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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 Although the growth kinetics of this strain has been
to reduce the caffeine content in food products and also studied [10], there are no reports on the degradation
for the treatment of caffeine-containing effluents that are kinetics of caffeine or the related methylxanthine. In this
toxic to the environment or for rendering coffee pulp study, the kinetics of the degradation of caffeine and the
and husk for other uses [5, 13, 15]. In this aspect, related methylxanthines have been studied along with
microbial degradation of caffeine (1,3,7-trimethylxan- the effect of various cofactors on the rate of degradation.
thine) and related methylxanthines has been the focus of The results obtained will be beneficial for the develop-
research in the recent past because of the major advan- ment of an economical bioprocess for caffeine removal
tages that it has over conventional techniques of deca- and also provide an insight on the nature of caffeine
ffeination [8]. Several microbial species have been demethylases.
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 Materials and Methods
such Pseudomonas strain isolated from the soil of a Chemicals. Pure caffeine (1,3,7 trimethylxanthine) was obtained from
coffee plantation area [9] and identified by 16S rRNA Merck; 3,7-dimethylxanthine, 7-methylxanthine, and xanthine were
analysis has been shown to degrade higher concentra- obtained from Sigma. All other reagents were of analytical grade.
tions of caffeine (15 g/L) by N-demethylation at a rate Bacterial Strain. Pseudomonas sp. previously isolated in our
higher than what has been reported for any strain so far laboratory from the soil of a coffee plantation area was maintained
[6]. 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 57

initial pH of the medium was 6.0. For preparation of induced cells, biocatalyst (induced cells) were found to be 8.0, 30°C,
optimized CAS medium with the following composition was used (g/ and 0.18 mg/mL, respectively. Under these optimal
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
conditions, the rate of degradation of caffeine was esti-
was adjusted to 7.8 with 1 N NaOH before sterilization. mated to be 0.47 mmol caffeine/g cell dry weight/min
and this rate is sufficiently higher than what has been
Preparation of Induced Cells of Pseudomonas sp. . Induced cells
reported so far [4]. It was also reported that optimal
were prepared as described earlier [11]. Briefly, three loopfuls of 36-h
grown culture from the CAS plate was transferred aseptically to 25 mL conditions obtained in this study was similar to other
nutrient broth medium and incubated to mid-log phase (OD600 nm1.4) reports on Pseudomonas strains (temperature: 30–35°C;
for 2.5 h at 180 rpm at 30°C. Twenty-five milliliters of sterile pH: 7.0–9.0) [7, 11, 14, 16]. In order to use this strain as
optimized CAS medium was inoculated with 6% (v/v) of the culture an effective biocatalyst for degrading caffeine and its
and incubated at 28°C and 190 rpm for 30 h. At 30 h, when caffeine
related methylxanthines, which are initial metabolites of
degradation was 90%, the bacterial cells were harvested by
centrifugation at 8000g for 15 min at 4°C and cells were washed the caffeine degradation pathway, kinetics of degrada-
with 10 mM potassium phosphate buffer (pH 7.0). The cells could be tion of caffeine and other related methylxanthines is
stored at )20°C for a maximum of 20 h, beyond which the cells lost the necessary.
caffeine degrading ability. Under these optimal conditions, the degradation
Degradation Assay. The standard reaction mixture consisted of 1 mM kinetics of caffeine and related methylxanthines (theo-
caffeine and 0.18 mg/mL of induced cells as the biocatalyst in 10 mM bromine and 7-methylxanthines) was studied at various
potassium phosphate buffer (pH 8.0). Degradation assays were initial substrate concentrations. The plot of initial rate of
performed in 5-mL glass vials that were set in gentle motion on a degradation versus substrate concentration followed
gel rocker at 30°C for proper mixing. After 10 min, the cells were
separated by centrifugation and the supernatant was assayed for
Michaelis–Menten kinetics (Fig. 1). Such an approach
caffeine content by reversed-phase high-performance liquid has been used to study the biodegradation of phenol by
chromatography (HPLC). The substrate concentrations and rates of immobilized cells of Pseudomonas putida [3]. The
degradation obtained were fitted to a Michaelis–Menten model and intrinsic kinetic parameters such as Km and Vmax were
kinetic parameters (i.e. Km and Vmax values were determined from the determined when caffeine, theobromine, and 7-meth-
curve by nonlinear regression analysis with a confidence level >95%
using GraphPad PRISMÒ Version 3 software).
ylxanthins were used as substrates using linear regression
The effect of cofactors was studied by adding NADH and fit (Table 1). As seen from Table 1, the values of Km and
NADPH to the reaction mixture at a concentration of 1 mM and per- Vmax were different for the different substrates used. The
forming the assay as described earlier. In order to study the effect of induced cells had lower substrate specificity for theo-
metal ions, Fe2+, Ca2+, Mg2+, Mn2+, Co2+, Cu2+, Zn2+, and Ni2+ were bromine compared to that for caffeine or 7-methylxan-
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
thine. However, these results clearly establish that the
5 H2O, ZnSO4 Æ H2O, NiSO4 Æ 6H2O) at a concentration of 1 mM and induced cells are not only able to use caffeine as the
the assay was performed as described earlier. In all of these experi- substrate but other methylxanthines as well. HPLC
ments, the initial concentrations of caffeine, theobromine, and 7- analysis of the reaction mixture after 10 min of reaction
methylxanthines were 5, 2.5, and 3 mM, respectively. showed theobromine, 7-methylxanthine, and xanthine as
Analytical Determination. Caffeine, 3,7 dimethylxanthine, and 7 products suggesting that the caffeine is degraded by the
methylxanthine were estimated by HPLC (Agilent 1100 series) demethylation route (data not shown), similar to the
equipment using a ZORBAX C-18 column with 10 mM ammonium pathway observed in growing cells of the same strain [6]
phosphate buffer (pH 2.5)/acetonitrile (4:1, v/v) as the mobile phase.
(Fig. 2a). Similarly, only 7-methylxanthine and xanthine
Pure caffeine, 3,7-dimethylxanthine, and 7-methylxanthine (Merck) at
2 g/L were used as standards. The retention time of caffeine, 3,7- was observed as products when theobromine was used as
dimethylxanthine, and 7-methylxanthine was found to be 4.9, 3.1, and the substrate (Fig. 2b) and only xanthine as the product
2.8 minute, respectively, at a flow rate of 1 mL/min and at 28°C. when 7-methylxanthine was the substrate (Fig. 2c) (data
Detection of caffeine and its metabolites was done at 254 nm using an not shown). These results clearly suggested N-demeth-
ultraviolet (UV) detector.
ylases are involved in the degradation of caffeine and its
Statistical Analysis. All of the experiments were performed in
related xanthines. Assays performed with dead cells of
duplicate and data are the mean of four independent measurements Pseudomonas sp. in a similar manner showed no de-
with significance P < 0.05. crease in concentration of caffeine and other methylx-
anthines, indicating that the decrease in substrate
concentration was solely due to degradation and not
Results and Discussion
absorption of substrates on the bacterial cells (data not
The degradation rates were calculated over a pH range shown). It has been suggested by various researchers that
of 5.0–9.0, temperature range of 25–40°C, and cell dry more than one enzyme is involved in caffeine demethy-
weight range of 0.036–1.96 g/L. Preliminary results lation to xanthine and their absolute requirement of co-
showed that optimal pH, temperature, and amount of 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
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
0.1
Ni2+, and drastically inhibited by Cu2+ and Zn2+
(Fig. 4a). This pattern was somewhat different for
theobromine, for which the degradation rate was greatly
0.0
0 1 2 3 4 5 enhanced in the presence of Mn2+ and Fe2+, unaffected
by Ca2+, and strongly inhibited by all other metal ions
7-methylxanthine concentration(mM)
(Fig. 4b) Similarly, the effect of metal ions on the
Fig. 1. Kinetics of degradation of (a) caffeine, (b) theobromine (3, 7- degradation rate of 7-methyl xanthine was different
dimethylxanthine), and (c) 7-methylxanthine by induced cells of when compared to that of caffeine and theobromine
Pseudomonas sp. Experiments were performed at various concentra-
tions of the substrates using 0.18 g/L dry weight of induced cells at
(Fig. 4c). In a previous report, it had been shown that
30°C and pH 8.0. Data showed that the degradation followed Micha- caffeine degradation was enhanced by Co2+ [12].
elis–Menten kinetics. All experiments were performed in duplicate However in this case, the rate was not influenced by the
under identical conditions and data points are mean € standard devi- presence of Co2+, indicating variation in the strain. The
ation of four independent measurements with a significance of conversion of caffeine to theobromine by Pseudomonas
P < 0.05.
was increased by Zn2+ [2]. The results obtained in this
study will be beneficial for the development of process
In order to further establish whether catabolism is in which the selective degradation of caffeine and its
due to a single enzyme or multiple enzymes, the effect related xanthines or production of any related xanthine
of NADH and NADPH on the degradation rate was from caffeine is the goal.
tested. It should be noted that NADH and NADPH for In conclusion, the induced cells of Pseudomonas sp.
these enzymes are already provided by the cell. How- are capable of degrading caffeine and related methylx-
ever, the effect of extraneous addition of these cofactors anthines and hence can be used as effective biocatalysts
was tested in this study. Results showed that the cofactor 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

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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.