FEMS Microbiology Letters 20 (1983) 337-341
Published by Elsevier
337
The conversion of cyanide to ammonia by extracts of a strain of
Pseudomonas fluorescens that utilizes cyanide as a source of
nitrogen for growth
(Cyanide; Pseudomonas fluorescens )
R a l p h E. H a r r i s a n d C h r i s t o p h e r J. K n o w l e s
*
Biological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ. England, U.K.
Received and accepted 18 July 1983
(Summary not submitted)
tained 1 1 medium and the rate of aeration was 1
1/min.
1. I N T R O D U C T I O N
2.2. Preparation of extracts
We have recently isolated a strain of Pseudomonas fluorescens that is able to utilize cyanide as
a source of nitrogen for growth [1]. Bacteria grown
on cyanide were able to degrade it with the appearance of stoichiometric amounts of ammonia in
the medium. 1 mol of oxygen was consumed per
mol of cyanide converted, and cyanide degradation did not proceed under anaerobic conditions.
In this communication we report the requirements
for cyanide degradation by cell-free extracts of the
bacterium.
Bacteria were harvested in the mid-exponential
(.4600 of 0.9 tO 1.1) by centrifugation at
20000 × g for 5 rain at 4°C, washed twice in 50
mM N a 2 H P O 4 / N a H 2 P O 4 buffer (pH 7.0) and
resuspended in 6 to 10 ml of phosphate buffer.
They were disrupted by sonication (4 x 10 s at
0°C, MSE sonicator model 150 W). Intact bacteria
and debris were removed by centrifugation at
35 000 × g for 10 min at 4°C. The cell-free extract
was separated into particulate and supernatant
fractions by centrifugation at 150000 × g for 90
min at 4°C. The particulate fraction was resuspended in 50 mM phosphate buffer (pH 7.0)
phase
2. M E T H O D S
2.3. 02 uptake measurements
2.1. Growth conditions
P. fluorescens NCIMB 11764 was grown at 30°C
in cyanide-limited fed-batch culture as described
previously [1] except that the growth vessel con-
* To whom correspondence should be sent.
02 uptake was determined with an 02 electrode
(Rank Bros., Bottisham, Cambridge). The incubation mixture consisted of 3 or 4 ml phosphate
buffer (50 mM, p H 7.0) containing 0.4 to 2.0 mg
supernatant fraction p r o t e i n / m l at 30°C. N A D H
and KCN to give the concentrations indicated
were added by injecting small volumes (20 to 80
/~1) of freshly made stock solutions.
0378-1097/83/$03.00 © 1983 Federation of European Microbiological Societies
338
2.4. Oxidation of N A D H and N A D P H
These were measured by the decrease in A340
using 1 cm cuvettes. The reaction mixture at 30°C
consisted of 1 ml phosphate buffer (50 mM, p H
7.0) containing supernatant fraction (0.4 to 2.0 mg
protein). N A D H , N A D P H and K C N to give the
concentrations indicated were added in 20 ~1 of
stock solution. Oxidation under anaerobic conditions was measured using a 3 ml cuvette attached
to a Thunberg tube fitted with a side-arm to
permit addition of KCN. The tube was repeatedly
evacuated and flushed with O2-free N 2. In each
case the endogenous rate of N A D H or N A D P H
oxidation was determined before K C N was added
to the reaction mixture.
2.5. Measurement of CO 2 formation
Phosphate buffer (2 ml, 50 mM, p H 7.0) containing supernatant fraction (7 to 15 mg protein)
was added to the main compartment of a 50 ml
flask fitted with a 2 ml centre well. Kl4CN solution (20/.tl, 100 mM, 17 m C i / m m o l ) was added to
the reaction mixture to give an initial cyanide
concentration of 1 mM, and the flask was sealed
with a serum cap. The flask was maintained at
30°C and the reaction initiated by injection of 40
t~l 100 mM N A D H into the reaction mixture via
the serum cap to give an initial N A D H concentration of 2 mM. When degradation of cyanide was
complete, as determined by a simultaneous control
experiment, 1 ml 2 M N a O H was injected into the
centre well and the medium acidified by injection
of 1 ml 4 M HC1. The flask was incubated overnight (18 h) to ensure that all the CO 2 was trapped
in the N a O H solution. CO 2 was precipitated by
adding the contents of the centre well to 1 ml
saturated Ba(OH)2 solution followed by 100 /xl
100 m M Na2CO 3. BaCO 3 was collected on Whatm a n 3 MM filter paper and washed twice with 10
ml water. The filter was placed in 4 ml phosphate
buffer (50 mM, p H 7.0) in an apparatus similar to
that of Wang [2]. Air was passed through the
vessel at 50 m l / m i n . CO 2 released by injection of
1 ml 4 M HC1 was trapped over a 15-min period in
a mixture of 3.3 ml ethanol and 6.7 ml ethanolamine. Samples (1 ml) were counted in 4 ml phase
combining system scintillation fluid (Amersham
Corp., Arlington Heights, IL, U.S.A.) in a scintillation counter.
2.6. Analytical methods
Cyanide was assayed by the method of Lambert
et al. [3]. Ammonia was assayed colorimetrically
[4].
3. RESULTS
No degradation of cyanide was observed in
cell-free extracts of cyanide-grown bacteria unless
N A D H or N A D P H was added to the reaction
mixture. Extracts of bacteria grown with ammonia
as the source of nitrogen had no detectable cyanide
degrading activity in the presence of N A D H .
Cyanide degrading activity in extracts of
cyanide-grown bacteria assayed in the presence of
I m M K C N and 2 m M N A D H was located in the
supernatant fraction, the specific activity of which
varied with the protein concentration used (see
below). No activity was detected in the particulate
fraction.
Cyanide degrading activity of the supernatant
fraction was associated with stimulation of oxygen
uptake (Fig. 1), which continued until all the
cyanide was exhausted. The molar ratio of K C N
used to 02 uptake was 1:0.96 + 0.09 S.D. (14
determinations). Oxidation of N A D H by the supernatant fraction was stimulated by addition of
K C N (Fig. 2). The molar ratio of K C N used to
N A D H oxidised was 1 : 1.00 _+ 0.09(34). N A D P H
was oxidised at about 40% of the rate of N A D H
oxidation. Oxidation of N A D H in the presence of
cyanide did not occur under anaerobic conditions.
Indeed, the low endogenous rate of N A D H oxidation was almost abolished on addition of KCN.
An increased rate of N A D H oxidation in the
presence of K C N occurred on reintroduction of
air into the reaction mixture.
Colorimetric measurements of ammonia release
following complete degradation of K C N (0.1 to 1
mM) in the presence of excess N A D H (0.2 to 2
mM) gave a molar ratio of 0.86 _+ 0.06(17) : 1, suggesting that some of the ammonia was further
339
80--
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~1ooI-
60
1
c
J
50
5min
I
Fig. 1. Stimulation of 02 uptake by cyanide. The oxygen
electrode at 30°C contained 4 ml 50 m M Na2 H P O 4 / N a H 2 P O 4
buffer (pH 7.0) with 0.84 mg supernatant fraction protein/ml.
N A D H (80 p,1, 10 mM) was injected (1) and the endogenous
rate of oxygen uptake determined. KCN (20 ~1, 20 mM) was
then injected (2). The arrow (3) indicates the point at which
cyanide became depleted.
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0.8
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Fig. 3. Effect of protein concentration on specific and total
activity. Reaction mixtures (1 ml) containing different amounts
of supernatant fraction protein were preincubated for 5 min at
30°C with 20/~1 10 m M NADH. After determination of the
endogenous rate of N A D H oxidation 20/zl 5 mM KCN was
added. The rate of KCN-dependent N A D H oxidation was
measured, which was equivalent to the rate of KCN degradation.
O.e
m
0.4
0.2
1rain
I
I
Fig. 2. Stimulation of N A D H oxidation by cyanide. A 1 ml
cuvette containing 1.9 mg supematant fraction protein was
preincubated for 5 rain at 30°C with 20 ~1 10 mM NADH.
After determination of the endogenous rate of N A D H oxidation 20/~1 5 mM KCN was added (1). The arrow (2) indicates
the point at which cyanide became depleted.
metabolised. When N A D H oxidation due to KCN
addition was measured in the presence of bovine
glutamate dehydrogenase (EC 1.4.1.3.) (Sigma type
III, 2.0 m g / m l ) and a-oxoglutarate (1 mM) to
continuously trap the ammonia produced the initial rate of N A D H oxidation was increased to
1.96 _-4-0.29(5) times the rate observed in the absence of glutamate dehydrogenase, suggesting that
ammonia was appearing as soon as cyanide was
degraded and that there was no appreciable build-
340
up of an intermediate. Addition of a-oxoglutarate
alone had no effect on the rate of N A D H oxidation or the rate of cyanide degradation.
Recovery of 14CO2 from K14CN (1 mM in the
presence of 2 mM N A D H ) was somewhat variable
(67 _+ 15%, n = 6). This was probably due to technical problems involved in trapping small quantities of CO 2. With boiled extracts only 3.2 _+ 1.7%(4)
of the K14CN added was recovered in the CO 2
trap.
Specific activity of KCN degradation by the
supernatant fraction, measured by the rate of
oxidation of N A D H , increased with the protein
concentration (Fig. 3). This suggested that there
might be more than one enzyme involved. This
view was reinforced by fractionation of the supernatant fraction with ( N H 4 ) 2 S O 4 (Table 1). There
was little cyanide degrading activity in the 0-35%,
saturated 35-70% or the 70% supernatant fractions alone but combination of the 0 - 3 5 % and
35-70% fractions resulted in KCN-dependent
N A D H oxidation. Thus there appear to be at least
two proteins involved in conversion of KCN to
CO: and ammonia, plus the associated oxidation
of NADH.
SDS-polyacrylamide gel electrophoresis of the
supernatant fractions of cyanide and ammoniagrown bacteria indicated that an extra protein was
present in the cyanide-grown bacteria. This was
present primarily in the 0-35% (NH4)SO 4 fraction
where it comprised the major staining band and
had an M r value of about 15000 to 17000.
4. DISCUSSION
Several possible routes for conversion of cyanide
to ammonia have been suggested [5]. Perhaps the
simplest is via cyanide hydratase [6], formamidase
and formate dehydrogenase. This pathway also
results in release of CO 2 and requires an electron
acceptor such as N A D +. There is no direct requirement for oxygen in the process but this route
has the potential advantage that it could involve
energy conservation by reoxidation of the electron
acceptor via linkage to a membrane-bound respiratory system. Alternative routes include conversion of cyanide to fl-cyanoalanine or an a-amino
nitrile, followed by hydrolysis of the products to
release ammonia and an acid [see ref 5]. There is
no direct requirement for 0 2 or NAD(P)H for
these processes, nor is CO 2 produced.
The results obtained with extracts of P. fluorescens suggest that none of these pathways is
operative in this bacterium. Although there was
not complete recovery of CO 2, the stoichiometry
of cyanide degradation indicates that the overall
process is:
N A D ( P ) H + 2 H + + HCN + 0 2
--~ C O 2 --}-NH~-
Table 1
Requirements for recombination of the (NH4)2SO 4 fractions
to obtain cyanide degrading activity
Fractions A (0-35% (NH4)2SO 4 pellet) and B (35-70%
(NH4)2SO 4 pellet) were resuspended in 1 ml buffer and desalted on a 10 ml bed volume Sephadex G-25 column. The
protein concentrations of the desalted fractions were 3.2 and
6.6 m g / m l , respectively. 200 p,1 of each fraction as shown was
added to I ml final volume reaction mixture containing 20 #1
10 m M N A D H . Mixtures were preincubated for 5 min at 30°C
to determine the endogenous rate of N A D H oxidation, then 20
p,1 5 m M K C N was added to give KCN-dependent N A D H
oxidation.
Fraction added
Activity (nmol cyanide
degraded min 1 m l - 1)
A
B
A+B
2.4
1.0
16.0
+ NAD(P) +
At least one step in this process is inducible.
These results provide preliminary evidence for a
cyanide oxygenase system. This could either be a
dioxygenase as given in the above equation or a
monooxygenase involving formation of an intermediate. Multicomponent, NAD(P)H-requiring dioxygenases, classified as EC 1.14.12. have been
reported; for example, benzene dioxygenase [7,8]
and toluene dioxygenase [9]. If the cyanide degrading system is a monooxygenase the process could
involve conversion to cyanate:
HCN + O z + N A D ( P ) H + H +
HOCN + N A D + +
H20
Cyanase (EC 3.5.5.3.) could then degrade the
341
cyanate [10-13]:
HOCN + H20 ~ CO 2 + NH 3
C y a n a t e d e g r a d i n g activity was p r e s e n t in extracts
of b o t h a m m o n i a - a n d c y a n i d e - g r o w n b a c t e r i a
(R.E. Harris, u n p u b l i s h e d o b s e r v a t i o n s ) .
ACKNOWLEDGEMENTS
W e wish to t h a n k Dr. A. B e a r d s m o r e a n d Dr.
K. Powell (ICI, B i l l i n g h a m ) for h e l p f u l d i s c u s s i o n s
a n d e n c o u r a g e m e n t . T h i s w o r k was s u p p o r t e d b y
the Science a n d E n g i n e e r i n g R e s e a r c h C o u n c i l a n d
I m p e r i a l C h e m i c a l I n d u s t r i e s via a C.A.S.E. a w a r d
to R.H.
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