Journal of Basic Microbiology 2008, 48, 227 – 233 227

Research Paper
Inducible nature of the enzymes involved in catabolism
of caffeine and related methylxanthines

Swati Sucharita Dash and Sathyanarayana N. Gummadi

Department of Biotechnology, Indian Institute of Technology-Madras, Chennai, India

Previously isolated strain of Pseudomonas sp. has the capability of utilizing caffeine as the sole
source of carbon and nitrogen and degrading caffeine at higher concentrations (> 10 g l–1). In
this study, an assay has been developed to study the enzymatic conversion of caffeine to
subsequent methylxanthines by cell free extracts of Pseudomonas sp., the activity of which has
been stabilized by use of stabilizers in the lysis buffer. Growth of the strain in various
methylxanthines and later enzyme assay demonstrated that the enzyme(s) involved in
degradation of caffeine and other methylxanthines were inducible in nature. The results also
indicated that more than one enzyme are involved in degradation of caffeine to xanthine,
which constitute the primary steps in bacterial caffeine catabolism.

Keywords: Caffeine degradation / Pseudomonas sp. / enzyme induction / demethylase / methylxanthine

Received: January 05, 2008; accepted: February 27, 2008

DOI 10.1002/jobm.200800004

*
Introduction
Microbial enzymes involved in bringing about the con-
version of caffeine (1,3,7-trimethylxanthine) to its me-
tabolites have been the focus of research in the last
decade. When compared to growing culture or induced
whole cells, enzymes offer the advantage of being more
specific. Therefore they prove suitable for applications
such as preparation of decaffeinated food products, the
demand of which has been on the rise due to the nu-
merous reports on the association of caffeine intake
with adverse health effects particularly on the cardio-
vascular system and health of women [1 – 4].
Previous studies have shown that two primary
groups of enzymes i.e. N-demethylases and oxidases are
involved in the initial steps of degradation of caffeine
by bacteria [5 – 9]. Recently, a new enzyme, caffeine
dehydrogenase has also been identified that converts
caffeine to trimethyluric acid [10]. Although caffeine
oxidases have been well studied, the information about
caffeine demethylation, which is the most common
Correspondence: Sathyanarayana N. Gummadi, Department of Bio-
technology, Indian Institute of Technology-Madras, Chennai 600 036,
India
E-mail: gummadi@iitm.ac.in
Phone: + 91-44-2257-4114
Fax: + 91-44-2257-4102
route of caffeine catabolism in bacteria, is relatively
limited. Growth studies and biochemical investigations
on caffeine degradation by Pseudomonas sp. [5, 11] do not
conclusively determine whether caffeine catabolism to
xanthine is mediated by a single enzyme or several
enzymes. However, few studies have shown that there
might be separate enzymes bringing about specific
demethylations in Pseudomonas sp. which act separately
[7, 12] or as a complex [13].
Previously we have isolated a strain of Pseudomonas,
closely resembling Pseudomonas putida, from the soil of
coffee plantation area that is capable of utilizing caf-
feine as sole source of carbon and nitrogen [14, 15].
Kinetic studies on this bacteria shows that it follows
substrate inhibition kinetics for caffeine degradation
[16]. The degrading ability of this strain was also found
to be better as it could bring about complete degrada-
tion of caffeine at an initial concentration of 5 g l–1 at
the rate of 0.1 g l–1 h–1, which further increased to
0.29 g l–1 upon optimization of medium components
and physical parameters [17]. It was of interest there-
fore to investigate the enzyme system involved in caf-
feine catabolism in this strain. The present paper deals
with the development of an assay for demethylase ac-
tivity in the strain. The results show that the cell free
extract of Pseudomonas sp. consist of enzyme(s) that

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228 S. S. Dash et al. Journal of Basic Microbiology 2008, 48, 227 – 233

bring about the catabolism of caffeine and other me- 50 mM potassium phosphate buffer (pH 8.0) as men-
thylxanthines to xanthine and the enzyme(s) are induc- tioned above. The cells were then disrupted by sonica-
ible in nature. tion over ice (4 cycles of 2 min each with adequate cool-
ing in between the cycles). The cell debris was
separated by centrifugation at 20000 × g at 4 °C for
Materials and methods 30 min. The supernatant was treated as the crude en-
zyme extract and was immediately used in assay. Mem-
Chemicals brane fractions were obtained after centrifugation of
Pure caffeine (1,3,7-trimethylxanthine) was obtained sonicated cells at 12 000 × g for 10 min at 4 °C and su-
from Merck. Theobromine (3,7-dimethylxanthine), pernatant was centrifuged again (diluted two times in
paraxanthine (1,7-dimethylxanthine), theophylline(1,3 50 mM potassium phosphate buffer, pH 8.0) at
dimethylxanthine), 7-methylxanthine, NADH and Di- 25 000 × g for 30 min at 4 °C over 5 ml-layer of 40%
thiotheritol (DTT) were obtained from Sigma. All other sucrose solution to get the pellet of membranes.
reagents were of analytical grade procured in India.
Assay for methylxanthine degrading activity
Bacteria and culture conditions
Bacterial caffeine degradation is primarily brought
Pseudomonas sp. NCIM 5235 was maintained on CAS
about by N-1 and N-3 demethylase enzymes, with
medium with the following composition (g l–1):
NADH as the cofactor, which release methyl groups
Na2HPO4, 0.12; KH2PO4, 1.3; CaCl2, 0.3; MgSO4. 7 H2O,
from position N-1 and N-3 of caffeine molecule result-
0.3; sucrose, 5; caffeine, 1.2; agar, 25. The initial pH of
ing in formation of theobromine (3,7-dimethylxan-
the medium was adjusted to 6.0. The strain was sub
thine) and paraxanthine (1,7-dimethylxanthine) respec-
cultured every alternate day.
tively as the first intermediates. Subsequent action of
Three loopfuls of 36 h grown culture from CAS plate
these enzymes results in monomethylxanthines which
was transferred aseptically to 25 ml nutrient broth
are ultimately converted to xanthine molecule. Hence
medium and incubated to mid-log phase (OD600nm ∼ 1.4)
methylxanthine degrading activity can be directly es-
for 2.5 h on a rotary shaker operating at 180 rpm at
timated by measuring the decrease in initial caf-
30 °C. 50 ml of sterile optimized CAS medium was in-
feine/methylxanthine concentration following enzy-
oculated with 6% (v/v) of the culture and incubated for
matic reaction using RP-HPLC.
~ 10 h (when about 90% caffeine degradation was
Basing on this principle, the methylxanthine degrad-
achieved) at 30 °C and 180 rpm in a rotary shaker. For
ing activity in cell free extracts of Pseudomonas sp.
induction studies, caffeine in CAS medium was substi-
was assayed by previously described method [7, 12]
tuted with equal amount (g l–1) of methylxanthines viz.
with some modifications. Enzyme activities were
theobromine (3,7-dimethylxanthine), paraxanthine
measured at 30 °C in reaction mixture consisting of
(1,7-dimethylxanthine), theophyline (1,3-dimethylxan-
50 mM potassium phosphate buffer (pH 8.0), sub-
thine) and 7-methylxanthine. The cells were harvested
strate dissolved in the same buffer, 1 mM NADH.
by centrifugation at 8000 × g for 10 min at 4 °C, fol-
The reaction was initiated by adding cell free lysate
lowed by three washings with 50 mM potassium phos-
and after 10 min the reaction was stopped by adding
phate buffer (pH 8.0) under the same conditions. The
10% (w/v) TCA. Reaction carried out with enzyme inac-
cells were immediately used for cell lysis or stored at
tivated with TCA prior to incubation served as blank for
– 20 °C until used.
the assay. The reaction mixture was then centrifuged at
20 000 × g at 4 °C for 15 min and the supernatant was
Preparation of fractions and cell free lysate
analyzed by HPLC. One unit of enzyme activity (U) was
for enzyme studies
defined as the μmol of substrate (caffeine or other me-
The various fractions viz. periplasmic fraction and cy-
thylxanthines) degraded per min of reaction. All assays
toplasmic fraction were obtained from Pseudomonas sp.
were repeated at least twice in duplicates and the re-
as per previously described protocol [18]. For prepara-
sults presented are the average of four separate data
tion of cell free lysate, the cells were suspended in lysis
values.
buffer (50 mM phosphate buffer (pH 8.0) containing
1 mM EDTA, 1 mM DTT, 20% glycerol and 10% ethanol)
with cell mass: buffer ratio of 1 : 2 i.e. 2 ml of lysis Analytical procedures
buffer for every 1 g wet weight of cells. This step was Protein was estimated by the method described by
carried out immediately after the last wash with Lowry et al. [19]. Caffeine was estimated by RP-HPLC

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Journal of Basic Microbiology 2008, 48, 227 – 233
(Jasco PU-2080 Plus equipment) using a HiQSil C-18
column with water: methanol (3 : 1, v/v) as mobile phase
at a flow rate of 1 ml min–1 and at 28 °C. Pure caffeine
and other methylxanthines (Sigma) at 2 g l–1 were used
as standards. Detection was done at 254 nm using UV
detector.
Results and discussion
Localization of caffeine degrading activity
in Pseudomonas sp.
In order to determine the localization of caffeine de-
grading activity in Pseudomonas sp., extracellular culture
supernatant and various fractions were tested for the
ability to degrade caffeine. No activity was detected
in the culture supernatant indicating that the caffeine
degrading activity in Pseudomonas sp. was not extra-
cellular, which is in accordance with previous reports
in this aspect. Sonication proved to be the most
effective method for lysis of cells as compared to
other method viz. mechanical disruption and chemical
lysis in terms of maintaining activity. The lysate
once formed was found to be active only for a few
hours. Stabilization was achieved by addition of
glycerol (20%), EDTA (1 mM) ethanol (10%) and DTT
(1 mM) to the buffer during lysis and afterwards.
However, when compared with the whole cells, the
activity of the cell lysate was decreased by 90% (Ta-
ble 1). No activity was also detected for the membrane
fraction indicat-ing that the caffeine degrading en-
zymes are present in the cell cytoplasm and are not
membrane bound. Therefore for further studies, the
cell free supernatant obtained after cell lysis was used
as the source of enzyme.
N-demethylation is the most common route of caf-
feine degradation reported in Pseudomonas sp. The
schematic diagram of caffeine to xanthine demethyla-
tion pathway is given in Fig. 1a. Theobromine-
Table 1. Activity of whole cells and fractions with caffeine as the
a
substrate.
Source Activity U (µmol min–1)
Whole cells 0.099 ± 0.002
Cell free lysate 0.015 ± 0.001
Periplasmic fraction Not detected
Membrane fraction Not detected
a monas strains [5, 20]. However, it is still not clear
All experiments were performed three times in duplicates
and the values presented are the average of six separate set
of data with ± 2% to ± 6% standard deviation.
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Enzyme involved in catabolism of caffeine 229
(3,7-dimethylxanthine) and paraxanthine (1,7-dimethyl-
xanthine) are the first formed metabolites which are
further converted to 7-methylxanthine and then to
xanthine. Previous studies have shown that Pseudomonas
sp. used in this study also degrades caffeine through
the demethylation route, as indicated by the chroma-
tographic analysis of metabolites formed during degra-
dation [14]. Studies on induced cells have also shown
that caffeine is sequentially demethylated to other
methylxanthines. Chromatographic analysis of the
samples after reaction showed the presence of one or
more methylxanthines that were otherwise absent in
the reaction with inactivated enzyme, indicating the
demethylating activity present in the cell free super-
natant (Fig. 1b).
Effect of caffeine concentration
and protein concentration on enzyme activity
In order to determine the best conditions for assay,
enzyme activity was estimated in varying caffeine and
protein concentration. It was observed the enzyme
activity of cell free supernatant increased with increase
in caffeine concentration upto 10 mM and then de-
creased gradually, with the lowest activity detected for
40 mM (Fig. 2a). The specific activity was highest for
50 µg of protein, indicating that the activity was more
or less same over the range of protein concentration
tested (Fig. 2b). From the results obtained from the
above experiments, further assays were carried out
with 7.5 mM caffeine and protein concentration of
50 µg.
Inducible nature of methylxanthine degrading
activities in Pseudomonas sp.
As it had been already established from previous
studies that the catabolism of caffeine to xanthine in
Pseudomonas sp. occurred through demethylation route
[14], it was of interest to see whether the strain
was capable of utilizing other methylxanthine, which
are the metabolites of caffeine breakdown, as nutrients.
Growth was observed in all the methylxanthines and
also HPLC analysis of the culture supernatant showed
the degradation of the methylxanthines with due
course of time (data not shown). This indicates that the
enzymes in Pseudomonas sp. could degrade not only
caffeine but other methylxanthines as well. This has
been also reported for other caffeine degrading Pseudo-
whether the catabolism of caffeine and other metabo-
lites to xanthine, which are the initial steps in bacterial
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230 S. S. Dash et al. Journal of Basic Microbiology 2008, 48, 227 – 233

a)

b)

Figure 1. (a) Schematic representation of caffeine demethylation pathway in bacteria. (b) Chromatographic profiles of enzyme assay with
cell free extract of Pseudomonas sp. using caffeine as substrate. The presence of demethylase activity is indicated by appearance of
metabolite peaks in test sample (ii) as compared to blank (i) where no peak is detected.

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Journal of Basic Microbiology 2008, 48, 227 – 233
a)
b)
Figure 2. Effect of (a) caffeine concentration and (b) protein
concentration on enzyme activity of cell free extract of Pseudo-
monas sp. All experiments were performed at least twice in
duplicates under identical conditions and the values presented are
the average of four separate set of data with ± 1 – ± 10% standard
deviation.
caffeine catabolism is by the same enzyme or different
enzymes are involved in the process.
In order to have some insight into the nature of the
enzymes involved in caffeine catabolism in Pseudomonas
strain, induction experiments were carried out and
then the activity of the lysate was tested for all the
metabolites of N-demethylation by the strain. As con-
trol, cells were grown in Nutrient Broth with-out any
methylxanthines. The results obtained were quite in-
teresting and clearly indicated the inducible nature of
methylxanthine degrading enzymes in the strain. The
cells grown in caffeine medium showed activity to-
wards caffeine and other methylxanthines as compared
to control (Fig. 3a) indicating that caffeine can act as
the inducer for the enzyme(s) catabolising caffeine and
its metabolites. The activity of caffeine induced cells
towards theophylline was however not much different
than the control and this is probably due to the fact
that theophylline is not a metabolite of caffeine catabo-
lism pathway of this strain.
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Enzyme involved in catabolism of caffeine 231
The activity of lysate obtained from cells grown in
theobromine (3,7-trimethylxanthine) was increased for
theobromine, paraxanthine and 7-methylxanthine as
compared to that of control. However, caffeine degrad-
ing activity was found to be less than the control
(Fig. 3b). It can be inferred that only the enzyme(s) re-
quired to degrade theobromine, paraxanthine and
monomethyl xanthines are induced but not for caffeine
when cells were induced with theobromine (Fig. 3b). On
the other hand, no activity was detected towards theo-
phylline as was the case when cells were induced with
caffeine. The differences in activity towards tested me-
thylxanthines upon induction with theobromine sug-
gest the possibility of occurrence of more than one
enzyme involved in caffeine degradation by isolate used
in this study. This finding is supported by previous
studies by other workers who have shown that there is
a different enzyme for catabolism of theobromine in
Pseudomonas putida [7].
A different case however was noticed when paraxan-
thine (1,7-dimethylxanthine) was used as the inducer.
In this case, the activity of cell free lysate was highest
for theobromine (3,7-dimethylxanthine) and the ac-
tivities for paraxanthine and 7-methylxanthine were
the same (Fig. 3c). Although the cell lysate showed
activity towards caffeine, since the activity was less
than the control, induction could be ruled out. No
activity was detected when theophylline was used
as the substrate, as seen in all the other cases. When
7-methylxanthine was used as the inducer, activity
of the cell free lysate towards caffeine was less that that
of the control and was enhanced for 7-methylxanthine
and theobromine as compared to control (Fig. 3d).
The activity towards paraxanthine and theophylline
was however not significant. This indicates that the
catabolism of 7-methylxanthine is carried out by a
separate enzyme system. This is in correlation to a
previous study where it has been shown that a sepa-
rate enzyme system brings about the catabolism of
7-methylxanthine [12].
Concluding remarks
The present study elucidates the inducible nature
of methylxanthine degrading activity in Pseudomonas
sp. It is also clear form the above study that more
than one enzyme was involved in the initial steps
of caffeine catabolism pathway in Pseudomonas sp.
Further work regarding the purification and charac-
terization of the enzyme(s) involved is under pro-
gress.
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232 S. S. Dash et al. Journal of Basic Microbiology 2008, 48, 227 – 233

a)

b)

c)

d)

Figure 3. Activity of cell free extracts of Pseudomonas sp. toward caffeine and other methylxanthines when grown in the presence of various
methylxanthines as compared to when grown in Nutrient medium. (a) Activity of caffeine-induced cell free extract towards caffeine and other
methylxanthines. (b) Activity of theobromine (3,7-dimethylxanthine)-induced cell free extract towards caffeine and other methylxanthines. (c)
Activity of paraxanthine (1,7-dimethylxanthine)-induced cell free extract towards caffeine and other methylxanthine. (d) Activity of
7-methylxanthine induced cell free extract towards caffeine and other methylxanthine. All experiments were performed twice in duplicates under
identical conditions and the values presented are the average of four separate set of data with ± 1% to ± 13% standard deviation.

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Journal of Basic Microbiology 2008, 48, 227 – 233
Acknowledgement
This work was supported by grant from Department of
Science and Technology, Government of India.
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