Journal of General Microbiology (1982), 128, 99 1-996.
Printed in Great Britain
99 1
More Than One Amine Oxidase is Involved in the Metabolism by Yeasts
of Primary Amines Supplied as Nitrogen Source
By J E F F R E Y G R E E N , G E O F F R E Y W . H A Y W O O D A N D
PETER J. LARGE*
Department of Biochemistry, University of Hull, Hull HU6 7RX, U.K.
(Received 10 September 1981 )
Twenty-seven yeast species were tested for growth on methylamine and n-butylamine as sole
nitrogen sources. Five species (including Saccharomyces cerevisiae) failed to grow on either
amine, and a further five grew only on n-butylamine. The remainder grew on both amines.
Amine oxidase activity was detected in extracts of all the strains which could grow on amines,
but was generally absent from cells grown on ammonia or nitrate. From measurements in
cell-free extracts of oxidase activity for a number of different amines, it is concluded that
most such strains have at least two amine oxidases of different substrate specificity, and that
the substrate specificity also varies between different yeast species. Based on these observations, four different groups of yeasts could be distinguished. Formaldehyde dehydrogenase
activity was elevated in cells grown on amines or nitrate compared with those grown on
ammonia, and activity was generally higher in cells grown on methylamine than in those
grown on n-butylamine or nitrate.
INTRODUCTION
That yeasts can grow on amines as sole nitrogen source has been known for many years,
and ethylamine utilization has been used as a diagnostic tool since it was proposed by Van
der Walt (1962). The extent of the capabilities of yeasts to utilize amines as sole nitrogen
source, however, was not fully realized until Van Dijken & Bos (198 1) showed that 86 % of
the 461 strains of yeast from various genera tested were able to use at least one amine as sole
nitrogen source, and 30% could use all of the eight amines tested.
It is generally assumed that utilization of amines for growth involves an amine oxidase, and
this has been shown to be true for growth of Trichosporon sp. on trimethylamine (Yamada et
al., 1966), growth of Candida utilis and Hansenula polymorpha on methylamine (Zwart et
al., 1980) and growth of Candida boidinii on a number of different amines (Haywood &
Large, 1981). We have examined 27 yeast strains in an attempt to answer the following
questions. (1) Are amine oxidases always present when yeasts are grown on primary aliphatic
amines as sole nitrogen source? (2) In yeasts grown on a single amine substrate, what other
amines can be oxidized by cell-free extracts? (3) How many different amine oxidases are
present during growth on any single amine substrate? (4) Does the nature or number of amine
oxidases present in a cell change when the growth substrate is changed? In addition, evidence
(Van Dijken et al., 1979; Zwart et al., 1980; Van Dijken & Bos, 1981; Haywood & Large,
1981) suggests that formaldehyde dehydrogenase (EC 1 .2.1.1) activity is elevated in cells
grown on methylated amines compared with cells grown on ammonium salts. We investigate
here whether this is a general phenomenon in yeasts and also whether it occurs when other
non-ammonium nitrogen sources are used.
0022-1287/82/0001-0169 $02.00 0 1982 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 13:36:37
992
J . G R E E N , G . W. H A Y W O O D A N D P . J . L A R G E
METHODS
Organisms used. With two exceptions, the yeast strains were gifts from Dr J. P. van Dijken (Delft, The
Netherlands); 18 of the 27 were type strains from the Centraalbureau voor Schimmelcultures (Yeast Division) in
Delft (CBS). The nomenclature of the strains used is that of Barnett et al. (1979) except that former Torulopsis
species have been transferred to the genus Candida as proposed by Yarrow & Meyer (1978). The designatory
abbreviations for other culture collections are: NCYC, National Collection of Yeast Cultures (Norwich, U.K.); G,
culture collection of Microbiology Laboratory, University of Groningen, The Netherlands.
Maintenance. Strains were maintained on slopes containing 1% (w/v) malt extract and 2% (w/v) agar.
Growth of cells. Yeasts were grown on the defined medium previously described (Haywood & Large, 19Sl),
with glucose ( 5 5 mM) as carbon source and one of the following as nitrogen source: 10 mM-methylamine.HC1,
5 mM-(NH,),SO,, 10 mM-KNO, or 10 mM-n-butylamine.HC1. In most cases, the cells were in the exponential
growth phase (As63between 1 and 2) when they were harvested. They were washed with 50mM-potassium
phosphate pH 7.0 and stored at -18 “C until used.
Preparation of extracts and enzyme assays. Extracts were prepared in the French press as described by
Haywood & Large (198 I), and enzymes were assayed by the following methods. Methylamine, rz-butylamine,
isobutylamine, benzylamine and putrescine oxidases were assayedby the peroxidase-coupled spectrophotometric
assay of Haywood & Large (1981), with the substrates at the following final concentrations: methylamine, 3 . 3
mM; n-butylamine, 1 mM; isobutylamine, 1 mM; benzylamine, 0.33 mM; putrescine, 1 mM. Formaldehyde
dehydrogenase (EC 1.2.1.1) and formate dehydrogenase (EC 1.2.1.2) were assayed by the method of Van
Dijken et al. (1976), and isocitrate dehydrogenase (NADP-linked, EC 1.1.1.42) by the method of Bergmeyer
(1974). The formaldehyde and formate dehydrogenase activities were corrected by addition of the NADH oxidase
activity measured at the same pH. In some cases (marked 3: in Table 1) this NADH oxidase activity was so high
that no dehydrogenase activity could be measured.
Poiyacrylarnide gel electrophoresis. Electrophoresis of crude extracts and staining for amine oxidase activity
were performed as described by Haywood & Large (198 1). This method can detect extremely low levels of amine
oxidase activity.
Protein. This was determined by the method of Bradford (1976).
RESULTS A N D DISCUSSION
The yeast strains tested were selected on the basis of the results of Van Dijken & Bos
(1981). They fell into three categories: (a) those which grew on both methylamine and
n-butylamine, (b) those which grew on n-butylamine but not on methylamine and (c) those
which failed to grow on either amine as sole nitrogen source. In all but three cases our
observations as to which amines could be used for growth agreed with those of Van Dijken &
Bos (198 1). In category (c)were Cryptococcus albidus CBS 142, Pichia tannicola CBS 6065,
Saccharomyces cerevisiae NCYC 240*, Schizosaccharomyces japonicus var. japonicus
CBS 354 and Candidu (ToruZopsis) glabrata CBS 2663*. (An asterisk, both here and in
Table 1 denotes a strain which is not a type strain.) This category (c) was not examined
further. The various enzyme activities examined were the oxidases for five primary amines
(methylamine, n-butylamine, isobutylamine, benzylamine and putrescine), formaldehyde
dehydrogenase, formate dehydrogenase and NADP-linked isocitrate dehydrogenase. The
latter was selected because it would not be expected to show significant variation when the
nitrogen source was changed.
Expression of amine oxidase activity by the yeast strains
During growth on nitrate or ammonium as nitrogen source, no amine oxidase activity
could be detected, except in two cases, Hansenula polymorpha and Candida nemodendra,
where low levels were detected in cells grown on ammonia (Table 1). This does not affect our
conclusion that ammonia and nitrate both repress amine oxidase activity. During growth
on amines, however, in all cases oxidase activity for at least one of the five amines tested was
found. In a few cases no activity could be detected with the amine which had been used as
nitrogen source (see below).
From work on Candida boidinii (Haywood & Large, 1981) it is known that yeasts may
possess more than one amine oxidase capable of oxidizing n-butylamine. In Candidu boidinii
one of these enzymes (‘benzylamine oxidase’) is much less effective in oxidizing methylamine,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 13:36:37
Multiple amine oxidases in yeasts
993
while the second (‘methylamine oxidase’) is much less effective in oxidizing benzylamine. The
strategy used was to grow the yeasts on n-butylamine and methylamine and to measure the
levels of oxidase activity for methylamine, benzylamine and n-butylamine. The increase of
n-butylamine oxidase activity in cells grown on n-butylamine and the increase of methylamine
oxidase in cells grown on methylamine in most of the strains tested (Table 1) suggests that
enzyme multiplicity occurs in almost all cases. The only paradoxical result occurs in the
genus Pichia where both activities are lower in cells grown on methylamine. The existence of
multiple amine oxidase enzymes was also borne out by the presence of multiple bands in
activity stains on polyacrylamide gels (Table l), although these data have to be treated with
caution because pure yeast amine oxidases are known to give multiple bands (Haywood &
Large, 1981). The activity stains, however, did allow detection of amine oxidase activity in
those extracts where no activity could be measured in the assays with substrates on which the
cells had been grown. There are five instances of this in Table 1. Such failure to measure
activity may have been due to having harvested the cells at the wrong stage in the growth
cycle, or to use of sub-optimal assay conditions, because all such experiments were repeated
at least once, giving the same result. The assay conditions used have been established as
optimal for Candida boidinii, but may of course be different in other species.
From the amine oxidase data in Table 1, we may divide the organisms into four classes:
(i) Species with high methylamine, benzylamine and n-butylamine oxidase activities during
growth on one or both amine substrates: Candida lipolytica, C. nagoyaensis, C. utilis,
Hansenula minuta, H . polymorpha, Pichia pastoris, P. pinus, Sporopachydemia cereana and
Trigonopsis variabilis.
(ii) Species with high methylamine and n-butylamine oxidase activities but low
benzylamine oxidase activity: Candida boidinii and C.nemodendra.
(iii) Species with high methylamine oxidase activity but low n-butylamine and benzylamine
oxidase activities :R hodotorula graminis and Sporobolomyces albo-rubescens.
(iv) Species with low methylamine oxidase activity but high n-butylamine and benzylamine
oxidase activities : Candida steaiolytica, Kluyveromycesfragilis, K . lactis, K. phaseolosporus,
and all the organisms in category (b)in Table 1.
Further investigation will be necessary to establish whether these groupings really reflect
genuine differences, but what is clearly shown is that not only are there multiple enzymes, but
that these enzymes differ in substrate specificity. This was confirmed by the finding of
putrescine oxidase activity in only five organisms: Candida nagoyaensis (grown on
methylamine), C. steaiolytica (grown on both amines), C. utilis (grown on butylamine),
Pichia pastoris (grown on butylamine) and Sporopachydemia cereana (grown on
methylamine). In methylamine-grown Candida steatolytica, putrescine oxidase was the only
oxidase activity which could be detected by activity measurement. In Carzdida boidinii it is
known (Haywood & Large, 198 1) that neither methylamine oxidase nor benzylamine oxidase
can oxidize putrescine. Further evidence of differences in substrate specificity is given by
activity with isobutylamine (only one of the two amine oxidases in Candida boidinii will
oxidize this substrate). Most of the organisms in group (i) (except in the two Hansenula
species) and all those in category (6) of Table 1 had detectable isobutylamine oxidase activity
(although high levels were only found in Candida nagoyaensis, C. boidinii, Sporopachydemia
cereana and Trigonopsis variabilis). It may be significant that all species without
isobutylamine oxidase activity were ascospore-forming strains. The occurrence of more than
one amine oxidase, already established for Candida boidinii (Haywood & Large, 1981) and
for the fungus Phycomyces blakesleeanus (Hofmann & Hilgenberg, 1979), thus proves to be a
general phenomenon in yeasts.
Although the organisms in category (b) had very low methylamine oxidase activity, this is
not the sole cause of their failure to grow on methylamine, because this was also true of
Candida steatolytica and the three Kluyveromyces spp. in category (a) which grew slowly on
methylamine. It may perhaps be coupled with inability to transport methylamine.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 13:36:37
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 13:36:37
H. polymorpha
CBS 4732
Hansenula minuta
CBS 1708
C. utilis
NCYC 321*
C. steatolytica
CBS 5839
C. nemodendra
CBS 6280
C. lipolytica
CBS 2074*
C . nagoyaensis
CBS 6853
Candida boidinii
CBS 5777*
Organism and strain
I
Methylamine
oxidase
Benzylamine
oxidase
n-Butylamine
oxidase
A
*
1430
180
182
178
30
$
177
383
*
I
*
5
98
98
172
176
236
45
16
230
48
852
$
57
77
25
Formaldehyde
dehydrogenase
Specific activity [nmol min-’ (mg protein)-’)
( a ) Species capable of growth on both n-butylamine and methylamine as nitrogen source
2
I20
7
238
Mt
B
1
31
0
154
A
0
0
0
N
0
0
0
M
37
0
13
B
0
58
68
224
17
24 3
Mt
B
142
188
507
A
0
0
0
N
0
0
0
M
1
43
23
0
B
14
0
34
0
A
0
23
0
1
0
0
0
Mt
1
0
12
17
Bt
A
0
0
0
M
1
82
0
67
1
50
24 1
50
Bt
A
0
0
0
N
0
0
0
2
M
4
11
9
B
1
7
11
23
N
0
0
0
M
3
28
118
66
B
1
0
23
33
A
6
0
1
N
0
0
0
Nitrogen
source for
growth
No. of
amine
oxidase
bands
on gel
\
51
68
150
191
344
38 1
39
52
33
9
32
118
80
129
139
155
103
114
97
45
115
53
67
51
70
49
65
Isocitrate
dehydrogenase
Cells were grown on n-butylamine (group b) or n-butylamine and methylamine (group a ) and disrupted as described in Methods. In a number of
cases cells were also grown on ammonium sulphate or potassium nitrate. Crude extracts were prepared with a French press and assayed spectrophotometrically for enzyme activities and protein concentration. Some extracts were also examined for amine oxidase activity on polyacrylamide gels after
electrophoretic separation (-, no data available). Abbreviations used to denote the various nitrogen sources for growth: A, ammonium sulphate;
B, n-butylamine; M, methylamine; N, potassium nitrate.
Table 1. Enzyme activities present in yeasts grown on primary arnines as sole nitrogen sources
B
Q
5El
?
r
4
cd
tl
U
P
2:
e
0
0
P
W
\o
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 13:36:37
Trichosporon melibiosaceum
CBS 6087
Candida insectalens
CBS 6036
Kluyveromyces wickerhamii
CBS 2745
Pichia media CBS 5521
P.polymorpha CBS 186
Sporopachydemia cereana
CBS 6644
Trigonopsis variabilis
CBS 1040
Sporobolomyces albo-rubescens
CBS 482
Pichia pastoris
CBS 704
P. pinus
CBS 5098*
R hodotorula graminis
NCYC 980;
K . lactis
CBS 2359;
K . phaseolosporus
CBS 2103
G 7 14*
Kluyveromyces fragilis
2
1
0
5
0
0
2
5
0
24
66
5
110
66
11
0
0
41
0
0
259
74
10
18
45
41
144
39
125
45
0
37
27
4
49
0
0
0
0
0
0
0
26
50
0
63
91
13
36
14
0
9
159
52
183
0
7
0
0
0
0
0
82
263
125
82
6
37
2
1
1
B
B
B
2
0
0
2
0
7
17
14
0
171
0
218
3: Activity lower than NADH oxidase and therefore undetectable.
t Putrescine oxidase activity was also found (see text).
* Not a type strain.
-
1
B
A
-
A
0
124
0
45
0
175
(b) Organisms capable of growth on n-butylamine but not methylamine as nitrogen source
B
1
0
0
22
B
M
B
Mi.
A
M
B
M
B
M
B
A
M
Bt
M
B
M
B
A
N
M
B
29
82
8
51
32
75
$
15
$
1540
288
146
14
39
73
140
102
184
162
120
$
126
224
110
52
220
64
84
42
18
120
58
41
81
31
146
95
81
100
56
72
61
32
51
52
53
85
105
92
59
111
102
49
52
191
64
120
175
996
J . G R E E N , G . W. H A Y W O O D A N D P . J . L A R G E
The data do not allow us to determine precisely how many amine oxidase isoenzymes exist
in the different strains. This will require either enzyme purification or more sophisticated
identification methods for crude extracts. Partial purification of putrescine oxidase activity
from Pichia pastoris and Candida utilis has shown that in both these organisms the same
enzyme can oxidize putrescine, n-butylamine and benzylamine (unpublished results).
Formaldehyde andformate dehydrogenase activity
The results in Table 1 support the suggestion of Van Dijken & Bos (1981) that enzymes
oxidizing formaldehyde are widespread in yeasts and are elevated in activity in cells grown on
methylated amines. Activities of formaldehyde dehydrogenase were generally significantly
higher in cells grown on methylamine than in n-butylamine-grown cells, which themselves had
activities higher than in cells grown on ammonia. The activities of formate dehydrogenase
were so low as to be in most cases below the level of NADH oxidase activity and so
undetectable (not shown in Table 1). This is to be expected since formate dehydrogenase
activity only rises during the stationary phase of cell growth on methylamine (Zwart et al.,
1980). That the derepression of formaldehyde dehydrogenase observed in most cases in cells
grown on n-butylamine compared with the levels of the enzyme in ammonia-grown cells is not
directly concerned with amine metabolism is shown by the fact that in most cases the enzyme
also seemed to be derepressed in cells grown on nitrate. Derepression of formaldehyde
dehydrogenase during growth on glucose plus ammonia at low dilution rates was shown for
Hansenulu polymorpha and Kloeckera sp. 220 1 by Egli et al. (1980). The relatively slight
changes in NADP-linked isocitrate dehydrogenase with nitrogen source in most cases
suggests that the changes observed in the other enzymes examined are indeed of physiological
significance.
We thank Dr J. P. van Dijken for valuable discussions and suggestions. This work was supported by grant
GR/B/23687 from the Science and Engineering Research Council, which is gratefully acknowledged.
REFERENCES
BARNEIT,J. A., PAYNE,R. W. & YARROW,D. (1979).
A Guide to Identifying and Classifying Yeasts.
Cambridge: Cambridge University Press.
BERGMEYER,
H. U. (editor) (1974). Methods of Enzymatic Analysis, 2nd English edn, pp. 479-480.
Weinheim: Verlag-Chemie.
BRADFORD,M. M. (1976). A rapid and sensitive
method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye
binding. Analytical Biochemistry 72, 248-254.
EGLI,TH., VANDIJKEN,J. P., VEENHUIS,M., HARDER,
W. & FIECHTER,
A. (1980). Methanol metabolism in
yeasts: regulation of the synthesis of catabolic
enzymes. Archives of Microbiology 124, 1 15- 12 1.
HAYWOOD,G. W. & LARGE,P. J. (1981). Microbial
oxidation of amines. Distribution, purification and
properties of two primary-amine oxidases from the
yeast Candida boidinii grown on amines as sole
nitrogen source. Biochemical Journal 199, 187201.
HOFMANN,F. & HILGENBERG,
W. (1979). Aminooxidasen in Phycomyces blakesleeanus und ihre
Bedeutung fur die Indol-3-essigsaurebiosynthesedes
Pilzes. Bericht der Deutschen botanischen
Gesellschaft 91, 6 1 1-622.
VAN DLIKEN,J. P. & Bos, P. (1981). Utilization of
amines by yeasts. Archives of Microbiology 128,
320-324.
VAN DIJKEN,J. P., OTTO, R. & HARDER,W. (1976).
Growth of Hansenula polymorpha in a methanollimited chemostat. Physiological responses due to
the involvement of methanol oxidase as a key
enzyme in methanol metabolism Archives of Microbiology 111, 137-144.
VANDLIKEN,J. P., VEENHUIS,
M., ZWART,K., LARGE,
P. J. & HARDER,W. (1979). Utilisation of methylamine by yeasts. Society for General Microbiology
Quarterly 6, 70.
VANDER WALT,J. P. (1962). Utilization of ethylamine
by yeasts. Antonie van Leeuwenhoek 28,9 1-96.
YAMADA,H., KUMAGAI,H., UWAJIMA,T. & OGATA,
K. (1966). Trimethylamine metabolism. I. Monomethylamine oxidase. Memoirs of the Research
Institute for Food Science, Kyoto University 27,
1-14.
YARROW,D. & MEYER,S. A. (1978). A proposal for
amendment of the diagnosis of the genus Candida
Berkhout nom.cons. International Journal of
Systematic Bacteriology 28, 6 1 1-6 15.
ZWART,K., VEENHUIS,M., VAN DIJKEN, J. P. &
HARDER, W. (1980). Development of amino
oxidase-containing peroxisomes in yeasts during
growth on glucose in the presence of methylamine as
the sole source of nitrogen. Archives of Microbiology 126, 117-126.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 13:36:37