Synthesis of morphiceptin (Tyr-Pro-Phe-Pro-NH2) by dipeptidyl aminopeptidase IV derived from Aspergillus oryzae

Article abstract of DOI:10.1021/jf050420d

Morphiceptin (Tyr-Pro-Phe-Pro-NH₂), tetrapeptide, was synthesized using dipeptidyl aminopeptidase IV (DP IV, EC 3.4.14.5) derived from Aspergillus oryzae RIB 915 as a catalyst. Tyr-Pro-OEt was incubated with Phe-Pro-NH₂ in the presence of DP IV under various conditions of temperature, concentrations of ethylene glycol, pH, reaction time, and others. Morphiceptin was obtained at 40% yield under the optimal reaction conditions: substrate, 4 mM Tyr-Pro-OEt·HCl and 20 mM Phe-Pro-NH₂· HCl; enzyme, DP IV, 0.275 nkat; solvent, 60% ethylene glycol containing 20 mM phosphate buffer at pH 7.0; amine, 4.2 mM diisopropylamine at 4°C for 24 h. Amino group protection was unnecessary for synthesis of morphiceptin by DP IV.

Full text of DOI:10.1021/jf050420d

6112 J. Agric. Food Chem. 2005, 53, 6112−6116
Synthesis of Morphiceptin (Tyr-Pro-Phe-Pro-NH ) by Dipeptidyl
2
Aminopeptidase IV Derived from Aspergillus oryzae
,
†
‡
§
†
|
TORU OTA,* AKI ITOH, HIROSHI TACHI, KEITA KUDOH, TATSUO WATANABE,
YUJI YAMAMOTO, TADAHIRO TADOKORO, AND AKIO MAEKAWA‡
‡
‡
Department of Human Life and Development, Nayoro City College, Nayoro, Hokkaido 096-8641,
Japan, Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo
University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan, Department of Brewing and
Fermentation, Junior College of Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502,
Japan, and School of Food and Nutritional Sciences, University of Shizuoka,
Shizuoka 422-8526, Japan
Morphiceptin (Tyr-Pro-Phe-Pro-NH
IV (DP IV, EC 3.4.14.5) derived from Aspergillus oryzae RIB 915 as a catalyst. Tyr-Pro-OEt was
incubated with Phe-Pro-NH in the presence of DP IV under various conditions of temperature,
2
), tetrapeptide, was synthesized using dipeptidyl aminopeptidase
2
concentrations of ethylene glycol, pH, reaction time, and others. Morphiceptin was obtained at 40%
yield under the optimal reaction conditions: substrate, 4 mM Tyr-Pro-OEt‚HCl and 20 mM Phe-Pro-
NH ‚HCl; enzyme, DP IV, 0.275 nkat; solvent, 60% ethylene glycol containing 20 mM phosphate
2
buffer at pH 7.0; amine, 4.2 mM diisopropylamine at 4 °C for 24 h. Amino group protection was
unnecessary for synthesis of morphiceptin by DP IV.
KEYWORDS: Morphiceptin; opioid peptide; tetrapeptide; dipeptidyl aminopeptidase IV (DP IV); Koji mold
(Aspergillus oryzae); enzymatic synthesis; proline-containing peptide
INTRODUCTION
the method of enzymatic synthesis of peptides has been
established. However, the synthesis of proline-containing pep-
tides is difficult because most proteases do not form a peptide
bond for proline. Enzymatic synthesis of proline containing has
been limited to di- or tripeptides such as Pro-Phe, Arg-Pro, Thr-
Pro, Asp-Pro, and Phe-Pro-Gly (10-13) except for amidated
enterostatin (Ala-Pro-Gly-Pro-Arg-NH2) by the use of dipeptidyl
aminopeptidase from Lactococcus lactis (14). Dipeptidyl ami-
nopeptidase IV (DP IV, EC 3.4.14.5) is a serine enzyme that
liberates X-Pro specifically. Because morphiceptin, tetrapeptide,
includes -X-Pro- in its amino acid sequence, we assumed that
morphiceptin may also be produced by DP IV. Recently, Tachi
et al. reported that DP IV is produced easily by the Koji mold
Many physiologically active peptides derived from milk
protein have been found and identified during the last 20 years.
Among them are opioid peptides, opioid peptide antagonists,
angiotensin-converting enzyme inhibitory peptides, macrophage-
activating peptides, and platelet aggregation peptides (1-5).
Morphiceptin is a physiologically active peptide obtained from
bovine casein by enzyme digestion treatment and has the highest
opioid activity among the â-casomorphins (6). Its amino acids
are Tyr-Pro-Phe-Pro-NH2. Furthermore, morphiceptin is highly
selective for µ receptor; the structure of tetrapeptide amide is
considered essential for opioid activity. Recently, morphiceptin
has been reported to inhibit oxidation of the cytochrome P450
substrate and spontaneous firing of locus coeruleus (7, 8).
However, many questions remain on the role of morphiceptin
in ViVo, detected in the intestine. To use morphiceptin as a food,
we must examine its absorbability, stability, long-term intake
effect, effective dose, and so on. A large amount of morphiceptin
is required for these studies.
(Aspergillus oryzae) during the soy-sauce brewing process (15).
This study screened Aspergilli with high DP IV activity, partially
purified DP IV from A. oryzae, and investigated the optimal
condition for morphiceptin synthesis using that DP IV.
MATERIALS AND METHODS
We have studied organic synthesis using enzymes (reaction
in reverse direction from usual breakdown) (9). Many proteases
activate peptide formation under a specific condition; thereby,
Materials. Tyr-Pro-OEt‚HCl and Gly-Pro-pNA were obtained from
Peptide Institute Inc. (Osaka, Japan). Morphiceptin and Phe-Pro-NH
HCl were purchased from Bachem AG (Bubendorf, Switzerland).
2
‚
Cultivation. Six strains of Aspergilli isolated from various Koji
specimens were used: A. oryzae 076 M, A. oryzae RIB 128, A. oryzae
RIB 430, A. oryzae RIB 915, A. sojae 333 M, and A. sojae 337 M.
Wheat bran Koji was prepared according to the method of Ichishima
(16). Six strains of Aspergilli were inoculated on sterilized wheat bran
and incubated at 30 °C for 72 h. Their enzyme activity was measured.
*
To whom correspondence should be addressed. Telephone: +81-1654-
2
-4194. Fax: +81-1654-3-3354. E-mail: ntoruota@nayoro.ac.jp.
†
Nayoro City College.
‡
Tokyo University of Agriculture.
§
Junior College of Tokyo University of Agriculture.
|
University of Shizuoka.
1
0.1021/jf050420d CCC: $30.25
© 2005 American Chemical Society
Published on Web 07/02/2005

Synthesis of Morphiceptin by DP IV
J. Agric. Food Chem., Vol. 53, No. 15, 2005 6113
Table 2. Effect of Temperature on Morphiceptin Synthesisa
Table 1. Activity of DP IV Isolated from A. oryzae and A. sojae
strain
DP IV (nkat/g of Koji)
temperature (
°
C)
yield (%)
temperature (
°
C)
yield (%)
A. oryzae 076M
A. oryzae RIB 128
A. oryzae RIB 430
A. oryzae RIB 915
A. sojae 333M
0.48
0.67
0.36
0.81
0.27
0.27
−
20
4
12.4
13.8
30
40
0.9
0.5
a
2
Reaction conditions: 40 mM Tyr-ProOEt
HCl, 25 L; DP IV, 0.325 nkat; diisopropylamine, 2.0 µ
5% in 20 mM phosphate buffer at pH 7.5; total volume, 500
‚
HCl, 50
µ
L; 400 mM Phe-Pro-
L; ethylene glycol,
L; reaction time, 24
NH
5
‚
µ
A. sojae 337M
µ
h.
Enzyme Assay. Hydrolytic activity of DP IV was determined by
the method of Tachi et al. (15). The enzyme solution (0.1 mL), prepared
as described below, was added to 0.5 mL of 0.45 mM Gly-Pro-pNA
and incubated at 30 °C for 5 min. After the reaction was stopped by
the addition of 0.1 M HCl (0.5 mL), the absorbance at 400 nm was
measured. The activity of DP IV was shown by mol/s of pNA liberated
at pH 7.5 at 30 °C, and that unit is 1 katal.
from A. oryzae RIB 915, 0.81 nkat/g of Koji. This strain was
selected for further experiments.
The presence of four major components was confirmed in
the products of the reaction between Tyr-Pro-OEt and Phe-Pro-
NH in the presence of partially purified DP IV. One of those
products (rt ) 26.0 min) was identified as morphiceptin by LC-
MS (Figure 1). The two peaks at m/z 522 and 544 shown in
2
Partial Purification. Wheat bran inoculated with Aspergilli and
cultured was added to a 10-fold volume of 20 mM phosphate buffer
(pH 7.5) and kept at 4 °C for 3 h; the crude enzyme was obtained by
the ESI mass spectrum of the product indicated two kinds of
filtration. Ammonium sulfate at 90% saturation was added to the crude
enzyme solution and let to stand at 4 °C for 1 day. The precipitate
obtained by salting out was collected and extracted again with 20 mM
phosphate buffer (pH 7.5). The enzyme was purified partially with
Sephacryl S-300 (Amersham Pharmacia) using 20 mM phosphate buffer
solution (pH 7.5) as an eluent.
+
+
adducted molecules, a [M + H] and a [M + Na] , respectively.
The fragment peaks, m/z 408, 359, 262, and 261, generated by
collision-induced dissociation of the protonated molecule (MS-
MS), indicated b3, y3, y2, and b2, respectively. The fragment
pattern was coincident with that of morphiceptin (17). From
these results, the structure of the product was confirmed as
morphiceptin. The morphiceptin yield was 13.8% at 4 °C but
less than 1% at 30 or 40 °C (Table 2).
Synthetic Reaction. Ethylene glycol (0-80%), amines, 20 mM
phosphate buffer (pH 6.0-7.5), and DP IV (0.250-0.375 nkat) were
added to 50 µL of 40 mM Tyr-Pro-OEt‚HCl and 25 µL of 400 mM
Phe-Pro-NH ‚HCl in a 2 mL microtube to make a final volume of 500
2
In the absence of ethylene glycol, Tyr-Pro-OEt was rapidly
hydrolyzed. The morphiceptin yield increased with the increase
of the ethylene glycol concentration. The yield was 19.5% in
the presence of 60% ethylene glycol (Figure 2). The presence
of amines such as triethylamine and 1,8-diazabicyclo-[5.4.0]-
µL. After the reaction for various times, peptides in 5 µL of the reaction
mixture were examined by HPLC using 4.6 × 250 mm TSK-GEL ODS-
8
0Ts column (Tosoh Corp., Tokyo, Japan). The column was eluted
with a linear gradient of acetonitrile (0-60%) containing 0.1% TFA.
The flow rate was 1.2 mL/min, and detection was made at 280 nm.
Yields of morphiceptin and Tyr-Pro-OEt were calculated from the peak
area of each component in comparison with that of authentic chemicals.
After the synthesis reaction, the peak that coincided with morphiceptin
was purified by MPLC using 0.1% TFA and acetonitrile as the eluent.
The purified compund was analyzed by LC-MS. The ion trap mass
spectrometer equipped with an electrospray ionization (ESI) source
7
-undecene (DBU) did not affect morphiceptin synthesis (data
not shown). Application of 0.28 µL of diisopropylamine (final
concentration of 4.2 mM) elicited a 31% augmentation in yield
even though a further increase in the amount of diisopropy-
lamine decreased the yield (Figure 3). The yield in the presence
of 4.2 mM diisopropylamine at optimal pH (7.0) was 40.0%
(Finnigan LCQ, Thermo Electoron Corporation, San Jose, CA) was
(Figure 4).
used for analyzing the structure of the product.
Figure 5 shows the effect of the Phe-Pro-NH2 concentration
on DP IV for morphiceptin synthesis. The increase in the Phe-
Pro-NH2 ratio to Tyr-Pro-OEt was followed by higher peptide
synthesis; the optimum level was reached for Phe-Pro-NH2/Tyr-
Pro-OEt ) 5:1. The morphiceptin yield increased with an
RESULTS
Table 1 shows the activity of DP IV isolated from the six
strains of Aspergilli. The highest activity was shown by DP IV
Figure 1. HPLC profile of the reaction mixture. We injected a reaction mixture catalyzed by DP IV (0.325 nkat/tube) using 50
OEt HCl and 25 L of 400 mM Phe-Pro-NH HCl as the substrate in 55% ethylene glycol containing 20 mM phosphate buffer (pH 7.5) at 4
Conditions for chromatograms: column, TSK gel ODS-80Ts (5 m); mobile phase, acetonitrile (0 60%) containing 0.1% TFA; flow rate, 1.2 mL/min;
detector, UV 280 nm; sample, 5 L.
µ
L of 40 mM Tyr-Pro-
‚
µ
2
‚
°
C.
µ
−
µ

6114 J. Agric. Food Chem., Vol. 53, No. 15, 2005
Ota et al.
Figure 4. Effect of pH on morphiceptin synthesis. Reaction conditions:
Figure 2. Effect of ethylene glycol on morphiceptin synthesis. Reaction
conditions: 40 mM Tyr-Pro-OEt HCl, 50 L; 400 mM Phe-Pro-NH HCl,
L; DP IV, 0.325 nkat; pH 7.5, diisopropylamine, 2.0 L; total volume,
L; reaction temperature, 4 C; reaction time, 24 h. Morphiceptin
), Tyr-Pro-OEt ( ).
4
0 mM Tyr-Pro-OEt
IV, 0.325 nkat; diisopropylamine, 0.28
phosphate buffers of various pH; total volume, 500
temperature, 4 C; reaction time, 24 h. Morphiceptin (
).
‚
HCl, 50
µ
L; 400 mM Phe-Pro-NH
L; ethylene glycol, 60% in
L; reaction
), Tyr-Pro-OEt
2
‚HCl, 25 µL; DP
‚
µ
2
‚
µ
2
5
5
00
µ
µ
µ
µ
°
°
b
(
b
2
(
2
Figure 3. Effect of diisopropylamine on morphiceptin synthesis. Reaction
conditions: 40 mM Tyr-Pro-OEt HCl, 50 L; 400 mM Phe-Pro-NH HCl,
L; DP IV, 0.325 nkat; ethylene glycol, 60% in 20 mM phosphate
buffer at pH 7.5; total volume, 500 L; reaction temperature, 4 C; reaction
time, 24 h. Morphiceptin ( ), Tyr-Pro-OEt ( ).
‚
µ
2
‚
25 µ
µ
°
Figure 5. Effect of substrate ratio on morphiceptin yield. Reaction
conditions: DP IV, 0.325 nkat; diisopropylamine, 0.28 L; ethylene glycol,
0% in 20 mM phosphate buffer at pH 7.0; total volume, 500 L; reaction
temperature, 4 C; reaction time, 24 h.
b
2
µ
6
µ
increasing reaction time, reaching 40.0% at 24 h, but a further
increase in the reaction time engendered only slight effects
°
(
Figure 6). The morphiceptin yield at this condition was 40.0%.
However, many proteolytic enzymes do not cut the peptide at
the position of Pro. DP IV has been found in mammals, yeast,
and bacteria (18-20); it is known to liberate dipeptide from
the peptide having Pro at the second position from the amino
terminal. We used Koji mold and screened strains having a high
activity for specific separation of X-Pro. DP IV used in this
study was a new enzyme isolated from mold. It had the same
molecular weight as DP IV from hog kidney (15).
The amount of DP IV within the range of 0.250-0.375 nkat
only slightly affected the reaction, but the concentration of Tyr-
Pro-OEt decreased with an increasing amount of DPIV (Figure
7
). Therefore, we judged 0.275 nkat of DP IV addition to 500
µL of reaction mixture as optimal.
DISCUSSION
The DP IV used in this study hydrolyzes Gly-Pro-pNA and
Arg-Pro-pNA specifically, but it does not hydrolyze substrates
having a protective amino group such as benzyloxycarbonyl,
e.g., Z-Gly-Pro-pNA (15). In this study, we used Tyr-Pro-OEt‚
HCl, which has no protective amino acid as an acyl donor.
Morphiceptin has an amidized carboxyl group as well as gastrin
Enzymatic synthesis of peptides has been studied for a long
time. Merits of this method are that (1) racemization does not
occur during the reaction, (2) protection of the side-chain
functional group is unnecessary, (3) side reactions are limited,
and (4) the reaction proceeds under mild conditions (13). In
fact, as shown in Figure 1, the reaction solution contained only
a few byproducts other than the substrates and a product.
(21), amylin (22, 23), and galanin (24). The compound with
-NH2 seemed effective as the protective group of R-carboxyl
Generally, the peptide bond that can be formed by enzymatic
synthesis is determined by the primary specificity of the enzyme.
for the synthetic reaction. We used Phe-Pro-NH2 as a nucleo-
phile.

Synthesis of Morphiceptin by DP IV
J. Agric. Food Chem., Vol. 53, No. 15, 2005 6115
at -20 °C. Nilsson et al. suggested that the effect of low
temperature is attributable to water activity but that mechanism
is not yet known (27). However, in the absence of ethylene
glycol, little Tyr-Pro-OEt was found in the reaction mixture.
On the other hand, the higher the concentration of ethylene
glycol, the larger the amount of morphiceptin synthesized.
Ethylene glycol is considered to suppress the hydrolysis of Tyr-
Pro-OEt to Tyr-Pro-OH and shift the reaction toward synthesis
because Tyr-Pro-OEt remained in the presence of ethylene
glycol.
Diisopropylamine was added to neutralize hydrochloride; it
was effective. Nevertheless, the application of an extra amount
of diisopropylamine suppressed the synthesis, probably affecting
pH. In fact, the amount of remaining Tyr-Pro-OEt was
influenced markedly by pH, suggesting the importance of pH
in morphiceptin synthesis. The Phe-Pro-NH2 concentration was
5
times higher than the Tyr-Pro-OEt in the optimum condition.
The morphiceptin yield did not increase at the level Phe-Pro-
NH2/Tyr-Pro-OEt ) 10:1. The lack of increase is attributable
to the consumption of all Tyr-Pro-OEt within 24-48 h.
Although the amount of enzyme (DP IV) added did not largely
affect the percentage of synthesis, it affected the hydrolysis of
Tyr-Pro-OEt. These results imply the following optimum
condition for morphiceptin synthesis: substrate, Phe-Pro-NH2/
Tyr-Pro-OEt ) 5:1 (20 mM/4 mM); enzyme, DP IV, 0.275 nkat;
solvent, 60% ethylene glycol in 20 mM phosphate buffer at pH
Figure 6. Effect of reaction time on morphiceptin synthesis. Reaction
conditions: 40 mM Tyr-Pro-OEt
‚
HCl, 50
L; DP IV, 0.325 nkat; diisopropylamine, 0.28
0% in 20 mM phosphate buffer at pH 7.0; total volume, 500
C. Morphiceptin ( ), Tyr-Pro-OEt ( ).
µ
L; 400 mM Phe-Pro-NH
L; ethylene glycol,
L; reaction
2
‚HCl,
25
µ
µ
6
µ
temperature, 4
°
b
2
7
.0; diisopropylamine, 0.28 µL (4.2 mM); total volume, 500
µL; reaction temperature, 4 °C; reaction time, 24 h. Under this
condition, the yield was about 40%.
The possibility of enzymatic synthesis of morphiceptin has
been reported (14) but has not been demonstrated. We synthe-
sized morphiceptin enzymatically in this study. A protective
amino group such as the benzyloxycarbonyl group has been
effective in many enzymatic approaches, but such a protective
group is unnecessary for enzymatic synthesis of peptides using
DP IV. This fact is an important merit of this method.
ACKNOWLEDGMENT
We thank Dr. Kenji Kobata, University of Shizuoka, for
measuring the LC-MS data and Dr. Eiji Ichishima, Soka
University, for advice and helpful discussion.
Figure 7. Effect of the amount of DP IV on morphiceptin synthesis.
Reaction conditions: 40 mM Tyr-Pro-OEt HCl, 50 L; 400 mM Phe-Pro-
NH HCl, 25 L; diisopropylamine; 0.28 L; ethylene glycol, 60% in 20
mM phosphate buffer at pH 7.0; total volume, 500 L; reaction
temperature, 4 C; reaction time, 24 h. Morphiceptin ( ), Tyr-Pro-OEt
).
‚
µ
µ
2
‚
µ
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(
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Received for review February 23, 2005. Revised manuscript received
June 3, 2005. Accepted June 7, 2005.
(
JF050420D