JP2001046075A - Salt tolerant glutaminase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new salt tolerant glutaminase gene which comprises a salt tolerant glutaminase gene containing a nucleotide encoding a specific amino acid sequence and is useful in the production or the like of salt tolerant glutaminase having properties needed to produce soy sauce or soybean paste. SOLUTION: This gene is a new salt tolerant glutaminase gene, which comprises a salt tolerant glutaminase gene containing a nucleotide encoding the amino acid sequence in the position of 405-591 of the formula and codes for a dimeric polypeptide containing a polypeptide encoded by the nucleotide as a small subunit, possesses properties capable of sufficiently exhibiting the effect without deactivating in the presence of a high concentration of salt and is useful for industrially in large quantities producing the salt tolerant glutaminase peculiarly acting on glutamine and sufficiently reacting with glutamine. The gene is obtained by treating a chromosome DNA prepared from Bacillus subtilis strain GT with a restriction enzyme, blotting a DNA separated by agarose electrophoresis on a nylon membrane and screening out it by a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding a polypeptide having glutaminase activity and having excellent salt tolerance, an expression vector containing the gene, a host cell, and a method for producing a salt-tolerant glutaminase.

[0002]

2. Description of the Related Art Various processes for decomposing protein materials to produce amino acid seasonings and the like have been performed for a long time. As one of them, there is known a method in which various proteases and peptidases are allowed to act on a protein raw material to obtain an amino acid as a final product via a peptide. This method is extremely important in that glutaminase converts free glutamine in the proteolysate into glutamic acid, a tasty amino acid.

[0003] On the other hand, soy sauce and miso, which are known as representatives of seasoned foods, are produced by prolonged decomposition of proteins under a high salt concentration. In order to efficiently convert glutamine to glutamic acid in the production of such foods, the used glutaminase does not inactivate even in the presence of high-concentration salt, has the property of sufficiently exerting its action, and is specifically specific for glutamine. It is necessary that it acts and of course sufficiently reacts with low-concentration glutamine (that is, the Km value is small).

There are many reports on salt-tolerant glutaminase. However, these reported glutaminases are all too low in salt tolerance and have all of the above-mentioned various properties required for the production of soy sauce and miso. Not something. For example, JP-A-3-232486 discloses a glutaminase (γ-glutamine transpeptidase) derived from Bacillus subtilis strain, but is not shown to have salt tolerance. JP-A-63-94975 discloses a salt-tolerant glutaminase, but the Km value and the substrate specificity for low-concentration glutamine cannot be satisfied. In addition, since this is an intracellular enzyme, mass production is difficult, and there is a practically serious problem in terms of the source of the enzyme. As described above, conventional glutaminase is not sufficient in terms of the properties required for its use, and there is a long-awaited demand in the art for salt-tolerant glutaminase having superior salt tolerance and other properties exceeding these properties. It is in.

Japanese Patent No. 2,843,937 discloses that 85% at pH 5.5 in the presence of high-concentration salt (25%, w / v).
Disclosed are salt-tolerant glutaminases that exhibit glutaminase activity of greater than or equal to%.

[0006] This glutaminase is obtained by culturing Bacillus subtilis GT. However, this bacterium produces a large amount of proteins other than glutaminase (such as proteases and amylases). Therefore, in order to separate glutaminase from these by-produced proteins in large amounts, many steps including column chromatography operations are required, and the yield of glutaminase is low. A method for purifying salt-tolerant glutaminase from the culture supernatant of Bacillus subtilis GT bacteria is described in, for example, Journal of the Japan Brewing Association, Vol. 86, No. 6, 441-446, (1991). It is not a method that can be applied. The present invention is to isolate the salt-tolerant glutaminase gene and determine its nucleotide sequence, thereby revealing the amino acid sequence within the glutaminase polypeptide that has led to the acquisition of its salt tolerance and substrate specificity. Achieve the goal. The present invention provides important knowledge of genetic engineering techniques and protein engineering techniques relating to salt-tolerant glutaminase.

[0007]

SUMMARY OF THE INVENTION The present invention is intended to solve the above-mentioned conventional problems, and makes it possible to mass-produce a salt-tolerant glutaminase having the properties required for the production of soy sauce and miso. . The present invention provides a polypeptide having a salt-tolerant glutaminase activity derived from the Bacillus subtilis GT strain, and a gene sequence having a nucleotide encoding the polypeptide.

[0008]

Means for Solving the Problems In order to clarify the amino acid sequence of the salt-tolerant glutaminase and the nucleotide sequence encoding the same, the inventors of the present invention prepared a strain of Bacillus subtilis GT, a bacterial strain that produces salt-tolerant glutaminase. As a result of intensive studies on the glutaminase gene, we finally succeeded in elucidating, for the first time, the entire nucleotide sequence and the nucleotide portion that imparts a polypeptide structure related to salt tolerance. Further, the present inventors provide a method for industrially producing a large amount of salt-tolerant glutaminase using a salt-tolerant glutaminase gene.

[0009] The present invention relates to a salt-tolerant glutaminase gene.
And encoding a dimer peptide having a polypeptide encoded by this nucleotide as a small subunit. Preferably, the salt-tolerant glutaminase gene comprises a nucleotide encoding the amino acid sequence of positions 35 to 591 of SEQ ID NO: 2. Furthermore, it preferably contains a nucleotide encoding the amino acid sequence of positions 1 to 591 of SEQ ID NO: 2.

[0010] In one aspect, the present invention relates to an expression vector containing the above-mentioned salt-tolerant glutaminase gene.

The present invention further relates to a host cell containing the expression vector.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

(1) First, Journal of the Japan Brewing Association, Vol. 86,
No. 6, 441-446 (1991), Bacillus subtilis GT is cultured, and then salt-tolerant glutaminase is isolated from the culture supernatant.

(2) Next, the N-terminal amino acid sequence of the purified salt-tolerant glutaminase is determined. The N-terminal amino acid sequence may be, for example, a purified salt-tolerant glutaminase, SD
After S-polyacrylamide gel electrophoresis, blotting is performed on a PVDF membrane, stained with CBB (Coomassie Brilliant Blue), the band is cut out, and determined using a gas-phase amino acid sequencer.

(3) Next, a synthetic DNA probe is designed based on the obtained information on the N-terminal amino acid sequence, and the salt-resistant glutaminase gene is cloned.

(4) The DNA probe can be synthesized, for example, by the phosphoramidite method or the like. Isolation and purification of the gene can be performed by agarose gel electrophoresis. The DNA sequence of the gene of the present invention is determined by the dideoxy method (Sa
nger, F .; Proc. Natl. Acad. S
ci. U. S. A. , 74, 5463-5467 (19
77)), etc., or by a method using a commercially available sequencing kit.

(5) As a method for selecting a target salt-tolerant glutaminase gene, a Southern hybridization method or a dot hybridization method (in each case, Southern, EM, J. Mol. Bio)
l. , 98, 503-517 (1975)). These techniques and various genetic engineering techniques for handling other genes are all available from molecular cloning (Maniatis, T., Fritsch,
E. FIG. F. And Sambrook, J. et al. , Molec
ullar Cloning A Laboratory
Manual / Second Edition, Cold Spring H
arbor Laboratory N.R. Y. (198
8)).

(6) The salt-tolerant glutaminase gene of the present invention can be prepared, for example, by the phosphite-triester method (Nature, 310,
105 (1984)). In addition, it can be produced by a known method using a known established glutaminase gene or γ-glutamyl transpeptidase gene or a gene encoding a precursor thereof, and modifying them according to the obtained genetic information.

(7) The salt-tolerant glutaminase gene of the present invention obtained as described above is typically characterized by having a gene sequence encoding the amino acid sequence represented by SEQ ID NO: 2. The gene of the present invention also comprises
One or more amino acids in the amino acid sequence represented by SEQ ID NO: 2 are characterized by having a gene sequence encoding a substituted or deleted amino acid sequence. Such methods of amino acid substitution, deletion or addition are known in the art.

Typically, the gene of the present invention has the nucleotide sequence shown in SEQ ID NO: 1. As a result of the degeneracy of the genetic code, a sequence that encodes the amino acid sequence represented by SEQ ID NO: 2 and is equivalent to the base sequence represented by SEQ ID NO: 1 is also included in the present invention. That is, the gene of the present invention typically has a length of 1941 base pairs, and 1773 base pairs (5
One large open reading frame (positions 68 to 1840) consisting of 91 amino acid residues). In this reading frame, the mature salt-tolerant glutaminase gene is located at positions 170 to 1840,
The upstream sequence is the signal sequence (corresponding to a total of 34 amino acid residues).

The salt-tolerant glutaminase gene of the present invention comprises:
Typically, the polypeptide encodes the amino acid sequence represented by SEQ ID NO: 2 and contains a nucleotide encoding the amino acid sequence of positions 405 to 591 of SEQ ID NO: 2, and the polypeptide encoded by this nucleotide is referred to as a small subunit. Encoding a dimeric polypeptide. Typically, the translation product of the salt-tolerant glutaminase gene of the present invention is 41.5.
It is a salt-tolerant glutaminase consisting of polypeptide units of kDa (kilodalton) and 24.5 kDa.

The salt-tolerant glutaminase gene of the present invention comprises:
A nucleotide coding for an amino acid sequence in which one or more amino acids in the amino acid sequence at positions 405 to 519 in SEQ ID NO: 2 have been substituted and deleted, or one or more amino acids have been added, has a translation product Includes as long as it constitutes a subunit of the body polypeptide.

In one embodiment, the present invention relates to an expression plasmid for the salt-tolerant glutaminase gene,
This expression vector comprises the above-described salt-tolerant glutaminase gene operably linked to a control sequence operable in a given host cell.

[0024] Host cells that can be used to express the salt-tolerant glutaminase gene of the present invention, and control sequences that can be used in the host cells, are known in the art. For example, host cells such as E. coli, Bacillus subtilis, yeast, etc. are available in the art and have the lacI and lacZ promoters operable in their respective host cells, T3 and T3.
7 promoter, gpt promoter, λPR promoter and λPL promoter, trp promoter,
Alcohol dehydrogenase promoters are known (see Molecular Cloning, supra).

The gene of the present invention can produce a salt-tolerant glutaminase by utilizing such a gene recombination technique known in the art. More specifically, a vector containing a recombinant gene capable of expressing the gene of the present invention in a host cell is prepared, introduced into a host cell, transformed, and the obtained transformant is cultured. To express a salt-tolerant glutaminase according to a conventional method, for example, salting out method, centrifugation method, osmotic shock method, ultrasonic crushing method, ultrafiltration method, gel filtration method, adsorption chromatography method, ion exchange chromatography method And the salt-tolerant glutaminase expressed by high performance liquid chromatography or the like can be separated and purified. Such methods are known to those skilled in the art,
For example, Davis et al., Basic Methods.
in Molecular Biology (198
It is described in many standard laboratory manuals such as 6). The resulting glutaminase is a salt-tolerant glutaminase.

As used herein, the term “salt-tolerant glutaminase” refers to the condition at 37 ° C. and pH 5.5.
In the presence of 18% (w / v) salt, about 90% or more of the relative activity in the absence of salt, and in the presence of 25% (w / v) salt, about 85% or more of the relative activity in the absence of salt. Glutaminase.

[0027]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Bacillus subtilis GT
Purification of Salt-Tolerant Glutaminase from Strain) Bacillus subtilis GT strain was replaced with 0.3% Glucose, 3.0%
Polypepton, 1.0% yeast extract, 0.5%
Using a medium consisting of NaCl and pH 7.0, the cells were cultured in a 90 L jar fermenter under the conditions of aeration rate of 35 L / min, rotation speed of 200 rpm and 38 ° C. for 33 hours.
All cultures were centrifuged at 10,000 xg for 15 minutes,
114 L of culture supernatant was obtained. To this culture supernatant, cold ethanol was added little by little at 4 ° C. to 80% (v / v), and the mixture was allowed to stand overnight, and then centrifuged at 10,000 × g for 15 minutes. Collected. The resulting precipitate is
A small amount of 20 mmol / L Tris-HCl buffer (pH 7.
It was dissolved in 5) and dialyzed against the same buffer. The dialysis sample was adsorbed on a DEAE cellulose column (5.6 × 27 cm) equilibrated with the same buffer, and 0.1 mol / L
After washing with the same buffer solution containing NaCl, 0.3 mol /
The salt-tolerant glutaminase was eluted with the same buffer containing L NaCl. Next, the glutaminase activity of each fraction was measured, and the active fraction was collected and dialyzed against a 20 mmol / L sodium phosphate buffer (pH 7.4). Then
The dialysate was adsorbed onto a hydroxylapatite column (2.8 × 20 cm) which had been equilibrated with the same buffer solution,
After washing with the same buffer, 20 mmol / L to 400 mmol
Salt tolerant glutaminase was eluted by linearly increasing the concentration of phosphate buffer (pH 7.4) to 1 / L. Glutaminase activity was divided into two peaks centered on fractions 85 and 130. Fraction 1 which is the main peak
Nos. 20 to 145 were collected and used as an active fraction. The obtained active fraction was subjected to a 20 mmol / L Tris-HCl buffer (pH
7.5), and then a DEAE Toyopearl column (1.6 ×) equilibrated with the same buffer in advance.
(7.5 cm), washed with the same buffer,
05 mol / L, 0.1 mol / L, 0.15 mol / L
L, 0.2mol / L, 0.25mol / L NaC
and eluted in a stepwise manner with the same buffer. Salt-tolerant glutaminase was present in the eluted fraction at 0.15 mol / L NaCl. The active fraction was collected and 0.2 mol / L Na
20 mmol / L Tris-HCl buffer containing Cl (pH
The mixture was placed on a Sephacryl S-200 column (2.8 × 41 cm) equilibrated in 7.5), and 2 mL was collected at a flow rate of 12 mL per hour. The glutaminase activity of each fraction was measured, and the active fraction was collected and dialyzed against a 10 mmol / L sodium phosphate buffer (pH 6.0) containing 25% (w / v) ammonium sulfate. This dialysate is adsorbed on a phenyl sepharose CL-4B column (1.6 × 5 cm) which has been equilibrated with the same buffer in advance.
After washing with the same buffer, the ammonium sulfate concentration was 25% (w
/ V) to 15% (w / v), while ethylene glycol was stepwise increased from 0% (w / v) to 20% (w / v) to elute salt-tolerant glutaminase. As a result of measuring the glutaminase activity of each fraction, the fraction was divided into a peak near No. 40 and a peak near No. 49. By recovering the former, which is the main peak, purified salt-tolerant glutaminase was obtained. Purified salt-tolerant glutaminase was confirmed to be electrophoretically single by disk electrophoresis. In addition, SDS with a gel concentration of 18%
Purified salt-tolerant glutaminase was found to have a molecular weight of 41.5 kDa and 24.5 by polyacrylamide gel electrophoresis.
Given two protein bands corresponding to kDa, it was confirmed that the protein was a heterodimer consisting of two large and small subunits.

Example 2 Determination of N-Terminal Amino Acid Sequence of Salt-Tolerant Glutaminase A large and small subunit of salt-tolerant glutaminase separated by SDS-polyacrylamide gel electrophoresis at a gel concentration of 18% was subjected to semidry blotting ( (TRANS BLOT SD) on a PVDF membrane,
Stained with Coomassie Brilliant Blue R250. The band obtained was cut out and a gas phase sequencer (PSQ-
According to 1), the amino acid sequence of the N-terminal 24 residues and 29 residues of each of the large and small subunits was determined. The amino acid sequence is shown below: N-terminal amino acid sequence of the large subunit, Phe-Ser-Tyr-Asp-Asp-Tyr
-Lys-Gln-Val-Asp-Val-Gly-
Lys-Asp-Gly-Met-Val-Ala-T
hr-Ala-His-Pro-Leu-Ala; and the N-terminal amino acid sequence of the small subunit, Thr-Th
r-His-Phe-Thr-Val-Ala-Asp
-Arg-Phe-Gly-Asn-Val-Val-
Ser-Tyr-Thr-Thr-Thr-Ile-G
lu-Gln-Leu-Phe-Gly-Ser-Gl
y-Ile-Met.

These correspond to residues 35 to 58 and residues 405 to 405 of the amino acid sequence shown in SEQ ID NO: 2, respectively.
433.

Example 3 Synthesis of Oligonucleotide Corresponding to N-Terminal Amino Acid Sequence of Salt-Tolerant Glutaminase
A mixture of 32 17-base oligonucleotides encoding 6 amino acid residues from the third Tyr to the eighth Gln of the N-terminal amino acid sequence of the large subunit of the salt-tolerant glutaminase revealed by amino acid sequencing ( Probe GTN) 5'-TAY GAY GAY
TAY AAR CA-3 '(Y represents T or C, R represents A or G) was synthesized by a phosphoramidite method using Gene Assembler Plus, a gene synthesizer manufactured by Amersham Pharmacia Biotech.

Example 4 Cloning of Salt-Tolerant Glutaminase of Bacillus subtilis GT Strain Chromosome by Southern Hybridization Method Chromosomal DNA prepared from Bacillus subtilis GT strain was subjected to restriction enzyme PstI.
After complete digestion with agarose gel electrophoresis,
The separated DNA was separated from the gel by a nylon membrane Hybo.
nd N (manufactured by Amersham). The 3′-end of the probe GTN was changed to 3′-oligolabe
lling and detection system
m-kit (Amersham) for fluorescent labeling. Using the fluorescence-labeled probe GTN, the blotted chromosomal DNA was analyzed by Southern hybridization. As a result, it was confirmed that the probe GTN hybridized to about 4 kbp of the fragment obtained by digesting the chromosomal DNA with PstI. Then, a PstI digested DNA fragment of this surrounding size was extracted and purified from an agarose gel. Next, a PstI digested fragment of the chromosomal gene was ligated to a PstI digest of a plasmid vector pUC118 that can be replicated in E. coli,
E. FIG. E. coli JM109. The obtained transformant was transferred to an LB agar medium (0.5% Yeast Ex).
tract, 1.0% Tryton, 1.0% NaC
l, 100 µg / mL ampicillin, 1.5% agar), smear it, culture at 37 ° C, take a replica using the same medium, and remove nylon membrane Hybond N (manufactured by Amersham) from the agar plate. Was colony blotted. The chromosomal gene of the blotted colony was detected by Southern hybridization using the probe GTN whose 3 ′ end was fluorescently labeled. As a result, positive transformants were obtained. The plasmid contained in this transformant was isolated and purified by an alkaline method to obtain pU8GT1. FIG. 3 shows pU8GT1.
The construction of

Example 5 Determination of Base Sequence of PstI Fragment on pU8GT1 Approximately 4.0 kbp on pU8GT1
The deletion mutant for the PstI insert of
-Sequence Deletion Kit (Takara Shuzo 14), and then dideoxy sequencing (Sanger, F. et al., Proc. Natl. A)
cad. Sci. U. S. A. , 74, 5463-54
67 (1997)), and the nucleotide sequence was determined by performing a sequencing reaction. This reaction is called AutoRead Se.
The analysis was performed using a quenching kit (manufactured by Amersham Pharmacia Biotech) and analyzed using an ALF DNA sequencer (manufactured by Amersham Pharmacia Biotech). As a result, 1773 was found in the approximately 4.0 kbp fragment.
One open reading frame consisting of bp (positions 68 to 1840 of the nucleotide sequence shown in SEQ ID NO: 1) was found. The sequence from Phe at position 35 to Ala at position 58 from the start codon of this open reading frame is the N of the large subunit whose amino acid sequence was determined.
It completely matched the terminal amino acid sequence. Furthermore, the sequence from Thr at position 405 to Met at position 433 was completely identical to the N-terminal amino acid sequence of the small subunit. From the above results, it was revealed that the large and small subunits of the salt-tolerant glutaminase were formed by cleavage after translation. The nucleotide sequence of the enzyme gene is shown in SEQ ID NO: 1 (FIG. 2), and the deduced amino acid sequence is shown in SEQ ID NO: 2 (FIG. 1).

When the obtained sequence is compared with the glutaminase gene sequence described in JP-A-3-232486, which does not have salt tolerance, 91 nucleotides in the base sequence and 13 amino acids in the deduced amino acid sequence (signal sequence) 2 amino acids, and 11 amino acids in the mature protein sequence). Therefore, 11 amino acids that differ in the mature protein sequence portion (SEQ ID NO: 2 in the sequence listing)
177, 212, 223, 248, 265
370, 411, 530, 550, 589
Ala, Asn, at position 590 and position 590, respectively.
Val, Thr, Gln, Asn, Ala, Glu, L
ys, Cys and Glu) are thought to contribute to the structure conferring salt tolerance.

Example 6 Analysis of Salt-Tolerant Glutaminase Gene Product BamHI restriction enzyme sites (underlined) corresponding to the DNA sequences of the 5 ′ and 3 ′ non-coding regions of the salt-tolerant glutaminase gene determined above. Oligo DNA primer GTN1 (5′-GAC GG
ATCC CATTCACACAGAAACCCC-
3 ′) and GTC1 (5′-TCG GGATCC GC
GGTGTGCTTTTCGC-3 ′) was synthesized. Next, using these primers, a chromosomal gene prepared from Bacillus subtilis GT strain was
CR was performed. The above PCR product of about 2.0 kbp was
It was digested with mHI and ligated to the BamHI digest of plasmid vector pUB110, which is replicable in Bacillus subtilis.
Then, Bacillus subtilis strain KY110 was transformed with the ligation mixture. A plasmid was isolated and purified from the obtained transformant to obtain pGT1 in which a salt-tolerant glutaminase gene was inserted into pUB110 (FIG. 3). As a result of determining the base sequence of the insert fragment of this plasmid, p
It completely matched the nucleotide sequence of U8GT1. The transformant is Bacillus sub.
btilis) KY110 (pGT1) (Accession No. F
ERM P-17489) July 28, 1999
It is deposited as a date with the Institute of Biotechnology and Industrial Technology, the National Institute of Advanced Industrial Science and Technology.

Next, the Bacillus subtilis KY110 strain having only the plasmid pGT1 or the plasmid pUB110 was transformed into a BHI medium (3.7% Brain Hea) containing 2.5 μg / mL kanamycin, respectively.
After culturing with shaking overnight at 37 ° C. in rt Infusion, the glutaminase activity of the culture supernatant was measured.

Glutaminase activity was measured according to Japanese Patent No. 284.
According to the method described in No. 3937, as described below, the measurement was performed by a method of quantifying the amount of ammonia produced by hydrolyzing L-glutamic acid.

That is, first, the final concentration in the reaction system is 2
0.8 ml of 0.5% L-glutamic acid, 0.1 ml of 1 M acetate buffer (pH 5.5), and 0.1 ml of culture supernatant were added with sodium chloride to a concentration of 5% (w / v).
A reaction system consisting of 1 ml was prepared. This reaction solution is kept at 37 ° C.
, And the reaction was stopped by adding 0.5 ml of a 0.75 N perchloric acid solution. The resulting reaction solution was neutralized by adding 0.25 ml of a 1.5 N aqueous sodium hydroxide solution. Next, 0.1 ml of the obtained reaction solution was taken, and the amount of ammonia in the reaction solution was determined using an F kit urea / ammonia determination kit (Boehringer Mannheim Yamanouchi). As a control, a solution obtained by adding 0.5 ml of a 0.75N perchloric acid solution to 0.1 ml of the culture supernatant and further adding glutamine, a buffer solution, and sodium hydroxide under the same conditions as above was used. used.

As a result, glutaminase activity could not be detected in the culture supernatant of Bacillus subtilis KY110 having only pUB110, but 1.1 U / m2 in Bacillus subtilis KY110 having pGT1.
1 was able to detect glutaminase activity. Further, the culture supernatant of the recombinant Bacillus subtilis KY110 (pGT1) was concentrated and dialyzed, and a crude enzyme solution having a glutaminase activity of 3.02 U / ml was purified from a glutaminase bulk powder derived from the DNA donor GT bacterium. It was confirmed that both exhibited the same salt tolerance using 10.0 U / g as a control.
Under the condition of 5.5), in the presence of 18% (W / V) salt, a relative activity of about 90% or more in the absence of salt was exhibited (Table 1).

[0040]

[Table 1]

As described above, pU8GT1 and pGT1
Was found to encode a salt-tolerant glutaminase derived from Bacillus subtilis GT bacterium. In addition, it was revealed that the introduction of pGT1 imparts a salt-tolerant glutaminase activity to a glutaminase non-producing bacterium.

[0042]

According to the present invention, there is provided a salt-tolerant glutaminase which does not inactivate in the presence of high-concentration salt and has a property capable of sufficiently exerting its action, and which acts specifically on glutamine and reacts sufficiently. An encoding gene is provided. Also,
The use of this gene provides a method for industrially producing salt-tolerant glutaminase in large quantities.

[0043]

[Sequence List] <110> DAIWA KASEI KK <120> Halotolerant Glutaminase Gene <130> J199120164 <140> <141> <150> <151> <160> 2 <170> PatentIn Ver. 2.0 <210> 1 <211> 1941 <212> DNA <213> Bacillus subtilis GT <220> <221> CDS <222> (68) .. (1840) <400> 1 gtcacttgtg aaaacagcac attttactta ctctataatt gtaagcggaa aacaaaggag 60 gcagact atg aaa aag aaa aag ttt atg aatct tgt ttt atc gtt ctg 109 Met Lys Lys Lys Lys Phe Met Asn Leu Cys Phe Ile Val Leu 1 5 10 ctc agt gct ttg ctc acg gcc ggc agc atc cct tat cac gcc cag gct 157 Leu Ser Ala Leu Leu Thr Ala Gly Ser Pro Tyr His Ala Gln Ala 15 20 25 30 aag aaa cac ccg ttt tcc tat gat gac tac aaa cag gta gat gtc ggc 205 Lys Lys His Pro Phe Ser Tyr Asp Asp Tyr Lys Gln Val Asp Val Gly 35 40 45 aaa gac ggc atg gtc gcc acc gcc cat cct ctc gct tca caa atc ggt 253 Lys Asp Gly Met Val Ala Thr Ala His Pro Leu Ala Ser Gln Ile Gly 50 55 60 gct gac gtg ctg aaa aaa ggc ggc aat gca att gac gct gcg gtt gcg 301 Ala Asp Val Leu Lys Lys Gly Gly Asn Ala Ile Asp Ala Ala Val Ala 65 70 75 att caa ttc gca tta aac gta act gaa ccg atg atg tca ggg atc ggc 349 Ile Gln Phe Ala Leu Asn Val Thr Glu Pro Met Met Ser Gly Ile Gly 80 85 90 ggc ggc gga ttt atg atg gtt tat gat gcg aag aca aaa gac acc acc 397 Gly Gly Gly Phe Met Met Val Tyr Asp Ala Lys Thr Lys Asp Thr Thr 95 100 105 110 att atc gac agc cgg gaa cgc gca ccg gca ggc gca aca ccg gat atg 445 Ile Ile Asp Ser Arg Glu Arg Ala Pro Ala Gly Ala Thr Pro Asp Met 115 120 125 ttc ctt gac gaa aac ggc aaa gcc att cct ttc tcc gaa cgg gtt aca 493 Phe Leu Asp Glu Asn Gly Lys Ala Ile Pro Phe Ser Glu Arg Val Thr 130 135 140 aaa ggg act gcc gtc ggt gtt ccg gga aca tta aaa ggc ctt gaa aaa 541 Lys Gly Thr Ala Val Gly Val Pro Gly Thr Leu Lys Gly Leu Glu Lys 145 150 155 gcg ctc gac aag tgg ggc aca cgc tcc atg aaa caact cct 589 Ala Leu Asp Lys Trp Gly Thr Arg Ser Met Lys Gln Leu Ile Thr Pro 160 165 170 tcc att gca ctt gcc tca aag ggc ttt ccg att gat tcg gtt tta gct 637 Ser Ile Ala Leu Ala Ser Lys Gly Phe Pro Ile Asp Ser V al Leu Ala 175 180 185 190 gat gcc atc tca gat tat aaa gac aaa tta tca cac act gct gca aaa 685 Asp Ala Ile Ser Asp Tyr Lys Asp Lys Leu Ser His Thr Ala Ala Lys 195 200 205 gac gtg ttt ctt ccg aac gga gaa cct ctg aaa gaa gga gac aca ctc 733 Asp Val Phe Leu Pro Asn Gly Glu Pro Leu Lys Glu Gly Asp Thr Leu 210 215 220 gtc caa aaa gac tta gcc aaa aca ttc act gcc att aag tat aaa ggc 78p Val Gln Lys Leu Ala Lys Thr Phe Thr Ala Ile Lys Tyr Lys Gly 225 230 235 aca aaa gca ttc tac gac gga gca ttc acc aaa aag ctt gct gaa aca 829 Thr Lys Ala Phe Tyr Asp Gly Ala Phe Thr Lys Lys Leu Ala Glu Thr 240 245245 250 gtg cag gaa ttc ggc ggc tca atg aca gaa caa gat att aaa aac ttt 877 Val Gln Glu Phe Gly Gly Ser Met Thr Glu Gln Asp Ile Lys Asn Phe 255 260 265 270 aac gta acg att gac gag ccg atc tgg gga cag ggc tat cat 925 Asn Val Thr Ile Asp Glu Pro Ile Trp Gly Asp Tyr Gln Gly Tyr His 275 280 285 atc gcg act gct cct cct cca agt tcc ggc ggt gtt ttc ctg tta caa 973 Ile Ala Thr Ala Pro Pro Pro Ser Ser G ly Gly Val Phe Leu Leu Gln 290 295 300 atg ctg aac ctt ctg gat gat ttt aag ctt tct caa tat gat atc cgt 1021 Met Leu Asn Leu Leu Asp Asp Phe Lys Leu Ser Gln Tyr Asp Ile Arg 305 310 315 tct tgg caa tat cag ctt ctc gct gaa aca atg cat tta gct tat 1069 Ser Trp Gln Lys Tyr Gln Leu Leu Ala Glu Thr Met His Leu Ala Tyr 320 325 330 gct gac cga gcc gca ttt gcc gga gat ccc gag ttc gtc aac gtt cct 1117 Asp Arg Ala Ala Phe Ala Gly Asp Pro Glu Phe Val Asn Val Pro 335 340 345 350 ctc aaa ggt ctc ttg aat cca gat tat atc aat gcc cgc aga cag ctg 1165 Leu Lys Gly Leu Leu Asn Pro Asp Tyr Ile Asn Ala Arg Gln Leu 355 360 365 ata gat att aat aaa gtg aat aaa aag ccg aaa gcc ggc gat cct tgg 1213 Ile Asp Ile Asn Lys Val Asn Lys Lys Pro Lys Ala Gly Asp Pro Trp 370 375 380 gcc tat cag gaa ggt tct gca aac aaa caa gtg gag cag ccg act 1261 Ala Tyr Gln Glu Gly Ser Ala Asn Tyr Lys Gln Val Glu Gln Pro Thr 385 390 395 gac aaa caa gaa ggc caa acg act cac ttc acg gta gcc gat cgc ttc 1309 Asp Lys Gln Glu ly Gln Thr Thr His Phe Thr Val Ala Asp Arg Phe 400 405 410 gga aat gtc gta tct tac acg aca acg att gaa cag ctg ttc ggt tcc 1357 Gly Asn Val Val Ser Tyr Thr Thr Thr Ile Glu Gln Leu Phe Gly Ser 415 420 425 430 ggc att atg gtt ccc gga tac ggc gtt gtt ttg aac aat gaa tta aca 1405 Gly Ile Met Val Pro Gly Tyr Gly Val Val Leu Asn Asn Glu Leu Thr 435 440 445 gac ttc gat gcg gtg ccg ggc gg gca cag ccg aat aaa 1453 Asp Phe Asp Ala Val Pro Gly Gly Ala Asn Glu Val Gln Pro Asn Lys 450 455 460 cgt ccg ctc agc agc atg acc ccg acc att tta ttc aaa aat aac gaa 1501 Arg Pro Leu Ser Ser Met Thr Pro Thr Ile Leu Phe Lys Asn Asn Glu 465 470 475 cct gtc ctg act gtc ggc tcc ccc ggc gga gca acg att atc tct tcc 1549 Pro Val Leu Thr Val Gly Ser Pro Gly Gly Ala Thr Ile Ile Ser Ser 480 485 490 490 gtt ctg caa acg atc ctg aac aaa gtc gaa tac ggc atg gat ctg aaa 1597 Val Leu Gln Thr Ile Leu Asn Lys Val Glu Tyr Gly Met Asp Leu Lys 495 500 505 510 gcg gcg gtc gaa gag ccg aga att tac aca aac agt atg 1645 Ala Ala Val Glu Glu Pro Arg Ile Tyr Thr Asn Ser Met Thr Ser Tyr 515 520 525 cga tat gaa gaa gga gtg ccg gaa gaa gcc cgc aca aaa ctg aac gaa 1693 Arg Tyr Glu Glu Gly Val Pro Glu Glu Ala Thr Leu Asn Glu 530 535 540 atg ggg cat aaa ttc ggc agc aag ccg gtt gat atc gga aac gtg caa 1741 Met Gly His Lys Phe Gly Ser Lys Pro Val Asp Ile Gly Asn Val Gln 545 550 555 agc ata cta atc gac cgt ggc acc ttc acc gga gtt gcc gac 1789 Ser Ile Leu Ile Asp Arg Glu Asn Gly Thr Phe Thr Gly Val Ala Asp 560 565 570 tca agt cga aac gga gcc gca atc ggc gta aac ctg aaa aaa tgt gaa 1837 Ser Ser Arg Asn Gly Ala Ala Ile Gly Val Asn Leu Lys Lys Cys Glu 575 580 585 585 590 aaa taacaagaca aaaagcctcg tttctcaagc tgagaaatga ggcttttgtt 1890 Lys tattctggcgc tgtattcaga tgaagttttt tcagatgtta acgat <br> <br> > 2 Met Lys Lys Lys Lys Phe Met Asn Leu Cys Phe Ile Val Leu Leu Ser 1 5 10 15 Ala Leu Leu Thr Ala Gly Ser Ile Pro Tyr His Ala Gln Ala Lys Lys 20 25 30 His Pro Phe Ser Tyr Asp Asp Tyr Lys Gln Val Asp Val Gly Lys Asp 35 40 45 Gly Met Val Ala Thr Ala His Pro Leu Ala Ser Gln Ile Gly Ala Asp 50 55 60 Val Leu Lys Lys Gly Gly Asn Ala Ile Asp Ala Ala Val Ala Ile Gln 65 70 75 80 Phe Ala Leu Asn Val Thr Glu Pro Met Met Ser Gly Ile Gly Gly Gly Gly 85 90 95 Gly Phe Met Met Val Tyr Asp Ala Lys Thr Lys Asp Thr Thr Ile Ile 100 105 110 Asp Ser Arg Glu Arg Ala Pro Ala Gly Ala Thr Pro Asp Met Phe Leu 115 120 125 Asp Glu Asn Gly Lys Ala Ile Pro Phe Ser Glu Arg Val Thr Lys Gly 130 135 140 Thr Ala Val Gly Val Pro Gly Thr Leu Lys Gly Leu Glu Lys Ala Leu 145 150 155 160 Asp Lys Trp Gly Thr Arg Ser Met Lys Gln Leu Ile Thr Pro Ser Ile 165 170 175 Ala Leu Ala Ser Lys Gly Phe Pro Ile Asp Ser Val Leu Ala Asp Ala 180 185 190 Ile Ser Asp Tyr Lys Asp Lys Leu Ser His Thr Ala Ala Lys Asp Val 195 200 205 Phe Leu Pro Asn Gly Glu Pro Leu Lys Glu Gly Asp Thr Leu Val Gln 210 215 220 Lys Asp Leu Ala Lys Thr Phe Thr Ala Ile Lys Tyr Lys Gly Thr Lys 225 230 235 240 Ala Ph e Tyr Asp Gly Ala Phe Thr Lys Lys Leu Ala Glu Thr Val Gln 245 250 255 Glu Phe Gly Gly Ser Met Thr Glu Gln Asp Ile Lys Asn Phe Asn Val 260 265 270 270 Thr Ile Asp Glu Pro Ile Trp Gly Asp Tyr Gln Gly Tyr His Ile Ala 275 280 285 Thr Ala Pro Pro Pro Ser Ser Gly Gly Val Phe Leu Leu Gln Met Leu 290 295 300 Asn Leu Leu Asp Asp Phe Lys Leu Ser Gln Tyr Asp Ile Arg Ser Trp 305 310 315 320 Gln Lys Tyr Gln Leu Leu Ala Glu Thr Met His Leu Ala Tyr Ala Asp 325 330 335 Arg Ala Ala Phe Ala Gly Asp Pro Glu Phe Val Asn Val Pro Leu Lys 340 345 350 Gly Leu Leu Asn Pro Asp Tyr Ile Asn Ala Arg Arg Gln Leu Ile Asp 355 360 365 Ile Asn Lys Val Asn Lys Lys Pro Lys Ala Gly Asp Pro Trp Ala Tyr 370 375 380 Gln Glu Gly Ser Ala Asn Tyr Lys Gln Val Glu Gln Pro Thr Asp Lys 385 390 395 400 Gln Glu Gly Gln Thr Thr His Phe Thr Val Ala Asp Arg Phe Gly Asn 405 410 415 Val Val Ser Tyr Thr Thr Thr Ile Glu Gln Leu Phe Gly Ser Gly Ile 420 425 430 Met Val Pro Gly Tyr Gly Val Val Leu Asn Asn Glu Leu Thr Asp Phe 435 440 445 Asp Ala V al Pro Gly Gly Ala Asn Glu Val Gln Pro Asn Lys Arg Pro 450 455 460 Leu Ser Ser Met Thr Pro Thr Ile Leu Phe Lys Asn Asn Glu Pro Val 465 470 475 480 Leu Thr Val Gly Ser Pro Gly Gly Ala Thr Ile Ile Ser Ser Val Leu 485 490 495 Gln Thr Ile Leu Asn Lys Val Glu Tyr Gly Met Asp Leu Lys Ala Ala 500 505 510 Val Glu Glu Pro Arg Ile Tyr Thr Asn Ser Met Thr Ser Tyr Arg Tyr 515 520 525 Glu Glu Gly Val Pro Glu Glu Ala Arg Thr Lys Leu Asn Glu Met Gly 530 535 540 His Lys Phe Gly Ser Lys Pro Val Asp Ile Gly Asn Val Gln Ser Ile 545 550 555 560 Leu Ile Asp Arg Glu Asn Gly Thr Phe Thr Gly Val Ala Asp Ser Ser 565 570 575 Arg Asn Gly Ala Ala Ile Gly Val Asn Leu Lys Lys Cys Glu Lys 580 585 590

[Brief description of the drawings]

FIG. 1A is a diagram showing the deduced amino acid sequence of the salt-tolerant glutaminase gene of the present invention.

FIG. 1B is a diagram showing the deduced amino acid sequence of the salt-tolerant glutaminase gene of the present invention. 1B shows a continuation of the amino acid sequence of FIG. 1A.

FIG. 1C is a diagram showing the deduced amino acid sequence of the salt-tolerant glutaminase gene of the present invention. 1B shows a continuation of the amino acid sequence of FIG. 1B.

FIG. 2A is a diagram showing a base sequence of a salt-tolerant glutaminase gene of the present invention.

FIG. 2B is a view showing the nucleotide sequence of the salt-tolerant glutaminase gene of the present invention. 2B shows a continuation of the nucleotide sequence of FIG. 2A.

FIG. 2C is a diagram showing the nucleotide sequence of the salt-tolerant glutaminase gene of the present invention. 2B shows a continuation of the nucleotide sequence shown in FIG. 2B.

FIG. 3 shows a construction diagram of plasmid pGT1.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat II (reference) C12N 9/78 C12N 5/00 A F term (reference) 4B024 AA03 AA05 BA11 CA03 CA04 DA05 EA04 GA11 HA01 4B050 CC03 DD02 LL02 LL05 4B065 AA19X AA19Y AB01 AC14 BA02 CA31 CA41

Claims (6)

[Claims] 1. A salt-tolerant glutaminase gene comprising a nucleotide encoding the amino acid sequence from position 405 to position 591 of SEQ ID NO: 2, wherein the polypeptide encoded by the nucleotide is a dimer polypeptide having a small subunit. And a salt-tolerant glutaminase gene. 2. The salt-tolerant glutaminase gene according to claim 1, comprising a nucleotide encoding the amino acid sequence of positions 35 to 591 of SEQ ID NO: 2. 3. A salt-tolerant glutaminase gene comprising a nucleotide encoding the amino acid sequence of positions 1 to 591 of SEQ ID NO: 2. An expression vector comprising the salt-tolerant glutaminase gene according to any one of claims 1 to 3. 5. An expression vector comprising the expression vector according to claim 4.
Recombinant host cells. 6. A method for producing a salt-tolerant glutaminase, comprising a step of culturing the recombinant host cell according to claim 5, and a step of collecting a polypeptide having glutaminase activity from the culture.