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DOI 10.1007/s10529-006-9196-2
REVIEW
Received: 6 July 2006 / Revised: 10 August 2006 / Accepted: 21 August 2006 / Published online: 29 September 2006
Springer Science+Business Media B.V. 2006
123
1994 Biotechnol Lett (2006) 28:1993–2002
effect of certain analgesics and antipyretic drugs. bone mineral density leading to osteoporosis
It also finds application as a cardiac, neurological particularly in women (Cooper et al. 1992).
and respiratory stimulant and as a diuretic Conventional decaffeination techniques like
(Mazzafera 2002). solvent extraction or use of supercritical carbon
Commercial coffee is obtained from coffee dioxide can be expensive, toxic to the environ-
cherries, 6% of which generate the coffee powder ment and non-specific. These methods often re-
whereas the remaining 94% constitute the by- sult in the removal of aroma and flavour
products such as husk, pulp water etc. Therefore, imparting substances and their precursors result-
caffeine-containing byproducts and effluents ing in an unpalatable product. To compensate for
generated from coffee and tea-processing plants this, artificial flavours and colours are often sup-
constitute a major part of the agro-industrial plemented to the decaffeinated product. The
wastes in coffee producing nations. Solid wastes application of genetically modified coffee plants
such as coffee pulp and husks pollute the sur- produced by silencing the genes coding for en-
rounding landmass by increasing toxicity and zymes responsible for synthesis of caffeine (Ogita
bacterial contamination (Adams and Dougan et al. 2003) may be unfavourable because of the
1981; Bressani 1987). The presence of caffeine in importance of caffeine as a part of the plant de-
soil can also affect soil fertility as it inhibits seed fence mechanism against predators (Nathanson
germination and growth of seedlings (Friedman 1984; Hollingsworth et al. 2002). Hence, there is a
and Waller 1983). Caffeine-containing effluents strong need for degradation of caffeine from
are often discharged to the surrounding water products and waste streams by alternative routes
bodies and, subsequently, caffeine has been de- other than conventional extraction techniques.
tected in surface water, ground water and waste The potential use of microorganisms and enzymes
water effluents at a high concentration (~10 g obtained from microbial system for developing
caffeine l–1) (Buerge et al. 2003; Glassmeyer et al. biological decaffeination techniques offer a much
2005). The ingestion of caffeine and its chlori- attractive alternative to the present existing
nated byproducts (derived during chlorination of techniques (Gokulakrishnan et al. 2005).
water during recycling) has severe adverse effect Knowledge of the enzymology of caffeine
on the physiological system (White and Rasmus- degradation by microorganisms is essential for the
sen 1998). Therefore, in order to free the natural development of biodecaffeination techniques.
waters and soil from this xenobiotic, decaffein- This review aims at providing a comprehensive
ation of the byproducts becomes a very necessary account of the mechanism of degradation of
step in treatment of coffee and tea wastes. Caf- caffeine in microorganisms with emphasis on the
feine removal from coffee pulp and husk has also various microbial enzymes bringing about the
been advocated for increasing the biotechnologi- conversion of caffeine to its metabolites that can
cal potential of these carbohydrate and protein be used as biocatalysts for decaffeination
rich by-products that can be otherwise used as purposes.
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 Catabolism of caffeine in microorganisms
of change in lifestyle, decaffeination of other food
products and beverages is being recommended, Caffeine-degrading ability has been reported in
keeping in view the adverse effects of chronic various bacterial and fungal species isolated from
caffeine consumption on the cardiovascular sys- the soil of coffee plantation areas or from coffee
tem (James 2004). Caffeine consumption during dumps. Since caffeine is generally considered to
pregnancy increases the risk of spontaneous be toxic to microbes, these special groups of
abortion and affects foetal growth (Fernandez microorganism have unique metabolic character-
et al. 1998). In addition, moderate to high caffeine istics and enzyme systems for the breakdown of
intake (>150 mg caffeine/day) imbalances the caffeine and utilization of the same as a source of
123
Biotechnol Lett (2006) 28:1993–2002 1995
energy. The catabolic routes, however, are dif- Gummadi 2006). The methyl group is removed
ferent in different groups of microbes reflecting either as formaldehyde (Blecher and Lingens
the complexity in microbial caffeine catabolism. 1977) or as methanol (Woolfolk 1975). A similar
In yeasts, caffeine degradation is brought about pathway of degradation was also observed in
by cytochrome P450 enzymes and therefore the other bacterial species like Serratia marcescens
degradation pathway would be more similar to isolated from soil around a coffee cultivation area
that in animals (Sauer et al.1982). The degrada- (Mazzafera et al. 1994).
tion pathway in filamentous fungi is different and Apart from demethylation, caffeine can also be
species from various fungal genera, e.g. Penicil- converted to metabolites by an oxidative route
lium, Aspergillus, Stemphylium, Rhizopus, Phan- which has been observed in a mixed culture
erochaete, Trichoderma and Fusarium, have been consortium containing Klebsiella and Rhodococ-
shown to catabolize caffeine (Mazzafera 2002; cus (Madyastha and Sridhar 1998) and in Alca-
Gokulakrishnan et al. 2005). In filamentous fungi ligenes sp. (Mohapatra et al. 2006). In this
the first major metabolite is 1,3-dimethylxanthine pathway, caffeine is oxidized at the C-8 position
(theophylline) that is produced by the demethy- resulting in the formation of 1,3,7-trimethyluric
lation in the 7th position of the purine ring. acid which is further degraded to 3,6,8-trimeth-
Further, 1,3-dimethylxanthine (theophylline) ylallantoin which, on further degradation, forms
undergoes a second demethylation in the 1st dimethylurea (Fig. 1).
position of the purine ring resulting in formation
of 3-methylxanthine. In some cases trace amounts
of 3,7-dimethylxanthine (theobromine) and Enzymatic aspect of caffeine degradation
1,7-dimethylxanthine (paraxanthine) were also
detected during the course of degradation (Hakil Bioconversion of caffeine to its methylxanthine
et al. 1998). Therefore, the degradation of caf- derivatives is an enzymatic process involving
feine in fungi primarily proceeds via the caffeine- more than one type of enzyme. Both whole cells
theophylline-3-methylxanthine route as shown in and purified enzymes of caffeine-degrading bac-
Fig. 1. The metabolites formed thereafter have terial strains have been explored in this aspect.
not been reported probably because the inter- Resting cells of Pseudomonas putida (Middleho-
mediates formed are rapidly metabolized (Hakil ven and Bakker 1982) and Pseudomonas alcalig-
et al. 1999). enes (Sarath Babu et al. 2005) are effective in
Bacterial caffeine degradation has been stud- degrading caffeine in both free and immobilized
ied in detail and the pathway of conversion of form. Whole cells are advantageous in the bulk
caffeine to its metabolites has been elucidated treatment of soil, wastewater and caffeine con-
clearly (Mazzafera 2004). Among the several taining by products as it is cheaper than using
bacterial strains capable of degrading caffeine, purified enzymes which require costly cofactors
Pseudomonas putida was studied extensively and whole cells can convert complex compounds
(Woolfolk 1975; Blecher and Lingens 1977; Asa- to non-contaminating compounds such as CO2
no et al. 1993; Yamaoka-Yano and Mazzafera and H2O. However, decaffeination using whole
1999; Dash and Gummadi 2006). In bacteria, cells can be non-specific and when applied to food
caffeine is first demethylated to dimethylxanth- products may remove other compounds rendering
ines like theobromine or paraxanthine which flavour and aroma to coffee and tea resulting in
undergo further demethylations to form 7-meth- an unpalatable product. Since several enzymes
ylxanthine. The dimethylxanthines and 7-meth- are involved in conversion of caffeine to its end
ylxanthines can form methyluric acids which products, whole cells will not be beneficial when
appear as separate end products. 7-Methylxan- the objective is to recover a particular methyl-
thine is then converted to xanthine which is then xanthine intermediate requiring a specific enzyme
further oxidized to glyoxalic acid and urea of caffeine catabolic pathway. Hence for these
(Fig. 1) (Blecher and Lingens 1977; Glück and applications, purified enzymes are more beneficial
Lingens 1987; Asano et al. 1993; Dash and than whole cells.
123
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
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
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 Biotechnol Lett (2006) 28:1993–2002
been used in many studies to estimate the activity xanthine. The free xanthine presumably entered
of caffeine demethylases, which can be directly the purine degradation pathway to be converted
correlated to the amount of formaldehyde formed to simpler compounds by the action of enzymes
(Blecher and Lingens 1977; Hohnloser et al. 1980; like xanthine dehydrogenase and uricase. The
Asano et al. 1994). However, the assay is not methanol formed was oxidized to CO2 through
suitable in the presence of acetaldehyde, which is formaldehyde by the sequential action of metha-
a common metabolic intermediate in bacteria. nol dehydrogenase, formaldehyde dehydrogenase
Therefore, use of this technique for the estima- and formate dehydrogenase (Woolfolk 1975). The
tion of enzymatic activity in culture filtrates will involvement of a demethylase enzyme in caffeine
not be accurate as formaldehyde accumulated as degradation by P. putida was further confirmed
a result of caffeine degradation may give erro- by Blecher and Lingens (1977). However, they
neous values. postulated that the demethylation was oxidative
The interconversion of NADH and NAD+ is rather than hydrolytic, as suggested by Woolfolk,
yet another method of estimating the activity of resulting in the formation of formaldehyde in-
methylxanthine demethylases which is assayed by stead of methanol (Fig. 2). Their studies also
following the decrease in absorbance at 340 nm showed that the demethylase enzymes were not
for the conversion of NADH to NAD+. Although constitutive but inducible by the addition of caf-
it is an effective method, when cell lysate is used feine and other methylxanthine that formed as a
as enzyme source the cellular NADH/NAD+ may result of degradation of caffeine. Similar caffeine-
interfere with the assay but this can easily be ta- demethylating activity was demonstrated in the
ken into account by carrying out appropriate crude extracts of P. putida, and was absolutely
control arrays. dependent upon NADH and NADPH as cofac-
Detection of caffeine degradation as a result of tors (Hohnloser et al. 1980). Attempts to purify
enzymatic activity by high performance liquid this enzyme were unsuccessful as this enzyme lost
chromatography (HPLC) is the most accurate its activity rapidly in partially purified extracts.
assay procedure. By the use of HPLC, not only Similar to caffeine demethylase, enzymes for
can the reduction of caffeine be monitored but demethylation of other methylxanthines have
also the presence of metabolites can be detected. been reported in the literature. Asano et al.
However, few reports are available in the litera- (1994) isolated a monooxygenase, i.e. theobro-
ture, where all the above-mentioned techniques mine demethylase, from cell free extracts of
were used to confirm the caffeine demethylase P. putida and reported that this enzyme specifically
activity (Glück and Lingens 1988; Asano et al. brought about the demethylation of theobro-
1994). mine. They also showed that the theobromine
demethylase enzyme was inhibited by Zn2+,
which generated the accumulation of theobro-
Bacterial caffeine-degrading enzymes mine (Asano et al. 1994). Another enzyme,
heteroxanthinedemethylase isolated from
N-Demethylases: P. putida has been shown to selectively convert
heteroxanthine (7-methylxanthine) to xanthine
The speculation that N-demethylation was the by oxidative demethylation with the formation
possible mechanism of conversion of caffeine to of formaldehyde (Glück and Lingens 1988). The
its methylated derivatives came from the study of enzyme was partially purified from cell-free ex-
Woolfolk who demonstrated methylxanthine tracts of P. putida and showed maximum activity
degrading activity in cell free extracts and sub- with NADH/NADPH as cofactors. It showed no
cellular fractions of P. putida (Woolfolk 1975). activity when either caffeine or dimethylxanth-
He suggested that the conversion of caffeine to its ines were used as substrates. Similar to caffeine
metabolites was brought about by an enzyme that demethylase, heteroxanthinedemethylase was
hydrolytically removed all the three-methyl also found to be unstable and loss of activity
groups with the production of methanol and free occurred upon storage.
123
Biotechnol Lett (2006) 28:1993–2002 1999
123
2000 Biotechnol Lett (2006) 28:1993–2002
Yamaoka-Yano and
Mazzafera (1999)
freeze drying techniques (Sideso et al. 2001) can
7.3
7.8
7.5
7.0
terial species, from genera such as Klebsiella,
–
30–35
20–30
11.4 lM 30
8.94 lM 35
169 lM 30
1.1 mM –
–
–
85
65
Caffeine and
above.
Substrate
Xanthine
Caffeine
Caffeine
Caffeine
Caffeine demethylase
Caffeine oxidase
putida
Alcaligenes sp.
Pseudomonas
Pseudomonas
Rhodococcus
3
4
6
7
123
Biotechnol Lett (2006) 28:1993–2002 2001
cific intermediates and also in food applications, Dash SS, Gummadi SN (2006) Biodegradation of caffeine
whereas decaffeination by whole cells is more by Pseudomonas sp. NCIM 5235. Res J Microbiol
1:115–123
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