FEMS Microbiology Letters 60 (1989) 289-294
Published by Elsevier
289
FEM 03645
Cyanide oxygenase and cyanase activities
of Pseudomonas fluorescens NCIMB 11764
P a t r i c k K. D o r r a n d C h r i s t o p h e r J. K n o w l e s *
Biological Laboratoo'. Universi(vof Kent. Canterbury, U.K.
Received 21 March 1989
Accepted 3 April 1989
Key words: Pseudomonas fluorescens; Nickel cyanide; (Nitrogen source)
1. S U M M A R Y
Pseudomonas fluorescens N C I M B 11764 is able
to utilise cyanide (both K C N and Ni(CN) 2-) as a
nitrogen source for growth. Under such conditions
cyanide oxygenase activity is induced. When
potassium cyanate ( K O C N ) is supplied as the sole
nitrogen source for growth, cyanase activity is
induced. It has been demonstrated that these two
enzymic activities are physiologically distinct, and
are not co-induced under any of the growth conditions tested.
2. I N T R O D U C T I O N
Pseudomonas fiuorescens N C I M B 11764 was
isolated by a cyanide vapour plate technique, designed to select for organisms capable of utilising
cyanide ( K C N = H C N ) as the sole source of
nitrogen for growth [1]. Growth of this organism
has been demonstrated in fed-batch culture with
K C N as the growth limiting nutrient and in N-
Correspondence to." C.J. Knowles, Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ, U.K.
limited continuous culture as well as in batch
culture with N i ( C N ) ] - as the nitrogen source
[1,2]. Under all these growth conditions, cyanide
oxygenase activity is induced. An intracellular,
multicomponent, soluble enzyme system is responsible for cyanide oxygenase activity [3]. The
overall stoichiometry of the process is consistent
with the enzyme system being a dioxygenase:
HCN
+ 0 2 ~
~t~,
CO 2 + N H 3
(1)
N A D ' .- H + N A D '
In a search for possible intermediates of this pathway a number of chemicals were tested. Low
amounts of cyanase activity were found in both
ammonia and cyanide limited fed-batch cultures.
This suggested that cyanide oxygenase could be a
monoxygenase:
H C N + 0 2 ~m°n°xygenase--....,,) H O C N + H~O.
NAD-4
(2)
H" NAD"
H O C N + H 2 0 cyanasc~co2 + N H 3
(3)
It is reported here that induction of cyanide
oxygenase by K C N or Ni(CN),~- does not induce
cyanase activity. Similarly induction of cyanase by
cyanate does not induce cyanide oxygenase activity.
0378-1097/89/$03.50 ~ 1989 Federation of European Microbiological Societies
290
3. M A T E R I A L S A N D M E T H O D S
3.1. Growth conditions
lnocula for all growth experiments were prepared as previously described [1], except 2 mM
N H a C I was used as the nitrogen source.
3.2. Growth of P. fluorescens on different nitrogen
sources in batch culture
All cultures were grown in 250-ml conical flasks
containing 100 ml medium with 10 mM glucose as
carbon source, M9 salts [4], 1 ml trace metals 1
[5], and either 2 mM NH4C1, 2 m M K O C N or 0.5
m M Ni(CN)4z- as the nitrogen source. All flasks
were incubated at 3 0 ° C in a gyratory shaker at
250 r e v / m i n .
3. 3. Fed-batch culture growth conditions
Cyanide ( K C N --- H C N ) limited fed batch cultures were set up as previously described [1].
3. 4. Harvesting bacteria
Bacteria from all cultures were harvested by
centrifugation and resuspension in 5") m M N a 2 H P O 4 / N a H z P O 4 p H 7.0 buffer as previously
described [2].
3.5. Assay of ~yanide oxygenase activity
Cyanide oxygenase activity was assayed by the
oxygen uptake method as previously described [2],
except that KCN was added to a final concentration of 200 # M in the oxygen electrode.
3.6. Assay of cyanase activity
Suspensions of bacteria were added to an assay
mixture in a magnetically stirred chamber with a
3 0 ° C water jacket to give a final A6oo of 0.2. The
assay mixture was as described by Anderson [6].
Cyanase activity was determined by measuring the
initial rate of ammonia formation from cyanate
hydrolysis in the assay mixture, after bacteria had
been removed by centrifugation.
3. 7. Assay of ammonia
A m m o n i a in the cell-free culture medium, and
in the cyanase assays was measured colorimetritally by the method of Fawcett and Scott [7].
3.8. Assay of cyanate
Cyanate in the cell-free culture medium was
determined using a modification of the spectrophotometric assay described by Guilloton and
Karst [8]. In their paper, they reported that alkaline or strongly buffered solutions interfered
with the assay. By lowering the pH of the culture
medium to 6.4, no interference of the cyanate
assay was observed. A m m o n i a formation due to
acid hydrolysis of cyanate was not observed at this
pH.
3.9. Assay of Nickel cyanide
Ni(CN) 2- in the cell-free culture medium was
assayed by the spectrophotometric assay as described by Rollinson et al. [2].
4. RESULTS
4.1. Growth of P. fluorescens in batch culture
As reported previously [1-3], P. fluorescens
grew in batch culture on 10 mM glucose with 2
mM NH4Ci or 0.5 mM N i ( C N ) ] - included in the
growth medium; growth on the latter compound
gave a somewhat lower growth yield. The
bacterium was also found to grow on 10 mM
glucose plus 2 m M K O C N , with a similar growth
yield to growth on NH4CI. In each case growth
terminated due to nitrogen depletion from the
growth medium.
Fig, 1. shows growth on K O C N as the source
of nitrogen. K O C N depletion from the medium
occurred prior to growth and was associated with
formation of ammonia, which was utilized during
growth. Cyanase activity was induced to a maxim u m of about 2.34 ~mol N H 3 f o r m e d / m i n / m g
dry wt cells, and was lost once the cyanate had
been depleted from the medium. Cyanide oxygenase activity was absent throughout the growth
cycle.
Fig. 2. shows growth on Ni(CN) 2 " as the source
of nitrogen. This compound was utilized throughout growth. Cyanide oxygenase activity was induced by N i ( C N ) ] - up to a maximum of about
38 nmol 0 2 c o n s u m e d / m i n / m g dr 5' wt cells. Activity was lost after depletion of Ni(CN)24 - from
the medium. Cyanase activity was not induced
( > 43 nmoi N H 3 f o r m e d / m i n / m g dry wt cells).
291
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(h)
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Fig. 1. The growth of P. fluorescens on cyanate as the source of nitrogen. Growth ( o ) , cyanate (ll), ammonia (zx) and cyanase activity
(A)
monia were detectable in the medium. To induce
cyanide oxygenase activity either 0.25 m M
N i ( C N ) ~ - or 1 mM K C N were when added to the
medium. It was found that Ni(CN)42- induced
higher specific activity of cyanide oxygenase than
KCN, although an equivalent concentration of
4.2. Induction of cyanide oxygenase and cyanase
actioities in stationary phase batch cultures
Cultures were grown for 24 h in batch culture
when 10 m M glucose and 2 m M NH4CI as the
sources of carbon and nitrogen, respectively. At
the end of this period neither glucose nor am-
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Fig. 2. The growth of P. fluorescens on Ni(CN)~- as the source of nitrogen. Growth (o), Ni(CN)~- (v) and cyanide oxygenase
activity (D).
292
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Fig. 3. Induction of cyanase activity in stationary phase culture. P. fluorescens was grown [or 24 h on glucose/N}la('l and 2 mM
KOCN then added. KOCN (1), growth to). ammonia (a), and cyanasc activity (,,).
cyanide had been added. The optimum concentration of N i ( C N ) 4
for induction of cyanide
oxygenase activity was found to be 0.5 mM. Activity was at its highest level five hours after the
addition of N i ( C N ) ] . N o induction of cyanase
occurred under these conditions. For induction of
cyanase activity, 2 m M K O C N was added to
parallel cultures grown for 24 h. (Fig. 3). N o
induction of cyanide oxygenase activity occurred
under these conditions. Once cyanate had been
lost from the medium, cyanase activity decreased.
4.3. Cyanide o x y g e n a s e a n d (yanase actit,ities in
K C N - l i m i t e d fed-batch culture
A 500 ml KCN-limited fed-batch culture of P.
f l u o r e s c e n s was set up, cyanide oxygenase and
cyanase activities were measured throughout
growth (Fig. 4).
Cyanide oxygenase was induced to a maximum
activity of 40 nmol O 2 c o n s u m e d / m i n / m g dry wt
cells at the mid-exponential phase of growth. Only
very low levels of cyanase were observed, which
showed no correlation with cyanide oxygenase
activity. Indeed, when cyanide oxygenase activity
was at its highest to cyanase activity was detected.
A variable low basal level of cyanase activity
was present in bacteria grown under such conditions. This activity was at its highest at the start of
growth, and after growth had terminated.
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Fig. 4. Cyanase and cyanide oxygenase activities m K C N l i m i t e d fed-hatch culture. Growth ( o ) , cyanide oxygenase a c t i v i t y (1) and cyanase activity O ' ) .
293
Pulsing mid exponential phase (A550 = 1.0)
KCN-limited fed-batch cultures with 1 m M K C N
resulted in rapid loss of cyanide and consequent
formation of a m m o n i a in the culture medium. N o
cyanate was detected prior to a m m o n i a formation
from cyanide.
5. D I S C U S S I O N
Cyanase and cyanide oxygenase activities were
never co-induced. Cyanide (either N I ( C N ) 2- or
K C N ) induced cyanide oxygenase activity, but not
cyanase activity. Conversely, cyanate induced
cyanase activity not cyanide oxygenase activity. It
is noteworthy that the induced activity of cyanase
was much higher than the induced cyanide
oxygenase activity.
The growth of a m m o n i a and cyanate containing batch cultures were strikingly similar, the major
difference being the extended lag period of K O C N
grown cultures. This extended lag period is probably due to the induction of cyanase activity to
provide a m m o n i a as the nitrogen source for
growth. During the exponential phase of growth,
both K O C N and N H a C I grown cultures utilised
a m m o n i a as the nitrogen source. The growth profile of cultures utilising N i ( C N ) 4 as nitrogen
source bears little similarity. N i ( C N ) 2- was
utilized during growth, with no formation of ammonia in the medium. Cyanide oxygenase activity
was low during the lag phase of growth, but
reaches a m a x i m u m at mid exponential phase. At
this phase of growth, cyanase activity in cyanate
grown cultures was at a basal level.
Induction of cyanide oxygenase and cyanase
activities in stationary phase cultures were also
mutually exclusive. Due to the difficulty in obtaining large quantities of cells induced for cyanide
oxygenase activity, such conditions for induction
may prove useful for purification purposes.
C y a n a s e has been extensively studied in
Escherichia coli [9-11]. It has been purified and
characterised fully [6,12-15], cloned [16] and
studied at a genetic level [17]. K u n z and Nagappan [18] using P. fluorescens N C 1 M B 11764 have
recently confirmed our earlier observation of the
presence of cyanase activity in this bacterium [3].
In this paper we have shown that a separate
cyanase enzyme is p r o b a b l y not involved in thc
utilisation of cyanide by P. fluorescens N C I M B
11764. This suggests that the p a t h w a y given in
equations (2) and (3) is p r o b a b l y not a route for
cyanide utilisation. However it is possible that
cyanatc is an intermediate of cyanide utilisation,
but is present as a b o u n d intermediate of the
cyanide oxygenase activity without release, thereby
not inducing a separate cyanase activity.
ACKNOWLEDGEM ENTS
We wish to thank Dr. Steve Bungard (I.C.I.
BioProducts) for his helpful discussion and encouragement. This work was supported by the
Science and Engineering Research Council and
Imperial Chemical Industries BioProducts Business via a C.A.S.E. award to P.K.D.
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