APPLICATIONS
AND
KINETICS
OF
IMMOBILIZED
ENZYMES
AND
COUPLED
ENZYME
REACTIONS
Elena
Dominguez!*,
Gyö gy
Ma ko-Va ga?,
Bä bel
Hahn-Häge dal'
and
Lo
Go on?.
‘Depa men
o
Applied
Mic obiology,
*
Depa men
o
Analy ical
Chemis y,
Uni e si y
o
Lund,
P.
O.
Box
124,
S-221
00
Lund,
Sweden.
SUMMARY
Immobiliza ion
o
enzymes
esul s
in
mo e
adequa e
eagen s
o
be
used
analy ically.
A e
immobiliza-
ion,
he
kine ic
pa ame e s
o
he
enzymes
a e
modi ied
and
no hing
can
be
p edic ed
abou
he
ac i i y
o
he
he e ogeneous
sys em.
In
coupled
enzyme
eac ions
wi h
co-immobilized
enzymes,
his
is
e en
mo e
impo an
owing
o
he
kine ic
dependence
on
each
consecu i e
eac ion.
The
de e mina ion
o
e hanol
and
ace aldehyde
is
conside ed
in
his
pape ,
using
wo
di e en
coupled
enzyme
sys ems.
Some
impo an
pa ame e s,
as
he
enzyme
cha ged,
he
a io
o
each
enzyme
in
he
sequence
and
he
immobiliza ion
yield,
a e
conside ed
in
e ms
o
con e sion
e iciency
inally
de e mining
he
sensi i i y
o
he
analysis.
INTRODUCTION
Enzymes
a e
used
in
chemical
analysis
because
o
hei
high
deg ee
o
selec i i y
and
hei
ca aly ic
abili y
o
speed
up
eac ion
a es.
The
numbe
o
compounds
ha
can be
enzyma ically
analyzed
is
li ui ed
by
he
physical
o
chemical
p ope ies
o
he
subs a es
and
p oduc s.
Enzyma ic
eac ions
which
equi e
co ac o s
open
he
possibili y
o
moni o ing
he
eac ion
ia he
ans o med
co ac o .
In
cases
whe e
none
o
he
subs a es
o
p oduc s
a e
measu able,
i
is
o en
possible
o
de e mine
one
o
hese
componen s
by
coupled
sequen ial
o
compe i i e
enzyme
eac ion
[1].
This
conside ably
inc eases
he
numbe
o
analy es
ha
can
be
measu ed
enzyma ically.
Fu he mo e,
by
he
use
o
coupled
enzyme
eac ions
addi ional
ad an ages
can
be
gained:
i)
compounds
in ol ed
in
un a o able
equilib ia
may
also
be
e icien ly
con e ed
by
shi ing
he
equilib ium
cons an
wi h
he
subsequen
eac ion,
esul ing
in
a
sys em
he modynamically
a o able
and
wi h
an
expanded
linea
esponse
ange;
ii)
he
selec i i y
o
he
analysis
may
be
imp o ed
and
he e o e
he
accu acy,
diminishing
he
isk
o
in e e ing
subs a es;
iii)
inhibi o y
p oduc s
may
be
con inously
emo ed
and
iiii)
he
sensi i i y
is
inc eased
i
any
o
he
specimen
(subs a es,
p oduc s
o
co ac o s)
en e s
in
a
cyclic
enzyme
sequence
wi h
he
possibili y
o
signal
ampli ica ion
and
eco e ing
o
co ac o s.
Endogenous
coupled
enzyme
eac ions
occu ing
in
subcellula
ac ions
and
mic oo ganisms
ha e
been
used
in
bioca aly ic
elec odes
[2].
These
endogenous
mul ienzyme
sys ems
mimic
na u e
and
d i e
he
*On
lea e
om
he
Depa men
o
Pha macy,
Nu i ion
and
Food
Analysis,
Uni e si y
o
Alcalé
de
Hena es
(Mad id),
Spain.
226
E.
Dominguez
e
al.
kine ic
pa ame e s
in
he
way
o
inc easing
he
eac ion
luxes
[3]
and
consequen ly
o e
’na u al”
he mo-
dynamically
a o able
sys ems
o
be used
analy ically.
Mo e
o en,
he
analys
es ablishes
he
coupled
enzyme
eac ions
acco ding
o
he
analy es
in
he
sample
and
uses
highly
ac i e,
pu i ied
and
comme cially
a ailable
enzymes.
The
ac i i y
in
each
successi e
s ep
should
be
kep
highe
han
in
he
p eceding
s ep
[4],
in
o de
o
d i e
he
sys em
owa ds
he
p oduc
side
and
o
achie e
comple e
con e sion
in
he
o e all
sys em.
In
soluble
sys ems,
his
is
ela i ely
easy
o con ol,
bo h
empi ical
and
heo e ically,
a e
knowing
he
kine ic
pa ame e s
(K,,,
,,,,
and
a e
cons an
)
o
he
ee
enzymes.
The
ela i e
ac i e
enzyme
concen a ions
in
he
sys em
can
hen
be
es ablished.
F equen ly
i
may
occu ,
unlike
“na u al”
coupled
sys ems,
ha
op imum
condi ions
(pH,
T,
ionic
s eng h,
ac i a o s)
o
each
enzyme
a e
no
he
same
and
a
comp omise
be ween
hem
has
o
be
made.
The
use
o
enzymes
as
analy ical
eagen s
in
low
sys em
[5]
is
nowadays
a
equen
p ac ice,
mos ly
in
he
o m
o
immobilized
enzymes
[6]
owing
o
he
ad an ages
gained
a e
immobiliza ion
[7].
By
co-
immobilizing
he
enzymes
on
he
same
suppo ,
a
close
coupling
will
be
achie ed
be ween
he
eac ion
si es
esul ing
in
a
highe
con e sion
e iciency
han
by
mixing
enzymes
sepa a ely
immobilized
[8].
Co-
immobilized
enzyme
eac o s
(CIMERs)
ha e
been
desc ibed
o
he
simul aneous
de e mina ion
o
di e en
analy es
in
low
injec ion
analysis
(FIA)
[9-11].
Unlike
soluble
sys ems,
he
use
o
co-immobilized
enzymes
in
coupled
eac ions
o e s
some
a iables
which
make
he
e iciency
o
he
sys em
unp edic able.
Conce ning
he
de e mina ion
o
e hanol
and
ace al-
dehyde and
using
wo
di e en
coupled
enzyme
eac ions,
some
a iables
in
co-immobilized
sys ems
a e
con-
side ed.
COUPLED
ENZYME
REACTIONS
FOR
THE
DETERMINATION
OF
ETHANOL
AND
ACETALDEHYDE
The
mos
common
enzyma ic
de e mina ion
o
e hanol
is
based
on
he
use
o
alcohol
dehyd ogenase
(ADH,
EC
1.1.1.1)
[12]
in
he
p esence
o
NAD*,
see
eac ion
(1).
ADH
e hanol
+
NAD*
—«g————_
ace aldehyde
+
NADH
+
H*
(1)
Because
o
he
un a o able
equilib ium
o
his
eac ion
Ke=
8.0
10°M
(phospha e
bu e
pH
7.0;
20°C),
ace aldehyde
has
o
be
emo ed
in
o de
o
shi
he
eac ion
owa ds
he
p oduc
side.
Aldehyde
dehyd oge-
nase
(AIDH,
EC
1.2.1.5)
oxidizes
ace aldehyde
o
ace ic
acid
in
he
p esence o
NAD*
in
an
i e e sible
eac ion
( eac .
2)
which
becomes
he
he modynamic
d i ing
o ce
o
he o e all
sys em,
AIDH
ace aldehyde
+
NAD*
+
H,O
ace a e
+
NADH
+
H*
(2)
Mo eo e ,
by
using
his
indica o
eac ion
he
sensi i i y
o
he
analysis
inc eases
(one
mole
o
e hanol
gi es
wo
moleso
NADH)
and
simul aneously
allows
he
de e mina ion
o
aldehydes.
A
second
al e na i e
is
based
on
he
use
o
alcohol
oxidase
(AOD,
EC
1.1.3.13),
ca alase
(CAT,
EC
1.11.1.6)
and
AIDH.
The
addi ion
o
ca alase
emo es
hyd ogen
pe oxide
which
may
oxidize
he
aldehyde
and
eco e s
he
molecula
oxygen
which
en e s
in
a
cyclic
sys em:
CAT
ne
H,O
+0,
_—
AOD
—_,
e hanol
ace aldehyde
NAD*
oO
ill
AIDH
ace ic
acid
+
H*
NADH
1)
Enzyme
cha ged
pe
g am
o
suppo .
Fou
di e en
amoun s
o
AIDH
we e
added
o
comme cially
glu a aldehyde
ac i a ed
silica
suppo
(Se a,
Si
500,
po e
diame e
500
A
and
pa icle
size
30
Lim).
The
loaded
suppo s
we e
packed
in
50
l
eac o s
and
s udied
in
he
FIA
mode
by
injec ions
o
50
ul
o
2mM
Applica ions
o
Immobilized
Enzymes
and
Coupled
Enzyme
Reac ions
227
p opionaldehyde
in o
he
ca ie
s eam
consis ing
o
2mM
NAD*,
4mM
2-me cap oe hanol,
and
0.15
M
KCl
in
0.1
M
py ophospha e
bu e
(pH
8.5).
The
low
a e
was
0.4
ml
min".
Fig.
1
shows
ha
a
high
amoun s
o
enzyme,
he
esponse
eaches
a
maximum
le el.
A
simila
esponse
pa e n
has
been
ound
wi h
enzyme
elec odes
[1].
150
2
z
=
100
=
oO
=
3
Cc
=
=
50
<
=
©
ao
o
T
T T
T T
=
o
500
1000
1500
2000
2500
3000
AIDH
(IU
cha ged
/
g
suppo )
Fig.
1.
E ec
o
di e en
amoun s
o
AIDH
cha ged
pe
g am
o
suppo
on
he
ac i i y
o
he
esul an
immobilized
enzyme
o
he
con e sion
o
p opionaldehyde.
2)
S udy
o
he
in luence
o
wo
di e en
enzyma ic
a ios
on
he
con e sion
o
e hanol
and
ace alde-
hyde.
2.1
ADH/AIDH
coupled
enzyme
sys em.
250
IU
o
ADH
and
2500IU
o
AIDH
pe
g am
o
suppo
we e
co-immobilized.
9500
IU
o
ADH
and
300
IU
o
AIDH
we e
also
cha ged
pe
g am
o
silica
suppo .
The
con e sion
e iciency
was
s udied
in
he
FIA
mode
wi h
he
co-immobilized
enzymes
packed
in
50
1
eac o
olume.
25
il
injec ions
o
125
1M
e hanol
o
125
UM
ace aldehyde
we e
made
in o
he
ca ie
s eam
con aining
2
mM
NAD*,
0.15
M
KCl
in
0.1
M
phospha e
bu e
a
pH
7.0.
The
con e sion
e iciency
is
es ima ed
in
ela ion
o
injec ions
o
s oichiome ic
amoun s
o
NADH.
®
50
50
=
UO
z
40
40
Oo
a=
30
30
ie
w
520
20
oO
ng
10
-
D
S
O-+
T
oo
T
T
DO;
T
5
O
T
T
TTTT
T
207704700708
,109.12
14
00.02
04
06
0:8.
1:0.
To s
FLOW
RATE
(ml/min)
FLOW
RATE
(ml/min)
Fig.
2.
Va ia ion
o
he
con e sion
e iciency
o
(m)
ace aldehyde
and
(1X)
e hanol
wi h
he
low
a e
o
he
ca ie
and
wi h
di e en
ADH/AIDH
a ios;
(A)
250/2500
and
(B)
9500/300.
Nei he
o
hese
co-immobiliza ions
esul ed
in
a
good
con e sion
o
e hanol.
The
ac
ha
he
ini ial
solu ions
(be o e
immobiliza ion)
con aining
bo h
enzymes
p esen ed
ac i i y
o
e hanol
(measu ed
a
ze o
228
E.
Dominguez
e
al.
o de
eac ion),
and
he
%
o
immobiliza ion
we e
in
all
cases
highe
han
88
%
indica e
changes
in
he
kine ic
pa ame e s
and/o
an
inc eased
inac i a ion
a e
cons an .
I
has
been
shown
[13]
o
immobilized
ho se
li e
ADH
ha
he
s abili y
o
he
immobilized
p epa a ion
depends
on
he
quan i y
o
bound
enzyme.
2.2
AOD/CAT/AIDH
coupled
enzyme
sys em.
10000
IU
o
AOD
and
1000
IU
o
AIDH
we e
co-
immobilized
pe
g am
o
CPG-10
(po e
diame e
500
A,
pa icle
size
37-74
im)
ia
glu a aldehyde
as
epo ed
p e iously
[11].
1000
IU
o
AOD
and
2500
IU
o
AIDH
we e
also
co-immobilized
unde
he
same
condi ions.
In
bo h
cases
160000
IU
o
ca alase
we e
also
cha ged
pe
g am
o
he
suppo .
The
expe imen al
condi ions
we e
he
same
as
ha
men ioned
abo e
o
he
ADH/AIDH
sys em
wi h
he
sole
di e ence
ha
he
ca ie
solu ion
was
sa u a ed
wi h
O,.
100
100
5
z
80 80
wu
UO
60
60
uw
oO
Ww
z
40 40
oO
2
20
20
Bi
3
oo
Oo
o 5
S
0
T
T
T
T
T
ie:
T
TI
0
1
T
T
T
T ¥
T
1
82.020400:
08.1.0.
1.2..21.40%.6702902.06808
1012.14
16
FLOW
RATE
(ml/min)
FLOW
RATE
(ml/min)
Fig.
3.
Va ia ion
o
he
con e sion
e iciency
o
(m)
ace aldehyde
and
(0)
e hanol
wi h
he
low
a e
o
he
ca ie
and
wi h
di e en
AOD/AIDH
a ios;
(A)
10000/1000
and
(B)
1000/2500.
These
esul s
clea ly
show
he
impo ance o
he
enzyme
a io
in
he e iciency
o
a
coupled
enzyme
sys em.
Wi h
a
su plus
o
AOD
(Fig.
3A)
he e
is
ampli ica ion
o
e hanol
due
o
he
cyclic
egene a ion
o
oO
2
Financial
suppo
om
he
Na ional
Ene gy
Adminis a ion
(STEV)
and
he
Swedish
Boa d
o
Thecnical
De elopmen
(STU)
is
g ea ully
acknowledged.
REFERENCES
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F.W.Schelle ,R.
Rennebe g
andF.
Schube ,
inK.
Mosbach
(ed.),
Me hods
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137,
Pa D,
Academic
P ess,
New
Yo k
1988,
pp.
29-43.
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I.
Ka ube
in
A.
P.
F.
Tu ne ,
I.
Ka ube
and
G.
S.
Wilson
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1987,
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13-
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Eu .
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Biochem.,
184
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561-566.
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P.
W.
Ca
and
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D.
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John
Wiley,
New
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1980,
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o
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1977,
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in
H. U.
Be gmeye
(ed.)
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o
Enzyma ic
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1984,
pp.
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