JP2001054390A - Glutamate decarboxylase gene, vector, transformant obtained by using the same and its utilization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new glutamate decarboxylase gene which comprises glutamate decarboxylase gene derived from koji mold Aspergillus oryzae containing a specific base sequence and permits the large scale production of the above glutamate decarboxylase useful in the high production or the like of γ-aminobutyric acid. SOLUTION: This glutamate decarboxylase gene is a new glutamate decarboxylase gene, which contains a specific base sequenge and is derived from Aspergillus oryzae, and is used in the manufacture or the like of glutamate decarboxylase permitting the high production of γ-aminobutyric acid which is a nerve inhibitory mediator for higher animals and is useful in the development or the like of medicines or food materials. This glutamate decarboxylase gene is obtained by extracting a chromosomal DNA from Aspergillus oryzae strain RIB 128 to prepare a phage chromosomal library, isolating a gene by means of plaque hybridization using a partial gene sequence produced by PCR as a probe, transducing this gene into a plasmid to migrate into the host cell strain and liquid-cultivating it.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a glutamate decarboxylase gene obtained from Aspergillus oryzae, a vector containing the gene, a transformant obtained by transferring the vector into Aspergillus oryzae, and use thereof. Glutamate decarboxylase is an enzyme that produces γ-aminobutyric acid using glutamic acid as a substrate, and the transformant obtained according to the present invention produces a large amount of the enzyme, resulting in high production of γ-aminobutyric acid. Becomes Therefore, the present invention can be applied to the production of foods enriched in γ-aminobutyric acid.

[0002]

2. Description of the Related Art γ-Aminobutyric acid is a neurotransmitter that inhibits nerves in higher animals, and is known to have various physiological actions such as suppression of blood pressure increase, promotion of healing of organ inflammation such as ulcers, and diuretic action. ing. Therefore, the development of pharmaceuticals and foodstuffs enriched in γ-aminobutyric acid having many physiological actions has been studied in recent years at various research institutions.

As described above, since γ-aminobutyric acid is produced from glutamic acid by glutamate decarboxylase, a method of increasing γ-aminobutyric acid is to increase the activity of glutamic acid decarboxylase. However, there are few reports on Aspergillus aspergillus oryzae glutamate decarboxylase itself, and no report on a DNA chain that produces glutamate decarboxylase from Aspergillus oryzae has been found.

[0004]

An object of the present invention is to clarify the glutamate decarboxylase gene of Aspergillus oryzae and to obtain an Aspergillus oryzae transformant having enhanced glutamate decarboxylase-producing ability. An object of the present invention is to produce γ-aminobutyric acid at a high level and to produce γ-aminobutyric acid-enriched food depending on the body.

[0005]

As a result of repeated studies to elucidate the glutamate decarboxylase gene, a chromosome DN encoding glutamate decarboxylase was obtained from a chromosome library of Aspergillus oryzae.
The A fragment was successfully cloned.

[0006] This DNA fragment was also successfully transferred into an Aspergillus oryzae host vector system, and it was confirmed that the resulting transformant significantly increased glutamate decarboxylase productivity. The transformant obtained here has been deposited as FERMP-17449 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

[0007] When the transformant obtained here was cultured, a remarkable amount of γ-aminobutyric acid was produced as compared with the conventional koji mold. Furthermore, since a considerable amount of γ-aminobutyric acid was produced even in solid culture using this transformant, the present invention can be applied to the production of foods enriched in γ-aminobutyric acid.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
The chromosomal DNA, mRNA and glutamate decarboxylase donor used in the present invention are Aspergillus oryzae, specifically RIB128 strain.

[0009] Escherichia coli
ia coli), Arabidopsis saliana (Ar
The homology of the amino acid sequences of glutamic acid decarboxylase of G. abidopsis thaliana was compared, and from the amino acid sequence region highly conserved between the two, primers were prepared in consideration of the codon degeneracy (sequence list, SEQ ID NO :) 4,
5). Using this, Aspergillus oryzae mRN
Using A as a template, RT-PCR high-plus-
Reverse transcription of RNA and obtained c
DNA polymerase chain reaction (PCR) was performed (R
T-PCR). The obtained PCR product was analyzed with a DNA sequencer (ALfred DNA sequencer, manufactured by Amersham Pharmacia Biotech) to decode the DNA base sequence. When this DNA sequence was translated into amino acids, Escherichia coli (Escherichia coli) was used.
ia coli), Arabidopsis saliana (Ar
It showed a very high homology with the amino acid sequence of G. glutamic acid decarboxylase of G. abidopsis thaliana, and was considered to be a part of the glutamate decarboxylase gene of Aspergillus oryzae. Therefore, this PCR product was used as a probe for plaque hybridization described below.

Also, Aspergillus oryzae RIB1
Chromosomal DNA was extracted from 28 strains to prepare a phage chromosome library. Glutamate decarboxylase gene was isolated from this phage chromosome library using the PCR product obtained above as a probe and plaque hybridization. The obtained DNA fragment had the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.

Based on the nucleotide sequence obtained above, an oligo DNA is synthesized based on a sequence deduced as a coding region of glutamate decarboxylase, and this is used as a primer to mR of Aspergillus oryzae RIB128 strain.
The nucleotide sequence of the glutamate decarboxylase cDNA was determined from the NA using a 5 '/ 3'RACE Kit (Boehringer Mannheim). The results are shown in SEQ ID NO: 2 in the sequence listing.

The glutamate decarboxylase gene obtained above was subcloned into pUC119 (manufactured by Takara Shuzo Co., Ltd.), and a gene (argB) complementary to arginine requirement was inserted into this. The obtained DNA was transferred to an argB-deficient strain of Aspergillus oryzae to obtain a transformant.

Glutamate decarboxylase can be efficiently produced by culturing the transformant obtained above in liquid.

Further, γ-aminobutyric acid can be highly accumulated in the culture of the transformant. In particular, if glutamic acid is added during the culturing of the transformant, further high production of γ-aminobutyric acid can be achieved.

Further, by using this transformant for solid culture, for example, preparation of rice koji, a remarkable amount of γ-aminobutyric acid can be produced. Therefore, the present invention can be applied to the production of foods enriched in γ-aminobutyric acid. Next, examples of the present invention will be described.

[0016]

Example 1 Determination of Partial Amino Acid Sequence of Glutamate Decarboxylase of Aspergillus oryzae Escherichia col
i), Arabidopsis saliana (Arabidop)
The homology of the amino acid sequences of glutamic acid decarboxylase of S. thaliana was compared, and primers were prepared from the highly conserved amino acid sequence region in consideration of codon degeneracy. The forward primer is shown in SEQ ID NO: 4 in the Sequence Listing and the reverse primer is shown in SEQ ID NO: 5 in the Reverse Primer Sequence Listing.

The total RNA of Aspergillus oryzae RIB128 was used as an RNA extraction reagent ISOGEN.
(Nippon Gene) according to the attached instructions, followed by Oligotex-dT30 <Su
per> (manufactured by Nippon Synthetic Rubber Co., Ltd. and Roche Co., Ltd.), using Nucleic Acids Research
h Symposium series No. 19,
61-64 (1988)
Was extracted. Using this mRNA as a template and the above two primers, RT-PCR high-plus
As a result of RT-PCR using s- (manufactured by Toyobo), a clear band of 210 bp was obtained.

The obtained band was cloned and DNA
The result of analyzing with a sequencer and decoding the DNA base sequence is shown in SEQ ID NO: 6 in the sequence listing. When this DNA base sequence was translated into amino acids, Escherichia coli (Es
cherichia coli, Arabidopsis thalian
It showed very high homology with the amino acid sequence of glutamate decarboxylase in a), and was considered to be a part of the glutamate decarboxylase gene of Aspergillus oryzae. Therefore, this PCR product is used for PCR DIG
Using a DIG-labeled probe with a Labeling Kit (Boehringer Mannheim) as a probe,
Cloning of the glutamate decarboxylase gene was performed.

[0019]

Example 2 Cloning of Chromosomal DNA Fragment Encoding Aspergillus oryzae Glutamate Decarboxylase A chromosome was isolated from Aspergillus oryzae strain RIB128, for example, from Agric. Biol. Chem51, 32
3-328 (1987).
A phage chromosome library using EMBL3 was
rectangular Cloning, Second E
d. , Cold Spring HarborLabo
rattery Press (1989).

Using the probe described above, a The DIG System U was obtained from a phage chromosome library.
ser's Guide for FilterHyb
ridization, Boehringer Man
Clones containing the glutamate decarboxylase gene were selected by the plaque hybridization method described in Nheim. Four clones hybridizing to the probe were obtained from about 15000 plaques.

A phage DNA was isolated from this clone, and a DNA fragment (approximately 5.5 kb HindIII fragment) that hybridized to the above-mentioned probe was isolated from pUC118.
(Takara Shuzo). The nucleotide sequence of this DNA fragment was determined by the dideoxy method using a DNA sequencer. This nucleotide sequence is shown as SEQ ID NO: 1 in the sequence listing.

[0022]

Example 3 Determination of the Nucleotide Sequence of the Glutamate Decarboxylase cDNA of the Transformant A primer (LAN) for the stop codon region deduced from the nucleotide sequence of the glutamate decarboxylase gene obtained in Experimental Example 2 (sequence list, sequence) Number 7),
A reverse primer (ASF) near 1470 bp of SEQ ID NO: 1 in the sequence listing (SEQ ID NO: 8) and a forward primer (WRS) near 1400 bp in SEQ ID NO: 1 of the sequence listing (SEQ ID NO: 9) Created.

5 ′ / 3 ′ RAC from mRNA of Aspergillus oryzae RIB128 strain extracted in Experimental Example 1
The full-length nucleotide sequence of the glutamate decarboxylase cDNA was determined using E Kit (Boehringer Mannheim). In the case of 5'RACE, first P
In the CR, the above primer LAN is connected to the second PC
For R, primer ASF was used, and for 3′RACE, primer WRS was used for PCR. The amplified products of these PCRs were analyzed with a DNA sequencer to decode the nucleotide sequence of the cDNA, and were compared with the nucleotide sequence of the glutamate decarboxylase gene obtained in Experimental Example 2. As a result, the start codon and the stop codon could be confirmed. Was. The glutamate decarboxylase gene of Aspergillus oryzae consists of 514 amino acids,
It was found to contain three introns. The nucleotide sequence of the cDNA from the start codon to the stop codon is shown in SEQ ID NO: 2 in the sequence listing. This base sequence was translated into an amino acid sequence (Sequence Listing, SEQ ID NO: 3), and Escherichia coli (Esc
herichia coli), Arabidopsis thaliana
When compared with the amino acid sequence of glutamic acid decarboxylase, it showed high homology with both amino acid sequences.

[0024]

Example 4 Aspergillus oryzae expressing a large amount of glutamate decarboxylase from Aspergillus oryzae
Preparation of Oryzae Transformant From the phage DNA described in Example 2, a DNA fragment (about 5.8 kb S
(acI fragment) was prepared and subcloned into pUC119. A gene (argB) that complements the arginine requirement of Aspergillus oryzae was inserted into this plasmid to prepare a plasmid pGAD2a shown in FIG.
The inserted SacI fragment had a coding region of glutamate decarboxylase of 1.5 kb and its upstream portion of 2.9.
kb and 1.2 kb of the downstream portion, and it is considered that all the regions required for the expression of glutamate decarboxylase in Aspergillus oryzae are included. Therefore, this plasmid was transferred to Aspergillus oryzae.

The M-2-3 strain which is an argB-deficient strain of Aspergillus oryzae (Agric. Biol. Che.
m. , 51, 2549-2555 (1987)) by the plasmid pGAD2a. Biol.
Chem. , 51, 2549-2555 (1987).
I got Using the above probe, transformant G-
1, and a plasmid p having the argB gene as a marker
As a result of Southern analysis of the transformant C-1 in which OCT1 (FIG. 2) was similarly transferred to the M-2-3 strain, it was found that only G-1 has a multicopy glutamate decarboxylase gene. It could be confirmed.

Total RNA was extracted from the transformants G-1 and C-1 in the same manner, and the DIG System User's
Guide for Filter Hybridi
zation, Boehringer Mannhei
m, Northern analysis was performed using the method described in
1, only a weak signal was detected, while G
At -1, a very strong signal was detected.

[0027]

Example 5 Measurement of Glutamate Decarboxylase Activity of Transformant The resulting transformant was subjected to a Zapex-Docs liquid medium (0.2% NaNO ₃ , 0.1%) containing 1% polypeptone.
% K ₂ HPO ₄ , 0.05% MgSO ₄ .7H ₂ O,
The cells were shake-cultured with 0.05% KCl, 0.001% FeSO ₄ , 2% glucose, pH 6.0) at 30 ° C. for 3 days.

Glutamate decarboxylase activity of the cultured cells was measured. Glutamate decarboxylase activity was measured as follows. The cultured cells were crushed with liquid nitrogen and suspended in a 0.01 M succinate buffer (pH 5.7) of about 8 times the weight of the wet cells. This suspension is heated at 4 ° C, 3
The mixture was shaken for 0 minutes and centrifuged at 3000 rpm for 30 minutes to remove crushed residues to obtain a crude enzyme solution.

First, a 5-fold amount of GAD ass was added to the crude enzyme solution.
ai Reagent (0.05M succinate buffer (p
H5.7), 100 mM glutamic acid, 500 μM pyridoxal phosphoric acid, 0.4% mercaptoethanol), and the reaction was carried out by shaking at 38 ° C. for 1 hour, and the reaction was stopped by placing it in boiling water for 3 minutes (reaction 1). ). Next, the following reaction was performed in order to measure the amount of γ-aminobutyric acid generated by Reaction 1 (Reaction 2). After the reaction solution of reaction 1 was filtered, it was diluted to an appropriate concentration with 0.1 M succinate buffer solution (pH 5.0) and added to 100 μl, and 400 μl of G
ABA assay Reagent (0.3 MTri
An s-HCl buffer (pH 8.9), 20 mM α-ketoglutaric acid, 2 mM NADP, 0.01% mercaptoethanol, 120 μg / ml GABAse) were added.
The reaction was carried out at 38 ° C. for 30 minutes, and 0.1M Tris-HC
The reaction was stopped by adding 2 ml of 1 buffer solution (pH 8.0). First, 0.1M Tris-HCl
Buffer (pH 8.0) was added, followed by GABA as
A sample in which the same reaction was performed with the addition of Say Reagent was also prepared. After the reaction, the fluorescence of these reaction solutions was measured (excitation wavelength 365 nm, fluorescence wavelength 4
70 nm) and the resulting NADPH was measured, and the amount of γ-aminobutyric acid generated by Reaction 1 was calculated. Glutamate decarboxylase activity, which produces 1 mg of γ-aminobutyric acid by reaction 1, was defined as 1 U, and the activity value per protein amount of the crude enzyme solution was calculated. Quantification of protein in crude enzyme solution
A Bio-Rad protein assay kit (Bio-Rad) was used.

The glutamate decarboxylase activity of the obtained transformant G-1 and the glutamate decarboxylase activity of the transformant C-1 obtained by transferring the plasmid pOCT1 having the argB gene as a marker into the M-2-3 strain were measured. It is shown in Table 1.

[0031]

[Table 1]

As described above, by transferring the glutamate decarboxylase gene into Aspergillus oryzae, the activity of glutamate decarboxylase is reduced to 10%.
A fold increase in transformants was obtained. This transformant G-1 was transformed into Aspergillus oryzae G
-1 was deposited with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology as FERMP-17449.

[0033]

Example 6 Production of γ-aminobutyric acid by transformant The above transformant was shake-cultured at 30 ° C. for 3 days in a medium (pH 6.0) in which 20 mM glutamic acid was added to a Zapex-Docs liquid medium containing 1% polypeptone. did. The amount of γ-aminobutyric acid in the culture solution was measured using a Hitachi ninhydrin method amino acid analysis system. Table 2 shows the results. As is clear from these results, G-1 is 1 of C-1.
0-fold or more of γ-aminobutyric acid was produced.

[0034]

[Table 2]

[0035]

Example 7 Production of γ-Aminobutyric Acid by Solid Culture of Transformant Using α rice for brewing, 0.5% of sodium glutamate was added to koji making water, and the spores of the transformant were inoculated. Rice koji was prepared according to the method. However, the koji making temperature was constant at 35 ° C., and the koji making was 50 hours. A 5-fold amount of 0.1 M succinate buffer (pH 4.0) was added to the obtained koji, and 4 ° C.
The mixture was shake-extracted for 5 minutes to extract γ-aminobutyric acid contained in the koji. Γ-Aminobutyric acid in the extract was measured using a Hitachi ninhydrin method amino acid analysis system. Table 3 shows the results. As is clear from these results, G-1 produced about 7 times as much γ-aminobutyric acid as C-1.

[0036]

[Table 3]

[0037]

According to the present invention, the glutamic acid decarboxylase gene of Aspergillus oryzae has been successfully cloned for the first time, and its nucleotide sequence has been elucidated. In the present invention, the host has been successfully transformed by inserting the cloned gene into a vector. Therefore, glutamate decarboxylase can be produced in a significant amount by culturing the obtained transformant.

[0038] In addition, by transforming the transformant that produces a significant amount of glutamate decarboxylase in liquid culture, a significant amount of γ-aminobutyric acid can be produced. Furthermore, by performing solid culture using this transformant, for example, preparing rice koji, a remarkable amount of γ-aminobutyric acid can be produced, and the present invention can be applied to the production of γ-aminobutyric acid-enriched foods. . Therefore, not only in the fermentation industry, but also in the food industry and the pharmaceutical industry, it is possible to develop products having a health-promoting function by enhancing γ-aminobutyric acid, and the present invention can be greatly expected.

[0039]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows a restriction map of plasmid pGAD2a.

FIG. 2 shows a restriction map of plasmid pOCT1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 9/88 9/88 C12P 13/00 C12P 13/00 C12N 5/00 A // ( C12N 15/09 ZNA C12R 1:69) (C12N 1/15 C12R 1:69) (C12P 13/00 C12R 1:69) (72) Inventor Masamichi Hara 1-8-6 Uozaki-Nishi-cho, Higashinada-ku, Kobe, Hyogo Prefecture No. F-term in Kiku Masamune Sake Brewery Co., Ltd. (Reference) 4B018 MD18 ME04 ME11 ME14 4B024 AA01 AA05 BA07 CA04 DA11 4B050 CC03 DD03 LL01 LL02 4B064 AE01 CA05 CA19 CA21 CB30 CC24 DA10 4B065 AA63X AA63Y AB01 CA02 CA16 CA27

Claims (6)

[Claims] (1) It comprises the nucleotide sequence of SEQ ID NO: 1 in the sequence listing,
Glutamate decarboxylase gene derived from Aspergillus oryzae, Aspergillus oryzae. 2. A vector containing the glutamate decarboxylase gene according to claim 1. 3. A transformant obtained by transferring the vector according to claim 2 into Aspergillus oryzae. 4. A method for producing glutamate decarboxylase, which comprises collecting glutamate decarboxylase from a culture by culturing the transformant according to claim 3. (5) γ-aminobutyric acid is collected by culturing the transformant according to (3).
-A method for producing aminobutyric acid. 6. A method for producing a γ-aminobutyric acid-enriched food using the transformant according to claim 3.