Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: www.tandfonline.com/journals/tbbb20

Microbial Production of Theobromine from

Caffeine

Yasuhisa Asano, Toshihiro Komeda & Hideaki Yamada

To cite this article: Yasuhisa Asano, Toshihiro Komeda & Hideaki Yamada (1993) Microbial
Production of Theobromine from Caffeine, Bioscience, Biotechnology, and Biochemistry, 57:8,
1286-1289, DOI: 10.1271/bbb.57.1286

To link to this article: https://doi.org/10.1271/bbb.57.1286

Published online: 12 Jun 2014.

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Biosci. Biotech. Biochem., 57 (8), 1286-1289, 1993

Microbial Production of Theobromine from Caffeine

Yasuhisa ASANO, t Toshihiro KOMEDA, * and Hideaki YAMADA *
Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, Kosugi, Toyama 939-03, Japan
and * Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan
Received January 18, 1993

Production of theobromine from caffeine by caffeine-degrading bacteria was studied. We found that
addition of metal ions such as Zn 2 + to intact cells of a caffeine-degrading isolate from soil, Pseudomonas
sp. No.6, resulted in a high theobromine accumulation from caffeine. We hypothesized that Zn 2 + acts as
a selective inhibitor of one of the theobromine-demethylating enzymes and further screened for
theobromine-producing activities in the presence of Zn 2 + among a number of caffeine-using microorganisms.
A strain identified taxonomically as Pseudomonas putida No. 352 showed the best productivity among 973
microorganisms of stock cultures and soil isolates. Culture conditions for the production of theobromine
from caffeine by P. putida No. 352 were studied. Under optimal conditions, nearly 20 g/liter of theobromine
was produced from caffeine in a yield of 92%.

Caffeine (l,3, 7-trimethylxanthine) is an alkaloid naturally caffeine in a high amount and yield.
occurring in coffee and cocoa beans, cola nuts, and tea
leaves, etc. It is one of a group of xanthine derivatives Materials and Methods
including theobromine (3,7-dimethylxanthine) and the- Materials. Caffeine, theobromine, and uric acid were purchased from
ophylline (l,3-dimethylxanthine).1) In mammals, ingested Wako Pure Chemicals, Osaka, Japan, theophylline, 1,3-dimethylbarbituric
caffeine is rapidly absorbed, metabolized, and excreted in acid, 1,3-dimethyl-3,4,5,6-tetrahydro-2(lH)-pyrimidine, I-methylimidaz-
the urine as methyl xanthine derivatives. It has a variety of ole, and I-methylhydantoin were from the Aldrich Chemical Co., Inc,
Milwaukee, Wisconsin, U.s.A. 1,3-Dimethyluracil was synthesized from
biological effects: it stimulates the central nervous system, uracil and dimethylsulfuric acid. 10) Other chemicals used were commercial
shows toxicity when fed excessively, and is even mutagenic products.
in vitro. 2 - 4 ) Although theobromine shows caffeine-like
pharmacological activities, it does not cause excessive Isolation of caffeine-degrading microorganisms. Caffeine-degrading
microorganisms were isolated from soil samples by an enrichment culture
physiological reactions as caffeine does, therefore it has been
technique using a medium containing 5 g caffeine, 2 g K2 HPO 4' 1g NaCI,
used as diuretic. Theobromine is obtained by an extraction 0.5 g MgSO4' 7H 0, 05 g yeast extract, 10 J,lg thiamine' HCI, 20 J,lg
2
from plants (such as coffee, cocoa, and tea) or by riboflavin, 20 J,lg pantothenate' HCl, 20 J,lg pyridoxine' HCI, 1J,lg biotin,
complicated chemical synthesis. 5) 10 J,lg p-aminobenzoate, and 20 J,lg nicotinic acid in 1 liter of tap water (PH
Lingens et al. investigated caffeine metabolism by a 7.0).
caffeine-degrading bacterium, Pseudomonas putida. They
Assay of theobromine production. Isolated strains were cultivated
proposed a pathway of caffeine degradation as an aerobically at 28°C for 15 h on a medium containing 3 g caffeine, 5 g
experimental result of successive formation of theobromine K 2HP04 , 0.02g FeS04 '7H 20, and various amounts of organic nutrients
followed by 7-methylxanthine from caffeine in the culture as described in the text in 1 liter of tap water (PH 7.0), to which was
broth of P. putida (Fig. 1).1) 7-Methylxanthine is further further added caffeine at one time to a concentration of 20 g/liter with or
demethylated by heteroxanthine demethylase 7) to produce without 1 mM metal ions, and incubated at 28°C with moderate shaking
for 1 h to 2 days. Activity of theobromine production was expressed as
xanthine, which then is metabolized by the cells to end the amount of theobromine formed in 1 h (g/liter· h) at 28°C with moderate
products such as urea and glyoxylic acid. Caffeine shaking, using a reaction mixture (4ml) containing 0.08g caffeine, 0.02g
demethylase which yields theobromine from caffeine, has K 2 HPO4' 0.08 mg FwSO 4' 7H 2 0, 0.02 g yeast extract, 1 mM Zn2+, and
also been isolated from the same strain and partially cells from 4 ml of culture broth in tap water (PH 7.0). After an addition
of 0.5 ml of 1 N HCI to stop the reaction, the amount of theobromine
characterized. 8 ) Although they derived mutants of P. putida formed in the mixture was measured with a Shimadzu LC-6A
that accumulate mostly 7-methylxantine with a trace high~performance liquid chromatography (HPLC), with an M&S pack
amoun t of theo bromine, 9) they were unable to isolate those CI8 column (reversed phase column, 4.6 x l50mm; M&S Instrument,
which exclusively accumulate theobromine. 7) Tokyo, Japan) at a flow rate of 1.5 ml/min, using a solvent system
In this paper, we describe the results of the first study on containing 1% acetic acid: methanol=80: 20 (v/v). Theobromine and
caffeine were detected spectrophotometrically at 273 nm. When theo-
the microbial production of theobromine selectively from bromine was precipitated in the reaction mixture due to its low solubility,
its concentration was estimated after dissolving it completely by an addition
o ~H3 0 ~H3 0 ~H3 0 H of2N NaOH.

H').?L~ "\ "'?L~

CH
3
HCHO CH
3
"\o:(JL-~ "\ol-X~ -
HCHO H HCHO H
Assay of caffeine-degrading activity. The degradation of caffeine was
monitored by thin-layer chromatography (TLC) using Silicagel 70
Plate-Wako (Wako Pure Chemical Industries, Osaka, Japan) with a solvent
Caffeine Theobromine 7-Methylxanthlne Xanthine
system containing ethyl acetate: methanol: aqueous ammonia (28 %) = 4 :
Fig. 1. Proposed Metabolic Pathway of Caffeine in Pseudomonas putida. 2: 1 (v/v). The strains isolated from soil were cultivated aerobically at 28°C

t Corresponding author.
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 Microbial Production of Theobromine 1287

for 3 days on the isolation medium. Strains from type culture collections roqueforti, Stremphylium sp., and Bacillus coagulans 12 )
tested included 33 strains of 29 genera of bacteria, 30 strains of 15 genera degrade caffeine, almost nothing is known about their
of yeasts, and 35 strains of 15 genera of molds. They were obtained from metabolism of caffeine. We examined caffeine-degrading
stock cultures of Toyama Prefectural University (TPU). The bacteria were
cultivated at 28°C for 3 days on a medium containing 5 g of caffeine, 3 g
abilities of various microorganisms. Bacterial cultures
of tryptone, 3 g of meat extract, 1g of yeast extract, and 1 g of glucose in excluding the genus Pseudomonas, yeast, and molds in the
1 liter of tap water (pH 7.0); the yeasts on a medium containing 5 g of stock culture of Toyama Prefectural University (TPU) were
caffeine, 5 g of peptone, 3 g of yeast extract, 3 g of malt extract, and 109 chosen to screen for the activity to degrade caffeine. None
of glucose in I liter tap water (pH 5.6); and the molds on a potato sucrose of the bacteria (33 strains), yeast (30 strains), and molds
medium containing 5 g caffeine in l·liter of this medium (pH 6.0). The
potato sucrose medium was prepared as follows: washed potatoes were (35 strains) of the stock cultures could degrade caffeine.
cleaned with tap water, peeled, and cut into l·cm cubes. Two hundred Next, theobromine production in the presence of 1 mM Zn 2 +
grams of potato cubes were boiled with 1 liter of tap water for 20 min. was tested among 973 isolated strains which are capable of
They were mashed and squeezed through a muslin bag. Then 20 g of growth on caffeine. Theobromine production ranged from
sucrose was added to the potato extract. The solution was made up to 1
liter and its pH adjusted.
o to more than 10 g/liter. The numbers of strains which
The culture broths were spotted on TLC plates and the degradation of accumulated theobromine were 893 strains (0 to 5 g/liter),
caffeine was visually estimated under UV light at 265 nm. For the screening 61 (5 to 10 g/liter), and 19 (more than 10 g/liter). Theo-
for high theobromine producers, the isolated strains were cultivated on bromine was not detected in the culture broth, when the
the basal medium containing 0.5% yeast extract. The cells were harvested latter 19 strains were cultivated in the absence of I mM
by centrifugation and washed once with 0.01 M potassium phosphate buffer,
Zn 2 +.
pH 7.0, and incubated in the presence of I mM of Zn2+ for 2 days under
the conditions as described above.
Identification of microorganism
Isolation of theobromine. The supernatant of the reaction mixture A bacterial isolate from soil, No. 352, was chosen as the
containing insoluble theobromine was removed by centrifugation. The best producer of theobromine, and identified taxonomical-
precipitated theobromine was dissolved in a small amount of 2 N NaOH,
and again centrifuged to remove the microbial cells. The supernatant
ly as follows. Non-fementative rod. Non spore-forming.
containing the theobromine was filtered through a cellulose nitrate filter Motile. Gram-negative. Nitrate reduction: negative. Indole
(0.22 f-Lm, Advantec, Tokyo, Japan), and put into the HPLC system. The production: negative. Catalase and oxidase: positive.
peak containing theobromine was collected and crystallized after Pigment production in King's B medium. No acid from
evaporation. Isolated theobromine was identified with an authentic sample glucose, and maltose. Assimilation of carbon compounds:
by the criteria of retention times in HPLC and mass and infrared spectra.
caprate, citrate, benzylamine, gluconate, glucose, malate,
mannose, and phenylacetate are assimilated; adipate,
Results and Discussion arabinose, inositol, maltose, N-acetylglucosamine are not
Inhibition of theobromine-degradation by metal ions assimilated. Arginine dihydrolase: positive. Urease: nega-
We found that almost no theobromine was produced tive. According to "Bergey's Manual of Systematic
when ca:t!eine was incubated with the intact cells of several Bacteriology",!3) the strain was identified as Pseudomonas
unidentified bacteria isolated from soil using caffeine as a putida.
sole source of carbon and nitrogen. We hypothesized that The effects of addition of Cu 2+, Mn2+, Pb 2+, Cd 2+,
the demethylation of caffeine is catalyzed by several enzymes Co 2+, Mg2 +, Ni 2+, and Zn 2+ (1 mM each) on the relative
and a selective inhibition of one of the enzymes may cause amount of theobromine accumulation in 2 days as described
an accumulation of theobromine in a high yield. A in Materials and Methods were 160, 168, 200, 13, 89, 116,
caffeine-degrading isolate from soil, Pseudomonas sp. No. 155, and 2630/0, respectively, as compared with the control
6, was randomly chosen among caffeine degraders from soil in which theobromine was accumulated up to 4.0 g/liter
and used to test effects of various metal ions on the (taken as 100%). Zn 2+ was found to be the most effective
accumulation of theobromine from caffeine. Pseudomonas additive, with the optimal concentration of 1 mM, as
sp. No. 6 was cultivated on the basal medium containing observed in Pseudomonas sp. No.6. However, the degree
0.5% yeast extract. The washed cells were incubated in the of the effects of addition of metal ions such as Cu 2+, Mn 2 +,
presence of various metal ions for 2 days under the and Mg2 + differed greatly between Pseudomonas sp. No.6
conditions described above. Effects of addition of Cu 2 +, and Pseudomonas putida No. 352, probably due to different
Fe 2 +, AI 3 +, Mn2+, Pb 2 +, Cd 2+, C0 2+, Mg2+, Sr 2 +, Ni 2+, sensitivity of their caffeine-degrading enzymes toward the
and Zn 2 + (1 mM each) for the theobromine accumulation metal ions.
were 31, 128, 101, 76, 46, II, 97, 0, 0, 147, and 238%,
respectively, as compared with the control in which Culture conditions for the theobromine production by P.
theobromine was accumulated up to 2.72 g/liter (taken as putida No. 352
100 % ). Although the degradation of caffeine may be started Culture conditions for the theobromine production by P.
by one of the three demethylation reactions as mentioned putida No. 352 were investigated.
above, only little is known about the enzymes catalyzing (i) Carbon and nitrogen sources. Effects of addition of
the reactions. The observation that caffeine degradation by glycerol, glucose, and fructose as carbon sources on
Pseudomonas sp. No.6 was inhibited with an accumulation theobromine production by P. putida No. 352 were studied.
of theobromine by Zn 2+ supports the hypothesis that Fructose was the most suitable carbon sources for cell growth
caffeine is actually demethylated to 7-methylxanthine by and theobromine production, as shown in Table I. The
more than two enzymes in Pseudomonas sp. optimal concentration of fructose was 1.0%. Addition of
glycerol stimulated the theobromine production, although
Screening for microorganisms producing theobromine glucose did not. Next, effects of various nitrogen sources
Although there are reports that yeasts,11) Penicillium on theobromine production by P. putida No. 352 wete
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 1288 Y. ASANO, T. KOMEDA, and H. YAMADA

Table I. Effects of Carbon and Nitrogen Sources on Theobromine

Production by P. putida No. 352
Activity - -0-. Growth
After P. putida No. 352 were cultivated in each medium, reactions in
~ -II- caffeine
the presence of Zn 2+ were done under the conditions as described in 8 .:. -t:J- ActlvHy
Materials and Methods. The reaction time was 1 h. a,
to
Theobromine Growth Theobromine
....
Carbon G rowth Nitrogen 0.6
source (OD 610 )
produced
(g/liter'h) source (
OD
610)
produced
(g/liter'h) 1.5 §
"0 -
'o;!/.
ea. Ci)
None 3.55 0.319 None 0.451 0.0271 c
Glycerol Yeast extract ~ 'i
:::as
.::
0.5%
1.0%
4.22
4.78
1.10
1.23
0.5%
2.0%
4.78
7.80
1.53
0.496
2
~ (,)

2.0% 2.99 0.249 Casitone

Glucose 0.5% 4.90 1.78 o
0.5% 4.08 0.543 2.0% 6.02 0.911 3 6 9 12 15 18 21
1.0% 4.40 0.332 Meat extract Cultivation time(hr)
2.0% 2.99 0.249 0.5% 3.11 0.763 Fig. 2. Course of Growth and Theobromine Production by P. putida
Fructose 2.0% 7.19 0.156 No. 352.
0.5% 5.26 1.40 Tryptone The cultivation and reaction conditions were the same as described in Materials
1.0% 5.56 1.53 0.5% 5.11 2.12 and Methods. TB stands for theobromine.
2.0% 6.10 1.21 2.0% 6.32 1.15

Table II. Effects of Various Metal Ions on the Cultivation of P. putida decreased by cultivating with Zn 2+, Ni 2+, Pb 2+, Cu 2+, and
No. 352 and the Production of Theobromine Mn2+, which appeared to inhibit theobromine degradation
Various metal ions were added to the medium shown in Materials and in a system using intact cells as described above. On the
Methods. After P. putida No. 352 was cultivated in each medium, reactions
other hand, when cultivated with Fe 2+, about 10 times more
in the presence of 1 mM Znz + were done for 1 h. Cell growth was expressed
as absorbance at 610 nm.
theobromine was produced than the control. The optimal
concentration of Fe 2 + was 0.004%. This result suggests
Concentration Growth
Theobromine that Fe 2+ may participate in the production of the
Metal ion produced demethylating enzymes or be important as a cofactor of the
(%) (OD610 )
(g/liter' h) demethylating enzyme(s).
None 4.49 0.27
(iv) Amino acids. Effects of various amino acids were
ZnCl z 0.002 1.66 0.02 investigated at concentrations of 0.1 and 0.2%. Addition
NiCl z 0.002 1.78 0.01 of L-arginine, L-aspartic acid, glycine, L-glutamic acid, and
PbCl z 0.002 4.76 0.17 L-threonine increased theobromine production by 1.1 to 1.8
CuCl z 0.002 3.98 0.05 times as compared with the control. However, the addition
MgS0 4 0.002 4.60 0.23 of L-cysteine, L-histidine, and L-serine decreased theo-
MnCl z 0.002 3.75 0.043
bromine production as well as the cell growth. L-Glutamic
FeS0 4 0.002 5.59 2.32
0.004 5.41 2.71
acid was found to be the most suitable additive for the
0.008 5.12 2.48 production of theobromine with its optimal concentration
0.010 5.13 2.14 of 0.1 0/0.
0.020 2.55 0.46 (v) Inducer. Since the enzyme or enzymes catalyzed
caffeine degradation appeared to be inducible, the inductive
effects of various compounds having an N-methyl group on
studied (Table I). Various nitrogen sources were added to theobromine production was investigated. Caffeine and
the basal medium. Tryptone (0.5 and 1.0%) was found to theobromine had an inductive effect, although structurally
be the most effective nitrogen source among various organic related theophylline, uric acid, 1,3-dimethylbarituric acid,
nutrients tested as compared with the control in which yeast 1,3-dimethyluracil, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-
extract was omitted from the basal medium. The optimal pyrimidine, I-methyl imidazole, and I-methylhydantoin
concentration of tryptone was 0.5%. Effects of other (each at 0.3% concentration) were all inert as inducers. The
nitrogen source such as yeast extract, Casitone and meat effects of concentration of caffeine and theobromine was
extract were also examined. Addition of yeast extract examined. Addition of 0.6% caffeine was about 1.3-fold
stimulated both the growth and theobromine production. more effective than 0.3 % caffeine. Addition of 0.3 %
Casitone was found to be also effective, followed by meat theobromine was effective to 0.8-fold that of 0.3% caffeine.
extract. (vi) Cultivation time. The medium for P. putida No. 352
(ii) Vitamins. Addition of various vitamins (thiamine' was thus optimized as follows; 5 g of caffeine, 5 g of
HCI, calcium pantothenate, pyridoxine' HCI, biotin, L- K 2HPO 4, 5 g of tryptone, 109 of fructose, I g of sodium
ascorbic acid, folic acid, nicotinic acid, nicotine amide), L-glutamate, and 0.04 g of FeS0 4 ' 7H 2 0 in l1iter tap water
at 20 J.lg/liter concentration each were not effective either (pH 7.0). The time course of theobromine production in
for cell growth or theobromine production. the cultivation of P. putida No. 352 on this medium was
(iii) Metal ions. Effects of addition of various metal ions shown in Fig. 2. The activity of theobromine production
are shown in Table II. Theobromine production was was found to be the highest after 15-18 h cultivation ...
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 Microbial Production of Theobromine 1289

degrading bacterium P. putida No. 352, as the best producer

:::i' 25 Theobromine of theobromine among caffeine-degrading microorganisms
C, Yield in a screening sysem containing Zn 2 +. Theobromine was
92%
C
o
20 obtained in higher amounts and yield when the intact cells
;::
o

-B
E 15
c
C
10
cultivated in the medium containing caffeine as an inducer
were incubated with caffeine in the presence of Zn 2 + .
Purification of the product, theobromine, was easy because
o most of the theobromine formed was precipitated in the
(.) 5
reaction mixture.
Oo-~~-'~'-~~~~-r~~~ This is the first report on the microbiological production
o 6 12 18 24 30 36 42 48
Reaction time(hr) of theobromine selectively from caffeine in a high yield.
Caffeine has various pharmacological activities, and high
Fig. 3. Course of Conversion of Caffeine to Theobromine by P. putida levels of caffeine are toxic. 2 ,3) Caffeine may be removed by
No. 352. a microbial method from some foods to produce de-
The caffeine and theobromine were measured by HPLC. Caffeine (~.) and theobro- caffeinated coffee. 14) Since caffeine is a cheap starting
mine (0).
material, it can be used as a feedstock for bacteria such as
P. putida in the production of theobromine as described in
Production of theobromine by P. putida No. 352 this study. Further studies to show a selective inhibition of
Theobromine was produced by P. putida No. 352 under one of the theobromine-degrading enzymes by Zn 2 + are in
the conditions optimized as described above. After the strain progress.
had been cultured at 28°C for 15 h, 1 mM of Zn 2 +, and
25 g/liter of caffeine was added to the culture broth, and References
incubated at 28°C with a moderate shaking. After 48 h of
1) T. W. Rall, in "The Pharmacological Basis of Therapeutics," 7th
reaction, 20.1 g/liter of theobromine was formed, as shown Ed., ed. by Goodman and Gilman, Macmillan, New York, 1984, pp.
in Fig. 3. The solubility of theobromine is so low (maximum, 589-603.
0.5 g/liter water) that the reaction mixture became muddy 2) D. M. Graham, Nutrition Rev., 36, 97-102 (1978).
with the precipitated theobromine. Infrared- and mass- 3) P. E. Stephenson, J. Am. Diet. Assoc., 71, 240-247 (1977).
4) J. Timson, Mut. Res., 47, 1-52 (1977).
spectra, and retention time in HPLC (2.3 min) proved that
5) W. Hutzenlaub and W. Pfieider, Liebigs. Ann. Chern., 11, 1847-1854
the isola~d theobromine was identical with the authentic (1979).
one. MS(mlz: 180 (M+, relative intensity 100%), 137 (8), 6) R. Blecher and F. Lingens, Hoppe-Seyler's Z. Physiol. Chern., 358,
109 (30); 82 (30), 67 (47), 55 (50). IR Vmax (KBr) cm -1: 807-817 (1977).
3450, 3175, 3075, 3000, 2850, 1680, 1550, 1410, 1220. The 7) M. GlUck and F. Lingens, Appl. Microbiol. Biotechnol., 28, 59-62
(1988).
yielq. with respect to the reacted caffeine was about 92%.
8) W. Hohnloser, B. Osswald, and F. Lingens, Hoppe-Seyler's Z.
Llng~ns et al. isolated a caffeine-degrading bacterium, P. Physiol. Chern., 361, 1763-1766 (1980).
putida . WS, and derived a mutant accumulating a mixture 9) M. Gluck and FI Lingens, Appl.. Microbiol. Biotechnol., 25, 334-340
of theobromine (0225 g/liter) and 7-methylxanthine (0.425 (1987).
g/liter, 50.2% from caffeine), when caffeine was completely 10) G. Bram, G. Decodts, Y. Bensaid, C. C. Farnoux, H. Galons, and
M. Miocque, Synthesis, 1985, 543-545.
consumed. 9 ) However, the mutant has drawbacks in that it
11) M. Sauer, O. Kappeli, and A. Fiechter, Developments in Biochem.,
accumulates a mixture of theobromine and 7-methyl- 23, 452-457 (1982).
xanthine' and has a tendency to rev:ert. They were unaware 12) S. Schwimmer, R. H. Kurtzmann, Jr., and E. Heftmann., Arch.
that metal ions may cause accumulation of theobromine from Biochem. Biophys., 147, 109-113 (1971).
caffei~e, nor did they optimize the conditions to yield 13) N. J. Palleroni, in "Bergey's Manual of Systematic Bacteriology,"
Vol. 1, ed. by N. R. Krieg and J. G. Holt, The Williams and Wilkins
theobromine. We are the first to show that a selective effect Co., Baltimore, 1984, pp. 141-199.
by Zn 2 + caused an accumulation of theobromine. Based 14) R. H. Kurtzmann, Jr. and S. Schwimmer, Experientia, 27, 481-482
on this observation, we further isolated the caffeine- (1971).

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