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Journal of Biotechnology 125 (2006) 319–327
Purification and characterization of a novel caffeine
oxidase from Alcaligenes species
B.R. Mohapatra ∗ , N. Harris, R. Nordin, A. Mazumder
Water and Watershed Research Program, Department of Biology, University of Victoria, Petch Building 116,
3800 Finnerty Road, Victoria, BC, Canada, V8P5C2
Received 13 November 2005; received in revised form 15 February 2006; accepted 13 March 2006
Abstract
Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The
optimal medium for caffeine oxidase production by this strain was (w/v) NaNO3 , 0.4%; KH2 PO4 , 0.15%; Na2 HPO4 , 0.05%;
FeCl3 ·6H2 O, 0.0005%; CaCl2 ·2H2 O, 0.001%; MgSO4 ·7H2 O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme
was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel
filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed
that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life
of 20 min at 50 ◦ C had maximal activity at pH 7.5 and 35 ◦ C. The purified caffeine oxidase had strict substrate specificity towards
caffeine (Km 8.94 ␮M and Vmax 47.62 U mg protein−1 ) and was not able to oxidize xanthine and hypoxanthine. The enzyme
activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium
arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca+2 , Mg+2 and Na+ ,
but was completely inhibited by Co+2 , Cu+2 and Mn+2 at 1 mM level. The novel caffeine oxidase isolated here from Alcaligenes
species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Alcaligenes sp.; Caffeine; Caffeine oxidase; Kinetics; Purification
1. Introduction
Sustaining the quality of freshwater for drinking,
recreation and irrigation has been emerging as a challenge due to unprecedented release of anthropogenic
∗ Corresponding author. Tel.: +1 250 472 4833;
fax: +1 250 721 7120.
E-mail address: [email protected] (B.R. Mohapatra).
0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2006.03.018
pollutants, such as discharge of domestic wastes, leaching from septic tanks, and improper management of
agro-industrial wastes (Vitousek et al., 1997). Appropriate counter measures against water pollution necessitate developing novel and efficient techniques to
detect, manage and mitigate these pollutants.
Caffeine (1,3,7-trimethylxanthine), a purine alkaloid occurs in more than 60 plant species including in
the seeds of coffee, cacao and cola tree and in the leaves
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B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327
of tea (Steffen, 2000). Caffeine is considered as one of
the major agro-industrial wastes generated from coffee and tea processing plants. It is also an important
constituent of a variety of beverages such as coffee,
tea, caffeinated cola and soda, and cocoa-derived food
products (chocolate, desserts and pastries). Coffee, tea,
cola and cacao contain about 100, 50, 40 and 10 mg of
caffeine per serving, respectively (Buerge et al., 2003).
Caffeine is also an important pharmaceutical ingredient. It accelerates the analgesic actions in anticold and antipyretic drugs. Caffeine is used as a cardiac, neurological and respiratory stimulant and as
a diuretic (Mazzafera, 2002). Considering its uptake
with beverages, foods, and medicines, caffeine is probably the most widely consumed drugs in the world
(Ogunseitan, 1996). It is added on the United States
Environmental Protection Agency list of high production volume of chemical (USEPA, 2002). The global
average consumption of caffeine is between 80 and
400 mg per person per day (Gokulakrishnan et al.,
2005). In humans, dietary caffeine has been partially
catabolized by hepatic cytochrome P450 1A2 (EC
1.14.14.1) through oxidative N-demethylation and/or
ring oxidation to produce paraxanthine, theophylline,
1,3,7-trimethyluric acid and other byproducts those are
excreted with unmetabolized caffeine in urine (Berthou
et al., 1992; Ogunseitan, 2002).
Because of its extensive use in food, beverages and
medicines, caffeine has been detected in surface water,
groundwater and wastewater effluents world wide
(Buerge et al., 2003; Weigel et al., 2004; Glassmeyer et
al., 2005). Recently, caffeine has also been proposed as
a potential chemical marker for tracking human fecal
waste input to source waters (Scott et al., 2002; Buerge
et al., 2003).
Decaffeination is being recommended for food,
beverages and wastewaters because of potential
chronic ingestion of caffeine and/or its byproduct 8chlorocaffeine (produced during chlorination) can have
adverse effects on the physiological systems (Gould
and Hay, 1982; White and Rasmussen, 1998). Decaffeination is usually performed by physico-chemical
treatments, such as water diffusion (Feldman and Katz,
1977), solvent extraction (Smith, 1999) and super critical carbon dioxide oxidation (Udayasankar et al.,
1986). However, these methods have high operational
costs and usually produce waste products that require
further treatment and/or disposal. In order to overcome
these problems, a specific decaffeination process is
necessary. The microbial bioprocessing is increasingly
being considered to be an efficient technique for the
caffeine abatement in environments (Gokulakrishnan
et al., 2005).
It has been reported that some bacteria belonging
to the genera Pseudomonas and Serratia, and fungi
belonging to the genera Aspergillus, Penicillium, Rhizopus and Stemphyllium have the ability to metabolize caffeine by enzymatic conversion (reviewed
by Mazzafera, 2002). However, the enzymatic pathways for caffeine metabolisms in microorganisms are
poorly understood (Madyastha and Sridhar, 1998).
The enzymatic catabolism of caffeine in microbes
is performed by N-demethylation and oxidation with
the enzymes demethylases and oxidases, respectively
(Gokulakrishnan et al., 2005). Caffeine oxidase, the
key enzyme responsible for direct oxidation of caffeine to 1,3,7-trimethyluric acid has been purified to
homogeneity only from mixed cultures of Klebsiella
and Rhodococcus species isolated from wastewaters
(Madyastha et al., 1999). Caffeine-oxidizing enzyme
can be useful in the treatment of caffeine in the agroindustrial wastes of coffee pulps and husks (Mazzafera,
2002), groundwaters and wastewaters (Ogunseitan,
1996), and also as a biosensing element similar to other
oxido-reductase enzymes (Eggins, 1996) for real-time
monitoring of caffeine in natural waters. In view of the
potential biotechnological application of caffeine oxidase, it is essential to study this enzyme from various
microbial sources.
This paper presents results on the purification and
partial biochemical characterization of a novel caffeine
oxidase from Alcaligenes species isolated from the surface waters.
2. Materials and methods
2.1. Bacteria and culture conditions
Caffeine oxidase-producing Alcaligenes species
CF8, was isolated from the surface waters of Round
lake, Smithers, British Columbia, Canada by spread
plate technique with caffeine supplemented mineral
medium (CMM) agar composed of (g l−1 ) NaNO3 ,
4.0; KH2 PO4 , 1.5; Na2 HPO4 , 0.5; FeCl3 ·6H2 O, 0.005;
CaCl2 ·2H2 O, 0.01; MgSO4 ·7H2 O, 0.2; glucose, 2.0;
B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327
caffeine, 0.5 and agar powder 15 in distilled water. The
isolated Alcaligenes sp. CF8 was routinely maintained
on CMM agar plates.
For production of caffeine oxidase, Alcaligenes
species CF8 was cultivated aerobically in 3-L batch
culture with CMM. Fermentation was carried out at
25 ◦ C for 72 h with agitation of 100 rpm on a rotary
shaker. The bacterial cells were harvested by centrifugation at 5500 × g for 30 min at 4 ◦ C. The resulting cell
pellets were resuspended in 0.2 M phosphate buffer (pH
7.5), and disrupted by sonication at 160 W for 20 min
with an ultrasonicator. The cell debris were removed
by centrifugation at 5500 × g for 30 min at 4 ◦ C. The
cell-free supernatant was designated as crude caffeine
oxidase.
2.2. Assay of caffeine oxidase
Caffeine oxidase activity was assayed by the
method of Madyastha et al. (1999) with some modifications. The standard assay mixture containing
0.5 ml of 0.2 M phosphate buffer (pH 7.5), 0.2 ml of
0.055 mM caffeine in the same buffer, 0.1 ml of 1.1 mM
2,6-dichlorophenolindophenol (DCIP) and 0.2 ml of
0.55 mM phenazine methosulfate (PMS). The reaction
was started by the addition of 0.1 ml of enzyme solution in a total volume of 1.1 ml, and the rate of decrease
in the absorbance at 600 nm was followed against a
blank in which 0.22 ␮m filtered distilled water was
added in place of enzyme. One unit (U) of caffeine oxidase activity was defined as the ␮mol of DCIP reduced
per minute per milliliter of enzyme solution. The caffeine oxidase was also assayed with molecular oxygen
(dissolved O2 ) as electron acceptor instead of PMS coupled DCIP by measuring the absorbance of the assay
mixture at 300 nm according to a spectrophotometric
method previously reported (Woolfolk, 1975). Protein
concentration was quantified by using the method of
Lowry et al. (1951) with bovine serum albumin as a
standard.
2.3. Culture conditions optimization
To study the effect of different carbon (fructose, dglucose, lactose, d-mannose, maltose, soluble starch
and sucrose), and nitrogen sources (ammonium chloride, ammonium sulfate, casein, sodium nitrate, sodium
nitrite, peptone, tryptone and yeast extract) on the caf-
321
feine oxidase production, 0.22 ␮m filter sterilized individual carbon and nitrogen compounds were added
to the 24 h old culture of Alcaligenes sp. CF8 growing in a 500 ml Erlenmeyer flask contained 100 ml of
CMM without glucose and sodium nitrate, respectively.
Incubation was carried out at 25 ◦ C with agitation of
100 rpm in an orbital shaker for another 48 h, and the
enzyme activity and protein concentration was determined as mentioned above.
2.4. Purification of caffeine oxidase
The crude caffeine oxidase was treated with 60%
(w/v) (NH4 )2 SO4 . The precipitate was collected by
centrifugation at 5500 × g for 30 min at 4 ◦ C. The precipitated enzyme was dissolved in 0.2 M phosphate
buffer (pH 7.5), followed by dialysis (10 kDa molecular
weight cut off) against the same buffer. The dialyzates
were passed through a DEAE (diethylaminoethyl)cellulose column pre-equilibrated with 0.2 M phosphate buffer (pH 7.5). The elution of enzyme was performed with a linear gradient from 0.1 to 1 M KCl. The
active enzyme fractions were pooled and further dialyzed with a 25 kDa dialysis membrane against 0.2 M
phosphate buffer (pH 7.5). The dialyzates were passed
through a Sephadex G-100 column previously equilibrated with 0.2 M phosphate buffer containing 0.15 M
NaCl (pH 7.5). Fractions of a milliliter were collected
per minute and estimated for protein concentration as
well as caffeine oxidase activity. The active caffeine
oxidase fractions were combined and dialyzed with
10 kDa cut off dialysis membrane against 0.2 M phosphate buffer (pH 7.5). The dialyzates were used for
further studies.
2.5. Characterization of caffeine oxidase
To evaluate the optimum temperature and pH for the
purified caffeine oxidase activity, the enzyme assay was
performed at different temperatures from 5 to 60 ◦ C
with 5 ◦ C increments and with buffers of different pH,
such as 0.2 M citrate (pH 3, 4 and 5), 0.2 M phosphate (pH 6.0, 7.0 and 7.5), 0.2 M Tris–HCl (pH 8),
0.2 M borate (pH 9–10) and 0.2 M borax–NaOH (pH
11–12). For the pH and thermal stability studies, the
purified caffeine oxidase solution was pre-incubated
for 2 h with the above-mentioned buffers of different
pH (3–12) and 30 min with above-mentioned temper-
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atures (from 5 to 60 ◦ C with 5 ◦ C increments), respectively and the residual enzyme activity was assayed
as mentioned above. The extinction coefficients of
DCIP at different pH were calculated from the data
of Armstrong (1964).
The effect of different metal ions and chemicals
were performed by pre-incubating the purified enzyme
solution with individual metal ions and chemicals for
15 min at 25 ◦ C. The residual caffeine oxidase activity
was estimated. To determine the effect of different analogues of caffeine on the caffeine oxidase activity, the
assay conditions were the same as mentioned above
except different caffeine analogues were used as the
substrate instead of caffeine. Separate blanks with individual metal ions, chemicals and caffeine analogues
were also prepared.
The kinetics parameters, such as Michaelis–Menten
constant (Km ) and maximum reaction velocity (Vmax )
values against caffeine as the substrate were calculated
using double reciprocal Lineweaver–Burk plot.
2.6. Molecular mass determination and purity
checking
The relative molecular mass and purity of the purified caffeine oxidase was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
(PAGE) by the methods of Laemmli (1970). Briefly,
the purified caffeine oxidase and low molecular weight
protein standard (Biorad Inc., US) were diluted with
1:4 and 1:20, respectively with Laemmli sample buffer
(1.6 ml glycerol, 0.4 ml ␤-mercaptoethanol, 1.6 ml
10% SDS, 1.0 ml 0.5 M Tris buffer pH 6.8, and 0.4 ml
0.5% bromophenol blue in a total volume of 8 ml). The
purified caffeine oxidase solution and protein standard
in Laemmli sample buffer were heated at 95 ◦ C for
4 min and loaded onto a 10% acrylamide gel. Electrophoresis was performed at 200 V constant voltages.
After electrophoresis, the gel was stained with BioSafetyTM Coomassie blue (Biorad Inc., US) for 1 h,
followed by destaining for 30 min in distilled water
and photographed.
3. Results and discussion
Table 1
Effect of different carbon source on caffeine oxidase production by
Alcaligenes sp. CF8
Carbon source (2 g l−1 )
Caffeine oxidase activitya
(U mg protein−1 )
Controlb
Soluble starch
Lactose
Maltose
Sucrose
Fructose
d-Glucose
d-Mannose
0.318 ± 0.029
0
0.113 ± 0.011
0.129 ± 0.008
0.156 ± 0.006
0.322 ± 0.014
0.613 ± 0.011
0.099 ± 0.006
a All the measurements were performed thrice and expressed as
mean ± standard deviation.
b Control culture was grown in the CMM without glucose.
eral medium supplemented with caffeine as the sole
source of carbon and/or nitrogen. To improve the
production of caffeine oxidase, the medium composition was optimized in shake flask. The maximum enzyme production was obtained at pH 7.5 and
25 ◦ C (data not shown). Various carbon sources were
added at a concentration of 2 g l−1 to the caffeine
supplemented cultivation medium (Table 1). Addition of glucose significantly increase the activity by
almost two-fold. A low level of enzyme secretion was
observed in the presence of disaccharides (lactose,
maltose and sucrose). The synthesis of caffeine oxidase was ceased with soluble starch. Among all the
nitrogen sources tested (4 g l−1 ) the caffeine oxidase
production ranged between 0.011 U mg protein−1 and
0.631 U mg protein−1 (Table 2). The optimum activity
Table 2
Effect of different nitrogen source on caffeine oxidase production by
Alcaligenes sp. CF8
Nitrogen source (4 g l−1 )
Caffeine oxidase activitya
(U mg protein−1 )
Controlb
Ammonium chloride
Ammonium sulfate
Casein
Sodium nitrate
Sodium nitrite
Peptone
Tryptone
Yeast extract
0.306
0.018
0.011
0.199
0.630
0.183
0.598
0.568
0.581
a
Alcaligenes species CF8 produced a significant
amount of caffeine oxidase when grown in the min-
±
±
±
±
±
±
±
±
±
0.029
0.006
0.002
0.013
0.037
0.023
0.016
0.012
0.029
All the measurements were performed thrice and expressed as
mean ± standard deviation.
b Control culture was grown in the CMM without sodium nitrate.
B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327
323
Table 3
A typical purification of caffeine oxidase isolated from Alcaligenes sp. CF8
Step
Total protein (mg)
Total activity (U)
Specific activity (U mg−1 )
Purification fold
Crude extract
60% (NH4 )2 SO4 (Dialysis Mr 12 kDa cut off)
DEAE-cellulose (Dialysis Mr 25 kDa cut off)
Sephadex G-100 (Dialysis Mr 12 kDa cut off)
3120
260
18.75
9.75
1965.6
985.4
580.88
388.34
0.630
3.79
30.98
39.83
1
6
49
63
(0.630 U mg protein−1 ) was achieved with the inorganic nitrogen compound sodium nitrate, followed
by the organic nitrogen compounds peptone, yeast
extract and tryptone. Enzyme production was substantially suppressed in the presence of ammonium sulfate and ammonium chloride. These results suggest
that the external carbon and nitrogen sources have
regulatory effect on caffeine oxidase biosynthesis by
Alcaligenes species. Furthermore, the enzyme synthesis is demonstrated to be modulated by induction and
repression mechanism. The potent inducers for caffeine
oxidase production were glucose and sodium nitrate.
The catabolite repressors for the enzyme synthesis
were starch and ammonium salts (ammonium sulfate
and ammonium chloride). Previous studies have also
demonstrated that the enzymatic pathways of caffeine
catabolism in bacteria are inducible. Glucose has been
found to be the effective inducers for mixed cultures of
Klebsiella and Rhodococcus species (Madyastha et al.,
1999), and fructose and tryptone for Pseudomonas sp.
No. 6 (Asano et al., 1993).
A typical purification of caffeine oxidase isolated
from Alcaligenes sp. CF8 is summarized in Table 3.
The crude extract obtained after centrifugation was
precipitated with 60% (w/v) ammonium sulfate. This
step yielded a six-fold purification step with a specific activity of 3.79 U mg protein−1 . This fraction was
chromatographed with DEAE-cellulose column and
caffeine oxidase activity was eluted between 0.4 and
0.6 M KCl. The purification fold was increased to
49-fold with specific activity of 30.98 U mg protein−1 .
Caffeine oxidase was purified further by gel filtration
chromatography with Sephadex G-100 column. The
major peak was eluted with 0.2 M phosphate buffer
(pH 7.5) containing 0.15 M NaCl. By this method,
the caffeine oxidase was purified 63-fold with a specific activity 39.83 U mg protein−1 . The homogeneity
of caffeine oxidase was checked with SDS-PAGE. The
relative molecular mass of the purified caffeine oxi-
dase was 65 kDa (Fig. 1). The enzyme also displayed a
molecular mass of 65 kDa when chromatographed with
Sephadex G-100 suggesting the enzyme is monomeric.
This molecular mass is found to be lower than the
Fig. 1. Negative image of electrophoretogram (SDS-PAGE) of purified caffeine oxidase from Alcaligenes sp. CF8 on 10% polyacrylamide gel. Lane A: purified caffeine oxidase. Lane B: crude caffeine oxidase. Lane C: low molecular weight markers: phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin
(45 kDa), carbonic anhydrase (31 kDa) and soybean trypsin inhibitor
(21.5 kDa).
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B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327
Fig. 2. Effect of pH on activity () and stability () of purified
caffeine oxidase from Alcaligenes sp. CF8.
value reported for the caffeine oxidase isolated from the
mixed cultures of Klebsiella and Rhodococcus species
(Madyastha et al., 1999) and xanthine oxidase isolated from human liver (Krenitsky et al., 1986) and
Arthrobacter species (Woolfolk and Downard, 1978).
The optimum pH for maximum caffeine oxidase
activity was found at pH 7.5 (Fig. 2). The pH optimum of purified caffeine oxidase isolated from mixed
consortium of Klebsiella and Rhodococcus species
(Madyastha et al., 1999) was pH 7.5, and xanthine
oxidase isolated from Pseudomonas putida L. was pH
7.0 (Yamaoka and Mazzafera, 1999). The enzyme lost
only 35% and 40% of the activity at pH 6 and 9,
respectively. The enzyme was stable (>80%) in the
pH range between pH 6 and 9 for at least 2 h incubation. Above pH 9, the stability sharply declined and
retained only 60% of its maximum activity at pH 10.
The effect of temperature on stability and activity of
purified caffeine oxidase is shown in Fig. 3. Caffeine
oxidase was quite stable at 35 ◦ C for almost 60 min
incubation. The enzyme activity rapidly decreased
above 40 ◦ C, and the enzyme revealed a half-life of
20 min at 50 ◦ C. The optimum temperature for maximum enzyme activity was found at 35 ◦ C. Nearly
complete inactivation of enzyme occurred at 55 ◦ C. At
45 and 50 ◦ C, the enzyme exhibited 70% and 46% of
its maximum enzyme activity, respectively. The activation and deactivation energies of the purified caffeine
oxidase estimated from an Arrhenius plot were 68.69
and 122.06 kJ mol−1 , respectively (Fig. 4). This optimum temperature was found to be higher than the value
reported for caffeine oxidase from mixed cultures of
Klebsiella and Rhodococcus species (Madyastha et al.,
Fig. 3. Effect of temperature on activity () and stability () of
purified caffeine oxidase from Alcaligenes sp. CF8.
1999) and xanthine oxidase isolated from Arthrobacter
species (Woolfolk and Downard, 1978) and P. putida
L. (Yamaoka and Mazzafera, 1999).
Caffeine oxidase of Alcaligenes sp. CF8 utilizes
molecular oxygen (O2 ) as electron acceptor for oxidation of caffeine with production of hydrogen peroxide in the assay mixture. Hydrogen peroxide was
determined spectrophotometerically by using the peroxidase method (Ngo and Lenhoff, 1980). Specific
activity (4.55 U mg protein−1 ) of caffeine oxidase with
O2 was eight times lower than the value of specific
activity (39.83 U mg protein−1 ) with DCIP as electron
acceptor. Molecular oxygen was also found to be a
poor electron acceptor compared to DCIP for caffeine oxidase isolated from Klebsiella and Rhodococcus species (Madyastha et al., 1999). The ultravioletvisible absorption spectrum of the purified caffeine
oxidase of Alcaligenes sp. CF8 showed maxima at 278,
Fig. 4. Arrhenius plot to estimate the activation and deactivation
energies of purified caffeine oxidase from Alcaligenes sp. CF8.
B.R. Mohapatra et al. / Journal of Biotechnology 125 (2006) 319–327
325
Table 4
Substrate specificity of purified caffeine oxidase from Alcaligenes
sp. CF8
Substrate (0.055 mM)
Relative activity (%)
Caffeine
Xanthine
Hypoxanthine
Theophylline
Theobromine
Ethyltheobromine
100
1.26
3.89
16.76
10.23
19.66
365, 450, 480 and 550 nm. The absorption spectrum of
the enzyme was similar to that of xanthine oxidase from
Arthrobacter sp. (Woolfolk and Downard, 1978).
The relative oxidizing efficiencies of purified caffeine oxidase of Alcaligenes sp. CF8 were compared
by measuring the reduction of DCIP with various substrates of caffeine analogues (Table 4). The enzyme
showed strict specificity to caffeine. The enzyme did
not catalyze xanthine and hypoxanthine. The DCIP
reduction was only 10%, 16% and 19% in the presence
of theobromine, theophylline and ethyltheobromine,
respectively. Similarly the caffeine oxidase isolated
from Klebsiella and Rhodococcus species did not show
specificity towards xanthine and theophylline, however, the enzyme had an affinity towards theobromine
and its derivatives ethyltheobromine (Madyastha et al.,
1999).
Kinetics parameters like the Michaelis–Menten
constant (Km ) and maximum reaction velocity
(Vmax ) were determined for purified caffeine oxidase with respect to caffeine and PMS at 35 ◦ C
by Lineweaver–Burk plots. Under the optimal conditions (35 ◦ C, pH 7.5) the enzyme exhibited
Michaelis–Menten type kinetics. The kinetic constant
(Km ) measured for caffeine was 8.94 ␮M and Vmax
was 47.62 U mg protein−1 (Fig. 5). The Km value for
phenazine methosulfate (PMS) was estimated to be
37.42 ␮M (Fig. 6). The Km value with caffeine as substrate was lower than the value (11.4 ␮M) reported
for the mixed cultures of Klebsiella and Rhodococcus
species at pH 7.8 and 30 ◦ C (Madyastha et al., 1999).
The effect of metal ions and chemicals on the purified caffeine oxidase is shown in Table 5. The enzyme
was not affected by the metal ions Ca+2 , Mg+2 and
Na+ at 1 mM, however, the enzyme was completely
inhibited by Co+2 , Cu+2 and Mn+2 . The metal chelating agents, EDTA and o-phenanthroline also inhibited
Fig. 5. Lineweaver–Burk plot of purified caffeine oxidase activity
against caffeine concentration in the presence of 0.55 mM PMS. S:
caffeine concentration; V: caffeine oxidase specific activity. Error
bars are standard deviation of triplicate measurements. Error bars
are shown when they exceed dimension of the symbol.
the enzyme activity at 1 mM concentration, indicating
the caffeine oxidase is a metalloenzyme, and requires
metal ions for its activity. Diphenyliodonium chloride
inhibited the caffeine oxidase activity, suggesting the
enzyme to be a flavoprotein. Further inhibition of caffeine oxidase by phenylmethylsulfonyl fluoride, pointing towards the participation of serine at the active site
of the enzyme. The non-inhibition of enzyme activity
by iodoacetamide and para-chloromercuribenzoic acid
precluding the involvement of sulfhydryl (–SH) group
at the active sites. The caffeine oxidase activity was
Fig. 6. Lineweaver–Burk plot of purified caffeine oxidase activity
against PMS concentration in the presence of 0.055 mM caffeine.
S: PMS concentration; V: caffeine oxidase specific activity. Error
bars are standard deviation of triplicate measurements. Error bars
are shown when they exceed dimension of the symbol.
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Table 5
Effect of different metal ions and chemical agents on purified caffeine
oxidase of Alcaligenes sp. CF8
Metal ions and chemicals
Ca+2
Mg+2
Na+
Co+2
Cu+2
Mn+2
EDTA
PCMB
Iodoacetamide
O-phenanthroline
DPI
PMSF
Salicylic acid
Sodium arsenite
N-methylmaleimide
Percent activity retained
0.1 mM
1 mM
100
100
100
2
15
12
8
100
98
11
3
18
97
96
94
100
100
100
0
3
0
0
93
96
3
0
1
95
92
91
PCMB: para-chloromercuribenzoic acid; PMSF: phenylmethylsulfonyl fluoride; DPI: diphenyliodonium chloride.
not inhibited by the xanthine oxidase inhibitors such as
n-methylmaleimide, salicylic acid and sodium arsenite
indicating the enzyme is not belonging to xanthine oxidase family (Woolfolk and Downard, 1978).
The novel caffeine oxidase isolated from the Alcaligenes species CF8 is thermostable, salt-tolerant and
has broad pH optima for its activity. Further it has
a lower Km value, and has strict substrate specificity
towards caffeine. These results suggest the superior
quality of this enzyme for its potential biotechnological utilization including waste treatment and biosensor
development. Future studies should be undertaken to
elucidate the structure of caffeine oxidase as well as
immobilization of caffeine oxidase on various matrices to increase its biocatalytic efficacy.
Acknowledgements
This research work was supported by the Natural
Sciences and Engineering Research Council of Canada
Industrial Research Chair Program in Environmental Management of Drinking Water, and the Canadian
Institute of Health Research Grant under Safe Food and
Water Initiative to AM. We also acknowledge the support from British Columbia Ministry of Environment
for collection of water samples.
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