Production of natural methyl anthranilate by microbial N-demethylation of N-methyl methyl anthranilate by the topsoil-isolated bacterium Bacillus megaterium

Article abstract of DOI:10.1021/jf0521395

Bacillus megaterium, isolated in a screening process from topsoil, was used for N-demethylation of natural N-methyl methyl anthranilate to produce natural methyl anthranilate. Maximal productivity of 70 mg/L/day was achieved under laboratory-scale conditions without further optimization. No byproducts were observed. Thus, production of natural methyl anthranilate using B. megaterium is a significant improvement over comparable already existing procedures.

Full text of DOI:10.1021/jf0521395

9586 J. Agric. Food Chem. 2005, 53, 9586−9589
Production of Natural Methyl Anthranilate by Microbial
N-Demethylation of N-Methyl Methyl Anthranilate by the
Topsoil-Isolated Bacterium Bacillus megaterium
MARCUS TAUPP,† DAG HARMSEN,‡ FRANK HECKEL,† AND PETER SCHREIER*,†
Lehrstuhl fu¨r Lebensmittelchemie, Universita¨t Wu¨rzburg, Am Hubland, 97074 Wu¨rzburg, Germany,
and Poliklinik fu¨r Paradontologie, Universita¨t Mu¨nster, Waldeyerstrasse 30, 48149 Mu¨nster, Germany
Bacillus megaterium, isolated in a screening process from topsoil, was used for N-demethylation of
natural N-methyl methyl anthranilate to produce natural methyl anthranilate. Maximal productivity of
70 mg/L/day was achieved under laboratory-scale conditions without further optimization. No
byproducts were observed. Thus, production of “natural” methyl anthranilate using B. megaterium is
a significant improvement over comparable already existing procedures.
KEYWORDS: Bacillus megaterium; bioflavor; biotransformation; methyl anthranilate; N-demethylation;
N-methyl methyl anthranilate
INTRODUCTION
many). All organic solvents used for extraction were distilled prior use.
Chemicals used for the preparation of liquid media were all of highest
purity.
Twenty years after the introduction of bioflavors into the
flavor market (1), the group of so-called “natural” flavor
compounds (2) has reached a well-defined position. Nowadays,
a number of industrially important natural flavor compounds
are settled on the market (3). The continuing demand by
consumers combined with the high economic value of natural
flavor substances have led to permanent improvements of
biotechnological processes, as well documentation of several
“key compounds” such as γ-decalactone or vanillin (4, 5).
Methyl anthranilate is the characteristic flavor compound of
Concord grapes and also appears in several essential oils such
as neroli and bergamot oils. The substance is also known as a
characteristic constituent of wood strawberry (6). Methyl
anthranilate is widely used in the industry for food flavorings
and perfume compositions. A consumption of 27 tons/year by
the flavor industry is reported but because of its very low
concentrations in plants, no methyl anthranilate “ex plant” is
available. A number of procedures to produce “natural” methyl
anthranilate have been reported (7-11). However, their ap-
plication is limited as the procedures suffer from low yields,
long reaction times, and formation of byproducts. In this paper,
we report an improvement of the microbiological production
of methyl anthranilate using Bacillus megaterium.
Microorganism. B. megaterium was isolated from topsoil in a
screening process and characterized by 16S rDNA and DNA/DNA
hybridization against the B. megaterium type strain ATCC 14581.
Isolation of Microorganisms from Topsoil. One gram of topsoil
(collected from garden soil in Karlstadt, Germany) was stirred in sterile
water for 20 min, and 100 µL of this solution was plated on Standard
Methods Agar (Bio Me´rieux), containing 10 µL of N,N-dimethylaniline,
and incubated at 30 °C. Nitrogen-adapted microorganisms were
singularized and cultivated on Standard Methods Agar plates. In total,
83 unknown cultures were obtained, and the isolated strains were used
for substrate acceptability tests by incubation in liquid media.
Substrate Acceptability Test with the Isolated Strains. Liquid
minimal medium (75 mL) was prepared according to the method of
Dworkin and Foster (20). A trace element solution (375 µL) was added,
and the culture medium was autoclaved (121 °C, 16 min) (21). The
culture was maintained under sterile conditions during the addition of
750 µL of a sterile glucose solution (50%). This liquid medium was
inoculated with freshly grown (on Standard Methods Agar plates)
bacteria, and the culture was pregrown for 18 h at 30 °C and 120 rpm.
Then, 100 µmol of N,N-dimethylaniline as substrate was added under
sterile conditions, and the mixture was shaken for a further 18 h at 30
°C and 120 rpm. After incubation, the culture was worked up by
sonication of the bacterial broth for 10 min and centrifugation at 15000g
for 20 min. The supernatant was extracted four times each with 40 mL
of diethyl ether; combined organic layers were dried over Na2SO4 and
filtered, and the organic solvent was evaporated under reduced pressure.
The residue was redissolved in 500 µL of dichloromethane and
submitted to gas chromatographic-mass spectrometric (GC-MS)
analysis to search for N-demethylated products. One strain, N-
demethylating N,N-dimethylaniline, was incubated in further experi-
ments with the substrate N-methyl methyl anthranilate.
MATERIALS AND METHODS
Chemicals. N-Methyl methyl anthranilate and methyl anthranilate
were obtained from Lancaster (Lancashire, U.K.). Natural N-methyl
methyl anthranilate was purchased from Aldrich (Schnelldorf, Ger-
* Author to whom correspondence should be addressed [telephone
+49-931-888-5481; fax +49-931-888-5484; e-mail schreier@
pzlc.uni-wuerzburg.de].
RIDOM 16S rDNA Sequencing of the Isolated Bacteria. DNA
preparation, DNA amplification, and DNA sequencing of the 16S
ribosomal RNA gene were performed as previously described by
Harmsen et al. (12). Briefly, the thermal cycling conditions consisted
† Lehrstuhl fu¨r Lebensmittelchemie.
‡ Poliklinik fu¨r Paradontologie.
10.1021/jf0521395 CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/29/2005

Microbial Production of Methyl Anthranilate
J. Agric. Food Chem., Vol. 53, No. 24, 2005 9587
of an initial denaturation (80 °C, 5 min) followed by 28 cycles of
denaturation (94 °C, 45 s), annealing (53 °C, 1 min), and extension
(72 °C, 90 s), with a single final extension (72 °C, 10 min). For
amplification, the broad-range primers 16S-27f (5′-AGA GTT TGA
TCM TGG CTC AG-3′) and 16S-907r (5′-CCG TCA ATT CMT TTR
AGT TT-3′) reported by Lane (13) were applied. The PCR product
was purified by an enzymatic method using exonuclease I (New
England Biolabs GmbH, Frankfurt-Hoechst, Germany) and shrimp
alkaline phosphatase (Amersham Biosciences, Freiburg, Germany) (14).
The amplicons were sequenced using the Prism BigDye Terminator v
3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems,
Weiterstadt, Germany). The sequencing reaction required 0.5 µL of
premix from the kit, 1.8 µL of Tris-HCl/MgCl2 buffer (400 mM Tris-
HCl; 10 mM MgCl2), 10 pmol of sequencing primer, and 2 µL of the
cleaned PCR product in a total volume of 10 µL. For 16S rDNA
sequencing either of the primers 16S-27f or 16S-519r (5′-GWA TTA
CCG CGG CKG CTG-3′) was used with an annealing temperature of
53 or 60 °C, respectively. All sequencing reactions were performed
using a T1 thermocycler (Whatman Biometra, Go¨ttingen, Germany)
with 25 cycles of denaturation (96 °C, 10 s), annealing (5 s), and
extension (60 °C, 4 min) and a thermal ramping of 1 °C/s. The
sequencing products were purified with MultiScreen HV plates
(Millipore, Billerica, MA) loaded with Sephadex G50 Superfine
columns (Amersham Biosciences) according to the instructions of the
manufacturer (Millipore Tech Note TN053), followed by preparation
for running onto the ABI Prism 3100 Avant Genetic Analyzer. The
region from base positions 54 to 510 (corresponding to Escherichia
coli 16S rDNA positions) for the 16S rDNA was analyzed using Ridom
TraceEdit Pro (version 1.0; Ridom GmbH, Wu¨rzburg, Germany)
software. Sequences from primer regions were therefore not included
in this analysis. Finally, a homology search of the sequence data against
the RIDOM database was performed (15, 16).
DNA/DNA Hybridization of the Isolated B. megaterium against
B. megaterium ATCC 14581 Type Strain (DSM 32T). The isolated
strain, identified as B. megaterium by 16S rDNA analysis, was subjected
additionally to a DNA/DNA hybridization analysis to reassess the
correctness of the given species name. B. megaterium ATCC 14581
(DSM 32T) type strain was purchased from DSMZ (Deutsche Sammlung
von Mikroorganismen und Zellkulturen, Braunschweig, Deutschland)
and cultivated in liquid media (medium 1, DSMZ) under the same
incubation conditions as described above. Briefly, DNA was isolated
using a French pressure cell (Thermo Spectronic) and purified by
chromatography on hydroxyapatite according to the method of Cashion
et al. (17). DNA/DNA hybridization was carried out as described by
De Ley et al. (18) considering the modifications described by Huss et
al. (19), using a Cary Bio UV-vis spectrophotometer equipped with a
Peltier-thermostated 6 × 6 multicell changer and a temperature
controller with in-situ temperature probe (Varian).
g; yeast extract, 3.0 g; beef extract, 1.5 g, MnSO4‚4 H2O, 1.0 µg);
Bacillus medium 2 [(NH4)2HPO4, 1.0 g; MgSO4‚7 H2O, 0.2 g, KCl,
0.2 g; yeast extract, 0.2 g]; Bacillus medium 3 (peptone, 10.0 g; lactose,
5.0 g; NaCl, 5.0 g; beef extract, 3.0 g; K2HPO4, 2.0 g). Finally, one
medium was checked that was recommended by the DSMZ for the
cultivation of B. megaterium (DSMZ medium 1: peptone, 5.0 g; meat
extract, 3.0 g); preparation of DMSZ medium 1 was carried out without
addition of MnSO4‚H2O. All media were used in the same volume of
75 mL as described above.
Extraction for Subsequent Analysis of Biotransformation Prod-
ucts. After shaking, the culture was worked up by sonication of the
bacterial broth for 10 min and centrifugation at 15000g for 20 min.
The supernatant was extracted four times each with 40 mL of diethyl
ether. The combined organic layers were dried over Na2SO4 (sicc) and
filtered, and the organic solvent was evaporated under reduced pressure.
The residue was redissolved in 2 mL of diethyl ether for subsequent
HRGC-MS analysis and quantification using a calibration curve.
High-Resolution Gas Chromatography-Mass Spectrometry
(HRGC-MS). HRGC-MS analysis was performed using an Agilent
6890 gas chromatograph (split injector 1:20) coupled to an Agilent
5973 mass selective detector. A J&W DB-Wax fused-silica capillary
column (30 m × 0.25 mm i.d., 0.25 µm film thickness) was employed.
The temperature program was 3 min isothermal at 50 °C, then raised
at 4 °C/min to 220 °C, using 1.0 mL/min helium constant flow. The
MS operating values were as follows: ionization voltage, 70 eV
(electron impact ionization); ion source and interface temperatures, 230
°C. Identification was carried out by comparison of mass spectrometrical
and retention data of authentic methyl anthranilate with those of the
sample.
RESULTS AND DISCUSSION
The used microorganism, isolated in a screening process from
topsoil, was identified as B. megaterium by standard 16S rDNA
analysis and DNA/DNA hybridization against B. megaterium
ATCC 14581. A direct comparison of the 16S rDNA partial
sequences indicated that the isolate belongs to the species B.
megaterium, showing a similarity of 99.6% for 456 base pairs
against the B. megaterium type strain (ATCC 14581, DSM 32T).
To complete the assignment of the isolate to the species B.
megaterium, DNA/DNA hybridization against the B. megate-
rium type strain was conducted. A mean DNA/DNA similarity
value of 83.2% was obtained. Therefore, the assignment of the
isolate to B. megaterium is justified, and the results of the 16S
rDNA analysis are supported by this technique.
B. megaterium was shown to be able to N-demethylate
N-methyl methyl anthranilate to yield methyl anthranilate
(Scheme 1). Byproducts, as previously reported in the course
of fungal demethylation of N-methyl methyl anthranilate (8),
were not observed.
Deposition of the Soil Isolate. The isolated B. megaterium strain
was deposited in the BCCM/LMG, Bacteria Collection, Laboratorium
voor Microbiologie, Universiteit Gent (RUG), B-9000 Gent, Belgium,
under the LMG no. 23147.
A series of studies was performed to check the biotransfor-
mation capacity of B. megaterium using different amounts of
N-methyl methyl anthranilate added to the incubation broth; they
varied from 10 to 100 mg/75 mL of incubation medium.
The variation of the substrate concentration led to the
following findings (Figure 1): Amounts of N-methyl methyl
anthranilate lower than 20 mg/75 mL did not heighten the yields
of methyl anthranilate; this means that a certain concentration
of substrate had to be present in the incubation medium to start
the microbial demethylation. Higher concentrations of N-methyl
methyl anthranilate, however, did not further increase the
Standard Incubation Method. Liquid minimal medium (75 mL)
was prepared according to the method of Dworkin and Foster (20). A
trace element solution (375 µL) was added, and the mixture was
autoclaved (121 °C, 16 min) (21). The culture was maintained under
sterile conditions during the addition of 750 µL of a sterile glucose
solution (50%). This liquid medium was inoculated with freshly grown
(on Standard Methods Agar plates) bacteria, and the culture was
pregrown for 18 h at 30 °C and 120 rpm. Various amounts of N-methyl
methyl anthranilate (10-100 mg) were added under sterile conditions,
and the mixture was shaken for a further 18 h at 30 °C and 120 rpm.
Control experiments without bacteria were carried out to verify the
stability and authenticity of the starting material.
Variation of Incubation Broth. All media tested were prepared
according to the method of Atlas and Parks (21). These were, in detail,
as follows (g/L): Bacillus broth one-fourth strength (yeast extract, 2.5
g; pancreatic digest of casein, 1.0 g); beef extract with NaCl (beef
extract, 10.0 g; NaCl, 5.0 g); diaminopimelic acid medium (pancreatic
digest of casein, 5.0 g; beef extract, 3.0 g; diaminopimelic acid, 0.050
g); Bacillus medium 1 (peptone, 6.0 g; pancreatic digest of casein, 3.0
Scheme 1

9588 J. Agric. Food Chem., Vol. 53, No. 24, 2005
Taupp et al.
Table 1. Advantages and Disadvantages of Already Reported Biotechnological Processes To Produce Natural Methyl Anthranilate (MA) Compared
with the B. megaterium Process [N-Methyl Methyl Anthranilate (NMMA)]
method
advantages
disadvantages
refs
enzymatic esterification of
anthranilic acid
other natural aromatic esters such as
methyl cinnamate or methyl
max yields of 10%; methanol used for
esterification denatures the enzyme;
10
salicylate can be obtained
expensiveness of pure enzymes
de novo biosynthesis of MA
large amounts of the biocatalysts can be
produced at anytime
short incubation times; up to 135 mg/L MA
in 10 min of incubation time by use
very low yields and long incubation times;
7, 9
11
max yield of MA in 5 days ) 18.7 mg/L
necessity of relatively pure enzymes;
production of byproducts
enzymatic N-demethylation
of NMMA
of pure soybean peroxidase
microbial N-demethylation
of NMMA by fungal
microorganisms
large amounts of biocatalysts can be
produced at anytime
low yields and long incubation times; production
of the byproduct N-formyl methyl
anthranilate; toxicity of NMMA and MA;
max yield of MA after 24 days of
8, 22
incubation
N-formyl methyl anthranilate
toxicity of NMMA and MA against
)
7 g/L, but production of
2.8 g/L
)
microbial N-demethylation
of NMMA by
B. megaterium
70 mg/L/day under lab-scale conditions;
fast-growing and easy to handle
microorganisms; no formation of
byproducts; short incubation times
this work
B. megaterium
production of methyl anthranilate; this is caused by the fact that
both, substrate and product, exhibit toxic effects toward B.
megaterium, as proven by measurement of the optical density
(data not shown). Therefore, 20 mg/75 mL of N-methyl methyl
anthranilate was the optimal substrate concentration for methyl
anthranilate production under the mentioned laboratory condi-
tions. Extension of the incubation time up to 48 h did not
increase the yield of methyl anthranilate.
In addition, different incubation media were tested to
investigate the influence of the medium on the N-demethylation
activity of B. megaterium toward N-methyl methyl anthranilate.
Among the media under study (see Material and Methods),
Bacillus broth one-fourth strength, beef extract with NaCl,
diaminopimelic acid medium, and DSMZ medium 1 were
especially recommended for the growth of B. megaterium,
whereas the others were media for Bacillus species in general
(21).
As shown in Figure 2, remarkable differences were observed
using various incubation media. At first glance, the differences
in optical density are obvious. Bacillus medium 1, Bacillus
medium 3, and DSMZ medium 1 provided good accommodation
for the nutrition of B. megaterium witnessed through fast growth.
Bacillus broth one-fourth strength and beef extract medium with
NaCl showed lower growth of bacterial cells. Finally, Bacillus
medium 2 led to descent of the bacterial cells maybe not having
the important growth nutrients necessary for reproduction. In
contrast to this, a low growth of bacterial cells resulted in a
high amount of methyl anthranilate using Bacillus broth one-
fourth strength. Interestingly, the highest growth of B. mega-
terium using Bacillus medium 1 yielded only 13% product. This
clearly indicates not only that the demethylation is dependent
on the number of bacterial cells but also that the composition
of the incubation medium plays an important role in this process.
The Bacillus broth one-fourth strength, containing only yeast
extract and pancreatic digest of casein, seems to have some
necessary content that is responsible for the microbial N-
demethylation. Unfortunately, this medium led to the formation
of a strong emulsion during solvent extraction. In the end, none
of the tested media were able to heighten the yield of MA during
the incubation process in comparison to the used defined
standard medium (Figure 1) containing only various salts and
glucose as carbon source. Advantageously, this medium is quite
cheap to prepare and shows a well-defined composition, and
the solvent extraction is easy to handle.
In comparison to other methods for the biotechnological
production of natural methyl anthranilate (Table 1), the N-
Figure 2. Influence of incubation medium on the production of methyl
anthranilate (MA) by B. megaterium. Cells were grown to an optical density
of 0.05. Optical density was measured 4 h (gray bars) and 24 h (black
bars) after addition of 20 mg/75 mL of N-methyl methyl anthranilate.
Figure 1. Influence of substrate concentration on yield of N-demethylation
of N-methyl methyl anthranilate (NMMA) by B. megaterium. The highest
yield of methyl anthranilate (MA) [
9 (mg); 2 (%)] was reached by addition
of 20 mg of substrate to the incubation broth (75 mL), resulting in 5 mg
of MA after 18 h.
Highest yields [
2 (%)] were achieved by using Bacillus broth one-fourth
strength medium. For incubation media, see Materials and Methods.

Microbial Production of Methyl Anthranilate
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demethylation of N-methyl methyl anthranilate by B. megate-
rium has some considerable advantages.
Anthranilic acid, used as a natural precursor of methyl
anthranilate, has been found in acid hydrolysates of casein and
peptone, being cheap byproducts of the food industry. By the
use of microbial enzymes in the presence of 10% methanol,
anthranilic acid was esterified to methyl anthranilate. However,
this enzymatic esterification of anthranilic acid to obtain methyl
anthranilate suffers from problems such as low yields and the
expensiveness of the biocatalysts. As the used methanol showed
denaturation effects of the protein, maximum yields of 10% of
MA were obtained. On the other hand, such a procedure allows
the production of other important compounds such as methyl
cinnamate or methyl salicylate (10).
The de novo synthesis of methyl anthranilate by two species
of fungi belonging to the Polyporaceae, Pycnoporus cinnabari-
nus and Poria cocos, has been reported. Unfortunately, this
method is afflicted with long incubation times and low yields.
Best results were obtained with P. cinnabarinus I-397 at a low
nitrogen concentration, the use of maltose as the carbon source,
uncontrolled pH, and 1-day-old spores as the source of inocu-
lum; 18.7 mg/L of methyl anthranilate was produced under these
conditions after 5 days of cultivation (7, 9).
In addition, different peroxidase preparations were used to
demethylate N-methyl methyl anthranilate (11). One clear-cut
benefit is the short incubation time using these enzyme
preparations. However, the use of relatively pure enzymes and
the lack of reusability of the biocatalyst are counterproductive
for largeup-scale processes.
The application of a microbial system for the N-demethylation
of N-methyl methyl anthranilate has also been reported (8).
However, this patented N-demethylation process using fungal
microorganisms such as Trametes and Polysporus species is
expensive, incubation times are long, the yields are low, and
an unwanted byproduct, N-formyl methyl anthranilate, is also
produced (22). Despite maximum yields of 7 g/L/24 days, this
process requires an additional time- and cost-expensive prein-
cubation period of 13 days with a subsequent incubation period
of a further 11 days. In contrast, the isolated B. megaterium is
a fast-growing and easy to handle bacterium.
As byproducts in the N-demethylation of N-methyl methyl
anthranilate by our isolated B. megaterium were not detectable
and the productivity of 70 mg/L/day is quite acceptable under
laboratory-scale conditions without any further optimization, the
herewith reported procedure seems to be very promising. Trials
to scale-up the process are under development.
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Received for review August 31, 2005. Revised manuscript received
September 30, 2005. Accepted September 30, 2005.
JF0521395