Summary_. The mode of electron transport associated with the dehydrogenase enzymes located on the cytoplasmic membrane in Gluconobacter ox/dans.
BIOTECHNOLOGY LETTERS volume 14 No.5 (may 1992) pp.391-396 Received as revised 25th March ELECTRON
TRANSPORT SYSTEM ASSOCIATED WITH DIRECT O X I D A T I O N IN G L U C O N O B A C T E R 0XYDANS N.
5harma,
R.
Parshad
and
G.
N.
GLUCOSE
fi)azi*
Genetic Engineering Unit Regional Research Laboratory J a m m u - T a w i - 1 8 0 001(INDIA). *Corresponding
author
Summary_ The mode of electron transport associated with the dehydrogenase enzymes located on the cytoplasmic m e m b r a n e in Gluconobacter o x / d a n s (ATCC 9937) has been postulated. High turnover of dehydrogenases under oxygen enrichment conditions is explained on the basis of a simplistic electron transport chain comprising cytochrome c553 (MW o3000) as a subunit of dehydrogenase and a cytochrome b562. The electron transport chain under low dissolved oxygen tension (DOT) is shown to comprise a n u m b e r of cytochrome c species with very low midpoint potential difference.
Introduction Gluconobacter through al.,
oxydans
intermediate
1991 ).
The
distinct
from
oxidation
pathway
is
1991).
direct
ox__~dans
(ATCC
Michaelis
Menton
yield
diketo
derivatives
system.
of
components
was
would et
al.,
with
earlier
study membrane
have
suboxydans
a cytochrome
components bound
of the
shift be
oxidation
produced
by
oxidation
by
rather
efficient
described
its
electron electron
comprising
G___t.
than
et al., 1990). and
G.
(Buse et al.,
induction (Buse
of
growing
keto-
A and
transport transport
a few species
'o' type terminal oxidase.
of direct
391
is
substrate
or chemostat
mainly
which
phosphorylated
gluconate an
through
1. I. I. 47).
enzyme
like
et
(E. C.
of the
reported
need
1987
mediated
on the glucose
through
(0azi
1.1.99.17)
selectively
DOT
obviously
c-type and
present
can
products
of Gluconobacter
of cytochrome
associated
mediated
oxidation
Ameyama
acid
is
environment
advantage
of high
kinetics
larger
or the
diketogluconate
2-ketogluconate
(E.C.
(Oazi et al., 1991)
influence 9937)
upon
Taking
to
dehydrogenase
direct
dependent
2,5-diketo-gluconic
Positive
and
oxidation
glucose
either
culture
glucose
dehydrogenase
of G__t oxydans.
oxydans_ in batch
the
of
oxidizes
gluconate
called
NADP-linked
operation
pathways,
9937) of
glucose
Selective
culture
steps so
NADP-independent
In
(ATCC
the
electron
glucose
transport
dehydrogenase
chain in
G.
oxydans
were
components DOT.
and
Based
membranes
assayed;
of
a
enhanced on
the
correlation
is
enzyme
activities
obtained
cytochrome
cultures
enrichment conditions,
direct
grown
species
under
drawn
isolated
oxygen
between under from
deficient
these
elevated the
and
cell
oxygen
a hypothesis on the simplified electron transport
chain is presented.
Materials a n d
Methods
Gluconobacter oxydans (ATCC 9937) was used throughout the investigation. The maintenance m e d i u m used for the culture contained mannitol 259, yeast extract 59, peptone 39, agar 159, in 1 I double distilled water. The organism w a s cultivated in a m e d i u m consisting of glucose 1009, glycerol 59, yeast extract 59, m a g n e s i u m sulphate 0.59, calcium carbonate 10g, in i 1 double distilled water (pH 6.0). The continuous cultivation w a s performed in a 3 1 working volume Airlift fermentor, the design & details of which are reported elsewhere (Tr~ger et al., 1990). The glucose concentration in the feed w a s kept at I00 m tool l - ~ n d a dilution rate of D=0.2 h - ~ a s maintained in the chemostat. Batch cultivation was m a d e in a i0 1 Airlift fermentor (Bioengineering, Switzerland) h a v i n g a central draught tube which w a s gassed with air and oxygen enriched air at the rate of 1.5 vvm. The cells were harvested by centrifugation at 10000g and then w a s h e d twice with double distilled water and once with 20ram phosphate buffer (pH 6.4) a n d subjected to ultrasonication (I00 w crn I , 22.5KHz, 4~ Cell free extracts were obtained after centrifuging the sonicated cells at 100009 for 30 rain. (55 34 rotor). The cell m e m b r a n e s were prepared by centrifuging the cell free extracts at 100,000g for i h. at 4~ (5F 36 rotor Dupont RC 28 5). The m e m b r a n e s thus obtained were w a s h e d with 20ram phosphate buffer pH 6.4 before extraction of the enzymes. For solubilisation of dehydrogenases and accompanying cytochromes the m e m b r a n e s were subjected to following tenside treatments: i. 1% Brij 58 and 2% T w e e n 80 in 10ram sodium acetate buffer(pH 5.0) 2. 1% Triton X i00 in 10ram sodium acetate (pH 5.0) 3. 2% Triton X i00 and IM KCI in 20ram phosphate buffer(pH 7.0) Partial purification of the cytochromes w a s performed on DEAE-cellulose as described by A m e y a m a et al., 1987. Absorption spectra of different cytochromes was taken by Wavelength Scanning programme (U3200 Hitachi spectrophotometer). Protein contents were determined according to L o w r y et a l . , 1951. S D S - P A G E w a s performed as described by King and Lamelli., 1971. Results a n d Discussion Table
1
shows
the
spectral
properties
cytochromes extracted from the cell m e m b r a n e s a chemostat under methods.
The
data
varied D O T shown
of
different
species
of
of the cultures g r o w n
in
conditions as described in materials a n d
in Table
392
1 reveal
that under
low
DOT
i.e.
2%
relative
'c' are
On
to air saturation
saturation
cell
solubilized
Kd
an
integral
high
DOT
conditions
only
c553
type
of cytochrome
Polyacrylamide
of the m e m b r a n e s
part
i.e.
gel
revealed
of the h o l o e n z y m e
and
type
100%
relative
could
be
to air
solubilized
electrophoresis
of
that the cytochrome
could
be identified
the c553
as
a 23
1:
T y p e of Cytochrome
Absorption Dithionite
Deficient DOT(2%)
High DOT (100%)
The
other
'b'
type
and
425
oxidized
Air Oxidized
416
405
2. Cyt.c*550
550
523
411
---
3. Cyt.c551
551
---
416
413
4. Cyt.c553
553
524
414
413
5. Cyt.b556
556
528
425
408
1.
Cyt.c553
553
524
414
---
2.
Cyt.b562
562
532
431
404
of
cytochromes
under
b562
low
reduced under
431 n m
DOT
in
conditions
state
high
found
and
DOT
at
organism
b556 408
conditions
in dithionite
the
reduced
nm
were
cytochrome
with
maxima
as 556,
528
in
oxidized
state;
and
having
absorption
state a n d
404
cytochrome
b
nm,
maxima
at
in the
air
state.
generation
different
Reduced
522
in
532 a n d
(nm)
550
i.e. nm
Maxima
i. Cyt.c550
type
oytochrome
The
of cytochrome
(Fig.l).
5rate
562,
species
under
membranes.
fractions
subunit
Table
hand
at 1 bar,
the
was
many
generated,
the other
from
at ! bar,
DOT
of
different
conditions
is
in
types
of
agreement
393
with
the
species
results
of
under Kita
et
al.,
1986.
In
b
species
cyt ochr ome binding of
G.
studies oxydans
form
413nm,
while
studying
identified. cytochrome
under
any
c553
(CO)
exhibits
properties
(Ameyama
et al~
binds
c553
types
from
maxima
The
at
(CO )
that CO
563,
species
of
cultures
revealed
with CO.
(C0)
two
monoxide
isolated
condition
absorption to
toll,
carbon
fractions
given
similar
E.
The
species except Cyt.c553
spectral
suboxydans
with
growing
i.e.
case
were
made
other cytochrome c553
their
no
bound
531
found
and
in
G.
1987).
o >6" ~o
~7
o
'
,
,
~z o
$
Fig.l Fig.l
45kd;
lane
lane
2. Cyt.c551,
Absolute
enzyme
absorption
cytochrome not
a
high
species
the
5ubunit
3.
25kd;
Myoglobin,
Cyt.c550,
6B
-
of
)
Air
in
generated
DOT
was
maintained
in
analysis
membrane
of
found
maintained (Fig.l) G.
OVA,
17.5kd)
4. Cyt.c553,
Oxidized
Table
invariably
steadily
M.W.
lane
(/k/~/~/k/)
states of C y t o c h r o m e
presented
c were
lane
M.W.67kd,
5.
complex spectra
(
(BSA,
was
was
the
M.W.
markers
c553
electrophoretic from
data
of cytochrome
tension
Protein
lane
Dithionite R e d u c e d From
i.
Chymotrypsin,
Dehydrogenase Fig. 2
6
Fig.2
5DS-PAGE(10%) M.W.
6
the
oxydans
structure.
394
the
1
it
during
reactor.
However,
to be p r o d u c e d in
the
cells
was
when
to
the
whether
a low
complex
found
that
three
reactor.
dehydrogenase
c553.
appears growth,
&
or
other oxygen
From
the
solubilized be
a
three
cytochrome
c553
appeared
to
be
the
integral
complex as it constitutes the second subunit
part
(M.W.
23Kd)
having cumulative molecular weight of about 75Kd. preparation c553.
shows
the spectral properties
of
this
enzyme
of the protein
Also the holoenzyme
characteristics of eytochrome
(,Fig.2)
ee(+135 revue-, c 5 5 1 - ' ~ c550 ~ c550 ~ b 5 5 6
?
LOW DOT
eDehydrogenase E n z y m e
e- /
Cyt. c553
e-
/
Terminal t,] oxidase
I
(+60 mY)
----~
Complex
G,DH / GADH / 2-KGADH
_ HIGH D O T
G
GA
2-KGA
(+125 mV)
e-
\2,5-DKG
|
H
02
Fig.3 Proposed Schematic Electron Transport Chain in G. 0xydans.
In
Fig.3,
we
postulate
with
direct
glucose
DOT
of the
growing
under much may other
low
that
the
electron
dehydrogenation culture.
0 2 tension,
many
in
Based
transport
system
G_._to x y d a n s
on
the
depends
finding,
species of cytochrome
associated upon
it is clear that
c type
having
difference in their mid point potentials are generated. be transported so
as
presence
to finally
of
one
dehydrogenase) conditions,
in a sequential m a n n e r
integrated
in
in terminal
cytochrome
cultures
of
the
direct
indicate
dehydrogenation
culminate
from
G.
oxydans
b
562
(as
In
contrast,
subunit
under
high
DOT
electrons
from
the
for terminal
oxidation.
latter condition only two sps. of cytochromes were generated and appropriate ~ E m types m a k e
(mid
the rapid
point
potential
difference),
elecron flow plausible.
395
The
to the
a
grown of
not
Electrons
cyt.c sp.
oxidation.
c553
transfer
site to cytechrome
one
the
the
two
of
In
due to
cytochrome
simplistic model
of
electron
transport
under
high
dissolved
high turnover rate of dehydrogenase 1990)
as compared
the fact that K m
found
tension
at high
DOT
to slow oxidation rate observed
for oxygen
saturation at 1 bar
oxygen
for GDH
explains
the
(Buse
et al.~
at low D O T
despite
is of the order of only 3% of air
(Oosterhuis et ai.,1985).
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E.
0nken,
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U.
Adachi,
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Farr,
A.L.
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