Enzymatic Synthesis of γ-Glutamylvaline to Improve the Bitter Taste of Valine

Article abstract of DOI:10.1021/jf0347564

The taste of several bitter amino acids is reduced, sourness produced, and preference increased by γ-glutamylization. An enzymatic method for synthesizing γ-Glu-Val involving bacterial γ-glutamyltranspeptidase (GGT) was developed. The optimum reaction conditions for the synthesis of γ-Glu-Val were 20 mM Gln, 300 mM Val, and 0.04 U/ml GGT, pH 10. After 3-hr incubation at 37 °C, 17.6 mM γ-Glu-Val was obtained, with the yield being 88%. γ-Glu-Val was purified on a Dowex 1 × 8 column and then identified by NMR.

Full text of DOI:10.1021/jf0347564

J. Agric. Food Chem. 2004, 52, 577−580 577
Enzymatic Synthesis of γ-Glutamylvaline to Improve the Bitter
Taste of Valine
HIDEYUKI SUZUKI,* KENJI KATO, AND HIDEHIKO KUMAGAI†
,
†
‡
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa,
Sakyo-ku, Kyoto 606-8502, Japan, and Department of Life and Biomaterials Science,
Faculty of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
The taste of several bitter amino acids is reduced, sourness produced, and preference increased by
γ-glutamylization. An enzymatic method for synthesizing γ-Glu-Val involving bacterial γ-glutamyl-
transpeptidase (GGT) was developed. The optimum reaction conditions for the synthesis of γ-Glu-
Val were 20 mM Gln, 300 mM Val, and 0.04 U/ml GGT, pH 10. After 3-hr incubation at 37 °C, 17.6
mM γ-Glu-Val was obtained, with the yield being 88%. γ-Glu-Val was purified on a Dowex 1 × 8
column and then identified by NMR.
KEYWORDS: γ-glutamyltranspeptidase; γ-glutamyl amino acid; γ-glutamylvaline; bitter taste; enzymatic
synthesis
INTRODUCTION
synthesized. In fact, aromatic amino acids and basic amino acids
are good acceptors. Unfortunately, however, branched-chain
amino acids are poor ones, as described previously (7), and we
have to overcome this problem to synthesize γ-glutamyl
branched-chain amino acids using bacterial GGT.
Some L-amino acids are known as bitter amino acids.
Branched-chain amino acids, basic amino acids, and aromatic
amino acids are bitter (1). Several essential amino acids for
human beings are bitter. Therefore, when an amino acid mixture
is administered orally, the bitterness of these amino acids is a
crucial problem. These days, various kinds and large amounts
of beverages called “supplements” are sold by several companies
in Japan. These beverages contain large amounts of branched-
chain amino acids, and some aromatic and/or basic amino acids.
Without the addition of sweeteners and flavoring, their taste
would be terrible. We discovered and reported that the
γ-glutamylization of bitter amino acids abolished or at least
reduced their bitterness and improved preference of the panel
members of the taste test (2). We have developed an enzymatic
method involving bacterial γ-glutamyltranspeptidase (GGT, EC
MATERIALS AND METHODS
Reagents and the Enzyme. The amino acids and γ-glutamyl amino
acids used were all of the L-form. Amino acids were purchased from
Nacalai Tesque (Kyoto, Japan), γ-Glu-Gln was from Sigma Chemical
(
(
St. Louis, MO), and γ-Glu-Val and γ-Glu-Leu were from Bachem
Bubendorf, Switzerland). Escherichia coli K-12 strain SH642, which
harbors pUC18 with the E. coli GGT gene, was grown at 20 °C (8) in
LB broth containing 100 µg/mL ampicillin, and the GGT over-produced
was purified from the periplasmic fraction (9) as described previously
(5).
Measurement of GGT Activity. GGT activity was measured as
2
.3.2.2) and various amino acids as substrates for synthesizing
described previously (7). One unit of enzyme was defined as the amount
of enzyme that released 1 µmole of p-nitroaniline per min from
γ-glutamyl-p-nitroanilide through the transpeptidation reaction.
Measurement of γ-Glutamyl Amino Acids and Gln. The concen-
trations of γ-Glu-Val, γ-Glu-Ile, γ-Glu-Gln, Gln, and Val were
measured with a high-performance liquid chromatograph (HPLC)
equipped with a Shim-pack Amino-Na column and a fluorescence
detector (model LC-9A; Shimadzu, Kyoto, Japan), with o-phthalalde-
hyde as the detection reagent, as described previously (10). That of
γ-Glu-Leu was measured by a reverse phase HPLC as described
previously (11).
γ-glutamyl amino acids, including γ-glutamylphenylalanine,
γ-glutamyltyrosine methylester, and γ-glutamylhistidine (2-
4
). The method is superior because (i) glutamine, which is less
expensive than glutathione, can be used as a γ-glutamyl donor
efficiently; (ii) GGT does not require any energy source such
as ATP and product inhibition by ADP is not a problem; (iii)
a great deal of GGT is readily available because both E. coli
and B. subtilis GGTs can be purified by simple two-step
purification procedures from overexpressing strains (5, 6); (iv)
the protection and deblocking of reactive groups of substrates
are not required, unlike for a chemical synthetic method; and
NMR Analysis. Purified γ-Glu-Val (10 mg) was dissolved in D O
2
and then analyzed with a Bruker 500 MHz spectrometer, the spectrum
obtained being compared with that obtained with γ-Glu-Val purchased
from a commercial source.
(v) the substrate specificity of GGT for γ-glutamyl acceptors
is broad and thus various γ-glutamyl compounds can be
RESULTS AND DISCUSSION
*
Author to whom correspondence should be addressed. Telephone: 81-
7
5-753-6278. Fax: 81-75-753-6275. E-mail: hideyuki@lif.kyoto-u.ac.jp.
†
Enzymatic Synthesis of γ-Glu-Val with GGT. The synthesis
of γ-Glu-Val from Gln and Val with GGT as the catalyst was
Graduate School of Biostudies, Kyoto University.
Faculty of Agriculture, Kyoto University.
‡
1
0.1021/jf0347564 CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/10/2004

578 J. Agric. Food Chem., Vol. 52, No. 3, 2004
Suzuki et al.
Figure 1. Enzymatic synthesis of γ-Glu-Val involving bacterial GGT. The
reaction was carried out with 20 mM Gln, 20 mM Val, and 0.04 U/ml
GGT at pH 10 and 37 °C. A part of the reaction mixture was withdrawn
at the times indicated and the concentrations of amino acids, γ-Glu-Val,
and γ-Glu-Gln were determined by HPLC as described under Materials
and Methods. Gln (closed square), Val (open square), γ-Glu-Val (closed
circle), and γ-Glu-Gln (open circle).
Figure 3. Effect of the GGT concentration on γ-Glu-Val synthesis. The
reaction was carried out with 20 mM Gln and 300 mM Val at pH 10 and
37 °C. The GGT concentrations were 0.02, 0.04, 0.08, 0.14, and 0.16
U/ml (open squares, closed squares, open circles, closed circles, and
open triangles, respectively). γ-Glu-Val was determined by HPLC as
described under Materials and Methods.
Figure 4. Effect of the feeding 20 mM Gln after 4 h of incubation on the
yield of γ-Glu-Val. The reaction was carried out with 20 mM Gln, 300
mM Val, and 0.04 U/ml GGT at pH 10 and 37 °C. γ-Glu-Val was
determined by HPLC as described under Materials and Methods.
Figure 2. Effect of the Val concentration on γ-Glu-Val synthesis. The
reaction was carried out with 20 mM Gln and 0.04 units/ml GGT at pH
10 and 37 °C for 3 h, the concentration of Val being varied as indicated
in the figure. γ-Glu-Val was determined by HPLC as described under
Materials and Methods.
the yield being 17.6 mM, and the conversion rate of Gln to
γ-Glu-Val being 88%. In conclusion, the yield of γ-Glu-Val
synthesis involving bacterial GGT was dramatically improved.
Although a large amount of Val remained after 3 h of incubation,
we speculated that the conversion rate of Val could be improved
by feeding Gln and/or GGT after a few hours incubation or by
circulating the substrate using an immobilized enzyme. To
confirm it, 20 mM of Gln was added to the reaction mixture
after 4 h of incubation. As shown in Figure 4, the yield of
γ-Glu-Val increased from 17 mM (without feeding) to 28 mM.
Isolation and Identification of γ-Glu-Val. A 50-mL reaction
mixture was prepared to synthesize γ-Glu-Val. The reaction
mixture was applied to a column (2.3 × 7.2 cm) of Dowex 1 ×
confirmed. Gln and Val were dissolved in water and then the
pH was adjusted to 10 with NaOH. Their concentrations in the
reaction mixture were 20 mM. GGT was added to this solution
(final, 0.04 U/ml) and then the reaction mixture (total volume,
2
mL) was incubated at 37 °C for 3 h. The reaction was
terminated by the addition of a 1/10 volume of 100% trichlo-
roacetic acid, and then the filtrate through the membrane filter
(
Millex-LH, Millipore) was subjected to HPLC analysis.
Synthesis of γ-Glu-Val was observed; however, its concentration
was only 1.0 mM after 3 h of incubation, and more γ-Glu-Gln
was formed (Figure 1).
Optimization of the Reaction Conditions for the Synthesis
of γ-Glu-Val with GGT. Because the yield of γ-Glu-Val was
far from satifactory, the reaction conditions for its synthesis were
investigated. First of all, the optimum ratio of Gln and Val was
investigated. The concentration of Gln was fixed at 20 mM,
while that of Val was varied. The highest amount of γ-Glu-Val
was formed when 300 mM Val was used (Figure 2). Because
the pH of the reaction mixture had a strong influence on the
synthesis of γ-glutamyl compounds involving GGT, the opti-
mum pH of the reaction mixture was investigated and deter-
mined to be 10 (data not shown). The concentration of GGT in
the reaction mixture was also varied, and its effect was
measured. The amount of γ-Glu-Val after 3 h of incubation
was not so different when 0.04-0.16 U/ml GGT was used
-
8 that was prepared as the CH3COO form. The column was
washed with water and 0.1 N CH3COOH, and then γ-Glu-Val
was eluted with 0.5 N CH3COOH. The fractions containing only
γ-Glu-Val were collected and lyophilized. The purification yield
was 36.4%.
The NMR spectra of the synthesized and commercial γ-Glu-
Val were identical (Figure 5). We concluded that the sample
we had synthesized and purified was in fact γ-Glu-Val.
γ-Glutamylization of Leu and Ile Involving Bacterial
GGT. Whether or not γ-Glu-Leu can be synthesized similarly
was examined using a reaction mixture comprising 20 mM Gln,
100 mM Leu, and 0.08 U/ml GGT, pH 9.5, with incubation at
37 °C for 7 h. It was found that 3.4 mM of γ-Glu-Leu was
synthesized under these reaction conditions.
(Figure 3). To keep the amount of GGT as low as possible,
0
.04 U/ml of GGT was determined to be the optimum
Similarly, γ-Glu-Ile was synthesized using a reaction mixture
comprising 20 mM Gln, 100 mM Ile, and 0.08 U/ml GGT, pH
9.5, with incubation at 37 °C. The amount of a new peak was
such that we speculate γ-Glu-Ile increased along with the
concentration. The optimum conditions for the synthesis of
γ-Glu-Val were thus determined to be 20 mM Gln, 300 mM
Val, 0.04 U/ml GGT, pH 10, and incubation at 37 °C for 3 h,

Improving the Bitter Taste of Valine
J. Agric. Food Chem., Vol. 52, No. 3, 2004 579
Figure 5. NMR spectra of commercial γ-Glu-Val (top) and the isolated sample (bottom). The NMR spectra were measured in D
2
O with a Bruker 500
MHz spectrometer.
incubation time (data not shown). However, we could not
identify the peak as γ-Glu-Ile because γ-Glu-Ile, which can be
used as an authentic sample, is not available from a commercial
source. Because the structures of γ-Glu-Val, γ-Glu-Leu, and
γ-Glu-Ile are quite similar, it is reasonable that GGT can
catalyze the synthesis of γ-Glu-Ile.

580 J. Agric. Food Chem., Vol. 52, No. 3, 2004
Suzuki et al.
The results presented in this report indicate that the enzymatic
synthesis of γ-glutamyl branched-chain amino acids involving
GGT are promising.
(6) Minami, H.; Suzuki, H.; Kumagai, H. Salt-tolerant γ-glutamyl-
transpeptidase from Bacillus subtilis 168 with glutaminase
activity. Enzyme Microb. Technol. 2003, 32, 431-438.
(
7) Suzuki, H.; Kumagai, H.; Tochikura, T. γ-Glutamyltranspeptidase
from Escherichia coli K-12: purification and properties. J.
Bacteriol. 1986, 168, 1325-1331.
ACKNOWLEDGMENT
(
8) Hashimoto, W.; Suzuki, H.; Yamamoto, K.; Kumagai, H.
Analysis of low temperature inducible mechanism of γ-glutamyl-
transpeptidase of Escherichia coli K-12. Biosci. Biotechnol.
Biochem. 1997, 61, 34-39.
We wish to thank Kazuhiro Irie, Kyoto University, for NMR
analysis of γ-Glu-Val.
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(
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Received for review July 10, 2003. Revised manuscript received
November 16, 2003. Accepted December 8, 2003. This work was
supported by a Grant-in-Aid for Scientific Research, No. 15580061 to
HS, from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan, and by research funds from the Kaiun Mishima
Memorial Foundation to HS.
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JF0347564