JP2002218986A - New glutaminase and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new glutaminase having a new property, to determine its amino acid sequence and gene sequence derived from the same, to prepare a transformant into which the gene was transduced, and to provide a method for producing the glutaminase by culturing the transformant. SOLUTION: A new glutaminase was obtained from a microorganism (Aspergillus oryzae strain KBN616 or A. sojae strain BA-104). A specific amino acid sequence encoding the enzyme or modified sequence and a gene sequence were determined to prepare a transformant into which these were transduced.

Description

【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION

【０００１】[0001]

【発明の属する技術分野】本発明は、新規なグルタミナ
ーゼ、該グルタミナーゼをコードする遺伝子系、及び該
グルタミナーゼの製造方法に関する。TECHNICAL FIELD The present invention relates to a novel glutaminase, a gene system encoding the glutaminase, and a method for producing the glutaminase.

【０００２】[0002]

【従来の技術】グルタミナーゼ(Glutaminase）〔EC3.
5. 1. 2〕はグルタミンからグルタミン酸を生成する酵
素であり、特に、麹菌が生成するグルタミナーゼは醤油
や味噌などの醸造中に旨味を高めるなどの重要な役割を
果たしている。（板倉辰六郎 編著、醤油の化学と技
術、ｐ．181 、日本醸造協会（1988））また、麹菌のグ
ルタミナーゼについては精製され、その性質が検討され
ている。（T. Yano, M. Ito,K. Tomita, H. Kumagai, a
nd T. Tochikura, J. Ferment. Technol., 66, 137-143
(1988)）しかし、分子生物学的手法を用いたアプロー
チは未だされていない。また、このグルタミナーゼは、
生産性が低い、等のため、工業的利用の面で限界があっ
た。2. Description of the Related Art Glutaminase [EC3.
5.1.2] is an enzyme that produces glutamic acid from glutamine. In particular, glutaminase produced by koji mold plays an important role such as enhancing the umami during brewing of soy sauce and miso. (Edited by Tatsurorou Itakura, Chemistry and Technology of Soy Sauce, p. 181, Japan Brewing Association (1988)) Also, glutaminase of Aspergillus is purified and its properties are studied. (T. Yano, M. Ito, K. Tomita, H. Kumagai, a
nd T. Tochikura, J. Ferment. Technol., 66, 137-143
(1988)) However, an approach using a molecular biological technique has not yet been performed. Also, this glutaminase is
Due to low productivity, etc., there was a limit in terms of industrial use.

【０００３】また、アスペルギルス・ソヤー（Aspergil
lus sojae)由来のグルタミナーゼ及びそれをコードする
遺伝子が特開2000-166547 公報に記載されている。しか
しながらそのアミノ酸配列は本発明のグルタミナーゼの
それと全く異る。Aspergillus soyer (Aspergil
Glutaminase derived from C. lus sojae) and a gene encoding the same are described in JP-A-2000-166547. However, its amino acid sequence is completely different from that of the glutaminase of the present invention.

【０００４】[0004]

【発明が解決しようとする課題】従って、本発明は、新
規なグルタミナーゼを提供しようとするものである。本
発明はさらに、上記のグルタミナーゼの製造方法を提供
し、またその手段として、該グルタミナーゼをコードす
る遺伝子系を提供するものである。Accordingly, an object of the present invention is to provide a novel glutaminase. The present invention further provides a method for producing the above-mentioned glutaminase, and as a means thereof, a gene system encoding the glutaminase.

【０００５】従って本発明は、（Ａ）次の性質： （１）作用：Ｌ−グルタミンに作用してＬ−グルタミン
酸を生成する； （２）至適pH：至適pHが約８．５である； （３）安定pH範囲：４℃にて２４時間インキュベートし
た場合、安定pH範囲はおよそ５〜１０である； （４）至適作用温度：pH８．０において測定した場合、
至適作用温度は約４５℃である； （５）温度安定性：pH８．０にて３０分間インキュベー
トした場合、４０℃以下の温度で安定である； （６）分子量：ゲル濾過により測定した場合、分子量は
約７１，０００である；を有するグルタミナーゼを提供
する。Accordingly, the present invention provides (A) the following properties: (1) action: acting on L-glutamine to form L-glutamic acid; (2) optimum pH: when the optimum pH is about 8.5. (3) Stable pH range: when incubated at 4 ° C. for 24 hours, the stable pH range is approximately 5 to 10; (4) Optimal operating temperature: when measured at pH 8.0,
(5) Temperature stability: stable at a temperature of 40 ° C. or less when incubated at pH 8.0 for 30 minutes; (6) Molecular weight: measured by gel filtration , The molecular weight of which is about 71,000;

【０００６】本発明はまた、（Ｂ）配列番号：１４に示
すアミノ酸配列において、アミノ酸１〜５８４のアミノ
酸配列を有するグルタミナーゼ；配列番号：１４に示す
アミノ酸配列中のアミノ酸番号１〜５８４のアミノ酸配
列において、１個又は複数個のアミノ酸の欠失、付加及
び／又は置換により修飾されたアミノ酸配列を有し、且
つグルタミナーゼ活性を有する修飾されたグルタミナー
ゼ；配列番号：５に示す塩基配列を有する核酸のエクソ
ン領域とストリンジエント条件下でハイブリダイズする
ことができる核酸によりコードされているグルタミナー
ゼ；並びに配列番号：１４に示すアミノ酸配列中のアミ
ノ酸番号３４〜７０３のアミノ酸配列に対して７０％以
上の相同性を有するアミノ酸配列を有するグルタミナー
ゼを提供する。The present invention also relates to (B) a glutaminase having the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 14; the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 14 A modified glutaminase having an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acids and having a glutaminase activity; a nucleic acid having a base sequence shown in SEQ ID NO: 5; Glutaminase encoded by a nucleic acid capable of hybridizing with an exon region under stringent conditions; and 70% or more homology to the amino acid sequence of amino acids 34 to 703 in the amino acid sequence shown in SEQ ID NO: 14 A glutaminase having an amino acid sequence having an amino acid sequence is provided.

【０００７】本発明はさらに、（Ｃ）配列番号：１６に
示すアミノ酸配列において、アミノ酸１〜５８４のアミ
ノ酸配列を有するグルタミナーゼ；配列番号：１６に示
すアミノ酸配列中のアミノ酸番号１〜５８４のアミノ酸
配列において、１個又は複数個のアミノ酸の欠失、付加
及び／又は置換により修飾されたアミノ酸配列を有し、
且つグルタミナーゼ活性を有する修飾されたグルタミナ
ーゼ；配列番号：７に示す塩基配列を有する核酸とスト
リンジエント条件下でハイブリダイズすることができる
核酸によりコードされているグルタミナーゼ；並びに配
列番号：１６に示すアミノ酸配列中のアミノ酸番号３４
〜７０３のアミノ酸配列に対して７０％以上の相同性を
有するアミノ酸配列を有するグルタミナーゼを提供す
る。The present invention further provides (C) a glutaminase having the amino acid sequence of amino acids 1 to 584 in the amino acid sequence of SEQ ID NO: 16; the amino acid sequence of amino acids 1 to 584 in the amino acid sequence of SEQ ID NO: 16 Has an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acids,
A modified glutaminase having a glutaminase activity; a glutaminase encoded by a nucleic acid capable of hybridizing under stringent conditions with a nucleic acid having a base sequence shown in SEQ ID NO: 7; and an amino acid shown in SEQ ID NO: 16 Amino acid number 34 in the sequence
A glutaminase having an amino acid sequence having 70% or more homology to the amino acid sequence of -703.

【０００８】本発明はまた、（Ｄ）配列番号：１７に示
すアミノ酸配列において、アミノ酸１〜５８４のアミノ
酸配列を有するグルタミナーゼ；配列番号：１７に示す
アミノ酸配列中のアミノ酸番号１〜５８４のアミノ酸配
列において、１個又は複数個のアミノ酸の欠失、付加及
び／又は置換により修飾されたアミノ酸配列を有し、且
つグルタミナーゼ活性を有する修飾されたグルタミナー
ゼ；配列番号：８に示す塩基配列を有する核酸のエクソ
ン領域とストリンジエント条件下でハイブリダイズする
ことができる核酸によりコードされているグルタミナー
ゼ；並びに配列番号：１７に示すアミノ酸配列中のアミ
ノ酸番号１〜５８４のアミノ酸配列に対して７０％以上
の相同性を有するアミノ酸配列を有するグルタミナーゼ
を提供する。The present invention also relates to (D) a glutaminase having the amino acid sequence of amino acids 1 to 584 in the amino acid sequence of SEQ ID NO: 17; the amino acid sequence of amino acids 1 to 584 in the amino acid sequence of SEQ ID NO: 17 A modified glutaminase having an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acids and having a glutaminase activity; a nucleic acid having a base sequence represented by SEQ ID NO: 8; Glutaminase encoded by a nucleic acid capable of hybridizing under stringent conditions with an exon region; and 70% or more homology to the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 17 A glutaminase having an amino acid sequence having an amino acid sequence is provided.

【０００９】本発明はさらに、（Ｅ）配列番号：１８に
示すアミノ酸配列において、アミノ酸１〜５８４のアミ
ノ酸配列を有するグルタミナーゼ；配列番号：１８に示
すアミノ酸配列中のアミノ酸番号１〜５８４のアミノ酸
配列において、１個又は複数個のアミノ酸の欠失、付加
及び／又は置換により修飾されたアミノ酸配列を有し、
且つグルタミナーゼ活性を有する修飾されたグルタミナ
ーゼ；配列番号：９に示す塩基配列を有する核酸のエク
ソン領域とストリンジエント条件下でハイブリダイズす
ることができる核酸によりコードされているグルタミナ
ーゼ；並びに配列番号：１８に示すアミノ酸配列中のア
ミノ酸番号１〜５８４のアミノ酸配列に対して７０％以
上の相同性を有するアミノ酸配列を有するグルタミナー
ゼを提供する。The present invention further provides (E) a glutaminase having the amino acid sequence of amino acids 1 to 584 in the amino acid sequence of SEQ ID NO: 18; the amino acid sequence of amino acids 1 to 584 in the amino acid sequence of SEQ ID NO: 18 Has an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acids,
And a modified glutaminase having a glutaminase activity; a glutaminase encoded by a nucleic acid capable of hybridizing under stringent conditions to an exon region of a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 9; and SEQ ID NO: 18 A glutaminase having an amino acid sequence having 70% or more homology to the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in (1).

【００１０】本発明はまた、前記（Ｂ）〜（Ｅ）に記載
のグルタミナーゼをコードするＤＮＡ、該ＤＮＡを含ん
で成るベクター、及び該ベクターにより形質転換された
宿主細胞を提供する。本発明はさらに、前記Ａに記載の
グルタミナーゼの製造方法において、アスペルギルス
（Ａｓｐｅｒｇｉｌｌｕｓ）属に属し、該グルタミナー
ゼを生産することができる微生物を培養し、培養物から
グルタミナーゼを採取することを特徴とする方法を提供
する。[0010] The present invention also provides a DNA encoding the glutaminase described in the above (B) to (E), a vector comprising the DNA, and a host cell transformed with the vector. The present invention further provides the method for producing glutaminase according to the above A, wherein a microorganism belonging to the genus Aspergillus and capable of producing the glutaminase is cultured, and the glutaminase is collected from the culture. I will provide a.

【００１１】前記微生物は、例えば、アスペルギルス・
ソヤー(Aspergillus sojae）又はアスペルギルス・オリ
ゼー（Aspergillus oryzae）である。本発明はまた、前
記（Ｂ）〜（Ｅ）に記載のグルタミナーゼの製造方法に
おいて、該グルタミナーゼをコードするＤＮＡを含んで
成る発現ベクターにより形質転換された宿主細胞を培養
し、該培養物から、該グルタミナーゼを採取することを
特徴とする方法を提供する。The microorganism is, for example, Aspergillus
Aspergillus sojae or Aspergillus oryzae. The present invention also provides the method for producing glutaminase according to any one of (B) to (E), wherein the host cell transformed with an expression vector comprising a DNA encoding the glutaminase is cultured, and There is provided a method comprising collecting the glutaminase.

【００１２】[0012]

【発明の実施の形態】（１）微生物 本発明の微生物、すなわち、本発明のグルタミナーゼの
発酵生産に利用する微生物、及び本発明のグルタミナー
ゼをコードする遺伝子の分離源としての微生物として
は、本発明のグルタミナーゼを生産することができる微
生物であればよいが、好ましくはアスペルギルス(Asper
gillus）属の微生物であり、さらに好ましくは、アスペ
ルギルス・ソヤー(Aspergillus sojae）又はアスペルギ
ルス・オリゼー(Aspergillus oryzae)である。BEST MODE FOR CARRYING OUT THE INVENTION (1) Microorganism The microorganism of the present invention, that is, the microorganism used for the fermentative production of glutaminase of the present invention, and the microorganism as a separation source of the gene encoding glutaminase of the present invention, are those of the present invention. Any microorganism can be used as long as it can produce glutaminase from Aspergillus (Aspergillus).
gillus), more preferably Aspergillus sojae or Aspergillus oryzae.

【００１３】これらの種の具体的な菌株としては、アス
ペルギルス・ソヤー(Aspergillus sojae) BA-104、アス
ペルギルス・オリゼー(Aspergillus oryzae) KBN616 を
挙げることができる。なお、アスペルギルス・ソヤー株
もしくは、アスペルギルス・オリゼー株は、醤油や味噌
の製造のために広く使用されまた市販されている麹菌を
単胞子分離等の常法に従って純化することにより容易に
得られる。また、アスペルギルス・オリゼー株は、清酒
用として広く使用されまた市販されている麹菌を単胞子
分離等の常法に従って純化することによっても容易に得
られる。[0013] Specific strains of these species include Aspergillus sojae BA-104 and Aspergillus oryzae KBN616. The Aspergillus soyer strain or the Aspergillus oryzae strain can be easily obtained by purifying a commercially available koji mold widely used for the production of soy sauce or miso according to a conventional method such as single spore separation. The Aspergillus oryzae strain can also be easily obtained by purifying a commercially available Aspergillus oryzae, which is widely used for sake, according to a conventional method such as isolation of single spores.

【００１４】（２）グルタミナーゼの精製 本発明のグルタミナーゼは、該グルタミナーゼを生産す
ることができる微生物、好ましくは上に記載した微生物
を常法に従って培養し、そして酵素の精製に用いられる
常法に従って培養物から回収（採取）することができ
る。培養は液体培地でも固体培地でもよく、例えばフス
マの固体培地において培養するのが好ましい。本発明の
グルタミナーゼの発酵生産方法の一例を、アスペルギル
ス・ソヤーＢＡ−１０４株を使用した場合について、実
施例１に具体的に記載する。(2) Purification of Glutaminase The glutaminase of the present invention is obtained by culturing a microorganism capable of producing the glutaminase, preferably the microorganisms described above, in accordance with a conventional method, and culturing in accordance with a conventional method used for purifying the enzyme. It can be collected (collected) from objects. The culture may be performed in a liquid medium or a solid medium. For example, the culture is preferably performed in a solid medium of bran. An example of the method of the present invention for producing glutaminase by fermentation is specifically described in Example 1 when Aspergillus soyer BA-104 strain is used.

【００１５】（３）グルタミナーゼの性質 （ａ）活性測定法及びユニットの定義 本発明のグルタミナーゼは、Ｌ−γ−グルタミニル−ｐ
−ニトロアニリドを基質として使用し、グルタミナーゼ
の作用により遊離するｐ−ニトロアニリンを比色定量す
ることにより活性測定することができる。(3) Properties of glutaminase (a) Activity measuring method and definition of unit The glutaminase of the present invention is L-γ-glutaminyl-p
Using -nitroanilide as a substrate, the activity can be measured by colorimetrically determining p-nitroaniline released by the action of glutaminase.

【００１６】具体的には、８００μｌの５０mM Tris-H
Cl緩衝液（pH８．０）と、該緩衝液中１mM Ｌ−γ−グ
ルタミル−ｐ−ニトロアニリド溶液２００μｌとを試験
管に入れ、これを３０℃にて３分間予熱した後、これに
２００μｌの被験酵素液を加えて６０分間反応せしめ、
４００μｌの１Ｍ酢酸を添加して反応を停止せしめ、４
０５nmの吸光度を測定する。盲検として、酵素液を加え
ずに同様の操作を行い、反応停止後に酵素液を加えて、
４０５nmの吸光度を測定する。上記の条件下で１分間に
１μmol のｐ−ニトロアニリンを生じさせる酵素量を１
ユニットと定義する。Specifically, 800 μl of 50 mM Tris-H
A Cl buffer (pH 8.0) and 200 μl of a 1 mM L-γ-glutamyl-p-nitroanilide solution in the buffer were placed in a test tube, which was preheated at 30 ° C. for 3 minutes, and 200 μl of this was added thereto. Add the test enzyme solution and react for 60 minutes.
The reaction was stopped by adding 400 μl of 1 M acetic acid.
Measure the absorbance at 05 nm. As a blind test, perform the same operation without adding the enzyme solution, add the enzyme solution after stopping the reaction,
The absorbance at 405 nm is measured. Under the above conditions, the amount of the enzyme that produces 1 μmol of p-nitroaniline per minute is 1
Defined as a unit.

【００１７】次に、本発明の酵素の１例として、実施例
１において得られた。アスペルギルス・ソヤーBA-104か
ら得られたグルタミナーゼの性質を次に示す。 （ｂ）至適pH 緩衝液９００μｌ（pH5.0 〜8.0 : 25mM Na₂HPO₄-KH₂PO
₄ ; pH8.0 〜 9.0 :25mM Tris-HCl)に酵素液１００μｌ
を加え、３０℃にて６０分間反応せしめ、最高活性を１
００として、各pHにおける相対活性を求める。結果を図
１に示す。この結果から明らかな通り、至適pHは約８．
５である。Next, an example of the enzyme of the present invention was obtained in Example 1. The properties of glutaminase obtained from Aspergillus soyer BA-104 are shown below. (B) 900 μl of optimal pH buffer (pH 5.0 to 8.0: 25 mM Na ₂ HPO ₄ -KH ₂ PO
₄ ; pH 8.0-9.0: 25 mM Tris-HCl)
And allowed to react at 30 ° C. for 60 minutes.
The relative activity at each pH is determined as 00. The results are shown in FIG. As is clear from this result, the optimum pH is about 8.
5

【００１８】（ｃ）pH安定性 ２５０μｌの緩衝液（pH3.0 〜5.0 : 25mM HCl-CH₃COON
a ; pH5.0 〜8.0 : 25mM Na₂HPO₄-KH₂PO₄ ; pH8.0 〜9.
0 : 25mM Tris-HCl;pH9.2 〜11.0 : 25mM Na₂B ₄P₇-NaO
H）に２５０μｌの酵素液を加え、４℃にて２４時間イ
ンキュベートし、50mM Tris-HCl （pH８．０）緩衝液中
で（ａ）に記載したようにして活性を測定し、最高活性
を１００として各インキュベーションpHにおける活性の
相対値を求める。結果を図２に示す。この結果から明ら
かな通り、上記の条件下でおよそpH５．０〜１０．０に
おいて安定である。(C) pH stability 250 μl of buffer solution (pH 3.0 to 5.0: 25 mM HCl-CH)_ThreeCOON
a; pH5.0 to 8.0: 25mM Na_TwoHPO_Four-KH_TwoPO_Four; pH 8.0 to 9.
0: 25 mM Tris-HCl; pH 9.2 to 11.0: 25 mM Na_TwoB _FourP₇-NaO
H), add 250 μl of the enzyme solution, and incubate at 4 ° C for 24 hours.
Incubate and in 50 mM Tris-HCl (pH 8.0) buffer
The activity was measured as described in (a) in
The activity at each incubation pH was taken as 100.
Find the relative value. FIG. 2 shows the results. It is clear from this result
As you can see, under the above conditions, the pH was adjusted to about 5.0 to 10.0.
Is stable.

【００１９】（ｄ）至適温度 ５０mM Tris-HCl（pH８．０）緩衝液中で２０℃〜５０
℃にて活性を測定し、最高値を１００として各温度にお
ける相対活性値を求める。結果を図３に示す。この結果
から明らかな通り、至適温度は約４５℃であり、４０℃
〜４５℃において高い活性を示す。(D) Optimum temperature: 20 ° C. to 50 ° C. in 50 mM Tris-HCl (pH 8.0) buffer.
The activity is measured at ° C, and the relative activity value at each temperature is determined with the maximum value as 100. The results are shown in FIG. As is apparent from this result, the optimum temperature is about 45 ° C,
Shows high activity at ~ 45 ° C.

【００２０】（ｅ）温度安定性 酵素を５０mM Tris-HCl（pH８．０）緩衝液中で２０℃
〜６０℃にて３０分間インキュベートした後、５０mM
Tris-HCl（pH８．０）緩衝液中３０℃にて活性を測定
し、３０分間のインキュベートなしのサンプルを１００
として各インキュベーション温度における相対活性値を
求める。結果を図４に示す。この結果から、本発明の酵
素は、上記の条件において、約４０℃以下において安定
である。(E) Temperature stability The enzyme was placed in a 50 mM Tris-HCl (pH 8.0) buffer at 20 ° C.
After incubating at 分 間 60 ° C. for 30 minutes, 50 mM
The activity was measured in Tris-HCl (pH 8.0) buffer at 30 ° C.
The relative activity value at each incubation temperature is determined. FIG. 4 shows the results. From these results, the enzyme of the present invention is stable at about 40 ° C. or lower under the above conditions.

【００２１】（ｆ）基質特異性 前記活性測定法（ａ）に記載した条件下で、反応液に基
質として各種試薬を添加して酵素活性を測定し、Ｌ−グ
ルタミンに対する活性を１００として相対活性を求めた
結果は次の表１に示す通りである。(F) Substrate specificity Under the conditions described in the activity measurement method (a), various reagents were added as substrates to the reaction solution, and the enzyme activity was measured. The relative activity was defined as the activity for L-glutamine as 100. Is as shown in Table 1 below.

【００２２】[0022]

【表１】 [Table 1]

【００２３】本酵素はＬ−グルタミン及びＬ−γ−グル
タミニル−ｐ−ニトロアニリドに対して高い反応性を示
し、Ｌ−テアニン及びグルタチオンに対してはＬ−グル
タミンに比べてそれぞれ約６４％及び６８％の反応性を
示すが、Ｌ−アスパラギン、Ｄ−アスパラギン、Glu-Gl
u,Glu-Asr 等に対しては実質的に反応性を示さない。The present enzyme shows high reactivity to L-glutamine and L-γ-glutaminyl-p-nitroanilide, and to L-theanine and glutathione about 64% and 68%, respectively, as compared to L-glutamine. % Reactivity, L-asparagine, D-asparagine, Glu-Gl
No substantial reactivity with u, Glu-Asr, etc.

【００２４】（ｈ）分子量 HPLCによるゲル濾過法（カラム：YMC-Pack Diol-200G６
mm×３００mm；移動相：５０mM Tris-HCl（pH７．０）
中０．１Ｍ NaCl；流速：０．５ml／分；温度：室温
（２３℃）；注入量：２０μｌ；標準蛋白質：ウシ血清
アルブミン（M.W.66,000）、オバルブミン（M.W.42,00
0）、大豆トリプシンインヒビター（M.W.20,000））に
おいて、約71,000の分子量を示す。(H) Molecular weight Gel filtration method by HPLC (column: YMC-Pack Diol-200G6
mm × 300 mm; mobile phase: 50 mM Tris-HCl (pH 7.0)
0.1 M NaCl in medium; flow rate: 0.5 ml / min; temperature: room temperature (23 ° C.); injection volume: 20 μl; standard proteins: bovine serum albumin (MW 66,000), ovalbumin (MW 42,00)
0), soybean trypsin inhibitor (MW 20,000)).

【００２５】上記の本発明の酵素（アスペルギルス・ソ
ヤーBA-104）とT.Yanoら、J. Ferment. Technol. Vol.
66, p.138 (1988)に記載されているアスペルギルス・オ
リゼーMA-27 由来のグルタミナーゼ（細胞内酵素及び細
胞外酵素）及び特開2000-166547 公報に記載されている
グルタミナーゼとを比較すれば次の表２に示す通りであ
る。The above enzyme of the present invention (Aspergillus soyer BA-104) and T. Yano et al., J. Ferment. Technol. Vol.
66, p. 138 (1988), the glutaminase derived from Aspergillus oryzae MA-27 (intracellular enzyme and extracellular enzyme) and the glutaminase described in JP-A-2000-166547 can be compared. As shown in Table 2.

【００２６】[0026]

【表２】 [Table 2]

【００２７】この表から明らかな通り、本発明のグルタ
ミナーゼは、公知のグルタミナーゼと比べて、分子量、
至適pH、pH安定性、至適温度、温度安定性等の点で明ら
かに異り、本発明の酵素は新規な酵素である。アスペル
ギルス・オリゼーMA-27 由来のグルタミナーゼのアミノ
酸配列及びそれをコードする塩基配列は不明であるが、
該グルタミナーゼの性質は本発明のアスペルギルス・ソ
ヤーBA-104由来のグルタミナーゼの性質と全く異り、特
に本発明の酵素は温度安定性及びpH安定性の点で優れて
いる。他方、特開2000-166547公報に記載されているア
スペルギルス・ソヤー及びBA-104由来のグルタミナーゼ
及びそれをコードする塩基配列は、本発明の酵素のそれ
と全く異り、化学物質として全く異るものである。As is clear from this table, the glutaminase of the present invention has a molecular weight,
The enzyme of the present invention is a novel enzyme, which is clearly different in terms of optimum pH, pH stability, optimum temperature, temperature stability and the like. The amino acid sequence of glutaminase derived from Aspergillus oryzae MA-27 and the nucleotide sequence encoding it are unknown,
The properties of the glutaminase are completely different from those of the glutaminase derived from Aspergillus soyer BA-104 of the present invention, and the enzyme of the present invention is particularly excellent in temperature stability and pH stability. On the other hand, the glutaminase derived from Aspergillus soyer and BA-104 and the nucleotide sequence encoding the same described in JP-A-2000-166547 are completely different from those of the enzyme of the present invention, and are completely different as chemical substances. is there.

【００２８】他方配列番号：１４，１６，１７及び１８
に示す本発明の酵素のアミノ酸配列は非常に高い相同性
を有する。従って、本発明は配列番号：１４，１６，１
７又は１８に示すアミノ酸配列を有するグルタミナーゼ
を提供する。On the other hand, SEQ ID NOs: 14, 16, 17, and 18
Have very high homology. Therefore, the present invention provides SEQ ID NOs: 14, 16, 1
A glutaminase having the amino acid sequence of 7 or 18 is provided.

【００２９】しかしながら、ある酵素がその酵素活性を
発揮するには、生来のアミノ酸配列のすべてがそのまま
必要なわけではなく、活性の発現に必須の領域以外の領
域においては、アミノ酸の欠失、付加、置換等によりア
ミノ酸配列が修飾されていても本来の酵素活性を発揮す
ることが知られている。従って本発明は、配列番号：１
４，１６，１７又は１８に記載のアミノ酸配列における
第１位のＭｅｔから第５８４位のＶａｌまでのアミノ酸
配列において、１又は複数個のアミノ酸の欠失、付加及
び／又は置換により修飾されているアミノ酸配列を有
し、且つグルタミナーゼ活性を維持している修飾型グル
タミナーゼを提供する。However, in order for a certain enzyme to exhibit its enzymatic activity, not all of the natural amino acid sequence is necessary as it is, and amino acids may be deleted or added in a region other than a region essential for the expression of the activity. It is known that even if the amino acid sequence is modified by substitution, etc., the original enzyme activity is exhibited. Therefore, the present invention provides SEQ ID NO: 1.
The amino acid sequence from Met at position 1 to Val at position 584 in the amino acid sequence of 4, 16, 17, or 18 is modified by deletion, addition and / or substitution of one or more amino acids. A modified glutaminase having an amino acid sequence and maintaining glutaminase activity is provided.

【００３０】上記の修飾の程度は、ＰＣＲ、部位特定変
異誘発等、周知技術により修飾できる程度であり、且つ
グルタミナーゼ活性を喪失しない範囲である。例えば、
配列番号：１４，１６，１７又は１８に記載の第１位の
Ｍｅｔから第５８４位のＶａｌまでのアミノ酸配列に対
して、７０％以上、８０％以上、８５％以上、９０％以
上、又は９５％以上の相同性を有するものである。従っ
て本発明はまた、配列番号：１４，１６，１７又は１８
に記載の第１位のＭｅｔから第５８４位のＶａｌまでの
アミノ酸配列に対して、７０％以上、８０％以上、８５
％以上、９０％以上、又は９５％以上の相同性を有する
アミノ酸配列を有し、且つグルタミナーゼ活性を維持し
ている修飾型グルタミナーゼを含む。The degree of the above modification is such that it can be modified by well-known techniques such as PCR and site-directed mutagenesis, and is within a range that does not cause loss of glutaminase activity. For example,
70% or more, 80% or more, 85% or more, 90% or more, or 95% of the amino acid sequence from Met at position 1 to Val at position 584 described in SEQ ID NO: 14, 16, 17, or 18 % Or more. Accordingly, the present invention also relates to SEQ ID NO: 14, 16, 17, or 18
70% or more, 80% or more, 85%
%, 90% or more, or 95% or more homologous amino acid sequences, and modified glutaminase that maintains glutaminase activity.

【００３１】一旦、酵素をコードする生来の生来のゲノ
ムＤＮＡ又はｃＤＮＡがクローニングされれば、該ゲノ
ムＤＮＡ特にそのエクソン部分もしくはｃＤＮＡ、又は
それらの一部分をプローブとして用いることにより、同
じ酵素活性を有する他の蛋白質をコードするＤＮＡを選
択することができる。従って、本発明は、配列番号：
５，７，８又は９に示す塩基配列を有する核酸、例えば
ＤＮＡ、特にエクソン部分の配列を有する核酸、例えば
ＤＮＡ、例えばゲノムＤＮＡ又はｃＤＮＡ、あるいはそ
れらの断片、例えば好ましくは１５塩基以上、さらに好
ましくは２０塩基以上、例えば３０塩基以上の長さの断
片とハイブリダイズすることができる核酸によりコード
されており、且つグルタミナーゼ活性を有する蛋白質を
も提供する。Once the native genomic DNA or cDNA encoding the enzyme has been cloned, the genomic DNA, particularly its exon or cDNA, or a portion thereof, can be used as a probe to provide another enzyme having the same enzymatic activity. Can be selected. Thus, the present invention provides
Nucleic acids having the base sequence shown in 5, 7, 8, or 9, for example, DNA, particularly nucleic acids having a sequence of exon part, for example, DNA, for example, genomic DNA or cDNA, or fragments thereof, for example, preferably 15 bases or more, more preferably Also provides a protein encoded by a nucleic acid capable of hybridizing with a fragment having a length of 20 bases or more, for example, 30 bases or more, and having a glutaminase activity.

【００３２】上記の場合のハイブリダイゼーション条件
は、例えば、野村慎太郎、稲澤譲治著「脱アイソトープ
実験プロトコール」ｐ．４０（秀潤社、１９９４）に記
載の条件の条件（５００mM ＮａＰｉ緩衝液（pH７．
２），７％ＳＤＳ，１mM ＥＤＴＡ）である。上記ハイ
ブリダイゼーションによるスクリーニングの対象となる
核酸は特に限定される。The hybridization conditions in the above case are described in, for example, Shintaro Nomura and Joji Inazawa, “Deisotope Experiment Protocol”, p. 40 (Shujunsha, 1994) (500 mM NaPi buffer (pH 7.0)).
2), 7% SDS, 1 mM EDTA). The nucleic acid to be screened by the above hybridization is not particularly limited.

【００３３】例えば合成ＤＮＡや、生来のＤＮＡを前記
のごとく修飾したものでもよいが、天然のＤＮＡ、例え
ば、ゲノムＤＮＡやｃＤＮＡのライブラリーが好まし
い。この様なＤＮＡライブラリーは、例えば真菌類、例
えばアスペルギルス属の微生物、例えばアスペルギルス
・オリゼー、アスペルギルス・ソヤー、アスペルギルス
・ニガー（Aspergillus niger ）等から、常法に従って
調製することができる。あるいは、そのようなライブラ
リーは、他の微生物、例えば細菌、動物細胞、植物細胞
等からも調製することができる。For example, synthetic DNA or natural DNA modified as described above may be used, but a library of natural DNA, for example, genomic DNA or cDNA is preferable. Such a DNA library can be prepared from a fungus, for example, a microorganism of the genus Aspergillus, for example, Aspergillus oryzae, Aspergillus soyer, Aspergillus niger, or the like according to a conventional method. Alternatively, such libraries can be prepared from other microorganisms, such as bacteria, animal cells, plant cells, and the like.

【００３４】本発明はまた、前記の生来のグルタミナー
ゼ、又はグルタミナーゼ活性を有する蛋白質をコードす
る核酸、特にＤＮＡに関する。典型的には、配列番号：
５，７，８又は９に示す塩基配列を有するゲノムＤＮＡ
又はそのエクソン部分のみから成るｃＤＮＡである。ゲ
ノムＤＮＡのクローニング方法の１例は、実施例３にお
いて具体的に記載する。The present invention also relates to a nucleic acid, particularly a DNA, encoding the above-mentioned native glutaminase or a protein having glutaminase activity. Typically, SEQ ID NO:
Genomic DNA having the base sequence shown in 5, 7, 8 or 9
Alternatively, it is a cDNA consisting only of the exon portion thereof. One example of a method for cloning genomic DNA is specifically described in Example 3.

【００３５】一旦、ゲノムＤＮＡがクローニングされれ
ば、該ゲノムＤＮＡ、又はその１部分、特にエクソン部
分のＤＮＡをプローブとして、ｃＤＮＡライブラリーを
スクリーニングすることにより本発明のグルタミナーゼ
をコードするｃＤＮＡを得ることができる。ｃＤＮＡラ
イブラリーの調製及びプローブハイブリダイゼーション
によるそのスクリーニングは常法に従って行うことがで
きる。ｃＤＮＡライブラリーは、例えば前記の出発材
料、例えばアスペルギルス属微生物、例えばアスペルギ
ルス・オリゼー、アスペルギルス・ソヤー、アスペルギ
ルス・ニガー、等から調製することができる。Once the genomic DNA has been cloned, cDNA encoding the glutaminase of the present invention can be obtained by screening a cDNA library using the genomic DNA, or a portion thereof, particularly the DNA of the exon portion, as a probe. Can be. Preparation of a cDNA library and its screening by probe hybridization can be performed according to a conventional method. A cDNA library can be prepared, for example, from the aforementioned starting materials, for example, a microorganism of the genus Aspergillus, for example, Aspergillus oryzae, Aspergillus soyer, Aspergillus niger, and the like.

【００３６】また、修飾されたグルタミナーゼをコード
するＤＮＡは、生来のゲノムＤＮＡ又はｃＤＮＡを基礎
とし、部位特定変異誘発、ＰＣＲ、ランダム変異、ジー
ンシャッフリング等の常法に従って行うことができる。
また、１又は複数個のアミノ酸が欠失した短縮型酵素を
コードするＤＮＡは、生来のＤＮＡに開始コドン及び／
又は終止コドンを導入することによっても得られる。さ
らに、適当な制限酵素により、生来のＤＮＡを切断する
ことによっても得られる。The DNA encoding the modified glutaminase can be obtained by using site-specific mutagenesis, PCR, random mutation, gene shuffling, etc., based on natural genomic DNA or cDNA.
In addition, DNA encoding a truncated enzyme in which one or more amino acids have been deleted has a start codon and / or
Alternatively, it can be obtained by introducing a stop codon. Further, it can be obtained by cutting native DNA with an appropriate restriction enzyme.

【００３７】本発明はまた、上記のＤＮＡを含んで成る
ベクター、特に発現ベクター、及び該ベクターにより形
質転換された宿主細胞に関する。グルタミナーゼを生産
するための宿主細胞としては、グルタミナーゼをコード
するＤＮＡがゲノムＤＮＡである場合にはスプライシン
グ活性を有する宿主細胞である必要があり、真核性細
胞、例えば真菌類、例えば酵母又は糸状真菌類、又は動
物細胞、さらには植物細胞が使用される。The present invention also relates to a vector comprising the above DNA, in particular, an expression vector, and a host cell transformed with the vector. The host cell for producing glutaminase must be a host cell having a splicing activity when the DNA encoding glutaminase is genomic DNA, and is a eukaryotic cell such as a fungus, such as a yeast or a filamentous fungus. Kinds, or animal cells, and even plant cells are used.

【００３８】酵母としては例えばサッカロミセス（Sacc
haromyces ）属酵母、例えばサッカロミセス・セービシ
エー（Saccharomyces cerevisiae）等が挙げられ、糸状
真菌類としては、例えばアスペルギルス属微生物、例え
ばアスペルギルス・オリゼー、アスペルギルス・ニガ
ー、等が挙げられる。動物細胞としては、昆虫細胞、例
えばカイコの細胞、哺乳類培養細胞、例えばＣＯＳ細胞
等が挙げられる。さらに植物細胞を用いることもでき
る。As yeast, for example, Saccharomyces (Sacc
yeasts such as Saccharomyces cerevisiae; and filamentous fungi include microorganisms of the genus Aspergillus, such as Aspergillus oryzae and Aspergillus niger. Animal cells include insect cells, such as silkworm cells, cultured mammalian cells, such as COS cells. Further, plant cells can also be used.

【００３９】さらに、グルタミナーゼをコードするＤＮ
ＡがｃＤＮＡである場合、上記の宿主の他に、原核性宿
主、例えば細菌宿主を用いることができる。細菌宿主と
しては、例えば大腸菌（Escherichia coli）、バシルス
（Bacillus）属細菌、例えばバシルス・ズブチリス(Bac
illus subtilis) 等の常用の宿主が用いられる。Furthermore, DN encoding glutaminase
When A is cDNA, a prokaryotic host such as a bacterial host can be used in addition to the above-mentioned hosts. Examples of the bacterial host include Escherichia coli and Bacillus bacteria such as Bacillus subtilis (Bac).
A common host such as illus subtilis) is used.

【００４０】発現ベクターは、宿主に応じて発現制御配
列、例えばプロモーター、ターミネーター等を含有する
必要がある。例えば、酵母用のプロモーターとしては解
糖系酵素、遺伝子のプロモーター、Ｇａｌプロモーター
等が挙げられ、糸状菌用プロモーターとしてはＡｍｙＡ
プロモーター等が挙げられ、動物細胞用プロモーターと
してはウイルスプロモーター等が用いられる。また、細
菌用プロモーターとしては、ウイルスベクター用プロモ
ーター等が用いられる。宿主としての細胞とそれに適合
するベクター系の使用はすでに常用技術となっており、
本発明においては、それらの既知の発現系を適宜選択し
て使用することができる。The expression vector needs to contain an expression control sequence, such as a promoter and a terminator, depending on the host. For example, yeast promoters include glycolytic enzymes, gene promoters, Gal promoters and the like, and filamentous fungal promoters include AmyA
Promoters and the like, and as a promoter for animal cells, viral promoters and the like are used. As the bacterial promoter, a viral vector promoter and the like are used. The use of cells as host cells and compatible vector systems has become a routine technique,
In the present invention, those known expression systems can be appropriately selected and used.

【００４１】本発明はまた、本発明のグルタミナーゼの
製造方法を提供する。第１の態様によれば、本発明のグ
ルタミナーゼを生産することができるアスペルギルス属
微生物を培養し、該培養物から、グルタミナーゼを採取
すればよい。このための生産菌としては、アスペルギル
ス・オリゼー、アスペルギルス・ソヤー等が挙げられ、
具体例としてアスペルギルス・オリゼーKBN616株及びア
スペルギルス・ソヤーBA-104株が挙げられる。The present invention also provides a method for producing the glutaminase of the present invention. According to the first aspect, a microorganism of the genus Aspergillus capable of producing the glutaminase of the present invention may be cultured, and glutaminase may be collected from the culture. Production bacteria for this purpose include Aspergillus oryzae, Aspergillus soyer, etc.
Specific examples include Aspergillus oryzae KBN616 strain and Aspergillus soyer BA-104 strain.

【００４２】グルタミナーゼの製造のための培養は、固
体培地及び液体培地のいずれでもよい。固体培地に培養
する場合には、例えばフスマ等の通気性のよい材料、好
ましくは植物材料に、必要に応じて、リン酸塩等の無機
物、又は米糖等の有機窒素源などを添加し、２０℃〜３
５℃、好ましくは約３０℃の温度で好気的に培養すれば
よい。また、液体培地に培養する場合には、アスペルギ
ルス属微生物が増殖することができる任意の液体培地を
使用することができる。The culture for producing glutaminase may be either a solid medium or a liquid medium. When culturing on a solid medium, for example, a material having good air permeability such as bran, preferably a plant material, if necessary, an inorganic substance such as phosphate, or an organic nitrogen source such as rice sugar is added, 20 ° C-3
Aerobic culture may be performed at a temperature of 5 ° C, preferably about 30 ° C. When culturing in a liquid medium, any liquid medium in which Aspergillus microorganisms can grow can be used.

【００４３】液体培地には、アスペルギルス属微生物が
資化し得る炭素源、例えば澱粉、マルトース、グルコー
ス等を０．１〜１０％、好ましくは１〜５％含有せし
め、またアスペルギルス属微生物が資化し得る窒素源、
例えば酵母エキス、マルトエキス（炭素源としても役立
つ）等を、例えば０．１〜５％の濃度で含有せしめるこ
とができる。さらに無機窒素源として、硝酸塩、アンモ
ニウム塩等を例えば０．０１％〜２％の濃度で含有せし
めることもできる。無機塩類として、リン酸塩、硫酸
塩、塩化物等の陰イオンと、ナトリウムイオン、カリウ
ムイオン、カルシウムイオン等の陽イオンとを添加する
こともできる。The liquid medium contains a carbon source that can be assimilated by Aspergillus microorganisms, for example, starch, maltose, glucose, etc. in an amount of 0.1 to 10%, preferably 1 to 5%, and the Aspergillus microorganism can assimilate. Nitrogen source,
For example, yeast extract, malt extract (also useful as a carbon source) and the like can be contained at a concentration of, for example, 0.1 to 5%. Further, as an inorganic nitrogen source, a nitrate, an ammonium salt or the like may be contained at a concentration of, for example, 0.01% to 2%. As inorganic salts, anions such as phosphates, sulfates and chlorides, and cations such as sodium, potassium and calcium ions can also be added.

【００４４】液体培養は、通気、撹拌及び／又は振とう
等の手段により好気的条件下で行う。培養温度は約２０
〜３５℃、好ましくは２８℃〜３０℃である。本発明の
第二の態様によれば、本発明のグルタミナーゼをコード
するＤＮＡを含んで成る発現プラスミドにより形質転換
された宿主を培養する。宿主としては詳細に前記したも
のを使用し、常法に従って培養、好ましくは液体培養す
ればよい。The liquid culture is carried out under aerobic conditions by means such as aeration, stirring and / or shaking. Culture temperature is about 20
To 35 ° C, preferably 28 ° C to 30 ° C. According to a second aspect of the present invention, a host transformed with an expression plasmid comprising a DNA encoding the glutaminase of the present invention is cultured. As the host, those described in detail above may be used, and the cells may be cultured according to a conventional method, preferably liquid culture.

【００４５】本発明の酵素は菌体外に分泌される。従っ
て、固体培養した場合には、培養物を水又は水性緩衝
液、例えばリン酸緩衝液等により抽出することにより酵
素含有水溶液が得られる。また、液体培養した場合に
は、濾過、遠心分離等の常法に従って菌体を除去するこ
とにより、酵素含有液が得られる。酵素含有液から酵素
を採取、精製するには、酵素の精製のための常法を用い
ればよい。例えば、塩析、ゲル濾過クロマトグラフィ
ー、イオン交換クロマトグラフィー、吸着クロマトグラ
フィー、高速液体クロマトグラフィー（HPLC）等を組合
せて使用することができる。The enzyme of the present invention is secreted extracellularly. Therefore, when the solid culture is performed, an enzyme-containing aqueous solution can be obtained by extracting the culture with water or an aqueous buffer such as a phosphate buffer. In the case of liquid culture, an enzyme-containing liquid can be obtained by removing cells according to a conventional method such as filtration or centrifugation. To collect and purify the enzyme from the enzyme-containing solution, a conventional method for purifying the enzyme may be used. For example, salting out, gel filtration chromatography, ion exchange chromatography, adsorption chromatography, high performance liquid chromatography (HPLC) and the like can be used in combination.

【００４６】[0046]

【実施例】次に、本発明を実施例によりさらに具体的に
説明する。実施例１．アスペルギルス・ソヤーBA-104からのグルタ
ミナーゼの製造 １０本の２Ｌ容三角フラスコに５００mlのＧＰ培地（N.
Kitamoto, J.Matsui,Y.Kawai, A.Kato, S.Yoshino, K.O
hmiya, and N.Tsukagoshi, Appl. Microbiol.Biotechno
l., 50, 85-92 (1998)）を入れ、殺菌後、アスペルギル
ス・ソヤー（Aspergillus sojae)BA-104を植菌し、３０
℃、９日間培養した。Next, the present invention will be described more specifically with reference to examples. Embodiment 1 FIG. Gluta from Aspergillus soyer BA-104
Production of Minase 500 ml of GP medium (N.
Kitamoto, J. Matsui, Y. Kawai, A. Kato, S. Yoshino, KO
hmiya, and N.Tsukagoshi, Appl.Microbiol.Biotechno
l., 50, 85-92 (1998)), and after sterilization, inoculated with Aspergillus sojae BA-104 and 30
C., and cultured for 9 days.

【００４７】その培養液をろ過し、４０％飽和になるよ
うに硫酸アンモニウムを加え、遠心分離した上清を４０
％飽和硫酸アンモニウムを加えた２０mM Tris-HCl緩衝
液（pH７．０）で平衡化したButyl Toyopearl 650M（50
φ×150mm 、東ソー製）のカラムに供与し、同緩衝液で
洗浄を行い、２０％飽和硫酸アンモニウムを加えた２０
mM Tris-HCl緩衝液（pH７．０）で溶出した。溶出した
活性画分を２０mM Tris-HCl緩衝液（pH７．０）に対
し、透析し、２０mM Tris-HCl緩衝液（pH７．０）で平
衡化したDEAE-Sepharose FF （３０φ×１３０mm、アマ
シャム ファルマシア製）のカラムに供与し、同緩衝液
で洗浄を行い、０．２Ｍ NaClを加えた同緩衝液で溶出
した。The culture was filtered, ammonium sulfate was added so as to be 40% saturated, and the supernatant obtained by centrifugation was added to 40% of the supernatant.
Butyl Toyopearl 650M (50%) equilibrated with 20 mM Tris-HCl buffer (pH 7.0) to which
φ150 mm, manufactured by Tosoh Corporation), washed with the same buffer, and added with 20% saturated ammonium sulfate.
Elution was carried out with mM Tris-HCl buffer (pH 7.0). The eluted active fraction was dialyzed against a 20 mM Tris-HCl buffer (pH 7.0) and equilibrated with a 20 mM Tris-HCl buffer (pH 7.0). DEAE-Sepharose FF (30 φ × 130 mm, manufactured by Amersham Pharmacia) ), Washed with the same buffer, and eluted with the same buffer to which 0.2 M NaCl was added.

【００４８】その溶出液の活性画分に４０％飽和になる
ように硫酸アンモニウムを加え、４０％飽和硫酸アンモ
ニウムを加えた１０mM酢酸ナトリウム−塩酸緩衝液（pH
５．０）で平衡化したPhenyl-5PW（８．０φ×７．５m
m、東ソー製）のＨＰＬＣカラムに供与し、４０％から
０％までの飽和硫酸アンモニウムのリニアグラジエント
で溶出した。その活性画分を２０mM Tris-HCl緩衝液
（pH７．０）で透析した後、同緩衝液で平衡化したPoro
s HQ/H（4.6 φ×50mm、ピーイーバイオシステムズ製）
のカラムに供与し、０Ｍから０．３ＭまでのNaClのリニ
アグラジエントで溶出した。Ammonium sulfate was added to the active fraction of the eluate so as to be 40% saturated, and a 10 mM sodium acetate-hydrochloric acid buffer (pH: 40%) to which 40% saturated ammonium sulfate was added.
Phenyl-5PW (8.0φ × 7.5m) equilibrated with 5.0)
m, manufactured by Tosoh Corporation) and eluted with a linear gradient of saturated ammonium sulfate from 40% to 0%. The active fraction was dialyzed against a 20 mM Tris-HCl buffer (pH 7.0) and then equilibrated with the same buffer.
s HQ / H (4.6 φ × 50mm, manufactured by PIE Biosystems)
And eluted with a linear gradient of 0M to 0.3M NaCl.

【００４９】その活性画分をウルトラセント−１０（東
ソー製）で濃縮後、０．２Ｍ ＮａＣｌを加えた２０mM
Tris-HCl緩衝液（pH７）で平衡化したYMC Dio1200G
（8.0φ×300mm 、YMC 製）のカラムに供与し、その活
性画分を得た。その活性画分を１０mM酢酸ナトリウム−
塩酸緩衝液（pH５．０）で透析後、Poros HQ/Hを用いた
ＨＰＬＣを用い、同緩衝液中で０Ｍから０．２Ｍまでの
NaClのリニアグラジエントで溶出した。The active fraction was concentrated with Ultracent-10 (manufactured by Tosoh Corporation), and then added with 20 mM of 0.2 M NaCl.
YMC Dio1200G equilibrated with Tris-HCl buffer (pH 7)
(8.0φ × 300 mm, manufactured by YMC) to give an active fraction. The active fraction was treated with 10 mM sodium acetate-
After dialysis against a hydrochloric acid buffer (pH 5.0), HPLC using Poros HQ / H was used to elute from 0M to 0.2M in the same buffer.
Elution was with a linear gradient of NaCl.

【００５０】その活性画分を１０mM酢酸ナトリウム−塩
酸緩衝液（pH４．２５）で透析後、Poros HQ/Hを用いた
ＨＰＬＣを使い、同緩衝液中で０Ｍから０．２Ｍまでの
NaClのリニアグラジエントで溶出した。この活性画分を
蒸留水に対して透析後、精製酵素とした。全精製工程で
の活性の回収率０．８９％、２８００倍に精製した。精
製過程を表３に示す。The active fraction was dialyzed against a 10 mM sodium acetate-hydrochloric acid buffer solution (pH 4.25), and was then subjected to HPLC using Poros HQ / H.
Elution was with a linear gradient of NaCl. This active fraction was dialyzed against distilled water and used as a purified enzyme. Purification was performed at a recovery rate of 0.89% of the activity in the entire purification step, 2800 times. Table 3 shows the purification process.

【００５１】[0051]

【表３】 この酵素は、前記の性質を有し、そして具体的には図１
〜図４に示す性質を有する。[Table 3] This enzyme has the properties described above and is specifically described in FIG.
4 to FIG.

【００５２】実施例２．アスペルギルス・オリゼーKBN6
16株からの、グルタミナーゼをコー ドするゲノムＤＮＡ
及びｃＤＮＡのクローニング （１）グルタミナーゼの部分アミノ酸配列の決定 実施例１で得た精製酵素の凍結乾燥品３０μｇを用い、
常法（マイクロシークエンスのための微量タンパク質精
製法、p.48-52 、羊土社、1992年）に準じて、酵素の断
片を得て、その配列を決定した。 Embodiment 2 FIG . Aspergillus oryzae KBN6
From 16 strains, glutaminase the co-sul genomic DNA
And cloning of cDNA (1) using the freeze-dried products 30μg of the purified enzyme obtained in determining the first embodiment of the partial amino acid sequence of the glutaminase,
Enzyme fragments were obtained and sequenced according to a conventional method (microprotein purification method for microsequencing, pp. 48-52, Yodosha, 1992).

【００５３】即ち、２５μｌの８Ｍ尿素・０．４Ｍ重炭
酸ナトリウム溶液を加え、２．５μｌの４５mMジチオス
レイトールを加え、５０℃、１５分間処理を行い、２．
５μｌの１００mMヨードアセトアミドを加え、１５分間
放置した。７０μｌの蒸留水を加え、３０μｌの１０μ
ｇ／mlエンドプロテイナーゼLys-C （ロッシュ・ダイア
グノスティックス製）を添加後、３７℃、２４時間イン
キュベートした。その断片化した酵素をＨＰＬＣにより
分離し、各酵素断片を取得した。その各断片をＮ末端配
列シークエンサー（島津製作所製PPSQ-21 ）を用いて解
析した。得られた配列は次のようになった。That is, 25 μl of 8 M urea / 0.4 M sodium bicarbonate solution was added, and 2.5 μl of 45 mM dithiothreitol was added, followed by treatment at 50 ° C. for 15 minutes.
5 μl of 100 mM iodoacetamide was added and left for 15 minutes. Add 70 μl of distilled water and add 30 μl of 10 μl
After adding g / ml endoproteinase Lys-C (manufactured by Roche Diagnostics), the mixture was incubated at 37 ° C for 24 hours. The fragmented enzyme was separated by HPLC to obtain each enzyme fragment. Each fragment was analyzed using an N-terminal sequence sequencer (PPSQ-21, manufactured by Shimadzu Corporation). The resulting sequence was as follows:

【００５４】断片（１）LeuGlyAlaValAlaSerGluSerAlaI
leCysSerArgHisGlyThrGluMetLeuLys（配列番号：１９） 断片（２）AspProThrTrpAlaIleAspPheAlaProAsnGlyThrA
rgLeuGlyLeuGlyAspThrIleThrArgLys（配列番号：２０） 断片（３）ValMetValProGluThrGlyIleIleMetAsnAsnGluM
etAspAspPhe（配列番号：２１） 断片（４）ArgProLeuSerSerIleThrProThrIleValThrHisG
lnAsnGlySerValPhePheValAlaGlySerAla （配列番号：２
２）Fragment (1) LeuGlyAlaValAlaSerGluSerAlaI
leCysSerArgHisGlyThrGluMetLeuLys (SEQ ID NO: 19) Fragment (2) AspProThrTrpAlaIleAspPheAlaProAsnGlyThrA
rgLeuGlyLeuGlyAspThrIleThrArgLys (SEQ ID NO: 20) Fragment (3) ValMetValProGluThrGlyIleIleMetAsnGluM
etAspAspPhe (SEQ ID NO: 21) Fragment (4) ArgProLeuSerSerIleThrProThrIleValThrHisG
lnAsnGlySerValPhePheValAlaGlySerAla (SEQ ID NO: 2
2)

【００５５】（２）ＤＮＡのクローニング全ＲＮＡの調
製 アスペルギルス・オリゼー（Aspergillus oryzae) ＫＢ
Ｎ６１６株をツァペック培地で３０℃、３日間振とう培
養後の菌糸からライフテックオリエンタル製TRIZOL Rea
gentを用い、そのプロトコールに従って抽出し、全ＲＮ
Ａを得た。(2) Cloning of DNA Preparation of total RNA
Made by Aspergillus oryzae KB
The strain N616 was cultured in a Tzapek medium at 30 ° C. for 3 days with shaking for 3 days.
gent, and extract according to the protocol.
A was obtained.

【００５６】グルタミナーゼ遺伝子ｃＤＮＡ断片の増幅 プライマーの設計 断片（３）からＰ１プライマー（配列番号：２３）を設
計した。Design of Amplification Primer for Glutaminase Gene cDNA Fragment A P1 primer (SEQ ID NO: 23) was designed from the fragment (3).

【化１】 Embedded image

【００５７】断片（４）のPro Thr Ile Val Thr His Gl
n の配列からアンチセンスでＰ２プライマー（配列番
号：２４）を設計した。 Ｐ２ TG(A/G)TG(A/G)GT(A/G)AC(A/G)AT(A/G)GT(A/G)GG 断片（３）からＰ３プライマー（配列番号：２５）を設
計した。Fragment (4) of Pro Thr Ile Val Thr His Gl
A P2 primer (SEQ ID NO: 24) was designed from the n sequence by antisense. P2 TG (A / G) TG (A / G) GT (A / G) AT (A / G) GT (A / G) GG Fragment (3) to P3 primer (SEQ ID NO: 25) ) Designed.

【化２】 Embedded image

【００５８】ｃＤＮＡ部分配列の取得 約１μｇの全ＲＮＡから3'-Full RACE Core Set （宝酒
造製）用いて、プロトコールに準じた条件でｃＤＮＡの
合成を行った。そのｃＤＮＡを鋳型ＤＮＡとしてプライ
マーに3site Adaptor Primer（宝酒造製）（配列番号：
２６）及びＰ１プライマーを用い、ＰＣＲ反応を３０サ
イクル行った。次に、１００倍希釈したＰＣＲ反応液を
鋳型ＤＮＡとしてプライマーにＰ１プライマー及びＰ２
プライマーをそれぞれ使用して、ＰＣＲ反応を３０サイ
クル行った。更に、１００倍希釈したＰＣＲ反応液を鋳
型ＤＮＡとしてプライマーにＰ２プライマー及びＰ３プ
ライマーをそれぞれ使用して、ＰＣＲ反応をさらに３０
サイクル行った。その結果、１３４bpのＤＮＡ断片が特
異的に増幅された。 Acquisition of cDNA partial sequence cDNA was synthesized from about 1 μg of total RNA using 3′-Full RACE Core Set (Takara Shuzo) under conditions according to the protocol. Using the cDNA as a template DNA, a 3 site Adapter Primer (manufactured by Takara Shuzo) as a primer (SEQ ID NO:
26) and the P1 primer was used to perform a PCR reaction for 30 cycles. Next, the P1 primer and P2 were used as primers with the PCR reaction solution diluted 100-fold as template DNA.
A PCR reaction was performed for 30 cycles using each of the primers. Further, using the PCR reaction solution diluted 100-fold as a template DNA and P2 and P3 primers as primers, the PCR
Cycled. As a result, a 134 bp DNA fragment was specifically amplified.

【００５９】１３４bpのＤＮＡ断片をｐＵＣ１８にサブ
クローニングし、大腸菌ＤＨ５α株を形質転換した。そ
して、サブクローニングされたＤＮＡ断片の塩基配列を
Ｔｈｅｒｍｏ Ｓｅｑｕｎａｓｅ蛍光シークエンサー用
サイクルシーケンシングキット（アマシャム ファルマ
シア バイオテク製）及びＤＮＡシーケンサーModel400
0LS（LI-COR製）を使用して決定した。この配列を配列
番号：１に示す。なお、この配列は配列番号：７に示す
塩基配列の第1288〜第1420塩基配列に相当する。配列番
号：１に示す塩基配列によりコードされるアミノ酸配列
を配列番号：１０に示す。なお、以下の配列決定は上記
の同様の方法によった。The 134 bp DNA fragment was subcloned into pUC18 to transform Escherichia coli DH5α strain. Then, the nucleotide sequence of the subcloned DNA fragment was analyzed by using a cycle sequencing kit for thermosequenase fluorescence sequencer (manufactured by Amersham Pharmacia Biotech) and a DNA sequencer Model400.
It was determined using 0LS (manufactured by LI-COR). This sequence is shown in SEQ ID NO: 1. This sequence corresponds to nucleotides 1288 to 1420 of the nucleotide sequence shown in SEQ ID NO: 7. The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 10. The following sequencing was performed by the same method as described above.

【００６０】配列番号：１からＰ４プライマー（配列番
号：２７）を設計した。 Ｐ４ CCAACGCATTTGGATACGTCCC 再度、ｃＤＮＡを用いて、プライマーに3site Adaptor
Primer（配列番号：２６）及びＰ４プライマーをそれぞ
れ使用して、ＰＣＲ反応を４０サイクル行った。その結
果、ポリＡ配列を除いて６００bpのＤＮＡ断片が特異的
に増幅された。増幅されたＤＮＡ断片を同様にpUC18 に
サブクローニングした後、その塩基配列を決定した。こ
の配列を配列番号：２に示す。これは、配列番号：７の
第1328〜第1927の塩基配列に相当する。なお、配列番
号：２の塩基配列によりコードされるアミノ酸配列を配
列番号：１１に示す。A primer from SEQ ID NO: 1 to P4 (SEQ ID NO: 27) was designed. P4 CCAACGCATTTGGATACGTCCC Again, using cDNA, 3 site Adapter
The PCR reaction was performed for 40 cycles using the Primer (SEQ ID NO: 26) and the P4 primer, respectively. As a result, a DNA fragment of 600 bp was specifically amplified except for the poly-A sequence. After the amplified DNA fragment was similarly subcloned into pUC18, its nucleotide sequence was determined. This sequence is shown in SEQ ID NO: 2. This corresponds to the nucleotide sequence from 1328 to 1927 of SEQ ID NO: 7. The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 11.

【００６１】グルタミナーゼ遺伝子の塩基配列決定 配列番号：２からＰ５プライマー（配列番号：２８）を
設計した。 Ｐ５ TGATCGTGAAGACGTGGTCTAG 配列番号：２からＰ６プライマー（配列番号：２９）を
設計した。 Ｐ６ TCTGATGCGTCACAATGGTTGG Determination of the base sequence of glutaminase gene A primer P5 (SEQ ID NO: 28) was designed from SEQ ID NO: 2. P5 TGATCGTGAAGACGTGGTCTAG A P6 primer (SEQ ID NO: 29) was designed from SEQ ID NO: 2. P6 TCTGATGCGTCACAATGGTTGG

【００６２】５’上流域のＤＮＡ断片の増幅は、次のよ
うにして行った。Ｒａｅｄｅｒらの方法（U. Raeder an
d P. Broda, Lett. Appl. Microb., 1, 17-20(1985))に
したがって調製したA.oryzae KBN616 株の染色体ＤＮＡ
を制限酵素Bgl IIで消化した後、宝酒造製LA-PCR in vi
tro Cloning Kit を用いて、cassette-ligation-mediat
ed PCRを行った。即ち、制限酵素処理断片にＳａｕ３Ａ
Ｉカセット（宝酒造製）を結合させ、このＤＮＡを鋳型
ＤＮＡとしてＣ１プライマー（宝酒造製）（配列番号：
３０）及びＰ５プライマーをそれぞれ使用して、ＰＣＲ
反応を３０サイクル行った。The amplification of the DNA fragment in the 5 ′ upstream region was performed as follows. Raeder et al. (U. Raeder an
d Chromosomal DNA of A.oryzae KBN616 strain prepared according to P. Broda, Lett. Appl. Microb., 1, 17-20 (1985))
Was digested with the restriction enzyme Bgl II, and then Takara Shuzo LA-PCR in vi
Using tro Cloning Kit, cassette-ligation-mediat
ed PCR was performed. That is, Sau3A was added to the fragment treated with the restriction enzyme.
I cassette (Takara Shuzo) was bound, and this DNA was used as a template DNA for C1 primer (Takara Shuzo) (SEQ ID NO:
30) and P5 primers, respectively.
The reaction was performed for 30 cycles.

【００６３】１００倍希釈した反応液を鋳型ＤＮＡとし
てＣ２プライマー（宝酒造製）（配列番号：３１）及び
Ｐ６プライマーを使用して、ＰＣＲ反応をさらに３０サ
イクル行った。その結果、2233bpのＤＮＡ断片が特異的
に増幅された。増幅されたＤＮＡ断片を同様に、pUC18
にサブクローニングした後、その塩基配列を決定した。
その塩基配列を配列番号：３に示す。この配列は配列番
号：５の第１〜第２２３３の塩基配列に相当する。な
お、配列番号：３に示す塩基配列によりコードされるア
ミノ酸配列を配列番号：１２に示す。Using the reaction solution diluted 100-fold as a template DNA, C2 primer (manufactured by Takara Shuzo) (SEQ ID NO: 31) and P6 primer, a PCR reaction was further carried out for 30 cycles. As a result, a 2233 bp DNA fragment was specifically amplified. The amplified DNA fragment was similarly treated with pUC18
After subcloning into the above, its nucleotide sequence was determined.
The nucleotide sequence is shown in SEQ ID NO: 3. This sequence corresponds to the 1st to 2233rd base sequences of SEQ ID NO: 5. The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 3 is shown in SEQ ID NO: 12.

【００６４】３’下流域のＤＮＡ断片の増幅は、次のよ
うにして行った。Raederらの方法にしたがって調製した
A.oryzae KBN616 株の染色体ＤＮＡを制限酵素Hind III
で消化した後、宝酒造製LA-PCR in vitro Cloning Kit
を用いて、cassette-ligation-mediated PCRを行った。
即ち、制限酵素処理断片にHind IIIカセット（宝酒造
製）を結合させた。このＤＮＡを鋳型ＤＮＡとしてＣ１
プライマー及びＰ４プライマーを使用して、ＰＣＲ反応
を３０サイクル行った。Amplification of the DNA fragment in the 3 'downstream region was performed as follows. Prepared according to Raeder et al.
A.oryzae KBN616 strain chromosomal DNA was digested with restriction enzyme Hind III
After digestion with Takara Shuzo LA-PCR in vitro Cloning Kit
Was used to perform cassette-ligation-mediated PCR.
That is, a Hind III cassette (Takara Shuzo) was ligated to the restriction enzyme-treated fragment. Using this DNA as a template DNA, C1
The PCR reaction was performed for 30 cycles using the primers and the P4 primer.

【００６５】１００倍希釈した反応液を鋳型ＤＮＡとし
てＣ２プライマー及びＰ４プライマーを使用して、ＰＣ
Ｒ反応をさらに３０サイクル行った。その結果、６５７
bpのＤＮＡ断片が特異的に増幅された。増幅されたＤＮ
Ａ断片を同様に、ｐＵＣ１８にサブクローニングした
後、その塩基配列を決定した。この配列を配列番号：４
に示す。この配列は配列番号：５の第２１３位〜第２７
９４位の塩基配列に相当する。なお配列番号：４に示す
塩基配列によりコードされるアミノ酸配列を配列番号：
１３に示す。The reaction solution diluted 100-fold was used as a template DNA and the C2 primer and the P4 primer were used.
The R reaction was performed for another 30 cycles. As a result, 657
A DNA fragment of bp was specifically amplified. Amplified DN
Similarly, after subcloning the A fragment into pUC18, its nucleotide sequence was determined. This sequence is represented by SEQ ID NO: 4.
Shown in This sequence corresponds to positions 213 to 27 of SEQ ID NO: 5.
This corresponds to the nucleotide sequence at position 94. The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4 is the same as SEQ ID NO: 4.
FIG.

【００６６】前記配列番号：３の配列と配列番号：４の
配列をつなぎ合わせて、グルタミナーゼをコードするゲ
ノムＤＮＡの塩基配列を決定した。この配列を配列番
号：５に示す。また、この塩基配列によりコードされる
アミノ酸配列を配列番号：１４に示す。配列番号：３か
らＰ７プライマー（配列番号：３３）を設計した。 Ｐ７ CCATGTATCGTTTTTTAAGGAATACGThe nucleotide sequence of genomic DNA encoding glutaminase was determined by joining the sequence of SEQ ID NO: 3 and the sequence of SEQ ID NO: 4. This sequence is shown in SEQ ID NO: 5. The amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 14. From SEQ ID NO: 3 to P7 primer (SEQ ID NO: 33) were designed. P7 CCATGTATCGTTTTTTAAGGAATACG

【００６７】ｃＤＮＡを鋳型ＤＮＡとして、プライマー
としてＰ６プライマー及びＰ７プライマーを使用して、
ＰＣＲ反応を３０サイクル行った。その結果、1423bpの
ＤＮＡ断片が特異的に増幅された。増幅されたＤＮＡ断
片を同様に、pUC18 にクローニングした後、その塩基配
列を決定した。この塩基配列を配列番号：６に示す。こ
の配列は、配列番号：７の第１位〜第１４２３位の塩基
配列に相当する。なお、この配列番号：６に示す塩基配
列によりコードされるアミノ酸配列を配列番号：１５に
示す。Using cDNA as a template DNA and P6 and P7 primers as primers,
The PCR reaction was performed for 30 cycles. As a result, a 1423 bp DNA fragment was specifically amplified. Similarly, the amplified DNA fragment was cloned into pUC18, and its nucleotide sequence was determined. This nucleotide sequence is shown in SEQ ID NO: 6. This sequence corresponds to the nucleotide sequence at positions 1 to 1423 of SEQ ID NO: 7. The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 15.

【００６８】配列番号：２の配列と配列番号：６の配列
をつなぎ合わせて、グルタミナーゼをコードするｃＤＮ
Ａを塩基配列を決定した。この塩基配列を配列番号：７
に示す。この配列番号：７に示す塩基配列によりコード
されるアミノ酸配列を配列番号：１６に示す。The sequence of SEQ ID NO: 2 and the sequence of SEQ ID NO: 6 are joined to form cDN encoding glutaminase.
A was sequenced. This nucleotide sequence is represented by SEQ ID NO: 7
Shown in The amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 7 is shown in SEQ ID NO: 16.

【００６９】実施例３．アスペルギルス・ソヤーBA-104
のグルタミナーゼ遺伝子のクローニ ング Ｐ７プライマーを１塩基変更し、Ｐ８（配列番号：３
３）を設計した。 Ｐ８ CCATGCATCGTTTTTTAAGGAATACG 配列番号：５からＰ９プライマー（配列番号：３４）を
設計した。 Ｐ９ CCTCTAGAGCATATCCACAAGATTTCTCTT Embodiment 3 FIG . Aspergillus soyer BA-104
The cloning P7 primers glutaminase gene 1 and base changes, P8 (SEQ ID NO: 3
3) was designed. P8 CCATGCATCGTTTTTTAAGGAATACG SEQ ID NO: 5 to P9 primer (SEQ ID NO: 34) were designed. P9 CCTCTAGAGCATATCCACAAGATTTCTCTT

【００７０】実施例１で使用したアスペルギルス・ソヤ
ー（A.sojae ）BA-104株からＲａｅｄｅｒらの方法（U.
Raeder and P. Broda, Lett. Appl. Microb., 1, 17-2
0 (1985)) にしたがって染色体ＤＮＡを単離精製し、そ
のＤＮＡを鋳型ＤＮＡとしてＰ８プライマー及びＰ９プ
ライマーを用いてＰＣＲ反応を３０サイクル行った。そ
の結果、約２．３kbp のＤＮＡ断片が得られた。From the Aspergillus sojae strain BA-104 used in Example 1, the method of Raeder et al.
Raeder and P. Broda, Lett.Appl. Microb., 1, 17-2
Chromosomal DNA was isolated and purified according to the method described in (0 (1985)), and the PCR reaction was carried out for 30 cycles using the DNA as a template DNA and P8 and P9 primers. As a result, a DNA fragment of about 2.3 kbp was obtained.

【００７１】得られたＤＮＡ断片をT4 DNAポリメラーゼ
で処理した後、制限酵素Xbalで消化し、その断片を制限
酵素EcoT22I で消化後、T4 DNAポリメラーゼで処理し、
その後、制限酵素Xbalで消化したベクターｐＴＦ１００
（N. Kitamoto, J. Matsui,Y. Kawai, Y. Kato, S. Yos
hino, K. Ohmiya, and N. Tsukagoshi, Appl. Microbio
l. Biotechnol., 50, 85-92 (1998))に組み込みグルタ
ミナーゼ遺伝子を含むベクターを作製した。そのベクタ
ーを大腸菌DH５αへ形質転換後、得られたベクター中に
は２種類のグルタミナーゼ遺伝子が存在していること
が、制限酵素HindIIIによる消化により判明した。その
２種類のベクター（pGT200及びpGT300）からグルタミナ
ーゼ遺伝子の配列を解析し、各グルタミナーゼの塩基配
列を決定した。After the obtained DNA fragment was treated with T4 DNA polymerase, it was digested with a restriction enzyme Xbal. The fragment was digested with a restriction enzyme EcoT22I, and then treated with T4 DNA polymerase.
Thereafter, the vector pTF100 digested with the restriction enzyme Xbal was used.
(N. Kitamoto, J. Matsui, Y. Kawai, Y. Kato, S. Yos
hino, K. Ohmiya, and N. Tsukagoshi, Appl. Microbio
l. Biotechnol., 50, 85-92 (1998)) to prepare a vector containing the integrated glutaminase gene. After transformation of the vector into Escherichia coli DH5α, digestion with the restriction enzyme HindIII revealed that two types of glutaminase genes were present in the resulting vector. The sequence of the glutaminase gene was analyzed from the two types of vectors (pGT200 and pGT300), and the nucleotide sequence of each glutaminase was determined.

【００７２】その結果、配列番号：８及び配列番号：９
に示す塩基配列が得られた。これらの塩基配列によりコ
ードされるアミノ酸配列をそれぞれ配列番号：１７及び
配列番号：１８に示す。両アミノ酸配列（配列番号：１
７、１８）には、実施例１で精製した酵素の部分アミノ
酸配列（配列番号：１９、２０、２１、２２）が完全に
含まれており、その酵素の遺伝子であることが確認でき
た。As a result, SEQ ID NO: 8 and SEQ ID NO: 9
Was obtained. The amino acid sequences encoded by these nucleotide sequences are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively. Both amino acid sequences (SEQ ID NO: 1
7, 18) completely contained the partial amino acid sequence (SEQ ID NO: 19, 20, 21, 22) of the enzyme purified in Example 1, and it was confirmed that the gene was a gene of the enzyme.

【００７３】実施例４．グルタミナーゼ遺伝子の麹菌で
の高発現 Raederらの方法にしたがって調製したアスペルギルス・
オリゼー（A.oryzae）KBN616株の染色体ＤＮＡを鋳型Ｄ
ＮＡとしてＰ８プライマー及びＰ９プライマーを使用し
て、ＰＣＲ反応を３０サイクル行った。増幅されたＤＮ
Ａ断片を制限酵素EcoT22I 及びXbalで消化した断片と、
制限酵素Sall及びEcoT22I で消化したpTF100（N. Kitam
oto, J. Matsui, Y. Kawai, Y. Kato, S. Yoshino, K.
Ohmiya,and N. Tsukagoshi, Appl. Microbiol. Biotech
nol., 50, 85-92 (1998))の断片を、制限酵素Sall及びX
balで消化したpND300（N. Kitamoto, T. Kimura, Y. Ki
to, K. Ohmiya, and N. Tsukagoshi, Biosci. Biotech.
Biochem., 59, 1795-1797 (1995)) に組み込みグルタ
ミナーゼ遺伝子高発現ベクターpTFGT100を作製した。 Embodiment 4 FIG . Aspergillus with glutaminase gene
Of Aspergillus prepared according to the method of high expression Raeder et al
Chromosomal DNA of A. oryzae KBN616 strain was used as template D
The PCR reaction was performed for 30 cycles using the P8 and P9 primers as NA. Amplified DN
A fragment obtained by digesting fragment A with restriction enzymes EcoT22I and Xbal,
PTF100 (N. Kitam) digested with restriction enzymes Sall and EcoT22I
oto, J. Matsui, Y. Kawai, Y. Kato, S. Yoshino, K.
Ohmiya, and N. Tsukagoshi, Appl. Microbiol. Biotech
nol., 50, 85-92 (1998)) with the restriction enzymes Sall and X.
pND300 digested with bal (N. Kitamoto, T. Kimura, Y. Ki
to, K. Ohmiya, and N. Tsukagoshi, Biosci. Biotech.
Biochem., 59, 1795-1797 (1995)) to prepare a glutaminase gene high expression vector pTFGT100.

【００７４】pTFGT100でアスペルギルス・オリゼー（A.
oryzae）KBN616-39 niaD- 株（N. Kitamoto, T. Kimur
a, Y. Kito, K. Ohmiya, and N. Tsukagoshi, Biosci.
Biotech. Biochem., 59, 1795-1797 (1995)) を形質転
換したところ、グルタミナーゼ高生産アスペルギルス・
オリゼーGT7 株を得ることができた。Aspergillus oryzae (A.
oryzae) KBN616-39 niaD- strain (N. Kitamoto, T. Kimur
a, Y. Kito, K. Ohmiya, and N. Tsukagoshi, Biosci.
Biotech. Biochem., 59, 1795-1797 (1995)) was transformed into Aspergillus
Oryzae GT7 strain was obtained.

【００７５】また、同様にアスペルギルス・ソヤー由来
のグルタミナーゼ遺伝子を含む上記のpGT200及びpGT300
をそれぞれ制限酵素Sall及びXbalで消化した断片を制限
酵素Sall及びXbalで消化したpND300に組み込みグルタミ
ナーゼ遺伝子高発現ベクターpTFGT200及びpTFGT300を作
製した。アスペルギルス・オリゼーKBN616-39 niaD-株
に形質転換したところ、アスペルギルス・ソヤー由来グ
ルタミナーゼ高生産アスペルギルス・オリゼーGT26株、
及びアスペルギルス・オリゼーGT33株を得ることができ
た。Similarly, the above pGT200 and pGT300 containing the glutaminase gene derived from Aspergillus soybean
Was digested with restriction enzymes Sall and Xbal, respectively, and incorporated into pND300 digested with restriction enzymes Sall and Xbal to prepare glutaminase gene high expression vectors pTFGT200 and pTFGT300. When transformed into Aspergillus oryzae KBN616-39 niaD- strain, Aspergillus oryzae high-producing Aspergillus oryzae strain GT26,
And Aspergillus oryzae GT33 strain were obtained.

【００７６】A.oryzae GT7株を用いてＧＰ培地（N. Kit
amoto, J. Matsui, Y. Kawai, Y. Kato, s. Yoshino,
K. Ohmiya, and N. Tsukagoshi, Appl. Microbiol. Bio
technol., 50, 85-92 (1998))１００mlを用い、３０
℃、１０日間静置培養を行ったところ、３１２ユニット
／ｌのグルタミナーゼ活性を得た。また、アスペルギル
ス・オリゼー（A.oryzae）GT26株及びアスペルギルス・
オリゼー（A.oryzae）GT33株を用いて同様に培養後、活
性測定を行ったところ、培養液当り、２００ユニット／
ｌ及び３４０ユニット／ｌのグルタミナーゼ活性を得
た。Using A.oryzae GT7 strain in a GP medium (N. Kit
amoto, J. Matsui, Y. Kawai, Y. Kato, s. Yoshino,
K. Ohmiya, and N. Tsukagoshi, Appl. Microbiol. Bio
technol., 50, 85-92 (1998))
After static culture at 10 ° C. for 10 days, a glutaminase activity of 312 units / l was obtained. In addition, Aspergillus oryzae (A.oryzae) GT26 strain and Aspergillus
After similarly culturing using the A. oryzae GT33 strain, the activity was measured.
1 and 340 units / l of glutaminase activity were obtained.

【００７７】[0077]

【配列表】 ＳＥＱＵＥＮＣＥ ＬＩＳＴＩＮＧ 〈１１０〉 Ichibiki Kabushiki Kaisha et al. 〈１２０〉 Novel glutaminase and process for production thereof 〈１６０〉 34[Sequence list] SEQUENCE LISTING <110> Ichibiki Kabushiki Kaisha et al. <120> Novel glutaminase and process for production there <160> 34

【００７８】 〈２１０〉 1 〈２１１〉 134 〈２１２〉 DNA 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of DNA coding for glutaminase 〈４００〉 1 tg aac aat gaa atg gat gac ttc tca aca ccc ggc tcg tcc aac gca 47 Met Asn Asn Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala 1 5 10 15 ttt gga tac gtc cca tcg gag gcg aac ttc att cga ccc ggg aag cga 95 Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg 20 25 30 cct ctg tct tct att act cca acc att gtg acg cat ca 134 Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 35 40 45 <210> 1 <211> 134 <212> DNA <213> Aspergillus oryzae KBN616 <223> A part of DNA coding for glutaminase <400> 1 tg aac aat gaa atg gat gac ttc tca aca ccc ggc tcg tcc aac gca 47 Met Asn Asn Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala 1 5 10 15 ttt gga tac gtc cca tcg gag gcg aac ttc att cga ccc ggg aag cga 95 Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg 20 25 30 cct ctg tct tct att act cca acc att gtg acg cat ca 134 Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 35 40 45

【００７９】 〈２１０〉 2 〈２１１〉 600 〈２１２〉 DNA 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of DNA coding for glutaminase 〈４００〉 2 cc aac gca ttt gga tac gtc cca tcg gag gcg aac ttc att cga ccc 47 Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro 1 5 10 15 ggg aag cga cct ctg tct tct att act cca acc att gtg acg cat cag 95 Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 20 25 30 aat gga tct gtc ttc ttc gtt gca ggt tcg gct ggg ggt agt cga atc 143 Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly Gly Ser Arg Ile 35 40 45 att aca gcg act gta cag aac atc atc cat gct gtc gat gag ggc ttg 191 Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val Asp Glu Gly Leu 50 55 60 tcg gca gcc gag gct ttg gct aga cca cgt ctt cac gat caa ttg atc 239 Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His Asp Gln Leu Ile 65 70 75 80 ccc aat cag gtc aga ttc gag tat acc tac aac aat gag acg gtt gct 287 Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala 85 90 95 ttc atg aaa tct cta ggt cac aat gtg acc tgg gtc gct cca gga gac 335 Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val Ala Pro Gly Asp 100 105 110 agc act gcg caa gca atc cgg gta cta ccg aac gga acg ttc gat gcc 383 Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly Thr Phe Asp Ala 115 120 125 gct ggg gag cct agg cag ctt gac tct ggt ggt ttc gcg gtc tga 428 Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe Ala Val 130 135 140 ggactacttt ctagctgcag ctatgcagtc tacgggctat ctgccatcac ggtcacaatg 488 atagatcaaa cctatctttt gacatatttc gagcatcgac aaacttatat gggaacttgg 548 aaagacatct gaagatatag aaagaaggct catcgattgc gaatacatat ac 600<210> 2 <211> 600 <212> DNA <213> Aspergillus oryzae KBN616 <223> A part of DNA coding for glutaminase <400> 2 cc aac gca ttt gga tac gtc cca tcg gag gcg aac ttc att cga ccc 47 Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro 1 5 10 15 ggg aag cga cct ctg tct tct att act cca acc att gtg acg cat cag 95 Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 20 25 30 aat gga tct gtc ttc ttc gtt gca ggt tcg gct ggg ggt agt cga atc 143 Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly Gly Ser Arg Ile 35 40 45 att aca gcg act gta cag aac atc atc cat gct gtc gat gag ggc ttg 191 Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val Asp Glu Gly Leu 50 55 60 tcg gca gcc gag gct ttg gct aga cca cgt ctt cac gat caa ttg atc 239 Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His Asp Gln Leu Ile 65 70 75 80 ccc aat cag gtc aga ttc gag tat acc tac aac aat gag acg gtt gct 287 Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala 85 90 95 ttc atg aaa tct cta ggt cac aat gtg acc tgg gtc gct cca gga gac 335 Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val Ala Pro Gly Asp 100 105 110 agc act gcg caa gca atc cgg gta cta ccg aac gga acg ttc gat gcc 383 Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly Thr Phe Asp Ala 115 120 125 gct ggg gag cct agg cag ctt gac tct ggt ggt ttc gcg gtc tga 428 Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe Ala Val 130 135 140 ggactacttt ctagctgcag ctatgcagtc tacgggctat ctgccatcac ggtcacaatg 488 atagatcaaa cctatctttt gacatatttc gagcatcgac aaacttatat gggaactgag gagtagtaggag katga tg

【００８０】 〈２１０〉 3 〈２１１〉 2233 〈２１２〉 DNA 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of DNA coding for glutaminase 〈４００〉 3 agatctggcg gtgttgttat ctctaggttg tacctctcct ttgccgggcg cagtcagtga 60 tagcctcctt atcttgtgtt ccatcgtatt ttttacactg tgtaccatct atctaattca 120 ttatgaaatc ctttcttcgg cttatatttt tgtcatattc cgttcctgtc ttcttatttt 180 tcaacatcac agatgcaaac cccacatcct aactcaatac gccggaatga gtttaaccaa 240 acccagtgaa ttggttggac caggggtcca agacatatcg ggccgagatg tggagaaaca 300 ggtagatata agttgctcgt tctcttgtcc tgacattgcg ccatctctgg gcattatttg 360 tattatttta gcagcagtac ctggttggtg accttgacag ccacttttaa cagcccgcga 420 ccttcagggc gccgaaccaa ccactggctg gccgagtgac gagacggaaa gaccctcaac 480 c atg tat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta gtt 529 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 ctg gcg ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 577 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ttc gag 625 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 673 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gac gcc gtaag 723 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala 65 70 75 tgatagtgac ttagtctctt agttatattg acctggctga gatacaatgc ag tta gtt 781 Leu Val 80 gcg aca ata ttc tgt gta ggg gtt gtt g gtgagataaa taaaagtagc gggga 834 Ala Thr Ile Phe Cys Val Gly Val Val G 85 90 agatatgtta actgacgggt accag ga atg tac cac agt ggg atc gct gga 885 ly Met Tyr His Ser Gly Ile Ala Gly 95 gga ggg ttt atg tta gtg aga gcc cct aac ggt tcg tac gaa ttc atc 933 Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr Glu Phe Ile 100 105 110 115 gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa atg ttc aag 981 Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu Met Phe Lys 120 125 130 aac aac acc gac gca tcc aca tct ggg ggt ttg gca ag gtaacaggca ctc 1032 Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Se 135 140 cctcaatcaa gccgaggaac aggagactaa tatgcaccaa g t ggc gtt cct ggt 1086 r Gly Val Pro Gly 145 gag gtg cga ggc ctg gaa tac ctt cac aag aag tat ggt tct ctt cca 1134 Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr Gly Ser Leu Pro 150 155 160 tgg tcg acc atc atg cag cca gcc atc cag aca gcc cgt caa ggt ttc 1182 Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala Arg Gln Gly Phe 165 170 175 180 ccg gtt ggt cgt gat ctc gtt cgt tat atg aac tcg gcc gtc ggg gat 1230 Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser Ala Val Gly Asp 185 190 195 ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct att gac ttt gcc 1278 Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala Ile Asp Phe Ala 200 205 210 cct aat gga act cgt ctc ggt ctt gga gat acc atc acc cgt aaa cgc 1326 Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile Thr Arg Lys Arg 215 220 225 tac gca gac acg ctg gag aca att gcc aac aac ggc ccg gat gct ttt 1374 Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly Pro Asp Ala Phe 230 235 240 tat acc ggt cca att gcg gaa acc atg ata cag gcg ctg caa gct gcg 1422 Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala Leu Gln Ala Ala 245 250 255 260 aac ggc aca atg acg cta gaa gac cta cgt aac tac acc att gct atc 1470 Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr Thr Ile Ala Ile 265 270 275 cga gac acc tcg cag att gac tat cga ggg tat aga gtt att gga act 1518 Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg Val Ile Gly Thr 280 285 290 agc gcg cca tcc agc ggc act gtc gcc ttg aac atc ctt aag gtt ttg 1566 Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile Leu Lys Val Leu 295 300 305 gat act tac gac agc ttc atg gta ccc gac aac gta aat ctt agc acg 1614 Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val Asn Leu Ser Thr 310 315 320 cat cgt ttg gat gaa gct ata cgt ttc gga tat gga ctg gtacgtagct gc 1665 His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly Leu 325 330 335 atccctttat attgacccca acacctagct aactcgatat caag aga acc gaa cta 1721 Arg Thr Glu Leu 340 ggt gat ccg tac ttc ctc gaa gga tta gat gaa tac cag aaa gat atg 1769 Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu Tyr Gln Lys Asp Met 345 350 355 ctg gaa cag tcg act att gat gag att cat ggg aaa att tcc gac ttg 1817 Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly Lys Ile Ser Asp Leu 360 365 370 cac aca cag aac gtg tct gct tat gat cct aaa ggc ctc gag agc ctt 1865 His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys Gly Leu Glu Ser Leu 375 380 385 gac ac gtaagcgcac actcgcaaat tctcatttac cggttgtact aaccgattca g 1921 Asp Th 390 c ccc gga acg tcg cat atc gct gct gtt gat cat act gga ctt act att 1970 r Pro Gly Thr Ser His Ile Ala Ala Val Asp His Thr Gly Leu Thr Ile 395 400 405 tcc act atc aca acc atc aac ttg ctt ttc ggc agt aaa gtc atg gtg 2018 Ser Thr Ile Thr Thr Ile Asn Leu Leu Phe Gly Ser Lys Val Met Val 410 415 420 cca gag acg gga ata att atg aac aat gaa atg gat g gttggtgatc tccc 2069 Pro Glu Thr Gly Ile Ile Met Asn Asn Glu Met Asp A 425 430 435 agaaagcctc tgctccagcc ctgaacctac ctgctgatac gattcag ac ttc tca 2124 sp Phe Ser aca ccc ggc tcg tcc aac gca ttt gga tac gtc cca tcg gag gcg aac 2172 Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn 440 445 450 ttc att cga ccc ggg aag cga cct ctg tct tct att act cca acc att 2220 Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile 455 460 465 470 gtg acg cat cag a 2233 Val Thr His Gln <210> 3 <211> 2233 <212> DNA <213> Aspergillus oryzae KBN616 <223> A part of DNA coding for glutaminase <400> 3 ctttcttcgg cttatatttt tgtcatattc cgttcctgtc ttcttatttt 180 tcaacatcac agatgcaaac cccacatcct aactcaatac gccggaatga gtttaaccaa 240 acccagtgaa ttggttggac caggggtcca agacatatcg ggccgagatg tggagaaaca 300 ggtagatata agttgctcgt tctcttgtcc tgacattgcg ccatctctgg gcattatttg 360 tattatttta gcagcagtac ctggttggtg accttgacag ccacttttaa cagcccgcga 420 ccttcagggc gccgaaccaa ccactggctg gccgagtgac gagacggaaa gaccctcaac 480 c atg tat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta gtt 529 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 ctg gcg ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 577 Leu Ala His Leu Pro Asp Val Le u Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ttc gag 625 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 673 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gac gcc gtaag 723 Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala 65 70 75 tgatagtgac ttagtctctt agttatattg acctggctga gatacaatgc ag tta gtt 781 Leu Val 80 gcg aca ata ttc tgt gta ggg gtt gtt g gtgagataagtag ag ag Ag Ag Ag Val Val G 85 90 agatatgtta actgacgggt accag ga atg tac cac agt ggg atc gct gga 885 ly Met Tyr His Ser Gly Ile Ala Gly 95 gga ggg ttt atg tta gtg aga gcc cct aac ggt tcg tac gaa ttc atc 933 Gly Gly Ply Val Arg Ala Pro Asn Gly Ser Tyr Glu Phe Ile 100 105 110 115 gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa atg ttc aag 981 Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu Met Phe Lys 120 125 130 aac aac acc gac gca tcc aca tct ggg ggt ttg gca ag gtaacaggca ctc 1032 Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Se 135 140 cctcaatcaa gccgag gtagggacta cct ggt 1086 r Gly Val Pro Gly 145 gag gtg cga ggc ctg gaa tac ctt cac aag aag tat ggt tct ctt cca 1134 Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr Gly Ser Leu Pro 150 155 160 tgg tcg acc atc atg cag cca gcc atc cag aca gcc cgt caa ggt ttc 1182 Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala Arg Gln Gly Phe 165 170 175 180 ccg gtt ggt cgt gat ctc gtt cgt tat atg aac tcg gcc gtc ggg gat 1230 Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser Ala Val Gly Asp 185 190 195 ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct att gac ttt gcc 1278 Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala Ile Asp Phe Ala 200 205 210 cct aat gga act cgt ctc ggt ctt gga gat acc atc acc cgt aaa cgc 1326 Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile Thr Arg Lys Arg 215 220 225 ta c gca gac acg ctg gag aca att gcc aac aac ggc ccg gat gct ttt 1374 Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly Pro Asp Ala Phe 230 235 240 tat acc ggt cca att gcg gaa acc atg ata cag gcg ctg cag gct gcg 1422 Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala Leu Gln Ala Ala 245 250 255 260 aac ggc aca atg acg cta gaa gac cta cgt aac tac acc att gct atc 1470 Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr Thr Ile Ala Ile 265 270 275 cga gac acc tcg cag att gac tat cga ggg tat aga gtt att gga act 1518 Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg Val Ile Gly Thr 280 285 290 agc gcg cca tcc agc ggc act gtc gcc ttg aac atc ctt aag gtt ttg 1566 Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile Leu Lys Val Leu 295 300 305 gat act tac gac agc ttc atg gta ccc gac aac gta aat ctt agc acg 1614 Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val Asn Leu Ser Thr 310 315 320 cat cgt ttg gat gaa gct ata cgt ttc gga tat gga ctg gtacgtagct gc 1665 His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly Leu 325 3 30 335 atccctttat attgacccca acacctagct aactcgatat caag aga acc gaa cta 1721 Arg Thr Glu Leu 340 ggt gat ccg tac ttc ctc gaa gga tta gat gaa tac cag aaa gat atg 1769 Gly Asp Pro Tyr Phe Leu Glu Glu 345 350 355 ctg gaa cag tcg act att gat gag att cat ggg aaa att tcc gac ttg 1817 Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly Lys Ile Ser Asp Leu 360 365 370 cac aca cag aac gtg tct gct tat gat cct aaa ggc ctc gag agc ctt 1865 His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys Gly Leu Glu Ser Leu 375 380 385 gac ac gtaagcgcac actcgcaaat tctcatttac cggttgtact aaccgattca g 1921 Asp Th 390 c ccc gga acg tcg cat atg gga ctt act att 1970 r Pro Gly Thr Ser His Ile Ala Ala Val Asp His Thr Gly Leu Thr Ile 395 400 405 tcc act atc aca acc atc aac ttg ctt ttc ggc agt aaa gtc atg gtg 2018 Ser Thr Ile Thr Thr Ile Asn Leu Leu Phe Gly Ser Lys Val Met Val 410 415 420 cca gag acg gga ata att atg aac aat gaa atg gat g gttggtgatc tccc 2069 Pro Glu Thr Gly Ile Ile Met Asn Asn Glu Met Asp A 425 430 435 agaaagcctc tgctccagcc ctgaacctac ctgctgatac gattcag ac ttc tca 2124 sp Phe Ser aca ccc ggc tcg tcc aac gca ttt gga tac gtc cca tcg gag Gcg Ag Ser G Ser G Ser G Ser G Ser G Ser G Ser G Ser G Pro Ser Glu Ala Asn 440 445 450 ttc att cga ccc ggg aag cga cct ctg tct tct att act cca acc att 2220 Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile 455 460 465 470 470 gtg acg cat cag a 2233 Val Thr His Gln

【００８１】 〈２１０〉 4 〈２１１〉 657 〈２１２〉 DNA 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of DNA coding for glutaminase 〈４００〉 4 cc aac gca ttt gga tac gtc cca tcg gag gcg aac ttc att cga ccc 47 Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro 1 5 10 15 ggg aag cga cct ctg tct tct att act cca acc att gtg acg cat cag 95 Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 20 25 30 aat gga tct gtc ttc ttc gtt gca ggt tcg gct ggg ggt agt cga atc 143 Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly Gly Ser Arg Ile 35 40 45 att aca gcg act gta cag aac atc atc cat gct gtc gat gag ggc ttg 191 Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val Asp Glu Gly Leu 50 55 60 tcg gca gcc gag gct ttg gct aga cca cgt ctt cac gat caa ttg atc 239 Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His Asp Gln Leu Ile 65 70 75 80 ccc aat cag gtc aga ttc gag tat acc tac aac aat gag acg gtt gct 287 Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala 85 90 95 ttc atg aaa tct cta ggt cac aat gtg acc tgg gtc gct cca gga gac 335 Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val Ala Pro Gly Asp 100 105 110 agc act gcg caa gca atc cgg gta cta ccg aac gga acg ttc gat gcc 383 Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly Thr Phe Asp Ala 115 120 125 gct ggg gag cct agg cag ctt gac tct ggt ggt ttc gcg gtc tga 428 Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe Ala Val 130 135 140 ggactacttt ctagctgcag ctatgcagtc tacgggctat ctgccatca cggtcacaat 487 gatagatcaa acctatcttt tgacatattt cgagcatcga caaacttata tgggaacttg 547 gaaagacatc tgaagatata gaaagaaggc tcatcgattg cgaatacata tacaaagaga 607 aatcttgtgg atatgcttga cgacgccgac tctccgccgg tcctaagctt 657<210> 4 <211> 657 <212> DNA <213> Aspergillus oryzae KBN616 <223> A part of DNA coding for glutaminase <400> 4 cc aac gca ttt gga tac gtc cca tcg gag gcg aac ttc att cga ccc 47 Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro 1 5 10 15 ggg aag cga cct ctg tct tct att act cca acc att gtg acg cat cag 95 Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 20 25 30 aat gga tct gtc ttc ttc gtt gca ggt tcg gct ggg ggt agt cga atc 143 Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly Gly Ser Arg Ile 35 40 45 att aca gcg act gta cag aac atc atc cat gct gtc gat gag ggc ttg 191 Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val Asp Glu Gly Leu 50 55 60 tcg gca gcc gag gct ttg gct aga cca cgt ctt cac gat caa ttg atc 239 Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His Asp Gln Leu Ile 65 70 75 80 ccc aat cag gtc aga ttc gag tat acc tac aac aat gag acg gtt gct 287 Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala 85 90 95 ttc atg aaa tct cta ggt cac aat gtg acc tgg gtc gct cca gga gac 335 Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val Ala Pro Gly Asp 100 105 110 agc act gcg caa gca atc cgg gta cta ccg aac gga acg ttc gat gcc 383 Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly Thr Phe Asp Ala 115 120 125 gct ggg gag cct agg cag ctt gac tct ggt ggt ttc gcg gtc tga 428 Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe Ala Val 130 135 140 ggactacttt ctagctgcag ctatgcagtc tacgggctat ctgccatca cggtcacaat 487 gatagatcaa acctatcttt tgacatattt cgagcatcga caaacttata tgggaacttg 547 gaaagacatc tgaagatata gaaagaaggc tcatcgattg cgaatacata tacaaagaga 607 aatcttgtgg atatgcttga cgacgccgac tctccgccgg tcctaagctt 657

【００８２】 〈２１０〉 5 〈２１１〉 2794 〈２１２〉 DNA 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 DNA coding for glutaminase 〈４００〉 5 agatctggcg gtgttgttat ctctaggttg tacctctcct ttgccgggcg cagtcagtga 60 tagcctcctt atcttgtgtt ccatcgtatt ttttacactg tgtaccatct atctaattca 120 ttatgaaatc ctttcttcgg cttatatttt tgtcatattc cgttcctgtc ttcttatttt 180 tcaacatcac agatgcaaac cccacatcct aactcaatac gccggaatga gtttaaccaa 240 acccagtgaa ttggttggac caggggtcca agacatatcg ggccgagatg tggagaaaca 300 ggtagatata agttgctcgt tctcttgtcc tgacattgcg ccatctctgg gcattatttg 360 tattatttta gcagcagtac ctggttggtg accttgacag ccacttttaa cagcccgcga 420 ccttcagggc gccgaaccaa ccactggctg gccgagtgac gagacggaaa gaccctcaac 480 c atg tat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta gtt 529 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 ctg gcg ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 577 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ttc gag 625 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 673 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gac gcc gtaag 723 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala 65 70 75 tgatagtgac ttagtctctt agttatattg acctggctga gatacaatgc ag tta gtt 781 Leu Val 80 gcg aca ata ttc tgt gta ggg gtt gtt g gtgagataaa taaaagtagc gggga 834 Ala Thr Ile Phe Cys Val Gly Val Val G 85 90 agatatgtta actgacgggt accag ga atg tac cac agt ggg atc gct gga 885 ly Met Tyr His Ser Gly Ile Ala Gly 95 gga ggg ttt atg tta gtg aga gcc cct aac ggt tcg tac gaa ttc atc 933 Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr Glu Phe Ile 100 105 110 gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa atg ttc aag 981 Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu Met Phe Lys 115 120 125 130 aac aac acc gac gca tcc aca tct ggg ggt ttg gca ag gtaacaggca ctc 1032 Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Se 135 140 cctcaatcaa gccgaggaac aggagactaa tatgcaccaa g t ggc gtt cct ggt 1086 r Gly Val Pro Gly 145 gag gtg cga ggc ctg gaa tac ctt cac aag aag tat ggt tct ctt cca 1134 Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr Gly Ser Leu Pro 150 155 160 tgg tcg acc atc atg cag cca gcc atc cag aca gcc cgt caa ggt ttc 1182 Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala Arg Gln Gly Phe 165 170 175 ccg gtt ggt cgt gat ctc gtt cgt tat atg aac tcg gcc gtc ggg gat 1230 Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser Ala Val Gly Asp 180 185 190 195 ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct att gac ttt gcc 1278 Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala Ile Asp Phe Ala 200 205 210 cct aat gga act cgt ctc ggt ctt gga gat acc atc acc cgt aaa cgc 1326 Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile Thr Arg Lys Arg 215 220 225 tac gca gac acg ctg gag aca att gcc aac aac ggc ccg gat gct ttt 1374 Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly Pro Asp Ala Phe 230 235 240 tat acc ggt cca att gcg gaa acc atg ata cag gcg ctg caa gct gcg 1422 Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala Leu Gln Ala Ala 245 250 255 aac ggc aca atg acg cta gaa gac cta cgt aac tac acc att gct atc 1470 Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr Thr Ile Ala Ile 260 265 270 275 cga gac acc tcg cag att gac tat cga ggg tat aga gtt att gga act 1518 Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg Val Ile Gly Thr 280 285 290 agc gcg cca tcc agc ggc act gtc gcc ttg aac atc ctt aag gtt ttg 1566 Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile Leu Lys Val Leu 295 300 305 gat act tac gac agc ttc atg gta ccc gac aac gta aat ctt agc acg 1614 Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val Asn Leu Ser Thr 310 315 320 cat cgt ttg gat gaa gct ata cgt ttc gga tat gga ctg gtacgtagct gc 1665 His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly Leu 325 330 335 atccctttat attgacccca acacctagct aactcgatat caag aga acc gaa cta 1721 Arg Thr Glu Leu 340 ggt gat ccg tac ttc ctc gaa gga tta gat gaa tac cag aaa gat atg 1769 Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu Tyr Gln Lys Asp Met 345 350 355 ctg gaa cag tcg act att gat gag att cat ggg aaa att tcc gac ttg 1817 Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly Lys Ile Ser Asp Leu 360 365 370 cac aca cag aac gtg tct gct tat gat cct aaa ggc ctc gag agc ctt 1865 His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys Gly Leu Glu Ser Leu 375 380 385 gac ac gtaagcgcac actcgcaaat tctcatttac cggttgtact aaccgattca g 1921 Asp Th 390 c ccc gga acg tcg cat atc gct gct gtt gat cat act gga ctt act att 1970 r Pro Gly Thr Ser His Ile Ala Ala Val Asp His Thr Gly Leu Thr Ile 395 400 405 tcc act atc aca acc atc aac ttg ctt ttc ggc agt aaa gtc atg gtg 2018 Ser Thr Ile Thr Thr Ile Asn Leu Leu Phe Gly Ser Lys Val Met Val 410 415 420 cca gag acg gga ata att atg aac aat gaa atg gat g gttggtgatc tccc 2069 Pro Glu Thr Gly Ile Ile Met Asn Asn Glu Met Asp A 425 430 435 agaaagcctc tgctccagcc ctgaacctac ctgctgatac gattcag ac ttc tca 2124 sp Phe Ser aca ccc ggc tcg tcc aac gca ttt gga tac gtc cca tcg gag gcg aac 2172 Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn 440 445 450 ttc att cga ccc ggg aag cga cct ctg tct tct att act cca acc att 2220 Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile 455 460 465 470 gtg acg cat cag aat gga tct gtc ttc ttc gtt gca ggt tcg gct ggg 2268 Val Thr His Gln Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly 475 480 485 ggt agt cga atc att aca gcg act gta cag aac atc atc cat gct gtc 2316 Gly Ser Arg Ile Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val 490 495 500 gat gag ggc ttg tcg gca gcc gag gct ttg gct aga cca cgt ctt cac 2364 Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His 505 510 515 gat caa ttg atc ccc aat cag gtc aga ttc gag tat acc tac aac aat 2412 Asp Gln Leu Ile Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn 520 525 530 gag acg gtt gct ttc atg aaa tct cta ggt cac aat gtg acc tgg gtc 2460 Glu Thr Val Ala Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val 535 540 545 550 gct cca gga gac agc act gcg caa gca atc cgg gta cta ccg aac gga 2508 Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly 555 560 565 acg ttc gat gcc gct ggg gag cct agg cag ctt gac tct ggt ggt ttc 2556 Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe 570 575 580 gcg gtc tga ggactacttt ctagctgcag ctatgcagtc tacgggctat ctgccatca 2614 Ala Val Stop 585 cggtcacaat gatagatcaa acctatcttt tgacatattt cgagcatcga caaacttata 2674 tgggaacttg gaaagacatc tgaagatata gaaagaaggc tcatcgattg cgaatacata 2734 tacaaagaga aatcttgtgg atatgcttga cgacgccgac tctccgccgg tcctaagctt 2794[0082] <210> 5 <211> 2794 <212> DNA <213> Aspergillus oryzae KBN616 <223> DNA coding for glutaminase <400> 5 agatctggcg gtgttgttat ctctaggttg tacctctcct ttgccgggcg cagtcagtga 60 tagcctcctt atcttgtgtt ccatcgtatt ttttacactg tgtaccatct atctaattca 120 ttatgaaatc ctttcttcgg cttatatttt tgtcatattc cgttcctgtc ttcttatttt 180 tcaacatcac agatgcaaac cccacatcct aactcaatac gccggaatga gtttaaccaa 240 acccagtgaa ttggttggac caggggtcca agacatatcg ggccgagatg tggagaaaca 300 ggtagatata agttgctcgt tctcttgtcc tgacattgcg ccatctctgg gcattatttg 360 tattatttta gcagcagtac ctggttggtg accttgacag ccacttttaa cagcccgcga 420 ccttcagggc gccgaaccaa ccactggctg gccgagtgac gagacggaaa gaccctcaac 480 c atg tat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta gtt 529 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 ctg gcg ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 577 Leu Ala Leu Ile His Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ttc gag 625 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 673 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gac gcc gtaag 723 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala 65 70 75 tgatagtgac ttagtctctt agttatattg acctggctga gatacaatgc ag tta gtt 781 Leu Val 80 gcg aca ata ttc tgt gta ggg gtt gtt g gtgagata Ava Gag Val Gag Thrag 85 90 agatatgtta actgacgggt accag ga atg tac cac agt ggg atc gct gga 885 ly Met Tyr His Ser Gly Ile Ala Gly 95 gga ggg ttt atg tta gtg aga gcc cct aac ggt tcg tac gaa ttc atc 933 Gly Gly Phe Ahe Met Pro Asn Gly Ser Tyr Glu Phe Ile 100 105 110 gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa atg ttc aag 981 Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Ala P he Glu Glu Met Phe Lys 115 120 125 130 aac aac acc gac gca tcc aca tct ggg ggt ttg gca ag gtaacaggca ctc 1032 Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Se 135 140 cctcaatcaa gccgaggaac aggagccc tatgcgccaa 1086 r Gly Val Pro Gly 145 gag gtg cga ggc ctg gaa tac ctt cac aag aag tat ggt tct ctt cca 1134 Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr Gly Ser Leu Pro 150 155 160 tgg tcg acc atc atg cag cca gcc atc cag aca gcc cgt caa ggt ttc 1182 Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala Arg Gln Gly Phe 165 170 175 ccg gtt ggt cgt gat ctc gtt cgt tat atg aac tcg gcc gtc ggg gat 1230 Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser Ala Val Gly Asp 180 185 190 195 ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct att gac ttt gcc 1278 Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala Ile Asp Phe Ala 200 205 210 cct aat gga act cgt ctc ggt ctt gga gat acc atc acc cgt aaa cgc 1326 Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile Thr Arg Lys Arg 215 220 225 tac gca gac acg ctg gag aca att gcc aac aac ggc ccg gat gct ttt 1374 Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly Pro Asp Ala Phe 230 235 240 tat acc ggt cca att gcg gaa acc atg ata cag gcg ctg caa gcg Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala Leu Gln Ala Ala 245 250 255 aac ggc aca atg acg cta gaa gac cta cgt aac tac acc att gct atc 1470 Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr Thr Ile Ala Ile 260 265 270 275 cga gac acc tcg cag att gac tat cga ggg tat aga gtt att gga act 1518 Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg Val Ile Gly Thr 280 285 290 290 agc gcg cca tcc agc ggc act gt gcc ttg aac atc ctt aag gtt ttg 1566 Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile Leu Lys Val Leu 295 300 305 gat act tac gac agc ttc atg gta ccc gac aac gta aat ctt agc acg 1614 Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val Asn Leu Ser Thr 310 315 320 cat cgt ttg gat gaa gct ata cgt ttc gga tat gga ctg gtacgtagct gc 1665 His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly Leu 325 330 335 at ccctttat attgacccca acacctagct aactcgatat caag aga acc gaa cta 1721 Arg Thr Glu Leu 340 ggt gat ccg tac ttc ctc gaa gga tta gat gaa tac cag aaa gat atg 1769 Gly Asp Pro Tyr Phe Leu Glu Glu Leu Asp 355 ctg gaa cag tcg act att gat gag att cat ggg aaa att tcc gac ttg 1817 Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly Lys Ile Ser Asp Leu 360 365 370 cac aca cag aac gtg tct gct tat gat cct aaa ggc ct gag agc ctt 1865 His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys Gly Leu Glu Ser Leu 375 380 385 gac ac gtaagcgcac actcgcaaat tctcatttac cggttgtact aaccgattca g 1921 Asp Th 390 c ccc gga acg tcg cat atc gtt gct gct gct act att 1970 r Pro Gly Thr Ser His Ile Ala Ala Val Asp His Thr Gly Leu Thr Ile 395 400 405 tcc act atc aca acc atc aac ttg ctt ttc ggc agt aaa gtc atg gtg 2018 Ser Thr Ile Thr Thr Ile Asn Leu Leu Phe Gly Ser Lys Val Met Val 410 415 420 cca gag acg gga ata att atg aac aat gaa atg gat g gttggtgatc tccc 2069 Pro Glu Thr Gly Ile Ile Met Asn Asn Glu Met Asp A 425 430 435 agaaagcctc tgctccagcc ctgaacctac ctgctgatac gattcag ac ttc tca 2124 sp Phe Ser aca ccc ggc tcg tcc aac gca ttt gga tac gtc cca tcg gag gcg Aac Ser AeG Pro G Ser A Ser G Pro G Ser A G Pro G Ser G A Ser G Pro G Pro Glu Ala Asn 440 445 450 ttc att cga ccc ggg aag cga cct ctg tct tct att act cca acc att 2220 Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile 455 460 465 470 gtg acg cat cag aat gga tct gtc ttc ttc gtt gca ggt tcg gct ggg 2268 Val Thr His Gln Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly 475 480 485 ggt agt cga atc att aca gcg act gta cag aac atc atc cat gct gtc 2316 Gly Ser Arg Ile Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val 490 495 500 gat gag ggc ttg tcg gca gcc gag gct ttg gct aga cca cgt ctt cac 2364 Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His 505 510 510 515 gat caa ttg atc ccc aat cag gtc aga ttc gag tat acc tac aac aat 2412 Asp Gln Leu Ile Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn 520 525 530 gag acg gtt gct t tc atg aaa tct cta ggt cac aat gtg acc tgg gtc 2460 Glu Thr Val Ala Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val 535 540 545 550 gct cca gga gac agc act gcg caa gca atc cgg gta cta ccg aac gga 2508 Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly 555 560 565 acg ttc gat gcc gct ggg gag cct agg cag ctt gac tct ggt ggt ttc 2556 Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe 570 575 580 gcg gtc tga ggactacttt ctagctgcag ctatgcagtc tacgggctat ctgccatca 2614 Ala Val Stop 585 cggtcacaat gatagatcaa acctatcttt tgacatattt cgagcatcga caaacttata 2674 tgggaacttg gaaagacatc tgaagatata gaaagaaggc tcatcgattg cgaatacata 2734 tacaaagaga aatcttgtgg atatgcttga cgacgccgac tctccgccgg tcctaagctt 2794

【００８３】 〈２１０〉 6 〈２１１〉 1440 〈２１２〉 DNA 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of DNA coding for glutaminase 〈４００〉 6 atg tat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta gtt 48 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 ctg gcg ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 96 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ttc gag 144 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 192 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gac gcc tta 240 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 gtt gcg aca ata ttc tgt gta ggg gtt gtt gga atg tac cac agt ggg 288 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 atc gct gga gga ggg ttt atg tta gtg aga gcc cct aac ggt tcg tac 336 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 gaa ttc atc gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa 384 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 atg ttc aag aac aac acc gac gca tcc aca tct ggg ggt ttg gca agt 432 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 ggc gtt cct ggt gag gtg cga ggc ctg gaa tac ctt cac aag aag tat 480 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 ggt tct ctt cca tgg tcg acc atc atg cag cca gcc atc cag aca gcc 528 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 cgt caa ggt ttc ccg gtt ggt cgt gat ctc gtt cgt tat atg aac tcg 576 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 gcc gtc ggg gat ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct 624 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 att gac ttt gcc cct aat gga act cgt ctc ggt ctt gga gat acc atc 672 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 acc cgt aaa cgc tac gca gac acg ctg gag aca att gcc aac aac ggc 720 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 ccg gat gct ttt tat acc ggt cca att gcg gaa acc atg ata cag gcg 768 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 ctg caa gct gcg aac ggc aca atg acg cta gaa gac cta cgt aac tac 816 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 acc att gct atc cga gac acc tcg cag att gac tat cga ggg tat aga 864 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 gtt att gga act agc gcg cca tcc agc ggc act gtc gcc ttg aac atc 912 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 ctt aag gtt ttg gat act tac gac agc ttc atg gta ccc gac aac gta 960 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 aat ctt agc acg cat cgt ttg gat gaa gct ata cgt ttc gga tat gga 1008 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 ctg aga acc gaa cta ggt gat ccg tac ttc ctc gaa gga tta gat gaa 1056 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 tac cag aaa gat atg ctg gaa cag tcg act att gat gag att cat ggg 1104 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 aaa att tcc gac ttg cac aca cag aac gtg tct gct tat gat cct aaa 1152 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 ggc ctc gag agc ctt gac acc ccc gga acg tcg cat atc gct gct gtt 1200 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 gat cat act gga ctt act att tcc act atc aca acc atc aac ttg ctt 1248 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 ttc ggc agt aaa gtc atg gtg cca gag acg gga ata att atg aac aat 1296 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 gaa atg gat gac ttc tca aca ccc ggc tcg tcc aac gca ttt gga tac 1344 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 gtc cca tcg gag gcg aac ttc att cga ccc ggg aag cga cct ctg tct 1392 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 tct att act cca acc att gtg acg cat cag a 1440 Ser Ile Thr Pro Thr Ile Val Thr His Gln 465 470 <210> 6 <211> 1440 <212> DNA <213> Aspergillus oryzae KBN616 <223> A part of DNA coding for glutaminase <400> 6 atg tat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta gtt 48 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 ctg gcg ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 96 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ttc gag 144 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 192 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gac gcc tta 240 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 gtt gcg aca ata ttc tgt gta ggg gtt gtt gga atg tac cac agt ggg 288 Val Ala Thr Ile Phe Cys Val Gly Va l Val Gly Met Tyr His Ser Gly 85 90 95 atc gct gga gga ggg ttt atg tta gtg aga gcc cct aac ggt tcg tac 336 Ile Ala Gly Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 gaa ttc atc gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa 384 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 atg ttc aag aac aac acc gac gca tcc aca tct ggg ggt ttg Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 ggc gtt cct ggt gag gtg cga ggc ctg gaa tac ctt cac aag aag tat 480 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 ggt tct ctt cca tgg tcg acc atc atg cag cca gcc atc cag aca gcc 528 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 cgt caa ggt ttc ccg gtt ggt cgt gat ctc gtt cgt tat atg aac tcg 576 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 gcc gtc ggg gat ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct 624 Ala Val Glyp Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 att gac ttt gcc cct aat gga act cgt ctc ggt ctt gga gat acc atc 672 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 acc cgt aaa cgc tac gca gac acg ctg gag aca att gcc aac aac ggc 720 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 ccg gat gct ttt tat acc ggt cca att gcg gaa acc atg cag gcg 768 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 ctg caa gct gcg aac ggc aca atg acg cta gaa gac cta cgt aac tac 816 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 acc att gct atc cga gac acc tcg cag att gac tat cga ggg tat aga 864 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 gtt att gga act agc gcg cca tcc agc ggc act gtc gcc ttg aac atc 912 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 ctt aag gtt ttg gat act tac gac agc ttc atg gta ccc gac aac gta 960 Leu Lys ValLeu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 aat ctt agc acg cat cgt ttg gat gaa gct ata cgt ttc gga tat gga 1008 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 ctg aga acc gaa cta ggt gat ccg tac ttc ctc gaa gga tta gat gaa 1056 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 tac cag aaa gat atg ctg gat cag gat gag att cat ggg 1104 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 aaa att tcc gac ttg cac aca cag aac gtg tct gct tat gat cct aaa 1152 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 ggc ctc gag agc ctt gac acc ccc gga acg tcg cat atc gct gct gtt 1200 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 395 400 gat cat act gga ctt act att tcc act atc aca acc atc aac ttg ctt 1248 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 ttc ggc agt aaa gtc atg gtg cca gag acg gga ata att atg aa c aat 1296 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 gaa atg gat gac ttc tca aca ccc ggc tcg tcc aac gca ttt gga tac 1344 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 gtc cca tcg gag gcg aac ttc att cga ccc ggg aag cga cct ctg tct 1392 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 tct att act cca acc att gtg acg cat cag a 1440 Ser Ile Thr Pro Thr Ile Val Thr His Gln 465 470

【００８４】 〈２１０〉 7 〈２１１〉 1927 〈２１２〉 DNA 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 DNA coding for glutaminase 〈４００〉 7 atg tat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta gtt 48 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 ctg gcg ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 96 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ttc gag 144 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 192 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gac gcc tta 240 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 gtt gcg aca ata ttc tgt gta ggg gtt gtt gga atg tac cac agt ggg 288 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 atc gct gga gga ggg ttt atg tta gtg aga gcc cct aac ggt tcg tac 336 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 gaa ttc atc gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa 384 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 atg ttc aag aac aac acc gac gca tcc aca tct ggg ggt ttg gca agt 432 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 ggc gtt cct ggt gag gtg cga ggc ctg gaa tac ctt cac aag aag tat 480 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 ggt tct ctt cca tgg tcg acc atc atg cag cca gcc atc cag aca gcc 528 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 cgt caa ggt ttc ccg gtt ggt cgt gat ctc gtt cgt tat atg aac tcg 576 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 gcc gtc ggg gat ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct 624 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 att gac ttt gcc cct aat gga act cgt ctc ggt ctt gga gat acc atc 672 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 acc cgt aaa cgc tac gca gac acg ctg gag aca att gcc aac aac ggc 720 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 ccg gat gct ttt tat acc ggt cca att gcg gaa acc atg ata cag gcg 768 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 ctg caa gct gcg aac ggc aca atg acg cta gaa gac cta cgt aac tac 816 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 acc att gct atc cga gac acc tcg cag att gac tat cga ggg tat aga 864 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 gtt att gga act agc gcg cca tcc agc ggc act gtc gcc ttg aac atc 912 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 ctt aag gtt ttg gat act tac gac agc ttc atg gta ccc gac aac gta 960 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 aat ctt agc acg cat cgt ttg gat gaa gct ata cgt ttc gga tat gga 1008 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 ctg aga acc gaa cta ggt gat ccg tac ttc ctc gaa gga tta gat gaa 1056 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 tac cag aaa gat atg ctg gaa cag tcg act att gat gag att cat ggg 1104 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 aaa att tcc gac ttg cac aca cag aac gtg tct gct tat gat cct aaa 1152 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 ggc ctc gag agc ctt gac acc ccc gga acg tcg cat atc gct gct gtt 1200 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 gat cat act gga ctt act att tcc act atc aca acc atc aac ttg ctt 1248 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 ttc ggc agt aaa gtc atg gtg cca gag acg gga ata att atg aac aat 1296 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 gaa atg gat gac ttc tca aca ccc ggc tcg tcc aac gca ttt gga tac 1344 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 gtc cca tcg gag gcg aac ttc att cga ccc ggg aag cga cct ctg tct 1392 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 tct att act cca acc att gtg acg cat cag aat gga tct gtc ttc ttc 1440 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 gtt gca ggt tcg gct ggg ggt agt cga atc att aca gcg act gta cag 1488 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 aac atc atc cat gct gtc gat gag ggc ttg tcg gca gcc gag gct ttg 1536 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 gct aga cca cgt ctt cac gat caa ttg atc ccc aat cag gtc aga ttc 1584 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 gag tat acc tac aac aat gag acg gtt gct ttc atg aaa tct cta ggt 1632 Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly 530 535 540 cac aat gtg acc tgg gtc gct cca gga gac agc act gcg caa gca atc 1680 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 cgg gta cta ccg aac gga acg ttc gat gcc gct ggg gag cct agg cag 1728 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 575 ctt gac tct ggt ggt ttc gcg gtc tga ggactacttt ctagctgcag ctatgca 1782 Leu Asp Ser Gly Gly Phe Ala Val 580 585 gtctacgggc tatctgccat cacggtcaca atgatagatc aaacctatct tttgacatat 1842 ttcgagcatc gacaaactta tatgggaact tggaaagaca tctgaagata tagaaagaag 1902 gctcatcgat tgcgaataca tatac 1927<210> 7 <211> 1927 <212> DNA <213> Aspergillus oryzae KBN616 <223> DNA coding for glutaminase <400> 7 atg tat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta gtt 48 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 ctg gcg ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 96 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ttc gag 144 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 192 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gac gcc tta 240 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 gtt gcg aca ata ttc tgt gta ggg gtt gtt gga atg tac cac agt ggg 288 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 atc gct gga gga ggg ttt atg tta gtg aga gcc cct aac ggt tcg tac 336 Ile Ala Gly Gly Gly Ply Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 gaa ttc atc gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa 384 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 atg ttc aag aac aac acc gac gca tcc aca tct ggg ggt ttg gca agt 432 Met Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 ggc gtt cct ggt gag gtg cga ggc ctg gaa tac ctt cac aag aag tat 480 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 ggt tct ctt cca tgg tcg acc atc atg cag cca gcc atc cag aca gcc 528 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 cgt caa ggt ttc ccg gtt ggt cgt gat ctc gtt cgt tat atg aac tcg 576 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 gcc gtc ggg gat ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct 624 Ala Val Gly Asp Gly Glu Asp Le u Ser Lys Asp Pro Thr Trp Ala 195 200 205 att gac ttt gcc cct aat gga act cgt ctc ggt ctt gga gat acc atc 672 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 acc cgt aaa cgc tac gca gac acg ctg gag aca att gcc aac aac ggc 720 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 ccg gat gct ttt tat acc ggt cca att gcg gaa acc atg ata cag 768 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 ctg caa gct gcg aac ggc aca atg acg cta gaa gac cta cgt aac tac 816 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 acc att gct atc cga gac acc tcg cag att gac tat cga ggg tat aga 864 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 gtt att gga act agc gcg cca tcc agc ggc act gtc gcc ttg aac atc 912 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 ctt aag gtt ttg gat act tac gac agc ttc atg gta ccc gac aac gta 960 Leu Lys Val Leu Asp Th r Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 aat ctt agc acg cat cgt ttg gat gaa gct ata cgt ttc gga tat gga 1008 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 ctg aga acc gaa cta ggt gat ccg tac ttc ctc gaa gga tta gat gaa 1056 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 tac cag aaa gat atg ctg ga atg gc gag atg gg att cat ggg 1104 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 aaa att tcc gac ttg cac aca cag aac gtg tct gct tat gat cct aaa 1152 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 ggc ctc gag agc ctt gac acc ccc gga acg tcg cat atc gct gct gtt 1200 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 gat cat act gga ctt act att tcc act atc aca acc atc aac ttg ctt 1248 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 ttc ggc agt aaa gtc atg gtg cca gag acg gga ata att atg aac aat 1296 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 gaa atg gat gac ttc tca aca ccc ggc tcg tcc aac gca ttt gga tac 1344 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 gtc cca tcg gag gcg aac ttc att cga ccc ggg aag cga cct ctg tct 1392 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 tct att act cca acc att gtg acg cat cag aat gga tct gtc ttc ttc 1440 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 gtt gca ggt tcg gct ggg ggt agt cga atc att aca gcg act gta cag 1488 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 aac atc atc cat gct gtc gat gag ggc ttg tcg gca gcc gag gct ttg 1536 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 gct aga cca cgt ctt cac gat caa ttg atc ccc aat cag gtc aga ttc 1584 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 gag tat acc tac aac aat gag acg gtt gct ttc atg a aa tct cta ggt 1632 Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly 530 535 540 cac aat gtg acc tgg gtc gct cca gga gac agc act gcg caa gca atc 1680 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 cgg gta cta ccg aac gga acg ttc gat gcc gct ggg gag cct agg cag 1728 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 570 575 ctt gac tct ggt ggt ttc gcg gtc tga ggactacttt ctagctgcag ctatgca 1782 Leu Asp Ser Gly Gly Phe Ala Val 580 585 gtctacgggc tatctgccat cacggtcaca atgatagatc aaacctatct tttgacat tgagacac taggaccat gagcacat 1

【００８５】 〈２１０〉 8 〈２１１〉 2289 〈２１２〉 DNA 〈２１３〉 Aspergillus sojae BA-104 〈２２３〉 DNA coding for glutaminase 〈４００〉 8 atg cat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta ctt 48 Met His Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Leu 1 5 10 15 ctg gca ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 96 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ctc gag 144 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Leu Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 192 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gat gcc gtaag 242 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala 65 70 75 taacagtgac ttagcctcct agttatattg acctggctga gatacaatgc ag tta gtt 300 Leu Val 80 gcg aca ata ttc tgt gta ggg gtt gtt g gtgagataca aaaaagtagc ggtga 353 Ala Thr Ile Phe Cys Val Gly Val Val G 85 90 atatatgtga actgacaaaa gggtaccag ga atg tac cac agt gga atc gct 405 ly Met Tyr His Ser Gly Ile Ala 95 gga gga ggg ttt atg tta gtg aga gct cct aac ggt tcg tac gaa ttc 453 Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr Glu Phe 100 105 110 atc gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa atg ttc 501 Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu Met Phe 115 120 125 130 aag aac aac acc gac gca tcc act tct ggg ggt ttg gca ag gtaacgggca 552 Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Se 135 140 ctccctcgat caaaccgagg aacagaggac taatatgcac caag t ggc gtt cct 606 r Gly Val Pro 145 ggc gag gtg cga ggc ctg gaa tac ctt cac aag aag tat ggt tct ctt 654 Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr Gly Ser Leu 150 155 160 cca tgg tcg acc gtc atg cag cct gcc atc cag aca gcc cgt caa ggt 702 Pro Trp Ser Thr Val Met Gln Pro Ala Ile Gln Thr Ala Arg Gln Gly 165 170 175 ttc cca gtt ggt cgt gat ctc gtt cgc tat atg aac tcg gcc gtc ggg 750 Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser Ala Val Gly 180 185 190 195 gat ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct att gac ttt 798 Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala Ile Asp Phe 200 205 210 gcc cct aat gga act cgt ctc ggt ctt gga gat acc atc acc cgt aaa 846 Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile Thr Arg Lys 215 220 225 cgc tac gca gac acg ctg gag aca att gcc aac aac ggc ccg gat gct 894 Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly Pro Asp Ala 230 235 240 ttt tat acc ggt cca att gcg gaa acc atg ata cag gcg ctg caa gcc 942 Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala Leu Gln Ala 245 250 255 gcg aac ggc aca atg acg cta gaa gac cta cgt aac tac acc att gct 990 Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr Thr Ile Ala 260 265 270 275 atc cga aac acc tcg cag att gac tat cga ggg tat aaa gtt att gga 1038 Ile Arg Asn Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Lys Val Ile Gly 280 285 290 act agc gcg ccc tcc agc ggc act gtt gct ttg aac atc ctt aag gtt 1086 Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile Leu Lys Val 295 300 305 ttg gat act tat gac agc ttc atg gta cct gac aac gta aac ctt agc 1134 Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val Asn Leu Ser 310 315 320 acg cat cgt ttg gac gaa gct ata cgt ttc gga tat gga ctg gtacgtgga 1185 Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly Leu 325 330 335 tgcatctctt tatagtgacc ccaacaccta gctaattcga catcaag aga gcc gaa 1241 Arg Ala Glu 340 ctg ggt gat ccg tac ttc ctt gaa gga ttg gat gaa tac cag aag gaa 1289 Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu Tyr Gln Lys Glu 345 350 355 atg ctg gaa cag tcg act att gat gag att cgt ggg aaa att tcc gat 1337 Met Leu Glu Gln Ser Thr Ile Asp Glu Ile Arg Gly Lys Ile Ser Asp 360 365 370 ttg cac aca cag aac gtg tct gct tat gat cct aaa ggc ctc gag agc 1385 Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys Gly Leu Glu Ser 375 380 385 ctt gac ac gtaagcgcac actcgctaat cctcatgaac cggttgtact aaccgattca 1443 Leu Asp Th 390 g c cct gga acg tcg cat atc gct gct gtt gat cat act gga ctt act 1490 r Pro Gly Thr Ser His Ile Ala Ala Val Asp His Thr Gly Leu Thr 395 400 405 att tcc act atc aca acc atc aac ttg ctt ttc ggc agc aaa gtc atg 1538 Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu Phe Gly Ser Lys Val Met 410 415 420 gtg cca gag acg gga ata att atg aac aat gaa atg gat g gttggtgatc 1588 Val Pro Glu Thr Gly Ile Ile Met Asn Asn Glu Met Asp A 425 430 435 acccagaaag cctctgctcc agccctgaac ctacatgctg atactattca g ac ttc 1644 sp Phe tca aca ccc ggc tcg tcc aac gca ttt gga tac gtc cca tcg gag gcg 1692 Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala 440 445 450 aac ttc att cga ccc ggg aag cga cct ctg tct tct att act cca acc 1740 Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr 455 460 465 att gtg acg cat cag aat gga tct gtc ttc ttc gtt gca ggt tcg gct 1788 Ile Val Thr His Gln Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala 470 475 480 485 ggg ggt agt cga atc att aca gcg act gta cag aac atc atc cat gct 1836 Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala 490 495 500 gtc gat gag ggc ttg tcg gca gcc gag gct ttg gct aga cca cgt ctt 1884 Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu 505 510 515 cac gat caa ttg atc ccc aat cag gtc aga ttc gag tat acc tac aac 1932 His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn 520 525 530 aat gag acg gtt gct ttc atg aaa tct cta ggt cac aat gtg acc tgg 1980 Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly His Asn Val Thr Trp 535 540 545 gtc gct cca gga gac agc act gcg caa gca atc cgg gta cta ccg aac 2028 Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn 550 555 560 565 gga acg ttc gat gcc gct ggg gag cct agg cag ctt gac tct ggt ggt 2076 Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly 570 575 580 ttc gcg gtc tga ggactacttt ctagctgcag ctatgcagtc tacgggctat ctgcc 2133 Phe Ala Val 585 atcgcggtca caatgataga tcaaacctat cttttgacat atttcgagca tcgacaaact 2193 tatatgggaa cctggaaaga catctgaaga tatagaaaga aggctcatcg attgcgaata 2253 catatacaaa gagaaatctt gtggatatgc tctaga 2289<210> 8 <211> 2289 <212> DNA <213> Aspergillus sojae BA-104 <223> DNA coding for glutaminase <400> 8 atg cat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta ctt 48 Met His Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Leu 1 5 10 15 ctg gca ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 96 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ctc gag 144 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Leu Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 192 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gat gcc gtaag 242 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala 65 70 75 taacagtgac ttagcctcct agttatattg acctggctga gatacaatgc ag tta gtt 300 Leu Val 80 gcg aca ata ttc tgt gta ggg gtt gtt g gtgagataca aaaaagtagc ggtga 353 Ala Thr Ile Phe Cys Val Gly Val Val G 85 90 atatatgtga actgacaaaa gggtaccag ga atg tac cac agt gga atc gct 405 ly Met Tyr His Ser Gly Ile Ala 95 gga gga ggg ttt atg tta cgt gaga g tcg tac gaa ttc 453 Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr Glu Phe 100 105 110 atc gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa atg ttc 501 Ile Asp Phe Arg Alu Thr Thr Ala Ala Phe Glu Glu Met Phe 115 120 125 130 aag aac aac acc gac gca tcc act tct ggg ggt ttg gca ag gtaacgggca 552 Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Se 135 140 ctccctcgat caaaccgag tacat gtt cct 606 r Gly Val Pro 145 ggc gag gtg cga ggc ctg gaa tac ctt cac aag aag tat ggt tct ctt 654 Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr Gly Ser Leu 150 155 160 cca tgg tcg acc gtc atg cag cct gcc atc cag aca gcc cgt caa ggt 702 Pro Trp Ser Thr Val Met Gln Pro Ala Ile Gln Thr Ala Arg Gln Gly 165 170 175 ttc cca gtt ggt cgt gat ctc gtt cgc tat atg aac tcg gcc gtc ggg 750 Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser Ala Val Gly 180 185 190 195 gat ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct att gac ttt 798 Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala Ile Asp Phe 200 205 210 gcc cct aat gga act cgt ctc ggt ctt gga gat acc atc acc cgt aaa 846 Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile Thr Arg Lys 215 220 225 cgc tac gca gac acg ctg gag aca att gcc aac aac ggc ccg gat gct 894 Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly Pro Asp Ala 230 235 240 ttt tat acc ggt cca att gcg gaa acc atg cag gcg ctg caa gcc 942 Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala Leu Gln Ala 245 250 255 gcg aac ggc aca atg acg cta gaa gac cta cgt aac tac acc att gct 990 Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr Thr Ile Ala 260 265 270 275 atc cga aac acc tcg cag att gac tat cga ggg tat aaa gtt att gga 1038 Ile Arg Asn Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Lys Val Ile Gly 280 285 285 290 act ag c gcg ccc tcc agc ggc act gtt gct ttg aac atc ctt aag gtt 1086 Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile Leu Lys Val 295 300 305 ttg gat act tat gac agc ttc atg gta cct gac aac gta aac ctt agc 1134 Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val Asn Leu Ser 310 315 320 acg cat cgt ttg gac gaa gct ata cgt ttc gga tat gga ctg gtacgtgga 1185 Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly Leu 325 330 335 tgcatctctt tatagtgacc ccaacaccta gctaattcga catcaag aga gcc gaa 1241 Arg Ala Glu 340 ctg ggt gat ccg tac ttc ctt gaa gga ttg gat gaa tac cag aag gaa 1289 Leu Gly Lep Glu Glu Asp G 345 350 355 atg ctg gaa cag tcg act att gat gag att cgt ggg aaa att tcc gat 1337 Met Leu Glu Gln Ser Thr Ile Asp Glu Ile Arg Gly Lys Ile Ser Asp 360 365 370 ttg cac aca cag aac gtg tct gct tat gat aaa ggc ctc gag agc 1385 Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys Gly Leu Glu Ser 375 380 385 ctt gac ac gtaagcgcac actcgctaat cctcatgaac cggttgtact aa ccgattca 1443 Leu Asp Th 390 gc cct gga acg tcg cat atc gct gct gtt gat cat act gga ctt act 1490 r Pro Gly Thr Ser His Ile Ala Ala Val Asp His Thr Gly Leu Thr 395 400 405 att tcc act atc aca acc atc aac ttg ctt ttc ggc agc aaa gtc atg 1538 Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu Phe Gly Ser Lys Val Met 410 415 420 gtg cca gag acg gga ata att atg aac aat gaa atg gat g gttggtgatc 1588 Val Pro Glu Thr Ile Met Asn Asn Glu Met Asp A 425 430 435 acccagaaag cctctgctcc agccctgaac ctacatgctg atactattca g ac ttc 1644 sp Phe tca aca ccc ggc tcg tcc aac gca ttt gga tac gtc cca tcg gag GgAg Ser G Ser G Ser G Ser Ser Ser Val Pro Ser Glu Ala 440 445 450 aac ttc att cga ccc ggg aag cga cct ctg tct tct att act cca acc 1740 Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr 455 460 465 att gtg acg cat cag aat gga tct gtc ttc ttc gtt gca ggt tcg gct 1788 Ile Val Thr His Gln Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala 470 475 480 485 ggg ggt agt cga atc att aca gcg act gta cag aac atc atc cat gct 1836 Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala 490 495 500 gtc gat gag ggc ttg tcg gca gcc gag gct ttg gct aga cca cgt ctt 1884 Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu 505 510 515 cac gat caa ttg atc ccc aat cag gtc aga ttc gag tat acc tac aac 1932 His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn 520 530 530 aat gag acg gtt gct ttc atg aaa tct cta ggt cac aat gtg acc tgg 1980 Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly His Asn Val Thr Trp 535 540 545 gtc gct cca gga gac agc act gcg caa gca atc cgg gta cta ccg aac 2028 Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn 550 555 560 565 gga acg ttc gat gcc gct ggg gag cct agg cag ctt gac tct ggt ggt 2076 Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly 570 575 580 ttc gcg gtc tga ggactacttt ctagctgcag ctatgcagtc tacgggctat ctgcc 2133 Phe Ala Val 585 atcgcggtca caatgataga tcaaacctat cttttgacat atttcgagca tcgacaact tatatgggaa cctggaaaga catctgaaga tatagaaaga aggctcatcg attgcgaata 2253 catatacaaa gagaaatctt gtggatatgc tctaga 2289

【００８６】 〈２１０〉 9 〈２１１〉 2289 〈２１２〉 DNA 〈２１３〉 Aspergillus sojae BA-104 〈２２３〉 DNA coding for glutaminase 〈４００〉 9 atg cat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta ctt 48 Met His Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Leu 1 5 10 15 ctg gca ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 96 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ctc gag 144 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Leu Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 192 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gat gcc gtaag 242 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala 65 70 75 taacagtgac ttagcctcct agttatattg acctggctga gatacaatgc ag tta gtt 300 Leu Val 80 gcg aca ata ttc tgt gta ggg gtt gtt g gtgagataca aaaaagtagc ggtga 353 Ala Thr Ile Phe Cys Val Gly Val Val G 85 90 atatatgtga actgacaaaa gggtaccag ga atg tac cac agt gga atc gct 405 ly Met Tyr His Ser Gly Ile Ala 95 gga gga ggg ttt atg tta gtg aga gct cct aac ggt tcg tac gaa ttc 453 Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr Glu Phe 100 105 110 atc gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa atg ttc 501 Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu Met Phe 115 120 125 130 aag aac aac acc gac gca tcc act tct ggg ggt ttg gca ag gtaacgggca 552 Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Se 135 140 ctccctcgat caaaccgagg aacagaggac taatatgcac caag t ggc gtt cct 606 r Gly Val Pro 145 ggc gag gtg cga ggc ctg gaa tac ctt cac aag aag tat ggt tct ctt 654 Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr Gly Ser Leu 150 155 160 cca tgg tcg acc gtc atg cag cct gcc atc cag aca gcc cgt caa ggt 702 Pro Trp Ser Thr Val Met Gln Pro Ala Ile Gln Thr Ala Arg Gln Gly 165 170 175 ttc cca gtt ggt cgt gat ctc gtt cgc tat atg aac tcg gcc gtc ggg 750 Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser Ala Val Gly 180 185 190 195 gat ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct att gac ttt 798 Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala Ile Asp Phe 200 205 210 gcc cct aat gga act cgt ctc ggt ctt gga gat acc atc acc cgt aaa 846 Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile Thr Arg Lys 215 220 225 cgc tac gca gac acg ctg gag aca att gcc aac aac ggc ccg gat gct 894 Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly Pro Asp Ala 230 235 240 ttt tat acc ggt cca att gcg gaa acc atg ata cag gcg ctg caa gcc 942 Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala Leu Gln Ala 245 250 255 gcg aac ggc aca atg acg cta gaa gac cta cgt aac tac acc att gct 990 Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr Thr Ile Ala 260 265 270 275 atc cga aac acc tcg cag att gac tat cga ggg tat aaa gtt att gga 1038 Ile Arg Asn Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Lys Val Ile Gly 280 285 290 act agc gcg ccc tcc agc ggc act gtt gct ttg aac atc ctt aag gtt 1086 Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile Leu Lys Val 295 300 305 ttg gat act tat gac agc ttc atg gta cct gac aac gta aac ctt agc 1134 Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val Asn Leu Ser 310 315 320 acg cat cgt ttg gac gaa gct ata cgt ttc gga tat gga ctg gtacgtgga 1185 Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly Leu 325 330 335 tgcatctctt tatagtgacc ccaaccacct agctaattcg acatcaag aga gcc gaa 1242 Arg Ala Glu 340 ctg ggt gat ccg tac ttc ctt gaa gga ttg gat gaa tac cag aag gaa 1290 Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu Tyr Gln Lys Glu 345 350 355 atg ctg gaa cag tcg act att gat gag att cgt ggg aaa att tcc gat 1338 Met Leu Glu Gln Ser Thr Ile Asp Glu Ile Arg Gly Lys Ile Ser Asp 360 365 370 ttg cac aca cag aac gtg tct gct tat gat cct aaa ggc ctc gag agc 1386 Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys Gly Leu Glu Ser 375 380 385 ctt gac ac gtaagcgcac actcgctaat cctcatgaac cggttgtact aaccgattca 1444 Leu Asp Th 390 g c cct gga acg tcg cat atc gct gct gtt gat cat act gga ctt act 1491 r Pro Gly Thr Ser His Ile Ala Ala Val Asp His Thr Gly Leu Thr 395 400 405 att tcc act atc aca acc atc aac ttg ctt ttc ggc agc aaa gtc atg 1539 Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu Phe Gly Ser Lys Val Met 410 415 420 gtg cca gag acg gga ata att atg aac aat gaa atg gat g gttggtgatc 1589 Val Pro Glu Thr Gly Ile Ile Met Asn Asn Glu Met Asp A 425 430 435 acccagaaag cctctgctcc agccctgaac ctacatgctg atactattca g ac ttc 1645 sp Phe tca aca ccc ggc tcg tcc aac gca ttt gga tac gtc cca tcg gag gcg 1693 Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala 440 445 450 aac ttc att cga ccc ggg aag cga cct ctg tct tct att act cca acc 1741 Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr 455 460 465 att gtg acg cat cag aat gga tct gtc ttt ttc gtt gca ggt tcg gct 1789 Ile Val Thr His Gln Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala 470 475 480 485 gga gga agt cga atc att aca gcg acc gtg cag aac atc atc cat gct 1837 Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala 490 495 500 gtt gat gag ggc ttg tcg gca gcc gaa gct ttg gct cga cca cgt ctt 1885 Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu 505 510 515 cac gat caa tta atc cct aac cag gtc aga ttc gag tat gcc tac aat 1933 His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe Glu Tyr Ala Tyr Asn 520 525 530 aat gaa acg gtt gct ttc atg aaa tct cta ggt cac aat gtg acc tgg 1981 Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly His Asn Val Thr Trp 535 540 545 gtc gct cca gga gac agc act gcg cag gca atc cgg gta tta ccg aac 2029 Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn 550 555 560 565 gga aca ttc gat gcg gct ggg gag cct agg cag ctt gac tct ggt ggt 2077 Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly 570 575 580 ttc gcg gtc tga ggacactctc tagctgcagc catgcagtct aagggctatc tgcca 2134 Phe Ala Val Stop 585 tgacggtcgc aatgatagag taaacctatc ttttgacatt tttcgagcat cgacaagctt 2194 atatgaaaac ttggaaagac acctgaagat atagatagaa ggttcttcaa ttgcgaatac 2254 atataggaag agaaatcttg tggatatgct ctaga 2289<210> 9 <211> 2289 <212> DNA <213> Aspergillus sojae BA-104 <223> DNA coding for glutaminase <400> 9 atg cat cgt ttt tta agg aat acg tta tcc ctc agc ttc ata cta ctt 48 Met His Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Leu 1 5 10 15 ctg gca ttg ata cat cta ccg gat gtg ctc tct tca cca cta gag ccg 96 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 tcg gct cac tac gac tcg cga agc gcc aat aca gtt gag cat ctc gag 144 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Leu Glu 35 40 45 gag ggc aag ctc gga gca gtg gct agc gag agt gct att tgc agt cgt 192 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 cat ggg aca gaa atg ctg aag ata ggc gga aat gct gcc gat gcc gtaag 242 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala 65 70 75 taacagtgac ttagcctcct agttatattg acctggctga gatacaatgc ag tta gtt 300 Leu Val 80 gcg aca ata ttc tgt gta ggg gtt gtt g gtgagataca aaaaagtagc ggtga 353 Ala Thr Ile Phe Cys Val Gly Val Val G 85 90 atatatgtga actgacaaaa gggtaccag ga atg tac cac agt gga atc gct 405 ly Met Tyr His Ser Gly Ile Ala 95 gga gga ggg ttt atg tta ctg g gt gt c gc gct gc gct gc gct tac gaa ttc 453 Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr Glu Phe 100 105 110 atc gat ttc cgc gaa aca gcg cct gca gcc gcg ttc gag gaa atg ttc 501 Ile Asp Phe Arg Gla Ala Pro Ala Phe Glu Glu Met Phe 115 120 125 130 aag aac aac acc gac gca tcc act tct ggg ggt ttg gca ag gtaacgggca 552 Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Se 135 140 ctccctcgat caaaccgagg aacagaggacagtagtatag cct 606 r Gly Val Pro 145 ggc gag gtg cga ggc ctg gaa tac ctt cac aag aag tat ggt tct ctt 654 Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr Gly Ser Leu 150 155 160 cca tgg tcg acc gtc atg ca cct gcc atc cag aca gcc cgt caa ggt 702 Pro Trp Ser Thr Val Met Gln Pro Ala Ile Gln Thr Ala Arg Gln Gly 165 170 175 ttc cca gtt ggt cgt gat ctc gtt cgc tat atg aac tcg gcc gtc ggg 750 Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser Ala Val Gly 180 185 190 195 gat ggg gaa gac ttc ctg tct aag gat ccg aca tgg gct att gac ttt 798 Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala Ile Asp Phe 200 205 210 gcc cct aat gga act cgt ctc ggt ctt gga gat acc atc acc cgt aaa 846 Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile Thr Arg Lys 215 220 225 cgc tac gca gac acg ctg gag aca att gcc aac aac ggc ccg gat gct 894 Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly Pro Asp Ala 230 235 240 ttt tat acc ggt cca att gcg gaa acc atg cag gcg ctg caa gcc 942 Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala Leu Gln Ala 245 250 255 gcg aac ggc aca atg acg cta gaa gac cta cgt aac tac acc att gct 990 Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr Thr Ile Ala 260 265 270 275 atc cga aac acc tcg cag att gac tat cga ggg tat aaa gtt att gga 1038 Ile Arg Asn Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Lys Val Ile Gly 280 285 285 290 act ag c gcg ccc tcc agc ggc act gtt gct ttg aac atc ctt aag gtt 1086 Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile Leu Lys Val 295 300 305 ttg gat act tat gac agc ttc atg gta cct gac aac gta aac ctt agc 1134 Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val Asn Leu Ser 310 315 320 acg cat cgt ttg gac gaa gct ata cgt ttc gga tat gga ctg gtacgtgga 1185 Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly Leu 325 330 335 tgcatctctt tatagtgacc ccaaccacct agctaattcg acatcaag aga gcc gaa 1242 Arg Ala Glu 340 ctg ggt gat ccg tac ttc ctt gaa gga ttg gat gaa tac cag aag gaa 1290 Leu Glu Glu Glu Glu Gly 345 350 355 atg ctg gaa cag tcg act att gat gag att cgt ggg aaa att tcc gat 1338 Met Leu Glu Gln Ser Thr Ile Asp Glu Ile Arg Gly Lys Ile Ser Asp 360 365 370 ttg cac aca cag aac gtg tct gct tat gat aaa ggc ctc gag agc 1386 Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys Gly Leu Glu Ser 375 380 385 ctt gac ac gtaagcgcac actcgctaat cctcatgaac cggttgtact a accgattca 1444 Leu Asp Th 390 gc cct gga acg tcg cat atc gct gct gtt gat cat act gga ctt act 1491 r Pro Gly Thr Ser His Ile Ala Ala Val Asp His Thr Gly Leu Thr 395 400 405 att tcc act atc aca acc atc aac ttg ctt ttc ggc agc aaa gtc atg 1539 Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu Phe Gly Ser Lys Val Met 410 415 420 gtg cca gag acg gga ata att atg aac aat gaa atg gat g gttggtgatc 1589 Val Pro Glu Thr Gly Ile Met Asn Asn Glu Met Asp A 425 430 435 acccagaaag cctctgctcc agccctgaac ctacatgctg atactattca g ac ttc 1645 sp Phe tca aca ccc ggc tcg tcc aac gca ttt gga tac gtc cca tcg gag GcG Ser A Ser G Ser G Ser G Ser G Ser G Ser Ser G Ser Val Pro Ser Glu Ala 440 445 450 aac ttc att cga ccc ggg aag cga cct ctg tct tct att act cca acc 1741 Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr 455 460 465 att gtg acg cat cag aat gga tct gtc ttt ttc gtt gca ggt tcg gct 1789 Ile Val Thr His Gln Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala 470 475 480 485 gga gga agt cga atc att aca gcg acc gtg cag aac atc atc cat gct 1837 Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala 490 495 500 gtt gat gag ggc ttg tcg gca gcc gaa gct ttg gct cga cca cgt ctt 1885 Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu 505 510 515 cac gat caa tta atc cct aac cag gtc aga ttc gag tat gcc tac aat 1933 His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe Glu Tyr Ala Tyr Asn 520 530 530 aat gaa acg gtt gct ttc atg aaa tct cta ggt cac aat gtg acc tgg 1981 Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly His Asn Val Thr Trp 535 540 545 gtc gct cca gga gac agc act gcg cag gca atc cgg gta tta ccg aac 2029 Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn 550 555 560 565 gga aca ttc gat gcg gct ggg gag cct agg cag ctt gac tct ggt ggt 2077 Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly 570 575 580 ttc gcg gtc tga ggacactctc tagctgcagc catgcagtct aagggctatc tgcca 2134 Phe Ala Val Stop 585 tgacggtcgc aatgatagag taaacctatc ttttgacatt tttcgagcat cga 2194 atatgaaaac ttggaaagac acctgaagat atagatagaa ggttcttcaa ttgcgaatac 2254 atataggaag agaaatcttg tggatatgct ctaga 2289

【００８７】 〈２１０〉 10 〈２１１〉 45 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of amino acid sequence of glutaminase 〈４００〉 10 Met Asn Asn Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala 1 5 10 15 Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg 20 25 30 Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 35 40 45 <210> 10 <211> 45 <212> PRT <213> Aspergillus oryzae KBN616 <223> A part of amino acid sequence of glutaminase <400> 10 Met Asn Asn Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala 1 5 10 15 Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg 20 25 30 Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 35 40 45

【００８８】 〈２１０〉 11 〈２１１〉 142 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of amino acid sequence of glutaminase 〈４００〉 11 Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro 1 5 10 15 Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 20 25 30 Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly Gly Ser Arg Ile 35 40 45 Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val Asp Glu Gly Leu 50 55 60 Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His Asp Gln Leu Ile 65 70 75 80 Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala 85 90 95 Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val Ala Pro Gly Asp 100 105 110 Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly Thr Phe Asp Ala 115 120 125 Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe Ala Val 130 135 140 <210> 11 <211> 142 <212> PRT <213> Aspergillus oryzae KBN616 <223> A part of amino acid sequence of glutaminase <400> 11 Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro 1 5 10 15 Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 20 25 30 Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly Gly Ser Arg Ile 35 40 45 Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val Asp Glu Gly Leu 50 55 60 Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His Asp Gln Leu Ile 65 70 75 80 Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala 85 90 95 Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val Ala Pro Gly Asp 100 105 110 Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly Thr Phe Asp Ala 115 120 125 Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe Ala Val 130 135 140

【００８９】 〈２１０〉 12 〈２１１〉 474 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of amino acid sequence of glutaminase 〈４００〉 12 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln 465 470 <210> 12 <211> 474 <212> PRT <213> Aspergillus oryzae KBN616 <223> A part of amino acid sequence of glutaminase <400> 12 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Il e Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr A sp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln 465 470

【００９０】 〈２１０〉 13 〈２１１〉 142 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of amino acid sequence of glutaminase 〈４００〉 13 Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro 1 5 10 15 Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 20 25 30 Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly Gly Ser Arg Ile 35 40 45 Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val Asp Glu Gly Leu 50 55 60 Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His Asp Gln Leu Ile 65 70 75 80 Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala 85 90 95 Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val Ala Pro Gly Asp 100 105 110 Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly Thr Phe Asp Ala 115 120 125 Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe Ala Val 130 135 140 <210> 13 <211> 142 <212> PRT <213> Aspergillus oryzae KBN616 <223> A part of amino acid sequence of glutaminase <400> 13 Ser Asn Ala Phe Gly Tyr Val Pro Ser Glu Ala Asn Phe Ile Arg Pro 1 5 10 15 Gly Lys Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln 20 25 30 Asn Gly Ser Val Phe Phe Val Ala Gly Ser Ala Gly Gly Ser Arg Ile 35 40 45 Ile Thr Ala Thr Val Gln Asn Ile Ile His Ala Val Asp Glu Gly Leu 50 55 60 Ser Ala Ala Glu Ala Leu Ala Arg Pro Arg Leu His Asp Gln Leu Ile 65 70 75 80 Pro Asn Gln Val Arg Phe Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala 85 90 95 Phe Met Lys Ser Leu Gly His Asn Val Thr Trp Val Ala Pro Gly Asp 100 105 110 Ser Thr Ala Gln Ala Ile Arg Val Leu Pro Asn Gly Thr Phe Asp Ala 115 120 125 Ala Gly Glu Pro Arg Gln Leu Asp Ser Gly Gly Phe Ala Val 130 135 140

【００９１】 〈２１０〉 14 〈２１１〉 584 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 Amino acid sequence of glutaminase 〈４００〉 14 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly 530 535 540 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 575 Leu Asp Ser Gly Gly Phe Ala Val 580 <210> 14 <211> 584 <212> PRT <213> Aspergillus oryzae KBN616 <223> Amino acid sequence of glutaminase <400> 14 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly 530 535 540 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 575 Leu Asp Ser Gly Gly Phe Ala Val 580

【００９２】 〈２１０〉 15 〈２１１〉 474 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of amino acid sequence of glutaminase 〈４００〉 15 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln 465 470 <210> 15 <211> 474 <212> PRT <213> Aspergillus oryzae KBN616 <223> A part of amino acid sequence of glutaminase <400> 15 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Il e Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr A sp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln 465 470

【００９３】 〈２１０〉 16 〈２１１〉 584 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 Amino acid sequence of glutaminase 〈４００〉 16 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly 530 535 540 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 575 Leu Asp Ser Gly Gly Phe Ala Val 580 <210> 16 <211> 584 <212> PRT <213> Aspergillus oryzae KBN616 <223> Amino acid sequence of glutaminase <400> 16 Met Tyr Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Val 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Phe Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Ile Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asp Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Arg 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Thr Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Asp Met Leu Glu Gln Ser Thr Ile Asp Glu Ile His Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly 530 535 540 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 575 Leu Asp Ser Gly Gly Phe Ala Val 580

【００９４】 〈２１０〉 17 〈２１１〉 584 〈２１２〉 PRT 〈２１３〉 Aspergillus sojae BA-104 〈２２３〉 Amino acid sequence of glutaminase 〈４００〉 17 Met His Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Leu 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Leu Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Val Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asn Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Lys 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Ala Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Glu Met Leu Glu Gln Ser Thr Ile Asp Glu Ile Arg Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly 530 535 540 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 575 Leu Asp Ser Gly Gly Phe Ala Val 580 <210> 17 <211> 584 <212> PRT <213> Aspergillus sojae BA-104 <223> Amino acid sequence of glutaminase <400> 17 Met His Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Leu 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Leu Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Val Met Gln Pro Ala Ile Gln Thr A la 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asn Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Lys 275 280 285 val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Ala Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Glu Met Leu Glu Gln Ser Thr Ile Asp Glu Ile Arg Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 Glu Tyr Thr Tyr Asn Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly 530 535 540 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 575 Leu Asp Ser Gly Gly Phe Ala Val 580

【００９５】 〈２１０〉 18 〈２１１〉 584 〈２１２〉 PRT 〈２１３〉 Aspergillus sojae BA-104 〈２２３〉 Amino acid sequence of glutaminase 〈４００〉 18 Met His Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Leu 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Leu Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Val Met Gln Pro Ala Ile Gln Thr Ala 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asn Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Lys 275 280 285 Val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Ala Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Glu Met Leu Glu Gln Ser Thr Ile Asp Glu Ile Arg Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys 370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 Glu Tyr Ala Tyr Asn Asn Glu Thr Val Ala Phe Met Lys Ser Leu Gly 530 535 540 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 575 Leu Asp Ser Gly Gly Phe Ala Val 580 <210> 18 <211> 584 <212> PRT <213> Aspergillus sojae BA-104 <223> Amino acid sequence of glutaminase <400> 18 Met His Arg Phe Leu Arg Asn Thr Leu Ser Leu Ser Phe Ile Leu Leu 1 5 10 15 Leu Ala Leu Ile His Leu Pro Asp Val Leu Ser Ser Pro Leu Glu Pro 20 25 30 Ser Ala His Tyr Asp Ser Arg Ser Ala Asn Thr Val Glu His Leu Glu 35 40 45 Glu Gly Lys Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg 50 55 60 His Gly Thr Glu Met Leu Lys Ile Gly Gly Asn Ala Ala Asp Ala Leu 65 70 75 80 Val Ala Thr Ile Phe Cys Val Gly Val Val Gly Met Tyr His Ser Gly 85 90 95 Ile Ala Gly Gly Gly Phe Met Leu Val Arg Ala Pro Asn Gly Ser Tyr 100 105 110 Glu Phe Ile Asp Phe Arg Glu Thr Ala Pro Ala Ala Ala Phe Glu Glu 115 120 125 Met Phe Lys Asn Asn Thr Asp Ala Ser Thr Ser Gly Gly Leu Ala Ser 130 135 140 Gly Val Pro Gly Glu Val Arg Gly Leu Glu Tyr Leu His Lys Lys Tyr 145 150 155 160 Gly Ser Leu Pro Trp Ser Thr Val Met Gln Pro Ala Ile Gln Thr A la 165 170 175 Arg Gln Gly Phe Pro Val Gly Arg Asp Leu Val Arg Tyr Met Asn Ser 180 185 190 Ala Val Gly Asp Gly Glu Asp Phe Leu Ser Lys Asp Pro Thr Trp Ala 195 200 205 Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly Leu Gly Asp Thr Ile 210 215 220 Thr Arg Lys Arg Tyr Ala Asp Thr Leu Glu Thr Ile Ala Asn Asn Gly 225 230 235 240 Pro Asp Ala Phe Tyr Thr Gly Pro Ile Ala Glu Thr Met Ile Gln Ala 245 250 255 Leu Gln Ala Ala Asn Gly Thr Met Thr Leu Glu Asp Leu Arg Asn Tyr 260 265 270 Thr Ile Ala Ile Arg Asn Thr Ser Gln Ile Asp Tyr Arg Gly Tyr Lys 275 280 285 val Ile Gly Thr Ser Ala Pro Ser Ser Gly Thr Val Ala Leu Asn Ile 290 295 300 Leu Lys Val Leu Asp Thr Tyr Asp Ser Phe Met Val Pro Asp Asn Val 305 310 315 320 Asn Leu Ser Thr His Arg Leu Asp Glu Ala Ile Arg Phe Gly Tyr Gly 325 330 335 Leu Arg Ala Glu Leu Gly Asp Pro Tyr Phe Leu Glu Gly Leu Asp Glu 340 345 350 Tyr Gln Lys Glu Met Leu Glu Gln Ser Thr Ile Asp Glu Ile Arg Gly 355 360 365 Lys Ile Ser Asp Leu His Thr Gln Asn Val Ser Ala Tyr Asp Pro Lys370 375 380 Gly Leu Glu Ser Leu Asp Thr Pro Gly Thr Ser His Ile Ala Ala Val 385 390 395 400 Asp His Thr Gly Leu Thr Ile Ser Thr Ile Thr Thr Thr Ile Asn Leu Leu 405 410 415 Phe Gly Ser Lys Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn 420 425 430 Glu Met Asp Asp Phe Ser Thr Pro Gly Ser Ser Asn Ala Phe Gly Tyr 435 440 445 Val Pro Ser Glu Ala Asn Phe Ile Arg Pro Gly Lys Arg Pro Leu Ser 450 455 460 Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly Ser Val Phe Phe 465 470 475 480 Val Ala Gly Ser Ala Gly Gly Ser Arg Ile Ile Thr Ala Thr Val Gln 485 490 495 Asn Ile Ile His Ala Val Asp Glu Gly Leu Ser Ala Ala Glu Ala Leu 500 505 510 Ala Arg Pro Arg Leu His Asp Gln Leu Ile Pro Asn Gln Val Arg Phe 515 520 525 Glu Tyr Ala Tyr Asn Asn Glu Thr Val Val Ala Phe Met Lys Ser Leu Gly 530 535 540 His Asn Val Thr Trp Val Ala Pro Gly Asp Ser Thr Ala Gln Ala Ile 545 550 555 560 Arg Val Leu Pro Asn Gly Thr Phe Asp Ala Ala Gly Glu Pro Arg Gln 565 570 575 Leu Asp Ser Gly Gly Phe Ala Val 580

【００９６】 〈２１０〉 19 〈２１１〉 20 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of amino acid sequence of glutaminase 〈４００〉 19 Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg His Gly Thr 5 10 15 Glu Met Leu Lys 20 <210> 19 <211> 20 <212> PRT <213> Aspergillus oryzae KBN616 <223> A part of amino acid sequence of glutaminase <400> 19 Leu Gly Ala Val Ala Ser Glu Ser Ala Ile Cys Ser Arg His Gly Thr 5 10 15 Glu Met Leu Lys 20

【００９７】 〈２１０〉 20 〈２１１〉 24 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of amino acid sequence of glutaminase 〈４００〉 20 Asp Pro Thr Trp Ala Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly 5 10 15 Leu Gly Asp Thr Ile Thr Arg Lys 20 <210> 20 <211> 24 <212> PRT <213> Aspergillus oryzae KBN616 <223> A part of amino acid sequence of glutaminase <400> 20 Asp Pro Thr Trp Ala Ile Asp Phe Ala Pro Asn Gly Thr Arg Leu Gly 5 10 15 Leu Gly Asp Thr Ile Thr Arg Lys 20

【００９８】 〈２１０〉 21 〈２１１〉 17 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of amino acid sequence of glutaminase 〈４００〉 21 Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn Glu Met Asp Asp 5 10 15 Phe <210> 21 <211> 17 <212> PRT <213> Aspergillus oryzae KBN616 <223> A part of amino acid sequence of glutaminase <400> 21 Val Met Val Pro Glu Thr Gly Ile Ile Met Asn Asn Glu Met Asp Asp 5 10 15 Phe

【００９９】 〈２１０〉 22 〈２１１〉 25 〈２１２〉 PRT 〈２１３〉 Aspergillus oryzae KBN616 〈２２３〉 A part of amino acid sequence of glutaminase 〈４００〉 22 Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly 5 10 15 Ser Val Phe Phe Val Ala Gly Ser Ala 20 25 <210> 22 <211> 25 <212> PRT <213> Aspergillus oryzae KBN616 <223> A part of amino acid sequence of glutaminase <400> 22 Arg Pro Leu Ser Ser Ile Thr Pro Thr Ile Val Thr His Gln Asn Gly 5 10 15 Ser Val Phe Phe Val Ala Gly Ser Ala 20 25

【０１００】 〈２１０〉 23 〈２１１〉 20 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 P1 Primer 〈４００〉 23 gtyatggtyc cygaracygg 20<210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P1 Primer <400> 23 gtyatggtyc cygaracygg 20

【０１０１】 〈２１０〉 24 〈２１１〉 20 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 P2 Primer 〈４００〉 24 tgrtgrgtra cratrgtrgg 20<210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P2 Primer <400> 24 tgrtgrgtra cratrgtrgg 20

【０１０２】 〈２１０〉 25 〈２１１〉 20 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 P3 Primer 〈４００〉 25 atgaayaayg aratggayga 20<210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> P3 Primer <400> 25 atgaayaayg aratggayga 20

【０１０３】 〈２１０〉 26 〈２１１〉 22 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 3 Site Adaptor Primer 〈４００〉 26 ctgatctaga ggtaccggat cc 22<210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 3 Site Adapter Primer <400> 26 ctgatctaga ggtaccggat cc 22

【０１０４】 〈２１０〉 27 〈２１１〉 22 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 P4 Primer 〈４００〉 27 ccaacgcatt tggatacgtc cc 22<210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> P4 Primer <400> 27 ccaacgcatt tggatacgtc cc 22

【０１０５】 〈２１０〉 28 〈２１１〉 22 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 P5 Primer 〈４００〉 28 tgatcgtgaa gacgtggtct ag 22<210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> P5 Primer <400> 28 tgatcgtgaa gacgtggtct ag 22

【０１０６】 〈２１０〉 29 〈２１１〉 22 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 P6 Primer 〈４００〉 29 tctgatgcgt cacaatggtt gg 22<210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> P6 Primer <400> 29 tctgatgcgt cacaatggtt gg 22

【０１０７】 〈２１０〉 30 〈２１１〉 35 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 C1 Primer 〈４００〉 30 gtacatattg tcgttagaac gcgtaatacg actca 35<210> 30 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> C1 Primer <400> 30 gtacatattg tcgttagaac gcgtaatacg actca 35

【０１０８】 〈２１０〉 31 〈２１１〉 35 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 C2 Primer 〈４００〉 31 cgttagaacg cgtaatacga ctcactatag ggaga 35<210> 31 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> C2 Primer <400> 31 cgttagaacg cgtaatacga ctcactatag ggaga 35

【０１０９】 〈２１０〉 32 〈２１１〉 26 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 P7 Primer 〈４００〉 32 ccatgtatcg ttttttaagg aatacg 26<210> 32 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> P7 Primer <400> 32 ccatgtatcg ttttttaagg aatacg 26

【０１１０】 〈２１０〉 33 〈２１１〉 26 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 P8 Primer 〈４００〉 33 ccatgcatcg ttttttaagg aatacg 26<210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> P8 Primer <400> 33 ccatgcatcg ttttttaagg aatacg 26

【０１１１】 〈２１０〉 34 〈２１１〉 30 〈２１２〉 ＤＮＡ 〈２１３〉 Artificial Sequence 〈２２０〉 〈２２３〉 P9 Primer 〈４００〉 34 cctctagagc atatccacaa gatttctctt 30<210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> P9 Primer <400> 34 cctctagagc atatccacaa gatttctctt 30

【図面の簡単な説明】[Brief description of the drawings]

【図１】図１は本発明のグルタミナーゼの至適pHを示す
グラフである。FIG. 1 is a graph showing the optimum pH of the glutaminase of the present invention.

【図２】図２は本発明のグルタミナーゼのpH安定性を示
すグラフである。FIG. 2 is a graph showing the pH stability of the glutaminase of the present invention.

【図３】図３は本発明のグルタミナーゼの至適温度を示
すグラフである。FIG. 3 is a graph showing the optimum temperature of the glutaminase of the present invention.

【図４】図４は本発明のグルタミナーゼの温度安定性を
示すグラフである。FIG. 4 is a graph showing the temperature stability of the glutaminase of the present invention.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.⁷ 識別記号 ＦＩ テーマコート゛(参考） Ｃ１２Ｎ 5/10 (Ｃ１２Ｎ 1/14 Ｂ 9/78 Ｃ１２Ｒ 1:66） //(Ｃ１２Ｎ 1/14 (Ｃ１２Ｎ 1/14 Ｂ Ｃ１２Ｒ 1:66） Ｃ１２Ｒ 1:69） (Ｃ１２Ｎ 1/14 (Ｃ１２Ｎ 9/78 Ｃ１２Ｒ 1:69） Ｃ１２Ｒ 1:66） (Ｃ１２Ｎ 9/78 Ｃ１２Ｎ 15/00 ＺＮＡＡ Ｃ１２Ｒ 1:66） 5/00 Ａ (72)発明者 北本 則行 愛知県名古屋市守山区大字下志段味字風越 1931番地の１ (72)発明者 安田 庄子 愛知県名古屋市中川区水里五丁目273番地 (72)発明者 伊藤 誉 愛知県一宮市笹野字宮北田９番地 (72)発明者 畔柳 孝 愛知県豊橋市中岩田２−４−21 ハイカム ール中岩田Ｂ−102 Ｆターム(参考） 4B024 AA03 AA05 BA11 CA04 CA06 DA11 EA04 GA11 HA01 4B050 CC01 CC03 DD03 LL02 LL05 4B065 AA60Y AA63X AA63Y AB01 BA02 BA22 CA31 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 (C12N 1/14 B 9/78 C12R 1:66) // (C12N 1/14 (C12N 1/14 B C12R 1:66) C12R 1:69) (C12N 1/14 (C12N 9/78 C12R 1:69) C12R 1:66) (C12N 9/78 C12N 15/00 ZNAA C12R 1:66) 5 / 00 A (72) Inventor Noriyuki Kitamoto, 1931, Kazukoshi, Shimozushi, Taji, Oji, Moriyama-ku, Nagoya City, Aichi Prefecture (72) Inventor Shoko 5-273 Mizusato, Nakagawa-ku, Nagoya City, Aichi Prefecture (72) Inventor Takashi Ito 9-29 Miyakita, Sasano, Ichinomiya-shi, Aichi Prefecture (72) Inventor Takashi Kuroyanagi 2-4-21 Naka-Iwata, Toyohashi-shi, Aichi Hikamuru Naka-Iwata B-102 F-term (reference) 4B024 AA03 AA05 BA11 CA04 CA06 DA11 EA04 GA11 HA01 4B050 CC01 CC03 DD03 LL02 LL05 4B065 AA60Y AA63X AA63Y AB01 BA02 BA22 CA31

Claims (26)

【特許請求の範囲】[Claims] 【請求項１】 次の性質： （１）作用：Ｌ−グルタミンに作用してＬ−グルタミン
酸を生成する； （２）至適pH：至適pHが約８．５である； （３）安定pH範囲：４℃にて２４時間インキュベートし
た場合、安定pH範囲はおよそ５〜１０である； （４）至適作用温度：pH８．０において測定した場合、
至適作用温度は約４５℃である； （５）温度安定性：pH８．０にて３０分間インキュベー
トした場合、４０℃以下の温度で安定である； （６）分子量：ゲル濾過により測定した場合、分子量は
約７１，０００である；を有するグルタミナーゼ。1. The following properties: (1) action: acts on L-glutamine to form L-glutamic acid; (2) optimal pH: optimal pH is about 8.5; (3) stable pH range: when incubated at 4 ° C. for 24 hours, the stable pH range is approximately 5-10; (4) Optimal operating temperature: when measured at pH 8.0,
(5) Temperature stability: stable at a temperature of 40 ° C. or less when incubated at pH 8.0 for 30 minutes; (6) Molecular weight: measured by gel filtration Glutaminase having a molecular weight of about 71,000. 【請求項２】 配列番号：１４に示すアミノ酸配列にお
いて、アミノ酸１〜５８４のアミノ酸配列を有するグル
タミナーゼ。2. A glutaminase having the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 14. 【請求項３】 配列番号：１４に示すアミノ酸配列中の
アミノ酸番号１〜５８４のアミノ酸配列において、１個
又は複数個のアミノ酸の欠失、付加及び／又は置換によ
り修飾されたアミノ酸配列を有し、且つグルタミナーゼ
活性を有する修飾されたグルタミナーゼ。3. The amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 14 having an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acids. And a modified glutaminase having glutaminase activity. 【請求項４】 配列番号：５に示す塩基配列を有する核
酸のエクソン領域とストリンジエント条件下でハイブリ
ダイズすることができる核酸によりコードされているグ
ルタミナーゼ。4. A glutaminase encoded by a nucleic acid capable of hybridizing under stringent conditions to an exon region of a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 5. 【請求項５】 配列番号：１４に示すアミノ酸配列中の
アミノ酸番号１〜５８４のアミノ酸配列に対して７０％
以上の相同性を有するアミノ酸配列を有するグルタミナ
ーゼ。5. 70% of the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 14
Glutaminase having an amino acid sequence having the above homology. 【請求項６】 配列番号：１６に示すアミノ酸配列にお
いて、アミノ酸１〜５８４のアミノ酸配列を有するグル
タミナーゼ。6. A glutaminase having the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 16. 【請求項７】 配列番号：１６に示すアミノ酸配列中の
アミノ酸番号１〜５８４のアミノ酸配列において、１個
又は複数個のアミノ酸の欠失、付加及び／又は置換によ
り修飾されたアミノ酸配列を有し、且つグルタミナーゼ
活性を有する修飾されたグルタミナーゼ。7. The amino acid sequence of amino acid numbers 1 to 584 in the amino acid sequence shown in SEQ ID NO: 16 having an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acids. And a modified glutaminase having glutaminase activity. 【請求項８】 配列番号：７に示す塩基配列を有する核
酸とストリンジエント条件下でハイブリダイズすること
ができる核酸によりコードされているグルタミナーゼ。8. A glutaminase encoded by a nucleic acid capable of hybridizing under stringent conditions with a nucleic acid having the nucleotide sequence of SEQ ID NO: 7. 【請求項９】 配列番号：１６に示すアミノ酸配列中の
アミノ酸番号１〜５８４のアミノ酸配列に対して７０％
以上の相同性を有するアミノ酸配列を有するグルタミナ
ーゼ。9. 70% of the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 16
Glutaminase having an amino acid sequence having the above homology. 【請求項１０】 配列番号：１７に示すアミノ酸配列に
おいて、アミノ酸１〜５８４のアミノ酸配列を有するグ
ルタミナーゼ。10. A glutaminase having the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 17. 【請求項１１】 配列番号：１７に示すアミノ酸配列中
のアミノ酸番号１〜５８４のアミノ酸配列において、１
個又は複数個のアミノ酸の欠失、付加及び／又は置換に
より修飾されたアミノ酸配列を有し、且つグルタミナー
ゼ活性を有する修飾されたグルタミナーゼ。11. In the amino acid sequence of amino acid numbers 1 to 584 in the amino acid sequence shown in SEQ ID NO: 17,
A modified glutaminase having an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acids and having glutaminase activity. 【請求項１２】 配列番号：８に示す塩基配列を有する
核酸のエクソン領域とストリンジエント条件下でハイブ
リダイズすることができる核酸によりコードされている
グルタミナーゼ。12. A glutaminase encoded by a nucleic acid capable of hybridizing under stringent conditions to an exon region of a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 8. 【請求項１３】 配列番号：１７に示すアミノ酸配列中
のアミノ酸番号１〜５８４のアミノ酸配列に対して７０
％以上の相同性を有するアミノ酸配列を有するグルタミ
ナーゼ。13. An amino acid sequence represented by SEQ ID NO: 17
A glutaminase having an amino acid sequence having homology of at least%. 【請求項１４】 配列番号：１８に示すアミノ酸配列に
おいて、アミノ酸１〜５８４のアミノ酸配列を有するグ
ルタミナーゼ。14. A glutaminase having the amino acid sequence of amino acids 1 to 584 in the amino acid sequence shown in SEQ ID NO: 18. 【請求項１５】 配列番号：１８に示すアミノ酸配列中
のアミノ酸番号１〜５８４のアミノ酸配列において、１
個又は複数個のアミノ酸の欠失、付加及び／又は置換に
より修飾されたアミノ酸配列を有し、且つグルタミナー
ゼ活性を有する修飾されたグルタミナーゼ。15. In the amino acid sequence of amino acids 1 to 584 in the amino acid sequence of SEQ ID NO: 18,
A modified glutaminase having an amino acid sequence modified by deletion, addition and / or substitution of one or more amino acids and having glutaminase activity. 【請求項１６】 配列番号：９に示す塩基配列を有する
核酸のエクソン領域とストリンジエント条件下でハイブ
リダイズすることができる核酸によりコードされている
グルタミナーゼ。16. A glutaminase encoded by a nucleic acid capable of hybridizing under stringent conditions to an exon region of a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 9. 【請求項１７】 配列番号：１８に示すアミノ酸配列中
のアミノ酸番号１〜５８４のアミノ酸配列に対して７０
％以上の相同性を有するアミノ酸配列を有するグルタミ
ナーゼ。17. An amino acid sequence represented by SEQ ID NO: 18
A glutaminase having an amino acid sequence having homology of at least%. 【請求項１８】 請求項２〜１７のいずれか１項に記載
のグルタミナーゼをコードするＤＮＡ。A DNA encoding the glutaminase according to any one of claims 2 to 17. 【請求項１９】 イントロンを含んで成る請求項１８に
記載のＤＮＡ。19. The DNA of claim 18, comprising an intron. 【請求項２０】 ゲノムＤＮＡである請求項１９に記載
のＤＮＡ。20. The DNA according to claim 19, which is a genomic DNA. 【請求項２１】 ｃＤＮＡである請求項１８に記載のグ
ルタミナーゼをコードするＤＮＡ。21. A DNA encoding the glutaminase according to claim 18, which is a cDNA. 【請求項２２】 請求項１８〜２１のいずれか１項に記
載のＤＮＡを含んで成るベクター。A vector comprising the DNA according to any one of claims 18 to 21. 【請求項２３】 請求項２２に記載のベクターにより形
質転換された宿主細胞。23. A host cell transformed by the vector according to claim 22. 【請求項２４】 請求項１に記載のグルタミナーゼの製
造方法において、アスペルギルス（Aspergillus ）属に
属し、該グルタミナーゼを生産することができる微生物
を培養し、培養物からグルタミナーゼを採取することを
特徴とする方法。24. The method for producing glutaminase according to claim 1, wherein a microorganism belonging to the genus Aspergillus and capable of producing the glutaminase is cultured, and the glutaminase is collected from the culture. Method. 【請求項２５】 前記微生物が、アスペルギルス・ソヤ
ー（Aspergillus sojae）又はアスペルギルス・オリゼ
ー（Aspergillus oryzae）である、請求項２４に記載の
方法。25. The method according to claim 24, wherein the microorganism is Aspergillus sojae or Aspergillus oryzae. 【請求項２６】 請求項２〜１７のいずれか１項に記載
のグルタミナーゼの製造方法において、該グルタミナー
ゼをコードするＤＮＡを含んで成る発現ベクターにより
形質転換された宿主細胞を培養し、該培養物から、該グ
ルタミナーゼを採取することを特徴とする方法。26. The method for producing glutaminase according to any one of claims 2 to 17, wherein a host cell transformed with an expression vector comprising a DNA encoding the glutaminase is cultured, and the culture is performed. A method of collecting the glutaminase from the above.