Appl Microbiol Biotechnol (2008) 81:1322

DOI 10.1007/s00253-008-1590-3

MINI-REVIEW

Synthesis and application of dipeptides; current status

and perspectives
Makoto Yagasaki & Shin-ichi Hashimoto

Received: 18 April 2008 / Revised: 22 June 2008 / Accepted: 23 June 2008 / Published online: 16 September 2008
# Springer-Verlag 2008

Abstract The functions and applications of L--dipeptides

(dipeptides) have been poorly studied compared with
proteins or amino acids. Only a few dipeptides, such as
aspartame (L-aspartyl-L-phenylalanine methyl ester) and Lalanyl-L-glutamine (Ala-Gln), are commercially used. This
can be attributed to the lack of an efficient process for
dipeptide production though various chemical or chemoenzymatic method have been reported. Recently, however,
novel methods have arisen for dipeptide synthesis including
a nonribosomal peptide-synthetase-based method and an Lamino acid -ligase-based method, both of which enable
dipeptides to be produced through fermentative processes.
Since it has been revealed that some dipeptides have unique
physiological functions, the progress in production methods
will undoubtedly accelerate the applications of dipeptides in
many fields. In this review, the functions and applications
of dipeptides, mainly in commercial use, and methods for
dipeptide production including already proven processes as
well as newly developed ones are summarized. As
aspartame and Ala-Gln are produced using different
industrial processes, the manufacturing processes of these
two dipeptides are compared to clarify the characteristics of
each procedure.

Introduction

Keywords Dipeptide . L-Amino acid -ligase . NRPS .

Aspartame . L-Alanyl-L-glutamine

The function of dipeptides can be considered from two

viewpoints, as a derivative of amino acid(s) and as the
dipeptide itself. The former viewpoint is easy to understand
because dipeptides and the constitutive amino acids have
different physicochemical properties but should share the same
physiological effects since dipeptides are degraded into the
individual amino acids in organisms. For example, L-glutamine
(Gln) is heat labile while the dipeptide L-alanyl-L-glutamine
(Ala-Gln) is much more tolerant to high temperature (Stehle
et al. 1984; Roth et al. 1988). Solubility is another obvious
example. Tyr is practically insoluble but Ala-Tyr can be

M. Yagasaki (*) : S.-i. Hashimoto

Technical Research Laboratories of
Kyowa Hakko Kogyo Co., Ltd.,
1-1 Kyowa-cho,
Hofu 747-8522, Japan
e-mail: m.yagasaki@kyowa.co.jp
S.-i. Hashimoto
e-mail: shashimoto@kyowa.co.jp

There have been numerous studies on the function,

application, and preparation of proteins and their components, amino acids. In contrast, L--dipeptides (dipeptides),
the simplest peptide bond product of two amino acids, have
been poorly investigated. One of the major reasons is due to
the low availability of dipeptides because of the lack of
cost-effective manufacturing processes. However, information on unique and interesting functions of dipeptides has
still been accumulating. Establishing an efficient process
for dipeptide production is expected to boost the exploration and development of the value of dipeptides. In this
article, the functions and applications of dipeptides are
summarized and current and newly developed technologies
for dipeptide production are reviewed. Though there are
dipeptides which contain unproteinogenic amino acids or are
cyclic structures (diketopiperazine) in nature (Hashimoto
2006), we will focus on linear dipeptides of proteinogenic
amino acids and their simple derivatives in this review.

Function and application of dipeptides

14

Appl Microbiol Biotechnol (2008) 81:1322

dissolved up to 14 g/L (Furst 2001). It is interesting to point

out that some dipeptides are much more soluble than each of
the constitutive amino acids; the solubilities of Ala and Gln
are 89 and 36 g/L, respectively, whereas that of Ala-Gln is
586 g/L (Furst 2001). Based on these properties and the fact
that Ala-Gln and Gly-Tyr are rapidly degraded into the
individual amino acids once taken into the human body
(Albers et al. 1988; Abumard et al. 1989; Frust et al. 1997),
they are used as components of patient infusions.
Some dipeptides have unique functions which cannot be
found in the constitutive amino acids. Dipeptides which
have commercial applications based on their unique
functions are listed in Table 1.
Carnosine (-alanyl-His) and the related dipeptide
anserine (-alanyl-N-methyl-His) have been found to exist
in a wide range of tissues of mammalian, bird, or fish origin
(Gulewitsch and Amiradzibi 1900; Hines and Sutfin 1956).
Many functions have been anticipated to these dipeptides,
such as antioxidation (Guiotto et al. 2005) and maintenance
of cellular pH (Begum et al. 2005). Reflecting these
possible functions, the dipeptides and their derivatives have
been used in several ways. They are employed in sport
nutrition based on the fact that the muscle of a fastswimming fish, the skipjack tuna, contains these dipeptides
in relatively high concentrations (Suzuki et al. 1987). Zinc
carnosine and N-acetyl carnosine are used as an antiulcer
drug (Cho et al. 1991) and as an agent for cataracts
(Babizhayev et al. 2001), respectively.
Research into the taste of dipeptides also has a long history.
The taste of synthetic dipeptides were examined and most of
them were reported to be bitter (Schiffman 1976; de Armas
et al. 2004). The relationship between bitterness and the
physicochemical properties of the dipeptides has captured
researchers interest. From the view point of commercial
applications, aspartame (Asp-Phe methyl ester) is the only
one of outstanding importance. More than 19,000 metric
tons of aspartame, which is 180 times as sweet as sugar
(Cloninger and Baldwin 1970; Ager et al. 1998), is used
annually around the world as a low-calorie sweetener.

Recently, the antihypertensive effect of dipeptides has

attracted researchers attention (Kitts and Weiler 2003).
Some extracts or hydrolysates of fish meat, seaweed, or
mushrooms have been reported to exert a blood-pressurelowering effect and the active agents were identified as
several kinds of dipeptides, such as Ile-Tyr, Lys-Trp, Val-Tyr,
and Ile-Trp. The antihypertensive effects of these dipeptides
have been demonstrated to be derived from their inhibitory
effect on angiotensin-I-converting enzyme (Kitts and Weiler
2003; Matsufuji et al. 1994; Sato et al. 2002; Yokoyama
et al. 1992). The extracts or hydrolysates containing these
dipeptides have been approved as foods for specified health
uses in Japan.
Apart from these industrially applied dipeptides, there
are several dipeptides not used practically but whose
functions are known. Kyotorphin (Arg-Tyr) was isolated
from bovine brain and shown to have analgesic effects
(Takagi et al. 1979). A synthetic dipeptide, Lys-Glu, was
reported to have antitumor activity (Khavinson and Anisimov
2000). Leu-Ile was described to have a neuroprotective
effect (Nitta et al. 2004). Tyr-Gly was shown to enhance
proliferation of peripheral blood lymphocytes (Kayser and
Meisel 1996). It should be also be mentioned that transport
mechanisms for dipeptides and amino acids in human
intestine are different (Adibi 1997). This implies that a
dipeptide and the corresponding amino acids may exert
different nutritional impacts on the human body when taken
orally.

Technologies for dipeptide synthesis

Various ways are known for producing a dipeptide or to
form a peptide bond. They are categorized into three
methods: chemical synthesis, chemoenzymatic synthesis,
and enzymatic synthesis (in this review, chemoenzymatic synthesis is defined as the method which uses an
enzyme and at least one protected amino acid as the
substrate).

Table 1 Commercially applied dipeptides and their unique functions

Compound

Usage

Commercial form

Reference

Aspartame
Ala-Gln
Gly-Tyr
Carnosine

Sweetener
Patient infusion
Patient infusion
Sport nutrition
Antiulcer (zinc salt)
Prevention of cataracts
Health food (antihypertensive)

Pure
Pure
Pure
Crude
Pure

Ager et al. 1998

Frust et al. 1997
Albers et al. 1988
Begum et al. 2005
Cho et al. 1991
Babizhayev et al. 2001
Sato et al. 2002

N-Acetyl carnosine
Val-Tyr

Crude extract

Appl Microbiol Biotechnol (2008) 81:1322

15

Fraenkel-Conrat (1937, 1938) first demonstrated the synthesis of well-defined peptides using proteolytic enzymes. Since
then, several hundred reports on this area have been
published. Enzymatic synthesis has many advantages over
chemical one, including strict stereoselectivity and more
mild conditions. To direct the order of the connection of the
amino acids and to drive the synthetic reaction, protection of
the substrate amino acids (at least one substrate) is required.
Two types of processes with different reaction mechanisms
are known, an equilibrium-controlled (thermodynamically
controlled) process or a kinetically controlled process
(Bordusa 2002; Sinisterra and Alcantara 1993; Kumar and
Bhalla 2005).
The equilibrium-controlled process is based on the
reverse reaction of a protease or an esterase (Fig. 1a). As
expected from the nature of the enzymes, the equilibrium of
the reaction is on the side of the hydrolysis products under
physiological conditions. To drive the equilibrium towards
peptide synthesis, some intervention is necessary. When the
solubility of the product is much less than those of
the substrates, precipitation can be used. Precipitation of
the dipeptide product removes the product from the reaction
equilibrium, promoting the synthetic direction. Other than
precipitation, several techniques, such as conducting the
reaction under a large excess of the substrate(s) or under
biphasic conditions in which the transfer of the product
from aqueous catalyst phase to immiscible phase promotes
synthetic reaction, have been reported (for review, Lombard
et al. 2005).
The kinetically controlled process depends on the fact
that a mildly activated C-terminal ester (or amide) rapidly
acylates a serine or cysteine protease. The acyl enzyme
intermediate undergoes a rate-limiting competitive deacylation by water and by an added nucleophile (the other
amino acid) to give a transient accumulation of the product
dipeptide (Fig. 1b). Since the protease slowly hydrolyzes

Chemical synthesis
While myriad methods are known (for review, Katsoyannis
and Ginos 1969; Nilsson et al. 2005), the principal scheme
of chemical synthesis of dipeptides is as follows (for
example, see Tables 3 and 4):
1. All of the functional groups except for those involved
in making the peptide bond of the amino acids are
protected.
2. The free carboxy group of the protected amino acid is
activated.
3. The activated amino acid is reacted with the other
protected amino acid.
4. All the protecting groups on the dipeptide are removed.
The advantages of chemical synthesis are summarized as
follows: (1) all kinds of dipeptides can be synthesized by
choosing appropriate protecting groups and activating
reagents; (2) the yield is usually high; (3) the procedure is
easy to carry out in a small scale. On the other hand, the
following disadvantages of chemical synthesis can be
pointed out. (1) The cost of synthesis is relatively expensive
because of the necessity of employing many reaction steps
and reagents. (2) There is a risk of racemization during
reactions. (3) A harmful reagent is sometimes needed.
Because of the balance of these advantages and disadvantages, chemical synthesis has been used mainly to fulfill the
demands of research laboratories.
Chemoenzymatic synthesis
Peptide-bond-hydrolyzing enzymes, such as proteases and
esterases, can be used to catalyze the reverse reaction, i.e.,
peptide bond formation between connection of two amino
acids. This approach extends back to late nineteenth century
(Henriques and Gjaldbak 1911). In the 1930s, Bergmann and
Fig. 1 a, b Schematic reactions
of chemoenzymatic dipeptide
synthesis

(a) Equilibrium-controlled process

P1

O
R1

OH

+ R2

R1

OH

P1

NH2

NH

O
NH

NH

OH
R2

(b) Kinetically controlled process

O
R1

H2 O

O
OX

R1

+ Enz-H

NH2

Enz

+ XOH

O
R1

OH + XOH

O
H2N

Enz-H

NH2

NH2

H2 O

OH
R2

H2N

OH
R2

O
R1

Enz
NH2

H2
N

R1

OH
R2

Enz-H

O
NH

NH2

OH
R2

16

the dipeptide formed, the accumulation of the dipeptide is

temporary. Because of this reaction mechanism, the product
yield depends on the velocities of the attack by water and
the nucleophile to the acyl enzyme and of degradation of
the dipeptide by the protease itself. Thus, as well as in
properties of the enzyme itself, the reaction conditions,
such as pH, ionic strength, and concentration of the
nucleophile, are crucially important. For more details,
please refer to the excellent reviews (Bongers and Heimer
1994; Morihara 1987; Schellenberger and Jakubke 1991).
Enzymatic synthesis
While the ribosome system is the ubiquitous equipment for
peptide formation in organisms, other enzymatic machineries which conduct peptide syntheses have been found in
nature. These include nonribosomal peptide synthetase
(NRPS, see below), polyglutamine synthase (Ashiuchi and
Misono 2002), cyanophycin synthetase (Aboulmagd et al.
2001), glutathione synthase (Meister 1974), D-alanine-Dalanine ligase (Ddl, Walsh 1989), and L-amino acid ligase (Lal, see below). Activities other than the ribosomal
system are specific for their own products. From the view
point of the way to activate the substrate amino acid(s),
these activities can be divided into two groups. One way is
via aminoacyl-adenosine monophosphate (AMP) and the
other way is via aminoacyl phosphate. The ribosomal
system and NRPS belong to the former group whereas
cyanophycin synthetase, glutathione synthase, Ddl, and Lal
belong to the latter group. Both ways have been proposed
for polyglutamine synthase (Ashiuchi and Misono 2002;
Candela and Fouet 2006). Since these naturally occurring
peptide-synthesizing activities use unprotected amino acids
as their substrates and catalyze only peptide-forming
reactions, they seem to be ideal catalysts for dipeptide
synthesis. The first such attempt was conducted in 1980.
Doel et al. (1980) expressed synthetic genes coding for a
protein consisted of about 150 repeats of Asp-Phe in
Escherichia coli. Unfortunately, this strategy was not
practical because of the low productivity and simultaneous
appearance of Phe-Asp along with Asp-Phe when the
produced polymer was cleaved by proteases. In the 1990s,
progress in a NRPS study provided researchers with
another approach. And recently, Lal has been demonstrated
to be useful for dipeptide production. These emerging
approaches and other possibilities are reviewed below.

Appl Microbiol Biotechnol (2008) 81:1322

enzymes are huge multifunctional proteins and are made

up of a series of modules, each of which takes charge of
adding one amino acid to a growing peptide. Each module
contains at least three enzymatic units called domains
(Fig. 2). An adenylation domain (A-domain) recognizes the
substrate amino acid and activates it as an aminoacyl-AMP
accompanied with the hydrolysis of adenosine triphosphate
(ATP) to AMP and pyrophosphate. Subsequently, the
activated amino acid is transferred to 4-phophopantetheine
moiety of the thiolation domain (T-domain) with the release
of AMP. Then the adjacent condensation domain (Cdomain) catalyzes the formation of the peptide bond.
Finally, the thioesterase domain (Te-domain) catalyzes the
release of the product peptide from the enzyme protein. For
more details on NRPS, please refer to the comprehensive
reviews (Finking and Marahiel 2004; Sieber and Marahiel
2005).
Modular manipulation of NRPS has been applied to
dipeptide synthesis. Doeckel and Marahiel designed artificial NRPSs by combining the A-domain, which recognizes
Ile, from the bacitracin-biosynthetic NRPS in Bacillus
licheniformis and the A-domain, which recognizes Leu,
from the tyrocidine-biosynthetic NRPS in Bacillus brevis
(Doekel and Marahiel 2000). The artificial dimodular
NRPS was expressed in E. coli simultaneously with 4phosphopantetheinyl transferase, which is necessary to
make NRPS holoenzyme. The purified enzyme was
demonstrated to produce Ile-Leu in the presence of Ile,
Leu, and ATP. A similar strategy was applied to create
NRPSs for Phe-Leu, Ile-Phe (Doekel and Marahiel 2000),

O
R1

OH

R1

+ ATP

O AMP

+ PPi

NH2

NH2

AMP

R1

T-domain

NH2

C-domain O
R2

NH2

Te-domain

R1

HN

NH2

R2

NRPS process
NRPSs have been found in various microorganisms such as
bacteria and fungi. They are responsible for the syntheses
of a wide array of therapeutically important peptides such
as vancomycin, gramicidin S, and cyclosporine. The

O
HO

HN
R2

Fig. 2 NRPS-catalyzed peptide bond formation

R1
NH2

Appl Microbiol Biotechnol (2008) 81:1322

17

Phe-Ala (Dieckmann et al. 2001), D-Phe-Pro (Keller and

Schauwecker 2003), and Asp-Phe (see the latter section).
From bioinformatics data and biochemical or structural
data, the selectivity-conferring nonribosomal code of the
A-domain has been determined (Stachelhaus et al. 1999;
Rausch et al. 2005), enabling researchers to design an
artificial enzyme for a desired peptide. But, practically,
there are still problems to overcome. One problem is Cdomain selectivity. While substrate specificity of NRPS is
basically determined by the A-domains, the C-domains also
have selectivity, which are important for the control of the
directionality of the peptide synthesis (Belshaw et al. 1999;
Linne and Marahiel 2004). Therefore, a suitable combination of A- and C-domains needs to be chosen. The other
problem is the low activities of the artificially created
enzymes. This may be the reason for the fact that there have
been a small number of reports on peptide production by
the living cells expressing engineered NRPS (Stachelhaus
et al. 1995; de Ferra et al. 1997; Symmank et al. 2002;
Mootz et al. 2002). Product yields in most of the living-cell
studies also remained quite low. How to fuse the domains
has been found to be important to optimize the interaction
of domains (Linne and Marahiel 2004). Thus, in contrast
with the succinctness of its principle, NRPS engineering
needs a lot of know-how to be applied practically.
Lal process
Lal was discovered by an in silico screening for a new
activity to catalyze a dipeptide formation (Tabata et al.
2005). The only gene found, ywfE in Bacillus subtilis, was
expressed in E. coli and confirmed to have the expected
activity, ligating Ala and Gln in an ATP-dependent manner.
From the amino acid sequence and biochemical data, the
enzyme was determined to belong to the ATP-dependent
carboxylate-aminethiol ligase superfamily, which uses
acyl phosphate as the reaction intermediate (Galperin and
Koonin 1997; Fig. 3). The following characteristics of the
O

O
R1

OH

R1

+ ATP

Pi

+ ADP

NH2

NH2

Lal

H2N

OH
R2

O
R1

O
NH

NH2
Fig. 3 Lal-catalyzed dipeptide formation

OH
R2

Pi

enzyme were reported. (1) It forms only dipeptides.

Tripeptides or longer peptides were never detected as the
reaction product. (2) It can take various kinds of amino
acids as the substrates but has certain selectivity. Acidic or
basic amino acids do not react. The order of the amino
acids is also directed, for example, Ala-Gln can be formed
but Gln-Ala cannot. Consequently, 44 kinds of dipeptides
were confirmed to be synthesized. (3) The enzyme dose not
accept D-amino acids.
Two types of processes for dipeptide production utilizing
Lal have been described, the resting cell reaction process
and the direct fermentation process.
The resting cell reaction process is a coupling reaction of
Lal and an ATP regeneration reaction. Detergent-treated E.
coli cells expressing Lal from B. subtilis and polyphosphate
kinase from Rhodobacter sphaeroides was reported to
produce several kinds of dipeptides (Ala-Met, Ala-Val,
Ala-Ile, Ala-Leu, Gly-Met, and Gly-Phe) by incubating the
corresponding amino acids and polyphosphate (Ikeda et al.
2006). Ala-Met gave the highest titer, 127.9 mM (28 g/L),
from 200 mM each of Ala and Met. Any dipeptides within
the product spectrum of Lal can be produced by changing
the substrate amino acids.
One can imagine that a Lal-expressing organism would
produce some dipeptides because Lal takes unprotected
amino acids. This is the conceptual idea of the direct
fermentation. But simply expressing Lal in E. coli resulted
in no accumulation of dipeptides (Tabata and Hashimoto
2007). This is attributed to two problems, first, the
relatively low affinity of Lal for amino acids and, second,
the dipeptide-degrading activity of the host cells. To
overcome these problems, some metabolic engineering,
such as enhancing the metabolic flux to the substrate
amino acids and reducing the degradation activity, are
necessary. Ala-Gln fermentation is a successful example
(see the latter section). Some other producer strains for
Ala-Met and Thr-Phe, respectively, were also reported
(Tabata and Hashimoto 2005). Obviously, the direct
fermentation method is the most cost-effective for dipeptide manufacturing since it does not need even the
substrate amino acids.
YwfE seemed to be an orphan enzyme since there has
been no clear homolog of YwfE in the public database
except for BacD from Bacillus amyloliquefaciens, which
encodes a protein that is 97% identical with YwfE.
Recently, however, several homologs have been found.
Proteins encoded by rsp1486 in Ralstonia solanacearum
and bl00235 in B. licheniformis were reported to have only
29% and 28% identity with YwfE, respectively, but have
Lal activities (Kino et al. 2006, 2007). A gene involved in
the biosynthesis of rhizocticin A, a peptidic antibiotic
produced by B. subtilis, was shown to possess Lal activity
(Kino et al. 2008). Interestingly, these newly found YwfE

18

Appl Microbiol Biotechnol (2008) 81:1322

condensation step 3, which has been reported to be 65~98%

(Ariyoshi et al. 1974a, b; Albini et al. 1985). The biggest
problem of the chemical synthesis is the by-production of
-Asp-PheOM, which exhibits a bitter taste (Albini et al.
1985). Much effort have been paid to reduce the formation
of this by-product (Albini et al. 1985; Yukawa et al. 1994;
Hill et al. 1991).
The chemoenzymatic process employs thermolysin from
Bacillus thermoproteolyticus (Lombard et al. 2005; Isowa
et al. 1979). The synthetic route is very similar to the
chemical synthesis. But, thanks to the selectivity of the
enzyme, the process is free from the -form and can use
DL-Phe instead of L-Phe as the starting material. N(benzoyloxycarbonyl)-Asp (Z-Asp) and DL-PheOM are
prepared and connected through the action of thermolysin.
In spite of the equilibrium-controlled mechanism, Z-AspPheOM synthesis by the enzyme proceeds efficiently
because the product Z-Asp-PheOMe forms insoluble salt
with the remaining D-PheOM (Oyama et al. 1987). A very
high yield (95%) in the condensation reaction has been
described (Nakanishi et al. 1985, 1990). After separating ZAsp-PheOM and D-PheOM, the latter compound is racemized to DL-PheOM and reused. While the overall process
is sophisticated, the decrease in the enzymatic activity is a
drawback of the process. To improve it, many attempts
have been investigated such as immobilization (Oyama
et al. 1987; Nakanishi et al. 1985, 1990), enzyme
engineering (Inouye et al. 2007), or use of a molecular
imprinted polymer (Ye et al. 1999). There have also been
reports on the enzymatic synthesis of aspartame from
unprotected Asp and PheOM (Table 3), but the yields
remained low (Francois et al. 1990).

homologs were shown to have different product spectrums

from that of YwfE from B. subtilis (Table 2).

Approaches for specific targets

Aspartame and Ala-Gln are the rare cases of commercialized dipeptides. Several manufacturing methods have
been proposed for each dipeptide. Comparing those will
help to understand the pros and cons of each process.
They will also show that the choice of an industrial
manufacturing process depends not only on the efficiency
of the dipeptide-forming reaction but also on the
controllability of by-product(s) and economics of the
total process.
Aspartame
Two processes have been commercially used, chemical
synthesis and chemoenzymatic synthesis (Table 3) while
there have been hundreds of patents and reports on
improvements or modifications of these principal processes.
The chemical synthesis of aspartame is basically carried
out as follows. (1) Phe is reacted with methanol to yield
Phe methyl ester (PheOM). (2) Asp is modified to an
N-protected aspartic anhydride such as N-carbobenzoxyaspartic anhydride or N-formyl-aspartic anhydride. Industrially, the cheaper formyl compound would be preferable.
(3) PheOM and N-protected aspartic anhydride are reacted
to form N-protected Asp-PheOM. (4) N-protected AspPheOM is treated with acid to get Asp-PheOM, aspartame.
The overall yield mainly depends on the yield of the

Table 2 Product spectrums of YwfE and its homologs

C-terminus

N-terminus

Gly
Ala
Ser
Cys
Thr
Leu
Met
Phe
Gln
His
Arg

Gly

Ala

Ser

Cys

Y
Y
Y

Y, R
Y, R

Y
Y, R
Y

Y
Y
Y, R
Y, R

Y
B

B
R, B
R
R
R
Z

B
R
R
Z

Thr

Val

Y
Y

Y
Y

R
R
R

Leu

Ile

Met

Phe

Tyr

Y
Y
Y
Y
Y

Y
Y
Y

Y
Y
Y
Y
Y

Y
Y
Y
Y

Y
Y
Y

Y, R

Y
R

Trp

Y
Y
Y

Gln

Asn

His

Y
Y
Y

Y
Y

Y
Y
Y

Arg

Y
Y

R
Z

Amino acids able to be accepted at the N-terminus are listed in the file. The ones able to be accepted at the C-terminus are listed in the line. Each
product were confirmed by HPLC or NMR analysis.
Y YwfE of B. subtilis, Z a gene product involved in rhizocticin biosynthesis, B protein encoded by bl00235 in B. licheniformis, R; protein encoded
by rsp1486 in R. solanacearum.

Appl Microbiol Biotechnol (2008) 81:1322

19

Table 3 Methods for aspartame production

Category

Schematic scheme
O

(a)
Chemical

Asp

O
N
H

O
N
H

H2N

CO2H
H
N

CO2H
H
N

CO2CH3

H2N

N
H
CO2H

N
H

N
H

formation of
-form
High yield, high
sterospecifcity

CO2H

O
O

H2N

1974a,b, Albini
et al. 1985
By-product

CO2CH3

CO2H

Ariyoshi et al.

CO2CH3

-form

Asp

High yield

O
O

Reference

CO2CH3

Phe

(b) Chemoenzymatic

Advantage /
disadvantage*

N
H

CO 2H
N

+H3N

CO 2CH3

CO 2H
H
N

CO2CH3
O

N
H

DL-Phe
H2 N

CO2 H
H
N

2005, Isowa et
al.

CO2CH3

1979,

Nakanishi et al.

CO2 CH 3

Lombard et al.

Decrease in
enzyme activity

1990

CO2CH 3

(c) Chemoenzymatic

Asp
H2N

CO2 CH 3

H2 N

CO2H
H
N

CO 2CH3

High
Francois et al.
stereospecificity, 1990
simple process

Phe

Low yield
(d)
Enzymatic

Asp + Phe

Asp-Phe

(+chemical)
a

Asp-PheOM

Cheap raw

Bachman et al.

materials

1976, Duerfahrt

in vitro level

et al. 2003

Upper section, advantage; lower section, disadvantage

Aspartame can be made in a different way, through the

selective esterification of Asp-Phe (Bachman et al. 1976).
As an attempt to this end, the enzymatic synthesis of AspPhe has been reported. Duerfahrt and coworkers created
artificial NRPSs containing the A-domain for Asp from
surfactin synthetase and the A-domain for Phe from
tyrocidine synthetase (Duerfahrt et al. 2003). Six artificial
genes with different fusion points and/or Te domains were
constructed. These NRPSs were purified and confirmed to
have the desired activity (Table 3). While all of them were
capable of synthesizing Asp-Phe, significant differences in
the activity depending on the fusion strategy were observed, indicating the importance of the design of the
artificial NRPS.
Ala-Gln
Since the effectiveness of Ala-Gln as a component of
patient infusions has been established (Furst et al. 1997;
Goeters et al. 2002), the dipeptide has been used in medical
fields. The following four processes (Table 4) or their
modified versions are used commercially.
While standard chemical synthetic methods can yield the
dipeptide, a chemical liquid-phase peptide synthesis via N-

carboxyanhydride intermediate (Furst et al. 1985) is used

for Ala-Gln production (Table 4). Ala is reacted with
carbonyl chloride to form N-carboxyalanine anhydride
followed by condensation with Gln. The Ala-Gln carbamate
formed is treated by an acid to yield Ala-Gln. As Ncarboxyalanine anhydride is highly reactive, the condensation rate is high. On the other hand, by-products such as
Ala-Ala-Gln and D-Ala-Gln are also formed. The other
problem of this method is the use of carbonyl chloride,
which is the famous poison gas, phosgene.
Sano et al. (2000) designed an alternative chemical
route. D-2-chloropropionic acid was subjected to the
Schottenn-Baumann reaction with Gln to yield D-2chloropropionyl-Gln. Ala-Gln was obtained by the ammonolysis reaction of the product (Table 4). Some by-products
including Ala-Glu were detected, but they could be
removed by recrystallization.
A chemoenzymatic process has also recently been
developed (Table 4). Yokozeki and Hara screened for the
activity to synthesize Ala-Gln from Ala methyl ester
(AlaOM) and Gln and found an enzyme from Empedobacter
brevis. The purified enzyme was demonstrated to produce
83 mM of Ala-Gln from 100 mM of AlaOM and 200 mM of
Gln (Yokozeki and Hara 2005). In the absence of Gln, the

20

Appl Microbiol Biotechnol (2008) 81:1322

Table 4 Methods for Ala-Gln production

Category

Schematic scheme

Advantage /

Reference

disadvantage*
(a) Chemical

High yield
COCl2
H2N

CO 2H

HM

NH 2

O
O

H
N

Gln

CONH 2

formation, Use

CO2 H

of phosgen

(b) Chemical

High
Cl

SOCl2 + Gln

Cl

Frust et al. 1985

Ala-Ala-Gln

CO 2H

H
N
O

CONH 2

NH3

NH 2

CO 2H

H
N

CONH 2
CO 2 H

Sano et al. 2000

stereospecificty
Ala-Glu
formation

(c) Chemoenzymatic

H 2N

CO 2H

H 2N

NH 2

H
N

CONH 2

Gln

CO 2H

Simple & easy

Yokozeki and

process

Hara 2005

Low yield,
Ala-Ala-Gln
formation
(d) Enzymatic
(fermentation)

NH 2

Glucose + NH3

H
N

CONH 2
CO2 H

Cheap raw

Tabata and

material, easy

Hashimoto 2005

process
Ala-Ala
formation

Upper section, advantage; lower section, disadvantage

enzyme hydrolyzed Ala-Gln, suggesting a kinetically controlled mechanism.

Recently, a Lal-based enzymatic process has been
established (Tabata and Hashimoto 2007). Tabata and
Hashimoto constructed a recombinant E. coli strain producing Ala-Gln without supplying Ala and Gln. To enhance
metabolic fluxes to the substrate amino acids, Gln biosynthesis was deregulated by destroying glnE and glnB genes
and alanine dehydrogenase (Ald) from B. subtilis was
coexpressed with Lal. To reduce dipeptide degradation,
genes for several dipeptidases (PepA, PepB, pepD, and
PepN) and the dipeptide import system (Dpp) were
disrupted in combination. Lal and Ald were expressed in
the host strain under a stationary-phase specific promoter
since the synthesis of Lal hampered cell growth. Fed batch
cultivation of the recombinant strain in a 5-L jar fermentor
on a glucose-ammonium medium resulted in the accumulation of Ala-Gln (100 mM) in the cultivation supernatant
(Table 4). No tripeptides or D-amino acid containing
dipeptides were detected. Ala-Ala was also produced, but
it can be separated by chromatography or crystallization.
Perspective
It is interesting that several production methods have been
employed for commercial production of aspartame or Ala-

Gln, illustrating researchers originality and ingenuity. Each

process has its own advantages and disadvantages (Table 3
and 4). These facts indicate that dipeptide manufacturing is
still in the early stages of the technology evolution, in
which processes converges to the most competitive methodology. Fermentative production of dipeptides based on
NRPS or Lal could become such the ultimate method
because it is likely the most cost-efficient and environmentally friendly. Since dipeptide fermentation has just
emerged, it needs to be more thoroughly studied to be
applied widely; including metabolic flow control for the
substrate amino acids and suppression of undesired byproducts. In addition, which enzyme of NRPS and Lal
should be used must be decided. The possible product
spectrum of the NRPS system is much wider than the Lal
system whereas the Lal system is easier to be practically
applied. Finding of Lal homologs suggests feature expansion of the product by the Lal-based fermentation.
Development of a new function or application and
development of an efficient production method are in a
mutually promoting relationship. The recent increase in the
interest in the functions of dipeptides and the appearance of
new processes for production imply that the exploration of
the dipeptide world both in applications and manufacturing technology will be much accelerated over the next
decade.

Appl Microbiol Biotechnol (2008) 81:1322

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