De elopmen
and
Applica ions
o
a
Fou -Channel
Enzyme
The mis o
Sys em
o
Biop ocess
Con ol
H.-G.
Hundeck,
U.
Hubne ,
A.
Lübbe ,
T.
Schepe ,
J.
Schmid ,
M.
Weiß
Ins i u
ü
Technische
Chemie,
Uni e si ä
Hanno e ,
Callins .
3,
D-3000
Hanno e
1
F.
Schube
Zen alins i u
ü
Mik obiologie,
Robe -Rössle-S .,
D-1115
Be lin-Buch
Summa y
A
la ge
numbe
o
pape s
on
biosenso s
ha e
been
published
in
he
las
ew
yea s.
Howe e ,
ew
o
hese
sophis ica ed
analysis
sys ems
ha e
been
used
o
moni o
eal
biop ocesses.
In
his
pape ,
a
newly
de eloped
ou -channel
enzyme
he mis o
sys em
and
i s
applica ion
o
bio echnological
p ocess
moni o ing
is
p esen ed
and
discussed.
Di e en
suga s
we e
de ec ed
simul aneously
and
on-
line
du ing
he
cul i a ion
o
Spodop e a
ugipe da
and
Bacillus
licheni o mis
in
echnical
media.
Immobilized
enzymes
and
en apped
mic oo ganisms
we e
used as
biological
compound
in
his
biosenso .
In
addi ion,
enan ioselec i e
analysis
was
pe o med
by
wo
enzyme
eac ions.
Fo
example,
he
de ec ion
o
D,L- acema es
o
aminoacid
es e s
was
p esen ed
in
an
aqueous
sys em.
Fu he mo e
he
possibili y
o
using
his
de ec ion
sys em
in
o ganic
sol en s
was
shown.
In oduc ion
On-line
moni o ing
echniques
a e impo an
o
op imal
biop ocess
con ol
and
au oma ion.
This
ype
o
measu emen
places
special
demands
on
he
ypes
o
senso s
in ol ed
(e.g.,
long
e m-
s abili y,
analy ical
accu acy,
and
au oma ed
analy ical
p ocedu e).
A
majo
ocus
o
bio echnological
analysis
is
he
cul i a ion
medium,
since
subs a es
and
p oduc s
a e
dissol ed
in
i .
The
en i onmen
o
he
cells
o
be
cul u ed
in luences
he
ac i i y
and
he
s a e
o
he
biomass
and
hus
o
he
whole
biop ocess.
O en,
he
analysis
o
he
cul i a ion
medium
is
pe o med
by
complex
o -line
me hods.
Biosenso s
p o ide
he
possibili y
o
measu ing
such
com-
pounds
wi h
on-line
sys ems,
and
g ea ly
inc ease
he
powe
o
au oma ed
biop ocess
moni o ing
and
con ol.
The
li e a u e
on
biosenso s
is
o e whelming
(1-6),
bu
he e
is
s ill
a
lack
o
applica ions
in
eal
indus ial
p ocesses
(7).
The
ou -channel
enzyme
he mis o
sys em
is
un
as a
s and-alone
de ice,
con olled
by
a
compu e .
The
simul aneous
analysis
o
di e en
mono-
and
disaccha ides
such
as
glucose,
mal ose,
suc ose,
and
lac ose
wi h
his
e sion
was
simply
322
H.-G.
Hundeck
e
al.
pe o med
a
cul i a ion
p ocesses
o e
pe iods
o
up
o
300
hou s
and
can
hus
be
used
as a
basis
o
p ocess
op imiza ion.
Ma e ial
and
Me hods
Ma e ials
Glucose
oxidase
(EC
1.1.3.4)
and
ca alase
(EC
1.11.1.6)
we e
used
o
glucose
analysis.
In e ase
(EC
3.2.1.26)
was
used
o
suc ose
analysis,
and
a-glucosidase
(EC
3.2.1.20)
was
employed
o
mal ose
analysis.
The
enzymes
we e
co alen ly
bound
on
oxi ane
ac ylic
suppo s
(VA
Epoxy
Biosyn h,
Riedel
de
Haen
AG,
W.
Ge many)
(8).
This
kind
o
immobiliza ion
is
mo e
e icien
han
he
usual
CPG
immobiliza ion
(9).
The
o ganisms
used
in
he
senso
(e.g.,
cells
o
Saccha omyces
ce e isiae)
we e
immobilized
in
calcium
algina e
(10).
The
Enzyme
The mis o
The
analy ical
sys em
is
based
on a
simple
isope ibol
low
calo ime e ,
in
which
he
hea
p oduced
is
measu ed
as
a
empe a u e
change
by
he mis o s
(11).
The
empe a u e
esolu ion
is
abou
10-5
K.
A
schema ic
diag am
o
an
enzyme
he mis o
is
shown
in
Figu e
1.
e e ence
enzyme
column
gold
ube
A—
LEA
ELL
LE
„a
a Y
A A
/
;
J Y
5
YZ
’
7
4}
7
|
7
7
he mis o
i)
]
a
hea
exchange
4 A
A
y
A
aluminium
cylinde
5 A
V
1
[A
insula ion
VLAZZLZ.
LAL
LZ]
a
©
1
4
sample
injec ion
bu e
Ki
ee
bu e
Fig.
1
Schema ic
diag am
o
an
enzyme
he mis o .
A
Fou -Channel
Enzyme
The mis o
Sys em
o
Biop ocess
Con ol
323
Bu e
is
pumped
con inuously
h ough
he
he mos a ed
aluminium
cylinde .
Be o e
en e ing
he
columns,
he
ca ie
low
passes
h ough
a
hea
exchange
coil.
The
empe a u e
a
he
inle
and
ou le
o
he
column
is
egis e ed
by
he mis o s
placed
on
gold
ubes.
One
o
he
columns
con ains
he
immobilized
biological
ma e ial
(e.g.,
enzymes),
while
he
o he
column
is
illed
wi h
inac i a ed
esin
ma e ial.
In
his
e e ence
column,
nonspeci ic
hea
e ec s
a e
measu ed.
Bu e
is
pumped
con inuously
h ough
he
analysis
sys em.
A
de ined
sample
olume
is
injec ed
by
an
au oma ed
al e
in o
he
bu e
s eam
acco ding
o
he
p inciples
o
low
injec ion
analysis
(12).
The
ca ie
low
anspo s
he
sample
h ough
he
columns;
he
eac ion
occu s
in
he
enzyme
column.
The
hea
p oduced
he e
is
measu ed
by
he
he mis o s.
analog
ampli ie
68000
mic op ocesso
Fig.
2
Compu e
con ol
sys em
o
he
ou -channel
enzyme
he mis o .
A
ou -channel
e sion
o
an
enzyme
he mis o
was
designed
and
buil
in
he
Ins i u
ü
Technische
Chemie
as
a
low
calo ime e ,
based
on
he
expe iences
wi h
Lund- ype
enzyme
he mis o s
(13-14).
I
can
analyze
ou
di e en
subs ances
simul-
aneously.
A e
changing
an
enzyme
column,
only
15
minu es
a e
necessa y
be o e
he
analysis
can
be
con inued.
Special
columns
ha e
been
designed
o
he
de ec ion
o
dissol ed
enzyme
ac i i ies.
In
324
H.-G.
Hundeck
e
al.
hese
columns,
subs a e
and
enzymes
a e
mixed
oge he
.
The
hea
measu ed
is
p opo ional
o
he
enzyme
ac i i y.In eg a ed
p e-
he mos a ing
c ea es
a
e y
s able
empe a u e
a
he
measu emen
cen e .
A
mic op ocesso
con ol
uni
is
u ilized
o
he
signal
de ec ion
(Figu e
2)
.
The
use
can
de ine he
analysis
channels
wi h
he
so wa e.
He
can
choose
a
e e ence
column,
a
measu emen
be ween
wo
he mis o s
o
he
same
column,
o
o he
special
measu emen
applica ions.
The
con ol
sys em
checks
he
measu emen
accu acy,
calib a ion,
and
da a
analysis
and
p ocessing.
The
68000
mic o-
p ocesso
sys em
wi h
a
mul i- asking
ope a ing
sys em
allows
he
implemen a ion
o
con o!
algo i hms
o
sophis ica ed
p ocess
con ol;
e.g.,
he
egula o
con ols
he
subs a e
eed
o
biop ocesses
ia
he
pump
low.
A
special
il a ion
sampling
p obe
was
in eg a ed
in o
he
whole
analy ical
sys em.
A
cell- ee
sample
can
be
wi hd awn
om
he
e men o
con inuously
o
injec ion
in o
he
enzyme
he mis o .
Dynamic
o
s a ic
dilu ion
s eps
a e
ini ia ed
by
he
compu e
i
necessa y.
The
sample
is
injec ed
in o
he
ca ie
bu e
s eam
ia
a
compu e -con olled
injec ion
al e.
Resul s
and
Discussion
Cul i a ion
o
Bacillus
licheni o mis
The
cul i a ion
o
Bacillus
licheni o mis
was
pe o med
o
p o ease
p oduc ion.
Ba ch
and
ed-ba ch
cul i a ions
in
complex
echnical
media
we e
in es iga ed.
The
e men a ion
media
con ained
hyd olyzed
co n
s a ch,
soy
meal,
casein,
and
co n
s eep
liquo
.The
in o ma ion
ob ained
in
he
ba ch
cul i a ions
p o ided
he
basis
o
he
ed-ba ch
in es iga ions.
In
he
beginning
o
he
p ocess,
biomass
is
p oduced
in
a
pu e
g ow h
phase.
The
p o ease
p oduc ion
phase
s a s
when
he
glucose
concen a ion
alls
below
a
ce ain
concen a ion
and
he
mic oo ganisms
exc e e
p o eases
in o
he
medium
o
assimila e
p o ein.
Suc ose
o
mal ose
can
be
added
du ing
he
p oduc ion
phase.
This
subs a e
eed
mus
be
con olled
o
op imal
p o ease
yield
wi hou
any
lag
phase.
I
he
suga
concen a ion
is
oo
high,
biomass
will
be
p oduced
and
he
p o ease
p oduc ion
will
dec ease.
On
he
o he
hand,
p o ease
p oduc ion
will
dec ease
when
he
subs a e
concen a ion
is
below
a
c i ical
le el
ha
esul s
in
dec easing
cell
ac i i y.
Figu e
3
shows
he
simul aneous
on-line
analysis
o
mal ose,
suc ose
and
glucose
wi h
he
ou -channel
enzyme
he mis o
o
a
ba ch
cul i a ion
o
Bacillus
licheni o mis.
A
compa ison
o
he
on-
line
da a
wi h
di e en
o -line
me hods
was
p esen ed
be o e
(15).
An
in e es ing
ela ion
be ween
he
suga
da a
and
he
oxygen
ans e
A
Fou -Channel
Enzyme
The mis o
Sys em
o
Biop ocess
Con ol
325
aD
T
T
T!
3
|
|
-
|
&
Pu
a
Ba
I
|||
ES
|
A:
pa ed
=
|
|
1
|
ane
=
a
5
35[628
P
®
3
742
2
3
15
E
D>
.
2
o
3
a
1
10
2
iq
45
5
{
|
8
0
05
10
1
3
e men a ion
ime
(h)
Fig.
3
Glucose(
o)-,
mal ose(
¢
)-
und
suc ose
concen a ion
(=)
du ing
a
ba ch
cul i a ion
o
B.
licheni o mis
compa ed
wi h
he
oxygen
ans e
a e
(OTR).
a e
(OTR)
can
be
obse ed.
In
a
i s
phase
he
suc ose
consump ion
was
no
co ela ed
wi h
a
change
in
he
OTR
amoun .
A e
he
comple e
consump ion
o
suc ose,
he
consump ion
o
glucose
wi h
a
linea
inc ease
o
he
OTR
s a .
A e
a
lag
phase
caused
by
he
comple e
glucose
consump ion,
he
OTR
da a
inc ease
is
longe .
A
ed-
ba ch
cul i a ion
moni o ed
wi h
he
ou -channel
enzyme
he mis o
is
p esen ed
in
Figu e
4.
In
compa ison
be ween
mal ose
concen a ion
and
cul u e
luo escence
signal
demons a es
an
impo an
di e ence
be ween
he
ba ch
and
he
ed-ba ch
p ocess.
In
he
ed-ba ch
p ocess
he
cul u e
luo escence
signal
inc eases
ea lie ,
and
eaches
highe
le el.
The
cul u e
luo escence
Signal
is
co ela ed
wi h
he
amoun
o
li ing
cells
and
hus
is
an
indica o
o
ceil
g ow h.
The
esul s
o
his
S udy
indica e
ha
mal ose
as
C-sou ce
is
esponsible
o
he
cell
g ow h,
because
he
cul u e
luo escene
inc eases
wi h
he
mal ose
consump ion.
A
small
peak
in
he
cul u e
luo escence
signal
can
be
obse ed
a e
he
comple e
consump ion
o
glucose,
and
indica es
he
change
om
glucose
o
mal ose
consump ion.
This
peak
appea s
ea lie
in
he
ed-ba ch
p ocess,
causing
a
smalle
amoun
o
glucose
in
he
s a
medium
and
indica es
he
apid
consump ion
o
glucose.
Du ing
ea lie
expe imen s
i
has
become
ob ious
ha
he
mul iple
addi ion
o
mal ose
has
no
posi i e
e ec
on
he
p oduc ion
o
p o eases.
Howe e ,
a
de ailed
analysis
wi h
he
enzyme
he mis o
was
success ully
pe o med
o
suc ose
analysis.
The
esul s
o
his

326
H.-G.
Hundeck
e
al.
s udy
indica ed
ha
he
addi ion
o
suc ose
should
imp o e
he
ane
|
|
®
oO
o
5
|
3
@
|
Ss
=
|
©
3
3
=
55,68
5
2
8
20
8 }5 2
<
42
3
15
E
mo
e a:
.
+10
2
5
4
0
+
-
o0z 0S
Ten:
ed-ba ch-cul i a ion
20
3
5
3
iss
[43
E
a
°,
0:
I?»
2
5
b
4
"0
0
0
5 10 15
e men a ion
ime
(h)
Fig.
4
Glucose(
no
)-,
mal ose(
©
)-
und
suc ose
concen a ion
(
=)
du ing
a
ba ch
and a
ed-ba ch
cul i a ion
o
B.
licheni o mis
compa ed
wi h
he
cul u e
luo escence
signal.
p o ease
p oduc ion.
Two
ed-ba ch
e men a ions
wi h
con ol
o
suc ose
addi ion
wi h
he
ou -channel
enzyme
he mis o
a e
p esen-
ed
in
Figu e
5.
I
he
suc ose
concen a ion
is
oo
high,
biomass
will
be
p oduced
while
he
p o ease
p oduc ion
dec eases.
The
p o ease
yield
was
measu ed
on-line
by
a
s opped- low-FIA
(16).
The
da a
o
he
p o ease
concen a ion
we e
i ed
and
di e en ia ed
o
ge
he
p o ease
p oduc ion
a e.
Immobilized
whole
cells
o
Saccha omyces
ce e isiae
we e
used
ins ead
o
he
enzyme
columns
o
moni o
he
concen a ion
o
assimilable
suga s
du ing
a
cul i a ion.
The
cells
we e
immobilized
in
calcium
algina e.
The
hea
p oduced
by
he
immobilized
cells
a e
addi ion
o
di e en
mono-
and
disaccha ides
is
shown
in
Figu e
6
as
empe a u e
change
e sus
concen a ion.
The
immobilized
cells
p oduce
hea
du ing
he
assimila ion
o
di e en
suga s
in
he
medium.
Thus,
his
mic obial
senso
doesn’
de ec
a
single
compound
bu
he
A
Fou -Channel
Enzyme
The mis o
Sys em
o
Biop ocess
Con ol
327
amoun
o
all
assimilable
subs ances
in
he
medium.
This
pa ame e
is
ie
80
7
:
2
a
©
i
«604
gh
:
oe
ae
io
|
&
e
-
y
}
a
Y
w e
ne
%
=;
a
°
ne
000°
u
ES
FIRE
Ea
oa
@)
Sa e
En
516
RR
one
5)
en
nl
:
10
20
a
e men a ion
ime
(h)
Fig.
5
Compa ison
o
on-line
da a
o
suc ose
de e mina ion
using
he
enzyme
he mis o
wi h
p o ease
p oduc ion
a e
du ing
wo
ed-
ba ch
cul i a ions
o
B.
licheni o mis.
<
10
E
©
oe
Ro}
=
4
6
4
=
©
4
glucose
7
44
*
suc ose
=
J
=
uc ose
=
23
mal ose
4
oF
2 3
4
concen a ion
(g/l)
Fig.
6
Hea
e olu ion
o di e en
suga s
measu ed
wi h
immobilized
cells
o
S.
ce e isiae.
highly
in e es ing
o
use
in
echnical
media,
in
which
he
concen a ion
dis ibu ion
o
di e en
subs a es
is
o en
nea ly
unde ined.
I
would
be
p e e able
o
use
he
same
o ganisms
in
he
mic obial
senso
as
in
he
cul i a ion
o
be
moni o ed.
Howe e ,
he
use
o
yeas
cells
demons a es
he
p inciple
and
po en ial
o
his
analy ical
me hod.
The
328
H.-G.
Hundeck
e
al.
da a
o
he
mic obial
senso
du ing
ba ch
cul i a ions
we e
shown
be o e
(15).
Enan ioselec i e
analysis
The
p inciple
o
de ec ion
o
enan iome ic
excess
is
based
on
wo
enzyme
eac ions
(Figu e
7).
One
o
he
used
enzymes
(a-
chymo ypsin)
can
only
eac
wi h
one
enan iome ,
p e e ably
he
L-
s e eospeci ic
compound.
The
o he
used
enzyme
eac s
wi h
bo h
s e eospeci ic
compounds.
Bo h
enzymes
eac
wi h
he
sample.
D,L-aminoacid
es e
+
0
ES
RCOO’+H"+
ROH
a-chymo ypsin
L-aminoacid
es e
+
H,O
>
RCOO’+
H'+
ROH
Fig.
7
Enzyme
eac ions
o
enan iome ic
analysis
The
hea
gene a ed
du ing
he
eac ion
is
measu ed
ia
he
enzyme
he mis o .
The
concen a ion
o
bo h
enan iome ic
compounds
was
ob ained
by
his
eac ion
and
he
enan iome ic
excess
was
calcula ed
om
his
da a.
The
empe a u e
signal
inc eases
in
a
linea
way
by
a
eac ion
o
a-chymo ypsin
wi h
di e en
concen a ions
o
a
50:50
acema e
o
D,L-es e s,
he
eac ion
wi h
he
es e ase
esul s
in
a
wice
as
high
empe a u e
signal
(Figu e
8).
Ss
40
E
SC
5
B
304
©
a
©
a
wee
§
3
104
04
-
T
0
10
20
phenylalanineme hyles e
(mM)
Fig.
8
Calib a ion
unc ion
o
eac ion
o
an
aminoacides e
1.)L-
(=)
und
D,L-es e s
(+)
wi h
a-chymo ypsin
2.)
L-
(a)
und
D,L-es e s
(
+
)
wi h
es e ase
A
Fou -Channel
Enzyme
The mis o
Sys em
o
Biop ocess
Con ol
329
By
using
he
L-es e
bo h
enzyme
eac ions
gi e
he
same
empe a u e
signal.
By
a ia ion
o
he
D-compound
in
a
mix u e
o
bo h
enan iome es
i
could
imp essi ely
be
shown
ha
he
empe a u e
signal
o
he
a-chymo ypsin
eac ion
depends
on
he
amoun
o
he
D-
compound
and
he
empe a u e
signal
o
he
es e ase
eac ion
was
cons an
caused
by
he
same
concen a ion
as
a
whole,
o
bo h
compounds
(Figu e
9).
By
combining
he
whole
concen a ion
o
D,L-
compounds,
ob ained
by
he
es e ase
eac ion
and
he
concen a ion
o
he
L-compound,
a ailable
om
he
a-chymo ypsin
eac ion,
i
is
possible
o
calcula e
he
enan iome ic
excess.
This
p ocedu e
is
a
new
p inciple
o
analyze
enan iome ic
mix u es.
The
same
eac ion
unc ioned
well
in
solu ions
ha
con ained
dime hyl
o mamide.
The
esul s
o
his
s udy
indica ed
ha
enan iospeci ic
de ec ion
is
possible
also
in
o ganic
sol en s.
40
y
=
31,405
-
4,2857e-4x
R*2
=
0,000
a
a
z
7
a
y =
27,362
-
0,27457x
R*2
=
0,996
empe a u e
signal
(mK)
wo
oO
1
10
J
0
20 40
60
80
100
ec
(%)
Fig.
9
Calib a ion
unc ion
o
a
eac ion
o
an
aminoacides e
acema e
by
a ia ion
o
he
D-compond
wi h
a-Chymo
ypsin
(
«
)
und
Es e ase
(
=
)
Summa y
A
biosenso
sys em
was
de eloped
and
sucess ully
applied
o
Cul i a ion
moni o ing
in
bio echnology.
To
achie e
high
eliabili y
and
long
e m
s abili y,
he
ou -channel
enzyme
he mis o
sys em
was
in eg a ed
in o
a
FIA
sys em.
The
en i e
biosenso
sys em
included
cell-
ee
sampling,
sample
condi ioning,
and
analysis.
The
sys em
was
au oma ed
and
could
be
un
as
a
s and-alone
e sion
o
on-line
moni o ing.
The
sys em
o e s
he
possibili y
o
ob ain
a
mo e
de ailed
insigh
in o
he
whole
biop ocesses
o
Spodop e a
ugipe da
and
Bacillus
licheni o mis
and
can
se e
as
a
basis
o
p ocess
con ol.