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Applied and Environmental Microbiology, August 1999, p. 3750-3753, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Production of a New D-Amino Acid
Oxidase from the Fungus Fusarium oxysporum
Matthias
Gabler and
Lutz
Fischer*
Institute of Biochemistry and Biotechnology,
Technical University of Braunschweig, 38106 Braunschweig, Germany
Received 16 February 1999/Accepted 25 May 1999
 |
ABSTRACT |
The fungus Fusarium oxysporum produced a
D-amino acid oxidase (EC 1.4.3.3) in a medium containing
glucose as the carbon and energy source and ammonium sulfate as the
nitrogen source. The specific D-amino acid oxidase activity
was increased up to 12.5-fold with various D-amino acids or
their corresponding derivatives as inducers. The best inducers were
D-alanine (2.7 µkat/g of dry biomass) and
D-3-aminobutyric acid (2.6 µkat/g of dry biomass). The
addition of zinc ions was necessary to permit the induction of
peroxisomal D-amino acid oxidase. Bioreactor cultivations
were performed on a 50-liter scale, yielding a volumetric
D-amino acid oxidase activity of 17 µkat
liter
1 with D-alanine as an inducer. Under
oxygen limitation, the volumetric activity was increased threefold to
54 µkat liter1 (3,240 U liter1).
| |
TEXT |
D-Amino acid oxidases
(D-AO) (EC 1.4.3.3) catalyze the enantioselective oxidation
of a broad variety of D-amino acids to their corresponding
-amino acids, which spontaneously hydrolyze to -keto acids and
ammonium. These enzymes are useful in several areas of biochemistry and
biotechnology (for a review, see reference 8). Most
important are the applications in qualitative and quantitative analyses
in either a soluble (12) or immobilized (11, 19)
manner, the oxidation of cephalosporin C (4, 24, 27), the
enantioselective conversion of D-contaminated
L-amino acid solutions (6, 22) or racemic
mixtures (15), and the production of keto acids (3,
5).
D-AO have been found in peroxisomes and microsomes of many
eucaryotic cells or tissues (16, 28). However, up to now the yeasts Trigonopsis variabilis (13, 17) and
Rhodotorula gracilis (23, 25) have seemed to be
the only microbial D-AO producers with satisfactory yields
of D-AO for commercial purposes. Although the gene of pig
kidney D-AO was expressed in Escherichia coli recently (26) and the three-dimensional structure of this
enzyme was solved by two groups (20, 21), pig kidney
D-AO is not appropriate for biotechnological processes due
to its low binding constant for flavin adenine dinucleotide and its
operational instability (8).
In a screening process for new microbial D-AO, the fungus
Fusarium oxysporum was isolated from a soil sample in Lower
Saxony (Germany) (10). It has been deposited at the Deutsche
Sammlung von Mikroorganismen, Braunschweig, Germany, as DSM 12646. Its D-AO accepts a broad range of D-amino acids,
including cephalosporin C, as substrates (7, 10). The enzyme
was purified and characterized (9). In order to find the
parameters important for D-AO production, we tested
different kinds of inducers, enantiomeric ratios, and inducer
concentrations as well as the influence of oxygen supply during
cultivation in a bioreactor.
Culture conditions (shaking flasks).
F. oxysporum was
cultivated in 500-ml flasks with baffles at 30°C and 100 rpm. The
standard medium (120 ml) contained glucose (18 g/liter),
K2HPO4 (4 g/liter),
(NH4)2SO4 (4 g/liter), yeast extract (4 g/liter), and metal salts
(MgSO4·7H2O [1 g/liter], CaCl2·2H2O [0.5 g/liter],
H3BO3 [0.1 g/liter], NaMoO4
[0.04 g/liter], ZnSO4·7H2O [0.04
g/liter], CuSO4·7H2O (0.045 g/liter],
FeSO4·7H2O [0.025 g/liter]). The pH was set
to 7.0 without further adjustment during cultivation.
The induction of D-AO was investigated by adding several
D-amino acids and D-amino acid derivatives at
concentrations of 5 and 30 mM, depending on whether the particular
compound was a substrate for the D-AO reaction (30 mM) or
not (5 mM). The D-AO activity of the cells was monitored
during cultivation by taking samples periodically (every 3 h) and
preparing a crude enzyme solution. For that, cells were harvested by
centrifugation, suspended in buffer, and disrupted with a bead mill
(13). After centrifugation, the clear supernatant was
analyzed. D-AO activity was determined by the
peroxidase-o-dianisidine assay (1, 7).
The highest
D-AO activities were usually measured at the
beginning of the stationary growth phase. The data are summarized
in
Table
1 (means of two separate
cultivations; the maximum deviation
was 7%). The best inducers for
D-AO activity in
F. oxysporum were
D-alanine (yield of dry biomass, 12.2 g/liter) and
D-3-aminobutyric
acid (yield of dry biomass, 10.5 g/liter).
Compared to the activity
in cells cultivated without any inducer, the
specific
D-AO activity
was increased about 13-fold.
Hörner et al. showed that with
N-carbamoyl-
D-alanine
as an inducer instead of
D-alanine, the yeast
T. variabilis produced
up
to 4.2-fold more
D-AO (
13). For
F. oxysporum, this inducer
was not suitable (Table
1). In general,
modification of the carboxy
or amino group of
D-amino acids
resulted in lower specific
D-AO
activities than unmodified
D-amino acids. When we examined just
the results obtained
with modified amino acids, protection of
the carboxy group resulted in
higher levels than protection of
the amino group. When we examined
protein-forming amino acids
as inducers for
D-AO in
F. oxysporum, the best results were obtained
with inducers
having small hydrophobic groups. The yeast
T. variabilis showed a similar correlation between
D-AO induction and the
character
of the
D-amino acid residue (
13,
18).
The dependence of
D-AO activity in
F. oxysporum
on
D-alanine as well as
D-3-aminobutyric acid
concentrations was investigated
(range, 0 to 150 mM). With
D-alanine, the specific
D-AO activity
increased
continuously with increasing inducer concentration.
Thus, the maximum
D-AO activity of approximately 4 µkat g of dry
biomass
1 was measured with 150 mM
D-alanine
(
Kinducer = 26 mM). With
D-3-aminobutyric
acid, the maximum
D-AO
activity (approximately 3 µkat g of dry
biomass
1) was
obtained at about 25 mM (
Kinducer = 2.7
mM).
An interesting finding regarding specific
D-AO induction by
D-alanine was obtained when various
D-alanine/
L-alanine ratios
were used to
supplement the standard medium. The concentration
of
D-alanine at the beginning of the cultivations was always
kept
at 30 mM, while the concentration of added
L-alanine
was varied
from 0 to 100 mM. The highest production of
D-AO
was measured
at a
D-alanine/
L-alanine ratio of
3:1. Without
L-alanine and at
a 1:1 ratio (racemate), the
specific
D-AO activities assayed were
nearly the same and
were about 50% the value obtained at a 3:1
ratio. If the
L
enantiomer was present in excess relative to the
D
enantiomer, the production decreased in inverse proportion to
the
excess
L enantiomer. With pure
L-alanine, no
D-AO induction
was recognized (Table
1). However, the
increased
D-AO production
obtained in the presence of
D-alanine/
L-alanine ratios of >1:1
is
surprising and is difficult to explain. For economic reasons,
further
cultivations were carried out with racemic inducers, as
was done by
others (
13,
25).
As with the yeast
T. variabilis (
13), there was a
significant influence of zinc ions on
D-AO induction.
Without additional
zinc sulfate in the standard medium containing
glucose, ammonium
sulfate, and
D-alanine, no induction of
D-AO was detected, although
the growth of the suspension
culture was as good as with additional
zinc sulfate. The zinc ion
concentration necessary for
D-AO induction
was tested in
the range of 20 to 420 µM. A zinc sulfate concentration
of >70 µM
resulted in a constant maximum specific
D-AO activity
(a
concentration of >280 µM caused a decrease in
growth).
Bioreactor cultivations.
F. oxysporum was cultivated in
a 50-liter bioreactor (Braun-Diesel Biotech, Melsungen, Germany) at
30°C with an air stream of 0.4 volume per volume of liquid per min
(v/vm). The stirrer used was a three-stage Rusthon turbine (400 rpm).
The standard medium (see above) plus the inducer DL-alanine
(80 mM) (Fig. 1) or
DL-3-aminobutyric acid (20 mM) was used. Determination of
the inducer concentration of the medium was done by capillary gas chromatography (CGC) analysis with a CGC-Chirasil-L-Val
column (Chrompack, Frankfurt, Germany) after derivatization of the
samples (6).
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FIG. 1.
D-AO production by F. oxysporum
(DSM 12646) in a 50-liter bioreactor with D-alanine as the
D-AO inducer. The standard medium (see text) plus a racemic
mixture of alanine (80 mM) was used (30°C; 0.4 v/vm; 400 rpm). t,
time.
|
|
The data obtained for cultivation with
DL-alanine as the
inducer are shown in Fig.
1 (means for at least two separate samples;
the maximum deviation was 5%). After 13 h of bioreactor
cultivation
with
DL-alanine, glucose was consumed and
L-alanine and
D-alanine
were taken up into the
cells to serve as energy sources.
L-Alanine
was metabolized
first, but
D-alanine uptake had already started
when about
25 mM
L-alanine (ca. 60%) was still present. When
D-alanine
uptake began, the induction of
D-AO
started and continued until
all the
D-alanine was consumed.
The specific
D-AO activity reached
its maximum after
approximately 16 h and, after 18 h, a volumetric
D-AO activity of approximately 18 µkat
liter
1 was present (yield of dry biomass, 14.2 g/liter;
maximum growth
rate, 0.11 h
1). Compared to the results
obtained in the shaking-flask experiment
(51 µkat
liter
1), only 35% of the volumetric activity was
reached.
A bioreactor cultivation with
DL-3-aminobutyric acid (20 mM) as the inducer was also performed under otherwise identical
conditions
(data not shown). In this case, the fungus took up the
inducer
after approximately 6 h, when more than 80% of the
glucose was
present. As expected, the induction of
D-AO
started at the same
time. After 20 h (yield of dry biomass, 10.5 g/liter; maximum
growth rate, 0.11 h
1), the maximum
volumetric
D-AO activity determined was 15 µkat
liter
1, corresponding to 55% the value obtained in the
shaking-flask
experiment (27.3 µkat/liter
1).
Bioreactor cultivations with limited oxygen supply.
The
bioreactor cultivations resulted in lower volumetric activities than
did the shaking-flask experiments with the same medium. Therefore, one
major difference between the systems, oxygen supply, could have been
responsible. The influence of a limited oxygen supply on the induction
of D-AO was investigated by repeating the bioreactor
cultivation shown in Fig. 1 with DL-alanine (80 mM). The
changed parameters for the cultivation were a decrease of the stirrer
speed from 400 to 300 rpm and a decrease of the gas flow rate from 0.4 to 0.2 v/vm. After 12 h, the pO2 in the medium was
decreased to <10% until the stationary phase was reached, after
approximately 18 h. Again, when D-alanine was taken up
by the fungus, D-AO induction occurred (after approximately
14 h) and volumetric D-AO activity increased rapidly
up to a maximum of 54 µkat liter1 (after approximately
18 h). At this time, the yield of dry biomass was 14.1 g
liter1. Due to the lower oxygen supply, a lower maximum
growth rate of 0.09 h1 was estimated, compared to that in
the previous cultivation (Fig. 1).
The influence of oxygen concentration on
D-AO production
was significant. Under oxygen limitation conditions, threefold-higher
specific
D-AO activity was achieved, compared to that
achieved
in a cultivation without this limitation. Because reoxidation
of the
D-AO cofactor flavin adenine dinucleotide depends on
oxygen
concentration, the fungus might compensate for the slower
turnover
of
D-amino acids in the presence of low oxygen
content by increasing
the total production of
D-AO. Thus,
the "bottleneck" for growth
would be
widened.
The volumetric activity of 54 µkat liter
1 gained on a
50-liter scale in a stirring bioreactor under oxygen limitation is one
of the highest reported for bioreactor productions of
D-AO
in
the literature. Higher
D-AO activity has only been
reported for
the yeast
T. variabilis (77 µkat
liter
1) (
13). The data reported by Huber et
al. (
14) and Biopure
Corporation (
2) indicated
about 112 µkat liter
1 (
T. variabilis CSB
4095) and about 250 µkat liter
1 (mutant strain of
T. variabilis), respectively, but in both cases,
the assays
were done under conditions significantly different
(substrate and
oxygen concentrations) from our conditions. Thus,
a comparison is not
fruitful (
7,
8).
The results described here establish that excellent production of
D-AO from
F. oxysporum is possible by bioreactor
cultivation,
thus enlarging the arsenal of microbial
D-AO
available to serve
as useful tools in analysis and
biotechnology.
| |
ACKNOWLEDGMENTS |
This work was financially supported by the Deutsche
Forschungsgemeinschaft, Bonn, Germany (Fi 655/1-1).
We express our appreciation to William F. Martin for linguistic advice.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biochemistry and Biotechnology, Technical University of Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany. Phone: 49-531-3915730. Fax: 49-531-3915763. E-mail: L.Fischer{at}tu-bs.de.
| |
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Applied and Environmental Microbiology, August 1999, p. 3750-3753, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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