Content uploaded by Victor Luque-Almagro
Author content
All content in this area was uploaded by Victor Luque-Almagro
Content may be subject to copyright.
Abstract
A new flow injection (FI) method for photomet-
ric
monitoring of cyanate in bioremediation processes us-
ing immobilised native cyanase is described. The method
is based on the catalytic reaction between cyanate and bi-
carbonate to produce ammonia and carbon dioxide in the
presence of an inducible native cyanase, immobilised in a
reactor packed with glass beads. Two degrees of purifica-
tion of the biocatalyst were used – heated cell-free extract
and purified extract of cyanase from Pseudomonas pseudo
-
alcaligenes CECT 5344. The ammonia produced by the
enzymatic reaction is finally monitored photometrically at
700 nm using a modification of the conventional Berthelot
method. The method furnishes different calibration curves
depending on the degree of purification of the cyanase,
with linear ranges between 1.23 and 616.50 µmol L
–1
(r
2
=0.9979, n=7) and between 1.07 and 308.25 µmol L
–1
(r
2
= 0.9992, n=7) for the heated cell-free extract and the
purified cyanase extract, respectively. No statistically sig-
nificant differences between the samples were found in
the precision study evaluated at two cyanate concentration
levels using one-way analysis of variance. A sampling
fre-
quency of 15 h
–1
was achieved. The method was used to
mon-
itor
cyanate consumption in a cyanate bioremediation tank
inoculated with Pseudomonas pseudoalcaligenes CECT
5344 strain. The correlation between cyanate degradation
and ammonia production was tested using a conventional
method. Finally, the method was applied to different sam-
ples collected from the bioremediation tank using the stan-
dard
addition method; recoveries between 85.9 and 97.4%
were obtained.
Keywords Cyanate · Cyanase · Purification · Flow
injection · Immobilised enzyme
Introduction
Cyanate occurs naturally in tissues as a result of non-en-
zymatic decomposition of carbamyl phosphate [1] and in
the environment as a result of the dissociation of urea and
photo-oxidation of cyanide [2]. Cyanate is one of the main
by-products of cyanide oxidation in the gold-processing
industry; it is converted to ammonia and metal-ammonia
complexes which are toxic to aquatic life [3]. Cyanate is
widely used as a herbicide, to prevent physiological reac-
tions in mammals [4, 5], and as a uremic toxin [6, 7]. The
toxicity of cyanate probably arises from its reactivity with
nucleophilic groups in proteins [8], which competes with
the carbonate/bicarbonate equilibrium [9].
Despite the toxicity of cyanate many living organisms
which are able to tolerate it and even use it as a nitrogen
source, due to their ability to induce cyanase enzyme
(cyanate hydratase, EC 4.2.1.104)
1
which transforms cyanate
into carbamate [10]. Cyanase has been widely studied in
E. coli [11] and Pseudomonas spp [12], which are the bac-
teria most able to produce cyanase. In this work, a new
strain CECT 5344 Pseudomonas pseudoalcaligenes, has
been used to produce a cyanase enzyme which has two
favourable features – thermostability and enzymatic activ-
ity in basic medium [13].
Two optical methods, photometric [14] and fluorimet-
ric [15], based on a similar derivatisation reaction using
2-aminobenzoic acid, have been reported for the determi-
V. M. Luque-Almagro · R. Blasco ·
J. M. Fernández-Romero · M. D. Luque de Castro
Flow-injection spectrophotometric determination of cyanate
in bioremediation processes by use of immobilised inducible cyanase
Anal Bioanal Chem (2003) 377 :1071–1078
DOI 10.1007/s00216-003-2152-2
Received: 9 May 2003 / Revised: 2 July 2003 / Accepted: 2 July 2003 / Published online: 11 September 2003
ORIGINAL PAPER
J. M. Fernández-Romero · M. D. L. de Castro (✉)
Department of Analytical Chemistry, Annex Edifice Marie Curie,
Campus of Rabanales, University of Córdoba,
14071 Córdoba, Spain
e-mail: qa1lucam@uco.es
R. Blasco
Department of Biochemistry, Molecular Biology and Genetics,
Veterinary School, University of Extremadura,
10071 Cáceres, Spain
V. M. Luque-Almagro
Department of Biochemistry and Molecular Biology,
Edifice Severo Ochoa, Campus of Rabanales,
University of Córdoba, 14071 Córdoba, Spain
© Springer-Verlag 2003
1
The actual inducible enzyme cyanase (EC 4.2.1.104) was denoted in
1972 as EC 3.5.5.3; this was changed in 1990 and 2001 to EC 4.3.99.1
and EC. 4.2.1.104, respectively (data from the IUBMB 2002, see Ref.
[11]).
nation of cyanate. In both cases, the applicability was lim-
ited by particular features – the former method only occurs
at pH 4.4, making it not applicable to cyanide samples,
and the second is a tedious method which requires labour-
intensive sample preparation including chemical derivati-
sation, liquid–liquid extraction, and chromatographic sep-
aration. Cyanate has also been determined by liquid chro-
matography with photometric [16], fluorimetric [15], or
potentiometric [17] detection. However, the applicability
of such methods to complex samples (i.e. solutions from
the gold mineral and gold-manufacturing industry) is lim-
ited by the drawbacks during the chromatography step, in
particular column deterioration [15] or saline interference
[18, 19, 20, 21, 22, 23, 24, 25, 26]). Although automatic
flow systems for determination of ammonium based on
pH indicators [27, 28, 29], ammonia selective electrodes
[30, 31], or the Berthelot reaction [32, 33, 34, 35] have
long been established, no automated methods for the de-
termination of cyanate have yet been proposed.
This research reports both the purification of an in-
ducible cyanase from the strain Pseudomonas pseudoal-
caligenes CECT 5344 and the development of a new au-
tomated flow-injection method for the determination of
cyanate in bioremediation processes. Enzyme purification
can be based on either thermal treatment only, or on this
step plus protein separation by anion-exchange chroma-
to
graphy. The automated flow injection method is based
on permanent immobilisation of the biocatalyst on con-
trolled-pore glass (CPG) and photometric monitoring of
the ammonium formed using the Berthelot derivatisation
procedure [36, 37, 38, 39]. The reaction sequence occurs
according to the following steps:
1.
Analytical reaction mediated by the immobilised cyanate
hydratase, which converts cyanate into carbamate; the
latter being rapidly transformed into ammonium and
carbon dioxide:
2. Derivatisation by a modified catalysed Berthelot reac-
tion (indophenol blue derivative reaction) which oc-
curs in three steps, as described below: chlorination of
ammonium, formation of 2-carboxyquinonechlorim-
ine, and addition of salicylate to produce 2,2-dicar-
boxyquinonechlorimine.
Finally, the condensation product is monitored photo-
metrically at 700 nm.
Experimental
Instruments and apparatus
A Unicam 8700 series UV–visible spectrophotometer furnished with
an 18-µL 178.012-QS photometric Hellma flow-cell and equipped
with a Knauer recorder and a Selecta thermostat was used. A
Gilson Minipuls-3, four channel peristaltic pump with a rate selec-
tor, a Rheodyne 5041 injection valve and Teflon tubing of 0.5-mm
i.d. were also used for construction of the flow-injection manifold.
Electrophoresis was performed with a vertical slab mini-gel ap-
paratus (Model Hoefer SE 250 Mighty Small II, Amersham Bio-
sciences). A bio-compatible Pharmacia FPLC system equipped
with an LCC-501 Plus controller, two P500 pumps, a dual path
Optical Monitor UV, an IMV-7 Motor Valve, a chart recorder, and
an anion-exchange Mono Q HR 5/5 column from Amersham Bio-
sciences were used for purification of cyanase by anion-exchange
chromatographic separation.
Reagents
All reagents were of analytical grade and all solutions were pre-
pared in bidistilled water of high purity obtained from a Millipore
Milli-Q plus system. Other reagents used are described in the cor-
responding section.
Carrier solution
An aqueous solution containing 50 mmol L
–1
dipotassium monohy-
drogen phosphate (Panreac, Barcelona, Spain) and 3 mmol L
–1
so-
dium bicarbonate (Panreac), at pH 8, adjusted with 100 mmol L
–1
hydrochloric acid (Panreac).
Reagent 1
Sodium salicylate (Sigma–Aldrich, St Wuentin Fallavier, France;
200 mg) and sodium nitroprusside (Sigma; 372 mg) were dissolved
in 250 mL water.
Reagent 2
Sodium hypochlorite (Panreac; 10%, 3.95 mL) and sodium hy-
droxide (Panreac; 2 g) diluted to 250 mL with distilled water.
Enzyme immobilisation
3-(Aminopropyl)triethoxysilane (Aldrich, No. 11,339–5), glutaral-
dehyde (Merck, No. 820603), and controlled-pore glass (Sigma
No. PG240–200) were used.
Cyanase production
Organisms used and growth conditions
The strain used was isolated from sludge by selective cultivation in
media with cyanide as the sole nitrogen source. In addition to cyanide
this strain can use cyanate as the sole nitrogen source. It has been
classified as Pseudomonas pseudoalcaligenes by comparison of its
16S RNA gene sequence, and deposited in the ‘Colección Es-
pañola de Cultivos Tipo’ – CECT Spanish Type Culture Collec-
tion – assignation number 5344 [13].
Cells were grown in 250-mL conical flasks containing 100 mL
mineral medium with the following composition: 0.050 mol L
–1
so-
dium acetate as carbon source, 0.042 mol L
–1
sodium monohydrogen-
phoshate, 0.025 mol L
–1
sodium dihydrogen phosphate, 8.5 mmol L
–1
sodium chloride, and 5 mmol L
–1
potassium cyanate as nitrogen
source. The pH was adjusted to 9.5 with 10 molL
–1
sodium hy-
droxide. All flasks were incubated at 30 °C in a rotary shaker at
250 rpm. Cell growth was monitored photometrically at 600 nm –
monitoring turbidity which is proportional to the cell number.
Cells were harvested in the exponential phase by centrifugation at
15,000×g for 10 min at 4 °C. The resulting cell pellet was then
washed twice with 0.050 mol L
–1
phosphate buffer (pH 8).
()
&12 + +&2 + 1&22
+ 2 1+ &2
→
→
1072
Preparation of cell-free extracts
The washed cell pellet was suspended in 0.050 mol L
–1
phosphate
buffer (pH 8) and broken by passage through a chilled French pres-
sure cell at 1000 psig twice. The cell debris was removed by cen-
trifugation at 10,000×g for 20 min. The clear supernatant, denoted
cell-free extract, was used for protein purification, before immo-
bilisation.
Enzyme purification
A simple purification step was based on a thermal treatment for re-
moval of thermolabile proteins. A more exhaustive purification
was performed in two stages, namely thermal treatment and anion-
exchange chromatographic separation. The effectiveness of the
process was tested by monitoring both protein concentration and
the cyanase activity. Additional information from the purification
process was also obtained by SDS–PAGE.
Thermal treatment
The cell-free extract was heated at 70 °C for 15 min. After cooling
to 4 °C, the solution was centrifuged at 20,000×g for 15 min.
Chromatographic separation
Separation of the thermostable proteins was performed using an
FPLC system (Pharmacia) which incorporated an anion-exchange
Mono Q HR 5/5 column (Pharmacia). Prior to injecting the heated
extract the chromatography column was equilibrated with 50 mmol
L
–1
Tris-HCl (Sigma) buffer (pH 8). Fractions (1mL) of the whole
heated extract were injected into the chromatographic system. Af-
ter loading, the proteins retained on the column were sequentially
removed by saline elution provided by a gradient program which
mixed sodium chloride and 50 mmol L
–1
Tris-chloride buffer ac-
cording to the steps depicted in Table 1. The chromatographic pro-
cess occurred in 25 min at a flow-rate of 1.0mL min
–1
. The chro-
matographic eluate was sequentially collected in 1-mL fractions
with a sampling rate of 1.0 mLmin
–1
. The protein concentration
was tested in all fractions by monitoring the absorbance at 280 nm
and the cyanase activity by a conventional procedure [40]. Due to the
restricted column-loading capacity, the procedure was developed
several times using aliquots of the original heated sample. Finally,
all fractions containing cyanase activity were pooled and concen-
trated by ultrafiltration using an Amicon Centricon YM-100 (Mil-
lipore Iberica, No. 4211) centrifugal filter device.
Electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS–PAGE) was performed according to the Laemmli procedure
[41] using acrylamide (Biorad) of 5% (w/v) and 14% (w/v) for
stacking and separating gels, respectively. Proteins were stained
with Coomassie brilliant blue R250 (Sigma). The molecular weight
was estimated by using protein markers (Biorad).
Cyanase activity determination
Cyanase was determined by monitoring ammonia formation at 30 °C
as described by Anderson [40]. The standard assay mixture contained
sodium bicarbonate (3 mmol L
–1
), potassium cyanate (2 mmol L
–1
)
potassium phosphate buffer (50 mmol L
–1
, pH 7.6), and the enzyme
in a final volume of 2.0 mL. Following enzyme addition, 0.5-mL
aliquots were removed and mixed with equal volumes of Nessler
reagent (Merck), which had been diluted 1:3 with water. The
amount of ammonia formed was determined by monitoring the ab-
sorbance at 420 nm within 1–5 min after addition of the Nessler
reagent [42].
One unit of cyanase is defined as the amount of enzyme which
catalyses the formation of 1 µmol of ammonia per minute under
the assay conditions described above.
Protein determination
Protein concentration was determined using a modified version of
the Lowry method developed by F.K. Shakir et al. [43].
Enzyme immobilisation
Cyanase extracts obtained by one- or two-stage purification were
used for immobilisation. The extracts were immobilised separately
on activated controlled-pore glass using the Masoom and Town-
shend method [44, 45]. Teflon tubing (0.5–mm i.d.) of different
lengths packed with the support-cyanase conjugated was used to
build the immobilised enzyme reactors (IMER).
The efficiency of the immobilisation was tested by SDS–
PAGE. When not in use, the IMER was stored refrigerated at 4 °C
in phosphate buffer (50 mmol L
–1
, pH 7). Under these conditions
the enzyme activity remained above 95% for at least 6 months.
Manifold and procedure
Figure 1 depicts the dynamic system used. A sample was injected
into the buffer solution and passed through the immobilised
1073
Table 1 Saline concentration
gradient for the anion-ex-
change chromatography
Step Fraction Elution conditions Duration
number (min)
1 1–5 Isocratic (50 mmolL
–1
Tris-HCl buffer pH 8) 5
2 6 Gradient from 0 to 0.3 mol L
–1
NaCl 1
4 7–10 Isocratic (0.3 mol L
–1
NaCl) 4
5 11–20 Gradient from 0.3 to 0.6 mol L
–1
NaCl 10
6 21 Gradient from 0.6 to 1.0 mol L
–1
NaCl 1
7 22–25 Isocratic (1.0 mol L
–1
NaCl) 4
Fig. 1 Manifold used for cyanate determination. P denotes peri-
staltic pump; IV, injection valve; T, thermostat; L
1
and L
2
, reactors;
a and b, merging points; IMER, cyanase immobilised reactor; D,
detector; W
1
and W
2
, wastes; S, sample solution; B, buffer solution;
and R
1
and R
2
, reagent solutions
cyanase reactor where the enzymatic reaction took place. Then, the
ammonium generated merged at point a with the salicylate-nitro-
prusside mixture (R
1
) and with the hypochlorite-NaOH mixture
(R
2
). The reactor L
1
facilitated the mixture between sample and
reagent R
1
, meanwhile the Berthelot reaction took place in the L
2
reactor. Finally, the 2,2-dicarboxyindophenol product was driven
to the detector for photometric monitoring at 700 nm. As can be
seen in Fig. 1, the IMER and the L
1
and L
2
reactors were ther-
mostatted at 50 °C.
Results and discussion
Purification of cyanase from P. pseudoalcaligenes
CECT 5344
Cyanase from P. pseudoalcaligenes CECT 5344 was par-
tially purified from cells that had been grown in a
cyanate-containing medium as the sole nitrogen source.
The purification efficiency, expressed as specific activity,
the purification factor and the final activity are sum-
marised in Table 2. Thermal treatment of the cell-free ex-
tract precipitated 70% of the cell proteins. Analysis of the
supernatant showed 60% of cyanase activity was retained
in comparison with the untreated cell-free extract; this
was equivalent to a significant increase of the specific ac-
tivity (at least twofold).
The purification step involving anion-exchange chro-
matographic separation using saline gradient as the mo-
bile phase was very effective. Figure 2 shows the chro-
matogram (monitoring wavelength 280 nm), which in-
cludes the cyanase activity monitored in each fraction and
the saline gradient programs used. Fractions number 14
and 15 contained 72% of the loaded activity with a spe-
cific activity of 107.5 mU mg
–1
protein.
The protein content of the extracts was monitored by
SDS–PAGE (Fig. 3, which includes: (A) native cell-free
extract, (B) extract from single-stage purification, (C)
fractions 14+15 from the two-stage purification, and (D)
the supernatant after immobilisation). As can be seen, the
only enriched polypeptide band appears at a migration
distance corresponding to the molecular mass of 17 kD,
which coincides with the molecular mass for the cyanases
described so far, namely 16.35 kD from E. coli [46] and
17.9 kD from Methylobacterium thiocyanatum [47].
Stability of the immobilised cyanase
The efficiency of the immobilisation procedure was tested
by determining the enzymatic activity in the extract be-
fore and after immobilisation. The cyanase activity yield
from the immobilised enzyme reactor was estimated as
95% by comparing the total activity of the extract used
prior to immobilisation and in the supernatant from the
immobilisation reaction mixture. This datum was also in
agreement with the results obtained from the SDS–PAGE
of the extract (Fig. 3; lanes C and D).
1074
Table 2 Purification of the
cyanase from Pseudomonas
pseudoalcaligenes CECT 5344
Step Protein Total activity Specific activity Purification Remaining
(mg) (mU) (mU mg
–1
) factor activity (%)
Crude cell extract 16 100 6.25 1 100
Heat-treated extract 4.8 60 12.5 2 60
Fractions 14 and 15 from 0.4 43 107.5 17.2 43
chromatographic separation
Fig. 2 Elution profile of the heated cell-free extracts from the
CECT 5344 strain by anion-exchange chromatography with Mono-
Q
HR 5/5 column. Open circles denotes absorbance at 280 nm; filled
circles, cyanase activity, and dashed lines saline; the gradient pro-
gram was used (for more details see the text)
Fig. 3 SDS–PAGE analysis of the proteins selected during differ-
ent stages of purification of the crude extract from the P. pseudoal-
caligenes CECT 5344: lane A. crude cell-free extract; lane B. heat
treated extract; lane C. mixture of active fractions (fraction 14–15
from the chromatographic eluate) before enzyme immobilisation;
lane D. the same mixture as lane C after immobilisation. The mo-
lecular masses of the marked standards are on the left (expressed in
kD). The white arrow indicates the polypeptide which is denoted
as the enzyme cyanase. All lanes were loaded with 15 µg proteins
(other electrophoretic conditions in the text)
One of the most remarkable features of immobilised
enzymes is the effective lifetime. For this reason, the sta-
bility of different IMER constructed using cyanase ex-
tracts (namely: (a) heated cell-free extract, (b) concen-
trated heated cell-free extract, and (c) purified and con-
centrated extract) were assayed in the FI arrangement for
800 h. Figure 4 depicts the stability curves expressed as a
percentage of the initial specific activity. As can be seen,
only the immobilised reactor prepared using the concen-
trated and two-stage-purified extract exhibits a consider-
able decrease of the activity, which is established at
cyanase activity values down to 40% in a week. However,
the high specific activity of the two-stage purified extract
(107.5 U g
–1
) as compared with the one-stage purified ex-
tract (12.5 U g
–1
) makes the use of the former advisable in
routine analysis.
Optimisation of the flow-injection approach
The variables affecting the performance of the semiauto-
mated flow injection system were optimised by a univari-
ate procedure. Table 3 depicts all the variables with poten-
tial influence on the method, grouped into physical, chem-
ical and dynamic variables. The table also includes the
ranges over which they were studied and the optimum val-
ues
found.
Physical variables
Changes in temperature have two effects on the chemical
system, because this variable influences both the enzy-
matic and the chemical derivatisation reactions. The effect
of temperature was studied between 25 and 80 °C. Ac-
cording to the well known thermostability of this enzyme,
it remains unaltered in solution over 60 °C. However,
when this biocatalyst is immobilised its activity decreases
rapidly over 55 °C (Fig. 5). A temperature of 50 °C was
selected for the best performance of the overall system
(including the enzymatic and derivatisation reactions).
Chemical variables
The chemical variables were classified into those affect-
ing the enzymatic reaction and those others which influ-
ence the derivatising reaction. An aqueous solution with so-
dium dihydrogen phosphate concentration of 50 mmol L
–1
was selected as buffer for the cyanase reaction. This
buffer also contained 3 mmol L
–1
sodium bicarbonate as
carbon substrate. The influence of pH was studied be-
tween 7 and 9. A pH of 8 gave the best medium for de-
veloping the cyanase reaction and also provided an appro-
priate medium for maximum absorbance of the derivatisa-
tion product.
1075
Fig. 4 Study of the stability of the immobilised enzyme reactors
using different cyanase extracts: (circles) concentrated and heated
cell-free extract; (triangles) heated cell-free extract; (squares) con-
centrated purified extract
Table 3 Optimisation of the
experimental variables for the
FI method
Type Variable Range Optimum
studied value
Physical Temperature (°C) 25–80 50
Hydrodynamic Flow-rate (mL min
–1
) 0.3–1.6 0.5
Sample injection volume (µL) 100–500 200
L
1
reactor length (cm) 50–500 100
L
2
reactor length (cm) 50–500 250
Chemical Buffer
Sodium dihydrogen phosphate (mmol L
–1
) 10–200 50
Disodium hydrogen phosphate (mmol L
–1
) 1–40 3
pH 7–9 8
Reagent 1
Sodium salicylate (mmol L
–1
) 1–50 5
Sodium nitroprusside (mmol L
–1
) 1–25 5
Reagent 2
Sodium hypochlorite (mmol L
–1
) 1–50 25
Sodium hydroxide (mmol L
–1
) – 200
The Berthelot reaction is often used for derivatisation
because it is a sensitive and relatively selective reaction
for ammonium ions. As previously reported [39] the opti-
mum temperature for this reaction is 100 °C, and maxi-
mum colour development occurs with relative rapidity,
but it fades rapidly due to degradation of the coloured
product. The use of a catalyst, e.g. nitroprusside, im-
proves the kinetics of the reaction and allows working at
lower temperatures, at which the colour remains stable for
long periods. Monochloramine is initially formed from
the reaction between ammonium and hypochlorite. The
addition of sodium salicylate as coupling agent increases
the rate of the second step of the Berthelot reaction by dis-
placing the equilibrium by formation of 2-carboxybenzo-
quinonechlorimine [38, 39], and, in the presence of the
catalyst, the reaction progresses rapidly through the third
and final steps of the reaction sequence in which 2,2-di-
carboxybenzoquinonechlorimine is formed. This last step
is the rate-determining step and the ingredients should be
added in excess in order to convert it in a pseudo first-or-
der reaction. Under these premises, the influence of ingre-
dients concentration was studied by grouping them in two
mixtures denoted reagent 1 (sodium salicylate and sodium
nitroprusside) and reagent 2 (sodium hypochlorite and so-
dium hydroxide), because this resulted in the highest ana-
lytical signal – absorbance values. All were studied within
the ranges shown in Table 3. Two solutions containing
5 mmol L
–1
sodium salicylate and 5 mmol L
–1
sodium ni-
troprusside for the first reagent; and 25 mmol L
–1
sodium
hypochlorite and 200 mmol L
–1
sodium hydroxide for the
second reagent, were selected as optimum.
Dynamic variables
Study of the flow-rate showed that high flow-rates re-
duced the response of the FI system dramatically, because
of the short contact-times, and reduced the residence-
times for both the enzymatic and the derivatising reac-
tions. In contrast, low flow-rates provided good contact
and long residence-times; sampling frequency, however,
was low. A flow-rate of 0.5 mL min
–1
was chosen as a com-
promise
between contact-time and sampling frequency. In
order to facilitate mixing and development of the Berth-
elot sequence of reactions the length of L
1
and L
2
were
optimised. Finally, reactor lengths of 100 and 250 cm, re-
spectively, were chosen as optimal. The effect of the in-
jection volume was studied in the range of 100–500 µL,
and an injection volume of 200 µL was selected as a com-
promise between sensitivity and sample frequency.
Features of the method
The features of the method were established using IMER
reactors which were prepared using cyanase extracts from
both the one-stage and two-stage purification procedures.
Two calibration graphs were constructed using the opti-
mum values of the variables established in the previous
section. Standard solutions containing cyanate concentra-
tions between 0.1 and 800 µmol L
–1
were injected in trip-
licate into the FI manifold. Table 4 summarises the figures
of merit, which include the equation parameters for the
linear relationship, range studied, detection and quantifi-
cation limits, the covariance and the y/x estimate of stan-
dard deviation. As can be seen, the method shows differ-
ent linear ranges; between 1.23 and 616.50 µmol L
–1
(r
2
=
0.9979, n= 7) and between 1.07 and 308.25 µmol L
–1
(r
2
=
0.9992, n=7) for use of the one-stage and two-stage puri-
fied extracts, respectively. The detection limits, estimated
as three times the standard deviation of 30 measurements
of a blank solution, was 0.519 µmol L
–1
for the two-stage
purified extract and slightly higher for the one-stage puri-
fied extract. However, the sensitivity, expressed as slope
1076
Fig. 5 Effect of temperature on cyanase in solution (E), and on
immobilised cyanase (N)
Table 4 Features of the
method
a
denotes absorbance and X
cyanate concentration
(µmol L
–1
)
b
Calculated as 3
σ
blank signal
deviation
c
Calculated as 10
σ
blank signal
deviation
Features Cell-free extract Purified cyanase
Equation
a
Y=6.71(±4.8)+0.64(±0.02)XY=7.52(±2.9)+1.54(±0.02)X
r
2
(n=7) 0.9979 0.9992
Linear range (µmol L
–1
) 1.23–616.5 1.073–308.25
Detection limit (µmol L
–1
)
b
0.750 0.519
Quantitation limit (µmol L
–1
)
c
2.15 1.73
Covariance 119.04 61.78
σ
x/y
4.86 3.51
of the calibration graph, was ca. twice as high for the pu-
rified extract (1.54 L µmol
–1
cm
–1
) compared with that pro
-
vided by the heated cell-free extract (0.64 L µmol
–1
cm
–1
);
this was in agreement with the relative cyanase activity
exhibited by the different enzymatic extracts in the immo-
bilisation section. As can also be seen in Table 4, the
lin
ear relationship between signal and concentration, ex-
pressed in terms of covariance and the y/x estimate of
standard deviation, correlates better for the most purified
extract than for the one-stage purified extract.
In order to determine the precision of the method, both
the within-laboratory reproducibility and repeatability of
the FI system were estimated with an IMER constructed
using the most purified extract, at two concentration lev-
els of cyanate – 12.33 and 308.25 µmol L
–1
. The calcula-
tion was made by ANOVA. The ANOVA table (Table 5)
shows the sum of squares, the degrees of freedom and the
mean square between and within groups. For two concen-
trations of cyanate, the F-ratios were 2.11 and 0.76, and
the p-values of the F-test were 0.095 and 0.604 (for the
high and low concentrations, respectively). The p-values
of the F-test were greater than 0.05, so there were no sta-
tistically significant differences between any pair of
means at the 95% confidence level.
No interference from cyano-compounds was tested,
because of the selectivity of the biocatalyst as cyanate in-
ducible enzyme. Other potential interferents such as am-
monium derivatives were rapidly consumed as N
2
-source
and thus their potential effects on the Berthelot derivatisa-
tion reaction were avoided. As a previous step to the use
of the enzyme in bioremediation processes, the stability of
the enzyme in the presence of metal cations was checked
using exhausted electroplating liquids as samples. No de-
crease in the activity of the enzyme reactors was detected
after a working week.
The estimated sampling frequency under the optimum
working conditions was 15 h
–1
.
Application of the method to monitoring
a bioremediation process
The effectiveness of the flow-injection approach proposed
here was tested by applying it to the discrete determina-
tion of cyanate in a bioremediation process with the strain
Pseudomonas pseudoalcaligenes CECT 5344. The biore-
actor worked with exhausted electroplating liquids from
the jewellery industry. Figure 6 shows the bacterial growth
using sodium cyanate as the source of nitrogen (in cir-
cles), the cyanate consumption (in triangles) and the am-
monia production (in squares). Monitoring of both bacte-
rial growth and the analytical parameters was conducted
at the intervals shown in the figure. As can be seen, the
bacterial growth at the exponential phase coincides with
two remarkable situations – effective cyanate depletion
and transient accumulation of ammonia.
The analytical applicability of the method was vali-
dated by applying it to the determination of cyanate in the
1077
Table 5 Results from analysis
of variance
Level Source Sum of Df Mean F-Ratio P-Value
squares square
Low Between groups 15.2638 6 2.54396 0.76 0.6040
Within groups 93.1785 28 3,3278
Total (correlation) 108.442 34
High Between groups 118.415 6 19,7358 2.11 0.0948
Within groups 196.133 21 9.33965
Total (correlation) 314.548 27
Table 6 Application of the
method
a
Cyanate concentration accord-
ing Fig. 6
b
From 12.3 and 246.6 µmol L
–1
for additions 1 and 2, respec-
tively
No. Bioremediation Dilution [CNO
–
] [CNO
–
] found Recovery (%)
b
processes factor (mmol L
–1
)
a
(µmol L
–1
)
time (h) Addition 1 Addition 2
1 24.5 250 0.307 123.3 97.4 95.3
2 32.0 125 0.038 308.3 85.9 93.5
Fig. 6 Continuous monitoring of the cyanate bioremediation
process using CECT 5344 Pseudomonas pseudoalcaligenes at
pH
9.5 and photometric monitoring of bacterial growth at 600 nm
(filled circles), cyanate depletion (mmol L
–1
, filled triangles), and
ammonia production (mmol L
–1
, filled squares)
previous bioremediation process and by studying the re-
covery afforded by the proposed method after addition of
cyanate (12.3 and 246.6 µmol L
–1
). As shown in Table 6,
recoveries of 85.9 and 97.4% were obtained, which re-
vealed the absence of matrix effects.
The non-inhibition of the enzyme in the presence of
metal ions was also ratified in this experiment.
Conclusion
The features of the proposed enzymatic flow-injection
method make it a useful tool for the development of a new
biochemical system for monitoring cyanate in bioremedi-
ation processes. The combination of a flow-injection sys-
tem with immobilised cyanase enhances the selectivity
and sensitivity as compared with conventional methods.
The advantages of the method thus proposed are the low
detection limit, the simplicity, and the potential for au-
tomation in order to develop rapid analyses of a large
number of samples.
In addition, the purification procedure for cyanase is
very simple and cheap, especially when the enzyme is not
available commercially. Both degrees of purified extracts
can be satisfactorily used in an FI system.
One other remarkable aspect is that use of the strain
Pseudomonas pseudoalcaligenes CECT 5344 allows the
cyanase to remain active in a wide range of temperature
and pH.
Acknowledgement The Dirección General de Investigación
Científica y Técnica (DGICyT) of Spain and the EU are thanked
for financial support (project No. 95–0270-OP and IFD97–0653).
VMLA expresses his gratitude to the Dirección General de En-
señanza Superior e Investigación Científica (DGEIC) for a schol-
arship.
References
1. Allen CM, Jones ME (1984) Biochemistry 3:1238–1247
2. Hagel P, Gerding JJT, Fieggen W, Bloemendal H (1971)
Biochem Biophys Acta 243:366–373
3. Water Environmental Federation (1995) In: Standard methods
for the examination of water and wastewater, 19th edn. Ameri-
can Public Health Association, Washington, DC, 1995, pp 18–
35
4. Lisanti LE (1963) Ann Speriment Agaria 17:45–51
5. Koshishi I, Imanari T (1990) J Pharmacobio-Dyn 13:254–258
6. Kraus LM, Elberger AJ, Handorf CR, Pabst MJ, Kraus AP
(1994) J Lab Clin Med 123:882–891
7. Kraus AP, Florendo KN, Kraus LM (1995) J Am Soc Neph
6:582–588
8. Mun KC, Golper TA (2000) Blood Purif 18:13–17
9. Kozliak EI, Fuchs JA, Guilloton MB, Anderson PM (1995)
J Bacteriol 177:3213–3219
10. Moss GP (2002) Recommendations of the Nomenclature Com-
mittee of the International Union of Biochemistry and Molecu-
lar Biology on the Nomenclature and Classification of En-
zyme-Catalysed
Reactions.http://www.chem.qmul.ac.uk/iubmb/ enzyme/
11. Walsh MA, Otwinowski Z, Perrakis A, Anderson PM,
Joachimiak A (2000) Structure 8:505–514
12. Kunz DA, Nagappan O (1989) Appl Environ Microbiol
55:256–258
13. Spanish patent No. 200 100 989
14. Guilloton M, Karst F (1985) Anal Biochem 149:291–295
15. Lundquist P, Backmangullers B, Kagedal B, Nilsson L, Ros-
ling H (1993) Anal Biochem 211:23–27
16. Fagan P, Paull B, Haddad PR, Dunne R, Kamar H (1997)
J Chromatogr A 770:267–272
17. Isildak I, Asan A (1999) Talanta 48:967–978
18. Barkley DJ, Dahms TE, Villeneuve KN (1987) J Chromatogr
395:631–640
19. Thielemann H (1970) Pharmazie 25:271–272
20. McCord BR (1994) Anal Chim Acta 288:43–56
21. Mehra MC, Pelletier C (1990) Chromatographia 30:337–338
22. Nonomura M (1987) Anal Chem 59:2073–2076
23. Nonomura M (1987) Metal Finish 85:15–17
24. Nonomura M, Hobo T (1989) J Chromatogr 465:395–401
25. Skelly NE (1982) Anal Chem 54:712–715
26. Black SB, Schulz RS (1999) J Chromatogr A 855:267–272
27. Ruzicka J, Hansen EH (1981) Flow injection analysis. Wiley,
New York, p 207
28. Wilanson SW, Johnson KS (1986) Mar Biol 91:285–290
29. Oms MT, Cerda A, Cladera A, Cerdá V, Forteza R (1996) Anal
Chim Acta 318:251–260
30. 30 Bratov A, Abramova J, Muñoz J, Domínguez C, Alegret S,
Batroli J (1997) J Electrochem Soc 144:617–621
31. 31 Shen H, Cardwell TJ, Cattrall RW (1998) Anal Chim Acta
367:193–199
32. Schulze G, Liu CY, Brodowski M, Elsholz W, Möller FJ
(1988) Anal Chim Acta 214:121–136
33. Cerdá A, Oms MT, Forteza R, Cerdá V (1992) Anal Chim Acta
311:165–173
34. de la Torre M, Luque de Castro MD, Valcárcel M (1992) Ta-
lanta 39:869–873
35. Daridon A, Sequeira M, Pennarum-Thomas G, Dirac H, Krog,
JP, Gravesen P, Lichtenberg J, Diamond D, Verpoorte E, de
Rooij NF (2001) Sensors Actuators B 76:235–243
36. Berthelot M (1859) Répertorie Chimie Pure Appliquée 284
37. Weatherburg MW (1967) Anal Chem 39:971–978
38. Searle PL (1984) Analyst 109:549–568
39. Gordon SA, Fleck A, Bell J (1978) Ann Clin Biochem 15:
270–275
40. Anderson PM (1980) Biochemistry 19:2882–2888
41. Laemmli UK (1970) Nature 227:680–685
42. Morrison GR (1971) Anal Biochem 43:527–532
43. Shakir FK, Audilet D, Drake AJ, Shakir KM (1994) Anal
Biochem 216:232–233
44. Yaqoob M, Nabi A, Masoom M (1997) J Biolumin Chemilu-
min 12:1–5
45. Masoom M, Townshend A (1984) Anal Chim Acta 166:111–
118
46. Taussing A (1960) Biochem Biophys Acta 44:510–519
47. Wood A, Kelly D, McDonald I, Jordan S, Morgan T, Khan S,
Murrell J, Borodina E (1998) Arch Microbiol 169:148–158
48. Gumus G, Demirata B, Apak R (2000) Talanta 53:305–315
1078
