Vol.
174,
No.
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
15, 1991
January
BIODEGRADATION OF TRIIODOPHENOL OF A PENTACHLOROPHENOL-DEGRADING
EXTRACTS
L. Xun
Department University Received
November
16,
43-45
BY CELL-FREE FLAVOBACTER/U/l.l
SP.”
and C. S. Orser
of Bacteriology and Biochemistry of Idaho, Moscow, ID 83843
1990
Pentachlorophenol (PCP) degrading Flavobacteriumsp. ATCC 39723 was found to degrade other polyhalogenated phenolic compounds, including triiodophenol, tribromophenol, and trichlorophenol. Each compound was able to induce the degradation of the other compounds. A PCP- Flavobacterium sp. mutant, F-2, was unable to degrade any of the halogenated compounds. The results suggest that all of the polyhalogenated phenols were degraded by the same enzyme system. This observation led us to exploit the sensitive leuco crystal violet assay, which measures the iodide released from triiodophenol. Cell free extracts from PCP-induced cells were able to release iodide from triiodophenol. The reaction required NADPH and oxygen. 0 1991 Academic mess, 1°C.
Several
organisms
(1,3,7,10,11,13,14), sp.
also
not
been
Although
demonstrating
Flavobacterium, released
@ID Agric.
Expt.
we from
Sta. Journal
or polybromophenols
phenols
Article
the
lack of a sensitive is
released
the sensitive
TIP in cell-free
2,4,6-
Here
we
(TBP)
report
has the
by the PCP-
39723.
The
iodide
Flavobacterium including
organisms.
in understanding
slow.
applied
This
phenols,
of polyiodophenols
of polyhalogenated
progress
that
sp. (7).
polychlorinated
PCP-degrading
sp. ATCC
has been
(PCP) have been isolated
(TIP) and 2,4,6-tribromophenol
dehalogenation
compounds,
dehalogenation
iodide
for
of 2,4,6-triiodophenol Flavobacterium
of such
other
reported
pentachlorophenol
is a Flavobacterium
(I 21. Degradation
previously
degrading
After
several
(TCP)
degradation
mineralize
one of which
degrades
trichlorophenol
which
extract
leuco
from crystal
assays.
No. 90513.
is essential
enzymology assay TIP violet
for degradation and
has been by
the
assay
genetics
of
one barrier.
PCP-degrading to measure
the
Vol.
174,
No.
MATERIALS
BIOCHEMICAL
1, 1991
AND
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
METHODS
Culture and Whole-cell Assav Conditions. Flavobacteriumsp. ATCC strain 39723 was cultured in mineral medium containing 4 g/liter of sodium glutamate as carbon source (6). PCP, TBP, TCP, and TIP degradation was induced by adding 40 ppm of the halogenated compound to early log phase cultures of the bacterium (7). Bacterial cells were collected by centrifugation in a microcentrifuge for 2 min at room temperature. The concentrations of PCP, TCP, TIP, and TBP in the supernatant were measured spectrophotometrically at 320 nm. Iodide concentration was measured calorimetrically by the leuco crystal violet method (2) with minor modifications. We proportionally reduced the assay from 100 ml to 1 ml. The method was based on the following principle: iodide is selectively oxidized to iodine by potassium peroxymonosulfate. The iodine produced reacts instantaneously with the colorless leuco crystal violet to produce the highly colored crystal violet dye. The color is stable and measured spectrophotometrically at a wavelength of 592 nm. Cell-free Extract Preoaration. Cell-free extracts were obtained by suspending 1 g of PCP-induced cells (wet weight) in 5 ml of 20 mM Tris (pH 8.0) and passing twice through an Aminco Model 4-3398 French Pressure cell (7,000 p.s.i.). A protein inhibitor, phenylmethylsulfonyl fluoride (Sigma), was added to the cell suspension to a final concentration of 5 mM just before breaking the cells. Unbroken cells were removed by centrifugation at 10,000 x g for 10 min. Protamine sulfate (Sigma) was added to a final concentration of 1 mg/ml, and the sample was centrifuged at 10,000 x g for IO min to remove nucleic acid-protamine complex. The supernatant, free of intact cells, were used for enzyme-activity assays. Enzvme Activity of Cell-free Extracts. The reaction mixture contained 1.2 mg of protein, 100 PM TIP, 50 mM Tris-acetate buffer, 10 mM MgSO,, and, if included, 0.5% Trition X-100 (Sigma), atmospheric oxygen, and 100 ,uM NADPH in a total volume of 1 ml. The samples were incubated at 24’C in the dark overnight. A standard curve of iodide was made in the same reaction mixture without TIP. Proteins were removed from the completed assays by acidifying 0.25 ml samples with citric buffer (0.05 ml), boiling for 3 min, and centrifuging for 2 min at 15,900 x g. The supernatant (0.25 ml) was diluted to 0.5 ml with distilled water, and 10 ~1 of potassium peroxymonosulfate solution and 10 /II of leuco crystal violet solution were added for measurement of free iodide. The O,-free assay was performed in a serum tube. Helium gas was passed through the assay mixture without TIP for 5 min, TIP was added, and the tube was sealed with a rubber stopper. At the end of the assay, the tube was immersed into a boiling water bath for 3 min and iodide release was assayed.
RESULTS AND
DISCUSSION
Degradation 39723
of TIP, TBP, and PCP was
in approximately
was
without
responsible
one enzyme
cultures
a lag period. for degradation
system
in Flavobacterium
40 min by each of the respective
added at 180 min to those degraded
induced
to remove
previously
This phenomenon
induced suggested
of all three halogenated different
halogens 44
substrates with
sp. ATCC (Fig. 1).
PCP
TIP or TBP was
also
that one enzyme compounds.
from otherwise
system
The ability of similar substrates
Vol.
174, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
80
70
10 0 0
80
120
160
200
2ko
2&io
Time (Min) Fig. 1. PCP-degradation by Flavobacterium sp. ATCC 39723 induced with TIP (W), TBP (-I-), or PCP (0). PCP concentration was monitored using absorbance at 320 nm. PCP (70 ppm) was added to all cultures at 180 min.
is not without
to produce
precedent.
A dioxygenase which dechlorinates 4-chlorophenylacetate
3,4-dihydroxyphenylacetate
bromophenylacetate
also uses 4-fluorophenylacetate
as substrate (5,6).
and 4-
Most chloroalkane and chloroalkanoic acid
dehalogenases also work on more than one halogen substitution (9).
Furthermore,
the PCP- Flavobacterium sp. mutant F-2, isolated for its inability to degrade PCP (II), was also unable to degrade TIP, TBP, or TCP. The Flavobacterium
sp. ATCC 39723 TIP-induced cells degraded PCP, TCP,
TBP and TIP at different rates (Fig. 2). The cells degraded TCP and TBP most rapidly, followed
by PCP, and finally TIP.
TCP is less chlorinated than PCP and faster
degradation was expected.
Although
substitution
enzyme
may interfere
with
TIP is less halogenated than PCP, iodine binding
as a consequence
of the halogen
size.
Bromine, having a smaller ionic radius may not interfere with enzyme binding, as it was degraded at a similar rate to TCP. Fig. 2 shows that equivalent molecule
amount degraded.
iodide production
of TIP degradation. Approximately
occurred at a rate expected
by an
Three iodides were released for every TIP
160 flmol
of iodide
TIP was degraded after 90 min of incubation.
was
released
and 53 prnol
of
The leuco crystal violet assay can
detect as little as 5 flmoles of iodide. Since the assay is very quick, accurate, and sensitive,
iodide
released
from
TIP can be used as a dehalogenating
assay. 45
enzyme
Vol.
174, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
170 160 9
150
3
140 130
@ "
120
G
110
2
100 90
-z
60
0
70
I
60
F
50
;;
40
n
30
2
20 10
0
20
40
60
60
100
Time (Min) Fig. 2. Degradation of polyhalogenated phenols by TIP-induced Flavobacterium sp. strain ATCC 39723 cells. After TIPinduction, the cells were harvested by centrifugation, resuspended in 100 mM Tris buffer, pH 8.0, adjusted to an optical density A,,, = 1 .OO, and supplied with 100 ,uM of TCP, TBP, TIP, or PCP. Concentrations of TCP (H), TBP ( + 1, TIP (A), or PCP (0) were monitored spectrophotometrically and iodide ( x 1 concentration was measured by the leuco crystal violet assay.
The
most
convenient
spectrophotometrically FAD
which
method
may
at 320 be added
and enzymatic
simple
method
however,
screening
of enzyme
to screen
many
the
simple
and
is the solution
HPLC
sample
leuco
attempts
to
as NADPH, can
and GC-MS,
have
violet
and
with
this
nm.
electrode;
considerable
phenols.
cost
for routine It is possible
of dehalogenating method
Another
high background
are not practical
of polyhalogenated
crystal
at 320
is
NADH
interfere
can produce
time for the presence
sensitive
concentration
use of a chlorine
and therefore
reactions
in a short
such
TIP
may also absorb
in the assay
for each
or
reactions,
PCP-degradation
degrading
samples
enzyme
including
PCP
cofactors,
end-products
methods,
10 min or longer
measure
However,
of chlorine
Other
to
to catalyze
of monitoring
concentrations.
using
nm.
reaction
the presence
and take
method
enzymes,
of measuring
iodide
concentration. Our
previous
Flavobacterium
sp. cell-free
PCP-degrading membrane soluble
activity bound
proteins
within
and soluble could
not
extracts
demonstrate had been
the cell was proteins orient
unsuccessful.
membrane
and therefore, correctly 46
dehalogenase
with
activity
with
We hypothesized
that
associated,
and required
when
the cells were
membrane
proteins.
both
broken, We
the
further
Vol.
174,
No.
BIOCHEMICAL
1, 1991
hypothesized
that
once
detergents,
the freed
and restore
some
to the assay
membrane
mixture
iodide.
gradient,
indicated
TIP
NADH was
enzyme
for detection
was
relatively
extracts
strongly Our
suggest
results
demonstrated converted
agree
that
with
a sucrose
PCP to TeCH.
suggested
that
demonstrated extracts
the
The
enzyme
that
TeCH
the
data
was
of a PCP-degrading
also
to
Rhodococcus
the
mechanism
of the conversion
to detect
the
conversion
of TeCH
ATCC
low
enzyme
First,
reasons.
Second,
NADPH.
The
oxidized
NADH
ceil-free
extract
the cell-free boiled, These
reaction.
NADPH
by the cell-free
was
much
is not clear
is required
39723
compounds
are
other
of both
cell-free are not
et
al.
and
(8).
They
activity
which
They
further
0,.
Haggblom
et al.
(4)
by cell-free
presence
of ascorbic
We have
acid.
not been
extract
from
Flavobacterium
extract
could
be due
the same
enzymes
able sp
smaller
than
TBP, same
which TCP, induced
the degradation
the
the
amount
of NADPH
oxidized cells
added
NADPH used
by the dehalogenase
with
A further
is that an additional
possibility
is reduced
by NADPH
and PCP were enzyme of the other 47
halogenated
NADPH
was TIP-
mixture. oxidizing cofactor
first.
degraded
system.
for
to the reaction other
rapidly
that NADPH
of NADPH
added
natural
effectively
We demonstrated
However,
so that that
also
and PCP-induced
TIP or PCP.
to several
as the enzymes’
which
PCP-uninduced
or without
at this moment.
by the induced
used
for NADPH
TIP,
the
we
proteins,
for TIP-degradation,
In summary,
from
for TIP-degradation.
by other
The level of competition proteins
with
substrate
oxidized
degradation
there
or NADPH
a required
rapidly
activity
the conditions
environment.
ATCC
for at least
containing
enzyme
is not known.
and
39723. The
was
mixture
Schenk
in the
that
NADPH,
1,2,4-trihydroxylbenzene sp.
However,
suggesting
indicated
be a monooxygenase.
converted
which
any free iodide.
contained
required
level of
the sample
reactions
by
X-l 00
acetonitrile
X-100,
an enzymatic
fraction
reaction could
was
an
detected
which
assay
reported
gradient
from
Triton
released,
reassociate
of Triton
using
did not generate
of iodide
could
of the assay
control
extracts
the release
HPLC
required
non-ionic
in a detectable
was
Incubation
The
with
proteins
of TIP disappeared
of any iodide
or no cell-free
by
intermediate
low.
dissolved
resulted
as analyzed
NADPH.
COMMUNICATIONS
In fact, the addition
The activity
replace
were
of 0.5%
12 PM
RESEARCH
and soluble
activity.
No accumulated
cannot
activity
results
proteins
dehalogenated.
required
inactivated
vesicles
concentration,
approximately
TIP was completely
4 hours
bound
BIOPHYSICAL
at a final concentration
42 ,YM iodide.
oxygen.
membrane
of the dehalogenating
released
released
the
AND
by Flavobacterium Each
of the compounds.
sp.
halogenated We have
Vol.
174,
No.
BIOCHEMICAL
1, 1991
used the simple and sensitive in both was
whole
detected
proteins
leuco crystal
cell and cell-free by this method.
involved
AND
extract
violet method
assays.
Currently,
in TIP-degradation.
BIOPHYSICAL
Enzymatic
RESEARCH
COMMUNICATIONS
to monitor
TIP-degradation
activity
of TIP-degradation
we are using this method
The same enzymes
to isolate
may also degrade
the
PCP.
ACKNOWLEDGMENTS Flavobacterium Crawford,
University
815250-01-O
sp. ATCC of Idaho.
strain
39723
was
kindly
This research
was
supported
from the Environmental
Protection
supplied
by Dr. Ron L.
in part by grant
R-
Agency.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Apajalahti, J.H.A., Karpanoja, P., and Salkinoja-Salonen, M.S. (1986) J. Syst. Bacterial. 36, 246-251. Black, A.P., and Whittle, G.P. (1967) J. Amer. Water Works Assoc. 59, 471490. Chu, J.P., and Kirsch, E.J. (1972) Appl. Microbial. 23, 1033-1035. Haggblom, M.M., Janke, K., and Salkinoja-Salonen, M.S. (1989) Appl. Environ. Microbial. 55, 516-519. Markus, A., Klages, U., Krauss, S., and Lingens, F. (I 984) J. Bacterial. 160, 618-621. Markus, A., Krekel, D., and Lingens, F. (1986) J. Biol. Chem. 261, 12883-12888. Saber, D.L., and Crawford, R.L. (1985) Appl. Environ. Microbial. 50, 1512-1518. Schenk, T., Muller, R., Morsberger, F., Otto, M.K., and Lingens, F. (1989) J. Bacterial. 171, 5487-5491. Scholtz, R., Leisinger, T., Suter, F., and Cook, A. (1987) J. Bacterial. 169, 5016-5021. Stanlake, G.J., and Finn, R.K. (1982) Appl. Environ. Microbial. 44, 1421-1427. Steiert, J.G., and Crawford, R.L. (1986) Biochem. Biophy. Res. Comm. 141, 825-830. Steiert, J.G., Pignatello, J.J., and Crawford, R.L. (1987) Appl. Environ. Microbial. 53, 907-910. Suzuki, T. (1977) J. Environ. Sci. Health Part B 12:113-l 27. Watanabe, I. (1973) Soil Sci. Plant Nutr. 19, 109-I 16.
48
174,
No.
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
15, 1991
January
BIODEGRADATION OF TRIIODOPHENOL OF A PENTACHLOROPHENOL-DEGRADING
EXTRACTS
L. Xun
Department University Received
November
16,
43-45
BY CELL-FREE FLAVOBACTER/U/l.l
SP.”
and C. S. Orser
of Bacteriology and Biochemistry of Idaho, Moscow, ID 83843
1990
Pentachlorophenol (PCP) degrading Flavobacteriumsp. ATCC 39723 was found to degrade other polyhalogenated phenolic compounds, including triiodophenol, tribromophenol, and trichlorophenol. Each compound was able to induce the degradation of the other compounds. A PCP- Flavobacterium sp. mutant, F-2, was unable to degrade any of the halogenated compounds. The results suggest that all of the polyhalogenated phenols were degraded by the same enzyme system. This observation led us to exploit the sensitive leuco crystal violet assay, which measures the iodide released from triiodophenol. Cell free extracts from PCP-induced cells were able to release iodide from triiodophenol. The reaction required NADPH and oxygen. 0 1991 Academic mess, 1°C.
Several
organisms
(1,3,7,10,11,13,14), sp.
also
not
been
Although
demonstrating
Flavobacterium, released
@ID Agric.
Expt.
we from
Sta. Journal
or polybromophenols
phenols
Article
the
lack of a sensitive is
released
the sensitive
TIP in cell-free
2,4,6-
Here
we
(TBP)
report
has the
by the PCP-
39723.
The
iodide
Flavobacterium including
organisms.
in understanding
slow.
applied
This
phenols,
of polyiodophenols
of polyhalogenated
progress
that
sp. (7).
polychlorinated
PCP-degrading
sp. ATCC
has been
(PCP) have been isolated
(TIP) and 2,4,6-tribromophenol
dehalogenation
compounds,
dehalogenation
iodide
for
of 2,4,6-triiodophenol Flavobacterium
of such
other
reported
pentachlorophenol
is a Flavobacterium
(I 21. Degradation
previously
degrading
After
several
(TCP)
degradation
mineralize
one of which
degrades
trichlorophenol
which
extract
leuco
from crystal
assays.
No. 90513.
is essential
enzymology assay TIP violet
for degradation and
has been by
the
assay
genetics
of
one barrier.
PCP-degrading to measure
the
Vol.
174,
No.
MATERIALS
BIOCHEMICAL
1, 1991
AND
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
METHODS
Culture and Whole-cell Assav Conditions. Flavobacteriumsp. ATCC strain 39723 was cultured in mineral medium containing 4 g/liter of sodium glutamate as carbon source (6). PCP, TBP, TCP, and TIP degradation was induced by adding 40 ppm of the halogenated compound to early log phase cultures of the bacterium (7). Bacterial cells were collected by centrifugation in a microcentrifuge for 2 min at room temperature. The concentrations of PCP, TCP, TIP, and TBP in the supernatant were measured spectrophotometrically at 320 nm. Iodide concentration was measured calorimetrically by the leuco crystal violet method (2) with minor modifications. We proportionally reduced the assay from 100 ml to 1 ml. The method was based on the following principle: iodide is selectively oxidized to iodine by potassium peroxymonosulfate. The iodine produced reacts instantaneously with the colorless leuco crystal violet to produce the highly colored crystal violet dye. The color is stable and measured spectrophotometrically at a wavelength of 592 nm. Cell-free Extract Preoaration. Cell-free extracts were obtained by suspending 1 g of PCP-induced cells (wet weight) in 5 ml of 20 mM Tris (pH 8.0) and passing twice through an Aminco Model 4-3398 French Pressure cell (7,000 p.s.i.). A protein inhibitor, phenylmethylsulfonyl fluoride (Sigma), was added to the cell suspension to a final concentration of 5 mM just before breaking the cells. Unbroken cells were removed by centrifugation at 10,000 x g for 10 min. Protamine sulfate (Sigma) was added to a final concentration of 1 mg/ml, and the sample was centrifuged at 10,000 x g for IO min to remove nucleic acid-protamine complex. The supernatant, free of intact cells, were used for enzyme-activity assays. Enzvme Activity of Cell-free Extracts. The reaction mixture contained 1.2 mg of protein, 100 PM TIP, 50 mM Tris-acetate buffer, 10 mM MgSO,, and, if included, 0.5% Trition X-100 (Sigma), atmospheric oxygen, and 100 ,uM NADPH in a total volume of 1 ml. The samples were incubated at 24’C in the dark overnight. A standard curve of iodide was made in the same reaction mixture without TIP. Proteins were removed from the completed assays by acidifying 0.25 ml samples with citric buffer (0.05 ml), boiling for 3 min, and centrifuging for 2 min at 15,900 x g. The supernatant (0.25 ml) was diluted to 0.5 ml with distilled water, and 10 ~1 of potassium peroxymonosulfate solution and 10 /II of leuco crystal violet solution were added for measurement of free iodide. The O,-free assay was performed in a serum tube. Helium gas was passed through the assay mixture without TIP for 5 min, TIP was added, and the tube was sealed with a rubber stopper. At the end of the assay, the tube was immersed into a boiling water bath for 3 min and iodide release was assayed.
RESULTS AND
DISCUSSION
Degradation 39723
of TIP, TBP, and PCP was
in approximately
was
without
responsible
one enzyme
cultures
a lag period. for degradation
system
in Flavobacterium
40 min by each of the respective
added at 180 min to those degraded
induced
to remove
previously
This phenomenon
induced suggested
of all three halogenated different
halogens 44
substrates with
sp. ATCC (Fig. 1).
PCP
TIP or TBP was
also
that one enzyme compounds.
from otherwise
system
The ability of similar substrates
Vol.
174, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
80
70
10 0 0
80
120
160
200
2ko
2&io
Time (Min) Fig. 1. PCP-degradation by Flavobacterium sp. ATCC 39723 induced with TIP (W), TBP (-I-), or PCP (0). PCP concentration was monitored using absorbance at 320 nm. PCP (70 ppm) was added to all cultures at 180 min.
is not without
to produce
precedent.
A dioxygenase which dechlorinates 4-chlorophenylacetate
3,4-dihydroxyphenylacetate
bromophenylacetate
also uses 4-fluorophenylacetate
as substrate (5,6).
and 4-
Most chloroalkane and chloroalkanoic acid
dehalogenases also work on more than one halogen substitution (9).
Furthermore,
the PCP- Flavobacterium sp. mutant F-2, isolated for its inability to degrade PCP (II), was also unable to degrade TIP, TBP, or TCP. The Flavobacterium
sp. ATCC 39723 TIP-induced cells degraded PCP, TCP,
TBP and TIP at different rates (Fig. 2). The cells degraded TCP and TBP most rapidly, followed
by PCP, and finally TIP.
TCP is less chlorinated than PCP and faster
degradation was expected.
Although
substitution
enzyme
may interfere
with
TIP is less halogenated than PCP, iodine binding
as a consequence
of the halogen
size.
Bromine, having a smaller ionic radius may not interfere with enzyme binding, as it was degraded at a similar rate to TCP. Fig. 2 shows that equivalent molecule
amount degraded.
iodide production
of TIP degradation. Approximately
occurred at a rate expected
by an
Three iodides were released for every TIP
160 flmol
of iodide
TIP was degraded after 90 min of incubation.
was
released
and 53 prnol
of
The leuco crystal violet assay can
detect as little as 5 flmoles of iodide. Since the assay is very quick, accurate, and sensitive,
iodide
released
from
TIP can be used as a dehalogenating
assay. 45
enzyme
Vol.
174, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
170 160 9
150
3
140 130
@ "
120
G
110
2
100 90
-z
60
0
70
I
60
F
50
;;
40
n
30
2
20 10
0
20
40
60
60
100
Time (Min) Fig. 2. Degradation of polyhalogenated phenols by TIP-induced Flavobacterium sp. strain ATCC 39723 cells. After TIPinduction, the cells were harvested by centrifugation, resuspended in 100 mM Tris buffer, pH 8.0, adjusted to an optical density A,,, = 1 .OO, and supplied with 100 ,uM of TCP, TBP, TIP, or PCP. Concentrations of TCP (H), TBP ( + 1, TIP (A), or PCP (0) were monitored spectrophotometrically and iodide ( x 1 concentration was measured by the leuco crystal violet assay.
The
most
convenient
spectrophotometrically FAD
which
method
may
at 320 be added
and enzymatic
simple
method
however,
screening
of enzyme
to screen
many
the
simple
and
is the solution
HPLC
sample
leuco
attempts
to
as NADPH, can
and GC-MS,
have
violet
and
with
this
nm.
electrode;
considerable
phenols.
cost
for routine It is possible
of dehalogenating method
Another
high background
are not practical
of polyhalogenated
crystal
at 320
is
NADH
interfere
can produce
time for the presence
sensitive
concentration
use of a chlorine
and therefore
reactions
in a short
such
TIP
may also absorb
in the assay
for each
or
reactions,
PCP-degradation
degrading
samples
enzyme
including
PCP
cofactors,
end-products
methods,
10 min or longer
measure
However,
of chlorine
Other
to
to catalyze
of monitoring
concentrations.
using
nm.
reaction
the presence
and take
method
enzymes,
of measuring
iodide
concentration. Our
previous
Flavobacterium
sp. cell-free
PCP-degrading membrane soluble
activity bound
proteins
within
and soluble could
not
extracts
demonstrate had been
the cell was proteins orient
unsuccessful.
membrane
and therefore, correctly 46
dehalogenase
with
activity
with
We hypothesized
that
associated,
and required
when
the cells were
membrane
proteins.
both
broken, We
the
further
Vol.
174,
No.
BIOCHEMICAL
1, 1991
hypothesized
that
once
detergents,
the freed
and restore
some
to the assay
membrane
mixture
iodide.
gradient,
indicated
TIP
NADH was
enzyme
for detection
was
relatively
extracts
strongly Our
suggest
results
demonstrated converted
agree
that
with
a sucrose
PCP to TeCH.
suggested
that
demonstrated extracts
the
The
enzyme
that
TeCH
the
data
was
of a PCP-degrading
also
to
Rhodococcus
the
mechanism
of the conversion
to detect
the
conversion
of TeCH
ATCC
low
enzyme
First,
reasons.
Second,
NADPH.
The
oxidized
NADH
ceil-free
extract
the cell-free boiled, These
reaction.
NADPH
by the cell-free
was
much
is not clear
is required
39723
compounds
are
other
of both
cell-free are not
et
al.
and
(8).
They
activity
which
They
further
0,.
Haggblom
et al.
(4)
by cell-free
presence
of ascorbic
We have
acid.
not been
extract
from
Flavobacterium
extract
could
be due
the same
enzymes
able sp
smaller
than
TBP, same
which TCP, induced
the degradation
the
the
amount
of NADPH
oxidized cells
added
NADPH used
by the dehalogenase
with
A further
is that an additional
possibility
is reduced
by NADPH
and PCP were enzyme of the other 47
halogenated
NADPH
was TIP-
mixture. oxidizing cofactor
first.
degraded
system.
for
to the reaction other
rapidly
that NADPH
of NADPH
added
natural
effectively
We demonstrated
However,
so that that
also
and PCP-induced
TIP or PCP.
to several
as the enzymes’
which
PCP-uninduced
or without
at this moment.
by the induced
used
for NADPH
TIP,
the
we
proteins,
for TIP-degradation,
In summary,
from
for TIP-degradation.
by other
The level of competition proteins
with
substrate
oxidized
degradation
there
or NADPH
a required
rapidly
activity
the conditions
environment.
ATCC
for at least
containing
enzyme
is not known.
and
39723. The
was
mixture
Schenk
in the
that
NADPH,
1,2,4-trihydroxylbenzene sp.
However,
suggesting
indicated
be a monooxygenase.
converted
which
any free iodide.
contained
required
level of
the sample
reactions
by
X-l 00
acetonitrile
X-100,
an enzymatic
fraction
reaction could
was
an
detected
which
assay
reported
gradient
from
Triton
released,
reassociate
of Triton
using
did not generate
of iodide
could
of the assay
control
extracts
the release
HPLC
required
non-ionic
in a detectable
was
Incubation
The
with
proteins
of TIP disappeared
of any iodide
or no cell-free
by
intermediate
low.
dissolved
resulted
as analyzed
NADPH.
COMMUNICATIONS
In fact, the addition
The activity
replace
were
of 0.5%
12 PM
RESEARCH
and soluble
activity.
No accumulated
cannot
activity
results
proteins
dehalogenated.
required
inactivated
vesicles
concentration,
approximately
TIP was completely
4 hours
bound
BIOPHYSICAL
at a final concentration
42 ,YM iodide.
oxygen.
membrane
of the dehalogenating
released
released
the
AND
by Flavobacterium Each
of the compounds.
sp.
halogenated We have
Vol.
174,
No.
BIOCHEMICAL
1, 1991
used the simple and sensitive in both was
whole
detected
proteins
leuco crystal
cell and cell-free by this method.
involved
AND
extract
violet method
assays.
Currently,
in TIP-degradation.
BIOPHYSICAL
Enzymatic
RESEARCH
COMMUNICATIONS
to monitor
TIP-degradation
activity
of TIP-degradation
we are using this method
The same enzymes
to isolate
may also degrade
the
PCP.
ACKNOWLEDGMENTS Flavobacterium Crawford,
University
815250-01-O
sp. ATCC of Idaho.
strain
39723
was
kindly
This research
was
supported
from the Environmental
Protection
supplied
by Dr. Ron L.
in part by grant
R-
Agency.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Apajalahti, J.H.A., Karpanoja, P., and Salkinoja-Salonen, M.S. (1986) J. Syst. Bacterial. 36, 246-251. Black, A.P., and Whittle, G.P. (1967) J. Amer. Water Works Assoc. 59, 471490. Chu, J.P., and Kirsch, E.J. (1972) Appl. Microbial. 23, 1033-1035. Haggblom, M.M., Janke, K., and Salkinoja-Salonen, M.S. (1989) Appl. Environ. Microbial. 55, 516-519. Markus, A., Klages, U., Krauss, S., and Lingens, F. (I 984) J. Bacterial. 160, 618-621. Markus, A., Krekel, D., and Lingens, F. (1986) J. Biol. Chem. 261, 12883-12888. Saber, D.L., and Crawford, R.L. (1985) Appl. Environ. Microbial. 50, 1512-1518. Schenk, T., Muller, R., Morsberger, F., Otto, M.K., and Lingens, F. (1989) J. Bacterial. 171, 5487-5491. Scholtz, R., Leisinger, T., Suter, F., and Cook, A. (1987) J. Bacterial. 169, 5016-5021. Stanlake, G.J., and Finn, R.K. (1982) Appl. Environ. Microbial. 44, 1421-1427. Steiert, J.G., and Crawford, R.L. (1986) Biochem. Biophy. Res. Comm. 141, 825-830. Steiert, J.G., Pignatello, J.J., and Crawford, R.L. (1987) Appl. Environ. Microbial. 53, 907-910. Suzuki, T. (1977) J. Environ. Sci. Health Part B 12:113-l 27. Watanabe, I. (1973) Soil Sci. Plant Nutr. 19, 109-I 16.
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