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Applied and Environmental Microbiology, May 1999, p. 2232-2234, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Biodegradation of Cyclohexylamine by Brevibacterium
oxydans IH-35A
Hiroaki
Iwaki,
Masatake
Shimizu,
Tai
Tokuyama, and
Yoshie
Hasegawa*
Department of Biotechnology, Faculty of
Engineering and High Technology Research Center, Kansai University,
Suita 564-8680, Japan
Received 16 November 1998/Accepted 24 February 1999
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ABSTRACT |
A bacterial strain capable of growing on cyclohexylamine (CHAM) was
isolated by using enrichment and isolation techniques. The strain
isolated, strain IH-35A, was classified as a member of the genus
Brevibacterium. The results of growth and enzyme studies are consistent with degradation of CHAM via cyclohexanone (CHnone), 6-hexanolactone, 6-hydroxyhexanoate, and adipate. Cell extracts obtained from this strain grown on CHAM contained CHAM oxidase, and the model for CHAM oxidation by this enzyme was similar to
the model for deamino oxidation of amine by amine oxidase.
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TEXT |
Cyclohexylamine (CHAM) is widely
used as an insecticide and antiseptic in various industries, and
considerable attention has been devoted to possible environmental
pollution by CHAM and CHAM toxicity to humans because CHAM is
suspected of being a weak carcinogen (6, 10, 11).
CHAM is a fairly strong base (pKa 10.6) and therefore
should be excreted mainly unchanged when it is administered
to animals (16). In fact, the metabolism of CHAM
has not been fully investigated. Renwick and Williams (12)
reported that CHAM was excreted largely unchanged and that only 4 to 5% of CHAM was metabolized to cyclohexanol, trans-cyclohexane-1,2-diol, and trans-3-,
cis-3-, trans-4-, and cis-4-aminocyclohexanols in rats, guinea pigs, and humans.
Microbial degradation of simple alicyclic compounds, such as
cyclohexane, cyclohexanol, cyclohexane 1,2-diol, and
cyclohexanecarboxylic acid, has been thoroughly investigated (1,
3, 4, 7, 9, 13-15). However, little is known about the
degradation of CHAM by microorganisms.
In this paper, we describe the isolation and properties of a pure
culture of a bacterium that grows on CHAM.
Isolation of the CHAM-utilizing bacterium.
An organism was
isolated from a soil sample from Osaka Prefecture by selective
enrichment by using minimal salts medium (MSM) containing yeast extract
(100 mg per liter) and CHAM · HCl (1.0 g per liter). It was
maintained on either liquid or solid mineral salts medium
containing CHAM · HCl as the sole carbon source. MSM contained
(per liter of distilled water) 1.0 g of
NH4NO3, 1.5 g of
KH2PO4, 1.5 g of
Na2HPO4, 0.5 g of MgSO4
· 7H2O, 0.01 g of CaCl2 · 2H2O, 0.005 g of FeSO4 · 7H2O, and 0.002 g of MnSO4 · 4H2O. The isolate was a gram-positive, rod-shaped
nonmotile, oxidase-negative, catalase-positive organism. This strain
had the following additional characteristics: gelatin was liquified, nitrate was not reduced to nitrite, 
-galactosidase was not
produced, and esculin was hydrolyzed. Identification and classification of this strain as a member of Brevibacterium oxydans were
confirmed by NCIMB Japan Co. Ltd. (Shizuoka, Japan); this strain was
designated strain IH-35A.
Growth substrates.
Growth of B. oxydans
IH-35A on CHAM was tested in MSM (Fig.
1). This strain also grew well in
nitrogen-free MSM (MSM without NH4NO3 and yeast
extract) containing 1.0% (wt/vol) (73.7 mM) CHAM · HCl as the
sole source of carbon, nitrogen, and energy. The ability of
B. oxydans IH-35A to grow on various substrates was tested in liquid media containing the carbon sources at a
concentration of 0.1% (wt/vol). It grew on
N-methycyclohexylamine, cyclohexanone (CHnone),
2-chlorocyclohexanone, and 6-hexanolactone but did not grow on
cyclohexanol, 4-aminocyclohexanol, or 2-, 3-, or 4-methylcyclohexanone. None of our 25 cyclohexanol-utilizing bacterial strains grew on CHAM.
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FIG. 1.
Degradation of CHAM by B. oxydans IH-35.
Increases in cell density ( ) were determined photometrically at 660 nm. The disappearance of CHAM ( ) was measured by GLC. The GLC
analysis was carried out with a gas chromatograph (model GC-14A;
Shimazu) equipped with a 30-m type DB-1 glass capillary column.
The column was operated at 60°C, and the temperature was programmed
to increase to 220°C at a rate of 10°C/min. The flow rate of the
carrier gas, He, was 1.36 ml/min. The CHAM peak (retention time, 2.55 min) was detected with a flame ionization detector. Accumulation of
CHnone ( ) was determined by HPLC as described in the text.
OD660, optical density at 660 nm.
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Transformation of CHAM by resting cell suspensions.
The amount
of ammonia that accumulated in the medium during transformation of CHAM
was measured by the method described by Fawcett and Scott
(5). Cells of strain IH-35A grown in MSM were washed and
suspended in 50 mM sodium-potassium phosphate buffer (pH 7.0)
containing CHAM. Stoichiometric amounts of ammonia were released into
the medium concomitant with degradation of CHAM, and then CHnone
accumulated in the medium. In addition, cyclohexanol,
trans-cyclohexane-1,2-diol, and 3- and
4-aminocyclohexanols were not detected in the medium (data not shown).
Enzyme activities in cell extracts of the CHAM-grown
Brevibacterium strain.
To confirm the major routes of
metabolism of CHAM by B. oxydans IH-35A, studies were
performed with crude cell extracts. The cell extracts were prepared by
ultrasonication with a Sonifire model 250 apparatus (Branson, Danbury,
Conn.) by using six 20-s bursts on ice.
The enzymatic reaction product was determined by
high-performance liquid chromatography (HPLC), gas-liquid
chromatography
(GLC), or thin-layer chromatography (TLC). HPLC was
performed
with a Capcell C
18 column (length, 250 mm;
diameter, 4.6 mm) by
using methanol as the mobile phase at a flow rate
of 1.0 ml/min.
CHnone was detected by photometric detection at
280 nm and was
identified by comparison with an authentic
standard. For GLC,
solutions of acidic reaction products in dry ether
were methylated
by using the procedure of Metcalf and Schmitz
(
8). GLC was
performed with a column (2 m by 3 mm) that was
packed with 10%
(wt/wt) methylsilicone gum on Chromosorb W. The
following conditions
were used: carrier gas, H
2 at a flow
rate of 25 ml/min; oven temperature,
150°C; injection temperature,
170°C; and thermal conductivity
detector temperature, 170°C. TLC of
the 2,4-dinitrophenyl-hydrazine
derivative was carried out on
0.25-mm-thick layers of Kieselgel
F
254 developed with
solvent A (hexane-ethyl formate, 4/1 [vol/vol])
or with solvent B
(benzene-tetrahydrofuran, 19/1 [vol/vol]). 2,4-Dinitrophenylhydrazone
was detected by direct visual observation. TLC of carboxylic acids
was carried out on 0.25-mm-thick layers of Kieselgel F
254
developed
with solvent C (benzene-ethyl acetate-formic acid, 25/25/2
[vol/vol/vol])
or with solvent D (benzene-dioxane-acetic acid,
40/8/4 [vol/vol/vol]).
Carboxylic acids were detected by
spraying thoroughly dried plates
with 0.1% (wt/vol) bromocresol green
in aqueous 95% (vol/vol)
ethanol adjusted to pH 6.0 with
NaOH.
Incubation of crude cell extracts (48,000-×-
g supernatants)
with CHAM demonstrated that CHAM-dependent stimulation of
O
2 consumption
occurred. Incubation of a
48,000-×-
g supernatant (1.26 mg of protein)
with CHAM under
aerobic conditions, followed by extraction and
identification of the reaction product by TLC
(
Rf with solvent
A, 0.69;
Rf with solvent B, 0.58) and HPLC
(retention time, 2.98
min), indicated that the product of this reaction
was CHnone.
When the reaction stoichiometry for CHAM was measured with
a Warburg
apparatus, ca. 0.5 µmol of O
2 was consumed per
µmol of CHAM oxidized
(Table
1).
Furthermore, investigation of the reaction stoichiometry
demonstrated
that oxidation of 1 µmol of CHAM was accompanied
by the formation of
ca. 1 µmol of NH
3 and ca. 1 µmol of CHnone
(Table
2).
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TABLE 1.
Reaction stoichiometry for consumption of oxygen during
deamination of CHAM by cell extracts of B. oxydans IH-35A
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TABLE 2.
Reaction stoichiometry for production of CHnone and
formation of ammonia during deamination of CHAM by cell
extracts of B. oxydans IH-35Aa
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On the basis of these results, we presume that the model for CHAM
oxidation by this enzyme is similar to the model for deamino
oxidation of amine by amine oxidase, which catalyzes oxidative
deamination of various amines to form the corresponding
aldehydes,
hydrogen peroxide, and ammonia according to the
following equilibrium:
R-CH
2NH-R' + H
2O + O
2 
R-CHO + R'-NH
2 + H
2O
2, where R is an alkyl
group or an aryl
group and R' is H or an aminoalkyl group. However,
the production of
H
2O
2 and the exact consumption of
O
2 are not
clear because of catalase contained in the cell
extracts of strain
IH-35A.
Several methods for assaying activity relating to CHnone degradation
were examined. When the 48,000-×-
g supernatant was
incubated
with NADPH, CHnone-stimulated consumption of O
2
occurred. No activity
was detected when NADPH was replaced by NADH. An
investigation
of the reaction stoichiometry demonstrated that oxidation
of 1
µmol of CHnone was accompanied by consumption of ca. 1 µmol of
NADPH and ca. 1 µmol of O
2, which is close to the
stoichiometry
theoretically required for a mixed-function
oxygenase. The enzyme
reaction product identified by TLC
(
Rf in solvent C, 0.37;
Rf in solvent D, 0.59) and GLC (retention time,
3.50 min) was 6-hydroxyhexanoate.
This observation is consistent
with the formation of 6-hydroxyhexanoate
from CHnone by cell extracts
of cyclohexanol-grown
Nocardia globerula CL1(9) or
Acinetobacter sp. strain NCIMB 9871(4) or
cyclohexane-grown
Xanthobacter species
(
15).
The presence of a 6-hexanolactone hydrolase in the
48,000-×-
g supernatant was demonstrated by incubation
of the 48,000-×-
g supernatant with 6-hexanolactone. The
residual 6-hexanolactone
content was measured by a procedure involving
alkaline hydroxamate
formation, followed by acidification
conversion into the ferric
hydroxamate and measurement of absorbance at
510 nm, as described
by Cain (
2). The expected product of
the reaction, 6-hydroxyhexanoate,
was identified by TLC
(
Rf with solvent C, 0.37;
Rf with solvent
D, 0.59) and GLC (retention
time, 3.50 min) after diethylether
extraction of a 1-h reaction mixture
containing 200 µmol of 6-hexanolactone
and 48,000-×-
g
supernatant (1.85 mg of
protein).
The 48,000-×-
g supernatant from CHAM-grown cells catalyzed
the reduction of NADP
+ in the presence of
6-hydroxyhexanoate. Addition of NAD
+ to the reaction
mixture did not result in any further increase
in absorbance at 340 nm.
The product of 6-hydroxyhexanoate oxidation
cochromatographed
with adipic acid in the TLC systems (
Rf
with
solvent C, 0.47;
Rf with solvent
D, 0.70) described by Donoghue
and Trudgill (
4).
The results of our studies with cell extracts of strain IH-35A are
consistent with the following main route for CHAM degradation:
CHAM
CHnone
6-hexanolactone
6-hydroxyhexanoate
adipate.
The dependence on monooxygenation for
ring cleavage was based
on the presence of a CHnone monooxygenase
and a 6-hexanolactone
hydrolase. Although strain IH-35A did not grow on
cyclohexanol,
CHnone is converted to adipate by enzymes in strain
IH-35A cell
extracts. When the CHnone degradative pathway is
considered, this
finding supports the results of Norris and
Trudgill (
9) and
Donoghue and Trudgill (
4)
obtained for oxidation of cyclohexanol
by
N. globerula
CL1 and
Acinerobacter sp. strain NCIMB
9871.
Induction of the enzymes catalyzing CHAM oxidation.
A
comparison of the activities of the enzymes responsible for CHAM
metabolism in crude extracts of Brevibacterium cells grown on CHAM, CHnone, or succinate is shown in Table
3. The results clearly demonstrate that
CHAM oxidase, CHnone monooxygenase, and 6-hexanolactone hydrolase
activities are induced by growth of B. oxydans IH-35A
on CHAM and are present in the cells at levels that are several
dozenfold greater than the levels in an extract of succinate-grown
cells. Furthermore, CHAM oxidase is not induced by growth on CHnone.
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TABLE 3.
pH optima, cofactor specificities, and activities of
enzymes involved in CHAM degradation by cell extracts of
B. oxydans IH-35A after growth on CHAM, CHnone,
and succinatea
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Finally, to the best of our knowledge, this is the first report of CHAM
metabolism by a
bacterium.
To gain a better understanding of degradation of CHAM by
B. oxydans IH-35A, a biochemical and genetic characterization study
of CHAM oxidase is in
progress.
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ACKNOWLEDGMENTS |
Financial support for this study was provided by Kansai University
research grants (Grant-in-Aid for Joint Research, 1998) and by the
Special Research Fund of The Institute of Industrial Technology, Kansai University.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biotechnology, Faculty of Engineering and High Technology Research
Center, Kansai University, Yamate-cho, Suita 564-8680, Japan.
Phone: (06) 6368-0909. Fax: (06) 6388-8609. E-mail:
yoshie{at}ipcku.kansai-u.ac.jp.
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Applied and Environmental Microbiology, May 1999, p. 2232-2234, Vol. 65, No. 5
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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