CURRENT MICROBIOLOGY Vol. 55 (2007), pp.

56–60
DOI: 10.1007/s00284-006-0588-2 Current
Microbiology
An International Journal
ª Springer Science+Business Media, LLC 2007

Degradation Kinetics of Caffeine and Related Methylxanthines by

Induced Cells of Pseudomonas sp.
Swati Sucharita Dash, Sathyanarayana N. Gummadi
Department of Biotechnology, Indian Institute of Technology-Madras, Chennai, 600 036, India

Received: 18 November 2006 / Accepted: 23 January 2007

Abstract. In this study, the kinetics of degradation of caffeine and related methylxanthines by induced
cells of Pseudomonas sp. was performed. The kinetics data showed that degradation of caffeine,
theobromine, and 7-methylxanthine followed Michealis–Menten kinetics. The values of Km are low for
caffeine and 7-methylxanthine and high for theobromine. Degradation of caffeine and theobromine was
enhanced in the presence of NADH and NADPH, whereas the degradation of 7-methylxanthine was
unaffected. Among the various metal ions tested, Fe2+ was found to enhance the rate of degradation for
all three substrates, whereas Zn2+ and Cu2+ inhibited the degradation of caffeine and theobromine but
not 7-methylxanthine. The differences in kinetic parameters and cofactor requirement suggest the
possibility of the involvement of more than one N-demethylases in the caffeine catabolic pathway in
Pseudomonas sp. The induced cells can serve as effective biocatalysts for the development of biode-
caffeination techniques.

Decaffeination is a necessary step in coffee processing
to reduce the caffeine content in food products and also
for the treatment of caffeine-containing effluents that are
toxic to the environment or for rendering coffee pulp
and husk for other uses [5, 13, 15]. In this aspect,
microbial degradation of caffeine (1,3,7-trimethylxan-
thine) and related methylxanthines has been the focus of
research in the recent past because of the major advan-
tages that it has over conventional techniques of deca-
ffeination [8]. Several microbial species have been
studied in this regard, and of them, bacterial strains
belonging to the genus Pseudomonas have been found to
be the most effective caffeine-degrading agents. One
such Pseudomonas strain isolated from the soil of a
coffee plantation area [9] and identified by 16S rRNA
analysis has been shown to degrade higher concentra-
tions of caffeine (15 g/L) by N-demethylation at a rate
higher than what has been reported for any strain so far
[6].
Although the growth kinetics of this strain has been
studied [10], there are no reports on the degradation
kinetics of caffeine or the related methylxanthine. In this
study, the kinetics of the degradation of caffeine and the
related methylxanthines have been studied along with
the effect of various cofactors on the rate of degradation.
The results obtained will be beneficial for the develop-
ment of an economical bioprocess for caffeine removal
and also provide an insight on the nature of caffeine
demethylases.
Materials and Methods
Chemicals. Pure caffeine (1,3,7 trimethylxanthine) was obtained from
Merck; 3,7-dimethylxanthine, 7-methylxanthine, and xanthine were
obtained from Sigma. All other reagents were of analytical grade.
Bacterial Strain. Pseudomonas sp. previously isolated in our
laboratory from the soil of a coffee plantation area was maintained
on nutrient agar medium at 4°C and was subcultured every 2 weeks.
The strain was deposited at the National Collection of Industrial
Microorganisms, Pune, India with the accession number NCIM 5235.

Media. For solid medium, the composition of CAS (caffeine and

Correspondence to: Sathyanarayana N. Gummadi; email: gummadi@ sucrose) medium was as follows (g/L): Na2HPO4, 0.12; KH2PO4, 1.3;
itm.ac.in CaCl2, 0.3; MgSO4 Æ 7H2O, 0.3; sucrose, 5; caffeine, 1.2; agar, 25. The
S.S. Dash and S.N. Gummadi: Microbial Degradation of Caffeine
initial pH of the medium was 6.0. For preparation of induced cells,
optimized CAS medium with the following composition was used (g/
L): Na2HPO4, 0.352; KH2PO4, 3.4; CaCl2, 0.3; MgSO4 Æ 7H2O, 0.3;
sucrose, 5; caffeine, 6.4; 0.075 % (w/v) Fe2+. The pH of the medium
was adjusted to 7.8 with 1 N NaOH before sterilization.
Preparation of Induced Cells of Pseudomonas sp. . Induced cells
were prepared as described earlier [11]. Briefly, three loopfuls of 36-h
grown culture from the CAS plate was transferred aseptically to 25 mL
nutrient broth medium and incubated to mid-log phase (OD600 nm1.4)
for 2.5 h at 180 rpm at 30°C. Twenty-five milliliters of sterile
optimized CAS medium was inoculated with 6% (v/v) of the culture
and incubated at 28°C and 190 rpm for 30 h. At 30 h, when caffeine
degradation was 90%, the bacterial cells were harvested by
centrifugation at 8000g for 15 min at 4°C and cells were washed
with 10 mM potassium phosphate buffer (pH 7.0). The cells could be
stored at )20°C for a maximum of 20 h, beyond which the cells lost the
caffeine degrading ability.
Degradation Assay. The standard reaction mixture consisted of 1 mM
caffeine and 0.18 mg/mL of induced cells as the biocatalyst in 10 mM
potassium phosphate buffer (pH 8.0). Degradation assays were
performed in 5-mL glass vials that were set in gentle motion on a
gel rocker at 30°C for proper mixing. After 10 min, the cells were
separated by centrifugation and the supernatant was assayed for
caffeine content by reversed-phase high-performance liquid
chromatography (HPLC). The substrate concentrations and rates of
degradation obtained were fitted to a Michaelis–Menten model and
kinetic parameters (i.e. Km and Vmax values were determined from the
curve by nonlinear regression analysis with a confidence level >95%
using GraphPad PRISMÒ Version 3 software).
The effect of cofactors was studied by adding NADH and
NADPH to the reaction mixture at a concentration of 1 mM and per-
forming the assay as described earlier. In order to study the effect of
metal ions, Fe2+, Ca2+, Mg2+, Mn2+, Co2+, Cu2+, Zn2+, and Ni2+ were
added to the reaction mixture in the form of salts (viz. FeSO4 Æ 7H2O,
CaCl2 Æ 2H2O, MgSO4 Æ 7H2O, MnSO4 Æ H2O, CoN2O6 Æ 6H2O, CuSO4 Æ
5 H2O, ZnSO4 Æ H2O, NiSO4 Æ 6H2O) at a concentration of 1 mM and
the assay was performed as described earlier. In all of these experi-
ments, the initial concentrations of caffeine, theobromine, and 7-
methylxanthines were 5, 2.5, and 3 mM, respectively.
Analytical Determination. Caffeine, 3,7 dimethylxanthine, and 7
methylxanthine were estimated by HPLC (Agilent 1100 series)
equipment using a ZORBAX C-18 column with 10 mM ammonium
phosphate buffer (pH 2.5)/acetonitrile (4:1, v/v) as the mobile phase.
Pure caffeine, 3,7-dimethylxanthine, and 7-methylxanthine (Merck) at
2 g/L were used as standards. The retention time of caffeine, 3,7-
dimethylxanthine, and 7-methylxanthine was found to be 4.9, 3.1, and
2.8 minute, respectively, at a flow rate of 1 mL/min and at 28°C.
Detection of caffeine and its metabolites was done at 254 nm using an
ultraviolet (UV) detector.
Statistical Analysis. All of the experiments were performed in
duplicate and data are the mean of four independent measurements
with significance P < 0.05.
Results and Discussion
The degradation rates were calculated over a pH range
of 5.0–9.0, temperature range of 25–40°C, and cell dry
weight range of 0.036–1.96 g/L. Preliminary results
showed that optimal pH, temperature, and amount of
57
biocatalyst (induced cells) were found to be 8.0, 30°C,
and 0.18 mg/mL, respectively. Under these optimal
conditions, the rate of degradation of caffeine was esti-
mated to be 0.47 mmol caffeine/g cell dry weight/min
and this rate is sufficiently higher than what has been
reported so far [4]. It was also reported that optimal
conditions obtained in this study was similar to other
reports on Pseudomonas strains (temperature: 30–35°C;
pH: 7.0–9.0) [7, 11, 14, 16]. In order to use this strain as
an effective biocatalyst for degrading caffeine and its
related methylxanthines, which are initial metabolites of
the caffeine degradation pathway, kinetics of degrada-
tion of caffeine and other related methylxanthines is
necessary.
Under these optimal conditions, the degradation
kinetics of caffeine and related methylxanthines (theo-
bromine and 7-methylxanthines) was studied at various
initial substrate concentrations. The plot of initial rate of
degradation versus substrate concentration followed
Michaelis–Menten kinetics (Fig. 1). Such an approach
has been used to study the biodegradation of phenol by
immobilized cells of Pseudomonas putida [3]. The
intrinsic kinetic parameters such as Km and Vmax were
determined when caffeine, theobromine, and 7-meth-
ylxanthins were used as substrates using linear regression
fit (Table 1). As seen from Table 1, the values of Km and
Vmax were different for the different substrates used. The
induced cells had lower substrate specificity for theo-
bromine compared to that for caffeine or 7-methylxan-
thine. However, these results clearly establish that the
induced cells are not only able to use caffeine as the
substrate but other methylxanthines as well. HPLC
analysis of the reaction mixture after 10 min of reaction
showed theobromine, 7-methylxanthine, and xanthine as
products suggesting that the caffeine is degraded by the
demethylation route (data not shown), similar to the
pathway observed in growing cells of the same strain [6]
(Fig. 2a). Similarly, only 7-methylxanthine and xanthine
was observed as products when theobromine was used as
the substrate (Fig. 2b) and only xanthine as the product
when 7-methylxanthine was the substrate (Fig. 2c) (data
not shown). These results clearly suggested N-demeth-
ylases are involved in the degradation of caffeine and its
related xanthines. Assays performed with dead cells of
Pseudomonas sp. in a similar manner showed no de-
crease in concentration of caffeine and other methylx-
anthines, indicating that the decrease in substrate
concentration was solely due to degradation and not
absorption of substrates on the bacterial cells (data not
shown). It has been suggested by various researchers that
more than one enzyme is involved in caffeine demethy-
lation to xanthine and their absolute requirement of co-
factors such as NADH or NADPH [1, 7, 12].
58 CURRENT MICROBIOLOGY Vol. 55 (2007)

A 0.3 Table 1. Kinetic parameters of degradation of caffeine and its

metabolites by induced cells of Pseudomonas sp.
rate (µ mol/min)
Substrate Vmax (lmol/min) Km (mM) R2
0.2
Caffeine 0.345 € 0.02 2.6 € 0.3 0.95
(1,3,7-trimethylxanthine)
Theobromine 0.773 € 0.1 7.4 € 2 0.986
0.1
(3,7- dimethylxanthine)
7-Methylxanthine 0.38 € 0.05 2.4 € 0.66 0.97

0.0
0.0 2.5 5.0 7.5 10.0
caffeine concentration (mM)

B 0.4 requirement differed for different substrates (Fig.3). For

caffeine, the degradation rate was enhanced from 0.3 to
0.36 lmol/min and 0.39 lmol/min with the addition of
r at e (µ mol/min)

0.3
NADH and NADPH, respectively, which was 1.2 and
1.3 times the original rate, respectively, indicating that
0.2 NADPH is a better cofactor for caffeine-degrading en-
zymes. In case of theobromine, NADH and NADPH had
0.1 the same effect by increasing the degradation rate from
0.11 to 0.17 lmol/min rate, which was1.5 times greater
0.0 compared to the control. However, the addition of co-
0 1 2 3 4 5 6 factors had no marked effect on the degradation rate of
theobromine (mM) 7-methylxanthine. This difference in cofactor require-
ment possibly indicates the diverse nature of the en-
C 0.3
zymes involved.
To confirm further, the effect of metal ions on the
rate (µ mol/min)

degradation rate of caffeine, theobromine, and 7-meth-

0.2
0.1
0.0
0 1 2 3
7-methylxanthine concentration(mM)
Fig. 1. Kinetics of degradation of (a) caffeine, (b) theobromine (3, 7-
dimethylxanthine), and (c) 7-methylxanthine by induced cells of
Pseudomonas sp. Experiments were performed at various concentra-
tions of the substrates using 0.18 g/L dry weight of induced cells at
30°C and pH 8.0. Data showed that the degradation followed Micha-
elis–Menten kinetics. All experiments were performed in duplicate
under identical conditions and data points are mean € standard devi-
ation of four independent measurements with a significance of
P < 0.05.
In order to further establish whether catabolism is
due to a single enzyme or multiple enzymes, the effect
of NADH and NADPH on the degradation rate was
tested. It should be noted that NADH and NADPH for
these enzymes are already provided by the cell. How-
ever, the effect of extraneous addition of these cofactors
was tested in this study. Results showed that the cofactor
ylxanthine was studied. It was found that the degrada-
tion rate of caffeine was enhanced in the presence of
Fe2+, Ca2+, and Mg2+, not affected by Co2+, Mn2+, and
Ni2+, and drastically inhibited by Cu2+ and Zn2+
(Fig. 4a). This pattern was somewhat different for
theobromine, for which the degradation rate was greatly
4 5 enhanced in the presence of Mn2+ and Fe2+, unaffected
by Ca2+, and strongly inhibited by all other metal ions
(Fig. 4b) Similarly, the effect of metal ions on the
degradation rate of 7-methyl xanthine was different
when compared to that of caffeine and theobromine
(Fig. 4c). In a previous report, it had been shown that
caffeine degradation was enhanced by Co2+ [12].
However in this case, the rate was not influenced by the
presence of Co2+, indicating variation in the strain. The
conversion of caffeine to theobromine by Pseudomonas
was increased by Zn2+ [2]. The results obtained in this
study will be beneficial for the development of process
in which the selective degradation of caffeine and its
related xanthines or production of any related xanthine
from caffeine is the goal.
In conclusion, the induced cells of Pseudomonas sp.
are capable of degrading caffeine and related methylx-
anthines and hence can be used as effective biocatalysts
for the decaffeination of caffeine-containing effluents
S.S. Dash and S.N. Gummadi: Microbial Degradation of Caffeine 59

O
O O
O
A CH 3 CH 3 CH3 H
H 3C N N
N N HN HN
N HN

N N O N N
O N N O N O HN
H
H 3C
CH 3
Theobromine 7-methylxanthine Xanthine
Caf f eine
(Substrate) (Products)
O
O
B O
CH 3
CH3 H
N N
N HN HN
HN

N N O N N
O N O HN
H
H 3C

Theobromine 7-methylxanthine Xanthine

(Substrate) (Products)

O
O
C CH3 H
N N
HN HN

Fig. 2. Schematic diagram of

N O N N
O HN degradation of (a) caffeine, (b)
H
theobromine, and (c) 7-
methylxanthine to respective
7-methylxanthine Xanthine products by the action of
(Substrate) (Product) N-demethylases.

0.5 enzyme is required to provide insight in the mechanism

Rate of degradation (µmols/min)

of caffeine demethylation to xanthine.

0.4 Control
NADH ACKNOWLEDGMENT
0.3 NADPH
This work was supported by a grant from the Department of Science
and Technology, India.
0.2

0.1
0 No. 352. Biosci Biotech Biochem 58:2303–2304
Caffeine
Fig. 3. Effect of NADH and NADPH on the degradation rates of
caffeine and related methylxanthines by induced cells of Pseudomonas
sp. Cofactors were added to the reaction mixture at a concentration of 1
mM. Initial concentration of caffeine, theobromine, and 7- methyl-
xanthine were 5, 2.5, and 3 mM, respectively. The reaction was carried
out for 10 min at pH 8.0 and 30°C. All experiments were performed in
duplicate under identical conditions and data points are mean € stan-
dard deviation of four independent measurements with a significance
of P < 0.05.
and food product for which the use of a purified enzyme
might not prove economical. The requirement of a co-
factor and metal ions clearly suggest more than one
enzyme is involved in the N-demethylation of caffeine
to xanthine. However, detailed study with a purified
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200

150

100

50

0
Control Fe2+ Ca2+ Mg 2+ Mn2+ Co2+ Cu2+ Ni2+ Zn2+
Metal ions

Fig. 4. Effect of metal ions on rate of degradation of (a) caffeine, (b)

theobromine (3, 7-dimethylxanthine), and (c) 7-methylxanthine by
induced cells of Pseudomonas sp. Metal ions were added to the
reaction mixture at a concentration of 1 mM. Data are represented as
the relative rates of degradation, where the rates taken as 100% are
0.24, 0.1, and 0.08 lmol/min for caffeine, theobromine, and 7-meth-
ylxanthine, respectively. The reaction was carried out for 10 min at pH
8.0 and 30°C. All experiments were performed in duplicate under
identical conditions and data points are mean € standard deviation of
four independent measurements with a significance of P < 0.05.