articulo sintesis y aplicacion de dipetidos.pdf

8/10/2019 ARTICULO sintesis y aplicacion de dipetidos.pdf

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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 withproteins or amino acids. Only a few dipeptides, such as

aspartame (L-aspartyl-L-phenylalanine methyl ester) and L-

alanyl-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 chemo-

enzymatic method have been reported. Recently, however,

novel methods have arisen for dipeptide synthesis including

a nonribosomal peptide-synthetase-based method and an L-

amino 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.

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

Aspartame . L-Alanyl-L-glutamine

Introduction

There have been numerous studies on the function,

application, and preparation of proteins and their compo-

nents, 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, informa-

tion on unique and interesting functions of dipeptides has

still been accumulating. Establishing an efficient process

for dipeptide production is expected to boost the explora-

tion 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

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

Appl Microbiol Biotechnol (2008) 81:1322

DOI 10.1007/s00253-008-1590-3

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

Technical Research Laboratories of

Kyowa Hakko Kogyo Co., Ltd.,

1-1 Kyowa-cho,

Hofu 747-8522, Japan

e-mail: [email protected]

S.-i. Hashimoto

e-mail: [email protected]
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dissolved up to 14 g/L (Furst2001). 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 (Furst2001). 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 Amiradzibi1900; Hines and Sutfin1956).

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 thesepossible 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 fast-

swimming 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 (Schiffman1976; 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-pressure-

lowering 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 inhibitoryeffect 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 neuroprotectiveeffect (Nitta et al. 2004). Tyr-Gly was shown to enhance

proliferation of peripheral blood lymphocytes (Kayser and

Meisel1996). 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, chemoenzy-

matic 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 Sweetener Pure Ager et al. 1998

Ala-Gln Patient infusion Pure Frust et al. 1997

Gly-Tyr Patient infusion Pure Albers et al.1988

Carnosine Sport nutrition Crude Begum et al. 2005

Antiulcer (zinc salt) Pure Cho et al. 1991

N-Acetyl carnosine Prevention of cataracts Babizhayev et al. 2001

Val-Tyr Health food (antihypertensive) Crude extract Sato et al. 2002

14 Appl Microbiol Biotechnol (2008) 81:1322
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Chemical synthesis

While myriad methods are known (for review, Katsoyannis

and Ginos1969; Nilsson et al. 2005), the principal scheme

of chemical synthesis of dipeptides is as follows (for

example, see Tables3 and 4):

1. All of the functional groups except for those involvedin 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, thefollowing 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 disadvan-

tages, chemical synthesis has been used mainly to fulfill the

demands of research laboratories.

Chemoenzymatic synthesis

Peptide-bond-hydrolyzing enzymes, such as proteases andesterases, 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 Gjaldbak1911). In the 1930s, Bergmann and

Fraenkel-Conrat (1937,1938) first demonstrated the synthe-

sis 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 ofthe 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 deacy-

lation 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

(a)

Kinetically controlled process

NH2

OX

O

R1 + Enz-H

NH2

Enz

O

R1 + XOH

H2O

NH2

OH

O

R1 + XOH

R2

OH

O

H2N

NH2

Enz

O

R1

R2

OH

OH2N

NH2

NH

O

R1

R2

OH

O

Enz-H +

R2

OH

O

H2N

+

NH2

OH

O

R2

N H

N H

O

R1

P1R2

O H

O

N H

O H

O

R1

P1

Enz-H+

H2O

Equilibrium-controlled process

(b)

Fig. 1 a, b Schematic reactions

of chemoenzymatic dipeptide

synthesis

Appl Microbiol Biotechnol (2008) 81:1322 15
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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; Morihara1987; Schellenberger and Jakubke 1991).

Enzymatic synthesis

While the ribosome system is the ubiquitous equipment for

peptide formation in organisms, other enzymatic machiner-

ies 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-D-alanine 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.

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

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, theactivated amino acid is transferred to 4-phophopantetheine

moiety of the thiolation domain (T-domain) with the release

of AMP. Then the adjacent condensation domain (C-

domain) 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 artifi-

cial NRPSs by combining the A-domain, which recognizesIle, 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 4-

phosphopantetheinyl 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),

NH2

HN

O

R1

R2

S

O

NH2

OH

O

R1 + ATPNH2

O

O

R1AMP + PPi

NH2

S

O

R1

NH2

S

O

R2

AMP

T-domain

C-domain

Te-domain

NH2

HN

O

R1

R2

HO

O

Fig. 2 NRPS-catalyzed peptide bond formation

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

Schauwecker2003), 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 C-domain 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 combina-

tion 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-cellstudies 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, ywfEin 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

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 notaccept 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.

colicells expressing Lal fromB.subtilisand 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 dipep-

tide 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

NH2

OH

O

R1 + ATP

NH2

O

O

R1Pi + ADP

Lal

R2

OH

O

H2N

NH2

NH

O

R1

R2

OH

O

+ Pi

Fig. 3 Lal-catalyzed dipeptide formation

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homologs were shown to have different product spectrums

from that of YwfE from B. subtilis (Table2).

Approaches for specific targets

Aspartame and Ala-Gln are the rare cases of commer-

cialized dipeptides. Several manufacturing methods havebeen 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) whilethere 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-carbobenzoxy-

aspartic anhydride or N-formyl-aspartic anhydride. Indus-

trially, 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 Asp-

PheOM is treated with acid to get Asp-PheOM, aspartame.

The overall yield mainly depends on the yield of the

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 fromBacillus 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-Asp-

PheOM 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 beendescribed (Nakanishi et al.1985,1990). After separating Z-

Asp-PheOM and D-PheOM, the latter compound is race-

mized 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).

Table 2 Product spectrums of YwfE and its homologs

C-terminus

Gly Ala Ser Cys Thr Val Leu Ile Met Phe Tyr Trp Gln Asn His Arg

N-terminus Gly Y Y Y Y Y Y Y

Ala Y Y, R Y Y Y Y Y Y Y Y Y Y Y Y Y

Ser Y Y, R Y, R Y, R Y Y Y Y Y Y Y Y Y Y

Cys Y Y, R Y Y Y Y Y Y Y

Thr Y Y Y Y YLeu B

Met B R, B B Y, R Y Y Y

Phe R R R R

Gln R R R

His R R R R R R

Arg Z Z Z 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.

YYwfE ofB.subtilis,Za gene product involved in rhizocticin biosynthesis, B protein encoded by bl00235 inB. licheniformis,R; protein encoded

by rsp1486 in R. solanacearum.

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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 Asp-

Phe 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 ob-

served, 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 N-

carboxyalanine anhydride is highly reactive, the condensa-

tion 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-2-

chloropropionyl-Gln. Ala-Gln was obtained by the ammo-

nolysis 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

Table 3 Methods for aspartame production

Category Schematic scheme Advantage /

disadvantage*

Reference

High yield(a)

Chemical

By-product

formation of

-form

Ariyoshi et al.

1974a,b, Albini

et al. 1985

High yield, high

sterospecifcity

(b) Chemo-

enzymatic

Decrease in

enzyme activity

Lombard et al.

2005, Isowa et

al. 1979,

Nakanishi et al.

1990

High

stereospecificity,

simple process

(c) Chemo-

enzymatic

Francois et al.

1990

CO2H

CO2H

NH

O

O

H2N CO2CH3

CO2-

NH

O

HN CO2CH3

O

O +H3N CO2CH3 CO2H

NH

O

HN CO2CH3

O

O

CO2H

H2N

O

HN CO2CH3

Asp

DL-Phe

AspH2N CO2CH3

Phe

CO2H

H2N

O

HN CO2CH3

O

NH

O

O

O

H2N CO2CH3

CO2H

NH

O

HN CO2CH3

O

CO2HNH

N

H

CO2CH3O

O

CO2H

H2N

O

HN CO2CH3

Asp

Phe

-form

Low yield

Cheap raw

materials

(d)

Enzymatic

(+chemical) in vitro level

Bachman et al.

1976, Duerfahrt

et al. 2003

Asp + Phe Asp-Phe Asp-PheOM

aUpper section, advantage; lower section, disadvantage

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enzyme hydrolyzed Ala-Gln, suggesting a kinetically con-

trolled mechanism.

Recently, a Lal-based enzymatic process has been

established (Tabata and Hashimoto 2007). Tabata and

Hashimoto constructed a recombinant E. coli strain pro-

ducing Ala-Gln without supplying Ala and Gln. To enhance

metabolic fluxes to the substrate amino acids, Gln biosyn-

thesis was deregulated by destroying glnEand 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 accumu-

lation 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 researchersoriginality and ingenuity. Each

process has its own advantages and disadvantages (Table3

and4). These facts indicate that dipeptide manufacturing is

still in the early stages of the technology evolution, in

which processes converges to the most competitive meth-

odology. Fermentative production of dipeptides based on

NRPS or Lal could become such the ultimate method

because it is likely the most cost-efficient and environmen-

tally 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 by-

products. 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 expan-

sion 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 manufactur-

ing technology will be much accelerated over the next

decade.

Table 4 Methods for Ala-Gln production

Category Schematic scheme Advantage /

disadvantage*

Reference

High yield(a) Chemical

Ala-Ala-Gln

formation, Use

of phosgen

Frust et al. 1985

High

stereospecificty

(b) Chemical

Ala-Glu

formation

Sano et al. 2000

(c) Chemo-

enzymatic

Simple & easy

process

Yokozeki and

Hara 2005

H2N CO2H

HM

OO

O

NH2 HN CONH2

O CO2HGln

COCl2

Cl

CO2H

ClHN CONH2

O CO2H

SOCl2+ Gln NH3NH2 H

N CONH2

O CO2H

H2N CO2H H2N O

O

Gln

NH2 HN CONH2

O CO2H

Low yield,

Ala-Ala-Gln

formation

Cheap raw

material, easy

process

(d) Enzymatic

(fermentation)

Ala-Ala

formation

Tabata and

Hashimoto 2005

Glucose + NH3

NH2 HN CONH2

O CO2H

aUpper section, advantage; lower section, disadvantage

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