L thuyt

Glucose oxidase (GOx), mt flavonozyme, c sn xut, tinh ch t Aspergillus niger t,

m khng dng trong qu trnh oxy ha glucose. phng x ti a t 0,6 n 0,31 U
mg-1. Nm kh thu c l ni bo v ngoi t bo. Sn xut ti u (4000 IU / L) t
c khi mi trng mui c bn c b sung thm sucrose (7.5%), peptone (1.5%)
phosphorus (0.2%) v MgSO 4 (0.2%) pH 5.7 sau 48 gi tc 250 vng/pht.
Enzyme c tinh ch t s kt tama sunfat ammonium (75%), lc gel, Q-Sepharose
v DEAE Sepharose c 30,08, 63,3% v 22,3% tinh khit,% phc hi v hot ng
ring bit. Enzyme c dimeric bao gm hai tiu n v vi trng lng phn t 80 kDa.
V hin th nhit v pH optima 25-30 C v 5.5-6.0 tng ng cho qu trnh oxy ha-
D-glucose. Enzym n nh 50 C trong 1 gi m khng c bt k vn g. Vmax,
v gi tr Km 17 Umg-1 v Km, 7,1 mM tng ng. GOx b c ch bi Cu2 + (56,5%), Ag2
+ (48%) ng k v mc thp hn bi NaF (25%). S c nh v c ca Covalent
vi cc ht oxirane t hiu qu hn v hot ng b mt enzyme, nhng cc phng php
by ca alginate natri v poly acrylamide c hiu qu cho qu trnh oxy ha glucose.

GII THIU

GOx (EC 1.1.3.4, -D- glucose oxy-oxidoreductase) l mt flavoprotein xc tc qu trnh

oxy ha -Dglucose bng oxy phn t n D-glucolactone v H2O2. N loi b hydro khi
glucose v b gim, hnh thc gim ca GOx s c oxy ha li bng oxy phn t, v
hydrogen peroxide sn xut c phn hy bng catalase n nc v oxy. D-
glucolactone thy phn t nhin vi axit gluconic. Di y l phng trnh phn ng

-D-Glucose + Enzyme-FAD Enzyme-FADH2+ D-glucono-1, 5-lactone Enzyme-

FADH2 + O2 Enzyme-FAD+ H2O2

GOx l mt sn phm quan trng v mt thng mi v c ng dng nh l mt thit b

cm bin sinh hc xc nh glucose,loi b glucose v / hoc oxy v t bo nhin liu
sinh hc [8,9,10]. Nm nm Aspergillus v Penicillium l nhng nh sn xut cng nghip
ca GOx. Sn xut v tng cng tnh cht ca GOx vn ang c ch , c l l do
cc ng dng hin ti v rng ri ca enzym ny. khai thc cc ngun nng lng mi
cho tim nng cng nghip trong sn xut GOx, cn phi nghin cu cc dng vi khun
mi v hiu mi quan h n nh gia cc enzyme quan trng ny. Nghin cu ny m
t vic sn xut, thanh lc, m t v c nh ca mt GOx mi t A. niger.

T chc v bo tr

Nm A.niger (MTCC 181) c s dng trong nghin cu c mua t b su tp

nui cy vi khun loi IMTech Chandigarh, Punjab (n ). Cc vi sinh vt c duy tr
trn mi trng khoai ty dextrose 4-60C v nui cy sau mi 20 ngy.

Vn ho trc A. niger
Cc bo t nm A. niger (7.5105/ Ml) c trng trong bnh Erlenmeyer 250 ml
cha 50 ml dung mi
Thnh phn l : (NH4) 2HPO4, 0,4 (g / l); KH2PO4, 0,2(g / l), MgSO4, 0,2(g / l);
Peptone, 10(g / l); Sucrose, 70(g / l) v pH 5.5.Dung mi c trong 24 gi
trong my lc quay tc 200 vng/pht ti 300C ny mm bo t ny
mm.

Ti u ha cc iu kin nui cy sn xut GOx c ti a

i vi vic ti u ha sn xut mui basmax (BSM) ca GOx c thay i cht t

c cha:
(NH4)2HPO4 0,4(g/l), KH2PO4 0,2(g/l), NaNO3 2,0(g/l), KCl 0,5(g/l), MgSO4
0,2(g/l) v pH 5,5 c s dng cho cc tham s quy trnh ti u ha cho
sn xut GOx ti a. Tt c cc th nghim tng trng c thc hin trong
bnh Erlenmeyer 250 ml cha 50 ml dung mi. dung mi kh trng (15 pht
15 psi / cm2) c cy vo cc bo t ny mm trc 24 gi v c trong
my lc qu o (250 vng / pht) 300 o C. Cc tham s quy trnh vt l v
ho hc khc nhau iu chnh qu trnh sn xut enzym c ti u ha
bng cc phng php c in v phng php ti u ha va. Khi lng si
nanh c thu hoch bng cch ly tm tc 7000xg trong 20 pht trong
my ly tm gn vi rotor gc c nh. Tng trn to thnh phn GOx ngoi bo
v i vi sinh khi bo t phn t trong t bo (trng lng t) 50 gm
c l lng trong 10 ml dung dch m natri citrate pH 5.75 (50mM). Nit lng
c thm vo nm sinh khi v sau khi bc hi nit, sinh khi b nghin nt
thnh bt trong m. Tng lp trong sut ca cht nn t bo b v to thnh
phn GOx ni bo.

Xt nghim enzyme v protein

Hot tnh GOx c xc nh bi Ciucu v Petroescu, c Markwell v cng s sa i.

Bng cch gim benzoquinone xung hydroquinone. Vic c lng hydroquinone c
thc hin 290 nm ( = 2.31 mM-1cm-1) trn my quang ph UV-VIS. Mt n v (IU)
hot tnh GOx c nh ngha l lng enzyme, lm gim 1,0 M benzoquinone ml-1
pht-1. Nng protein c xc nh bng phng php hp th . Protein (mg / ml) =
1,55 A280 - 0,76 A260

Thanh lc GOx

Thnh phn enzyme c ly tm 9000xg trong 10 pht v protein sau c kt ta

bng dung dch amoni sulfat (75% phn on) 40C. Protein kt ta c ly tm trong
45 pht (10.000 xg 40C) v vin bi c l lng trong 10 ml m v lc gel (Sephadex
G-100) c a ra lm mui ra v chit xut GOx trn c s khi lng phn t.
Phn s vi hot ng enzyme ti a c kt hp v tinh ch thm vi Q-Sepharose
(anion trao i mnh) v DEAE-Sepharose (anion trao i yu) bng sc k trao i ion.
Protein gii hn c ra bng gradient tuyn tnh 500 ml t 50 n 500 mM NaCl
trong dung dch Tris m 20mM Tris (pH 7,2) tc dng chy 2 ml-1, vi b su tp
cc phn s 05 ml. Protein v GOx Hot ng xc nh cho mi phn s vi phng php
kho nghim cp trn.

c tnh ca GOx
Xc nh trng lng phn t Xc nh trng lng phn t GOx bng phng
php SDS-PAGE vi 12% acrylamide gel s dng t bo in di Mini-Gel
(Genei). GOx tinh khit c np v cc protein c hnh dung bng
Coomassie Brilliant Blue R-250. Trng s phn t gn ng c tnh ton

Nhit v pH ti u

B m kho nghim GOx c cn bng trong 10 pht nhit khc nhau (15 n
40C) trong bn nc iu khin nhit , trc khi bt u phn ng vi vic b sung
GOx (0.15Uml-1). pH ca GOx c thc hin trong cc h thng m khc nhau c
cha 50 mM conc. iu chnh n cc gi tr pH ca mc li sut dao ng t 4,0-8,5.

Th nghim Nhit n nh
S n nh ca GOx tinh khit c xc nh 30-60C, v nhit ny tng quan vi
cc ng dng tim nng ca enzyme. Lm sch GOx nng 1,25 IUml-1 c
chun b v trong bn tm trc cn bng cc nhit khc nhau, 30-60 C. Mu
(100 l) c loi b sau khong thi gian khc nhau cho phn tch hot ng ca
GOx v so snh vi mu ban u c thc hin khi bt u th nghim.

Xc nh c hiu v xc nh hng s ng hc

c hiu ca cht tinh khit ca GOx tinh khit c kim tra trn D-glucose, maltose,
fructose, galactose v arabinose cc kt khc nhau Ca 1-40 mmol l-1 cho mi
ng. Th nghim enzyme c thc hin trong iu kin pH v nhit ti u. Mt
mng Lineweaver-Burke c v t 1/(v) (hot ng ca enzyme) Vs 1/(S) (b mt
conc) v t cc l Km v Vmax c tnh ton. Gi tr Km l nng cht nn m
ti enzyme cho thy hot tnh ti a ca n.

c ch enzym tinh khit

S c nh ha cng ha tr ca GOx c vn chuyn bng cch s dng

Eupergit C qua cc nhm oxirane phn ng ca polymer. Enzyme tr nn bt
hot trong qu trnh c nh c nh bng ng ho tr v vy cc phng php
by ca phng php natri alginat v polyacrylmide vi s ci bin c s
dng by GOx. Sau khi bt ng, cc thng s nh pH, nhit , c tnh
ng hc v n nh nhit c nghin cu cho qu trnh oxy ha glucose.
 KT QU V THO LUN

Trc khi nui cy nm A. niger sau 24 gi (7 x 105 spores / ml) c tim BSM cc
t l khc nhau (5, 10, 15, v 20%) v sn lng GOx ti a (240 IU / L) bng bo t
tui 24 gi t c so vi 30 gi khi lng dung dch c thay i t 5-15% (Hnh
1a). Cc ngun cacbon khc nhau glucose, fructose, galactose, lactose, sucrose v tinh
bt c s dng quan st nh hng ca chng i vi sn xut v tng trng ca
nm A.xiger. Vi sinh vt c th pht trin trn mi ngun carbon nhng hot ng GOx ti
a l vi sucrose. Nng sucrose khc nhau (4,0-10,0%) c b sung trong BSM. Sn
lng GOx tng khi nng sucrose tng t 4,0-7,5% (w / v), nhng trn khng thy
tng sn xut GOx. C sn lng ti a mc 7,5% sucrose. (Bng 1) Zetelaky v Vas
[19] s dng nng sucrose 5-7% i vi sn xut GOx t hn so vi nng ti u.
Hatzinikolaou v Macris s dng 8% sucroza cho sn xut GOx, so vi cc kt qu
cp trn (7,5%) Peptone, chit xut t nm men v chit xut t tht b l ngun nit
tt nht, nhng peptone nh mt ngun nit c s dng trc y sn xut
GOx, nng peptone khc nhau c s dng ti u ha nng peptone tt nht.
Tng sn lng GOx v FDW vi nng peptone tng t 0,5 ln 2,0%, sn xut ti a
3200 IU / L nng 2,0% (hnh 1b). Nng ny thp hn so vi nng c s
dng cho sn xut GOx [23] l 3%, v nhiu hn so vi nng c s dng bi
Hatzinikolaou v Macris [20], tc l 1% i vi sn xut GOx t A. niger. (NH4) 2HPO4 l
ngun photpho tt nht so vi NH4) 2H2PO4 v KH2PO4, tng sn xut GOx khi nng
NH4 / 2HPO4 tng t 0.1% 0.2 v cao hn sn lng enzyme l hng s (Hnh 1b).
CaCO3 tng cng sn xut GOx nng 0,5-2,0% conc (4000 IU / L), nhng trn
nng ny c s gim sinh khi v sn phm GOx trn (hnh 2a). Tng l do nh hng
trc tip ca pha mui rn ln c ch hot ng ca enzim [20]. Cc pH khc nhau (4,5,
5,0, 5,5, 5,75, 6,0, 6,5 v 7,0) c hiu qu sn xut enzym v prodution ti u pH 5.5-
6.5 c pH optima 5.75 (hnh 2b) tng ng vi pH dng cho sn xut GOx. Nhit
khc nhau (25, 30, 35 v 40 0 C) nh hng n sn xut GOx vi sn xut ti u l 30
0 C (Hnh 2c) lin quan n nhit tng trng c s dng cho sn xut GOx.
[13,26]. T l pha trn khc nhau (50, 100, 200 v 250 vng / pht) c p dng cho sn
xut GOx v sn lng GOx ti a l 250 vng Min-1. C sn lng GOx ti a (4000 IU /
L) sau 48 gi ln men v khng i ln n 60 gi, sau l s suy gim enzyme sn
xut (Hnh 2d).

Lm sch v trng lng phn t ca GOx

GOx t A.niger c tinh ch bng bn bc: lng ma sunfat ammonium (75%) v

c kh mui bng lc gel. Sau khi lc bng tinh th vi Q-Sepharose v DEAE
Sepharose c tinh ch 30.08 ln, phc hi 63,3% v hot ng c hiu 22.3 (Bng
2). tinh khit ca enzym sau khi sc k DEAE cellulose c hn 30 ln, khi so
snh vi cc chit xut th ca siu nhin t. GOx tinh khit n tnh ng nht c
xc nhn bi SDS-PAGE. Trng lng phn t ca GOX bn a t A .niger xp x 160
kDa trong khi SDS-PAGE lm cho hai di c trng lng phn t khong 80 kDa, cho
thy GOx c th l mt protein homo-dimeric gm hai n v ph bng nhau.

Nhit ti u v hot ng ca pH Gox

GOX t A. niger hot ng ti u 25C v th hin trn 90% hot ng ti a

gia 20-35 C, nhng trn 45C hot tnh gim nhanh (Hnh 3a). GOx duy tr
hot ng cao 90% 37C, khi so snh vi hot ng ti u ca enzym ny
trong khong t 25 n 30C. iu ny cho thy enzim thch hp cho hot
ng nhit ny sau khi n nh. GOx hot ng ti u pH 5 v hin th
mt cu hnh pH hp gia pH 5,5-6,0. Di mc hot pH 5.0 gim mnh,
duy tr ch hot ng 20% pH 4 (Hnh 3b).

Nhit Tnh n nh ca enzym tinh khit

Hot tnh tn d ca tinh khit GOx vn khng thay i trong 10 gi 25 C,

trong khi trng by mt na. Tui th khong 30 pht 50 C. Enzym n
nh ln n 40 C v s n nh gim mc cao hn. Nhit . Khi GOx t P.
ostreatus n nh 70 C trong 120 pht. Thi hn s dng ca tinh khit
GOx khng c thm cht n nh chi ph l mt tnh nng hp dn i vi cc
ng dng lin quan n kinh doanh enzyme

c tnh v m t c hc:

Hot tnh ca tinh khit GOx c kim tra trn D-glucose, fructose
maltoza, galactose v arabinoza v enzyme rt c hiu cho D Glucose. Cc
loi ng khc c oxy ha tc thp (maltoza v fructose) hoc khng
(galactose v arabinoza) (Bng 3). Cc kt qu tng t c bo co cho
sn xut GOx t A. niger [28] v P. ostreatus [27]. Mi quan h gia hot ng
ca enzim v nng cht nn l ca Michaelis-Menten loi. Km v Vmax c
xc nh t l Lineweaver-Burk ln lt l 7,1 mM v 1,7U (Bng 3). Cc gi tr
ca ng glucose (7,1mM) thp hn so vi cc ng khc, theo gi tr ti liu
cho cc loi nm GOx khc (9,6-110 mM). S doanh thu (kcat) ca GOx c
xc nh l 641 s-1. Hng s c hiu (kcat / Km) i vi GOx c xc nh l
90 s-1mM-1.

nh hng ca cc cht c ch enzym ln hot tnh c ch

t l tinh ch c ch tinh sch GOx tinh khit ln lt l 56,5 v 48% do Cu2 +

v Ag2 + tng ng nh bo co cho enzyme t Aspergillus v Penicillium
spp. [34,35] (Hnh 4). S c ch GOx bi cc ion Ag2 + l do phn ng ca Ag2
+ vi nhm enzyme thiol cn thit cho hot ng enzym, gn vi vng gn kt
FAD ca protein [34]. GOx t P. chrysosporium cng b c ch bi Ag2 +, tng
t nh GOx t A. niger [35].