U.S. patent application number 10/500251 was filed with the patent office on 2006-10-19 for pyroglutamyl peptidase and its gene.
Invention is credited to Keietsu Abe, Osamu Akita, Kiyoshi Asai, Katsuya Gomi, Akira Hosoyama, Taishin Kin, Satoru Kuhara, Masayuki Machida, Hideki Nagasaki, Naotake Ogasawara, Chiaki Saitoh, Motoaki Sano, Akihiro Senoh, Itaru Toda, Chikara Tokunaga.
Application Number | 20060234320 10/500251 |
Document ID | / |
Family ID | 19190421 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060234320 |
Kind Code |
A1 |
Machida; Masayuki ; et
al. |
October 19, 2006 |
Pyroglutamyl peptidase and its gene
Abstract
The present invention provides DNA encoding novel pyroglutamyl
peptidase derived from Aspergillus oryzae, pyroglutamyl peptidase
which is produced by using the DNA, and a method for producing a
protein lysate with a good flavor at a high hydrolysis rate.
Inventors: |
Machida; Masayuki; (Ibaraki,
JP) ; Abe; Keietsu; (Miyagi, JP) ; Gomi;
Katsuya; (Miyagi, JP) ; Asai; Kiyoshi; (Tokyo,
JP) ; Sano; Motoaki; (Ibaraki, JP) ; Kin;
Taishin; (Tokyo, JP) ; Nagasaki; Hideki;
(Tokyo, JP) ; Hosoyama; Akira; (Tokyo, JP)
; Akita; Osamu; (Hiroshima, JP) ; Ogasawara;
Naotake; (Nara, JP) ; Kuhara; Satoru;
(Fukuoka, JP) ; Tokunaga; Chikara; (Ibaraki,
JP) ; Toda; Itaru; (Ibaraki, JP) ; Saitoh;
Chiaki; (Ibaraki, JP) ; Senoh; Akihiro;
(Tokyo, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Family ID: |
19190421 |
Appl. No.: |
10/500251 |
Filed: |
December 26, 2002 |
PCT Filed: |
December 26, 2002 |
PCT NO: |
PCT/JP02/13627 |
371 Date: |
October 29, 2004 |
Current U.S.
Class: |
435/7.31 ;
435/219; 435/254.3; 435/484; 435/68.1; 435/69.1; 530/388.5;
536/23.2 |
Current CPC
Class: |
C12P 21/06 20130101;
C12N 9/48 20130101; C12N 9/80 20130101; C12N 9/0022 20130101 |
Class at
Publication: |
435/007.31 ;
435/068.1; 435/069.1; 435/219; 435/254.3; 435/484; 536/023.2;
530/388.5 |
International
Class: |
G01N 33/569 20060101
G01N033/569; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 9/50 20060101 C12N009/50; C12N 15/74 20060101
C12N015/74; C12N 1/16 20060101 C12N001/16 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 2001 |
JP |
2001-403261 |
Claims
1. A polypeptide selected from the group consisting of the
following (a) to (c): (a) a polypeptide comprising an amino acid
sequence shown in SEQ ID NO: 2, (b) a polypeptide comprising an
amino acid sequence substantially identical to the amino acid
sequence shown in SEQ ID NO: 2, and having a pyroglutamyl peptidase
activity, and (c) a polypeptide comprising an amino acid sequence
wherein one or more amino acid residues are deleted, substituted or
added in the amino acid sequence shown in SEQ ID NO: 2, and having
a pyroglutamyl peptidase activity.
2. DNA comprising a nucleotide sequence encoding the polypeptide
according to claim 1.
3. DNA selected from the group consisting of the following (a) to
(c): (a) DNA comprising a nucleotide sequence shown in SEQ ID NO:
1, (b) DNA comprising a nucleotide sequence shown in SEQ ID NO: 5,
and (c) DNA which hybridizes with DNA comprising a nucleotide
sequence complementary to the nucleotide sequence shown in SEQ ID
NO: 1 or 5 under stringent conditions, and comprises a nucleotide
sequence encoding a polypeptide having a pyroglutamyl peptidase
activity.
4. DNA selected from the group consisting of the following (a) to
(c): (a) DNA comprising a nucleotide sequence shown in SEQ ID NO:
3, (b) DNA which comprises a partial sequence of the nucleotide
sequence shown in SEQ ID NO: 3 consisting of 100 or more
nucleotides and functions as a promoter, and (c) DNA which
hybridizes with DNA comprising a nucleotide sequence complementary
to the nucleotide sequence shown in SEQ ID NO: 3 under stringent
conditions and functions as a promoter.
5. DNA selected from the group consisting of the following (a) to
(c): (a) DNA comprising a nucleotide sequence complementary to a
nucleotide sequence shown in SEQ ID NO: 4, (b) DNA which comprises
a partial sequence of a nucleotide sequence complementary to the
nucleotide sequence shown in SEQ ID NO: 4 consisting of 15 or more
nucleotides, and (c) DNA which hybridizes with DNA comprising the
nucleotide sequence shown in SEQ ID NO: 4 under stringent
conditions.
6. The DNA according to any one of claims 2 to 5, wherein DNA is
genomic DNA.
7. An oligonucleotide comprising a nucleotide sequence consisting
of 15 or more contiguous nucleotides of the nucleotide sequence of
the DNA according to any one of claims 2 to 5 or a nucleotide
sequence complementary thereto.
8. A recombinant DNA comprising the DNA according to claims 2 or
3.
9. A transformant comprising the recombinant DNA according to claim
8.
10. A process for producing the polypeptide according to claim 1,
which comprises culturing a microorganism having an ability to
produce the polypeptide in a medium, so as to produce and
accumulate the polypeptide in a culture, and recovering the
polypeptide from the culture.
11. The process according to claim 10, wherein the microorganism is
a transformant comprising a recombinant DNA comprising DNA
comprising a nucleotide sequence encoding a polypeptide selected
from the group consisting of the following (a) to (c): (a) a
polypeptide comprising an amino acid sequence shown in SEQ ID NO:
2, (b) a polypeptide comprising an amino acid sequence
substantially identical to the amino acid sequence shown in SEQ ID
NO: 2, and having a pyroglutamyl peptidase activity, and (c) a
polypeptide comprising an amino acid sequence wherein one or more
amino acid residues are deleted, substituted or added in the amino
acid sequence shown in SEQ ID NO: 2, and having a pyroglutamyl
peptidase activity.
12. The process according to claim 10, wherein the microorganism is
filamentous fungus.
13. The process according to claim 12, wherein the filamentous
fungus belongs to one genus selected from a group consisting of
Aspergillus, Penicillium, Humicola, Trichoderma, Mucor, and
Fusarium.
14. The process according to claim 13, wherein the filamentous
fungus belonging to Aspergillus belongs to one species selected
from a group consisting of Aspergillus oryzae, Aspergillus sojae,
Aspergillus niger, Aspergillus awamori, Aspergillus kawachii,
Aspergillus parasiticus, Aspergillus flavus, Aspergillus nomius,
Aspergillus fumigatus, and Aspergillus nidulans.
15. A process for producing a protein hydrolysate, which comprises
adding the polypeptide according to claim 1 and a protein hydrolase
to a raw material containing a protein, and degrading the
protein.
16. A process for producing a protein hydrolysate, which comprises
adding a culture containing the polypeptide according to claim 1
which is obtained by culturing a microorganism having an ability to
produce the polypeptide according to claim 1 in a medium, or a
treated product thereof, and a protein hydrolase, to a raw material
containing a protein, and degrading the protein.
17. The process according to claim 16, wherein the microorganism is
a transformant comprising a recombinant DNA comprising DNA
comprising a nucleotide sequence encoding a polypeptide selected
from the group consisting of the following (a) to (c): (a) a
polypeptide comprising an amino acid sequence shown in SEQ ID NO:
2, (b) a polypeptide comprising an amino acid sequence
substantially identical to the amino acid sequence shown in SEQ ID
NO: 2, and having a pyroglutamyl peptidase activity, and (c) a
polypeptide comprising an amino acid sequence wherein one or more
amino acid residues are deleted, substituted or added in the amino
acid sequence shown in SEQ ID NO: 2, and having a pyroglutamyl
peptidase activity.
18. The process according to claim 16, wherein the microorganism is
filamentous fungus.
19. The process according to claim 18, wherein the filamentous
fungus belongs to one genus selected from a group consisting of
Aspergillus, Penicillium, Humicola, Trichoderma, Mucor, and
Fusarium.
20. The process according to claim 19, wherein the filamentous
fungus belonging to Aspergillus belongs to one species selected
from a group consisting of Aspergillus oryzae, Aspergillus sojae,
Aspergillus niger, Aspergillus awamori, Aspergillus kawachii,
Aspergillus parasiticus, Aspergillus flavus, Aspergillus nomius,
Aspergillus fumigatus, and Aspergillus nidulans.
21. A protein hydrolysate which is produced by the process
according to any one of claims 15 to 20.
22. An antibody which specifically binds to the polypeptide
according to claim 1.
23. A method of detecting or quantifying the polypeptide according
to claim 1 which comprises using the antibody according to claim
22.
24. An oligonucleotide comprising a nucleotide sequence consisting
of 15 or more contiguous nucleotides of the nucleotide sequence of
the DNA according to claim 6 or a nucleotide sequence complementary
thereto.
25. The process according to claim 10, wherein the microorganism is
a transformant comprising a recombinant DNA comprising DNA selected
from the group consisting of the following (a) to (c): (a) DNA
comprising a nucleotide sequence shown in SEQ ID NO: 1, (b) DNA
comprising a nucleotide sequence shown in SEQ ID NO: 5, and (c) DNA
which hybridizes with DNA comprising a nucleotide sequence
complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 5
under stringent conditions, and comprises a nucleotide sequence
encoding a polypeptide having a pyroglutamyl peptidase
activity.
26. The process according to claim 16, wherein the microorganism is
a transformant comprising a recombinant DNA comprising DNA selected
from the group consisting of the following (a) to (c): (a) DNA
comprising a nucleotide sequence shown in SEQ ID NO: 1, (b) DNA
comprising a nucleotide sequence shown in SEQ ID NO: 5, and (c) DNA
which hybridizes with DNA comprising a nucleotide sequence
complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 5
under stringent conditions, and comprises a nucleotide sequence
encoding a polypeptide having a pyroglutamyl peptidase
activity.
27. A protein hydrolysate which is produced by the process of claim
26.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pyroglutamyl peptidase
used in production of protein hydrolysates and DNA encoding the
pyroglutamyl peptidase.
BACKGROUND ART
[0002] Among filamentous fungi, in particular, Koji molds including
Aspergillus oryzae has been used for a long time in the field of
fermented food production in our country, which produces sake, bean
paste, soy sauce, Japanese sweet rice wine for cooking, etc., and
thus, this fungus has been directly eaten. Koji molds are safe gene
sources that have been listed up as GRAS (Generally Recognized as
Safe) by the FDA (Food and Drug Administration) of USA. Thus,
filamentous fungi, and especially, Koji molds are considered to be
a treasure trove of genes with extremely high utility value in
terms of safety.
[0003] Protein hydrolysates can be produced by hydrolysis of a raw
material comprising a protein with acid. From the viewpoint of the
use of protein hydrolysates as natural seasonings, a production
process of protein hydrolysates by enzymolysis is being studied, in
addition to the production method thereof by acidolysis. As protein
hydrolysates produced by enzymolysis, enzymatic hydrolysate of
albumen (Japanese Published Unexamined Patent Application No.
68773/73), enzymatic hydrolysate of defatted soybeans (Japanese
Published Unexamined Patent Application No. 70852/76), enzymatic
hydrolysate of cheese whey as a raw material (Japanese Published
Unexamined Patent Application No. 151155/87), and enzymatic
hydrolysate of corn gluten meal (Japanese Published Examined Patent
Application No. 295437/90) have been reported.
[0004] However, there may be a case where a generated peptide has a
bitter taste depending on properties of a protein and enzymes used
in proteolysis and therefore it is not functionally preferred.
Thus, a hydrolysate having a high degradation ratio in hydrolysis
and excellent functional properties has been desired. In order to
improve such a degradation rate, techniques applied to various
enzymes such as glutaminase derived from Koji molds (WO99/60104,
Japanese Published Unexamined Patent Application No. 166547/2000,
and Japanese National Publication of International Patent
Application No. 511746/2002) are being considered. As shown in the
fermentation of soy sauce and bean paste, in the case of using the
conventional Aspergillus culture products, although the hydrolysis
of proteins requires enormous manpower and time, it results in a
low releasing rate of amino acid. In particular, it results in a
low releasing rate of glutamic acid, which is contained in the
largest amount in soy protein and is important for flavoring.
[0005] In the mean time, there are a large number of proteins and
peptides whose N terminus is protected by an L-pyroglutamic acid
residue. Moreover, when a protein or peptide is hydrolyzed,
glutamine or glutamic acid at a newly generated amino terminus is
nonenzymatically cyclized to form a pyroglutamic acid residue in
many cases, and such a residue is detected also in food. Since
these proteins or peptides whose N terminus is protected by an
L-pyroglutamic acid residue are not directly subjected to
hydrolysis by amino peptidase, they require an operation to
eliminate the L-pyroglutamic acid residue. Pyroglutamyl peptidase
is an enzyme for specifically releasing an L-pyroglutamic acid
residue located at an amino terminus of these proteins or peptides.
It has been known that this enzyme widely exists in a wide range of
areas such as the brain, lung, serum or pituitary gland of various
animals, plants, or microorganisms. It has been reported that a
protein hydrolysate obtained by reacting a protein with protein
hydrolase is further reacted with pyroglutamyl peptidase, so as to
produce a protein hydrolysate with good flavoring properties
(Japanese Published Unexamined Patent Application No.
252075/96).
[0006] An enzyme derived from Bacillus amyloliquefaciens has been
known as pyroglutamyl peptidase derived from microorganisms [J.
Biochem., 84, 467 (1978)]. With regard to this enzyme, its gene has
been isolated, and a production method thereof has been reported
(Japanese Published Unexamined Patent Application No. 137572/93).
Moreover, with regard to an enzyme with high heat resistance
derived from Pyrococcus furiosus also, its gene has been isolated
(Japanese Published Unexamined Patent Application No. 298881/95).
Furthermore, an enzyme derived from Bacillus subtilis has been
known (Japanese Published Unexamined Patent Application No.
252075/96). However, pyroglutamyl peptidase derived from
filamentous fungi and a gene thereof have not been isolated yet.
Still further, in case of using pyroglutamyl peptidase together
with a culture product of Koji mold as a protein hydrolase source
in the production of protein hydrolysates, pyroglutamyl peptidase
derived from heterologous organisms other than Koji molds, such as
Bacillus subtilis, has been used. It is considered that it results
in high cost and deteriorated flavor.
DISCLOSURE OF THE INVENTION
[0007] The present invention provides: a novel pyroglutamyl
peptidase derived from filamentous fungi, which can be used in the
production of a protein hydrolysate having a higher releasing rate
of amino acid, and especially, a higher releasing rate of glutamic
acid, than those of the conventional protein hydrolysates that are
obtained by hydrolysis with enzymes; and a pyroglutamyl peptidase
gene that can be used in the production of the pyroglutamyl
peptidase.
[0008] That is to say, the present invention relates to
polypeptides of the embodiments described below, and DNAs encoding
the polypeptides. Among the polypeptides and DNAs of the present
invention, those derived from Aspergillus oryzae which is a
microorganism listed up as GRAS by the FDA of USA, have extremely
high utility value in terms of safety and economical
efficiency.
[0009] The present invention provides the following (1) to
(23):
(1) A polypeptide selected from the group consisting of the
following (a) to (c):
[0010] (a) a polypeptide comprising an amino acid sequence shown in
SEQ ID NO: 2, [0011] (b) a polypeptide comprising an amino acid
sequence substantially identical to the amino acid sequence shown
in SEQ ID NO: 2, and having a pyroglutamyl peptidase activity, and
[0012] (c) a polypeptide comprising an amino acid sequence wherein
one or more amino acid residues are deleted, substituted or added
in the amino acid sequence shown in SEQ ID NO: 2, and having a
pyroglutamyl peptidase activity.
[0013] The amino acid sequence shown in SEQ ID NO: 2 is the amino
acid sequence of pyroglutamyl peptidase of Aspergillus oryzae.
[0014] The term "an amino acid sequence substantially identical to
the amino acid sequence shown in SEQ ID NO: 2" is used herein to
mean an amino acid sequence showing an average homology of
approximately 30% or more, preferably approximately 50% or more,
more preferably approximately 80% or more, and particularly
preferably approximately 90% or more with the amino acid sequence
shown in SEQ ID NO: 2, when the two amino acid sequences are
aligned to compare the entirety of the sequences.
[0015] The term "an amino acid sequence wherein one or more amino
acid residues are deleted, substituted or added in the amino acid
sequence shown in SEQ ID NO: 2" is used herein to mean an amino
acid sequence wherein preferably approximately 1 to 20, more
preferably approximately 1 to 10, and further more preferably
several amino acid residues are deleted, substituted or added, or
amino acid sequence obtained by combining these amino acid
sequences.
[0016] The pyroglutamyl peptidase activity of a polypeptide can be
determined as follows. A 50 mmol/l phosphate buffer solution (pH
7.5) containing 5 mmol/l pyroglutamic acid-paranitroanilide is
prepared as a substrate solution. A sample solution containing
polypeptides is prepared. 20 .mu.l of the sample solution is added
to 100 .mu.l of the substrate solution, and the mixture is reacted
at 37.degree. C. for 10 minutes. Thereafter, an absorbance at 405
nm is determined using a spectrophotometer. The amount by which
paranitroaniline has released per minute of reaction time is
calculated from the molar absorption coefficient of
paranitroaniline (10500). An activity to release 1 micromole of
paranitroaniline at 37.degree. C. for 1 minute is defined as 1
unit.
[0017] The above polypeptide comprising an amino acid sequence
substantially identical to the amino acid sequence shown in SEQ ID
NO: 2, or polypeptide comprising an amino acid sequence wherein one
or more amino acid residues are deleted, substituted or added in
the amino acid sequence shown in SEQ ID NO: 2, can be easily
produced by appropriately combining methods known to a person
skilled in the art, such as a site-directed mutagenesis, gene
homologous recombination, primer extension or PCR method.
[0018] In order that the produced polypeptide has substantially
identical functions, it seems to be effective to substitute
homologous amino acids (e.g., polar/nonpolar amino acids,
hydrophobic/hydrophilic amino acids, positively charged/negatively
charged amino acids, aromatic amino acids, etc.) with each other,
among amino acids constituting the polypeptide. Moreover, in order
to maintain the substantially identical functions, amino acids in
functional domains contained in each polypeptide of the present
invention are desirably retained.
[0019] An example of the polypeptide comprising an amino acid
sequence wherein one or more amino acid residues are deleted,
substituted or added in the amino acid sequence shown in SEQ ID NO:
2 and having a pyroglutamyl peptidase activity includes a
polypeptide comprising an amino acid sequence shown in SEQ ID NO:
10, in which 41 amino acid residues are added to the N terminus of
the amino acid sequence shown in SEQ ID NO: 2.
(2) DNA comprising a nucleotide sequence encoding the polypeptide
according to (1).
(3) DNA selected from the group consisting of the following (a) to
(c):
[0020] (a) DNA comprising a nucleotide sequence shown in SEQ ID NO:
1, [0021] (b) DNA comprising a nucleotide sequence shown in SEQ ID
NO: 5, and [0022] (c) DNA which hybridizes with DNA comprising a
nucleotide sequence complementary to the nucleotide sequence shown
in SEQ ID NO: 1 or 5 under stringent conditions, and comprises a
nucleotide sequence encoding a polypeptide having a pyroglutamyl
peptidase activity.
[0023] DNAs according to (2) and (3) have a function as a region
which encodes a polypeptide having a pyroglutamyl activity. An
example of an amino acid sequence encoded by these DNAs is shown in
SEQ ID NO: 2. The amino acid sequence shown in SEQ ID NO: 2 has
been determined from various information (ORF, exon/intron region,
expressed sequence tag (EST), etc.) to identify a gene region
(location of gene) on the basis of the genomic nucleotide sequence
of Aspergillus oryzae RIB 40 (FERM P-18273 (transferred to FERM
BP-7935)). The nucleotide sequence shown in SEQ ID NO: 1 is the
nucleotide sequence of genomic DNA which comprises a pyroglutamyl
peptidase gene of the Aspergillus oryzae RIB 40 and a promoter
region thereof. The nucleotide sequence shown in SEQ ID NO: 5 is a
nucleotide sequence obtained by removing 5' and 3' non-translation
regions and introns from the nucleotide sequence shown in SEQ ID
NO: 1, and it matches the nucleotide sequence of a region which
encodes the pyroglutamyl peptidase of pyroglutamyl peptidase cDNA
of the Aspergillus oryzae RIB 40.
[0024] In the present specification, the term "under stringent
conditions" is used to mean conditions in which hybrids are
specifically formed only between nucleotide sequences having a high
homology, such as an average homology of approximately 80% or more,
preferably approximately 90% or more, and more preferably
approximately 95% or more. More specifically, an example of such
stringent conditions includes conditions of a temperature between
60.degree. C. and 68.degree. C., a sodium concentration of 150 to
900 mmol/l, and preferably 600 to 900 mmol/l, and pH of 6 to 8.
[0025] Hybridization can be carried out according to methods known
in this field, such as the method described in Current Protocols in
Molecular Biology, John Wiley & Sons (1987), or methods which
are similar thereto. In addition, when a commercially available
library is used, hybridization can be carried out according to the
method described in instructions attached thereto. Examples of DNA
which hybridizes with DNA comprising a nucleotide sequence
complementary to the nucleotide sequence shown in SEQ ID NO: 1 or 5
under stringent conditions, and having a nucleotide sequence
encoding a polypeptide having a pyroglutamyl peptidase activity,
include genomic DNA and cDNA which encode pyroglutamyl peptidase
derived from filamentous fungi other than Aspergillus oryzae.
(4) DNA selected from the group consisting of the following (a) to
(c):
[0026] (a) DNA comprising a nucleotide sequence shown in SEQ ID NO:
3, [0027] (b) DNA which comprises a partial sequence of the
nucleotide sequence shown in SEQ ID NO: 3 consisting of 100 or more
nucleotides and functions as a promoter, and [0028] (c) DNA which
hybridizes with DNA comprising a nucleotide sequence complementary
to the nucleotide sequence shown in SEQ ID NO: 3 under stringent
conditions and functions as a promoter.
[0029] These DNA sequences correspond to regions which encode the
polypeptide of the present invention in the genomic DNA of
filamentous fungi, such as a region located 5' upstream of a
nucleotide sequence corresponding to 1001 to 1111 nucleotides of
the nucleotide sequence shown in SEQ ID NO: 1.
[0030] In some of these sequences, EST has actually been confirmed
in a coding region located in a 3' downstream region thereof
(Expression conditions: any one of a culture in a complete liquid
medium containing 2% glucose; a culture in a complete liquid medium
containing 2% maltose; a culture in synthetic liquid medium
containing no carbon sources; a culture at a high temperature (37
.degree. C., but in other cultures, a temperature of 30.degree. C.
is applied unless otherwise specified) in a complete liquid medium
containing 2% glucose; a solid culture (using wheat bran); a
culture in an synthetic alkaline medium (pH 10) containing 2%
glucose; a culture which was carried out immediately after
germination of spores which had been cultured at 28.degree. C. for
8 days in a potato dextrose agar medium; and a solid culture at
25.degree. C. for 3 hours which was carried out after culturing for
34 hours in a mixed medium of soybeans and wheat). As is clear from
this fact, some of these sequences comprise a promoter region (a
sequence interacting with various polymerase, a general
transcription factor, or a transcription factor, such as a core
promoter or basic promoter and upstream promoter elements).
[0031] Accordingly, a sequence corresponding to preferably 200 or
more base pairs, more preferably 500 or more base pairs, further
more preferably 800 or more base pairs, and particularly preferably
900 or more base pairs from the 3' side of each of the above
sequences, is suitable as a partial sequence that can comprise the
above promoter region. Otherwise, a partial sequence having base
pairs corresponding to the above length in a suitable intermediate
portion in each of the above sequences can also be used.
[0032] It can be confirmed whether or not DNA functions as a
promoter, for example, by the following method. DNA to be tested is
ligated upstream of DNA encoding a polypeptide as a reporter, so as
to prepare new DNA. The obtained DNA is inserted into a suitable
vector comprising a transformation marker gene such as an
acetamidase gene of Aspergillus oryzae or nitrate reductase gene
[J. Ferment. Bioeng., 74, 389 (1992), Mol. Gen. Genet., 218, 99-104
(1989)], so as to produce a recombinant vector. Aspergillus oryzae
is transformed with the obtained recombinant vector by the method
described in the publications [J. Ferment. Bioeng., 74, 389 (1992),
Mol. Gen. Genet., 218, 99-104 (1989)]. The level of a polypeptide
as a reporter is measured in the obtained transformant. When such a
polypeptide is detected, it is confirmed that the DNA ligated
upstream of the DNA encoding the polypeptide acting as a reporter
functions as a promoter. Examples of a polypeptide as a reporter
may include .beta.-glucuronidase of Escherichia coli, a green
fluorescent protein, and .beta.-galactosidase of Escherichia coli.
The .beta.-glucuronidase, green fluorescent protein and
.beta.-galactosidase in the transformant or a culture supernatant
thereof can be detected according to the method described in the
publications [Appl. Environ. Microbiol. 61, 2482 (1995), Eur. J.
Biochem. 266, 252 (1999), Mol. Microbiol. 8, 211 (1993)].
[0033] The DNA according to (4) is also useful, for example, as a
region into which a foreign gene or the like is inserted and
expressed.
(5) DNA selected from the group consisting of the following (a) to
(c):
[0034] (a) DNA comprising a nucleotide sequence complementary to a
nucleotide sequence shown in SEQ ID NO: 4, [0035] (b) DNA which
comprises a partial sequence of a nucleotide sequence complementary
to the nucleotide sequence shown in SEQ ID NO: 4 consisting of 10
or more nucleotides, and [0036] (c) DNA which hybridizes with DNA
comprising the nucleotide sequence shown in SEQ ID NO: 4 under
stringent conditions.
[0037] This DNA is DNA of a 3' non-translation region comprising
nucleotides 1902 to 2201 of the nucleotide sequence shown in SEQ ID
NO: 1 in the genomic DNA of Aspergillus oryzae RIB 40. In
particular, when the DNA is hybridized with mRNA transcribed from a
pyroglutamyl peptidase gene comprising the nucleotide sequence
shown in SEQ ID NO: 1 and the mRNA is detected, this DNA can be
used as a probe for the detection. The region encoding pyroglutamyl
peptidase in the above mRNA, which corresponds to the nucleotide
sequence shown in SEQ ID NO: 5, often comprises sequences having a
high homology with other mRNAs in association with the functions of
polypeptides encoded thereby. In contrast, the nucleotide sequence
of this region comprises a highly arbitrary sequence that is
totally irrelevant to SEQ ID NO: 5. Accordingly, using the DNA
according to (5) as a probe, it is possible to differentially
detect and quantify mRNA of pyroglutamyl peptidase with extremely
high properties from a population of RNAs extracted from cells.
Moreover, it is also possible to produce primers for PCR having the
sequence of this region and to carry out such differential
detection and quantification by PCR.
[0038] A portion corresponding to preferably 300 base pairs, more
preferably 200 base pairs, and particularly preferably
approximately 100 base pairs from the 5' side of the above sequence
is suitable for the DNA of the present invention used as a probe,
or a region corresponding to a partial sequence thereof. In
addition, the length of such a partial sequence can be
appropriately selected by a person skilled in the art, depending on
intended use or the like. In general, it is preferred that the
length of the sequence is longer within the above ranges in terms
of detectability and quantitative sensitivity.
(6) The DNA according to any one of (2) to (5), wherein DNA is
genomic DNA.
(7) An oligonucleotide comprising a nucleotide sequence consisting
of 15 or more continuous nucleotides of the nucleotide sequence of
the DNA according to any one of (2) to (6) above or a nucleotide
sequence complementary thereto.
(8) A recombinant DNA comprising the DNA according to (2) or
(3).
(9) A transformant comprising the recombinant DNA according to
(8).
[0039] (10) A process for producing the polypeptide according to
(1), which comprises culturing a microorganism having an ability to
produce the polypeptide in a medium, so as to produce and
accumulate the polypeptide in a culture, and recovering the
polypeptide from the culture.
(11) The process according to (10), wherein the microorganism is
the transformant according to (9).
(12) The process according to (10), wherein the microorganism is
filamentous fungus.
(13) The process according to (12), wherein the filamentous fungus
belongs to one genus selected from a group consisting of
Aspergillus, Penicillium, Humicola, Trichoderma, Mucor, and
Fusarium.
[0040] (14) The process according to (13), wherein the filamentous
fungus belonging to Aspergillus belongs to one species selected
from a group consisting of Aspergillus oryzae, Aspergillus sojae,
Aspergillus niger, Aspergillus awamori, Aspergillus kawachii,
Aspergillus parasiticus, Aspergillus flavus, Aspergillus nomius,
Aspergillus fumigatus, and Aspergillus nidulans.
(15) A process for producing a protein hydrolysate, which comprises
adding the polypeptide according to (1) and a protein hydrolase to
a raw material containing a protein, and degrading the protein.
[0041] (16) A process for producing a protein hydrolysate, which
comprises adding a culture containing the polypeptide according to
(1) which is obtained by culturing a microorganism having an
ability to produce the polypeptide according to (1) in a medium, or
a treated product thereof, and a protein hydrolase, to a raw
material containing a protein, and degrading the protein.
(17) The process according to (16), wherein the microorganism is
the transformant according to (9).
(18) The process according to (16), wherein the microorganism is
filamentous fungus.
(19) The process according to (18), wherein the filamentous fungus
belongs to one genus selected from a group consisting of
Aspergillus, Penicillium, Humicola, Trichoderma, Mucor, and
Fusarium.
[0042] (20) The process according to (19), wherein the filamentous
fungus belonging to Aspergillus belongs to one species selected
from a group consisting of Aspergillus oryzae, Aspergillus sojae,
Aspergillus niger, Aspergillus awamori, Aspergillus kawachii,
Aspergillus parasiticus, Aspergillus flavus, Aspergillus nomius,
Aspergillus fumigatus, and Aspergillus nidulans.
(21) A protein hydrolysate which is produced by the process
according to any one of (15) to (20).
(22) An antibody which specifically binds to the polypeptide
according to (1).
(23) A method of detecting or quantifying the polypeptide according
to (1), which comprises using the antibody according to (22).
[0043] Embodiments of the present invention will be described in
detail below.
(1) Production of DNA of the Present Invention
[0044] With regard to DNA of the present invention, for example,
DNA comprising a nucleotide sequence shown in SEQ ID NO: 1 can be
produced, for example, by the shut gun cloning method described in
Examples, using the genome of Aspergillus oryzae as a starting
material. In the production, each fragmented chromosomal DNA is
ligated to a suitable cloning vector such as a plasmid vector or
phage, depending on its length or the like. Thereafter, a suitable
host cell such as Escherichia coli is transformed therewith by an
appropriate method such as the electroporation, so that a clone
library for cloning the above fragmented chromosomal DNA can be
produced.
[0045] Moreover, according to known methods such as a chemical
decomposition method (Maxam-Gilbert method) or dideoxy method, the
nucleotide sequence of each fragmented chromosomal DNA obtained
from the clone library can be determined.
[0046] Otherwise, the DNA of the present invention can also be
produced by amplification by PCR using, as primers, oligo DNA
comprising a nucleotide sequence consisting of 15 or more
contiguous nucleotides of the nucleotide sequence shown in SEQ ID
NO: 1 or 5 or a nucleotide sequence complementary thereto. For
example, using oligo DNA comprising nucleotide sequences shown in
SEQ ID NOS: 8 and 9 as a primer set, PCR is carried out using the
cDNA of Aspergillus oryzae as a template, so that DNA comprising
the nucleotide sequence shown in SEQ ID NO: 5 can be amplified and
isolated.
[0047] Moreover, PCR is carried out using, as a primer set, oligo
DNA comprising a nucleotide sequence consisting of 15 or more
contiguous nucleotides of the nucleotide sequence of the DNA of the
present invention and oligo DNA comprising a nucleotide sequence
consisting of 15 or more contiguous nucleotides of a nucleotide
sequence complementary to the nucleotide sequence of the DNA of the
present invention, so that the DNA fragment of the present
invention can be amplified, and can be detected or isolated. With
regard to primers, a region to be amplified is selected, and DNA
comprising a sequence of 15 to 50 nucleotides of the 5' terminus of
the region at the 3' terminus and DNA comprising a sequence
complementary to a sequence of 15 to 50 nucleotides of the 3'
terminus of the region at the 3' terminus are preferably produced
and used as primers. As a template, for example, chromosomal DNA or
cDNA of a microorganism, and preferably of filamentous fungus, can
be used. Examples of preferred filamentous fungi include
microorganisms belonging to any one genus selected from
Aspergillus, Penicillium, Humicola, Trichoderma, Mucor and
Fusarium. Of these, filamentous fungi belonging to Aspergillus may
be particularly preferred. Examples of filamentous fungi belonging
to Aspergillus include Aspergillus oryzae, Aspergillus fumigatus,
Aspergillus flavus, Aspergillus sojae, Aspergillus parasiticus,
Aspergillus nomius, Aspergillus niger, Aspergillus awamori,
Aspergillus kawachii, and Aspergillus nidulans. Of these,
filamentous fungi belonging to the flavi section are preferred.
Examples of filamentous fungi of Aspergillus belonging to the flavi
section include Aspergillus oryzae, Aspergillus sojae, Aspergillus
parasiticus, Aspergillus flavus, and Aspergillus nomius. Of these,
for example, Aspergillus oryzae RIB 40 (ATCC No. 42149) was
deposited with the National Institute of Advanced Industrial
Science and Technology, an Independent Administrative Institution
under the Ministry of Economy, Trade and Industry, at the AIST,
Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan) under
accession No. FERM P-18273 on Mar. 28, 2001. It was then
transferred to an international deposition on Mar. 4, 2002, and
received an accession No. FERM BP-7935.
[0048] DNA comprising a nucleotide sequence shown in SEQ ID NO: 3
or 4 is a partial fragment of DNA having the nucleotide sequence
shown in SEQ ID NO: 1, and it can be amplified and isolated by PCR
using the genomic DNA of Aspergillus oryzae as a template and a
primer set that is based on the nucleotide sequences shown in SEQ
ID NOS: 3 and 4.
[0049] PCR can be carried out by applying conditions and means
known to a person skilled in the art. As an example of PCR reaction
conditions, after heating at 94.degree. C. for 2 minutes, a
reaction cycle consisting of 94.degree. C. for 10 seconds,
55.degree. C. for 20 seconds, and 72.degree. C. for 2 minutes is
repeated 30 times, and finally the reaction product is heated at
72.degree. C. for 5 minutes. As another example of PCR reaction
conditions, after heating at 94.degree. C. for 5 minutes, a
reaction cycle consisting of 94.degree. C. for 2 minutes,
56.degree. C. for 30 seconds, and 72.degree. C. for 1 minute 30
seconds is repeated 30 times. A common thermal cycler such as 9600
manufactured by Perkin Elmer or Program Temp Control System PC-700
manufactured by Astec Inc. can be used as a thermal cycler. As
heat-resistant DNA polymerase, a commercially available product
such as Taq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.)
or ExTaq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.) is
used, and the composition of a reaction solution can be determined
in accordance with an instruction manual attached to the
product.
[0050] The length of each primer consisting of nucleotides used for
the above primer set for DNA amplification is not particularly
limited, but it can be appropriately selected by a person skilled
in the art depending on intended use or the like. A primer has a
length of generally 15 to 50 nucleotides, and preferably 20 to 30
nucleotides. As the number of primers, a minimum number can be
determined taking into consideration the degree of relationship
between a strain containing DNA to be amplified and Aspergillus,
and a mixed degree thereof. The number of primers is at least 1 set
(2 primers), and preferably 2 to 4 sets. Moreover, in order to
design primers, the length and properties of a sequence to be
amplified are considered. Oligo DNA can be produced by chemical
synthesis known to a person skilled in the art, for example, by
using a DNA Synthesizer manufactured by Applied Biosystems.
[0051] A partial fragment of the DNA of the present invention, or
oligo DNA comprising the nucleotide sequence of the DNA of the
present invention or a nucleotide sequence consisting of 15 or more
contiguous nucleotides of a nucleotide sequence complementary to
the nucleotide sequence of the DNA of the present invention, is
labeled with radioisotope, digoxigenin, biotin or the like, and it
is used as a probe. Hybridization is carried out using such a
probe, so as to detect the DNA of the present invention or mRNA
encoding the polypeptide of the present invention. The partial
fragment of the DNA of the present invention can be produced by PCR
as described above, and oligo DNA can be produced by chemical
synthesis or using a DNA synthesizer as in the case of the oligo
DNA used as primers. The length of a probe can be appropriately
selected by a person skilled in the art, considering a detection
target or the like. The probe has a length of generally 15 to 3000
nucleotides, and preferably 20 to 1000 nucleotides.
[0052] To carry out hybridization, for example, DNA or mRNA is
transferred from electrophoresis gel or colonies onto a
nitrocellulose or nylon membrane. Thereafter, it is reacted at
80.degree. C. for 2 hours in a vacuum or subjected to ultraviolet
radiation treatment, so as to immobilize DNA onto the membrane.
During this process, denaturation with an alkaline solution
containing 0.5 mol/l NaOH and 1.5 mol/l NaCl, and neutralization
with a solution containing 0.5 mol/l Tris-HCl (pH 7.5) and 3 mol/l
NaCl, are carried out as necessary. The obtained membrane is
prehybridized at 42.degree. C. for 2 hours in a hybridization
solution containing 50% formamide, 4.times.SSC, 50 mM HEPES-NaOH
(pH 7.0), 10.times. Denhardt's solution and 100 .mu.g/ml salmon
sperm DNA. Thereafter, hybridization is carried out at 42.degree.
C. over day and night in the same above hybridization solution to
which the above probe is added. The resultant membrane is washed
for 2 minutes with 2.times.SSC solution containing 0.1% SDS at room
temperature 3 times, and then washed for 2 hours with a
0.1.times.SSC solution containing 0.1% SDS at 50.degree. C. 3
times. The washed membrane is air-dried, and it is exposed to an X
ray film at -70.degree. C. for a period of time between 2 hours and
over day and night, and then developed for visualization.
Alternatively, the DNA of the present invention or mRNA encoding
the polypeptide of the present invention can also be detected by
using a DNA chip in which oligo DNA or a DNA fragment is
immobilized onto a substrate, and hybridizing the DNA chip with a
labeled mRNA or DNA, followed by detecting the above DNA or mRNA as
dots [Genome Res., 6, 639 (1996)].
(2) Production of Polypeptide of the Present Invention
[0053] The polypeptide of the present invention can be produced,
for example, by the method described below, which involves
introducing recombinant DNA comprising DNA encoding the polypeptide
of the present invention into a host cell to produce a
transformant, and culturing the transformant. The methods described
in Molecular Cloning: A Laboratory Manual, 3.sup.rd edition, Cold
Spring Harbor Laboratory (2001), Current Protocols in Molecular
Biology, John Wiley & Sons (1987) or the like, can be applied
as examples of gene manipulations.
[0054] From the DNA of the present invention obtained in (1), DNA
with an appropriate length comprising a region encoding the
polypeptide of the present invention is produced. In addition, DNA
is produced, as necessary, by substituting nucleotides of a
nucleotide sequence corresponding to the region encoding the
polypeptide of the present invention with other nucleotides, so as
to obtain codons optimal for expression in a host cell.
[0055] A recombinant vector is produced by inserting the above DNA
downstream of a promoter of a suitable expression vector.
[0056] The obtained recombinant vector is introduced into a host
cell suitable for the expression vector.
[0057] Any host cell can be used as long as the desired nucleic
acid sequence can be expressed therein, and examples of such a host
cell include bacteria, yeasts, filamentous fungi, animal cells,
insect cells, and plant cells.
[0058] As an expression vector, a vector, which can autonomously
replicate in the above host cells or be integrated into a
chromosome and comprises a promoter at a position which enables
transcription of DNA encoding the polypeptide of the present
invention, can be used.
[0059] When a prokatyote such as bacterium is used as a host cell,
the recombinant vector comprising DNA encoding the polypeptide of
the present invention is preferably a vector, which has such a
structure that a promoter, a ribosome binding sequence, DNA
encoding the polypeptide of the present invention and a
transcription termination sequence are ligated, as well as being
capable of autonomously replicating in the prokatyote. A gene for
regulating the promoter may also be contained therein.
[0060] Examples of an expression vector include pGEMEX-1
(manufactured by Promega), pQE-30 (manufactured by Qiagen), pKYP200
[Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem.,
53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306
(1985)], pTrS30 [produced from Escherichia coli JM109/pTrS30 (FERM
BP-5407)], pGEX-5X-3 (manufactured by Amersham Bioscience), pET14
(manufactured by Novagen), pPROTet.E (manufactured by Clontech),
and pRSET C (manufactured by Invitrogen).
[0061] Any promoter can be used as long as it functions in a host
cell. Examples of such a promoter include promoters derived from
Escherichia coli, phage or the like, such as a trp promoter
(P.sub.trp), lac promoter, P.sub.L promoter, P.sub.R promoter or T7
promoter. In addition, artificially designed and modified promoters
such as a promoter (P.sub.trp.times.2) in which the two P.sub.trps
are combined in tandem, tac promoter, lacT7 promoter or letI
promoter may also be used.
[0062] A plasmid in which a distance between a Shine-Dalgarno
sequence as a ribosome binding sequence and an initiation codon is
appropriately adjusted (e.g., 6 to 18 nucleotides) is preferably
used.
[0063] The recombinant vector of the present invention does not
necessarily require a transcription termination sequence for the
expression of the DNA of the present invention. However, such a
transcription termination sequence is preferably located
immediately downstream of the structural gene.
[0064] Examples of a host cell include microorganisms belonging to
Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium,
Microbacterium, Pseudomonas, etc. Specific examples of such
microorganisms include Escherichia coli XL1-Blue, Escherichia coli
XL2-Blue, Escherichia coli BL21, Escherichia coli DH1, Escherichia
coli MC1000, Escherichia coli KY3276, Escherichia coli W1485,
Escherichia coli JM109, Escherichia coli HB101, Escherichia coli
No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichia
coli GI698, Escherichia coli TB1, Serratia ficaria, Serratia
fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus
subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes,
Brevibacterium immariophilum ATCC14068, Brevibacterium
saccharolyticum ATCC14066, Brevibacterium flavum ATCC14067,
Brevibacterium lactofermentum ATCC13869, Corynebacterium glutamicum
ATCC13032, Corynebacterium glutamicum ATCC13869, Corynebacterium
acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354,
Pseudomonas putida, and Pseudomonas sp. D-0110.
[0065] Any method of introducing DNA into the above-described host
cell can be used as a method of introducing a recombinant vector
therein. Examples of such a method include a method of using
calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the
protoplast method (Japanese Published Unexamined Patent Application
No. 2483942/1988), and methods described in Gene, 17, 107 (1982) or
Molecular & General Genetics, 168, 111 (1979).
[0066] In the case of using a yeast strain as a host cell, examples
of an expression vector include YEP13 (ATCC37115), YEp24
(ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
[0067] Any promoter can be used as long as it functions in a yeast
strain. Examples of such a promoter include a promoter of gene in
glycolytic pathway such as hexose kinase, a PHO5 promoter, a PGK
promoter, a GAP promoter, an ADH promoter, a gal1 promoter, a gal10
promoter, a heat shock polypeptide promoter, an MF.alpha.1
promoter, and a CUP1 promoter.
[0068] Examples of a host cell include microorganisms belonging to
genus of Saccharomyces, Schizosaccharomyces, Kluyveromyces,
Trichosporon, Schwanniomyces, Pichia, or Candida. Specific examples
of such microorganisms include Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon
pullulans, Schwanniomyces alluvius, and Candida utilis.
[0069] Any method of introducing DNA into a yeast strain can be
used as a method of introducing a recombinant vector. Examples of
such a method include the electroporation method [Methods Enzymol.,
194, 182 (1990)], the spheroplast method [Proc. Natl. Acad. Sci.
USA, 75, 1929 (1978)], the lithium acetate method [J. Bacteriology,
153, 163 (1983)], and a method described in Proc. Natl. Acad. Sci.
USA, 75, 1929 (1978).
[0070] In the case of using filamentous fungus as a host cell,
examples of an expression vector include pPTRI (manufactured by
Hakutsuru Sake Brewing Co., Ltd.), pPTRII (manufactured by
Hakutsuru Sake Brewing Co., Ltd.), and pAUR316 (manufactured by
Takara Shuzo Co., Ltd.)
[0071] Any promoter can be used as long as it functions in
filamentous fungi. Examples of such a promoter include an amyB
promoter, an enoA promoter, a gpd promoter, and a melO
promoter.
[0072] Examples of a host cell include Aspergillus, Penicillium,
Tricoderma, Fusarium, Humicola, and Mucor genus.
[0073] Any method of introducing DNA into filamentous fungi may be
used as a method of introducing a recombinant vector. The
protoplast method [GENETICS of ASPERGILLUS NIDULANS: EMBO Practical
Course Manual, 8 (1988)] is an example of such a method.
[0074] In the case of using an animal cell as a host, examples of
an expression vector include pEGFP-C2 (manufactured by Clontech),
pAGE107 (Japanese Published Unexamined Patent Application 22979/91;
Cytotechnol., 3, 133 1990), pAS3-3 (Japanese Published Unexamined
Patent Application No. 227075/90), pCDM8 [Nature, 329, 840 (1987)],
pCMV-Tag1 (manufactured by Stratagene), pcDNA3.1(+) (manufactured
by Invitrogen), pREP4 (manufactured by Invitrogen), pMSG
(manufactured by Amersham Bioscience), and pAMo [J. Biol. Chem.,
268, 22782 (1993)].
[0075] Any promoter can be used as long as it functions in an
animal cell. Examples of such a promoter include a promoter of a
cytomegalovirus (CMV) IE (immediate early) gene, an SV40 early
promoter, a retrovirus promoter, a metallothionein promoter, a heat
shock promoter and an SR.alpha. promoter. In addition, an enhancer
of a human CMV IE gene may also be used together with a
promoter.
[0076] Examples of an animal host cell include: Namalwa cell which
is derived from human; COS cell which is derived from monkey; CHO
cell which is derived from Chinese hamster; and HBT5637 (Japanese
Published Unexamined Patent Application No. 299/88).
[0077] Any method of introducing DNA into an animal cell can be
used as a method of introducing a recombinant vector into an animal
cell. Examples of such a method include the electroporation method
[Cytotechnology, 3, 133 (1990)], the calcium phosphate method
(Japanese Published Unexamined Patent Application No. 227075/90),
and the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413
(1987), and Virology, 52, 456 (1973)].
[0078] In the case of using an insect cell as a host, polypeptides
can be expressed, for example, by methods described in Current
Protocols in Molecular Biology, John Wiley & Sons (1987),
Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman
and Company (1992), or Bio/Technology, 6, 47 (1988).
[0079] That is to say, insect cells are cotransfected with a vector
comprising a recombinant gene and baculovirus, so as to obtain a
recombinant virus in the culture supernatant of the insect cells.
Thereafter, insect cells are infected with the recombinant virus,
so that the cells are allowed to express polypeptides.
[0080] Examples of a vector for gene transfer used in this method
include pVL1392, pVL1393, pBlueBac4.5 (all of which are
manufactured by Invitrogen), and pBacPAK9 (manufactured by
Clontech).
[0081] Autographa californica nuclear polyhedrosis virus, which is
a virus infecting Noctuidae insects, can be used as an example of
baculovirus.
[0082] Examples of an insect cell include: Sf9 and Sf21, which are
ovarian cells of Spodoptera frugiperda [Baculovirus Expression
Vectors: A Laboratory Manual, W. H. Freeman and Company (1992)];
and High 5, which is ovarian cell of Trichoplusia ni (manufactured
by Invitrogen).
[0083] Examples of a method of cotransfecting insect cells with the
above recombinant vector for gene transfer and the above
baculovirus, for preparing recombinant virus, include the calcium
phosphate method (Japanese Published Unexamined Patent Application
No. 227075/90) and the lipofection method [Proc. Natl. Acad. Sci.
USA, 84, 7413 (1987)].
[0084] In the case of using a plant cell as a host cell, examples
of an expression vector include a Ti plasmid and a tobacco mosaic
virus vector.
[0085] Any promoter can be used as long as it functions in a plant
cell. Examples of such a promoter include a cauliflower mosaic
virus (CaMV) 35S promoter and a rice actin 1 promoter.
[0086] Examples of host cell include plant cells from tobacco,
potato, tomato, carrot, soybean, oilseed rape, alfalfa, rice, wheat
and barley.
[0087] Any method of introducing DNA into plant cells may be used
as a method of introducing a recombinant vector. Examples of such a
method include the Agrobacterium method (Japanese Published
Unexamined Patent Application Nos. 140885/84 and 70080/85, and
WO94/00977), the electroporation method (Japanese Published
Unexamined Patent Application No. 251887/85), and a method of using
a particle gun (Japanese Patent Nos. 2606856 and 2517813).
[0088] The transformant of the present invention obtained as above
is cultured in a medium, and the polypeptide of the present
invention is produced and accumulated in the obtained culture, and
then, it is recovered from the culture. Thus, the polypeptide of
the present invention can be produced.
[0089] The transformant of the present invention can be cultured by
a conventional method used in the culture of a host.
[0090] Where the transformant of the present invention is obtained
using, as a host, a prokaryote such as Escherichia coli or
eukaryote such as a yeast or filamentous fungus, a medium in which
the transformant is cultured may be either a natural medium or
synthetic medium, as long as it contains a carbon source that can
be assimilated by the transformant, nitrogen source or inorganic
salts, and the transformant can be efficiently cultured
therein.
[0091] Any carbon source can be used as long as it can be
assimilated by the transformant. Examples of such a carbon source
include carbonhydrates such as glucose, fractose, sucrose, molasses
containing these carbonhydrates, starch or starch hydrolysate,
organic acids such as acetic acid or propionic acid, and alcohols
such as ethanol or propanol.
[0092] Examples of a nitrogen source used herein include ammonium
salts of inorganic or organic acids such as ammonia, ammonium
chloride, ammonium sulfate, ammonium acetate or ammonium phosphate,
other nitrogen-containing compounds, peptone, meat extract, yeast
extract, corn steep liquor, casein hydrolysate, wheat protein,
wheat protein hydrolysate, soybean cake, soybean cake hydrolysate,
various types of fermentative bacteria, and digests thereof.
[0093] Examples of an inorganic salt used herein include potassium
dihydrogenphosphate, dipotassium hydrogenphosphate, magnesium
phosphate, magnesium sulfate, sodium chloride, ferrous sulfate,
manganese sulfate, cupric sulfate, and calcium carbonate.
[0094] Culturing is carried out under aerobic conditions such as
shaking culture or submerged spinner culture under aeration. The
culture temperature is generally between 15.degree. C. and
40.degree. C., and the culture time is generally between 16 hours
and 7 days. The pH for culture is preferably maintained between 3.0
and 9.0. The pH is adjusted using inorganic or organic acid, an
alkali solution, urea, calcium carbonate, ammonia, etc.
[0095] In addition, antibiotics such as ampicillin or tetracycline
may be added to the medium during the culture, as necessary.
[0096] In the case of culturing microorganisms transformed with a
recombinant vector comprising an inducible promoter, an inducer may
be added to a medium, as necessary. For example, in the case of
culturing microorganisms transformed with a recombinant vector
comprising a lac promoter, isopropyl-.beta.-D-thiogalactopyranoside
may be added to a medium. In the case of culturing microorganisms
transformed with a recombinant vector comprising a trp promoter,
indoleacrylic acid may be added to a medium.
[0097] Examples of a medium in which a transformant obtained from
an animal cell as a host is cultured include RPMI 1640 medium [J.
Am. Med. Assoc., 199, 519 (1967)], Eagle's MEM (minimum essential
medium) [Science, 122, 501 (1952)], Dalbecco's modified Eagle's
medium [Virology, 8, 396 (1959)], and 199 medium [Proc. Soc. Exp.
Biol. Med., 73, 1 (1950)], which are commonly used, and a medium
obtained by adding fetal bovine serum or the like to these
media.
[0098] The culturing is usually carried out at pH 6 to 8 at 30 to
40.degree. C. for 1 to 7 days in the presence of 5% CO.sub.2.
[0099] In addition, antibiotics such as kanamycin or penicillin may
also be added to the medium during the culturing, as necessary.
[0100] Examples of a medium in which a transformant obtained from
an insect cell as a host is cultured include TNM-FH medium
(manufactured by Pharmingen), Sf-900 II SFM medium (manufactured by
Invitrogen), ExCell400 and ExCell405 (both of which are
manufactured by JRH Bioscience), and Grace's insect medium [Nature,
195, 788 (1962)], which are commonly used.
[0101] The culturing is usually carried out at pH 6 to 7 at 25 to
30.degree. C. for 1 to 5 days.
[0102] In addition, antibiotics such as gentamicin may also be
added to the medium during the culturing, as necessary.
[0103] The transformant obtained from a plant cell as a host can be
cultured directly or after differentiating into plant cells or
organs. Examples of a medium in which the transformant is cultured
include Murashige and Skoog (MS) medium and White medium, which are
commonly used, and a medium obtained by adding plant hormone such
as auxin or cytokinin to these mediums.
[0104] The culturing is usually carried out at pH 5 to 9 at 20 to
40.degree. C. for 3 to 60 days.
[0105] In addition, antibiotics such as kanamycin or hygromycin may
also be added to the medium during the culturing, as necessary.
[0106] As stated above, a transformant derived from microorganisms,
animal cells or plant cells comprising a recombinant vector into
which DNA encoding the polypeptide of the present invention is
incorporated is cultured by an ordinary culturing method, the
polypeptide is produced and accumulated in the culture, and the
polypeptide is then recovered from the culture. Thus, the
polypeptide of the present invention can be produced.
[0107] When the polypeptide of the present invention is expressed
using yeast strains, filamentous fungi, animal cells, insect cells
or plant cells, a polypeptide to which sugar or sugar chain is
added can be obtained.
[0108] Processes for producing the polypeptide of the present
invention include a method of allowing a host cell to produce it
inside the cell, a method of allowing a host cell to secrete it out
of the cell, and a method of allowing a host cell to produce it on
an outer membrane thereof. Such a method can be selected depending
on the type of a host cell to be used or the type of the structure
of a polypeptide to be produced.
[0109] Even where the polypeptide of the present invention is
produced inside a host cell or on the outer membrane of the host
cell, it is possible to actively secrete the polypeptide out of the
host cell by applying the method of Paulson et al. [J. Biol. Chem.,
264, 17619 (1989)], the method of Lowe et al. [Proc. Natl. Acad.
Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or the
method described in Japanese Published Unexamined Patent
Application No. 336963/1993, WO94/23021, and the like.
[0110] That is to say, the polypeptide of the present invention is
expressed by gene recombination such that a signal peptide is added
just before a polypeptide comprising an active site of the
polypeptide of the present invention, so that the polypeptide of
the present invention can be actively secreted out of a host
cell.
[0111] Moreover, the production amount of polypeptide can be
increased by applying the method described in Japanese Published
Unexamined Patent Application No. 227075/1990, using a gene
amplification system comprising a dihydrofolate reductase gene or
the like.
[0112] Furthermore, the polypeptide of the present invention can be
produced by applying the known method [J. Biomol. NMR, 6, 129
(1998), Science, 242, 1162 (1988), J. Biochem., 110, 166 (1991)],
using an in vitro transcription and translation system. This is to
say, DNA encoding the polypeptide of the present invention is
ligated downstream of a promoter such as SP6, T7 or T3, and it is
then reacted with RNA polymerase specific for such a promoter, so
that a large amount of RNA encoding the polypeptide of the present
invention is synthesized in vitro. Thereafter, the polypeptide of
the present invention can be produced using a cell-free translation
system such as a translation system using a rabbit reticulocyte
lysate or wheat germ extract.
[0113] In order to isolate and purify the polypeptide produced by
the transformant of the present invention, an ordinary method of
isolating and purifying enzymes can be used. For example, where the
polypeptide of the present invention is expressed in a state
dissolved in cells, after completion of the culturing, cells are
recovered by centrifugal separation, and the recovered cells are
suspended in a water-based buffer. Thereafter, the cells are
disintegrated using an ultrasonic disintegrator, French press,
Manton Gaulin homogenizer or Dyno mill, so as to obtain a cell-free
extract. A supernatant is obtained by centrifuging the cell-free
extract, and then, a purified sample can be obtained from the
supernatant by applying, singly or in combination, the following
ordinary enzyme isolation and purification methods: the solvent
extraction, the salting-out method using ammonium sulfate, the
desalting method, the precipitation method using an organic
solvent, the anion exchange chromatography using resins such as
diethylaminoethyl (DEAE) sepharose or DIAION HPA-75 (manufactured
by Mitsubishi Chemical Corp.), the cation exchange chromatography
using resins such as S-Sepharose FF (manufactured by Pharmacia),
the hydrophobic chromatography using resins such as butyl sepharose
or phenyl sepharose, the gel filtration method using a molecular
sieve, the affinity chromatography, the chromatofocusing method,
and the electrophoresis such as isoelectric focusing. Where the
above polypeptide is tagged and then expressed using a pRSET vector
(manufactured by Invitrogen) or pGEX vector (manufactured by
Amersham Bioscience), affinity purification can be carried out by
using an appropriate carrier such as a nickel resin or glutathione
sepharose.
[0114] Where the above polypeptide is expressed with formation of
an insoluble form in cells, the cells are recovered, disintegrated
and centrifuged in the same manner as described above, so that the
insoluble form of polypeptide is recovered as a precipitation
fraction. Thereafter, the insoluble form of the recovered
polypeptide is solubilized using a polypeptide denaturing agent.
The obtained solubilized solution is diluted or dialyzed, so that
the concentration of the polypeptide denaturing agent contained in
the above solubilized solution is decreased. The polypeptide is
thus returned to a normal three-dimensional structure. After this
operation is performed, the same above isolation and purification
method is applied to the obtained polypeptide, so as to obtain a
purified sample.
[0115] Where the polypeptide of the present invention or a
derivative thereof such as a polypeptide obtained by adding a sugar
chain to the above polypeptide is extracellularly secreted, the
above polypeptide or a derivative thereof can be recovered from a
culture supernatant. This is to say, the culture is treated by the
same above techniques such as centrifugal separation, so as to
obtain a culture supernatant. Thereafter, a purified sample can be
obtained from the culture supernatant by the isolation and
purification method as mentioned above.
[0116] Examples of the polypeptide of the present invention
obtained as above include a polypeptide comprising an amino acid
sequence shown in SEQ ID NO: 2 and a polypeptide comprising an
amino acid sequence shown in SEQ ID NO: 10.
[0117] Moreover, the polypeptide of the present invention can be
produced by chemical synthesis methods such as the Fmoc method
(fluorenylmethyloxycarbonyl method) or the tBoc method
(t-butyloxycarbonyl method). Furthermore, the polypeptide of the
present invention can also be synthesized using peptide
synthesizers that are available from Applied Biosystems, Advanced
ChemTech, Shimadzu Corporation, etc.
[0118] Still further, the polypeptide of the present invention can
also be produced by culturing a microorganism having an ability of
producing the polypeptide of the present invention. Any
microorganism can be used as long as it has an ability of producing
the polypeptide of the present invention. Preferred examples of
such a microorganism include filamentous fungus. More preferred
examples include filamentous fungus belonging to a genus selected
from a group consisting of Aspergillus, Penicillium, Humicola,
Trichoderma, Mucor, and Fusarium. Further more preferred example
include filamentous fungus belonging to Aspergillus, such as
filamentous fungus belonging to a species selected from a group
consisting of Aspergillus oryzae, Aspergillus sojae, Aspergillus
niger, Aspergillus awamori, Aspergillus kawachii, Aspergillus
parasiticus, Aspergillus flavus, Aspergillus nomius, Aspergillus
fumigatus, and Aspergillus nidulans. The microorganism having an
ability of producing the polypeptide of the present invention may
be any of a wild strain, a transformant and a mutant, but it is
preferably a microorganism that has been subjected to
transformation, mutation or the like and thereby has an increased
ability of producing the polypeptide of the present invention.
Examples of such mutation treatment include ultraviolet irradiation
and treatment with mutagenic agents such as
N-methyl-N'-nitro-N-nitrosoguanidine. Culturing of the
microorganism and purification of the polypeptide can be carried
out in the same manner as in the above-described culturing of a
transformed microorganism and purification of a polypeptide.
(3) Process for Producing Protein Hydrolysate
[0119] The polypeptide of the present invention having a
pyroglutamyl peptidase activity and protein hydrolase are added to
a raw material containing a protein, and the mixture is blended,
and then reaction is carried out generally between 20.degree. C.
and 60.degree. C., preferably between 30.degree. C. and 50.degree.
C., for 24 to 264 hours, preferably for 48 to 240 hours, so as to
produce a protein hydrolysate. Otherwise, protein hydrolase is
first added to a raw material containing a protein, and the mixture
is blended, and then reaction is carried out generally between
20.degree. C. and 60.degree. C., preferably between 30.degree. C.
and 50.degree. C., for 24 to 264 hours, preferably for 48 to 240
hours for the hydrolysis of the protein. Thereafter, the
polypeptide of the present invention having a pyroglutamyl
peptidase activity is added to the reaction product, and the
mixture is blended, and then reaction is carried out generally
between 20.degree. C. and 60.degree. C., preferably between
30.degree. C. and 50.degree. C., for 5 to 96 hours, preferably for
12 to 72 hours, so as to produce a protein hydrolysate. In the
latter case, when the polypeptide of the present invention having a
pyroglutamyl peptidase activity is added to the reaction product,
protein hydrolase may also be added thereto. During the reaction,
any pH value may be applied as long as the polypeptide of the
present invention having a pyroglutamyl peptidase activity and
protein hydrolase can act in the pH. The pH is preferably adjusted
to pH 5 to 8.
[0120] As a polypeptide having a pyroglutamyl peptidase activity
used in this production method, a polypeptide purified by the
method described in (2) above can be used. Alternatively, a
polypeptide may not be purified, and a culture product or treated
product thereof containing the polypeptide of the present invention
having a pyroglutamyl peptidase activity, which is obtained by
culturing in a medium the above-described transformant containing
recombinant DNA comprising DNA encoding the polypeptide of the
present invention having a pyroglutamyl peptidase activity
described in (2) above, or microorganism having an ability of
producing the polypeptide of the present invention having a
pyroglutamyl peptidase activity, may also be used.
[0121] Examples of protein hydrolase include Flavorzyme
(manufactured by Novo Nordisk) and a culture product of Koji molds.
Any type of Koji molds may be used as long as it is used in the
brewing industry. Examples of such Koji molds include Aspergillus
oryzae and Aspergillus sojae. The type of a protein contained in a
raw material used in the production method of the present invention
is not particularly limited. A protein containing a large amount of
glutamic acid is preferable. Moreover, any type of raw material may
be used as long as it contains a large amount of protein. It is not
necessarily a purified protein. Examples of such a material include
wheat gluten, defatted soybeans, and a separated soybean protein.
After completion of the reaction, unreacted raw material protein,
fungus bodies or the like are removed out, and the supernatant is
concentrated and dried, as necessary, so that a protein hydrolysate
at a high hydrolysis rate can be obtained.
(4) Production of Antibody Which Specifically Recognizes the
Polypeptide of the Present Invention
(a) Production of Polyclonal Antibody
[0122] A polyclonal antibody which specifically binds to the
polypeptide of the present invention can be produced by
immunization of animals, using, as an antigen, a purified sample
from the full-length polypeptide of the present invention obtained
by the method described in (2) above or a partial fragment thereof,
or a peptide having an amino acid sequence of a part of the peptide
of the present invention. As an immunization method, an antigen may
be directly administered into the subcutis, vein or abdominal
cavity of an animal, but it is preferable to bind a highly
antigenic carrier protein with an antigen for administration, or to
administer an antigen together with a suitable adjuvant.
[0123] A peptide used as an antigen can be chemically synthesized
by applying chemical synthesis methods such as the Fmoc method
(fluorenylmethyloxycarbonyl method) or the tBoc method
(t-butyloxycarbonyl method), or using peptide synthesizers that are
available from Applied Biosystems, Advanced ChemTech, Shimadzu
Corporation, etc.
[0124] Examples of a carrier protein include keyhole limpet
hemocyanin, bovine serum albumin, and bovine thyroglobulin.
Examples of an adjuvant include Complete Freund's Adjuvant,
aluminum hydroxide gel, and pertussis vaccine.
[0125] Examples of an animal to be immunized include non-human
mammals such as a rabbit, goat, mouse, rat or hamster.
[0126] Antigen is administered 3 to 10 times every 1 to 2 weeks
after the first administration. On the third to seventh day after
completion of each administration, a blood sample is collected from
the venous plexus of the fundus oculi to prepare serum. Thereafter,
it is confirmed by enzyme linked immunoassay (ELISA) [Koso Meneki
Sokutei Ho (Enzyme linked Immunoassay) 3.sup.rd edition,
Igaku-Shoin Ltd. (1987); Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (1988)] or the like that the
prepared serum is reacted with the antigen that has been used for
the immunization. The dosage of the antigen is preferably 50 to 200
.mu.g per animal per administration.
[0127] The total serum is obtained from animals whose serum shows a
sufficient antibody titer to the antigen used in immunization, and
the obtained serum is then separated and purified, so as to obtain
a polyclonal antibody. Examples of such separation and purification
methods include centrifugal separation, salting-out using 40% to
50% saturated ammonium sulfate, caprylic acid precipitation
[Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, (1988)], or methods in which chromatographies using a
DEAE-sepharose column, anion exchange column, protein A or G
column, or gel filtration column are used singly or in
combination.
(b) Production of Monoclonal Antibody
(i) Preparation of Antibody-Producing Cells
[0128] A mouse or rat whose serum shows a sufficient antibody titer
to the antigen used in immunization in (a) above is used as a
source for supplying antibody-producing cells.
[0129] On the third to seventh day after the final administration
of an antigenic substance to the mouse or rat whose serum shows a
sufficient antibody titer, the spleen is extirpated therefrom. The
spleen is cut finely in MEM (Minimum Essential Medium), and is
taken apart to pieces with tweezers, followed by performing
centrifugal separation thereon at 1,200 rpm for 5 minutes.
Thereafter, the obtained supernatant is discarded. Spleen cells
contained in the thus obtained precipitation fractions are treated
with Tris-ammonium chloride buffer solution (pH 7.65) for 1 to 2
minutes, so as to eliminate erythrocytes. Thereafter, the residue
is washed with MEM 3 times. The obtained spleen cells are used as
antibody-producing cells.
(ii) Preparation of Myeloma Cells
[0130] A cell line established from a mouse or rat is used as
myeloma cells. For example, 8-azaguanine resistant mouse (BALB/c
derived) myeloma cell lines such as P3-X63Ag8-U1 [Curr. Topics
Microbiol. Immunol., 81, 1 (1978), Eur. J. Immunol., 6, 511
(1976)], SP2/0-Ag14 [Nature, 276, 269 (1978)], P3-X63-Ag8653 [J.
Immunol., 123, 1548 (1979)], or P3-X63-Ag8 [Nature, 256, 495
(1975)] can be used. These cell lines are subcultured in a
8-azaguanine medium [obtained by adding 1.5 mmol/l glutamine, 50
.mu.mol/l 2-mercaptoethanol, 10 .mu.g/ml gentamicin and 10% fetal
bovine serum to an RPMI 1640 medium (hereinafter referred to as a
normal medium), and further adding 15 .mu.g/ml 8-azaguanine
thereto]. The cell lines are cultured in a normal medium 3 to 4
days before cell fusion, and the 2.times.10.sup.7 cells are used
for the fusion.
(iii) Preparation of Hybridomas
[0131] The antibody-producing cells obtained in (i) above and the
myeloma cells obtained in (ii) above are well washed with MEM or
PBS (1.83 g/l disodium phosphate, 0.21 g/l monopotassium phosphate,
7.65 g/l NaCl, pH 7.2), and these cells are mixed such that the
number of cells becomes the antibody-producing cells:the myeloma
cells=5 to 10:1. The mixture is then centrifuged at 1,200 rpm for 5
minutes, and the obtained supernatant is discarded.
[0132] A mass of cells contained in the obtained precipitation
fractions is well loosen. Thereafter, to the cells, 0.2 to 1 ml of
a solution obtained by mixing 2 g of polyethylene glycol-1000, 2 ml
of MEM and 0.7 ml of dimethyl sulfoxide is added per 108
antibody-producing cells at 37.degree. C., while stirring.
Thereafter, 1 to 2 ml of MEM is further added thereto several times
every 1 or 2 minutes. After completion of the addition, MEM is
added to the obtained mixture to the total amount of 50 ml. The
thus prepared solution is centrifuged at 900 rpm for 5 minutes, and
the obtained supernatant is then discarded.
[0133] Cells contained in the obtained precipitation fractions are
gently taken apart to pieces, and the cells are gently suspended in
100 ml of an HAT medium [obtained by adding 0.4 mmol/l
hypoxanthine, 15 .mu.mol/l thymidine and 0.4 .mu.mol/l aminopterin
to a normal medium] by sucking and blowing with a measuring pipet.
The suspension is poured into each hole of a 96-hole culture plate
in an amount of 100 .mu.l/hole, and it is then cultured at
37.degree. C. for 7 to 14 days in a 5% CO.sub.2 incubator.
[0134] After completion of the culturing, an aliquot is taken from
the culture supernatant, and antibodies binding to the polypeptide
of the present invention are detected in the culture supernatant by
ELISA, so as to select hybridomas which produce the monoclonal
antibodies specifically binding to the polypeptide of the present
invention.
[0135] The following method may be a specific example of ELISA. A
polypeptide or peptide used as an antigen in immunization is coated
on an appropriate plate. It is reacted with the hybridoma culture
supernatant or a purified antibody obtained in (iv) described below
as a primary antibody. Thereafter, it is reacted with an anti-mouse
immunoglobulin antibody (in a case where antibody-producing cells
are derived from a rat, then an anti-rat immunoglobulin antibody is
used) labeled with an enzyme such as horseradish peroxidase, as a
secondary antibody. A substrate that develops color by a labeling
enzyme is added to the above reaction product, and reaction is
carried out, so that a primary antibody binding to the antigen is
detected.
[0136] The above hybridomas are cloned twice by limiting dilution
method [for the first cloning, an HT medium (obtained by
eliminating aminopterin from the HAT medium) is used, and for the
second cloning, a normal medium is used]. Hybridomas which stably
show a high antibody titer are selected as a hybridoma cell line
which specifically binds to the polypeptide of the present
invention.
(iv) Preparation of Monoclonal Antibodies
[0137] The hybridoma cells obtained in (iii) above that produce a
monoclonal antibody specifically binding to the polypeptide of the
present invention are injected in an amount of 5 to
20.times.10.sup.6 cells/mouse into the abdominal cavity of 8 to 10
week old mice or nude mice which are previously administered
intraperitoneally with 0.5 ml of pristane
(2,6,10,14-tetramethylpentadecane) and then grown for 2 weeks. 10
to 21 days later, the hybridomas turn cancerous in the ascites. The
ascites is collected from the mice with the thus cancerous ascites,
and it is then centrifuged at 3,000 rpm for 5 minutes to eliminate
solids. Monoclonal antibodies can be purified and obtained from the
obtained supernatant by the same method as applied in the
purification of polyclonal antibodies.
[0138] The subclass of antibodies is determined using a mouse
monoclonal antibody typing kit or rat monoclonal antibody typing
kit. The amount of protein is determined by the Lowry method, or it
is calculated from an absorbance at 280 nm.
(5) Detection and Quantification of Polypeptide of the Present
Invention Using Antibody Specifically Binding to Polypeptide of the
Present Invention
[0139] Using the antibody obtained in (4) above which specifically
binds to the polypeptide of the present invention, an
antigen-antibody reaction is carried out, so that the polypeptide
of the present invention can be immunologically detected and
quantified. As a measurement sample, a cell extract, a culture
supernatant and the like is used.
[0140] Examples of an immunological detection and quantification
method include the fluorescent antibody technique, ELISA, the
radioimmunoassay (RIA), the immunohistochemistry or
immunocytochemistry, the immunoblotting, the dot blotting, the
immunoprecipitation method, and the sandwich ELISA [Tan Clone Kotai
Jikken Manual (Monoclonal Antibody Experimental Manual) (Kodansha
Scientific) (1987), Zoku Seikagaku Jikken Koza 5 (Follow-Up
Biochemical Experiment Seminars 5); Meneki Seikagaku Kenkyu Ho
(Research Methods of Immunobiochemistry) (Tokyo Kagaku Dojin Co.,
Ltd.) (1986)].
[0141] The fluorescent antibody technique is a method involving
reacting a measurement sample with an antibody specifically binding
to the polypeptide of the present invention, then reacting the
measurement sample with an antibody binding to the above antibody
(e.g, in a case where the antibody specifically binding to the
polypeptide of the present invention is a mouse antibody, it is an
anti-mouse IgG antibody or fragment thereof), which has been
labeled with a fluorescent substance such as fluorescin isocyanate
(FITC), and measuring the fluorescent dye with a flow cytometer, so
as to detect the polypeptide of the present invention.
[0142] The ELISA is a method involving reacting a measurement
sample with an antibody specifically binding to the polypeptide of
the present invention, then reacting the measurement sample with an
antibody binding to the above antibody, which has been labeled with
an enzyme such as peroxidase, adding thereto a substrate that
develops color by addition of a labeling enzyme, performing
reaction, and measuring a coloring dye with a spectrophotometer, so
as to detect the polypeptide of the present invention.
[0143] The RIA is a method involving reacting a measurement sample
with an antibody specifically binding to the polypeptide of the
present invention, then reacting the measurement sample with an
antibody binding to the above antibody, which has been labeled with
a radioactive label, and measuring radioactivity with a
scintillation counter or the like, so as to detect the polypeptide
of the present invention.
[0144] The immunohistochemistry and immunocytochemistry is a method
involving reacting a measurement sample such as a cell or tissue
section with an antibody specifically binding to the polypeptide of
the present invention, then reacting the measurement sample with an
antibody binding to the above antibody, which has been labeled with
a fluorescent substance such as FITC, peroxidase or biotin, and
observing the sample with a microscope, so as to detect the
polypeptide of the present invention.
[0145] The immunoblotting (Western blotting) is a method involving
fractionating a measurement sample by SDS-PAGE [Antibodies--A
Laboratory Manual, Cold Spring Harbor Laboratory, (1988)], blotting
the gel onto a PVDF membrane or nitrocellulose membrane, reacting
the membrane with an antibody specifically binding to the
polypeptide of the present invention, then reacting it with an
antibody binding to the above antibody, which has been labeled with
a fluorescent substance such as FITC, peroxidase or biotin, and
carrying out a reaction depending on the labeling substance, so as
to detect the polypeptide of the present invention.
[0146] The dot blotting is a method involving blotting a
measurement sample onto a nitrocellulose membrane, reacting the
membrane with an antibody specifically binding to the polypeptide
of the present invention, then reacting it with an antibody binding
to the above antibody, which has been labeled with a fluorescent
substance such as FITC, peroxidase or biotin, and carrying out a
reaction depending on the labeling substance, so as to detect the
polypeptide of the present invention.
[0147] The immunoprecipitation method is a method involving
reacting a measurement sample with an antibody specifically binding
to the polypeptide of the present invention, and adding thereto a
carrier having an ability of specifically binding to
immunoglobulin, such as protein A-sepharose, to form an
antigen-antibody complex, followed by separation, so as to detect
the polypeptide of the present invention.
[0148] The sandwich ELISA is a method involving reacting a
measurement sample with a plate onto which an antibody specifically
binding to the polypeptide of the present invention is adsorbed,
then reacting the measurement sample with an antibody specifically
binding to the polypeptide of the present invention and having an
antigen recognition site different from that of the above antibody,
which has been labeled with an enzyme such as peroxidase, adding
thereto a substrate that develops color by a labeled enzyme, and
measuring a coloring dye with a spectrophotometer, so as to detect
the polypeptide of the present invention.
[0149] A solution containing a certain concentration of a purified
sample of the polypeptide of the present invention which can be
prepared by the method described in (2) above is prepared, and the
solution is diluted stepwise to obtain sample dilutions. These
samples are measured by the above detection methods. A calibration
curve is prepared from the measurement values of samples with
different concentrations. Thus, the polypeptide of the present
invention can be quantified by comparing the measurement values of
measurement samples.
[0150] The present invention will be further specifically described
in the following examples, but the scope of the present invention
is not limited thereby. Various gene manipulations described in the
examples were carried out in accordance with methods described in
Current Protocols in Molecular Biology, John Wiley & Sons
(1987).
BEST MODE FOR CARRYING OUT THE INVENTION
EXAMPLE 1
Method of Producing Whole Genome Shot Gun Library
1. Production of Insert DNA
(1) Obtainment f Chromosomal DNA
[0151] Spores of filamentous fungus Aspergillus oryzae RIB 40 (ATCC
No. 42149) were inoculated into a YPD medium (0.5% yeast extract,
1% peptone, 2% glucose), and the mixture was subjected to a shaking
culturing at 30.degree. C. overnight. Thereafter, genomic DNA was
extracted in accordance with the method of Iimura [Agric. Biol.
Chem., 51, 323 (1987)]. Mitochondrial DNA that had existed with the
genomic DNA was purified by cesium chloride ultracentrifugation in
accordance with the method of Watson et al. [Methods Enzymol., 118,
57 (1986)], so as to obtain only chromosomal DNA.
(2) Fragmentation of Chromosomal DNA
[0152] Using a random DNA fragmentation device HydroShear (Tomy
Seiko Co., Ltd.), the obtained pure chromosomal DNA was degraded
into fragments with a size of approximately 1 to 2 kb.
(3) Treatment of Fragmented DNA Termini
[0153] The fragmented chromosomal DNA was treated with BAL31
nuclease (Takara Shuzo Co., Ltd.) and then treated with a Klenow
fragment (Takara Shuzo Co., Ltd.), so that the termini were
blunted.
(4) Addition of Adaptor to Termini
[0154] DNA fragments comprising nucleotide sequences shown in SEQ
ID NOS: 6 and 7, 5' termini of which had been phosphorylated, were
used as adaptors. The adaptors were ligated to both termini of the
blunt-ended chromosomal DNA fragment, using T4 DNA ligase (Takara
Shuzo Co., Ltd.).
2. Insertion of Insert DNA into Vector and Transformation
[0155] pUC19 was cleaved with a restriction enzyme SalI (Takara
Shuzo Co., Ltd.), and a thymidine residue was then inserted into
the SalI cleavage site with Taq DNA polymerase (Roche Diagnostics).
The thus prepared plasmid was treated with alkaline phosphatase
(Takara Shuzo Co., Ltd.) for dephosphorylation, and it was then
used as a vector. The vector was ligated to the above-prepared
chromosomal DNA fragment using T4 DNA ligase, and then, Escherichia
coli DH10B (Gibco) was transformed therewith by the
electroporation.
3. Nucleotide Sequencing
[0156] The transformed Escherichia coli was cultured in a
2.times.YP medium at 37.degree. C. for 10 hours. After collecting
cell bodies, they were subjected to heat treatment in a sterilized
water at 99.degree. C. for 10 minutes. The obtained supematant was
used as a template DNA aqueous solution. The full length insert
fragment containing a region to be annealed with a primer for
sequencing was amplified by PCR consisting of 30 cycles of 20
seconds at 98.degree. C. and 2 minutes at 68.degree. C. The
obtained DNA fragment was used as a template for the Sanger method.
An M13 Universal primer or M13 Reverse primer and a PRISM
Dye-Terminator sequencing kit manufactured by Perkin Elmer were
used, and a sequencing reaction was carried out in accordance with
an instruction manual attached to the kit. An unreacted
Dye-terminator was eliminated from the sequencing reaction product
by the gel filtration method or the like. Thereafter, the
nucleotide sequence of the DNA fragment was determined using a 3700
DNA Sequencer manufactured by Perkin Elmer. The waveform data
outputted from the 3700 DNA Sequencer were reanalyzed with Phred
(Phil Green). The vector and the adaptor sequence were eliminated,
and the remaining sequences were assembled using SPS Phrap
(Southwest Parallel Software), so as to construct contigs of the
nucleotide sequence of the genomic DNA of Aspergillus oryzae RIB
40.
EXAMPLE 2
Identification of Gene
[0157] Identification of gene from the nucleotide sequence of the
genomic DNA was carried out using, in combination, a gene finding
system GeneDecoder based on the algorithm according to Kiyoshi Asai
et al. [Pacific Symposium on Biocomputing, 98, 228 (1998)] and a
gene finding system ALN based on the algorithm according to Osamu
Goto [Bioinformatics, 16, 190-202 (2000)] to the contigs of the
nucleotide sequence of the genomic DNA, while taking into
consideration the EST sequence information that had already been
obtained, and information regarding homology to the known protein
amino acid sequence database, by applying the methods according to
the following (1) to (7).
(1) Extraction of BLAST Homologous Gene Candidate Regions
[0158] A region having a high homology with the amino acid sequence
of a known protein is extracted from the contigs of the nucleotide
sequence of the genomic DNA. The homology of an amino acid sequence
can be determined by the algorithm BLAST according to Karlin and
Altschul [Proc. Natl. Acad. Sci. USA, 87, 2264 (1990), Proc. Natl.
Acad. Sei. USA, 90, 5873 (1993)]. A program called BLASTX had been
developed based on this algorithm [J. Mol. Biol., 215, 403-410,
(1990)], and when the nucleotide sequence of genomic DNA is
translated into an amino acid sequence, a region with high homology
can be directly searched by the program. Specific techniques of
these analysis methods are known (http://www.ncbi.nlm.nih.gov). In
this technique, SWISSPROT version 39 [Nucleic Acids Res., 28, 45
(2000)] and NRaa are searched as database by BLASTX using contig of
the nucleotide sequence of genomic DNA as query. Thus, regions in
which an index of homology in the BLAST algorithm, E-value, is
10.sup.-30 or lower (the lower the E-value, the higher the homology
is) are extracted. Gene candidate regions obtained by
preferentially extracting regions with higher homology from the
above regions without overlapping with one another were defined as
BLAST homologous gene candidate regions.
(2) Extraction of ALN Gene Candidate Regions
[0159] The gene finding system ALN was applied to contig sequences
to select candidate regions of homologous genes extracted by BLAST,
using a region having homology to a region corresponding to 90% or
more of the full-length amino acid sequence of a protein that is a
target of homology as a core. The extracted gene candidate region
was defined as an ALN gene candidate region. The ALN specifies a
splice site by making the full-length amino acid sequence of a
protein that is a target of homology align against the contigs, so
as to predict gene regions.
(3) Extraction of GD Homologous Gene Candidate Regions
[0160] The gene finding system GeneDecoder was applied to contig
sequences to select candidate regions of homologous genes extracted
by BLAST, using a region having homology to a region corresponding
to 20% or more to less than 90% of the length of residues of the
amino acid sequence of a protein that is a target of homology as a
core. The extracted gene candidate region was defined as a GD
homologous gene candidate region. The GeneDecoder integrates the
E-value of BLASTX with the dicodon bigram values that is an index
of the orientation of a protein encoding region, and further takes
into consideration the position-dependent 1.sup.st order Markov
model score of a splice site, so as to predict gene regions.
(4) Extraction of EST-GD Gene Candidate Regions
[0161] With regard to regions in which gene expression has been
confirmed by EST corresponding to contig sequences, the GeneDecoder
is applied to contig sequences around the regions. Thus, not only
gene regions determined by the sequences of EST, but also gene
candidate regions extracted by predicting the entire gene regions,
are defined as EST-GD gene candidate regions.
(5) Extraction of General GD Gene Candidate Regions
[0162] The GeneDecoder is applied to contig sequences that are not
contained in the gene candidate regions defined in (1) to (4)
above. Thus, gene candidate regions extracted by predicting gene
regions are defined as general GD gene candidate regions.
(6) Extraction of tRNA Gene Candidate Regions
[0163] A tRNA-scan is applied to all the contigs, and the extracted
tRNA gene candidates are defined as tRNA gene candidate
regions.
(7) Integration of Gene Candidate Regions
[0164] The gene candidate regions defined in (2) to (6) are
integrated by the following procedure: First, from the gene
candidate regions defined in (2) to (6), those predicting gene
regions which contradict splice sites determined by EST, are
eliminated. The remaining gene candidate regions are integrated by
removing those overlapping with each other. In this operation, the
gene candidate regions are integrated preferentially in the order
of the tRNA gene candidate regions, the ALN homologous gene
candidate regions, the GD homologous gene candidate regions, the
GD-EST gene candidate regions, and the general GD gene candidate
regions. The thus integrated gene candidate region is defined as a
set of predicted genes. The nucleotide sequence shown in SEQ ID NO:
1 is one of the thus obtained predicted genes.
[0165] By the above procedure, in terms of homology, it is assured
that a gene having homology to the full length of a known protein,
a gene having a certain level of homology to the known protein, and
a gene having no homology to the known protein are specified with
reliability in this order. Moreover, in terms of confirmation of
expression, genes are specified with reliability in the order of a
gene whose expression has been confirmed by EST and a gene whose
expression has not been confirmed by EST. Furthermore, it is
assumed that all the candidate genes do not contradict splice sites
specified by EST.
[0166] All the used techniques adopts algorithm that does not allow
a termination codon to be contained in a protein encoding region,
and accordingly, it is unlikely to predict a pseudogene as a gene
of interest.
[0167] The functions of the predicted gene region were determined
by performing homology search against database Nraa using BLAST,
and setting homology sufficient to specify the functions (an
E-value of 10.sup.-30) as a threshold value.
[0168] Furthermore, in order to extract pyroglutamyl peptidase from
these predicted genes with high precision, the nucleotide sequence
of a known pyroglutamyl peptidase gene was searched using the
BLASTX against database including query and the above predicted
gene set. As a result, it was found that human pyroglutamyl
peptidase I (GenBank registration No. AJ278828) and a predicted
gene comprising the nucleotide sequence shown in SEQ ID NO: 1 had
homology with each other, showing an E-value of
6.1.times.10.sup.-5. Accordingly, it was considered that DNA
comprising the nucleotide sequence shown in SEQ ID NO: 1 encodes
pyroglutamyl peptidase derived from Aspergillus oryzae. In
addition, it was also considered that an intron exists in a portion
of the nucleotide sequence shown in SEQ ID NO: 1 and that the
intron encodes the amino acid sequence shown in SEQ ID NO: 2. The
nucleotide sequence of a 5' non-translation region of the
nucleotide sequence shown in SEQ ID NO: 1, which is considered to
contain a region to function as a promoter, was shown in SEQ ID NO:
3. Likewise, the nucleotide sequence of a 3' non-translation region
thereof was shown in SEQ ID NO: 4. Moreover, the nucleotide
sequence of a coding region from which the sequences of the intron
and the non-translation regions are eliminated was shown in SEQ ID
NO: 5.
EXAMPLE 3
Cloning of Pyroglutamyl Peptidase cDNA of Aspergillus Oryzae
(1) Preparation of cDNA of Aspergillus Oryzae RIB 40 Strain
[0169] Aspergillus oryzae RIB 40 strain was cultured under the
following conditions. This is to say, this strain was inoculated
into 60 ml of a DPY medium (2% dextrin, 2% polypeptone, 0.5% yeast
extract, 0.5% monopotassium phosphate, 0.05% magnesium sulfate 7
hydrates), and the mixture was placed in a 300 ml Erlenmeyer flask
with a baffle, followed by a shaking culturing at 30.degree. C. at
150 rpm for 2 days. Thereafter, the culture was filtrated, and 1 g
of the obtained wet cell bodies was transferred into a mortar
containing liquid nitrogen, so that the cell bodies were frozen by
the liquid nitrogen. Thereafter, the cell bodies were ground with a
pestle into fine powders.
[0170] Total RNA was obtained from this fine powders, using an
RNeasy Midi Kit manufactured by Qiagen.
[0171] From the obtained total RNA, 100 .mu.L of 0.6 .mu.g/ml mRNA
solution was obtained, using an Oligotex.TM.-dT30 <Super>
mRNA Purification Kit manufactured by Takara Shuzo Co., Ltd. 10
.mu.l of 3 mol/l sodium acetate solution (pH 5.2) and 250 .mu.l of
99.5% ethanol were added to this solution. The obtained mixture was
intensively stirred, and it was then left at rest at -20.degree. C.
for 2 hours. Thereafter, the mixture was centrifuged at 12000 rpm
for 20 minutes, and the precipitate was then washed with 200 .mu.l
of 70% ethanol. Thereafter, it was dissolved in 6 .mu.l of a
diethylpyrocarbonate-treated water.
[0172] The first strand cDNA and the second strand cDNA were
synthesized from the recovered mRNA using a SUPERSCRIPT Plasmid
System with GATEWAY.TM. Technology for cDNA Synthesis and Cloning
manufactured by Invitrogen, and they were used as templates for
PCR.
(2) Cloning of Pyroglutamyl Peptidase cDNA of Aspergillus Oryzae by
PCR, and Construction of Plasmid Used for Expression of
Pyroglutamyl Peptidase
[0173] Primers comprising nucleotide sequences shown in SEQ ID NOS:
8 and 9 were designed from the nucleotide sequence shown in SEQ ID
NO: 1, and were synthesized.
[0174] Using the above primers, Aspergillus oryzae cDNA prepared in
(1) above as a template, and Premix Taq (manufactured by Takara
Shuzo Co., Ltd.), PCR was carried out with a Program Temp Control
System PC-700 (manufactured by Astec). After heating at 94.degree.
C. for 5 minutes to denature template DNA, a cycle consisting of 2
minutes at 94.degree. C., 30 seconds at 56.degree. C., and 1.5
minutes at 72.degree. C. was repeated 30 times. The reaction
solution was electrophoresed on 0.8% agarose gel. As a result, a
DNA fragment with a size of approximately 850 bp was detected.
[0175] The DNA fragment was purified using a GENECLEAN Kit
(manufactured by Q-Biogene) and then cleaved with restriction
enzymes PstI and EcoRI. The cleaved fragment was then ligated to a
plasmid vector pRSET C for expression of prokaryotes (manufactured
by Invitrogen), which had also been cleaved with PstI and EcoRI.
The ligation was carried out using a Ligation Kit Version 2
(manufactured by Takara Shuzo Co., Ltd.) Escherichia coli
DH5.alpha. (manufactured by Takara Shuzo Co., Ltd.) was transformed
with the obtained plasmid-containing solution by the calcium
chloride method [J. Mol. Biol., 53, 159 (1970)], and transformants
were selected in an LB agar plate medium (1% trypton, 0.5% yeast
extract, 0.5% NaCl, 1.5% agar) containing 50 .mu.g/ml
ampicillin.
[0176] The growing cells (transformants) on this medium were
subjected to a liquid culturing by common methods. Thereafter,
plasmid DNA was extracted by common methods, and the plasmid was
cleaved with PstI and EcoRI. Thereafter, it was subjected to
agarose gel electrophoresis, so that inserted fragments were
confirmed. As a result, approximately 850 bp inserted fragments in
addition to 2.9 kbp DNA fragments of the plasmid pRSET C were
detected. This plasmid was named pPGP. The pPGP is a plasmid for
expressing, under the control of a T7 promoter, a polypeptide
comprising the amino acid sequence shown in SEQ ID NO: 10, in which
41 amino acid residues comprising a polyhistidine tag were added to
the N terminus of pyroglutamyl peptidase of Aspergillus oryzae.
EXAMPLE 4
Production of Pyroglutamyl Peptidase by Escherichia Coli
[0177] Escherichia coli BL21 was transformed with the plasmid pPGP
for expression of pyroglutamyl peptidase, which had been
constructed in Example 3(2), by the calcium method [J. Mol. Biol.,
53, 159 (1970)], and transformants were selected in an LB agar
plate medium (1% trypton, 0.5% yeast extract, 0.5% NaCl, 1.5% agar)
containing 50 .mu.g/ml ampicillin.
[0178] The obtained transformants were inoculated into 60 ml of an
LB liquid medium (1% trypton, 0.5% yeast extract, 0.5% NaCl) and
then allowed to grow at 25.degree. C. up to the logarithmic range.
Thereafter, isopropyl-.beta.-D-thiogalactopyranoside (hereinafter
abbreviated as IPTG) was added thereto to a final concentration of
1 mmol/l. Thereafter, the mixture was further cultured at
25.degree. C. for 3 hours.
[0179] After completion of the culture, cell bodies were collected
and suspended in 1 ml of a phosphate buffer solution (50 mmol/l
Na.sub.2HPO.sub.4, 0.5 mol/l NaCl, pH 8). Thereafter, the cells
were disrupted with ultrasonic wave, and were then centrifuged, so
that insoluble fractions were eliminated and thus, a crude protein
extract solution was obtained. The crude protein extract solution
was mixed with 1 ml of an equilibrated Ni-NTA resin (manufactured
by Invitrogen) with a concentration of 50%. The obtained mixture
was incubated at 4.degree. C. for 30 minutes and then introduced
into a column. The Ni-NTA resin was washed twice with 8 ml of a
phosphate buffer solution containing 0.02 mol/l imidazole, and it
was then eluted with 1 ml of a phosphate buffer solution containing
0.25 mol/l imidazole, so that approximately 35 kDa protein was
purified as an almost single band on SDS-PAGE. Moreover, when the
amount of the recovered protein was measured with a protein assay
kit (manufactured by Biorad), it was found to be 91.8 .mu.g.
[0180] Using pyroglutamic acid-paranitroanilide (hereinafter
abbreviated as PCA-pNA) as a substrate, the activity of
pyroglytamyl peptidase was measured. PCA-pNA was added to a 50
mmol/l phosphate buffer solution (pH 7.5) to a final concentration
of 5 mmol/l, so as to prepare a substrate solution. 20 .mu.l of an
enzyme solution was added to 100 .mu.l of the substrate solution.
The mixture was reacted at 37.degree. C. for 10 minutes, and an
absorbance at 405 nm was measured. The amount of free
para-nitroaniline was calculated at a molar absorbance coefficient
of 10500. 1 unit (U) was defined as an amount by which 1 .mu.mol of
para-nitroaniline is released at 37.degree. C. for 1 minute. The
activity of a purified protein solution was measured by the
above-described method. As a result, it was found that it had an
activity of 6.6 mU/ml. On the other hand, when the activity of a
solution obtained by performing the same above purification
operation on cell bodies containing no IPTG was measured, it was
found to be one fiftieth of the above obtained activity. Thus, it
was found that pyroglutamyl peptidase activity was dependent on
expression induction by IPTG. Accordingly, it was confirmed that a
DNA fragment that has been inserted into pPGP encodes pyroglutamyl
peptidase and it can thereby be used in production of pyroglutamyl
peptidase, and that a polypeptide comprising the amino acid
sequence shown in SEQ ID NO: 10 (an amino acid sequence in which 41
amino acid residues were added to the N terminus of the amino acid
sequence shown in SEQ ID NO: 2) has a pyroglutamyl peptidase
activity.
EXAMPLE 5
Production of Protein Hydrolysate by Action of Pyroglutamyl
Peptidase
[0181] 1.0 g of Flavorzyme (manufactured by Novo Nordisk) was added
to 200 ml of an aqueous solution containing 10% wheat gluten
(Promic GT manufactured by Kyowa Hakko Kogyo Co., Ltd.), and the
mixture was subjected to enzymolysis at 48.degree. C. for 3 days.
Thereafter, the resultant product was filtrated and then treated by
heating at 90.degree. C., so as to obtain a protein
hydrolysate.
[0182] Escherichia coli BL21 into which pPGP had been introduced
was cultured in the same manner as in Example 4, so as to prepare a
purified protein solution. The obtained purified protein solution
having 0.5 U pyroglutamyl peptidase activity and a solution
containing 0.1 g of Flavorzyme were added to 20 ml of a protein
hydrolysate that had been filtrated using a membrane filter with a
pore size of 0.2 .mu.m, and the mixture was subjected to
enzymolysis at 40.degree. C. for 2 days (Test A). The amount of
total nitrogen, the amount of free amino acid, and the degradation
rate of the above hydrolysed product were analyzed, and the
obtained data were compared with those of a hydrolyzed product to
which a solution having no pyroglutamyl peptidase activity and
Flavorzyme had been added (Test B). The results are shown in Table
1. TABLE-US-00001 TABLE 1 A B Total nitrogen (g/dl) 1.31 1.27 Free
amino acid (g/dl) 0.56 0.49 Degradation rate (%) 68.9 62.0
[0183] As is clear from the above results, a protein lysate with a
high hydrolysis rate can be obtained by addition of the
pyroglutamyl peptidase of the present invention.
INDUSTRIAL APPLICABILITY
[0184] The present invention provides DNA encoding novel
pyroglutamyl peptidase derived from Aspergillus oryzae, and
pyroglutamyl peptidase which is produced by using the DNA. Using
the pyroglutamyl peptidase, a protein lysate with a good flavor can
be obtained at a high hydrolysis rate.
[Sequence Listing Free Text]
[0185] SEQ ID NO: 6--an adaptor [0186] SEQ ID NO: 7--an adaptor
[0187] SEQ ID NO: 8--a primer for amplification of pyroglutamyl
peptidase cDNA of Aspergillus oryzae [0188] SEQ ID NO: 9--a primer
for amplification of pyroglutamyl peptidase cDNA of Aspergillus
oryzae [0189] SEQ ID NO: 10--an amino acid sequence, which
comprises an addition of 41 amino acids comprising a polyhistidine
tag to the N terminus of the amino acid sequence shown in SEQ ID
NO: 2
Sequence CWU 1
1
10 1 2201 DNA Aspergillus oryzae Inventor Machida, Masayuki; Abe,
Keietsu; Gomi, Katsuya; Inventor Asai, Kiyoshi; Sano, Motoaki; Kin,
Taishin Inventor Nagasaki, Hideki; Hosoyama, Akira; Akita, Osamu
Inventor Ogasawara, Naotake; Kuhara, Satoru; Tokunaga, Chikara
Inventor Toda, Itaru; Saitoh, Chiaki; Senoh, Akihiro 1 tcaagatctg
aatgataaca gatttcctag tccattgtaa cacatcgatg gggttgacgt 60
ggtaatagaa tcccaagact gatggaaatc gttctaggtg aagtcaaagt ctagatgatg
120 aataaacaac cagactctag gaaaatgctg gtctagaccc ttgggcgaga
aggaatgtgc 180 tgataacgtc tcgctgcctt tcagcggtaa cgctaatcta
aaagatcaac aaacaatcca 240 ggagcaacca gagcaatcgg tgcgtgttca
gtaagtgagc agtgagtgca caggagcact 300 cacgtgctaa ccttacaaaa
gcagcggcac ccatatcaaa caggaagaag tgggccgtac 360 ggtagttcta
ggatgacata ccgaaacccc ttatttgttc gcttaaatag atccctgccc 420
agctttactg atggatttct aatcgcaaag taattgggtg aaataccatc ggtattaacc
480 tagtgaatgg tgattctcaa ccatcgagta caagtcattc tcactattga
actttccaaa 540 aagccccgtg aacaagcagt ctgcggtttg ccccggctga
agcaaggggg aaattgtcgg 600 tcaggactcg gaacttcgga agcgaagcag
aatcggcggt ggccaaaagg catgcgacgt 660 gacagcacct cacatcattc
cgggacaata acataggttc aattgcacaa ttgtctcaag 720 aacatgggtg
attgtcagat tgatacgtca atcaagcttt gtgggcggtc aagatgaggg 780
gaggtcatgt gccttatcac cttatcgata tcgatatcgc gtgatgccaa gacctgcatg
840 cggtggttgt aatgcggggg aagctccgtc gatatcttga atatatcttt
agtccctcct 900 ctctatcctt tttgtggcgt acatagctac cgtgtatata
cgaagtaaag gcgttgttcc 960 caccactgat tcctagcttg ccttgaccta
tccactagcc atg gga gac ttt ggc 1015 Met Gly Asp Phe Gly 1 5 cca ccg
gtg cca ata ccc gag acg gag gta att ggt ctt gct tcg tca 1063 Pro
Pro Val Pro Ile Pro Glu Thr Glu Val Ile Gly Leu Ala Ser Ser 10 15
20 tct ttg aca gat cca gaa gag gtc tcg gta ctg gtg aca ggg ttc ggt
1111 Ser Leu Thr Asp Pro Glu Glu Val Ser Val Leu Val Thr Gly Phe
Gly 25 30 35 gtaagtttat ctgtcttatg cttggcttat gtttctttga ccgtcttggc
ttctgattcg 1171 gccctcaaat gctaaaatat actag cca ttc aag acc aac cta
gtc aat gcc 1223 Pro Phe Lys Thr Asn Leu Val Asn Ala 40 45 tcg tat
ttg att gcc tca tct ctg cca gag tcg ctt gac ctt cct tcg 1271 Ser
Tyr Leu Ile Ala Ser Ser Leu Pro Glu Ser Leu Asp Leu Pro Ser 50 55
60 gcg aag ccg tct gga tcc ggg cct act tct cgt cgg att tca att cat
1319 Ala Lys Pro Ser Gly Ser Gly Pro Thr Ser Arg Arg Ile Ser Ile
His 65 70 75 gtc cat cca tcg ccc att ccc gtc gct tac tca aca gtg
cgg aca act 1367 Val His Pro Ser Pro Ile Pro Val Ala Tyr Ser Thr
Val Arg Thr Thr 80 85 90 att cca acc atc cta gag gat tac gcc aag
tcc cat gga ggt cga cga 1415 Ile Pro Thr Ile Leu Glu Asp Tyr Ala
Lys Ser His Gly Gly Arg Arg 95 100 105 110 cca gac att gta ctc cat
atg gga ata gcg gct aca aga tcg tac tac 1463 Pro Asp Ile Val Leu
His Met Gly Ile Ala Ala Thr Arg Ser Tyr Tyr 115 120 125 tcg att gag
acc aag gcg cat cga gat tct tac cac ttg tcc gat atc 1511 Ser Ile
Glu Thr Lys Ala His Arg Asp Ser Tyr His Leu Ser Asp Ile 130 135 140
aaa ggc aga atc ggt tat gaa gat ggg gag aag gtt tgg agg gag cag
1559 Lys Gly Arg Ile Gly Tyr Glu Asp Gly Glu Lys Val Trp Arg Glu
Gln 145 150 155 cag ctc ccg cca gta ctc cag gct ggt cct gcg gcg gat
tcc aca gac 1607 Gln Leu Pro Pro Val Leu Gln Ala Gly Pro Ala Ala
Asp Ser Thr Asp 160 165 170 gta gta cgg aaa gtt ctc cac ccc cag ccg
ccc aat gac gac ttt ctc 1655 Val Val Arg Lys Val Leu His Pro Gln
Pro Pro Asn Asp Asp Phe Leu 175 180 185 190 aac acg tgg aag tcg ttt
gta tct cct gga gca gac gtc cgg ata tcc 1703 Asn Thr Trp Lys Ser
Phe Val Ser Pro Gly Ala Asp Val Arg Ile Ser 195 200 205 gag gac gct
gga cgc tac ctc tgc gag ttc atc ttt tat aca agt ctg 1751 Glu Asp
Ala Gly Arg Tyr Leu Cys Glu Phe Ile Phe Tyr Thr Ser Leu 210 215 220
gcc cag gcg ttt caa caa ggc cag cac cga aac gtc gtt ttc ttc cat
1799 Ala Gln Ala Phe Gln Gln Gly Gln His Arg Asn Val Val Phe Phe
His 225 230 235 gtg cct gga tct tgc gcc gac gag gac atc gag aga ggc
acg gat att 1847 Val Pro Gly Ser Cys Ala Asp Glu Asp Ile Glu Arg
Gly Thr Asp Ile 240 245 250 gca gct gga ttg atc aaa gct ctt gta aga
tgt tgg gtt agc gag cag 1895 Ala Ala Gly Leu Ile Lys Ala Leu Val
Arg Cys Trp Val Ser Glu Gln 255 260 265 270 gta tag agcggcatgc
aggttgctgg tatcgttttg caaagcaaga gcatgggcac 1951 Val tggacgatat
atatacttgc atttctatgg cgcggtgcac tatctgggtt ccggatgcgc 2011
ttttagctgc agtcactcgt gatcatttat ttatagggga cttctgtccc cggtcttttc
2071 aggttgagtt atacatgttt cacaggtttt ggatacacta tttaccctct
gactactatc 2131 gatgaatata gacagttgtc aagcatgata ttgggttcta
ccgtattcgt atatgtgtag 2191 aatttcccgt 2201 2 271 PRT Aspergillus
oryzae 2 Met Gly Asp Phe Gly Pro Pro Val Pro Ile Pro Glu Thr Glu
Val Ile 1 5 10 15 Gly Leu Ala Ser Ser Ser Leu Thr Asp Pro Glu Glu
Val Ser Val Leu 20 25 30 Val Thr Gly Phe Gly Pro Phe Lys Thr Asn
Leu Val Asn Ala Ser Tyr 35 40 45 Leu Ile Ala Ser Ser Leu Pro Glu
Ser Leu Asp Leu Pro Ser Ala Lys 50 55 60 Pro Ser Gly Ser Gly Pro
Thr Ser Arg Arg Ile Ser Ile His Val His 65 70 75 80 Pro Ser Pro Ile
Pro Val Ala Tyr Ser Thr Val Arg Thr Thr Ile Pro 85 90 95 Thr Ile
Leu Glu Asp Tyr Ala Lys Ser His Gly Gly Arg Arg Pro Asp 100 105 110
Ile Val Leu His Met Gly Ile Ala Ala Thr Arg Ser Tyr Tyr Ser Ile 115
120 125 Glu Thr Lys Ala His Arg Asp Ser Tyr His Leu Ser Asp Ile Lys
Gly 130 135 140 Arg Ile Gly Tyr Glu Asp Gly Glu Lys Val Trp Arg Glu
Gln Gln Leu 145 150 155 160 Pro Pro Val Leu Gln Ala Gly Pro Ala Ala
Asp Ser Thr Asp Val Val 165 170 175 Arg Lys Val Leu His Pro Gln Pro
Pro Asn Asp Asp Phe Leu Asn Thr 180 185 190 Trp Lys Ser Phe Val Ser
Pro Gly Ala Asp Val Arg Ile Ser Glu Asp 195 200 205 Ala Gly Arg Tyr
Leu Cys Glu Phe Ile Phe Tyr Thr Ser Leu Ala Gln 210 215 220 Ala Phe
Gln Gln Gly Gln His Arg Asn Val Val Phe Phe His Val Pro 225 230 235
240 Gly Ser Cys Ala Asp Glu Asp Ile Glu Arg Gly Thr Asp Ile Ala Ala
245 250 255 Gly Leu Ile Lys Ala Leu Val Arg Cys Trp Val Ser Glu Gln
Val 260 265 270 3 1000 DNA Aspergillus oryzae source (1)..(1000)
/organism="Aspergillus oryzae" /strain="RIB 40" 3 tcaagatctg
aatgataaca gatttcctag tccattgtaa cacatcgatg gggttgacgt 60
ggtaatagaa tcccaagact gatggaaatc gttctaggtg aagtcaaagt ctagatgatg
120 aataaacaac cagactctag gaaaatgctg gtctagaccc ttgggcgaga
aggaatgtgc 180 tgataacgtc tcgctgcctt tcagcggtaa cgctaatcta
aaagatcaac aaacaatcca 240 ggagcaacca gagcaatcgg tgcgtgttca
gtaagtgagc agtgagtgca caggagcact 300 cacgtgctaa ccttacaaaa
gcagcggcac ccatatcaaa caggaagaag tgggccgtac 360 ggtagttcta
ggatgacata ccgaaacccc ttatttgttc gcttaaatag atccctgccc 420
agctttactg atggatttct aatcgcaaag taattgggtg aaataccatc ggtattaacc
480 tagtgaatgg tgattctcaa ccatcgagta caagtcattc tcactattga
actttccaaa 540 aagccccgtg aacaagcagt ctgcggtttg ccccggctga
agcaaggggg aaattgtcgg 600 tcaggactcg gaacttcgga agcgaagcag
aatcggcggt ggccaaaagg catgcgacgt 660 gacagcacct cacatcattc
cgggacaata acataggttc aattgcacaa ttgtctcaag 720 aacatgggtg
attgtcagat tgatacgtca atcaagcttt gtgggcggtc aagatgaggg 780
gaggtcatgt gccttatcac cttatcgata tcgatatcgc gtgatgccaa gacctgcatg
840 cggtggttgt aatgcggggg aagctccgtc gatatcttga atatatcttt
agtccctcct 900 ctctatcctt tttgtggcgt acatagctac cgtgtatata
cgaagtaaag gcgttgttcc 960 caccactgat tcctagcttg ccttgaccta
tccactagcc 1000 4 300 DNA Aspergillus oryzae source (1)..(300)
/organism="Aspergillus oryzae" /strain="RIB 40" 4 agcggcatgc
aggttgctgg tatcgttttg caaagcaaga gcatgggcac tggacgatat 60
atatacttgc atttctatgg cgcggtgcac tatctgggtt ccggatgcgc ttttagctgc
120 agtcactcgt gatcatttat ttatagggga cttctgtccc cggtcttttc
aggttgagtt 180 atacatgttt cacaggtttt ggatacacta tttaccctct
gactactatc gatgaatata 240 gacagttgtc aagcatgata ttgggttcta
ccgtattcgt atatgtgtag aatttcccgt 300 5 816 DNA Aspergillus oryzae
source (1)..(816) /organism="Aspergillus oryzae" /strain="RIB 40" 5
atg gga gac ttt ggc cca ccg gtg cca ata ccc gag acg gag gta att 48
Met Gly Asp Phe Gly Pro Pro Val Pro Ile Pro Glu Thr Glu Val Ile 1 5
10 15 ggt ctt gct tcg tca tct ttg aca gat cca gaa gag gtc tcg gta
ctg 96 Gly Leu Ala Ser Ser Ser Leu Thr Asp Pro Glu Glu Val Ser Val
Leu 20 25 30 gtg aca ggg ttc ggt cca ttc aag acc aac cta gtc aat
gcc tcg tat 144 Val Thr Gly Phe Gly Pro Phe Lys Thr Asn Leu Val Asn
Ala Ser Tyr 35 40 45 ttg att gcc tca tct ctg cca gag tcg ctt gac
ctt cct tcg gcg aag 192 Leu Ile Ala Ser Ser Leu Pro Glu Ser Leu Asp
Leu Pro Ser Ala Lys 50 55 60 ccg tct gga tcc ggg cct act tct cgt
cgg att tca att cat gtc cat 240 Pro Ser Gly Ser Gly Pro Thr Ser Arg
Arg Ile Ser Ile His Val His 65 70 75 80 cca tcg ccc att ccc gtc gct
tac tca aca gtg cgg aca act att cca 288 Pro Ser Pro Ile Pro Val Ala
Tyr Ser Thr Val Arg Thr Thr Ile Pro 85 90 95 acc atc cta gag gat
tac gcc aag tcc cat gga ggt cga cga cca gac 336 Thr Ile Leu Glu Asp
Tyr Ala Lys Ser His Gly Gly Arg Arg Pro Asp 100 105 110 att gta ctc
cat atg gga ata gcg gct aca aga tcg tac tac tcg att 384 Ile Val Leu
His Met Gly Ile Ala Ala Thr Arg Ser Tyr Tyr Ser Ile 115 120 125 gag
acc aag gcg cat cga gat tct tac cac ttg tcc gat atc aaa ggc 432 Glu
Thr Lys Ala His Arg Asp Ser Tyr His Leu Ser Asp Ile Lys Gly 130 135
140 aga atc ggt tat gaa gat ggg gag aag gtt tgg agg gag cag cag ctc
480 Arg Ile Gly Tyr Glu Asp Gly Glu Lys Val Trp Arg Glu Gln Gln Leu
145 150 155 160 ccg cca gta ctc cag gct ggt cct gcg gcg gat tcc aca
gac gta gta 528 Pro Pro Val Leu Gln Ala Gly Pro Ala Ala Asp Ser Thr
Asp Val Val 165 170 175 cgg aaa gtt ctc cac ccc cag ccg ccc aat gac
gac ttt ctc aac acg 576 Arg Lys Val Leu His Pro Gln Pro Pro Asn Asp
Asp Phe Leu Asn Thr 180 185 190 tgg aag tcg ttt gta tct cct gga gca
gac gtc cgg ata tcc gag gac 624 Trp Lys Ser Phe Val Ser Pro Gly Ala
Asp Val Arg Ile Ser Glu Asp 195 200 205 gct gga cgc tac ctc tgc gag
ttc atc ttt tat aca agt ctg gcc cag 672 Ala Gly Arg Tyr Leu Cys Glu
Phe Ile Phe Tyr Thr Ser Leu Ala Gln 210 215 220 gcg ttt caa caa ggc
cag cac cga aac gtc gtt ttc ttc cat gtg cct 720 Ala Phe Gln Gln Gly
Gln His Arg Asn Val Val Phe Phe His Val Pro 225 230 235 240 gga tct
tgc gcc gac gag gac atc gag aga ggc acg gat att gca gct 768 Gly Ser
Cys Ala Asp Glu Asp Ile Glu Arg Gly Thr Asp Ile Ala Ala 245 250 255
gga ttg atc aaa gct ctt gta aga tgt tgg gtt agc gag cag gta tag 816
Gly Leu Ile Lys Ala Leu Val Arg Cys Trp Val Ser Glu Gln Val 260 265
270 6 16 DNA Artificial adaptor 6 cgagagcggc cgctac 16 7 13 DNA
Artificial adaptor 7 gtagcggccg ctc 13 8 29 DNA Artificial primer
for amplification of Aspergillus oryzae pyroglutamyl peptidase cDNA
8 gggctgcaga tgggagactt tggcccacc 29 9 30 DNA Artificial primer for
amplification of Aspergillus oryzae pyroglutamyl peptidase cDNA 9
ggggaattcc tatacctgct cgctaaccca 30 10 312 PRT Artificial amino
acid sequence wherein 41 amino acids containing polyhistidine-tag
are added at N-terminal of amino acid sequence of SEQ ID NO2 10 Met
Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10
15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30 Arg Trp Ile Arg Pro Arg Asp Leu Gln Met Gly Asp Phe Gly
Pro Pro 35 40 45 Val Pro Ile Pro Glu Thr Glu Val Ile Gly Leu Ala
Ser Ser Ser Leu 50 55 60 Thr Asp Pro Glu Glu Val Ser Val Leu Val
Thr Gly Phe Gly Pro Phe 65 70 75 80 Lys Thr Asn Leu Val Asn Ala Ser
Tyr Leu Ile Ala Ser Ser Leu Pro 85 90 95 Glu Ser Leu Asp Leu Pro
Ser Ala Lys Pro Ser Gly Ser Gly Pro Thr 100 105 110 Ser Arg Arg Ile
Ser Ile His Val His Pro Ser Pro Ile Pro Val Ala 115 120 125 Tyr Ser
Thr Val Arg Thr Thr Ile Pro Thr Ile Leu Glu Asp Tyr Ala 130 135 140
Lys Ser His Gly Gly Arg Arg Pro Asp Ile Val Leu His Met Gly Ile 145
150 155 160 Ala Ala Thr Arg Ser Tyr Tyr Ser Ile Glu Thr Lys Ala His
Arg Asp 165 170 175 Ser Tyr His Leu Ser Asp Ile Lys Gly Arg Ile Gly
Tyr Glu Asp Gly 180 185 190 Glu Lys Val Trp Arg Glu Gln Gln Leu Pro
Pro Val Leu Gln Ala Gly 195 200 205 Pro Ala Ala Asp Ser Thr Asp Val
Val Arg Lys Val Leu His Pro Gln 210 215 220 Pro Pro Asn Asp Asp Phe
Leu Asn Thr Trp Lys Ser Phe Val Ser Pro 225 230 235 240 Gly Ala Asp
Val Arg Ile Ser Glu Asp Ala Gly Arg Tyr Leu Cys Glu 245 250 255 Phe
Ile Phe Tyr Thr Ser Leu Ala Gln Ala Phe Gln Gln Gly Gln His 260 265
270 Arg Asn Val Val Phe Phe His Val Pro Gly Ser Cys Ala Asp Glu Asp
275 280 285 Ile Glu Arg Gly Thr Asp Ile Ala Ala Gly Leu Ile Lys Ala
Leu Val 290 295 300 Arg Cys Trp Val Ser Glu Gln Val 305 310
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References