Biotechnol Lett (2006) 28:1993–2002
DOI 10.1007/s10529-006-9196-2
REVIEW
Catabolic pathways and biotechnological applications
of microbial caffeine degradation
Swati Sucharita Dash Æ
Sathyanarayana N. Gummadi
Received: 6 July 2006 / Revised: 10 August 2006 / Accepted: 21 August 2006 / Published online: 29 September 2006

Springer Science+Business Media B.V. 2006
Abstract Catabolism of caffeine (1,3,7-trimeth-
ylxanthine) in microorganisms commences via
two possible mechanisms: demethylation and
oxidation. Through the demethylation route, the
major metabolite formed in fungi is theophylline
(1,3-dimethylxanthine), whereas theobromine
(3,7-dimethylxanthine) is the major metabolite in
bacteria. In certain bacterial species, caffeine has
also been oxidized directly to trimethyl uric acid
in a single step. The conversion of caffeine to
its metabolites is primarily brought about by
N-demethylases (such as caffeine demethylase,
theobromine demethylase and heteroxanthin-
edemethylase), caffeine oxidase and xanthine
oxidase that are produced by several caffeine-
degrading bacterial species such as Pseudomonas
putida and species within the genera Alcaligenes,
Rhodococcus and Klebsiella. Development of
biodecaffeination techniques using these enzymes
or using whole cells offers an attractive alterna-
tive to the present existing chemical and physical
methods removal of caffeine, which are costly,
toxic and non-specific to caffeine. This review
mainly focuses on the biochemistry of microbial
caffeine degradation, presenting recent advances
S. S. Dash Æ S. N. Gummadi (&)
Department of Biotechnology, Indian Institute of
Technology-Madras, Chennai 600 036, India
e-mail: gummadi@iitm.ac.in
and the potential biotechnological application of
caffeine-degrading enzymes.
Keywords Caffeine degradation Æ Caffeine
demethylases Æ Caffeine oxidase Æ Demethylation Æ
Enzymes Æ Oxidation Æ Xanthine oxidase
Introduction
Caffeine (1,3,7-trimethylxanthine) is a commer-
cially important alkaloid, which belongs to family
of purine alkaloids synthesized by plants. In nat-
ure, caffeine is distributed in the leaves and fruits
of 13 different orders of plants, including coffee
(Coffea arabica), tea (Camellia sinensis), mate
(Ilex paraguariensis), guarana (Paullinia copla-
nar), cola (Cola nitida) and cacao (Theobroma
cacao) (Ashihara and Crozier 2001). Caffeine is
an active psychostimulant which increases alert-
ness and sustains concentration by overcoming
fatigue (Nehlig 1999; Smith 2002; Lorist and Tops
2003). This makes caffeine one of the most widely
consumed dietary chemicals. Caffeine enters the
human system in the form of beverages like tea,
coffee and caffeinated soft drinks and numerous
food products like chocolates and desserts, with
the global consumption ranging from 80 to
400 mg caffeine per person per day (Gokula-
krishnan et al. 2005). Caffeine is widely used in
pharmaceutical preparations as it enhances the
123
1994
effect of certain analgesics and antipyretic drugs.
It also finds application as a cardiac, neurological
and respiratory stimulant and as a diuretic
(Mazzafera 2002).
Commercial coffee is obtained from coffee
cherries, 6% of which generate the coffee powder
whereas the remaining 94% constitute the by-
products such as husk, pulp water etc. Therefore,
caffeine-containing byproducts and effluents
generated from coffee and tea-processing plants
constitute a major part of the agro-industrial
wastes in coffee producing nations. Solid wastes
such as coffee pulp and husks pollute the sur-
rounding landmass by increasing toxicity and
bacterial contamination (Adams and Dougan
1981; Bressani 1987). The presence of caffeine in
soil can also affect soil fertility as it inhibits seed
germination and growth of seedlings (Friedman
and Waller 1983). Caffeine-containing effluents
are often discharged to the surrounding water
bodies and, subsequently, caffeine has been de-
tected in surface water, ground water and waste
water effluents at a high concentration (~10 g
caffeine l–1) (Buerge et al. 2003; Glassmeyer et al.
2005). The ingestion of caffeine and its chlori-
nated byproducts (derived during chlorination of
water during recycling) has severe adverse effect
on the physiological system (White and Rasmus-
sen 1998). Therefore, in order to free the natural
waters and soil from this xenobiotic, decaffein-
ation of the byproducts becomes a very necessary
step in treatment of coffee and tea wastes. Caf-
feine removal from coffee pulp and husk has also
been advocated for increasing the biotechnologi-
cal potential of these carbohydrate and protein
rich by-products that can be otherwise used as
animal feed or in ethanol production, mushroom
cultivation and production of enzymes and or-
ganic acids (Pandey et al. 2000). An increase in
the number of cardiovascular disorders as a result
of change in lifestyle, decaffeination of other food
products and beverages is being recommended,
keeping in view the adverse effects of chronic
caffeine consumption on the cardiovascular sys-
tem (James 2004). Caffeine consumption during
pregnancy increases the risk of spontaneous
abortion and affects foetal growth (Fernandez
et al. 1998). In addition, moderate to high caffeine
intake (>150 mg caffeine/day) imbalances the
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Biotechnol Lett (2006) 28:1993–2002
bone mineral density leading to osteoporosis
particularly in women (Cooper et al. 1992).
Conventional decaffeination techniques like
solvent extraction or use of supercritical carbon
dioxide can be expensive, toxic to the environ-
ment and non-specific. These methods often re-
sult in the removal of aroma and flavour
imparting substances and their precursors result-
ing in an unpalatable product. To compensate for
this, artificial flavours and colours are often sup-
plemented to the decaffeinated product. The
application of genetically modified coffee plants
produced by silencing the genes coding for en-
zymes responsible for synthesis of caffeine (Ogita
et al. 2003) may be unfavourable because of the
importance of caffeine as a part of the plant de-
fence mechanism against predators (Nathanson
1984; Hollingsworth et al. 2002). Hence, there is a
strong need for degradation of caffeine from
products and waste streams by alternative routes
other than conventional extraction techniques.
The potential use of microorganisms and enzymes
obtained from microbial system for developing
biological decaffeination techniques offer a much
attractive alternative to the present existing
techniques (Gokulakrishnan et al. 2005).
Knowledge of the enzymology of caffeine
degradation by microorganisms is essential for the
development of biodecaffeination techniques.
This review aims at providing a comprehensive
account of the mechanism of degradation of
caffeine in microorganisms with emphasis on the
various microbial enzymes bringing about the
conversion of caffeine to its metabolites that can
be used as biocatalysts for decaffeination
purposes.
Catabolism of caffeine in microorganisms
Caffeine-degrading ability has been reported in
various bacterial and fungal species isolated from
the soil of coffee plantation areas or from coffee
dumps. Since caffeine is generally considered to
be toxic to microbes, these special groups of
microorganism have unique metabolic character-
istics and enzyme systems for the breakdown of
caffeine and utilization of the same as a source of
Biotechnol Lett (2006) 28:1993–2002
energy. The catabolic routes, however, are dif-
ferent in different groups of microbes reflecting
the complexity in microbial caffeine catabolism.
In yeasts, caffeine degradation is brought about
by cytochrome P450 enzymes and therefore the
degradation pathway would be more similar to
that in animals (Sauer et al.1982). The degrada-
tion pathway in filamentous fungi is different and
species from various fungal genera, e.g. Penicil-
lium, Aspergillus, Stemphylium, Rhizopus, Phan-
erochaete, Trichoderma and Fusarium, have been
shown to catabolize caffeine (Mazzafera 2002;
Gokulakrishnan et al. 2005). In filamentous fungi
the first major metabolite is 1,3-dimethylxanthine
(theophylline) that is produced by the demethy-
lation in the 7th position of the purine ring.
Further, 1,3-dimethylxanthine (theophylline)
undergoes a second demethylation in the 1st
position of the purine ring resulting in formation
of 3-methylxanthine. In some cases trace amounts
of 3,7-dimethylxanthine (theobromine) and
1,7-dimethylxanthine (paraxanthine) were also
detected during the course of degradation (Hakil
et al. 1998). Therefore, the degradation of caf-
feine in fungi primarily proceeds via the caffeine-
theophylline-3-methylxanthine route as shown in
Fig. 1. The metabolites formed thereafter have
not been reported probably because the inter-
mediates formed are rapidly metabolized (Hakil
et al. 1999).
Bacterial caffeine degradation has been stud-
ied in detail and the pathway of conversion of
caffeine to its metabolites has been elucidated
clearly (Mazzafera 2004). Among the several
bacterial strains capable of degrading caffeine,
Pseudomonas putida was studied extensively
(Woolfolk 1975; Blecher and Lingens 1977; Asa-
no et al. 1993; Yamaoka-Yano and Mazzafera
1999; Dash and Gummadi 2006). In bacteria,
caffeine is first demethylated to dimethylxanth-
ines like theobromine or paraxanthine which
undergo further demethylations to form 7-meth-
ylxanthine. The dimethylxanthines and 7-meth-
ylxanthines can form methyluric acids which
appear as separate end products. 7-Methylxan-
thine is then converted to xanthine which is then
further oxidized to glyoxalic acid and urea
(Fig. 1) (Blecher and Lingens 1977; Glück and
Lingens 1987; Asano et al. 1993; Dash and
1995
Gummadi 2006). The methyl group is removed
either as formaldehyde (Blecher and Lingens
1977) or as methanol (Woolfolk 1975). A similar
pathway of degradation was also observed in
other bacterial species like Serratia marcescens
isolated from soil around a coffee cultivation area
(Mazzafera et al. 1994).
Apart from demethylation, caffeine can also be
converted to metabolites by an oxidative route
which has been observed in a mixed culture
consortium containing Klebsiella and Rhodococ-
cus (Madyastha and Sridhar 1998) and in Alca-
ligenes sp. (Mohapatra et al. 2006). In this
pathway, caffeine is oxidized at the C-8 position
resulting in the formation of 1,3,7-trimethyluric
acid which is further degraded to 3,6,8-trimeth-
ylallantoin which, on further degradation, forms
dimethylurea (Fig. 1).
Enzymatic aspect of caffeine degradation
Bioconversion of caffeine to its methylxanthine
derivatives is an enzymatic process involving
more than one type of enzyme. Both whole cells
and purified enzymes of caffeine-degrading bac-
terial strains have been explored in this aspect.
Resting cells of Pseudomonas putida (Middleho-
ven and Bakker 1982) and Pseudomonas alcalig-
enes (Sarath Babu et al. 2005) are effective in
degrading caffeine in both free and immobilized
form. Whole cells are advantageous in the bulk
treatment of soil, wastewater and caffeine con-
taining by products as it is cheaper than using
purified enzymes which require costly cofactors
and whole cells can convert complex compounds
to non-contaminating compounds such as CO2
and H2O. However, decaffeination using whole
cells can be non-specific and when applied to food
products may remove other compounds rendering
flavour and aroma to coffee and tea resulting in
an unpalatable product. Since several enzymes
are involved in conversion of caffeine to its end
products, whole cells will not be beneficial when
the objective is to recover a particular methyl-
xanthine intermediate requiring a specific enzyme
of caffeine catabolic pathway. Hence for these
applications, purified enzymes are more beneficial
than whole cells.
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1996 Biotechnol Lett (2006) 28:1993–2002

Fig. 1 Catabolism of O O O
H 3C CH3 CH 3
caffeine in H 3C H
N N N
N N HN
microorganisms. O O O
Catabolism of caffeine in N O HN N
O HN N
O HN H H
fungi (indicated by H

broken arrows) takes 1 methyl uric acid 1,7 dimethyl uric 7 methyl uric acid
place by theophylline acid
O
route and is similar to the O
CH 3
H 3C H N
pathway operating in N O HN
N H 3C CH3
plants. In case of bacteria, N
N N
catabolism results in O HN N O HN

theobromine as the first O HN N

1 methyl xanthine 7 methyl xanthine
formed metabolite
(indicated by straight Paraxanthine
arrows). The conversion (1,7 dimethyl xanthine)
O
of caffeine to O
CH3 O
methylxanthines to H 3C H
N H3C N
CH3
N
xanthine is mostly by the N N HN

action of demethylase N N
O N N O N
enzymes. Xanthine is CH 3
O N CH3
oxidized by xanthine H3C
Theobromine
Theophylline
oxidase to form simpler Caffeine
compounds. In certain (1,3 dimethyl xanthine) O
(3,7 dimethyl xanthine)
O
bacterial species like H
H
N
O
H 3C HN CH3
Rhodococcus and N
N
N
HN
Klebsiella caffeine is O
N O
O HN
directly oxidized to O N N
H O N N
H
methlyuric acids primarily CH 3 Xanthine CH3

by caffeine oxidase 1,3 dimethyl uric 3,7 dimethyl uric

enzymes and this can be acid O O O acid O
CH3 H H H
considered as a minor H 3C N N N
HN
N
N HN HN
pathway of degradation O O O

(indicated by dotted lines) O N N

O HN N O N N O N N
H H H
CH3 CH 3 CH 3

1,3,7 trimethyl uric acid Uric acid 3 methyl xanthine 3 methyl uric acid
H
O CH3 H +
CH3 O H NH
N N
HN 4 3 NH 2 H N H
8 2 O
NH 2 COO2
O
7 5 O Urea +
O N N
O HN N NH
6 H O HN
CH3 1 H CO2
H2O Glyoxylate
3, 6, 8 trimethyl allantoin Allantoin Allantoate

Mode of action of caffeine-degrading enzymes N-1 and N-3 demethylase enzymes which release
methyl groups from position N-1 and N-3 of caf-
In bacteria, two major classes of enzymes bring feine molecule resulting in formation of theoph-
about the conversion of caffeine to its derivatives: ylline (3,7-dimethylxanthine) and paraxanthine
(1,7-dimethylxanthine), respectively, as the first
(i) N-demethylases, and
intermediates. Subsequent action of these en-
(ii) Oxidases which include caffeine oxidases and
zymes results in monomethylxanthines which are
xanthine oxidases.
ultimately converted to xanthenes. The methyl
N-Demethylases are possibly involved during the groups so split off are generally further oxidized
initial steps of caffeine degradation that bring by molecular oxygen to formaldehyde (Fig. 2A)
about the sequential demethylation of three or hydrolyzed to methanol (Fig. 2B), which is then
N-methyl groups present in caffeine. In bacteria, converted to CO2 and H2O. NADH/NADPH are
caffeine degradation is primarily brought about by used as cofactors for enzymatic activity in most of

123
Biotechnol Lett (2006) 28:1993–2002 1997

(A) H3C
O
H
O N
CH3 N
H3C
N
N
O HN N

O HN N
1-methylxanthine
1,7 dimethyl xanthine
O
NAD(P)H NAD(P)+ NAD(P) +
O NAD(P)H H NAD(P)H NAD(P)+
N O
CH3 HN
H
H3C N N
N N HN
O O N
CH3 CH3
N O HN N
O N N
HN
CH3 + O2 3-methylxanthine
CH3 HCHO
O N N Xanthine
O
CH3
Caffeine CH3
N
3,7 dimethyl xanthine HN

O HN N

7-methylxanthine

(B) H3C
O
H
O N
CH3 N
H3C
N
N
O HN N

O HN N
1-methylxanthine
1,7 dimethyl xanthine
O
NAD(P)H NAD(P)+
O NAD(P)H NAD(P)+ H NAD(P)H NAD(P)+ O
N
CH3 HN
H
H3C N N
N N HN
O O N
CH3 CH3
N O HN N
O N N
HN
CH3 + H2O CH3OH 3-methylxanthine
CH3
N Xanthine
O N O
Caffeine CH3
CH3
N
3,7 dimethyl xanthine HN

O HN N

7-methylxanthine

Fig. 2 Schematic representation of bacterial N-demethylation of caffeine by (A) oxidation and (B) hydrolysis

the cases. The overall scheme of N-demethylase Assay for caffeine degrading enzymes
activity is represented in Fig. 2.
Oxidases, on the other hand, operate in a dif- Based on the scheme given in Fig. 2, the activity
ferent mechanism resulting in the formation of of caffeine degrading enzymes can be assayed by
uric acids from the methylxanthines. Two such the following three methods:
oxidases viz., caffeine oxidase (1,3,7-trimethyl-
(i) By estimating formaldehyde or methanol
xanthine oxidase) and xanthine oxidase have
formed as a result of oxidation of the re-
been implicated in the catabolism of caffeine in
leased methyl groups.
bacteria. These enzymes directly bring about the
(ii) By analyzing the conversion of NADH to
oxidation of methylxanthines, resulting in the
NAD+ spectrophotometrically.
formation of methyluric acids, which are then
(iii) By measuring the decrease in initial caffeine
converted into uric acids that enter the normal
concentration after enzymatic reaction using
purine catabolic pathway for further degradation.
chromatographic techniques.
Unlike N-demthylases, which utilize NAD and
NADP as electron acceptors, dichlorophenol, The formaldehyde produced as a result of oxi-
indophenol and cytochrome c serve as electron dative demethylation can be estimated colori-
acceptors for caffeine oxidase (Madyastha et al. metrically using the Hantzsch reaction (Nash
1999). 1953). This assay is simple and specific and has

123
1998
been used in many studies to estimate the activity
of caffeine demethylases, which can be directly
correlated to the amount of formaldehyde formed
(Blecher and Lingens 1977; Hohnloser et al. 1980;
Asano et al. 1994). However, the assay is not
suitable in the presence of acetaldehyde, which is
a common metabolic intermediate in bacteria.
Therefore, use of this technique for the estima-
tion of enzymatic activity in culture filtrates will
not be accurate as formaldehyde accumulated as
a result of caffeine degradation may give erro-
neous values.
The interconversion of NADH and NAD+ is
yet another method of estimating the activity of
methylxanthine demethylases which is assayed by
following the decrease in absorbance at 340 nm
for the conversion of NADH to NAD+. Although
it is an effective method, when cell lysate is used
as enzyme source the cellular NADH/NAD+ may
interfere with the assay but this can easily be ta-
ken into account by carrying out appropriate
control arrays.
Detection of caffeine degradation as a result of
enzymatic activity by high performance liquid
chromatography (HPLC) is the most accurate
assay procedure. By the use of HPLC, not only
can the reduction of caffeine be monitored but
also the presence of metabolites can be detected.
However, few reports are available in the litera-
ture, where all the above-mentioned techniques
were used to confirm the caffeine demethylase
activity (Glück and Lingens 1988; Asano et al.
1994).
Bacterial caffeine-degrading enzymes
N-Demethylases:
The speculation that N-demethylation was the
possible mechanism of conversion of caffeine to
its methylated derivatives came from the study of
Woolfolk who demonstrated methylxanthine
degrading activity in cell free extracts and sub-
cellular fractions of P. putida (Woolfolk 1975).
He suggested that the conversion of caffeine to its
metabolites was brought about by an enzyme that
hydrolytically removed all the three-methyl
groups with the production of methanol and free
123
Biotechnol Lett (2006) 28:1993–2002
xanthine. The free xanthine presumably entered
the purine degradation pathway to be converted
to simpler compounds by the action of enzymes
like xanthine dehydrogenase and uricase. The
methanol formed was oxidized to CO2 through
formaldehyde by the sequential action of metha-
nol dehydrogenase, formaldehyde dehydrogenase
and formate dehydrogenase (Woolfolk 1975). The
involvement of a demethylase enzyme in caffeine
degradation by P. putida was further confirmed
by Blecher and Lingens (1977). However, they
postulated that the demethylation was oxidative
rather than hydrolytic, as suggested by Woolfolk,
resulting in the formation of formaldehyde in-
stead of methanol (Fig. 2). Their studies also
showed that the demethylase enzymes were not
constitutive but inducible by the addition of caf-
feine and other methylxanthine that formed as a
result of degradation of caffeine. Similar caffeine-
demethylating activity was demonstrated in the
crude extracts of P. putida, and was absolutely
dependent upon NADH and NADPH as cofac-
tors (Hohnloser et al. 1980). Attempts to purify
this enzyme were unsuccessful as this enzyme lost
its activity rapidly in partially purified extracts.
Similar to caffeine demethylase, enzymes for
demethylation of other methylxanthines have
been reported in the literature. Asano et al.
(1994) isolated a monooxygenase, i.e. theobro-
mine demethylase, from cell free extracts of
P. putida and reported that this enzyme specifically
brought about the demethylation of theobro-
mine. They also showed that the theobromine
demethylase enzyme was inhibited by Zn2+,
which generated the accumulation of theobro-
mine (Asano et al. 1994). Another enzyme,
heteroxanthinedemethylase isolated from
P. putida has been shown to selectively convert
heteroxanthine (7-methylxanthine) to xanthine
by oxidative demethylation with the formation
of formaldehyde (Glück and Lingens 1988). The
enzyme was partially purified from cell-free ex-
tracts of P. putida and showed maximum activity
with NADH/NADPH as cofactors. It showed no
activity when either caffeine or dimethylxanth-
ines were used as substrates. Similar to caffeine
demethylase, heteroxanthinedemethylase was
also found to be unstable and loss of activity
occurred upon storage.
Biotechnol Lett (2006) 28:1993–2002
Recovery of caffeine demethylase in a pure
form and its characterization has not yet been
reported. This provides a challenging and signifi-
cant area of research since methylxanthine inter-
mediates of caffeine catabolism obtained by the
action of N-demethylases have many applications.
In medicine, theobromine and theophylline are
used as diuretics, vasodilators, and myocardial
stimulants. Monomethylxanthines can be con-
verted to effective caffeine derivatives by chemi-
cal derivatization (Goering 1982) and hence can
serve as interesting alternatives to caffeine. Xan-
thine also finds pharmaceutical application in
drugs for treatment of asthma. The biotechno-
logical potential of N-demethylases therefore lies
not only in general decaffeination purposes but
also in specific product recovery from caffeine.
Caffeine oxidases
In certain species of bacteria, instead of caffeine
being demethylated to methylxanthines, it is oxi-
dized to 1,3,7-trimethyluric acid. The structure of
1,3,7-trimethyluric acid is shown in Fig. 3. En-
zymes catalyzing this reaction have been desig-
nated as caffeine oxidases. Two such caffeine
oxidases have been purified (Madyastha et al.
1998; Mohapatra et al. 2006). Caffeine oxidase
from Rhodococcus and Klebsiella is a 85 kDa
flavoprotein that catalyzes the formation of 1,3,7-
trimethyluric acid directly from caffeine in a sin-
gle step. This enzyme showed substrate specificity
toward caffeine and various analogues of theo-
bromine (Madyastha et al. 1998). The other caf-
feine oxidase isolated from an Alcaligenes sp. was
a serine-type metalloprotease with strict substrate
O
CH3
H3C
N
N enzymes
O
O N N
H ganisms have been identified in the recent past,
H3C
1,3,7-trimethyluric acid
Fig. 3 Structure of 1,3,7-trimethyluric acid
1999
specificity towards caffeine (Mohapatra et al.
2006). From the biotechnological point of view,
caffeine oxidase enzymes would prove to be more
useful in treatment of caffeine-containing efflu-
ents or the wastes of coffee processing industries
as these are more stable and can be purified to
homogeneity without loss of activity. However,
these enzymes will not be useful in recovery of
the methylxanthine intermediates that are phar-
maceutically important since caffeine is directly
converted to trimethyluric acid without the for-
mation of methylxanthine intermediates.
Xanthine oxidases
The methylxanthines formed during the course of
caffeine breakdown can be further converted to
the corresponding methyluric acids (Woolfolk
1975; Blecher and Lingens 1977) and this con-
version is brought about by a group of enzymes
called xanthine oxidases. Several researchers
have purified this particular enzyme from micro-
organisms (Bradshaw and Barker 1960; Smith
et al. 1967; Woolfolk et al. 1970; Sin 1975). Iso-
lation of the activities from diverse sources (Bray
1965) has revealed that these enzymes are all
fundamentally similar with regard to molecular
properties and prosthetic group content, although
they may differ considerably in utilization of
electron acceptors and their role in the purine
catabolism pathway in the cells from which they
are obtained. Xanthine oxidase isolated from
P. putida has three subunits (71, 66 and 62 kDa)
and could bring about the conversion of various
methylxanthines to methyluric acids (Yamaoka-
Yano and Mazzafera 1999). The bacterial caffeine
degrading enzymes and their properties are listed
in Table 1.
Bottlenecks in the study of caffeine-degrading
Although numerous caffeine-degrading microor-
reports on the enzymatic aspects of caffeine
degradation are few. The labile nature of N-de-
methylases hinders the purification and charac-
terization of these enzymes. Use of chemicals
123
2000 Biotechnol Lett (2006) 28:1993–2002

such as 20% (v/v) glycerol, BSA, thioagents

Glück and Lingens (1988)

Mohapatra et al. (2006)

Madyastha et al. (1999)
Hohnloser et al. (1980)
(Glück and Lingens 1988) or cryoprotectants and

Yamaoka-Yano and
Mazzafera (1999)
freeze drying techniques (Sideso et al. 2001) can

Asano et al. (1994)

Sideso et al. (2003)

confer some degree of stability but more re-
search in this area is required. In comparison,
Opt. Temp. Opt. pH Reference

caffeine oxidases are more stable and can be

utilized as biocatalysts for decaffeination pur-
poses. However oxidation of caffeine directly to
1,3,7-trimethyluric acid due to caffeine oxidase
activity has been reported in relatively few bac-
6.0

7.3

7.8

7.5
7.0
terial species, from genera such as Klebsiella,
–

Rhodococcus, and Alcaligenes (Madyastha et al.

1998; Mohapatra et al. 2006). There is no report
22–24

30–35
20–30

on this oxidative pathway operating in P. putida,

(C)

11.4 lM 30

8.94 lM 35
169 lM 30
1.1 mM –

the species most commonly associated with caf-

feine degradation. In this species, caffeine
Molecular weight (kDa) Km

catabolism occurs primarily by demethylation.

–

–
–

This indicates that caffeine oxidase-producing

bacterial strains are relatively rare. Caffeine-
oxidizing enzymes will be more relevant for the
bulk decaffeination of soil, caffeine containing
43.5 and 36.6
Table 1 Important enzymes involved in caffeine degradation in bacteria and their properties

agro-industrial wastes like coffee pulp and husk,

groundwater and wastewaters and in develop-
ment of biosensors for caffeine detection on
192
41

85

65

natural water bodies (Eggins 1996). Isolation of

–

putida WS Heteroxanthine demethylase 7-methylxanthine –

more of such strains will prove beneficial from

Theobromine
Theobromine

Caffeine and

the biotechnological point of view as mentioned

analogues

above.
Substrate

Xanthine
Caffeine

Caffeine

Caffeine

Conclusion and future perspective

Theobromine demethylase

Biological decaffeination provides an attractive

putida C1 Caffeine demethylase

Caffeine demethylase

alternative to the conventional methods of

Pseudomonas putida L. Xanthine oxidase
Caffeine oxidase

Caffeine oxidase

decaffeination. Keeping in mind the increasing

demand for decaffeinated products, such meth-
complex

ods can be carried out at a much larger scale with

Enzyme

the proper understanding of the biological sys-

tem involved in this conversion. Biodecaffein-
ation can be used effectively for the treatment of
solid caffeine wastes such as husk and pulp for
putida

putida

use as animal feed (Roussos et al. 1995; Maz-

and Klebsiella
Sl. No. Microorganism

Alcaligenes sp.

zafera 2002), for removal of caffeine in sewage

Pseudomonas
Pseudomonas

Pseudomonas
Pseudomonas

Rhodococcus

(Ogunseitan 1996), for the production of other

No. 352

commercially important methylxanthines (Glück

and Lingens 1988; Asano et al. 1993) and also for
the production of decaffeinated beverages and
other food products. Enzymatic degradation of
caffeine is useful in converting caffeine to spe-
1
2

3
4

6
7

123
Biotechnol Lett (2006) 28:1993–2002
cific intermediates and also in food applications,
whereas decaffeination by whole cells is more
economic in treating sewage and other wastes
containing caffeine. The isolation, purification
and characterization of new caffeine-degrading
enzymes and development of methods for
increasing the stability of existing ones are some
of the areas where more studies are required.
Further, identification of genes involved in caf-
feine catabolism will open up new avenues for
gaining insight on the regulatory mechanism of
these enzymes, and provide scope for their
manipulation.
Acknowledgements Authors acknowledge S. Gokula-
krishnan, research scholar in Biotechnology Department,
IIT-Madras for his help in preparing the manuscript.
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