn vng vi nhit , pH hn
31
SVTH: Nguyn Ngc Trm
so vi ht chitosan thng (gi tr 4% silica). Silica gel lm cho cu trc l tng ln

nn c cht c th vo ra d dng (gi tr 4% silica) v kt qu hot tnh ca enzyme
tng hn so vi ht chitosan khng b sung silica gel (gi tr 0% silica).
a) Kho st lng silica thm vo alginate
Hiu sut c nh (%)
54
53
52
51
50
49
48
47
46
45
44
silica/alginate (%)
1.5
4.5
7.5
Hnh 4.6: Kho st lng silica gel thm vo alginate

Trong , silica/alginate l t l khi lng silica gel thm vo trong 2g dung
dch alginate 3%. Vi 0% tng ng vi dung dch alginate 3% nguyn cht, khng
b sung silica gel.
Hiu sut enzyme c nh, silica-chitosan cao nht ch t 48.77%, trong khi
l 53,48% i vi silica-alginate.
Ti gi tr silica/alginate 7,5%, s hiu din ca silica gel cng nhiu th s lm
cho kch thc l ht b hp li v tr lc gy ra ln. Ti gi tr silica/alginate 6%,
hiu sut enzyme c nh t cc i, cao hn c enzyme c c nh trn ht
alginate 3%. iu ny c th do s lin kt gia ht silica trong mng li ca
alginate nn lm tng kch thc l gia silica v cc phn t polymer ti vng tip
gip vi silica.
32
SVTH: Nguyn Ngc Trm
(a)
(b)
(c)
(d)
(e)
(g)
Hnh 4.7: Ht silica-alginate

Trong , a, b, c, d, e, g tng ng ln lt vi t l silica/alginate l 0%, 1,5%, 3%, 4,5%,
6%, 7,5%.
T y ta c th kt lun, silica gel b sung vo trong dung dch chitosan v

alginate vi mt lng thch hp s gy ra hiu ng kh tt, gip ni rng kch
thc l ht, lm gim tr lc ca dng trao i cht m vn c th gi enzyme bn
trong khng b ra tri ra ngoi. V lng silica gel thch hp b sung vo trong
dung dch chitosan 3% v alginate 3% ln lt l 4% v 6%. Tc 0,12g silica/3g
chitosan v 0,12g silica/2g alginate. Ta s dng kt qu ny tip tc tin hnh cc
bc nghin cu tip theo.
4.3.1.2.
Kho st lng Termamyl c nh
a) Kho st lng Termamyl c nh silica-chitosan

Hnh 4.8 biu din mi tng quan gia hiu sut c nh protein v hiu sut
hot tnh vi lng enzyme cho vo c nh trn silica-chitosan. Theo , hm
lng protein c c nh tng dn khi tng lng enzyme t 0,1 0,5ml v
cn tip tc tng cho n khi ht gel t trng thi bo ha enzyme (lng enzyme
ti a m ht gel c th c nh c). Tuy nhin, hot tnh enzyme li khng t l
33
SVTH: Nguyn Ngc Trm
thun vi hm lng protein c c nh. C th, hot tnh ca enzyme c nh t

ti a gi tr 0,2ml (enzyme) v gim dn cc gi tr 0,3 - 0,5ml. Theo nh nh
ngha mc 3.3.3. th hiu sut c nh enzyme c xc nh theo cng thc:
Hiu sut c nh enzyme =
V mt ton hc, nu lng enzyme ban u cng ln th mu s cng ln s
lm cho hiu sut c nh enzyme gim mc d hot tnh ca enzyme c nh tng
theo lng enzyme cho vo (t s tng). V vy, c th kt lun rng, hm lng
protein c c nh cng cao th hot tnh ca enzyme c nh cng tng, tuy nhin
hiu sut c nh enzyme c th gim.
%
100
Hiu sut c
nh protein
(%)
75
Hiu sut
hot tnh
(%)
50
25
0
0.1
0.2
0.3
0.4
0.5
Lng enzyme
(ml)
Hnh 4.8: Kho st lng Termamyl c nh trn silica-chitosan

Gii thch v mt sinh hc, lng enzyme cho vo tng, tc phn trm protein
c c nh tng, tuy nhin hiu sut c nh enzyme gim. V enzyme c lin
kt chng cht trn b mt ht silica-chitosan dn n khng gian cho c cht chen
vo trung tm hot ng ca enzyme b hn ch. Bn cnh , v trn b mt ht gel
c s lin kt chng cht ca cc enzyme vi ht gel silica-chitosan nn xy ra hin
tng cnh tranh v c cht. Ngoi ra, enzyme cn thm thu vo bn trong mng
li ca ht silica-chitosan v c to lin kt. , kh nng tip xc vi c cht
34
SVTH: Nguyn Ngc Trm
b hn ch so vi b mt bn ngoi nn tuy lng protein c c nh tng i

cao nhng hiu sut c nh gim.
V vy, tit kim chi ph v enzyme, ta chn gi tr 0,2 ml l lng enzyme
thch hp, va tit kim chi ph v enzyme, va mang li hiu sut c nh cao hn
hn nhng gi tr khc.
b) Lng Termamyl c nh trn silica-alginate
Lng Termamyl c chn l 0,2ml cho vo c nh trong 2g dung dch
alginate 3% v c to ht trong CaCl2 3%. Phng php by nht enzyme trong
gel silica-alginate, enzyme b ra tri khng ng k nn xem nh hiu sut c nh
protein l 100%.
4.3.2. iu kin phn ng
4.3.2.1.
Kho st nhit ti u
a) Kho st nhit ti u ca Termamyl c nh trn silica-chitosan

Hot tnh (UI)
1
0.9
0.8
0.7
0.6
0.5
0.4
40
50
60
70
Nhit (oC)
Hnh 4.9: Kho st nhit ti u ca Termamyl c nh trn silica-chitosan
35
SVTH: Nguyn Ngc Trm
Theo hnh 4.9, khi nhit tng t 40 60oC th hot tnh ca enzyme c nh
tng v t nh ti 60oC. So vi enzyme t do, enzyme c nh hot ng tng
i n nh t 50 70oC. Vng hot ng ti u ca enzyme c nh l 50 70oC
trong khi enzyme t do c khong nhit hot ng l 80 90oC.
b) Kho st nhit ti u ca Termamyl c nh trn silica-alginate
Hot tnh (UI)

1.8
1.75
1.7
1.65
1.6
1.55
1.5
1.45
1.4
40
50
60
70
Nhit (oC)
Hnh 4.10: Kho st nhit ti u ca Termamyl c nh trn silica-alginate

Hnh 4.10, c mt s tng nh v hot tnh ca enzyme c nh khi nhit
tng t 40 70oC v cao nht 70oC. Mt khc, ht silica-alginate khi c nh
im nhit 70oC b trng n nhiu hn so vi im nhit 50oC. iu ny
cho thy nhit 70oC tuy c hot tnh ca enzyme c nh cao hn, nhng cu
trc ht li khng bn. N lm nh hng tiu cc n vic ti s dng ht gel c
nh sau ny.
4.3.2.2.
Kho st pH ti u
a) Kho st pH ti u ca Termamyl c nh trn silica-chitosan

Hnh 4.11, hot tnh ca enzyme c nh c xu hng tng dn theo gi tr pH
t 7 8 v gim pH 8,5. V chitosan bn trong mi trng c pH kim v khng
36
SVTH: Nguyn Ngc Trm
bn trong mi trng c pH thp. Kh nng chu ng ca enzyme c nh ph

thuc nhiu vo cht mang c nh chng. Mt khc, enzyme Termamyl 120L l
enzyme thng mi c kh nng chu nhit v pH trong phm vi rng hn vng
hot ng ti u ca chng nn ti pH 8, enzyme vn cn hot ng tt. Tuy nhin,
iu kin pH 8,5 (qu kim) lm nh hng n cu trc ca enzyme v enzyme
bt u b bt hot.
Hot tnh (UI)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
pH
7.5
8.5
Hnh 4.11: Kho st pH ti u ca Termamyl c nh trn silica-chitosan

Mc d hot tnh ca enzyme t do gim theo pH, nhng khi c c nh
trong cht mang silica-chitosan, kh nng chu ng ca n c tng ln v c th
chu c pH kh cao (pH 8). C th ni, silica-chitosan lm ci thin ng k
kh nng chu pH ca Termamyl.
b) Kho st pH ti u ca Termamyl c nh trn silica-alginate
Hnh 4.12, hot tnh ca enyzme c nh kh n nh, hu nh khng khc bit
nhau lm cc gi tr pH. Trong c hai gi tr c hot tnh cao hn hn l gi
tr pH 7 v pH 5,5. Khi quan st hnh thi ht v mu ca dung dch khi cho dung
dch m vo th ta thy c mt hin tng nh sau. mu cha dung dch 5; 5,5; 6
dung dch vn trong. Tuy nhin, dung dch 6,5 v 7 th dung dch b c. y l
mt hin tng bo hiu ht alginate khng bn trong mi trng c pH kim. V
37
SVTH: Nguyn Ngc Trm
vy, m bo vic ti s dng c nhiu ln v ht gel t b nh hng th ta

chn pH 5,5 l pH ti u ca Termamyl c nh trn silica-alginate.
Hot tnh (UI)
0.6
0.58
0.56
0.54
0.52
0.5
0.48
0.46
0.44
0.42
0.4
pH
5
5.5
6.5
Hnh 4.12: Kho st pH ti u ca Termamyl c nh trn silica-alginate

Vy, ta chn pH 8 l pH ti u ca Termamyl c nh trn silica-chitosan v
pH 5,5 l pH ti u ca Termamyl c nh trn silica-alginate tip tc thc hin
cc nghin cu tip theo.
4.3.3. Kho st kh nng ti s dng ca -amylase c nh
4.3.3.1. Kho st kh nng ti s dng ca Termamyl trn silica-chitosan
Trong qu trnh ti s dng, v phn ng c thc hin theo m nn lng
ng kh to ra mi m sau mi ln ti s dng l khng n nh. Thnh thong
c xut hin mt vi gi tr di 50%. Tuy nhin, ln ti s dng sau hot tnh
li tng vt trn 50%. Nguyn nhn l do vic thc hin phn ng trong iu kin
tnh (khng lc) nn xung quanh b mt ht gel hm lng ng kh bao quanh l
kh cao. N cn tr khng cho c cht mi thm thu vo trong v tip tc phn
ng. Mc khc, lng ng kh c to ra cha kp thot ra ngoi cng gy ra
hin tng ny.
38
SVTH: Nguyn Ngc Trm
Bng 4.3: S ln ti s dng Termamyl c nh trn silica-chitosan

S ln
Ban
u
X (%)
100
S ln
11
X (%)
73.66
S ln
22
X (%)
56.83
10
92.81 67.05 61.01 67.63 62.16 59.86 58.56 69.92 60.43 65.46
12
13
14
15
16
17
18
19
20
21
61.72 65.32 66.04 72.80 62.15 59.13 43.74 64.31 46.47 60.71
23
24
25
26
27
28
29
30
31
32
41.29 63.45 69.49 61.86 56.69 54.82 59.27 53.38 47.62 38.71
Trong , X l hiu sut phn ng ca ln ti s dng th n so vi ban u.

Trong X c tnh theo cng thc sau:
Hot tnh ca enzyme c nh ln th n
X (%) =
Hot tnh ca enzyme c nh ban u
Ht silica-chitosan c nh Termamyl hot ng kh n nh. Phn trm hot

tnh nhng ln ti s dng so vi ban u tuy khng cao, ch nm trong khong t
50 n 70%, nhng n kh n nh v ht gel silica-chitosan c nh Termamyl c
kh nng ti s dng ln n 30 ln. Sau khi ti s dng, ht gel vn khng c du
hiu ca s tan hoc b ph v cu trc do mi trng v iu kin phn ng (nhit
, pH).
Nhn chung, silica-chitosan c kh nng c nh tt Termamyl v hot ng
mt cch hiu qu v n nh iu kin nhit 60oC v pH 8. Mt khc, chitosan
cng l mt cht mang kh r, d kim v phng php c nh enzyme cng d
dng c thc hin, cng thm s ln ti s dng c th ln ti 30 ln bin
silica-chitosan c mt tim nng ln trong vic ng dng trong quy m cng
nghip.
39
Hiu sut phn ng (%)

100
90
80
70
60
50
40
30
20
10
S ln
0
0
10
12
13
14
16
17
21
22
24
26
27
28
30
31
Hnh 4.13: Kh nng ti s dng ca Termamyl c nh trn silica-chitosan
40
SVTH: Nguyn Ngc Trm
Sau hng lot cc th nghim kho st v kh nng c nh Termamyl trn ht

silica-chitosan thu c nhng kt qu nh sau: lng silica gel trong vo 3g
chitosan l 0.12g hay 4% (w/w), lng enzyme thch hp c nh l 0.2ml, nhit
thch hp l 60oC, pH ti thch l pH 8, s ln ti s dng lin tc iu kin ti
u l 30 ln.
4.3.3.2.
Kho st kh nng ti s dng ca Termamyl c nh trn silicaalginate iu kin nhit 50oC v pH 5,5
Alginate l mt cht mang c nhiu u im vt tri hn hn cc cht mang

khc. N c cu trc lin kt kh bn, tan trong iu kin pH thng, bn trong
khong pH t 4,5 n 6,5. Mt khc, nh vo u im c th tan iu kin thng
v d dng to gel khi gp mi trng c ion calci, n c xem l mt cht
mang thch hp by, nht enzyme trnh tn tht, tht thot enzyme trong qu
trnh phn ng so vi cc phng php b mt khc. Cng v l do m kh nng
ti s dng ca n cng cao hn hn. C th trong nghin cu ny, Termamyl c
nh trn gel silica-alginate 6% c kh nng ti s dng n 42 ln, mi ln phn
ng l 30 pht.
Nhn chung, hot tnh ca Termamyl c nh trn silica-alginate tng i n
nh. Tuy hiu sut phn ng nhng ln ti s dng thp hn ban u kh nhiu
nhng hot tnh ca n dao ng trong khong t 50% - 80%. Cng ging nh
enzyme c nh trn ht silica-chitosan, y cng xut hin mt vi im c hiu
sut phn ng thp hn 50% (ln ti s dng th 6, 8, 44). Tuy nhin, im ti s
dng tip theo th hiu sut phn ng li tng tr li trn 50% v c khi cn ln ti
70%.
Bng 4.4: S ln ti s dng Termamyl c nh trn silica-alginate 50oC, pH 5,5
S ln
10
11
X (%) 64.87 62.09 67.65 61.16 63.63 59.45 73.35 62.70 71.80 73.04 69.95
S ln
12
13
14
15
16
17
18
19
20
21
22
41
SVTH: Nguyn Ngc Trm
X (%) 70.11 66.71 77.98 70.57 64.70 42.47 74.74 49.26 65.78 64.24 57.29
S ln
23
24
25
26
27
28
29
30
31
32
33
X (%) 75.51 70.26 71.03 74.58 69.03 78.75 73.97 76.90 71.34 84.46 58.53
S ln
34
35
36
37
38
39
40
41
42
43
44
X (%) 58.68 71.19 73.81 72.27 71.19 58.22 68.10 55.44 68.41 43.42 34.81
iu kin ti u c chn i vi phng php nht Termamyl trong sodium
alginate 3%, CaCl2 3% v 0,2ml enzyme gip gi c enzyme c nh c th ti
s dng 42 ln v cu trc ht gel vn nguyn vn. Ht silica-alginate c t ra
trong nghin cu ny l mt k thut tng i n gin v r cho enzyme c nh
so vi vic ph calcium alginate bn ngoi ht chitosan trong nhng nghin cu
khc [12, 30].
Theo nghin cu ca Zoe Konsoula (2006) ht silica-alginate ca h c iu
kin ti u l 2% silica gel c b sung vo dung dch alginate 2%, to ht trong
dung dch CaCl2 5% v nhit phn ng t 50 70oC trong m phosphate 7,0
[34]. Phn ng c thc hin trong 8 gi v sau ht gel c ra sch bng
m v em i bo qun lnh cho gel phc hi. Kt qu ht silica-alginate ca h c
kh nng ti s dng c 20 ln vi tng lng thi gian phn ng l 160 gi. Mt
vi nguyn nhn dn n s khc nhau gia nghin cu ca Zoe Konsoula (2006)
v nghin cu ny.
42
Hiu sut phn ng (%)

100
75
50
25
S ln
0
0
10
12
14
16
20
22
24
26
28
30
32
34
36
38
40
42
44
Hnh 4.14: Kh nng ti s dng ca Termamyl c nh trn silica-alginate

43
SVTH: Nguyn Ngc Trm
- Mt l ngun nguyn liu. Nguyn liu h dng u t cc nh sn xut hng

u ti Anh cung cp, t cht lng tt hn nhiu so vi ngun nguyn liu ta s
dng (hu ht t Trung Quc).
- Hai l, sau mi m phn ng, h bo qun lnh cho ht gel c thi gian phc
hi sau mt qung thi gian di phn ng. iu ny mang li h qu tt hn
nhng ln phn ng tip theo.
i vi nghin cu ny, phn ng c tin hnh lin tc khng ngng ngh,
a iu kin th nghim tin gn n iu kin sn xut theo quy m cng nghip
kho st kh nng ti s dng ca ht silica-alginate n u. V kt qu 42 ln
ti s dng l t yu cu.
4.4.
ng c nh glucoamylase v Termamyl trn silica-alginate

Theo nghin cu trc ca TS. Hunh Ngc Oanh (2010) v t kho st thc
nghim cho thy kh nng to ng kh ca glucoamylase kh tt. N c th phn

ct cc lin kt -1,4 v -1,6 glucoside ca chui oligosaccharide thnh nhng gc
ng kh vi kch thc ngn, ch yu l isomaltose v glucose. Nhng sn phm
ny c ng dng rt nhiu trong thc phm. Tuy nhin, vic c nh
glucoamylase trn silica gel cng cho kt qu khng kh quan lm. Tnh trng ra
tri ca glucoamylase c nh cng tng t vi Termamyl (xem ph lc 3b). Mt
khc, theo nhng nghin cu v enzyme ng c nh, s ng c nh
glucoamylase v Termamyl mang li hiu qu kh cao, cao hn hn vic ch s
dng mt trong hai loi enzyme [11, 16, 32]. Bn cnh , hai cht mang silicachitosan v silica-alginate, kt qu cho thy vic c nh enzyme trn silica-alginate
tt hn trn silica-chitosan. V th, chng ti thc hin ng c nh
glucoamylase v Termamyl trn vt liu silica-alginate.
4.4.1. Kho st nhit ti u
Hnh 4.15 cho thy hot tnh ca enzyme ng c nh cc mc nhit kh
n nh. Khi tng nhit t 50 70oC, hot tnh ca enzyme ng c nh c xu
hng gim dn. Nguyn nhn do glucoamylase kh nhy cm vi nhit . Theo
ph lc 3b, glucoamylase hot ng mnh nhit phng. Khi nhit tng dn
44
SVTH: Nguyn Ngc Trm
t 50oC tr ln th hot tnh ca glucoamylase bt du gim. V th tc dng h tr

Termamyl trong vic thy phn h tinh bt gim.
Hot tnh (UI)
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
40
50
60
70
Nhit (oC)
Hnh 4.15: Kho st nhit ti u ca glucoamylase v Termamyl ng c nh

trn silica-alginate
4.4.2. Kho st pH ti u
Tng t nh Termamyl c nh trn silica-alginate, y enzyme ng c
nh trn silica-alginate hot ng tt nht pH 5,5 l pH m silica-alginate hot
ng n nh nht. Gi tr pH cng tng th cu trc ca silica-alginate khng bn v
lm nh hng ng k n hot tnh ca hai enzyme ng c nh.
45
SVTH: Nguyn Ngc Trm
Hot tnh (UI)

1
0.9
0.8
0.7
0.6
0.5
0.4
5
5.5
6.5
pH
Hnh 4.16: Kho st pH ti u ca Termamyl v glucoamylase ng c nh trn

silica-alginate
4.4.3. Ti s dng hai enzyme ng c nh trn silica-alginate
Nhn chung, hot tnh ca enzyme ng c nh trn silica-alginate kh n
nh v duy tr c hot tnh trong khong thi gian di. Do thi gian th nghim
c hn, khng cho php thc hin phn ng lin tc nn s ln ti s dng c tin
hnh kho st trong 4 ngy. Nhng ln phn ng u tin ca mt ngy mi cho
hiu sut phn ng cao hn nhng ln tip theo trong ngy (c th l ln ti s dng
th 10, 25, 34 v 44). Nguyn nhn v ht silica-alginate khi phn ng lu 50oC c
xu hng b trng n bi nhit. Sau mt ngy phn ng, mu c ra sch bng
nc ct v bo qun 4oC. Trong khong thi gian bo qun, di tc dng ca
nhit thp, nhng vi l bn trong cu trc ht gel c xu hng co li, hi phc v
trng thi ban u. Nhng c cht cn bn trong s tip tc c enzyme phn ct.
iu ny gy nn s dao ng (tng t ngt) trong hiu sut phn ng ca enzyme
khi phn ng ln u tin ca ngy k tip. Tuy nhin, nhng ln phn ng ngay sau
, hiu sut phn ng ca enzyme ng c nh li tr li n nh v t dao ng.
46
Hiu sut phn ng (%)

100
75
50
25
S ln
0
0
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
40
42
44
46
Hnh 4.17: Kh nng ti s dng ca glucoamylase v Termamyl ng c nh trn silica-alginate
47
SVTH: Nguyn Ngc Trm
Bng 4.5: S ln ti s dng enzyme ng c nh trn silica-alginate 50oC, pH

5,5
S ln
10
X (%) 88.20 95.28 92.33 67.56 75.82 69.15 69.15 74.34 64.90 73.63
S ln
11
12
13
14
15
16
17
18
19
20
X (%) 58.05 64.65 63.48 65.36 66.07 64.18 66.42 53.45 59.23 42.24
S ln
21
22
23
24
25
26
27
28
29
30
X (%) 57.81 42.48 54.04 51.09 59.35 60.17 54.16 56.16 56.28 59.35
S ln
31
32
33
34
35
36
37
38
39
40
X (%) 54.16 60.41 53.21 62.53 50.97 53.80 54.86 55.45 56.28 54.86
S ln
41
42
43
44
45
46
X (%) 49.44 57.46 56.28 61.12 48.73 36.34

Enzyme ng c nh c kh nng ti s dng (44 ln) nhiu hn so vi vic
c nh ch Termamyl (42 ln). N cho thy kh nng h tr xc tc ca hai enzyme
glucoamylase v Termamyl khi ng c nh trn silica-alginate.
48
SVTH: Nguyn Ngc Trm
KT LUN V KIN NGH

Kt lun
- Termamyl c nh trn silica gel c hot ha glutaraldehyde c hiu qu hn
khng hot ha vi glutaraldehyde. Tuy nhin s ci thin khng ng k, t l
enzyme b ra tri kh cao. Nhit v pH ti u ca Termamyl c nh trn silica
gel ln lt l 100oC v 5,5. S ln ti s dng tng i thp, ch c 2 ln.
- Hm lng silica gel thch hp b sung vo 3g dung dch chitosan 3% l
0,12g, tng ng vi t l silica/chitosan l 4% (w/w). Lng Termamyl cho vo c
nh l 0,2ml. Termamyl c nh trn silica-chitosan c nhit ti u l 60oC, pH
ti u l pH 8 v ti s dng c 30 ln.
- Hm lng silica gel thch hp b sung vo 2g dung dch alginate 3% l
0,12g, tng ng vi t l silica/chitosan l 6% (w/w). Lng Termamyl cho vo c
nh l 0,2ml. Silica-alginate c nh Termamyl c nhit v pH ti u ln lt l
l 60oC v pH 8. Kh nng ti s dng 42 ln.
- Vic ng c nh co-enzyme glucoamylase v Termamyl trn silica-alginate
cho kt qu rt kh quan, 44 ln ti s dng nhit 50oC v pH 5,5.
Vic c nh ng thi hai enzyme Termamyl v glucoamyase trn silicaalginate s gip ch cho vic thc hin mt dy chuyn phn ng lin tc bi nhiu
enzyme trong qu trnh chuyn ha. Mt khc, ta hon ton c th iu khin c
cc yu t nh thi gian phn ng, nhit v pH trong qu trnh sn xut.
Kin ngh
Vic c nh amylase trn vt liu phi hp silica gel v alginate t hiu qu
kh cao. Tuy nhin, do thi gian thc hin nghin cu c hn nn cn nghin cu
su hn mt s yu t nh:
- Kho st cc iu kin ng c nh glucoamylase v Termamyl theo iu
kin lc.
- Kho st iu kin ng c nh glucoamylase v Termamyl mt ct v hai
ct phn ng cng nh ti u cc iu kin nhit , pH ti u, s ln ti s dng,
49
SVTH: Nguyn Ngc Trm
iu khin sn phm to thnh v thc hin ti u ha bng phn mm Design

Expert vi cc gi tr n bin c kho st nghin cu ny.
- Kho st c nh cc enzyme khc trn vt liu phi hp vi silica gel.
- Phng php c nh amylase trn silica gel c thc hin trong nghin cu
ny khng thnh cng (phng php hp ph). Ta c th khc phc bng cch c
nh enzyme bng phng php cng ha tr bng cch: silan ha vi 3-APTES (3amino propyl triethoxysilane) v glutaraldehyde hoc ng hot ha vi 2 cht hot
ha l PEI (poly ethylen imine) v glutaraldehyde. (xem thm ph lc 7b)
50
TI LIU THAM KHO

1. Phm Th Thanh Chu, Phan Tun Ngha, 2007. Cng Ngh Sinh Hc, tp 3,
Enzyme V ng dng, trang 143. NXB gio dc.
2. Hunh Ngc Oanh, 2010. Nghin cu qu trnh c nh enzyme thy phn
carbonhydrate. Lun n tin s. Trng i hc Khoa Hc T Nhin thnh
ph H Ch Minh.
3. Ali Karout et al. (2007). Immobilization of a lipoxygenase in silica gels for
application in aqueous media. Molecular Catalysis B: Enzymatic 44 , 117127.
4. Aline M. Pascoal, Sydnei Mitidieri, Ktia F. Fernandes. (2011). Food and
bioproducts processing 89, 300-306.
5. Aliya Riaz at al. (2009). Immobilization of a thermostable -amylase on calcium
alginate beads from Bacillus subtilis KIBGE-HAR. Basic and Applied Sciences 3
(3), 2883-2887. ISSN 1991-8178.
6. Alper Akkaya, Arif Hikmet Uslan. (2010). Sequential immobilization of urease to
glycidyl methacrylate grafted sodium alginate. Molecular Catalysis B: Enzymatic
67, 195-201.
7. Antonella Petri et al. (2004). Covalent immobilization of chloroperoxidase on silica
gel and properties of the immobilized biocatalyst. Molecular Catalysis B:
Enzymatic 27, 103106.
8. Arpana Kumari, Arvind M. Kayastha. (2011). Immobilization of soybean (Glycine
max) -amylase onto Chitosan and Amberlite MB-150 beads: Optimization and
characterization. Molecular Catalysis B: Enzymatic 69, 8-14.
9. Aziz Tanriseven, Zehra Olcer. (2008). A novel method for the immobilization of
glucoamylase onto polyglutaraldehyde-activated gelatin. Biochemical Engineering
Journal 39, 430-434.
10. Aziz Tanriseven, Zehra Olcer. (2010). Co-immobilization of dextransucrase and
dextranase in alginate. Process Biochemistry 45, 1645-1651.
11. Bent W. Sigurskjold et al. (2000). Glucoamylase: structure/function relationships,
and protein engineering. Biophysica Acta 1543, 275-293.
12. Bladino A, Macias M, Cantero D. (2002). Glucose oxidase release from calcium
alginate gel capsules. Enzyme Microb Technol 27, 319-324.
13. Boadi DK, Neufeld RJ (2001). Encapsulation of tannase for the hydrolysis of tea
tannins. Enzyme Microb Technol 28, 590-595.
51
14. C. Vieille, G.J. Zeikus (2001). Hyperthermophilic enzymes: sources, uses and
molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1-43.
15. Chia-Hung Kuo et al. (2012). Optimum conditions for lipase immobilization on
chitosan-coated Fe3O4 nanoparticles. Carbohydrate Polymers 87, 2538 2545.
16. Cristina Lpez et al. (2006). Enzymatic inhibition and thermal inactivation in the
hydrolysis of chestnut pure with an amylases mixture. Enzyme and Microbial
Technology 39, 252-258.
17. Daewon Park et al. (2005). Immobilization of starch-converting enzymes on
surface-modified carriers using single and co-immobilized systems: properties and
application to starch hydrolysis. Process Biochemistry 40, 5361.
18. Dilek Kl Apar, Belma zbek. (2004). Process Biochemistry 39, 11371144.
19. Hertzberg S et al. (1992). Alginate as immobilization matrix and stabilizing agent
in two-phase liquid system: application in lipase-catalyzed reactions. Enzyme
Microb Technol 14, 42-47..
20. Jian Qin Zhou, Jian Wen Wang. (2009). Immobilization of alliinase with a water
solubleinsoluble reversible N-succinyl-chitosan for allicin production. Enzyme
and Microbial Technology 45, 299-304.
21. Jolanta Bryjak et al. (2007). Evaluation of man-tailored cellulose-based carriers in
glucoamylase immobilization. Carbohydrate Research 342, 11051109.
22. Jun-ichi Ida, Tatsushi Matsuyama, Hideo Yamamoto (2000). Immobilization of
glucoamylase on ceramic membrane surfaces modified with a new method of
treatment utilizing SPCPCVD. Biochemical Engineering Journal 5, 179-184.
23. M. Vezir Kahraman et al. (2007). -amylase immobilization on functionalized glass
beads by covalent attachment. Food Chemistry 104, 1385-1392.
24. Mayur G. Sankalia et al. (2007). Reversed chitosanalginate polyelectrolyte
complex for stability improvement of alpha-amylase: Optimization and
physicochemical characterization. Pharmaceutics and Biopharmaceutics 65, 215232.
25. P. Nigam, D. Singh (1995). Enzyme and microbial systems involved in starch
processing. Enzyme Microb. Technol. 17, 770-778.
26. Roy I, Gupta MN. (2004). Hydrolysis of starch by a mixture of glycoamylase and
pullulanase entrapped individually in calcium alginate beads. Enzyme Microb
Technol 34, 26-32.
27. Seung Won Park et al. (2002). Covalent immobilization of GL-7-ACA acylase on
silica gel through silanization. Reactive & Functional Polymers 51 , 7992.
52
28. Thayyath Screenivasan Anirudhan, Sreenivasan Rijith (2009). Glutaraldehyde

cross-linked epoxyaminated chitosan as an adsorbent for the removal and recovery
of copper (II) from aqueous media, Physicochem. Physicochem. Eng. Aspects 351,
52-59.
29. The amylase research society of Japan. (1988). Handbook of amylases and related
enzymes. Pergamon Press, Oxford, United Kingdom.
30. Tiourina OP, Sukhorukov GB. (2002). Multilayer alginate/protamine micro- sized
capsules: encapsulation of -chymotrypsin and controlled release study. Int J
Pharm 242, 155-161.
31. Y.M. Yang, J.W. Wang, R.X. Tan. (2004). Immobilization of glucose oxidase on
chitosanSiO2 gel. Enzyme and Microbial Technology 34, 126-131.
32. W. Gerhartz, 1990. Enzyme in industry
33. Z. Findrik. (2009). Starch hydrolysis by the synergistic action of amylase and
glucoamylase. New Biotechnology 25S. Doi: 10.1016/j.nbt.2009.06.1003
34. Zoe Konsoula, Maria Liakopoulou, Kyriakides. (2006). Starch hydrolysis by the
action of an entrapped in alginate caps ules -amylase from Bacillus subtilis.
Biochemistry 41, 343-349.
35. http://www.cyberchemvn.com/cyberchemvn/index.php?option=com_content
&view=article&id=67:lp-v-thong-minh-cho-phep-phong-thich-cht-cha-bentrong-nhit-tuy-chn&catid=39:theorical&Itemid=2
36. www.novozymes.com
37. http://www.sbu.ac.uk/water/hydro.html
38. http://vi.wikipedia.org/wiki/Silica_gel
53
PH LC A: CC PHNG PHP PHN TCH

nh ngha hot tnh ca enzyme
Mt n v hot (UI) ca Termamyl l s mg glucose sinh ra khi cho 1ml enzyme
thy phn tinh bt trong 1 pht iu kin nhit v pH thch hp.
S thy phn tinh bt.
1ml tinh bt 1% cng vi 2ml m pH thch hp c cho vo 1g mu enzyme c nh
v 0,2ml enzyme t do. Phn ng c thc hin trong ng nghim, yn trong 30 pht
nhit kho st. Sau dung dch sau phn ng s c em i tin hnh xc nh hm
lng ng kh. Ht sau khi phn ng c ra sch bng nc ct v c bo qun lnh.
Ph lc 1a: Phng php xc nh hot tnh
DNS (dinitro saliclic acid) l phng php xc nh hot tnh ca enzyme -amylase
da vo hm lng ng kh. Dung dch ng c pha long v cho tng ng 3ml mu
v 1ml DNS. Tip n mu c un trong nc si trong 5 pht, ngui v tin hnh o
quang bc sng 540nm. Lng ng kh c suy trc tip t phng trnh ng
chun vi D-glucose l cht chun.
Phng trnh ng chun DNS:
a = 0.407
vi r2 = 0.988
OD + 0.033
Hm lng ng kh c xc nh theo cng thc sau:

H=
(UI)
(1)
Trong :
a: hm lng ng kh theo ng chun (mg/ml); n: h s pha long; H: hot tnh
enzyme (UI); V: th tch dung dch thy phn (c cht, m v enzyme) (ml); l: lng
enzyme tham gia phn ng (ml) i vi enzyme t do v khi lng gel c nh enzyme (g)
i vi enzyme c nh; t: thi gian phn ng (30 pht).
Hiu sut c nh
enzyme (%)
Hot tnh ca enzyme c nh

=
Hot tnh ca enzyme t do
Ph lc 2a: Phng php xc nh hm lng protein

Bradford l phng php xc nh hm lng protein ca enzyme Termamyl da vo
kh nng bt mu ca protein vi dung dch Bradford. Vi mi 1ml mu cho tng ng 2ml
dung dch Bradford v lc u, yn trong 2 pht v tin hnh o quang bc sng
54
595nm. Lng protein c suy trc tip t phng trnh ng chun vi albumin l cht
chun.
Phng trnh ng chun Bradford: a = 1993
OD 1.253
vi r2 = 0.997
Lng protein (C) c trong 1g hoc 1ml nguyn liu c tnh theo cng thc:
C = a x n (mg)/1 (g hoc ml)
Tng lng protein trong mu (M)
M = a x n x m (mg)
Vi a: hm lng protein theo ng chun (mg/ml); n: h s pha long; m: khi lng
hoc th tch nguyn liu (g hoc ml)
Tng lng protein ban u = nng protein trong dung dch ban u x lng ch
phm enzyme ban u em s dng (ml)
Hm lng protein c nh = hm lng protein ban u - hm lng protein cn li.
Hiu sut c nh
protein (%)
Hm lng protein ca enzyme c nh

=
Hm lng protein ca enzyme t do
Ph lc 3a: To bt silica gel

u tin, cho dung dch (NH4)2SO4 1M vo cc. Tip n cho dung dch Na2SiO3 1M
vo cc trn, va cho va khuy u trong 1 gi. Sau dung dch c yn qua m
nhit phng. Dung dch c ra sch vi nc ct v c sy kh. Ta thu c silica gel
th.
Silica gel th c x l vi dung dch HNO3 5% trung ha lng base trong gel.
Sau , gel c hot ha vi glutaraldehyde 10% trong 3 gi 4oC. Tip theo, enzyme c
cho vo gel thc hin qu trnh c nh. Thi gian c nh enzyme l 3 gi
55
Dung dch (NH4)2SO4

1M
Dung dch Na2SiO3 1M
To sol
To gel
Nc ct
Lc, ra
Na2 SiO3
1
= v th t ch
(NH4 )2 SO4
1
Khuy nhit phng
Tip tc khuy nhit
phng trong 30 pht
yn qua m nhit
phng
B nc ra
Phn trn phu

Sy kh
Silica gel th
Dung dch HNO3 5%
Trung ha
un si trong 1gi
Sy kh
Dung dch glutaraldehyde
Hot ha
Ngm trong 3 gi 4oC
10%
Bt silica gel
Hnh 1: Quy trnh to bt silica gel
56
PH LC B:
BNG TNH TON
Ph lc 1b: Cc thng s ca Termamyl v glucoamylase t do

Bng 1: Hot tnh ca Termamyl v glucoamylase t do
Cc thng s
OD
OD trung bnh
Lng glucose sinh ra (mg)
H s pha long
Th tch dung dch (ml)
Thi gian phn ng (pht)
Lng enzyme tham gia (ml)
Hot tnh (UI)
Termamyl
1.003
0.994
0.981
0.993
0.437
50
3.2
30
0.2
11.657
glucoamylase
0.566
0.559
0.581
0.569
0.264
50
6.2
30
0.2
13.663
i vi Termamyl, th tch dung dch gm c 3ml h tinh bt 1% v 0.2ml enzyme. i

vi glucoamylase, th tch dung dch gm 1ml h tinh bt 3%, 5ml nc ct v 0.2ml
enzyme.
Bng 2: Hm lng protein ca Termamyl v glucoamylase t do
Cc thng s
OD
OD trung bnh
Hm lng protein (mg)
H s pha long
Lng protein c trong 1ml
Termamyl
0.046
0.045
0.045
0.045
0.051
50
12.829
glucoamylase
0.043
0.040
0.039
0.041
0.080
500
199.489
Ph lc 2b: Cc thng s ca Termamyl c nh trn silica gel

Bng 3: Hm lng protein
Cc thng s
OD
OD trung bnh
Hm lng protein (mg)
H s pha long
Lng protein c trong 1ml (1g
silica)
Termamyl c nh
0.028
0.02
0.024
0.029
0.047
1000
Termamyl t do
0.032
0.028
0.026
0.041
0.056
500
46.579
139.7
57
Tng lng protein c trong 5ml

enzyme*
Hiu sut c nh protein (%)
232.9
698.5
66.66
* Trong nghin cu ny, chng ti dng 5ml Termamyl cho c nh trn 1g silica gel. tnh
hiu sut c nh, chng ti quy v hm lng protein tng.
Bng 4: Hot tnh ca Termamyl c nh
Termamyl c nh
0.400
0.381
0.404
0.395
0.194
10
6
30
Cc thng s
OD
OD trung bnh
H s pha long
Lng enzyme tham gia (ml)
Hot tnh (UI)
Hiu sut c nh enzyme (%)
1.183
5.12
Termamyl t do
0.199
0.196
0.186
0.194
0.112
40
6.2
30
0.2
4.622
Bng 5: Kho st nh hng ca nhit

Cc thng s
Nhit (oC)
OD
OD trung bnh
H s pha long
Hot tnh (UI)
Termamyl c nh
Nhit phng
70
85
0.400
0.176
0.224
0.381
0.146
0.243
0.404
0.180
0.231
0.395
0.167
0.233
0.194
0.101
0.128
10
20
20
6
6
6
30
25
25
0.388
0.485
0.613
100
0.427
0.431
0.420
0.426
0.206
20
6
25
0.991
Bng 6: Kho st nh hng ca pH

Cc thng s
pH
OD
OD trung bnh
Lng glucose sinh ra
5
0.179
0.197
0.192
0.189
0.110
5.5
0.297
0.301
0.269
0.289
0.151
Termamyl c nh
6
6.5
0.25
0.2
0.24
0.208
0.234
0.216
0.241
0.208
0.131
0.118
7
0.214
0.218
0.218
0.217
0.121
58
(mg)
H s pha long
Hot tnh (UI)
20
6
30
0.440
20
6
30
0.602
20
6
30
0.525
20
6
30
0.471
20
6
30
0.485
Ph lc 3b: Cc thng s ca glucoamylase c nh trn silica gel

Bng 7: Hm lng protein
Th tch enzyme cho vo

c nh (ml)
OD
OD trung bnh
Hm lng protein (mg)
H s pha long
Lng protein c trong
1ml (1g silica)
Tng lng protein (mg)
Hiu sut c nh protein
(%)
Enzyme
t do
Enzyme c nh
Cc thng s
4
0.2
0.027
0.025
0.028
0.027
0.052
1250
0.031
0.026
0.027
0.028
0.055
1000
0.025
0.029
0.03
0.028
0.055
833
0.033
0.03
0.034
0.0323
0.063
714
0.042
0.046
0.042
0.0433
0.085
625
0.043
0.040
0.039
0.041
0.056
500
64.87
54.55
45.46
45.13
53.19
199.489
259.47
272.76
272.76
315.94
425.55
67.48
72.65
77.21
77.38
73.33
Bng 8: Hot tnh glucoamylase c nh trn silica gel

Enzyme c nh
Cc thng s
Th tch enzyme (ml)
OD
OD trung bnh
H s pha long
Hot tnh (UI)
Hiu sut c nh enzyme
(%)
4
0.841
0.868
0.838
0.849
0.379
10
6
30
1.39
5
0.593
0.525
0.585
0.568
0.264
20
6
30
1.55
6
0.781
0.736
0.761
0.759
0.342
20
6
30
1.67
7
0.615
0.543
0.625
0.594
0.275
20
6
30
1.15
8
0.649
0.737
0.683
0.690
0.314
20
6
30
1.15
0.757
1.056
1.368
1.100
1.255
Enzyme
t do
0.2
0.566
0.559
0.581
0.569
0.264
50
6.2
30
13.663
59
Bng 9: Nhit v pH ti u kho st theo Design Expert

Glucoamylase c nh trn silica gel
Cc thng s
mu
10
11
12
13
nhit ( C)
80
80
70
80
90
80
80
80
65
80
90
70
94
pH
6.5
3.8
3.34
3.8
0.094
0.036
0.211
0.095
0.986
0.228
0.268
0.377
0.454
0.412
0.57
0.286
0.484
0.086
0.046
0.224
0.126
1.058
0.209
0.182
0.427
0.456
0.379
0.563
0.271
0.484
0.12
0.03
0.215
0.136
0.954
0.273
0.205
0.415
0.444
0.331
0.584
0.294
0.491
OD trung bnh
0.100
0.037
0.217
0.119
0.999
0.237
0.218
0.406
0.451
0.374
0.572
0.284
0.486
0.074
0.048
0.121
0.081
0.440
0.129
0.122
0.198
0.217
0.185
0.266
0.148
0.231
H s pha long
40
40
40
40
40
40
40
40
40
40
40
40
40
30
30
30
30
30
30
30
30
30
30
30
30
30
Hot tnh (UI)
0.590
0.386
0.969
0.651
3.518
1.035
0.975
1.587
1.734
1.482
2.128
1.188
1.848
OD
60
Ph lc 4b: Termamyl c nh trn silica-chitosan

Bng 10: Kho st lng silica thm vo chitosan
Mu
0.03
0.06
0.09
0.12
0.15
0.214
0.203
0.213
0.210
0.118
25
0.207
0.189
0.213
0.203
0.115
25
0.203
0.229
0.196
0.209
0.118
25
0.232
0.221
0.213
0.222
0.123
25
0.239
0.248
0.232
0.240
0.131
25
0.219
0.212
0.221
0.217
0.121
25
3.2
30
30
30
30
30
30
30
1.01
1.03
1.05
1.04
1.02
Khi lng silica

gel (g)
Khi lng
alginate 3% (g)
OD
OD TB
Lng ng kh
H s pha long
Th tch dung
dch (ml)
Thi gian (pht)
Khi lng mu
(g)
Lng enzyme t
do
Hot tnh (UI)
Hiu sut (%)
Enzyme c nh
2
3
Enzyme
t do
0.509
0.517
0.511
0.512
0.241
25
0.2
0.296
45.97
0.286
44.42
0.286
44.48
0.294
45.67
0.314
48.77
0.297
46.16
0.643
Bng 11: Hiu sut c nh protein

Termamyl c nh
0.2
0.031
0.037
0.033
0.034
0.047
50
0.3
0.041
0.035
0.036
0.037
0.048
33.33
0.4
0.043
0.047
0.046
0.045
0.051
25
0.5
0.043
0.04
0.041
0.041
0.050
20
Enzyme
t do
0.2
0.046
0.045
0.045
0.045
0.051
50
11.990 11.368
5.235
3.144
1.911
12.829
1.02
6.538
1.02
1.02
59.192 75.490
1.04
85.103
Cc thng s
Th tch enzyme (ml)
OD
OD trung bnh
Hm lng protein (mg)
H s pha long
Lng protein c trong 1ml (1g
silica)
Khi lng mu (g)
Hiu sut c nh protein (%)
0.1
-0.031
-0.037
-0.035
-0.034
0.020
60
1.03
11.383
61
Bng 12: Hot tnh Termamyl c nh trn silica-chitosan

Termamyl c nh
Cc thng s
Th tch enzyme (ml)
OD
OD trung bnh
H s pha long
Khi lng mu (g)
Hot tnh (UI)
Hiu sut c nh enzyme
(%)
0.1
0.357
0.346
0.308
0.337
0.170
25
3
30
1.02
0.417
0.2
0.819
0.841
0.796
0.819
0.366
25
3
30
1.02
0.898
0.3
0.997
1.106
1.196
1.100
0.481
25
3
30
1
1.202
0.4
1.228
1.154
1.042
1.141
0.497
25
3
30
1
1.244
0.5
1.305
1.243
1.16
1.236
0.536
25
3
30
1.01
1.327
35.78
38.51
34.36
26.67
22.76
Enzyme
t do
0.2
1.012
0.993
0.973
0.993
0.437
50
3.2
30
0.417
35.78
Bng 13: Kho st nh hng ca nhit ln Termamyl c nh trn silica-chitosan

Cc thng s
Nhit (oC)
OD
OD trung bnh
H s pha long
Khi lng mu (g)
Hot tnh (UI)
40
0.271
0.237
0.245
0.251
0.135
50
3
30
0.98
0.69
Termamyl c nh
50
60
70
0.426
0.421
0.375
0.411
0.382
0.386
0.433
0.405
0.363
0.423
0.403
0.375
0.205
0.197
0.186
50
50
50
3
3
3
35
30
30
1.02
1.06
1.06
0.86
0.93
0.88
Bng 14: Kho st nh hng ca pH ln Termamyl c nh trn silica-chitosan

Cc thng s
pH
OD
OD trung bnh
7
0.278
0.263
0.274
0.272
0.144
Termamyl c nh
7.5
8
8.5
0.299
0.271
0.251
0.283
0.283
0.248
0.291
0.285
0.237
0.291
0.280
0.245
0.151
0.147
0.133
62
H s pha long
Khi lng mu (g)
Hot tnh (UI)
25
3
30
1
0.359
25
3
30
1.02
0.371
50
3
30
1.01
0.728
50
3
30
1.04
0.638
Ph lc 5b: Cc thng s ca Termamyl c nh trn silica-alginate

Bng 15: Kho st lng silica thm vo alginate
Mu
Khi lng silica
gel (g)
Khi lng
alginate 3% (g)
OD
OD TB
Lng ng kh
H s pha long
Th tch dung
dch (ml)
Thi gian (pht)
Khi lng mu
(g)
Lng enzyme t
do
Hot tnh (UI)
Hiu sut (%)
Enzyme c nh
2
3
0.03
0.06
0.09
0.12
0.15
0.506
0.489
0.494
0.496
0.235
25
0.501
0.505
0.462
0.489
0.232
25
0.547
0.504
0.526
0.247
25
0.541
0.55
0.516
0.536
0.251
25
0.563
0.572
0.489
0.541
0.253
25
0.461
0.488
0.461
0.475
0.226
25
1.01
0.444
50
3.2
30
30
30
30
30
30
30
1.01
1.02
1.02
1.01
Enzyme
t do
0.99
1.03
0.2
1.163
49.123
1.138
48.051
1.210
51.098
1.255
52.993
1.267
53.48
1.119
47.265
2.368
Bng 16: Kho st nh hng ca nhit ln Termamyl c nh trn silica-alginate

Cc thng s
Nhit (oC)
OD
OD trung bnh
H s pha long
40
0.690
0.684
0.678
0.684
0.311
25
3
30
Termamyl c nh
50
60
0.207
0.184
0.181
0.176
0.183
0.183
0.190
0.181
0.338
0.324
25
25
3
3
30
30
70
0.193
0.172
0.176
0.180
0.359
25
3
30
63
Khi lng mu (g)

Hot tnh (UI)
1
1.557
1.01
1.673
0.99
1.638
1.01
1.775
Bng 17: Kho st nh hng ca pH ln Termamyl c nh trn silica-alginate

Cc thng s
pH
OD
OD trung bnh
H s pha long
Khi lng mu (g)
Hot tnh (UI)
5
0.250
0.250
0.245
0.248
0.103
25
3
30
1
0.515
Termamyl c nh
5.5
6
6.5
0.207
0.184
0.193
0.181
0.176
0.172
0.183
0.183
0.176
0.190
0.181
0.180
0.110
0.107
0.106
25
25
25
3
3
3
30
30
30
1.04
1.03
1.03
0.530
0.518
0.516
7
0.192
0.176
0.201
0.190
0.110
25
3
30
1.01
0.546
Ph lc 6b: ng c nh Termamyl v glucoamylase trn silica-alginate

Bng 18: Kho st nh hng ca nhit ln enzyme ng c nh trn silica-alginate
Cc thng s
Nhit (oC)
OD
OD trung bnh
H s pha long
Khi lng mu (g)
Hot tnh (UI)
40
1.114
1.037
1.026
1.059
0.464
25
3
30
1
2.320
Enzyme c nh
50
60
0.994
1.023
1.121
1.042
1.147
0.993
1.087
1.019
0.4754 0.4477
25
25
3
3
30
30
1
1.01
2.377
2.217
70
0.874
0.784
0.894
0.851
0.3794
25
3
30
0.99
1.916
Bng 19: Kho st nh hng ca pH ln enzyme ng c nh trn silica-alginate

Cc thng s
pH
OD
OD trung bnh
H s pha long
5
0.309
0.328
0.313
0.317
0.162
25
Enzyme c nh
5.5
6
0.341
0.301
0.359
0.322
0.327
0.277
0.342
0.300
0.172
0.155
25
25
6.5
0.279
0.284
0.259
0.274
0.145
25
64

Khi lng mu (g)
Hot tnh (UI)
3
30
1
0.810
3
30
0.97
0.888
3
30
1.01
0.768
3
30
0.99
0.730
Ph lc 7b: Hot ha silica gel vi PEI v glutaraldehyde
Hnh 2: Hot ha silica gel
65
Ph lc 8b: Ti s dng
Bng 20: Kho st kh nng ti s dng Termamyl c nh trn silica gel
Cc thng s
Hot ha glutaraldehyde
S ln ti s dng
OD
OD trung bnh
Lng glucose sinh ra
(mg)
H s pha long
Hot tnh (UI)
Hiu sut phn ng (%)
Termamyl c nh
1
0.217
0.23
0.198
0.215
Khng hot ha
2
3
0.145
0.087
0.143
0.098
0.136
0.11
0.141
0.098
1
0.467
0.521
0.516
0.501
C hot ha
2
3
0.431
0.312
0.428
0.319
0.423
0.293
0.427
0.308
4
0.837
0.839
0.831
0.836
4
0.869
0.873
0.858
0.867
0.121
0.090
0.073
0.373
0.237
0.207
0.158
0.386
20
6
30
0.482
100
20
6
30
0.362
75.007
20
6
30
0.292
60.484
3
6
30
0.224
22.660
20
6
30
0.948
100
20
6
30
0.827
87.287
20
6
30
0.633
66.843
3
6
30
0.232
23.426
Bng 21: Kho st kh nng ti s dng glucoamylase c nh trn silica gel

Cc thng s
Hot ha glutaraldehyde
S ln ti s dng
OD
Glucoamylase c nh
Khng hot ha
C hot ha
1
2
3
1
2
0.511
0.354
0.443
0.523
0.443
0.521
0.353
0.45
0.534
0.453
0.523
0.378
0.451
0.521
0.439
3
0.513
0.514
0.521
66
OD trung bnh
Lng glucose sinh ra
(mg)
H s pha long
Hot tnh (UI)
Hiu sut phn ng (%)
0.5183
0.362
0.448
0.526
0.445
0.516
0.244
0.180
0.215
0.247
0.214
0.243
20
6
30
0.975
100
20
6
30
0.721
73.960
10
6
30
0.431
44.158
20
6
30
0.988
100
20
6
30
0.856
86.657
10
6
30
0.486
49.176
Bng 22: Kh nng ti s dng ca Termamyl c nh trn silica-chitosan

S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
Khi lng mu (g)
Hot tnh (UI)
Hiu sut phn ng
0
0.197
0.113
25
3
30
0.97
0.292
100
1
0.177
0.105
25
3
30
0.97
0.271
92.81
2
0.152
0.095
20
3
30
0.97
0.196
67.05
S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
12
0.348
0.175
10
3
30
13
0.373
0.185
10
3
30
14
0.378
0.187
10
3
30
Termamyl c nh trn silica-chitosan

3
4
5
6
7
8
0.131
0.154
0.135 0.127
0.326 0.405
0.086
0.096
0.088 0.085
0.166 0.198
20
20
20
20
10
10
3
3
3
3
3
3
30
30
30
30
30
30
0.97
0.97
0.97
0.97
0.97
0.97
0.178
0.197
0.181 0.175
0.171 0.204
61.01
67.63
62.16 59.86
58.56 69.92
15
16
17
18
19
20
0.425
0.351
0.33
0.223
0.366 0.242
0.206
0.176
0.167 0.124
0.182 0.131
10
10
10
10
10
10
3
3
3
3
3
3
30
30
30
30
30
30
9
0.339
0.171
10
3
30
0.97
0.176
60.43
10
0.374
0.185
10
3
30
0.97
0.191
65.46
11
0.431
0.208
10
3
30
0.97
0.215
73.66
21
0.341
0.172
10
3
30
22
0.314
0.161
10
3
30
23
0.206
0.117
10
3
30
67
Khi lng mu (g)

Hot tnh (UI)
Hiu sut phn ng
0.97
0.180
61.72
0.97
0.191
65.32
0.97
0.193
66.04
S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
Khi lng mu (g)
Hot tnh (UI)
Hiu sut phn ng
24
0.36
0.180
10
3
30
0.97
0.185
63.45
25
0.402
0.197
10
3
30
0.97
0.203
69.49
26
0.349
0.175
10
3
30
0.97
0.180
61.86
0.97
0.212
72.80
0.97
0.97
0.97
0.97
0.97
0.181
0.172 0.128
0.188 0.136
62.15
59.13 43.74
64.31 46.47
27
28
29
30
31
32
0.313
0.3
0.331
0.29
0.25
0.188
0.160
0.155
0.168 0.151
0.135 0.110
10
10
10
10
10
10
3
3
3
3
3
3
30
30
30
30
30
30
0.97
0.97
0.97
0.97
0.97
0.97
0.165
0.160
0.173 0.156
0.139 0.113
56.69
54.82
59.27 53.38
47.62 38.71
0.97
0.177
60.71
0.97
0.166
56.83
0.97
0.120
41.29
9
0.384
0.189
10
3
30
0.97
0.195
71.80
10
0.392
0.193
10
3
30
0.97
0.198
73.04
11
0.372
0.184
10
3
30
0.97
0.190
69.95
21
0.335
22
0.29
23
0.408
Bng 23: Kh nng ti s dng ca Termamyl c nh trn silica-alginate

S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
Khi lng mu (g)
Hot tnh (UI)
Hiu sut phn ng
0
0.178
0.105
25
3
30
0.97
0.272
100
1
0.129
0.086
20
3
30
0.97
0.176
64.87
2
0.12
0.082
20
3
30
0.97
0.169
62.09
3
0.138
0.089
20
3
30
0.97
0.184
67.65
S ln ti s dng
OD TB
12
0.373
13
0.351
14
0.424
15
0.376
Termamyl c nh trn silica-alginate

4
5
6
7
8
0.117
0.125 0.304
0.394 0.325
0.081
0.084 0.157
0.193 0.165
20
20
10
10
10
3
3
3
3
3
30
30
30
30
30
0.97
0.97
0.97
0.97
0.97
0.166
0.173 0.162
0.199 0.170
61.16
63.63 59.45
73.35 62.70
16
17
18
19
20
0.338
0.194 0.403
0.238 0.345
68
lng ng kh
h s pha long
Thi gian (pht)
Khi lng mu (g)
Hot tnh (UI)
Hiu sut phn ng
0.185
10
3
30
0.97
0.191
70.11
S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
Khi lng mu (g)
Hot tnh (UI)
Hiu sut phn ng
23
24
25
26
0.374
0.185
10
3
30
0.97
0.191
70.26
0.379
0.187
10
3
30
0.97
0.193
71.03
0.402
0.197
10
3
30
0.97
0.203
74.58
0.366
0.182
10
3
30
0.97
0.188
69.03
35
36
37
38
39
40
41
42
43
44
0.38
0.188
10
3
30
0.97
0.193
71.19
0.397
0.195
10
3
30
0.97
0.201
73.81
0.387
0.191
10
3
30
0.97
0.196
72.27
0.38
0.188
10
3
30
0.97
0.193
71.19
0.296
0.153
10
3
30
0.97
0.158
58.22
0.36
0.180
10
3
30
0.97
0.185
68.10
0.278
0.146
10
3
30
0.97
0.151
55.44
0.362
0.180
10
3
30
0.97
0.186
68.41
0.287
0.150
10
3
30
0.97
0.154
43.42
0.214
0.120
10
3
30
0.97
0.124
34.81
S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
Khi lng mu (g)
Hot tnh (UI)
Hiu sut phn ng
0.176
10
3
30
0.97
0.181
66.71
0.206
10
3
30
0.97
0.212
77.98
0.186
10
3
30
0.97
0.192
70.57
0.171
0.112 0.197
0.130 0.173
10
10
10
10
10
3
3
3
3
3
30
30
30
30
30
0.97
0.97
0.97
0.97
0.97
0.176
0.115 0.203
0.134 0.179
64.70
42.47 74.74
49.26 65.78
27
28
29
30
31
0.429
0.398 0.417
0.381 0.466
0.208
0.195 0.203
0.188 0.223
10
10
10
10
10
3
3
3
3
3
30
30
30
30
30
0.97
0.97
0.97
0.97
0.97
0.214
0.201 0.209
0.194 0.230
78.75
73.97 76.90
71.34 84.46
0.169
10
3
30
0.97
0.175
64.24
0.151
10
3
30
0.97
0.156
57.29
0.199
10
3
30
0.97
0.205
75.51
32
33
34
0.298
0.154
10
3
30
0.97
0.159
58.53
0.299
0.155
10
3
30
0.97
0.159
58.68
0.374
0.185
10
3
30
0.97
0.191
70.26
69
Bng 24: Kh nng ti s dng ca Termamyl v glucoamylase ng c nh trn silica-alginate

Enzyme ng c nh trn silica-alginate
3
4
5
6
7
8
0.232
0.148
0.176 0.212
0.212 0.234
0.127
0.093
0.105 0.119
0.119 0.128
25
25
25
20
20
20
3
3
3
3
3
3
30
30
30
30
30
30
0.97
0.97
0.97
0.97
0.97
0.97
0.328
0.240
0.270 0.246
0.246 0.264
92.33
67.56
75.82 69.15
69.15 74.34
15
16
17
18
19
20
0.479
0.463
0.482 0.372
0.421 0.277
0.228
0.221
0.229 0.184
0.204 0.146
10
10
10
10
10
10
3
3
3
3
3
3
30
30
30
30
30
30
0.97
0.97
0.97
0.97
0.97
0.97
0.235
0.228
0.236 0.190
0.211 0.150
66.07
64.18
66.42 53.45
59.23 42.24
S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
Khi lng mu (g)
Hot tnh (UI)
Hiu sut phn ng
0
0.258
0.138
25
3
30
0.97
0.356
100
1
0.218
0.122
25
3
30
0.97
0.314
88.20
2
0.242
0.131
25
3
30
0.97
0.339
95.28
S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
Khi lng mu (g)
Hot tnh (UI)
Hiu sut phn ng
12
0.467
0.223
10
3
30
0.97
0.230
64.65
13
0.457
0.219
10
3
30
0.97
0.226
63.48
14
0.473
0.226
10
3
30
0.97
0.232
65.36
S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
23
24
25
26
27
28
29
30
0.352
0.176
10
3
30
0.422
0.205
10
3
30
0.429
0.208
10
3
30
0.378
0.187
10
3
30
0.395
0.194
10
3
30
0.396
0.194
10
3
30
0.422
0.205
10
3
30
0.378
0.187
10
3
30
9
0.194
0.112
20
3
30
0.97
0.231
64.90
10
0.231
0.127
20
3
30
0.97
0.262
73.63
11
0.411
0.200
10
3
30
0.97
0.206
58.05
21
0.409
0.199
10
3
30
0.97
0.206
57.81
22
0.279
0.147
10
3
30
0.97
0.151
42.48
23
0.377
0.186
10
3
30
0.97
0.192
54.04
31
32
33
34
0.431
0.208
10
3
30
0.37
0.184
10
3
30
0.449
0.216
10
3
30
0.352
0.176
10
3
30
70
Khi lng mu (g)

Hot tnh (UI)
Hiu sut phn ng
0.97
0.182
51.09
0.97
0.211
59.35
0.97
0.214
60.17
S ln ti s dng
OD TB
lng ng kh
h s pha long
Thi gian (pht)
Khi lng mu (g)
Hot tnh (UI)
Hiu sut phn ng
35
36
37
38
39
40
41
42
0.351
0.176
10
3
30
0.97
0.181
50.97
0.375
0.186
10
3
30
0.97
0.191
53.80
0.384
0.189
10
3
30
0.97
0.195
54.86
0.389
0.191
10
3
30
0.97
0.197
55.45
0.396
0.194
10
3
30
0.97
0.200
56.28
0.384
0.189
10
3
30
0.97
0.195
54.86
0.338
0.171
10
3
30
0.97
0.176
49.44
0.406
0.198
10
3
30
0.97
0.204
57.46
0.97
0.97
0.97
0.97
0.97
0.97
0.193
0.200
0.200 0.211
0.193 0.215
54.16
56.16
56.28 59.35
54.16 60.41
0.97
0.189
53.21
0.97
0.222
62.53
0.97
0.182
51.09
43
44
45
46
0.396
0.194
10
3
30
0.97
0.200
56.28
0.437
0.211
10
3
30
0.97
0.217
61.12
0.332
0.168
10
3
30
0.97
0.173
48.73
0.227
0.125
10
3
30
0.97
0.129
36.34
71

khảo sát điều kiện cố định amylase trên silica-chitosan và silica-alginate

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khảo sát điều kiện cố định amylase trên silica-chitosan và silica-alginate

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C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

GVHD: TS. Hunh Ngc Oanh

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

GVHD: TS. Hunh Ngc Oanh

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

GVHD: TS. Hunh Ngc Oanh

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

1.1. L do chn ti ........................................................................................ 1

TNG QUAN .................................................................................. 3

2.1. Khi nim v c nh enzyme ..................................................................... 3

u, nhc im ca enzyme c nh ................................................ 5

2.2. Vt liu c nh .......................................................................................... 6

Silica gel ........................................................................................... 8

2.3. Hot ha vt liu bng glutaraldehyde ...................................................... 15

2.5. iu kin phn ng khi tin hnh xc nh hot enzyme ...................... 20

Cc phng php xc nh hot enzyme .................................... 20

Mt s lu khi xc nh hot enzyme ...................................... 21

VT LIU V PHNG PHP ................................................... 22

3.1. Nguyn vt liu ........................................................................................ 22

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

3.4. Phng php nghin cu .......................................................................... 23

Chun b ht chitosan ...................................................................... 24

Chun b ht silica-chitosan ............................................................ 24

Hot ha ht silica-chitosan vi glutaraldehyde .............................. 24

C nh Termamyl ln cc cht mang ............................................. 24

Kho st nhit v pH ti u ........................................................ 25

4.1. Enzyme t do............................................................................................ 27

Hiu sut c nh Termamyl trn silica gel ..................................... 27

Kho st pH ti u ca Termamyl c nh trn silica gel ................ 28

Ti s dng Termamyl c nh trn silica gel.................................. 29

4.3. Termamyl c nh trn silica-chitosan v silica-alginate ........................... 30

iu kin phn ng ......................................................................... 35

Kho st kh nng ti s dng ca -amylase c nh ..................... 38

4.4. ng c nh glucoamylase v Termamyl trn silica-alginate................... 44

Kho st nhit ti u .................................................................. 44

Ti s dng hai enzyme ng c nh trn silica-alginate ............... 46

KT LUN V KIN NGH................................................................................ 49

BNG TNH TON ................................................................... 57

GVHD: TS. Hunh Ngc Oanh

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

Hnh 4.14: Kh nng ti s dng ca Termamyl c nh trn silica-alginate .......... 43

GVHD: TS. Hunh Ngc Oanh

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

GVHD: TS. Hunh Ngc Oanh

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

(-amylase), lin kt -1,4 v lin kt -1,6 glucoside (glucoamylase) v to sn

GVHD: TS. Hunh Ngc Oanh

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

GVHD: TS. Hunh Ngc Oanh

C nh amylase trn vt liu phi hp silica gel

SVTH: Nguyn Ngc Trm

Khi nim v c nh enzyme

Enzyme c nh (immobilized) hay enzyme khng ha tan (insoluble enzyme)