Characterization of a new enzyme oxidizing ω-amino group of aminocarboxyric acid, aminoalcohols and amines from Phialemonium sp. AIU 274

Article abstract of DOI:10.1016/j.molcatb.2013.06.012

A new enzyme exhibiting oxidase activity for ω-aminocarboxylic acids, ω-aminoalcohols, monoamines and diamines was found from a newly isolated fungal strain, Phialemonium sp. AIU 274. The enzyme also oxidized aromatic amines, but not l- and d-amino acids. The V_max/K_m value for hexylamine was higher than those for 6-aminoalcohol and 6-aminhexanoic acid in the aliphatic C₆ substrates. In the aliphatic amines, the higher V_max/K_m values were obtained by the longer carbon chain amines. Thus, the enzyme catalyzed oxidative deamination of the ω-amino group in a wide variety of the ω-amino compounds and preferred medium- and long-chain substrates. The oxidase with such broad substrate specificity was first reported here. The enzyme contained copper, and the enzyme activity was strongly inhibited by isoniazid, iproniazid and semicarbazide, but not by clorgyline and pargyline. The enzyme was composed of two identical subunits of 75 kDa.

Full text of DOI:10.1016/j.molcatb.2013.06.012

Journal of Molecular Catalysis B: Enzymatic 96 (2013) 89–95
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Characterization of a new enzyme oxidizing ␻-amino group of
aminocarboxyric acid, aminoalcohols and amines from Phialemonium
sp. AIU 274
Kimiyasu Isobea,∗, Tomoko Sasakia, Yuusuke Aigamia, Miwa Yamadaa,
Shigenobu Kishinob, Jun Ogawab
a Department of Biological Chemistry and Food Science, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan
b Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new enzyme exhibiting oxidase activity for ␻-aminocarboxylic acids, ␻-aminoalcohols, monoamines
and diamines was found from a newly isolated fungal strain, Phialemonium sp. AIU 274. The enzyme also
oxidized aromatic amines, but not l- and d-amino acids. The Vmax/Km value for hexylamine was higher
than those for 6-aminoalcohol and 6-aminhexanoic acid in the aliphatic C6 substrates. In the aliphatic
amines, the higher Vmax/Km values were obtained by the longer carbon chain amines. Thus, the enzyme
catalyzed oxidative deamination of the ␻-amino group in a wide variety of the ␻-amino compounds
and preferred medium- and long-chain substrates. The oxidase with such broad substrate specificity was
first reported here. The enzyme contained copper, and the enzyme activity was strongly inhibited by
isoniazid, iproniazid and semicarbazide, but not by clorgyline and pargyline. The enzyme was composed
of two identical subunits of 75 kDa.
Received 11 April 2013
Received in revised form 21 June 2013
Accepted 22 June 2013
Available online 5 July 2013
Keywords:
Medium-chain amine
Aminocarboxylic acid
Aminoalcohol
␻-Amino compound
Phialemonium
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
from a fungal strain belonging to the genus of Phialemonium and
revealed that the enzyme was a new copper-containing oxidase.
6-Aminohexanoic acid (6-AHA) is a derivative and analog of
l-lysine, and used for treatment of excessive postoperative bleed-
ing. The compound is also known as an intermediate in the
polymerization of Nylon-6, and used for production of poly(ester
amide)s, which have recently been developed as degradable mate-
rials with application as commodities. The enzymes for degradation
of 6-AHA-containing polymers or 6-AHA dimer were reported
[1–4], but those for degradation of 6-AHA have not been reported
yet.
In the studies of microbial and enzymatic oxidation of l-lysine
derivatives and analogs, we isolated some microorganisms, which
produced an enzyme catalyzing oxidation of 6-AHA. Further-
more, we found that certain microorganisms in these isolates
produced an enzyme catalyzing oxidation of ␻-aminocarboxylic
acids, ␻-aminoalcohols, aliphatic monoamines and diamines. The
oxidase exhibiting such broad substrate specificity has not been
reported yet, although it was known that some microbial amine
oxidases catalyzed oxidation of aliphatic monoamines, diamines
and/or aromatic amines [5–8]. We therefore purified the enzyme
The present paper describes production, purification and some
remarkable properties of the oxidase produced by a newly isolated
strain, Phialemonium sp. AIU 274.
2. Materials and methods
2.1. Chemicals
Monoamines from C2 to C8, diamines from C2 to C10, ␻-
aminocarboxylic acids from C2 to C8, ␻-aminoalcohols from
C2 to C8, benzylamine, tyramine, 2-phenylethylamine, 4-
phenylbutylamine, l-lysine, d-lysine and 2-aminoadipic acid
were purchased from Wako Pure Chemicals (Osaka, Japan). N␣-
Benzyloxycarbonyl-l-lysine (N␣-Z-l-lysine) and N␣-Z-d-lysine
were from Watanabe Chemical Industries (Hiroshima, Japan).
Peroxidase was gift from Amano Enzyme (Nagoya, Japan). All
other chemicals used were of analytical grade and commercially
available.
2.2. Screening of microorganism
∗
Microorganisms were isolated by triple-enrichment culture
using a 6-AHA medium, pH 7.0, consisting of 0.3% 6-AHA, 0.5%
Corresponding author. Tel.: +81 019 621 6155; fax: +81 019 621 6155.
E-mail address: kiso@iwate-u.ac.jp (K. Isobe).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.06.012

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K. Isobe et al. / Journal of Molecular Catalysis B: Enzymatic 96 (2013) 89–95
glucose, 0.2% KH2PO4, 0.1% Na2HPO4, and 0.05% MgSO4·7H2O. The
isolates were then incubated in a test tube containing 5 ml of the 6-
AHA medium at 30 ◦C for 3 days with shaking (115 strokes/min),
after which a cell-free extract was prepared by disrupting the
cells using a Multi-beads shocker (Yasui Kikai, Osaka, Japan) below
5 ◦C for 8 min. In the next step, the oxidase activity for 6-AHA
was assayed using the cell-free extract from each isolated strain,
and five strains were selected as producers of oxidase for 6-AHA.
Then, each crude enzyme solution from the five selected strains
was applied to a DEAE-Toyopearl column and the enzyme was
eluted by a linear gradient with 10 mM potassium phosphate buffer,
pH 7.0 and 0.3 M NaCl. The enzyme activity of the eluates was
then assayed using 6-AHA, hexylamine, 6-aminohexanol and 1,6-
diaminohexane as substrates. The fungal strain, which produced
the enzyme exhibiting high activity for these four substrates, was
selected and used in this study.
2.6. Purification of enzyme
Purification of enzyme was carried out at 5–10 ◦C using mycelia
obtained from 32 l culture broth of the 6-AHA medium.
In the first step, mycelia (48 g of wet weight) were suspended
with 600 ml of 10 mM potassium phosphate buffer, pH 7.0, and
disrupted by a Multi-beads shocker (Yasui Kikai, Osaka). The super-
natant solution was obtained by centrifugation at 10,000 × g for
10 min. The cell pellets were resuspended with 500 ml of a newly
prepared 10 mM potassium phosphate buffer, pH 7.0, and the
supernatant solution was obtained by centrifugation. The cell pel-
lets were suspended again with 500 ml of a newly prepared 10 mM
potassium phosphate buffer, pH 7.0, and the cell-disruption was
carried out two times under the same conditions as described
above. The each supernatant solution obtained was mixed and used
as a crude enzyme solution for purification. To the 1730 ml of super-
natant solution, 419 g of solid ammonium sulfate was added to
reach 40% saturation, and allowed to stand for 12 h. The result-
ing precipitate was collected by centrifugation at 10,000 × g for
10 min, dissolved with 10 mM potassium phosphate buffer, pH 7.0,
and dialyzed against the same buffer solution, pH 7.0.
The deionized enzyme solution was applied to a GigaCap Q-
Toyopearl column (20 cm × 2.5 cm diameter) equilibrated with
10 mM potassium phosphate buffer, pH 7.0, and the column was
washed with same buffer solution. The adsorbed enzyme was then
eluted by a linear gradient with 10 mM potassium phosphate buffer,
pH 7.0, and 10 mM potassium phosphate buffer, pH 7.0, containing
0.15 M NaCl (500 ml each). The active fractions were collected, and
solid ammonium sulfate was added to 0.5 M.
2.3. Taxonomic studies of selected strain
Identification of a newly isolated strain was performed at Tech-
noSuruga Laboratory Co., Ltd. (Shizuoka, Japan) as follows. The
isolated strain was incubated on a potato-dextrose agar plate
(Nihon seiyaku, Tokyo, Japan), 2% malt agar plate and an oatmeal
agar plate (Becton Dickinson, MD, USA) at 25 ◦C in the dark, and
the morphological characteristics were observed with both a com-
pound microscope and a stereomicroscope. The sequence of 28S
rDNA-D1/D2 was analyzed using an ABI BigDye Terminator v3.1
Kit (Applied Biosystems, Foster City, CA, USA), and a ABI PRISM
3100 Genetic Analyzer System (Applied Biosystems, Foster City,
CA, USA). The sequence alignment and calculation of the homol-
ogy levels were carried out using the database of Gen Bank, DDBJ
and EMBL.
The enzyme solution was then applied to a Phenyl-Toyopearl
column (20 cm × 2.5 cm diameter) equilibrated with 10 mM potas-
sium phosphate buffer, pH 7.0, containing 0.5 M ammonium sulfate.
After the column was washed with same buffer solution, the
adsorbed enzyme was eluted by a linear gradient with 10 mM
potassium phosphate buffer, pH 7.0, containing 0.5 M ammonium
sulfate and 10 mM potassium phosphate buffer, pH 7.0 (500 ml
each). The active fractions were collected, and deionized by ultra-
filtration.
2.4. Cultivation of selected strain
A newly isolated strain, Phialemonium sp. AIU 274, was first
incubated in a 500-ml shaker flask containing 150 ml of the 6-AHA
medium, pH 7.0, at 30 ◦C for 4 days with shaking (115 strokes/min).
The culture (100 ml) was then transferred into a 3-l culture flask
containing 2 l of the 6-AHA medium. After the cultivation was car-
ried out at 30 ◦C for 30 h, the mycelia were harvested by filtration,
washed with 10 mM potassium phosphate buffer, pH 7.0, and then
stored at −30 ◦C until use.
The deionized enzyme solution was applied to
a DEAE-
Toyopearl column (20 cm × 2.5 cm diameter) equilibrated with
10 mM potassium phosphate buffer, pH 6.3, and the column was
washed with same buffer solution. The adsorbed enzyme was then
eluted by a linear gradient with 10 mM potassium phosphate buffer,
pH 6.3, and 10 mM potassium phosphate buffer, pH 6.3, containing
0.06 M NaCl (500 ml each). The active fractions were collected, and
the purity was analyzed.
2.7. Identification of reaction products
2.5. Assay of enzyme activity
The purified enzyme (13.6 mU) was incubated with 0.1 mmol
of 6-AHA, hexylamine, 6-amino-1-hexanol or 1,6-diaminohexane
in 1.0 ml of 0.1 M potassium phosphate buffer, pH 7.0, at 30 ◦C for
1 h, and the reaction was stopped by boiling for 3 min. The reaction
productions were analyzed using following methods.
Formation of hydrogen peroxide was assayed using 50 ␮l of the
above reaction mixture by the color development method with 4-
AA, TOOS and peroxidase as described in the section of assay of
enzyme activity.
Formation of ammonia was assayed by the glutamate dehydro-
genase method under the following conditions. The above reaction
mixture (50 ␮l) was incubated with 3 ␮mol of ␣-ketoglutarate,
0.36 ␮mol of NADPH and 20 units of glutamate dehydrogenase
at pH 8.0, in a final volume of 1.0 ml. The formation of glutamic
acid, which is compatible with ammonia amounts released from
substrate in the previous reaction, was spectrophotometrically fol-
lowed at 30 ◦C by measuring the absorbance at 340 nm.
The oxidase activity for 6-AHA was assayed by measuring
the rate of hydrogen peroxide formation at pH 7.0 as fol-
lows, because the enzyme was stable at pH 7.0. The standard
reaction mixture contained 20 ␮mol of 6-AHA, 0.6 ␮mol of
4-aminoantipyrine (4-AA), 1.94 ␮mol of N-ethyl-N-(2-hydroxy-3-
sulfopropyl)-3-methylaniline sodium salt dihydrate (TOOS), 6.7
units of peroxidase, 0.1 mmol of potassium phosphate, pH 7.0,
and an appropriate amount of enzyme, in a final volume of
1.0 ml. The assay of enzyme activity was started by addition of
enzyme solution, and formation of hydrogen peroxide was spec-
trophotometrically followed at 30 ◦C for 5 min by measuring the
absorbance at 555 nm. One unit of enzyme activity was defined
as the amount of enzyme catalyzing the formation of one micro-
mole of hydrogen peroxide per min. The molar absorptivity value of
16.5 × 103 M−1 cm−1 was used for calculation of the enzyme activ-
ity.

K. Isobe et al. / Journal of Molecular Catalysis B: Enzymatic 96 (2013) 89–95
91
Table 1
The oxidation product from 6-AHA was analyzed by HPLC with a
TSKgel DEAE-5PW column (Tosoh, Tokyo, Japan). The product was
eluted by a linear gradient (0–100%) with water and 0.3 M NaCl
solution for 10 min, and followed by 0.3 M NaCl solution for 10 min
at a flow rate of 0.8 ml/min. The elution was monitored at 210 nm.
Identification of aldehyde group was carried out by the method
of Paz et al. [9] using 3-methyl-2-benzothiazolinone hydrazone
(MBTH) as follows. Derivative 1; The 50 ␮l of reaction mixture
or eluate from a TSKgel DEAE-5PW column was incubated with
1.0% MBTH solution (0.3 ml) and 0.2 M glycine–HCl buffer, pH 4.0
(0.75 ml), at 25 ◦C for 20 min. Derivative 2; The 50 ␮l of reaction
mixture or eluate from a TSKgel DEAE-5PW column was incubated
with 1.0% MBTH solution (0.30 ml) and 0.2 M glycine–HCl buffer,
pH 4.0 (0.15 ml) at 25 ◦C for 20 min. Then, 0.75 ml of 0.2 M FeCl3
solution was added into the reaction mixture and incubation was
continued at 25 ◦C for 10 min. The absorption spectra of derivative
1 and derivative 2 were analyzed.
Effect of nitrogen source on production of ␻-amino compound-oxidizing enzyme
by Phialemonium sp. AIU 274.
Nitrogen
pH of culture Cell growth
Enzyme activity
(mU/100 ml broth)
broth
(mg/100 ml broth)
6-Aminohexanoic
acid
l-Lysine
6.44
73
20.8
6.21
395
376
0.8
0
Ammonium sulfate 4.23
The spore suspension (1.0 ml) of Phialemonium sp. AIU 274 was inoculated into
150 ml of the medium containing 0.3% indicated nitrogen source and it was incu-
bated at 30 ◦C for 4 days. Mycelia of 150 ml culture broth were disrupted by a
Multi-beads shocker at 2200 rpm for 8 min. The oxidase activity was assayed under
standard assay conditions using crude enzyme solution and 20 mM 6-AHA. Phiale-
monium sp. AIU 274 did not grow with the medium containing 6-aminohexanol,
1,6-diaminohexane or hexylamine as a sole nitrogen source.
from the vegetative hyphae. Adelophialides were mainly recog-
nized. Conidia were phialidic and oval, and its surface was smooth.
On the basis of above genetic and morphological characteristics, we
concluded that the selected strain is imperfect fungus belonging to
the genus Phialemonium and named Phialemonium sp. AIU 274.
2.8. Other analytical methods
Protein concentration was spectrophotometrically determined
1%
1 cm
by measuring the absorbance value at 280 nm. The E
value of
10. 0 was used throughout this work.
3.3. Enzyme induction
SDS-PAGE was performed according to the method of Laemmli
[10]. Proteins were stained with Coomassie Brilliant Blue R-250.
Molecular mass of denatured enzyme was estimated by SDS-PAGE
using standard markers of molecular mass (Sigma, Japan, Tokyo).
Molecular mass of intact enzyme was estimated by gel filtration on
a TSK gel G3000SWXL column equilibrated with 50 mM potassium
phosphate buffer, pH 7.0 containing 0.3 M NaCl.
The copper concentration was analyzed using Inductively
Coupled Plasma-Atomic Emission Spectrometry (Shimadzu ICPE
spectrometry-9000) under the following conditions. Radio fre-
quency power, 1.2 kW; plasma gas flow rate, 10.0 l/min.
The isoelectric point was determined with an isoelectric focus-
ing apparatus (Nihon Eido, Tokyo, Japan) under conditions of 1%
Pharmalyte, pH 3.5–10 (GE Healthcare Japan, Tokyo, Japan), with
a sucrose gradient at 400 V for 2 days at 4 ◦C. 1-ml fractions were
collected, and the pH was measured at 4 ◦C.
Effects of nitrogen sources on enzyme production were
investigated using 150 ml of the medium containing 6-AHA,
6-aminohexanol, 1,6-diaminohexane, hexylamine, l-lysine or
ammonium sulfate as a sole nitrogen source in a 500-ml shaker
flask. Phialemonium sp. AIU 274 grew well in the medium contain-
ing 6-AHA, l-lysine or ammonium sulfate, but not in the medium
containing 6-aminohexanol, 1,6-diaminohexane or hexylamine.
The oxidase activity for 6-AHA was detected in the mycelia grown in
the medium containing 6-AHA or l-lysine, and enzyme productivity
of the 6-AHA medium was higher than that of the l-lysine medium.
No oxidase activity for 6-AHA was detected in the mycelia grown
with the ammonium sulfate medium (Table 1). These results indi-
cated that compounds containing the amino group and carboxyl
group might be essential for induction of the enzyme. Then, the
strain was incubated with 2 l of the 6-AHA medium in a 3-l culture
flask at 30 ◦C for 4 days according to the method described in the
section of cultivation of selected strain, and the oxidase activity for
6-AHA was assayed each day. The oxidase activity reached max-
imum between 24 and 36 h of cultivation (Fig. 1). Therefore, the
mycelia harvested after 30 h of incubation were used for purifica-
tion of the enzyme.
3. Results
3.1. Isolation of microorganisms
In the first step, five strains exhibiting oxidase activity for 6-AHA
were isolated from soil samples after triple-enrichment culture
using the 6-AHA medium containing 0.3% 6-AHA as a sole nitrogen
source. Then, substrate specificity of the enzymes from these five
strains was analyzed using each partially purified enzyme solution
eluted from a DEAE-Toyopearl column. Of these strains selected,
an enzyme from a fungal strain, No. 274, exhibited high activity for
6-AHA, hexylamine, 6-aminohexanol and 1,6-diaminohexane. We,
therefore, selected this strain and used in the following studies.
30
25
20
15
10
5
7.5
7.0
6.5
6.0
0.3
0.2
0.1
0
3.2. Identification of isolated strain
The sequence of 28S rDNA-D1/D2 of the selected strain was
99.1% identical to that of Phialemonium aff. dimorphosporum II-
056.3b (AY188371). When the selected strain was incubated at
25 ◦C for 10 days, it grew well on the agar plates of potato-dextrose,
malt and oatmeal. The colonies were velvet with a color of grayish
yellow-yellowish white on the potato-dextrose agar plate, grayish
yellow-yellowish gray on the malt agar plate and olive brown-
yellowish gray on the oatmeal agar plate. The hyphae had septa and
conidiophores did not develop or very short conidiophores arose
0
0
12
24
36
48
Cultivation time (hr)
Fig. 1. Effect of cultivation time on enzyme production by Phialemonium sp. AIU
274. Phialemonium sp. AIU 274 was incubated in a 3-l culture flask containing 2 l
of the 6-AHA medium at 30 ◦C with shaking (115 strokes/min). The 6-AHA oxidase
activity was assayed each day under standard assay conditions at pH 7.0. Closed
circles, oxidase activity for 6-AHA; open circles, cell growth; closed triangles, pH of
culture broth.

92
K. Isobe et al. / Journal of Molecular Catalysis B: Enzymatic 96 (2013) 89–95
Table 2
Summary of purification of ␻-amino compound-oxidizing enzyme from Phialemonium sp. AIU 274.
Step
Activity (unit)
Protein (mg)
Specific activity (unit/mg protein)
Recovery (%)
100
80
63
54
28
Purification (fold)
Cell-free extract
14.4
11.5
9.11
7.78
4.05
8650
4640
60.1
9.90
3.93
0.0017
0.0025
0.152
0.786
1.03
1.0
1.5
89
462
606
Ammonium sulfate
GigaCap Q-Toyopearl
Phenyl-Toyopearl
DEAE-Toyopearl
Purification of enzyme was carried out using mycelia obtained from 32 l culture broth of the 6-AHA medium. Enzyme activity was assayed under standard assay conditions
using 20 mM 6-AHA. Specific activity was expressed as units per milligram of protein.
3.4. Purification and molecular mass
substrates tested, and oxidation speeds of monoamines were faster
than those of aminocarboxylic acids, aminoalcohols and diamines
in the aliphatic substrates from C4 to C6. The enzyme also oxidized
aromatic amines such as benzylamine, 2-phenylethylamine and
4-phenylbutylamine, and the reaction rates were also affected by
the side-chain length. Thus, the enzyme oxidized a wide variety of
the ␻-amino compounds with different functional groups, and the
reaction speed was affected by the functional groups and carbon
chain length of substrates.
Effect of substrate concentration on enzyme activity was
analyzed under standard assay conditions using 6-AHA, 6-amino-
1-hexanol, hexylamine and 1,6-diaminohexane, and the kinetic
values for these aliphatic C6 substrates were calculated by a plot
of s/v against s. The Km value for hexylamine was lowest in these
substrates, while that for 1,6-diaminohexane was not calculated,
because the speed of 1,6-diaminohexane oxidation exhibited a
sigmoidal curve against its concentrations. The Km value for 6-
AHA was higher than those for 6-aminohexanol and hexylamine
(Table 4). Thus, the binding affinity between enzyme and substrate
was strongly affected by another functional group in the ␻-amino
compounds.
A 600-fold purification of the enzyme was achieved by a pro-
cedure summarized in Table 2. The purified enzyme showed a
single protein band on native-PAGE (line A of Fig. 2). The dena-
tured enzyme also showed a single protein band on SDS-PAGE, and
the molecular mass was estimated to be 75 kDa (line B of Fig. 2). The
molecular mass of the native enzyme was estimated to be 150 kDa
by a TSK gel G3000SWXL column chromatography. These results
indicated that the enzyme consisted of two identical subunits.
The purified enzyme solution exhibited absorption maximum at
275 nm and a gentle absorption curve without marked absorbance
maximum in visible region (Fig. 3). These results indicated that the
enzyme did not contain a flavin as a prosthetic group. The pres-
ence of one copper atom per enzyme protein was identified by a
Shimadzu ICPE spectrometry-9000, which was similar to that of
amine oxidase-I from Aspergillus niger AKU 3302 [6].
3.5. Substrate specificity and kinetic values
The enzyme oxidized ␻-aminocarboxylic acids, ␻-
aminoalcohols, aliphatic monoamines, diamines and aromatic
Effect of carbon chain length of substrate on the binding affinity
to enzyme was analyzed using aliphatic amines from C2 to C8. In
these aliphatic amines, the Km values became lower by increasing
␧
amines, but did not oxidize l-ornithine, l-lysine, d-lysine, N -
␧
Z-l-lysine and N -Z-d-lysine (Table 3). Thus, the enzyme was
specific to the ␻-amino compounds. In addition, the oxidation
rates became faster by increasing the carbon chain length in all
Fig. 2. Native- and SDS-PAGE of purified enzyme. Line A, native-PAGE of intact
enzyme; Line B, SDS-PAGE of denatured enzyme; Line C, SDS-PAGE of standard
proteins.
Fig. 3. Absorption spectrum of purified enzyme solution. The purified enzyme solu-
tion with specific activity of 1.03 units/mg protein was used. UV/visible spectrum of
the enzyme was measured with a Shimadzu UV-2450 spectrophotometer.

K. Isobe et al. / Journal of Molecular Catalysis B: Enzymatic 96 (2013) 89–95
93
Table 3
Table 5
Substrate specificity of ␻-amino compound-oxidizing enzyme from Phialemonium
Effects of chemicals and metals on oxidase activity for 6-AHA.
sp. AIU 274.
Chemical
Relative activity (%)
Substrate
Relative
activity (%)
Substrate
Relative
activity (%)
None
100
0
0
0
0
Hydroxylamine
Hydrazine
Phenylhydrazine
Semicarbazide
Iproniazid
Isoniazid
Clorgyline
Pargyline
o-Phenanthroline
␣,␣ꢀ-Dipyridyl
8-Hydroxyquinoline
EDTA
Aminocarboxylic acids
2-Aminoacetic acid
Monoamines
Ethylamine
Propylamine
Butylamine
Pentylamine
Hexylamine
Heptylamine
Octylamine
0
0
0
7
42
3-Aminopropanoic acid
4-Aminobutyric acid
5-Aminopentanoic acid
6-Aminohexanoic acid
7-Aminoheptanoic acid
8-Aminooctanoic acid
112
188
192
299
359
5
0
2
100
146
140
80
60
44
57
6
90
80
22
90
64
72
65
90
90
39
24
82
Aminoalcohols
2-Aminoethanol
3-Amino-1-propanol
4-Amino-1-butanol
5-Amino-1-pentanol
6-Amino-1-hexanol
Diamines
10
83
87
138
161
1,2-Diaminoethane
1,3-Diaminopropane
1,4-Diaminobutane
1,5-Diaminopentane
1,6-Diaminohexane
1,7-Diaminoheptane
1,8-Diaminooctane
1,10-Diaminodecane
0
1
9
10
25
31
71
131
NaN3
N-Ethylmaleimide
Monoiodo acetic acid
KCN
MgCl2
NiCl2
CoCl2
MnCl2
CuCl2
FeCl2
Amino acids and derivatives
l-Ornithine
Aromatic amines
Benzylamine
2-Phenylethylamine
4-Phenylbutylamine
4-Hydroxyphenyl ethylamine 65
Tryptamine
5-Hydroxytryptamine
Histamine
Dopamine
0
0
1
0
0
2
0
54
75
188
l-Lysine
N˛-Z-l-lysine
Nε-Z-l-lysine
d-Lysine
FeCl3
Enzyme activity was assayed under standard assay conditions with 1 mM chemicals
or metals, except for 0.1 mM clorgyline. Relative activity was obtained as percentage
of enzyme activity without chemicals.
10
0
12
0
N˛-Z-d-lysine
Nε-Z-d-lysine
eluted at 17.1 min from the column, and the peak area compara-
bly increased by the incubation time (data not shown). When 0.1 M
hexylamine, 6-aminohexanol or 1,6-diaminohexane was incubated
in the same conditions, formation of 0.70, 0.92 and 0.44 ␮mol
of hydrogen peroxide and 0.70, 0.82 and 0.49 ␮mol of ammonia
were determined, respectively. Thus, it was demonstrated that the
enzyme catalyzed oxidative deamination of the ␻-amino group of
aminocarboxylic acids, aminoalcohols, monoamines and diamines
according to schemes in Fig. 4.
Enzyme activity was assayed under standard assay conditions using 0.01 U/ml of
enzyme activity for 6-AHA. Relative activity is repressed as percent of the oxidase
activity for 6-AHA.
carbon chain length. In contrast, the Vmax values became higher in
the longer carbon chain amines (Table 4). These results indicated
that the enzyme preferred the medium- and long-chain amines in
the ␻-amino compounds tested.
3.6. Reaction products
3.7. Effect of compounds on enzyme activity
When 0.1 mmol 6-AHA was incubated with 13.6 mU of puri-
fied enzyme at 30 ◦C for 1 h at pH 7.0, formation of 1.2 ␮mol of
hydrogen peroxide and 0.98 ␮mol of ammonia was confirmed.
Formation of oxidation product with the aldehyde group was
confirmed by the MBTH method. Then, the incubation time was
prolonged up to 3 h, and the oxidation product was analyzed by
HPLC with a TSKgel DEAE-5PW column. The oxidation product was
Effect of compounds on enzyme activity was investigated
under standard assay conditions by addition of 1 mM chemicals
or metals. The enzyme activity was strongly inhibited by carbonyl
reagents such as hydroxylamine, hydrazine or phenylhydrazine
and inhibitors of copper-containing amine oxidase such as isoni-
azid or iproniazid. The enzyme activity was also strongly inhibited
by semicarbazide but not by clorgyline and pargyline (Table 5).
We further confirmed that the enzyme activity was completely
inhibited by addition of 0.1 ␮M phenylhydrazine, 1 ␮M hydroxy-
lamine or 10 ␮M hydrazine, and 97%, 95% and 90% of the enzyme
activity were inhibited by addition of 0.1 mM of isoniazid, semi-
carbazide and iproniazid, respectively (Fig. 5). Chelating reagents
such as 8-hydroxyquinoline, o-phenanthroline and ␣,␣ꢀ-dipyridyl
also inhibited the oxidase activity. In the metals tested, FeCl2 and
CuCl2 inhibited the oxidase activity (Table 5). These results indi-
cated that copper might play an important role in the oxidation
reactions.
Table 4
Kinetic properties of ␻-amino compound-oxidizing enzyme from Phialemonium sp.
AIU 274.
Substrate
Km (mM)
Vmax (␮mol/min/mg protein)
Vmax/Km
Ethylamine
Propylamine
Butylamine
Pentylamine
Hexylamine
Heptylamine
Octylamine
1,6-Diaminohexane
6-Amino-1-hexanol
6-Aminohexanoic acid
113
0.334
0.621
0.924
1.39
1.39
1.97
2.57
n.d.
0.579
0.915
0.00295
0.0301
0.307
1.97
2.53
5.20
7.36
–
0.251
0.0295
20.7
3.01
0.705
0.550
0.379
0.349
n.d.
3.8. N-terminal amino acid sequence
2.31
31.0
The amino acid sequence of the intact enzyme was determined
using Shimadzu gas-phase protein sequencer equipped with an
on-line reverse-phase chromatography system for identification
of PTH-amino acids. However, the N-terminal sequence of the
intact protein was not identified, indicating that N-terminus of this
enzyme might be blocked.
The oxidase activity was assayed under standard assay conditions using indicated
substrates. The Km and Vmax values were calculated using 20–200 mM of ethylamine,
2.0–80 mM propylamine, 1.0–20 mM butylamine, 0.025–10 mM of pentylamine,
0.025–10 mM of hexylamine, 0.025–5.0 mM of heptylamine, 0.025–5.0 mM of octy-
lamine, 1.0–20 mM of 6-amino-1-hexanol and 5.0–80 mM of 6-AHA. n.d., Km value
was not determined, because the speed of 1,6-diaminohexane oxidation exhibited
a sigmoidal curve against its concentrations.

94
K. Isobe et al. / Journal of Molecular Catalysis B: Enzymatic 96 (2013) 89–95
1. NH2(CH2)nCOOH + O2 + H2O
OHC(CH2)n-1COOH + NH3 + H2O2
2. NH2(CH2)nOH + O2 + H2O
3. NH2(CH2)nCH3 + O2 + H2O
4. NH2(CH2)nNH2 + O2 + H2O
5. NH2(CH2)nNH2 + 2O2 + 2H2O
OHC(CH2)n-1OH + NH3 + H2O2
OHC(CH2)n-1CH3 + NH3 + H2O2
OHC(CH2)n-1NH2 + NH3 + H2O2
OHC(CH2)n-2CHO + 2NH3 + 2H2O2
Fig. 4. Reaction scheme for oxidation of ␻-amino compounds by enzyme from Phialemonium sp. AIU 274.
3.9. Isoelectric point
[18]. However, oxidation of aliphatic ␻-aminocarboxylic acids and
␻-aminoalcohols by amine oxidases has not been reported yet.
Here, we first demonstrated that Phialemonium sp. AIU 274 pro-
duced a new enzyme catalyzing oxidative deamination of a wide
variety of ␻-amino compounds such as ␻-amincarboxylic acids, ␻-
aminoalcohols, aliphatic amines, aliphatic diamines and aromatic
amines. The enzyme efficiently oxidized medium- and long-chain
␻-amino compounds, and the oxidation speeds of aliphatic amines
were faster than those of aminoalcohols, aminocarboxylic acids
and diamines. The Km value for aliphatic amine was lower than
those for aminoalcohol and aminocarboxylic acid in the ␻-amino
C6 compounds tested. In addition, the higher Vmax/Km values
were obtained by the longer carbon chain amines in alphatic
amines tested. Thus, the Phialemonium enzyme efficiently oxidized
medium- and long-chain amines in the ␻-amino compounds. We
further revealed that copper played an important role in the above
oxidation reactions by the Phialemonium enzyme. In addition, the
enzyme was strongly inhibited by semicarbazide but not by clorgy-
line (inhibitor of minoamine oxidase A) and pargyline (inhibitor of
minoamine oxidase B). These results indicated that the Phialemo-
nium enzyme might be classified into the semicarbazide-sensitive
amine oxidase in the copper-containing amine oxidases. The puri-
fied enzyme solution from Phialemonium sp. AIU 274 did not exhibit
the marked absorbance maximum in visible region, whereas that
of the copper-containing amine oxidase I from A. niger AKU 3302
exhibited absorbance maximum at 490 nm [6]. These results indi-
cated that the binding states of copper or existence of another
cofactor might be different in both enzymes. The pI value and N-
terminal amino acid were also different in both enzymes, although
the molecular mass of subunit was similar to each other.
A single peak of the enzyme activity at pH 5.7 was observed after
isoelectric focusing with carrier Pharmalyte, pH 3.5–10.
4. Discussion
The l-amino acid oxidases and amine oxidases are well known
as enzymes capable of oxidizing certain compounds with amino
group. In these enzymes, l-amino acid oxidases (EC 1.4.3.2) contain
flavin as the prosthetic group, and catalyze oxidative deamnination
of the ␣-amino group of ␣-amincarboxylic acids to the corre-
sponding ␣-keto acids with the subsequent release of ammonia
and hydrogen peroxide [11–15]. Some other l-amino acid oxidases
also catalyze oxidative deamnination the ␧-amino group of l-lysine
and/or N␣-acyl-l-lysine [16,17]. However, those l-amino acid oxi-
dases do not oxidize ␻-aminocarboxylic acids, ␻-aminoalcohols
and aliphatic amines. In contrast, amine oxidases, which are classi-
fied into two groups, flavin-containing amine oxidase (EC 1.4.3.4)
and copper-containing amine oxidase (EC 1.4.3.21), catalyze oxida-
tion of a wide range of amines to the corresponding aldehydes with
the subsequent release of ammonia and hydrogen peroxide, but
do not oxidize free l-amino acids. Recently, we demonstrated that
copper-containing amine oxidase from A. niger AKU 3302 oxidized
not only aliphatic amines but also N␣-Z-l-lysine and N␣-Z-d-lysine
100
80
Studies of gene cloning and DNA sequence analysis of the
Phialemonium enzyme are now in progress to reveal the unique
characteristics of the enzyme in more detail. Studies of enzymatic
production of aldehyde carboxylic acids, aldehyde alcohols, alde-
hyde amines are also underway using the Phialemonium enzyme
and the ␻-amino compounds.
60
40
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