JPS6075280A - Production of aspergillus by protoplast fusion - Google Patents

Production of aspergillus by protoplast fusion

Info

Publication number
JPS6075280A
JPS6075280A JP58180798A JP18079883A JPS6075280A JP S6075280 A JPS6075280 A JP S6075280A JP 58180798 A JP58180798 A JP 58180798A JP 18079883 A JP18079883 A JP 18079883A JP S6075280 A JPS6075280 A JP S6075280A
Authority
JP
Japan
Prior art keywords
aspergillus
strain
glutaminase
medium
protease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
JP58180798A
Other languages
Japanese (ja)
Other versions
JPH0371877B2 (en
Inventor
Shigeomi Ushijima
牛島 重臣
Tadanobu Nakadai
中台 忠信
Kinji Uchida
内田 金治
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kikkoman Corp
Original Assignee
Kikkoman Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kikkoman Corp filed Critical Kikkoman Corp
Priority to JP58180798A priority Critical patent/JPS6075280A/en
Publication of JPS6075280A publication Critical patent/JPS6075280A/en
Publication of JPH0371877B2 publication Critical patent/JPH0371877B2/ja
Granted legal-status Critical Current

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Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/02Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
    • C12N15/04Fungi

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  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Soy Sauces And Products Related Thereto (AREA)
  • Molecular Biology (AREA)

Abstract

PURPOSE:To obtain an aspergillus strain having high activity of produce protease and glutaminase, by preparing a protoplast fusion cell of a strain having high activity to produce protease and belonging to Aspergillus genus and a strain having high activity to produce glutaminase, and culturing the obtained fusion cell in a hypertonic regeneration medium. CONSTITUTION:A protoplast is obtained from a strain having high activity to produce protease and belonging to Aspergillus genus (e.g. Aspergillus sojae ATCC 42249) and a strain having high activity to produce glutaminase [e.g. Aspergillus sojae 262 (FERM-P No.2128)] by the use of a cell wall lyase (e.g. a combination of beta-1,3-glucanase and chitinase). The fused cell obtained by the protoplast fusion is cultured in a hypertonic regeenration medium (e.g. a medium obtained by adding agar to the soup of malted rice containing sorbitol), and an aspergillus strain capable of highly producing both protease and glutaminase is separated from the cultured product.

Description

【発明の詳細な説明】 本発明は、プロトプラスト融合による高プロテアーゼ生
産能を有し且つ高グルタミナーゼ生産能を有する麹菌の
製造法に関する。
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing Aspergillus oryzae having a high protease-producing ability and a high glutaminase-producing ability by protoplast fusion.

醤油は、アミノ酸の旨味を主体とする調味料であるので
、醤油の醸造に於いては窒素利用率の向上とグルタミン
酸生成量の向上に多大の考慮が払われており、このよう
なことから醤油醸造業界にとって強力にプロテアーゼと
グルタミナーゼを生産する麹菌を得ることは極めて重要
である。
Soy sauce is a seasoning whose flavor is mainly derived from amino acids, so when brewing soy sauce, great consideration is given to improving the nitrogen utilization rate and increasing the amount of glutamic acid produced. It is extremely important for the brewing industry to obtain koji molds that produce potent protease and glutaminase.

従来、このような優良な麹菌を得る方法の1手段として
人工変異により酵素生産能を高くする方法が知られてい
るが、取得される変異株はプロテアーゼ或いはグルタミ
ナーゼのうち一方について親株に比べ高くとも他方は逆
に低い場合が多く、両方とも親株に比べ高い菌株を取得
することは困難であった。
Conventionally, one method of obtaining such excellent koji molds is to increase the enzyme production ability by artificial mutation, but the obtained mutant strain has at least a higher level of protease or glutaminase than the parent strain. On the other hand, in many cases the values were low, and it was difficult to obtain strains with both values higher than the parent strain.

そこで、本発明者らは、高プロテアーゼ生産能を有し且
つ高グルタミナーゼ生産能を有する麹菌を得る目的で種
々検討を重ねた結果、アスペルギルス属に属する、高プ
ロテアーゼ生産株と高グルタミナーゼ生産株とをプロト
プラスト融合させ、該融合細胞を高張再生培地に培養し
たところ、そこから高プロテアーゼ生産能を有し且つ高
グルタミナーゼ生産能を有する麹菌が取得できることを
知シ、この知見に基いて本発明を完成した。
Therefore, as a result of various studies aimed at obtaining Aspergillus aspergillus having high protease-producing ability and high glutaminase-producing ability, the present inventors have developed a high-protease-producing strain and a high-glutaminase-producing strain belonging to the genus Aspergillus. When protoplasts were fused and the fused cells were cultured in a hypertonic regeneration medium, the inventors discovered that Aspergillus oryzae having a high protease-producing ability and a high glutaminase-producing ability could be obtained from the fused cells, and based on this knowledge, they completed the present invention. .

すなわち本発明は、アスペルギルス属に属する、高プロ
テアーゼ生産株と高グルタミナーゼ生産株のそれぞれの
培養細胞からプロトプラストを得、これらをプロトプラ
スト融合させ、該融合細胞を高張再生培地に培養し、培
養物から高プロテアーゼ生産能を有し且つ高グルタミナ
ーゼ生産能を有する麹菌を採取することを特徴とする麹
菌の製造法である。
That is, the present invention obtains protoplasts from cultured cells of a high protease-producing strain and a high glutaminase-producing strain belonging to the genus Aspergillus, fuses these with protoplasts, cultures the fused cells in a hypertonic regeneration medium, and extracts hypertonic cells from the culture. This is a method for producing Aspergillus oryzae, which is characterized by collecting Aspergillus oryzae having protease production ability and high glutaminase production ability.

以下、本発明の詳細な説明する。The present invention will be explained in detail below.

先ず本発明で使用される、アスペルギルス属に属する高
プロテアーゼ生産株としては、アスペルギルス属に属し
、プロテアーゼ生産能の高い菌株であればいずれでもよ
く、醤油、味噌、清酒等の製造に用いられる醸造用麹菌
の中からプロテアーゼ生産能の高い菌株を検索すること
によシ容易に入手することができる。その具体例として
はアスペルギルス・ソーヤ ATCC42249、同4
2250、同42251等が挙げられるが、更にこれら
の変種又は変異株も使用することができる。
First, the high protease-producing strain belonging to the genus Aspergillus used in the present invention may be any strain as long as it belongs to the genus Aspergillus and has a high protease-producing ability. It can be easily obtained by searching for a strain of koji mold with high protease production ability. A specific example is Aspergillus sojae ATCC42249, ATCC4
2250, 42251, etc., but variants or mutant strains of these can also be used.

また、アスペルギルス属に属する高グルタミナーゼ生産
株としては、アスペルギルス属に属し、グルタミナーゼ
生産能の高い菌株であればいずれでもよく、例えば醸造
用麹菌の中からグルタミナーゼ生産能の高い菌株を検索
することにより容易に入手することができる。その具体
例としては、アスペルギルス・ソーヤ262(FE几M
−P2128)、同2165(FB’RM P−7Zl
rO)等が挙げられるが、更にこれらの変種又は変異株
も使用することができる。
In addition, the high glutaminase producing strain belonging to the genus Aspergillus may be any strain as long as it belongs to the genus Aspergillus and has a high glutaminase producing ability. can be obtained. As a specific example, Aspergillus sawyer 262 (FE几M
-P2128), 2165 (FB'RM P-7Zl
rO) and the like, but variants or mutant strains of these can also be used.

次に上記生産株の培養細胞を得る方法としては該生産株
の分生胞子を栄養培地に培養することにより得られる。
Next, cultured cells of the above production strain can be obtained by culturing conidia of the production strain in a nutrient medium.

ここに用いられる栄養培地としては、麹菌が必要とする
栄養源を含有するものであればいずれでもよく、合成培
地、半合成培地または天然培地が用いられる。合成培地
としては炭嬌源としてグルコース、サッカロース等、窒
素源として硫安・1.硝安等が用いられる。その具体例
としては例えばツアペック(Czapck)培地に酵母
エキス0.5%及びカザミノ酸0.2%を加え、P H
6,0に調製したものを水で10倍に稀釈した培地(以
下、ツアペック変形培地と略記する)が挙げられる。ま
た天然培地の組成としては、炭素源として割砕小麦、皺
等、窒素源としてスキムミルク、脱脂大豆粉等が挙げら
れる、また上記培地にリン酸、カリウム、マグネシウム
、カルシウム等の適当な無機塩類を適宜使用することが
でき、さらに必要に応じて菌の生育に必要なアミノ酸、
ビタミン類等を培地に添加使用することができる。
The nutrient medium used here may be any medium as long as it contains the nutrients required by Aspergillus oryzae, and a synthetic medium, a semi-synthetic medium, or a natural medium may be used. As a synthetic medium, glucose, sucrose, etc. can be used as a carbon source, and ammonium sulfate can be used as a nitrogen source.1. Ammonium nitrate etc. are used. As a specific example, 0.5% of yeast extract and 0.2% of casamino acids are added to Czapck medium, and P H
Examples include a medium prepared at a concentration of 6.0 and diluted 10 times with water (hereinafter abbreviated as Czapek's modified medium). In addition, the composition of the natural medium includes cracked wheat, wrinkles, etc. as a carbon source, skim milk, defatted soybean flour, etc. as a nitrogen source, and appropriate inorganic salts such as phosphoric acid, potassium, magnesium, and calcium are added to the medium. Can be used as appropriate, and if necessary, amino acids necessary for bacterial growth,
Vitamins and the like can be added to the medium.

培養方法としては、上記の適当な培地、例えばツアペッ
ク変形培地を常法によυ加熱殺菌処理しろ菌子の長さを
合わせたもの)の長さかもとの胞子(培養開始時)外径
の5〜15倍に生育するのに充分な5〜15時間、静置
、振盪、通気培養等、好気的に培養する。また固体培養
を行う場合は、皺、小麦などの炭素源、大豆などの窒素
源、その他の有機質あるいは無機質を原料とし、これら
を蒸煮など殺菌処理を行った固体培地に種菌を接種混合
し、25〜38℃で、発芽胞子の長さかもとの胞子外径
の5〜15倍に生育するのに充分な時間、無菌的に培養
するのが好ましい。
As for the culture method, use the above-mentioned suitable medium, such as Czapek's modified medium, and heat sterilize it in a conventional manner. Cultivate aerobically by standing, shaking, aerating, etc. for 5 to 15 hours, which is sufficient to double the size. In addition, when performing solid culture, use carbon sources such as wrinkles, wheat, nitrogen sources such as soybeans, and other organic or inorganic materials as raw materials, inoculate and mix the inoculum into a solid medium that has been sterilized by steaming, etc. It is preferable to culture aseptically at ~38°C for a time sufficient to grow the germinated spores to a length of 5 to 15 times the original spore outer diameter.

本発明において、発芽胞子の長さかもとの胞子外径の5
〜15倍に生育した培養細胞を用いることは重要である
。即ち5倍未満であるときは麹菌のプロトプラストを殆
んど形成することができず、また反対に15倍を越える
と、麹菌のプロトプラストの形成率が非常に低くなるの
で好ましくない。
In the present invention, the length of germinated spores and the outer diameter of 5
It is important to use cultured cells that have grown ~15 times. That is, when it is less than 5 times, it is hardly possible to form protoplasts of Aspergillus oryzae, and on the other hand, when it exceeds 15 times, the formation rate of protoplasts of Aspergillus oryzae becomes extremely low, which is not preferable.

次に、こうして得られた培養細胞からプロトプラストを
得るには、上記各々の発芽胞子について、106〜10
8個/コ(高張液)の濃度の発芽胞子懸濁液を11il
製し、各調製懸濁液を等量混合し、遠心分離して集菌し
たのち、これに予じめ無菌処理した細胞壁溶解酵素を添
加し処理するか、又は各々の発芽胞子懸濁液を遠心分離
して集菌したのち、これを最初、縄胞壁溶解酵素を添加
して処理を行い、次いで各処理液を等量混合することに
よシ得られる。
Next, in order to obtain protoplasts from the cultured cells thus obtained, for each of the above germinated spores, 106 to 10
11 il of germinated spore suspension with a concentration of 8 spores/child (hypertonic solution)
After mixing equal amounts of each prepared suspension and collecting the bacteria by centrifugation, either add a cell wall lytic enzyme that has been sterilized in advance and treat it, or treat each germinated spore suspension. After collecting bacteria by centrifugation, this is first treated by adding a follicle wall lytic enzyme, and then equal amounts of each treatment solution are mixed.

ここに用いられる細胞壁溶解酵素としては、麹菌の細胞
壁を溶解する活性を有するものであればいずれでもよく
、セルラーゼ、β−1,3−グルカナーゼ及びキチナー
ゼ等を単独、または混合して用い、或いは一般に細胞壁
溶解酵素として用いられている混合酵素等を用いること
ができる。本発明者らが実験したところ、これらの酵素
のうちβ−1,3−グルカナーゼとキチナーゼを併用す
ると、麹菌のプロトプラストの形成率を著しく高めるこ
とが判明したので、上記2つの酵素を併用することが好
ましい。
The cell wall lytic enzyme used here may be any enzyme as long as it has the activity of lysing the cell wall of Aspergillus oryzae, and cellulase, β-1,3-glucanase, chitinase, etc. may be used alone or in combination, or in general. Mixed enzymes used as cell wall lytic enzymes can be used. As a result of experiments conducted by the present inventors, it was found that the combination of β-1,3-glucanase and chitinase among these enzymes significantly increases the protoplast formation rate of Aspergillus oryzae. is preferred.

上記キチナーゼとしては、キチナーゼ活性を有する酵素
剤が用いられ、例えば米国ICN社製キチナーゼ Jに
100466.米国シグマ社製キチナーゼ 75C−6
137、生化学工業社販売キチナーゼ・ファンガ/’ 
(f u n g aI ) IFh 100290等
が挙げられる。
As the chitinase, an enzyme agent having chitinase activity is used, such as Chitinase J and 100466. Chitinase 75C-6 manufactured by Sigma, USA
137, Chitinase Fanga sold by Seikagaku Corporation/'
(fun g aI ) IFh 100290 and the like.

またβ−1,3−グルカナーゼとしてはキリンビール社
製、ザイモリアーゼ(Zymolyase)5000、
同60000及び本発明者らが微生物バチラス・サーキ
ュランス IAM1165及びストレプトマイセス 0
143(FEBMP−7,27J)のそれぞれの培養液
から調製し7た2つの粗酵素等が挙げられるが、特に後
者の2つの粗酵素が好ましい。
In addition, as β-1,3-glucanase, Zymolyase 5000 manufactured by Kirin Brewery,
60,000 and the microorganisms Bacillus circulans IAM1165 and Streptomyces 0
143 (FEBMP-7, 27J), and the like, and the latter two crude enzymes are particularly preferred.

上記バチラス・サーキュランス IAM 1165から
調製した粗酵素及びストレプトマイセス0143株から
調製した粗酵素は以下のようにして得られたものである
The crude enzyme prepared from Bacillus circulans IAM 1165 and the crude enzyme prepared from Streptomyces strain 0143 were obtained as follows.

〔工〕ハチラス・サーキュランス IAMII65から
の粗酵素調製法 肉エキス1%、ポリペプトン1%、麹菌湿潤菌体2%及
び水からなる培地(PH7,2)1/を51容三角フラ
スコに入れ、高圧滅菌したものに、麹菌湿潤菌体を入れ
ない以外は上記と全く同じ培地で前培養(30℃で48
時間)したハチラス・サーキュランス IAM 116
5の種菌液10mを接種し、30℃で48時間、振盪培
養(170r、 p、 rll) した。こうして得た
培養液から粗酵素を得るには、これを遠心分離して上7
H液を分離取得し、これに硫安を加えて常法により塩析
し、生成した沈戯区分を採取した。これを水25Mに溶
解し、セロファンチー−プに入れ、1夜5℃で蒸留水を
用いて透析処理し、得られた透析内液を凍結乾燥し、粗
酵素(以下、「バチラス・サーキュランス起源の粗酵素
」と称す)を得だ。
[Engineering] Preparation of crude enzyme from Hachilas Circulans IAMII65 1% of meat extract, 1% of polypeptone, 2% of wet bacterial cells of Aspergillus oryzae and 1/2 of a medium (pH 7,2) of water was placed in a 51-volume Erlenmeyer flask and heated under high pressure. Pre-cultivate in the same medium as above except that the wet cells of Aspergillus oryzae are not added to the sterilized medium (48°C at 30°C).
Time) Hachilas Circulans IAM 116
5 was inoculated with 10 m of the inoculum solution, and cultured with shaking (170r, p, rll) at 30°C for 48 hours. To obtain the crude enzyme from the culture solution obtained in this way, centrifuge it and
The H solution was separated and obtained, ammonium sulfate was added thereto and salted out by a conventional method, and the resulting silt fraction was collected. This was dissolved in 25M water, placed in a cellophane Cheap, and dialyzed against distilled water overnight at 5°C. The original crude enzyme) was obtained.

[I’l ストレプトマイセス o143がらの粗酵素
調製法 培地組成 1 i< l−l2P 040.2 % 酵母エキス o、o59g KO,l O,05% グルコース 0.25% Mg504j 7 l−1200,1%ネオペプトン 
0.1 % NaC10,05% NaN0.、 0.1 % 麹菌湿潤菌体 2.1 % 蒸留水 1.0! P H5,0 上記組成の培地1(PH5,0)に、これと同一組成の
培地にて前培養したストレプトマイセス0143 (F
 E几M−P −727A ) ノ種菌液10プを接種
し、30°Gで培養液のP IIが7.0に上昇する迄
(7日間)、振盪培養(170r、p、 nl) した
。以下、上記バチラス・サーキュランスの培養液から粗
酵素を得る方法と全く同様にして、培養液から粗酵素(
以下、「ストレプトマイセス起源の粗酵素」と称す)を
得た。
[I'l Crude enzyme preparation method from Streptomyces o143 carcasses Medium composition 1 i<l-l2P 040.2% Yeast extract o, o59g KO,l O,05% Glucose 0.25% Mg504j 7 l-1200,1 %neopeptone
0.1% NaC10.05% NaN0. , 0.1% Aspergillus wet cells 2.1% Distilled water 1.0! PH5,0 Streptomyces 0143 (F
10 copies of the inoculum solution of E (MP-727A) were inoculated and cultured with shaking (170 r, p, nl) at 30°G until the P II of the culture solution rose to 7.0 (7 days). Hereinafter, the crude enzyme (crude enzyme) was obtained from the culture solution in exactly the same manner as the above-mentioned method for obtaining crude enzyme from the culture solution of Bacillus circulans.
Hereinafter referred to as "crude enzyme of Streptomyces origin") was obtained.

尚、上記で得られる2つの粗酵素は、β−1,3−グル
カンを基質として、常法によりβ−1,3−グルカナー
ゼ活性の有無を調べた結果、強力に基質を分解すること
が確認され、β−1,3−グルカナーゼとして使用でき
ることが判明した。
The two crude enzymes obtained above were tested for β-1,3-glucanase activity using β-1,3-glucan as a substrate by a conventional method, and it was confirmed that they strongly degraded the substrate. It was found that it could be used as β-1,3-glucanase.

まだ、洗滌し、ホモゲナイズされた麹菌湿潤菌体を含む
寒天平板培地に上記の両組酵素は作用し、透明なハロー
を形成することから、該粗酵素はいずれも麹菌菌体の細
胞壁を溶解することも確認された。
Both enzymes mentioned above still act on the agar plate medium containing the washed and homogenized wet cells of Aspergillus oryzae and form a transparent halo, so both of the crude enzymes dissolve the cell wall of the Aspergillus cells. This was also confirmed.

次に、麹菌の発芽胞子と細胞壁溶解酵素との処理条件に
ついて説明する。すガわち、細胞壁溶解酵素の使用成度
は、麹菌発芽胞子の細胞壁を溶解し、完全なプロトプラ
ストを得るのに充分な濃度とすることが好ましく、例え
ばバチラス・サーキュランス起源の粗酵素の場合、10
〜50 mg / ml、好ましくは25〜35+++
g/dである。まだキチナーゼを併用する場合、その使
用濃度は1−10■/m、好ましくは3〜5mg/ゴで
ある。酵素処理の時間は麹菌の細胞壁を溶解し、プロト
プラストを得るのに充分な時間とすることが必要で、通
常30分〜6時間が好ましい。またPHは6.0〜7.
0、特に6.5が好ましい。
Next, conditions for treating germinated spores of Aspergillus oryzae with a cell wall lytic enzyme will be explained. In other words, it is preferable that the cell wall lytic enzyme used be at a concentration sufficient to dissolve the cell wall of the germinating spores of Aspergillus oryzae and obtain a complete protoplast; for example, in the case of a crude enzyme derived from Bacillus circulans. , 10
~50 mg/ml, preferably 25-35+++
g/d. If chitinase is also used, the concentration used is 1-10 mg/m, preferably 3-5 mg/m. The enzyme treatment time must be sufficient to dissolve the cell wall of the koji mold and obtain protoplasts, and is usually preferably 30 minutes to 6 hours. Also, the pH is 6.0-7.
0, especially 6.5 is preferred.

次いで、酵素処理が終了したら、菌体を保獲する洗滌液
、例えば0.7〜1.5Mソルビトール或いは0.5〜
1.0M塩化カリウム溶液等で、菌体を洗滌し、麹菌の
細胞壁が溶解除去されたプロトプラストを得る。
Next, after the enzyme treatment is completed, a washing solution for retaining bacterial cells, such as 0.7-1.5M sorbitol or 0.5-1.5M sorbitol, is added.
The bacterial cells are washed with a 1.0 M potassium chloride solution or the like to obtain protoplasts in which the cell wall of Aspergillus oryzae has been dissolved and removed.

次に、ここで得られたプロトプラストの融合処理及び融
合プロトプラストの再生は、例えば、Annp等の方法
(J、Gen、 Mi c r ob i 。
Next, the fusion treatment of the protoplasts obtained here and the regeneration of the fused protoplasts are performed, for example, by the method of Annp et al. (J, Gen, Microbi.

1、.92,413(1976)’]に準じて行うこと
ができる。
1. 92, 413 (1976)'].

即ち、0.7〜1.5M・ソルビトール、5〜10Q 
m M −Ca 6−2及び30〜80mMグリシンを
含む15〜33%ポリエチレングリコール6000(P
H7,5)中に、上記で得られた高プロテアーゼ生産株
と高グルタミナーゼ生産株のそれぞれの洗滌プロトプラ
ストを添加混合し、20〜30℃で15〜45分処理す
ることによりプロトプラスト融合細胞を得ることができ
る。
That is, 0.7-1.5M sorbitol, 5-10Q
15-33% polyethylene glycol 6000 (P
H7, 5), add and mix the washed protoplasts of the high protease producing strain and the high glutaminase producing strain obtained above, and process at 20 to 30°C for 15 to 45 minutes to obtain protoplast fused cells. I can do it.

次に、こうして得られたプロトプラスト融合細胞の再生
は、プロトプラストを保設しつつ細胞壁を再生すること
が可能な高張再生培地、例えば0.7〜1.5Mのソル
ビトールを含む米麹汁(又は麦芽汁)に外大2%を含有
させだ高張再生寒天培地(PH6,5)及び、0.7〜
1.5Mのソルビットをツアペック液体培地に寒天2%
を含有させた高張最少再生寒天培地(P)I6.5)に
上記で得られたプロトプラスト融合細胞を移し、25℃
〜35℃で2〜10日間培養する方法により行われる。
Next, the protoplast-fused cells obtained in this way are regenerated using a hypertonic regeneration medium that can regenerate the cell wall while preserving the protoplasts, such as rice malt juice (or malt) containing 0.7 to 1.5 M sorbitol. Hypertonic regenerated agar medium (pH 6.5) containing 2% Sokadai (soup) and 0.7~
1.5M sorbitol in Czapek liquid medium with 2% agar
The protoplast fused cells obtained above were transferred to a hypertonic minimal regeneration agar medium (P)I6.5) containing
This is carried out by culturing at ~35°C for 2 to 10 days.

こうして、プロトシラストの融合細胞は、その周囲に細
胞壁が再生し、培地上で=盛求生背して再生コロニーを
形成する。
In this way, the protocylast fused cells regenerate a cell wall around them and grow on the medium to form a regenerated colony.

次に、こうして得られた再生コロニーから、プロトプラ
スト融合細胞の選別は、予じめその親株である高プロテ
アーゼ生産株と、親株である高グルタミナーゼ生産株の
洗滌プロトプラストについて、それぞれ融合細胞の場合
と同様に再生させ、得られた再生コロニーの形態学的性
質(色相、色彩、明度、光σ(、形状、大きさ等)及び
生理学的性質等の特徴を把・i屋しておくことによって
、それらとは異なる特徴を佃ijえた+Ij生コロニー
として、選別採取することができる。
Next, from the regenerated colonies obtained in this way, protoplast fusion cells are selected in advance by washing the protoplasts of the parent strain, the high protease producing strain, and the parent strain, the high glutaminase producing strain, in the same manner as for the fused cells. By keeping track of the morphological properties (hue, color, brightness, light σ, shape, size, etc.) and physiological properties of the resulting regenerated colonies, It is possible to select and collect +Ij live colonies that have characteristics different from those of the above.

尚、上記高プロテアーゼ生産株と高グルタミナーゼ生産
株のそれぞれの親株の分生胞子に、人工変異処理(X線
、紫外線等の照射、ニトロソグアニジン、エチルメチル
ザルフィート等の化学変異処理)を施して、それぞれ親
株の分生胞子に特徴ある色や各種の栄養要求性、例えば
アラニン、メチオニン等のアミノ酸要求性、ビオチン、
ニコチン酸、パラアミノ安息香+’i2等の要求性を付
与した変異株を得、この変異株を親株として上記と同様
に酵素処理、プロトプラスト融合処理を行い、次いで再
生し、再生コロニーとすると、親株である変異株は特徴
ある色を有し、この色を有しないプロトプラスト融合細
胞を明確に区別し、採取することができる。まだ栄養要
求変異株のプロトプラストは再生の際、特定の栄養源が
存在しないと生育出来ないので、特定の栄養源が存在し
なくても生育できるプロトプラスト融合細胞を容易に区
別し採取することができる。このように両親法に色及び
栄養要求性等のマーカーを付しておくと、目的とする融
合細胞の分離操作が極めて容易となる。
In addition, the conidia of the parent strains of the high protease producing strain and the high glutaminase producing strain were subjected to artificial mutation treatment (irradiation with X-rays, ultraviolet rays, etc., chemical mutation treatment with nitrosoguanidine, ethylmethylsulfite, etc.). , the characteristic color and various nutritional requirements of the conidia of the parent strain, such as amino acid requirements such as alanine and methionine, biotin,
A mutant strain with requirements for nicotinic acid, para-aminobenzoic acid + 'i2, etc. is obtained, and this mutant strain is used as a parent strain and subjected to enzyme treatment and protoplast fusion treatment in the same manner as above, and then regenerated to form a regenerated colony. Certain mutant strains have a characteristic color, and protoplast fusion cells that do not have this color can be clearly distinguished and collected. Protoplasts, which are still auxotrophic mutants, cannot grow unless a specific nutrient source is present during regeneration, so protoplast fusion cells that can grow even in the absence of a specific nutrient source can be easily distinguished and collected. . By attaching markers such as color and auxotrophy to the biparental method in this way, the separation operation of the desired fused cells becomes extremely easy.

上記で得られる高プロテアーゼ生産株の変異株22−9
1(FERP−7λ77)が挙げられ、また高グルタミ
ナーゼ生産株の変異株としては、例えば分生胞子が黄色
で、ニコチン酸チ念f要求性の付与されたアスペルギル
ス・ソーヤM12−2−61(FE)LM P−72ざ
/)等が挙げられる。
Mutant strain 22-9 of the high protease producing strain obtained above
1 (FERP-7λ77), and as a mutant strain of a high glutaminase producing strain, for example, Aspergillus sojae M12-2-61 (FE )LM P-72za/), etc.

次に、こうして高張再生培地で再生したプロトプラスト
融合株は親株に戻シ易いヘテロカリオンである場合が多
く、不安定である。そこで、これらへテロカリオン株の
分生胞子に、小出等の方法(Agric、Biol、C
hem、、27,758(1963))に従って、紫外
線照射(距離39ぼ、1〜5分)を施し、検定すること
によって、安定な2倍体、即ち目的とする麹菌菌体を得
ることができる。
Next, the protoplast fusion strain thus regenerated in a hypertonic regeneration medium is often a heterokaryon that easily reverts back to the parent strain and is therefore unstable. Therefore, we used the method of Koide et al. (Agric, Biol, C.
By applying ultraviolet irradiation (distance: 39 m, 1 to 5 minutes) and assaying according to the method described in Hem. .

例t ハ、アスペルギルス・ソーヤ D−78は、こう
して得た高プロテアーゼ生産能を有し且つ高グルタミナ
ーゼ生産能を有する安定な2倍体株であり、工業技術院
微生物工業技術研究所に微工研菌寄第72ツタ号として
寄託されている。
Example t C. Aspergillus sojae D-78 is a stable diploid strain with high protease-producing ability and high glutaminase-producing ability obtained in this way, and is affiliated with the National Institute of Microbiology, Agency of Industrial Science and Technology. It has been deposited as Tsuta No. 72.

本発明の利点は次の通りである。従来一般に採用されて
いた紫外線照射等の変異処理による変異株の分離は、極
めて偶然性が高く、スクリーニングに膨大な労力を要す
るなど効率が極めて悪く、まだ、たまたま酵素生産性に
すぐれた変異株が得による変異株の取得法で最も大きな
欠点は、取得される変異株はプロテアーゼ或いはグルタ
ミナーゼのうち一方については親株に比べ高くとも、他
方は逆に低い場合が多く、両方とも親株に比べ高い菌株
を取得することは困難であったことである。
The advantages of the invention are as follows. Isolation of mutant strains by mutation treatments such as ultraviolet irradiation, which have been commonly used in the past, is highly random and requires a huge amount of effort for screening, making it extremely inefficient, and it is still difficult to obtain mutant strains that happen to have excellent enzyme productivity. The biggest drawback of this method for obtaining mutant strains is that even if the obtained mutant strain has a higher level of protease or glutaminase than the parent strain, the other is often lower than the parent strain; It was difficult to do so.

これに対し、本発明によれば、高プロテアーゼ生産能を
有し且つ高グルタミナーゼ生産能を有する麹菌を効率よ
く採取し、製造することができる。
In contrast, according to the present invention, Aspergillus oryzae having a high protease-producing ability and a high glutaminase-producing ability can be efficiently collected and produced.

また、本発明で得られだ麹菌を醤油麹原料に接種培養し
て麹をつくり、この麹を用いて仕込みを行い醤油を醸造
すると、醤油原料は良く分解されて窒素利用率が約3〜
10%向上するばかりか、グルタミン酸が著しく増加す
るので、非常に旨味の強い醤油が得られるという、産業
上画期的な効果を奏する。、(実験例1、第1表の結果
参照)、麹菌のプロトプラストの形成率が低くプロ)・
ブラスト融合技術を用いる研究の大きな障害となってい
たが、培養細胞として、発芽胞子の長さかもとの胞子外
径の5〜15倍の発芽胞子を用いることにより、麹菌の
プロトプラストの形成率を著しく高めることが可能とな
り、本発明方法によれば目的とする麹菌菌体を効率良く
得ることができる。(実験例2の嬉2表の結果参照) 以下、本発明の効果を実験例を挙げて説明する。
In addition, when the koji mold obtained in the present invention is inoculated and cultured in soy sauce koji raw material to make koji, and this koji is used to prepare and brew soy sauce, the soy sauce raw material is well decomposed and the nitrogen utilization rate is about 3~3.
Not only is it improved by 10%, but the glutamic acid content is also significantly increased, resulting in a soy sauce with extremely strong flavor, which is an industrially groundbreaking effect. (Refer to the results in Experimental Example 1 and Table 1), the protoplast formation rate of Aspergillus aspergillus is low.
This has been a major obstacle in research using blast fusion technology, but by using germinated spores that are 5 to 15 times longer than the original spore outer diameter as cultured cells, we can significantly increase the protoplast formation rate of Aspergillus oryzae. According to the method of the present invention, the desired koji mold cells can be efficiently obtained. (Refer to the results in Table 2 of Experimental Example 2) The effects of the present invention will be described below with reference to experimental examples.

実験例1 下記実施例2で得られた高プロテアーゼ生産能を有し且
つ高グルタミナーゼ生産能を有する麹菌アスへルキルス
・ソーヤ D−78(FEI(MP−7279)を、脱
脂大豆300gと炒蒸割砕小麦300gからなる醤油原
料に接種し、麹−1壷ににて通當の醤油麹製造法に従っ
て製麹し、得られた麹を25%食塩水1200mに仕込
み、常法の諸法発酵管理を行い、30℃で3ケ月発酵熟
成させた。また対照方法として、両親法を用いて同様に
熟成醤油諸味を得た。こうして得られた諸法液汁につい
て総屋素(’rNと略記する)、グルタミン酸(Q ]
 u )、Q l u / T N及び窒素利用率を測
定した。その請来を第1表に示す。
Experimental Example 1 The Aspergillus aspergillus D-78 (FEI (MP-7279)), which has a high protease production ability and a high glutaminase production ability obtained in Example 2 below, was roasted and steamed with 300 g of defatted soybeans. A soy sauce raw material consisting of 300 g of crushed wheat was inoculated, and koji was made in a koji pot according to the current soy sauce koji manufacturing method.The resulting koji was poured into 1200 m of 25% salt water, and fermentation was controlled using conventional methods. was fermented and aged at 30°C for 3 months.As a control method, aged soy sauce moromi was obtained in the same manner using both methods.Soyamoto (abbreviated as 'rN) , glutamic acid (Q)
u), Qlu/TN and nitrogen utilization were measured. The requests are shown in Table 1.

第 1 表 実験例2 後記実施例1において、培養細胞として発芽胞子の長さ
がいろいろと異なる発芽胞子を用いる以外は、実施例1
と全く同様にして、麹菌のグロトソリストを得、その形
成率を調べた。
Table 1 Experimental Example 2 Example 1 was performed except that in Example 1 described later, germinated spores with various lengths of germinated spores were used as cultured cells.
In exactly the same manner as above, glotosolists of Aspergillus oryzae were obtained and their formation rate was investigated.

その結果を第2表に・示す。The results are shown in Table 2.

第 2 表 注1=結果はアスペルギルス・ソーヤ2165菌を使用 注2:プロトプラスト形成率は発芽胞子数に対するプロ
トプラスト数の割合を示す。
Table 2 Note 1 = Results used Aspergillus sawjae 2165 bacteria Note 2: Protoplast formation rate indicates the ratio of the number of protoplasts to the number of germinated spores.

注3二発芽胞子数及びプロドブシスト数の計数は位相差
光学顕微鏡にコン社製)によ り測定してめた。
Note 3: The number of germinated spores and prodobocysts was determined using a phase-contrast optical microscope (manufactured by Kon Co., Ltd.).

実施例1 高プロテアーゼ生産株(アスペルギルス・ソーヤ AT
CC42251)及び、高グルタミナーゼ生産株〔アス
ペルギルス・ソーヤ 2165(FERM P−72♂
O)〕を、それぞれ30℃で66時間培養した米麹汁ス
ラント培地より分生胞子を採取し、これをそれぞれ0.
01%ソルビタン脂肪酸エステル溶液[ソルゲンTW−
60(第1工業製薬社製)」に分散し、1〜5×10個
/dの胞子懸濁液を調製した。
Example 1 High protease producing strain (Aspergillus sojae AT
CC42251) and high glutaminase producing strain [Aspergillus sojae 2165 (FERM P-72♂
Conidia were collected from rice malt juice slant medium that had been cultured for 66 hours at 30°C.
01% sorbitan fatty acid ester solution [Sorgen TW-
60 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.)" to prepare a spore suspension of 1 to 5 x 10 spores/d.

次いで、この胞子懸濁液1dをツアペック変形培地3Q
mlに接種し、150 ml容三角フラスコ内で30℃
で12時間振盪培養し、発芽胞子の長さかもとの胞子外
径の10倍の発芽胞子(培養細胞)懸濁液を得た。
Next, this spore suspension 1d was added to Czapek modified medium 3Q.
ml and inoculated at 30°C in a 150 ml Erlenmeyer flask.
The cells were cultured with shaking for 12 hours to obtain a suspension of germinated spores (cultured cells) whose length and outer diameter were 10 times the original spore outer diameter.

次いで、発芽胞子懸濁液を遠心分離(4500g、20
分)して発芽胞子を集め、これを充分水洗滌し、水切シ
したのち、細胞壁溶解酵素1 mlと混和し、27℃で
2時間、ゆるやかに振盪(毎分50〜60回往復)して
酵素処理し、それぞれ麹菌ノプロトプラストを得た。
The germinated spore suspension was then centrifuged (4500 g, 20
Collect the germinated spores, wash thoroughly with water, drain the water, mix with 1 ml of cell wall lytic enzyme, and shake gently (50 to 60 times per minute) at 27°C for 2 hours. After enzyme treatment, Aspergillus oryzae noprotoplasts were obtained.

ここに用いた細胞壁溶解酵素は、バチラス・サーキーラ
ンス起源の粗酵素と市販のキチナーゼ(米国ICN社製
)をそれぞれ溶液1ゴに30mg及び5II1gの割合
で溶解し得られたものである。
The cell wall lytic enzyme used here was obtained by dissolving a crude enzyme originating from Bacillus circilians and a commercially available chitinase (manufactured by ICN, USA) at a ratio of 30 mg and 1 g of 5II to 1 g of solution, respectively.

次に、こうして得たプロドブシストをG−3規格のガラ
スフィルターにより分離し、これを高張液(0,8Mソ
ルビトール溶液)で数回繰シ返し洗滌し、得られた洗滌
プロトプラストを高張液1dに移し、懸濁する。
Next, the protoplasts thus obtained were separated using a G-3 standard glass filter, washed several times with hypertonic solution (0.8M sorbitol solution), and the washed protoplasts obtained were transferred to hypertonic solution 1d. , suspend.

次いで、それぞれプロトプラストが懸濁された高張液を
混合し、20℃で遠心分離(700g、15分)して、
混合プロトプラストのペレットを採取した。これを0.
8 Mソルビトール、10mMCa1l、及び50mM
グリシンを含む20%ポリエチレングリコール6000
(PH7,5)溶液1mlと混合し、25°Cで30分
プロトプラスト融合反応を行い、プロトプラスト融合細
胞を得だ。
Next, the hypertonic solutions in which protoplasts were suspended were mixed and centrifuged at 20°C (700g, 15 minutes).
A pellet of mixed protoplasts was collected. This is 0.
8M sorbitol, 10mM Ca1l, and 50mM
20% polyethylene glycol 6000 containing glycine
(PH7.5) solution was mixed with 1 ml of the solution, and a protoplast fusion reaction was performed at 25°C for 30 minutes to obtain protoplast fused cells.

次いで、プロトプラスト融合細胞の再生は、該融合細胞
を高張液で適当な細胞の濃度まで稀釈しこれを予じめシ
ャーレ内に流し固めた高張再生寒天培地(0,8Mソル
ビトールを含む糖濃度8度の米麹汁寒天培地(寒天2%
)〕に播種し、その上から、寒天0,5%以外は上記と
全く同じ高張再生寒天培地を流し固化重層させ、30℃
で2〜5日間培養し、プロトプラスト融合細胞の再生コ
ロニーをイ↓↑た。
Next, the protoplast fused cells are regenerated by diluting the fused cells with a hypertonic solution to an appropriate cell concentration, pouring this into a petri dish in advance, and hardening it on a hypertonic regeneration agar medium (containing 0.8 M sorbitol at a sugar concentration of 8 degrees Celsius). rice malt juice agar medium (agar 2%
)], and then pour the same hypertonic regeneration agar medium as above except for 0.5% agar over it, solidify it, and incubate at 30°C.
The cells were cultured for 2 to 5 days, and regenerated colonies of protoplast fused cells were obtained.

次に、ここで得られた再生コロニーの中から、両親法の
プロトプラストが形成するコロニーとをよ4なる形y4
を有する再生コロ−を選び、そこからプロトプラスト融
合再生株を採取した、次いで、採取したプロトプラスト
融合再生株を米旬汁スラント培地に接種し、30℃で4
日培養して分生胞子を得、この胞子懸濁液に39Gの距
離から紫外線を1分間照射し、得られた胞子を米麹汁平
板培地に塗布し、30℃で4日培養し、生じたコロニー
の中から、本発明の目的とする高プロテアーゼ活性を有
し且つ高グルタミナーゼ活性を有する#mm体〔アスペ
ルギルス・ソーヤD−24(FEB、M P−7コ7t
)〕を採取した。
Next, from among the regenerated colonies obtained here, the colonies formed by the protoplasts of the biparental method were divided into 4 forms y4.
A regenerated colo possessing this was selected, and a protoplast-fused regenerated strain was collected from it.Then, the collected protoplast-fused regenerated strain was inoculated into rice shunjitsu slant medium and incubated at 30°C for 4 hours.
Conidia were obtained by culturing for 1 day, and this spore suspension was irradiated with ultraviolet rays for 1 minute from a distance of 39 G. The obtained spores were applied to a rice malt juice plate medium and cultured at 30°C for 4 days. Among the colonies, Aspergillus sojae D-24 (FEB, M P-7 7t
)] was collected.

得られた本発明1菌菌体とその両親法の酵素生産能につ
いて調べた。
The enzyme production ability of the obtained Invention 1 bacterial cells and their parent methods was investigated.

その結果を43表に示す。The results are shown in Table 43.

第3表 麹菌の酵素生産能 実施例2 ルス・ソーヤW2−91(FERM P−7,277)
音用b1高グルタミナーゼ生産株として、分生胞子が黄
色で、ニコチン酸勇年4要求性の付与されたアスペルギ
ルス・ソーヤM12−2−61(FEBM P−721
7)を用い、“グロトブラスト融合細胞の高張再生培地
として、高張最少再生寒天培地(0,8Mソルビトール
を含むツアペック寒天培地〕を用いる以外は実施例1と
全く同様にしてプロトプラスト融合再生採音採取した。
Table 3 Enzyme production ability of Aspergillus Aspergillus Example 2 Russ Sawyer W2-91 (FERM P-7,277)
Aspergillus sawya M12-2-61 (FEBM P-721), which has yellow conidia and a nicotinic acid requirement, is used as a b1 high glutaminase producing strain.
7), protoplast fusion regeneration samples were collected in exactly the same manner as in Example 1, except that hypertonic minimal regeneration agar medium (Zapek agar medium containing 0.8 M sorbitol) was used as the hypertonic regeneration medium for glotoblast fusion cells. .

米 次に、ここで得られt再生株ヲオ麹汁スラント培地に接
種し、30℃で4日間培養して、緑色、黄色、白色の胞
子をもつヘテロカリオン株を得、得られたヘテロカリオ
ン株の胞子に、39菌の距離から紫外線を1分間照射し
、得られた・胞子を再び最少寒天平板培地に塗布し、3
0℃で4日間培養して、緑色のみの胞子から成る、安定
なコロニーを形成する麹菌菌体を得た。この麹菌菌体の
中から高グロテアーゼ生産能を有し且つ高グルタミナー
ゼ生産能を有する一菌株、アスペルギルス・ソーヤD−
78(FERM P−7,27F)を得た次に、こうし
て得られた本発明麹菌菌体とその両親法の酵素生産能に
ついて調べた。
Yoneji then inoculated the regenerated strain obtained here into a koji juice slant medium and cultured it at 30°C for 4 days to obtain a heterokaryon strain with green, yellow, and white spores. The spores were irradiated with ultraviolet rays for 1 minute from a distance of 39 bacteria, and the obtained spores were again applied to the minimal agar plate medium.
After culturing at 0° C. for 4 days, Aspergillus aspergillus cells forming stable colonies consisting of only green spores were obtained. Among these koji mold cells, one strain, Aspergillus sojae D-, has a high grotease production ability and a high glutaminase production ability.
After obtaining 78 (FERM P-7, 27F), the enzyme production ability of the Aspergillus aspergillus of the present invention thus obtained and its parent methods was investigated.

その結果を第4表に示す。The results are shown in Table 4.

第4表 N菌の酵素生産能 注1):第4表におけるプロテアーゼ活性に、脱脂大豆
301と炒蒸割砕小麦30f’jt−原料として、直径
15傷のシャーレ内で、通常の醤油麹の製造法と同様に
製麹し、得られた麹について、水10倍量を用いて2時
間抽出し九間に1μmolのチロシンを生成する活性を
1単位として表示した。またグルタミナーゼ活性は前記
抽出残青金ホモジナイズして得られた液とL−グルタミ
ンt−30℃で1時間振盪反応させ、濾過後、F液のグ
ルタミン酸量を測定してめた。グルタミナーゼ活性は、
麹12当り1分間に1μmolのグルタミン酸量を生成
する活性を1単位として表示した。 ′第1表及び第2
表の結果から、プロテアーゼが高い株はグルタミナーゼ
活性が低く、又反対にグルタミナーゼ活性が高い株にプ
ロテアーゼ活性が低いのが一般的麹菌の特徴であったが
、本発明方法によれば、プロテアーゼ活性とグルタミナ
ーゼ活性が共に高い麹菌Mが得られることが判る。
Table 4 Enzyme production capacity of N bacteria Note 1): The protease activity in Table 4 was determined by using defatted soybeans 301 and roasted cracked wheat 30f'jt as raw materials in a petri dish with 15 scratches in diameter, and using normal soy sauce koji. Koji was made in the same manner as in the production method, and the obtained koji was extracted for 2 hours using 10 times the amount of water, and the activity of producing 1 μmol of tyrosine in 9 hours was expressed as 1 unit. The glutaminase activity was determined by reacting the solution obtained by homogenizing the extracted residual blue gold with L-glutamine by shaking at -30° C. for 1 hour, and after filtration, measuring the amount of glutamic acid in solution F. Glutaminase activity is
The activity of producing 1 μmol of glutamic acid per minute per 12 pieces of koji was expressed as one unit. 'Table 1 and 2
From the results in the table, it is found that strains with high protease have low glutaminase activity, and conversely, strains with high glutaminase activity have low protease activity, which is a characteristic of Aspergillus aspergillus. It can be seen that Aspergillus Aspergillus M having high glutaminase activity can be obtained.

特許出願人 キッコーマン株式会社Patent applicant: Kikkoman Corporation

Claims (2)

【特許請求の範囲】[Claims] (1)アスペルギルス属に属する、高プロテアーゼ生産
株と高グルタミナーゼ生産株のそれぞれの培養細胞から
グロトプラストを得、これらをプロトプラスト融合させ
、該融合細胞を高張再生培地に培養し、培養物から高プ
ロテアーゼ生産能を有し且つ高グルタミナーゼ生産能を
有する麹菌を採取することを特徴とする麹菌の製造法、
(1) Obtain grotoplasts from cultured cells of a high protease-producing strain and a high glutaminase-producing strain belonging to the genus Aspergillus, fuse these with protoplasts, culture the fused cells in a hypertonic regeneration medium, and produce high protease from the culture. A method for producing Aspergillus oryzae, characterized by collecting Aspergillus oryzae having the ability to produce high glutaminase,
(2)培養細胞が、発芽胞子の長さかもとの胞子外径の
5〜15倍の発芽胞子である特許請求の範囲第1項記載
の製造法。
(2) The production method according to claim 1, wherein the cultured cells are germinated spores whose length is 5 to 15 times the original spore outer diameter.
JP58180798A 1983-09-30 1983-09-30 Production of aspergillus by protoplast fusion Granted JPS6075280A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP58180798A JPS6075280A (en) 1983-09-30 1983-09-30 Production of aspergillus by protoplast fusion

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP58180798A JPS6075280A (en) 1983-09-30 1983-09-30 Production of aspergillus by protoplast fusion

Publications (2)

Publication Number Publication Date
JPS6075280A true JPS6075280A (en) 1985-04-27
JPH0371877B2 JPH0371877B2 (en) 1991-11-14

Family

ID=16089522

Family Applications (1)

Application Number Title Priority Date Filing Date
JP58180798A Granted JPS6075280A (en) 1983-09-30 1983-09-30 Production of aspergillus by protoplast fusion

Country Status (1)

Country Link
JP (1) JPS6075280A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60188057A (en) * 1984-03-09 1985-09-25 Kikkoman Corp Koji mold having high protease preparation ability and high glutaminase preparation ability and its breeding
JPH01120282A (en) * 1987-11-02 1989-05-12 Nakano Vinegar Co Ltd Regeneration of spheroplast of bacterium belonging to genus acetobacter
JPH01168290A (en) * 1987-12-25 1989-07-03 Nakano Vinegar Co Ltd Electrical fusion of acetic acid bacteria spheroplast
JPH01168289A (en) * 1987-12-25 1989-07-03 Nakano Vinegar Co Ltd Fusion of acetic bacteria spheroplast

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60188057A (en) * 1984-03-09 1985-09-25 Kikkoman Corp Koji mold having high protease preparation ability and high glutaminase preparation ability and its breeding
JPH0368672B2 (en) * 1984-03-09 1991-10-29 Kikkoman Corp
JPH01120282A (en) * 1987-11-02 1989-05-12 Nakano Vinegar Co Ltd Regeneration of spheroplast of bacterium belonging to genus acetobacter
JPH01168290A (en) * 1987-12-25 1989-07-03 Nakano Vinegar Co Ltd Electrical fusion of acetic acid bacteria spheroplast
JPH01168289A (en) * 1987-12-25 1989-07-03 Nakano Vinegar Co Ltd Fusion of acetic bacteria spheroplast

Also Published As

Publication number Publication date
JPH0371877B2 (en) 1991-11-14

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