JP2886551B2 - Method for producing 5'-inosinic acid by fermentation method - Google Patents
Method for producing 5'-inosinic acid by fermentation methodInfo
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- JP2886551B2 JP2886551B2 JP13383189A JP13383189A JP2886551B2 JP 2886551 B2 JP2886551 B2 JP 2886551B2 JP 13383189 A JP13383189 A JP 13383189A JP 13383189 A JP13383189 A JP 13383189A JP 2886551 B2 JP2886551 B2 JP 2886551B2
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Description
【発明の詳細な説明】 産業上の利用分野 本発明は、発酵法による５′−イノシン酸の製造法に
関する。５′−イノシン酸は調味料などとして広く用い
られ、食品工業分野において有用な物質である。Description: TECHNICAL FIELD The present invention relates to a method for producing 5′-inosic acid by a fermentation method. 5'-Inosinic acid is widely used as a seasoning and the like, and is a useful substance in the field of food industry.
従来の技術 従来、糖類より直接５′−イノシン酸を発酵生産する
方法として、コリネバクテリウム属に属し、アデニン要
求性を有し、デコイニンまたはサルファ剤に耐性を有
し、さらに５′−イノシン酸生産能を有する微生物を培
養し、培地中に蓄積された５′−イノシン酸を採取する
方法（特開昭55−150899）、コリネバクテリウム属に属
し、アデニン要求性を有し、発酵培地の加圧蒸煮殺菌の
影響を受けない菌株を取得、培養し培養中に５′−イノ
シン酸を生成蓄積させる方法（特公昭58−46319）など
が知られている。2. Description of the Related Art Conventionally, as a method for directly producing 5′-inosic acid from sugars by fermentation, it belongs to the genus Corynebacterium, has an adenine requirement, has resistance to decoinin or sulfa drugs, and further has 5′-inosic acid production. A method of culturing a microorganism having the ability to collect 5'-inosinic acid accumulated in the medium (Japanese Patent Laid-Open No. 55899/1979), which belongs to the genus Corynebacterium, has an adenine-requiring property, and is added to a fermentation medium. There is known a method of obtaining and culturing a strain which is not affected by autoclaving and producing and accumulating 5'-inosic acid during culturing (Japanese Patent Publication No. 58-46319).
浸透圧に対して耐性を有する菌株を用いて、５′−イ
ノシン酸を製造する方法は知られていない。There is no known method for producing 5'-inosinic acid using a strain resistant to osmotic pressure.
なお、本明細書中では従来ブレビバクテリウム・アン
モニアゲネスと命名されている菌株をコリネバクテリウ
ム・アンモニアゲネスに変更して記載する。本菌種名の
変更はインターナショナル・ジャーナル・オブ・システ
マティックバクテリオロジー（International Journal
of Systematic Bacteriology）442〜443,10月（1987）
の記載にもとづくものである。In the present specification, the strain conventionally referred to as Brevibacterium ammoniagenes is described as being changed to Corynebacterium ammoniagenes. The name of this strain has been changed by the International Journal of Systematic Bacteriology (International Journal
of Systematic Bacteriology) 442-443, October (1987)
It is based on the description of.
発明が解決しようとする課題 本発明の目的は、調味料として広く用いられている
５′−イノシン酸を直接発酵法により工業的に安価に効
率よく製造する方法を提供することにある。An object of the present invention is to provide a method for industrially and efficiently producing 5'-inosic acid, which is widely used as a seasoning, by a direct fermentation method.
課題を解決するための手段 本発明によれば、コリネバクテリウム属に属し、2000
mOSMOL/KG以上の浸透圧に耐性を有しかつ５′−イノシ
ン酸生産能を有する微生物を培地に培養することによ
り、培養物中に５′−イノシン酸を生成蓄積させ、該培
養物から５′−イノシン酸を採取することができる。Means for Solving the Problems According to the present invention, belonging to the genus Corynebacterium, 2000
By culturing a microorganism having an osmotic pressure of at least mOSMOL / KG and capable of producing 5'-inosinic acid in a culture medium, 5'-inosic acid is produced and accumulated in the culture, and 5'-inosinic acid is produced and accumulated in the culture. '-Inosinic acid can be collected.
以下に本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
用いられる微生物としては、コリネバクテリウム属に
属し、2000mOSMOL/KG以上の浸透圧に耐性を有しかつ
５′−イノシン酸生産能を有する微生物であれば野生
株、変異株のいずれでもよい。上記したような変異株
は、コリネバクテリウム属に属する５′−イノシン酸生
産菌に2000mOSMOL/KG以上の浸透圧耐性を付与したり、
コリネバクテリウム属に属し2000mOSMOL/KG以上の浸透
圧に耐性を有している菌株に５′−イノシン酸生産能を
付与したりすることにより得ることができる。The microorganism to be used may be a wild type or a mutant as long as it belongs to the genus Corynebacterium, has a resistance to osmotic pressure of 2000 mOSMOL / KG or more and has 5'-inosinic acid-producing ability. The mutant strain as described above imparts osmotic resistance of 2000 mOSMOL / KG or more to 5′-inosinic acid-producing bacteria belonging to the genus Corynebacterium,
It can be obtained by imparting 5'-inosinic acid-producing ability to a strain belonging to the genus Corynebacterium and having an osmotic pressure of 2000 mOSMOL / KG or more.
たとえば本発明の実施例で用いられる変異株は、コリ
ネバクテリウム・アンモニアゲネス（Corynebacterium
Ammoniagenes）KY13184（FERM P−3790）（以下、KY131
84株という。）を親株として、これにＮ−メチル−Ｎ′
−ニトロ−Ｎ−ニトロソグアニジン、紫外線、Ｘ線など
の処理による通常の変異処理を施すことにより得られ
る。For example, the mutant strain used in Examples of the present invention is Corynebacterium ammoniagenes (Corynebacterium
Ammoniagenes) KY13184 (FERM P-3790) (hereinafter KY131
84 shares. ) As a parent strain, to which N-methyl-N ′
-Nitro-N-nitrosoguanidine, ultraviolet rays, X-rays, and the like, and can be obtained by performing a usual mutation treatment.
高い浸透圧を有する変異株を選択するには、選択的培
地にNaCl、KCl、硫酸アンモニウム、コハク酸ナトリウ
ムなどの塩類、ソルビトール、グルコース、シュクロー
スなどの糖類を添加する。添加量は20〜100g/が好ま
しい。In order to select a mutant strain having a high osmotic pressure, salts such as NaCl, KCl, ammonium sulfate and sodium succinate, and saccharides such as sorbitol, glucose and sucrose are added to the selective medium. The addition amount is preferably 20 to 100 g /.
以下に本発明で用いられる変異株の具体的取得方法を
示す。Hereinafter, a specific method for obtaining the mutant strain used in the present invention will be described.
親株としてKY13184株を用い、該菌株をＮ−メチル−
Ｎ′−ニトロ−Ｎ−ニトロソグアニジン100μg/mlで30
℃、30分処理した後、親株が生育阻害を示す濃度（70g/
ml）のNaClを含む最少培地寒天平板上（グルコース20g/
、塩化アンモニウム2g/、KH2PO4 0.1g/、K2HPO4
0.3g/、MgCl2・2H2O 0.3g/、FeSO4・7H2O 10mg/
、MnSO4・４〜6H2O 1mg/、ZnSO4・7H2O 1mg/、Cu
SO4・5H2O 0.2mg/、CaCl2・2H2O 10mg/、Ｌ−シス
テイン40mg/、Ｌ−アスパラギン0.5g/、サイアミン
塩酸塩10mg/、パントテン酸カルシウム20mg/、ビオ
チン60μg/、ニコチン酸3g/、アデニン20mg/、グ
アニン20mg/、寒天20g/、pH7.2）上に塗布する。30
℃で７〜10日間培養後、生育してくる変異株のうち、
５′−イノシン酸の生産能がとくに優れている菌株を選
ぶ。コリネバクテリウム・アンモニアゲネスＨ−7464
（以下、Ｈ−7464株という。）はそのうちの一株であ
る。Using the KY13184 strain as a parent strain, the strain was N-methyl-
N'-nitro-N-nitrosoguanidine 100 μg / ml
After treatment at 30 ° C for 30 minutes, the concentration at which the parent strain shows growth inhibition (70 g /
ml) on a minimal medium agar plate containing NaCl (20 g /
, Ammonium chloride 2 g /, KH 2 PO 4 0.1 g /, K 2 HPO 4
0.3g /, MgCl 2 · 2H 2 O 0.3g /, FeSO 4 · 7H 2 O 10mg /
, MnSO 4 · 4~6H 2 O 1mg /, ZnSO 4 · 7H 2 O 1mg /, Cu
SO 4 · 5H 2 O 0.2mg / , CaCl 2 · 2H 2 O 10mg /, L- cysteine 40 mg /, L-asparagine 0.5 g /, thiamine hydrochloride 10 mg /, calcium pantothenate 20 mg /, biotin 60 [mu] g /, nicotinic acid 3 g /, adenine 20 mg /, guanine 20 mg /, agar 20 g /, pH 7.2). 30
After culturing at 7 ° C for 7 to 10 days,
A strain having a particularly excellent ability to produce 5'-inosinic acid is selected. Corynebacterium ammoniagenes H-7644
(Hereinafter referred to as the H-7644 strain) is one of them.
また、NaClのかわりにKClを用いる以外は上記と同様
の方法をおこなうことにより５′−イノシン酸の生産が
優れた菌株を得ることができる。コリネバクテリウム・
アンモニアゲネスＨ−7467（以下、Ｈ−7467株とい
う。）はそのうちの一株である。By performing the same method as described above except that KCl is used instead of NaCl, a strain excellent in 5'-inosic acid production can be obtained. Corynebacterium
Ammoniagenes H-7467 (hereinafter referred to as strain H-7467) is one of them.
Ｈ−7466株およびＨ−7467株は、平成元年５月18日付
で、工業技術院微生物工業技術研究所にそれぞれ微工研
条寄第2427号（FERM BP−2427）および第2428号（FERM
BP−2428）としてブダペスト条約に基づいて寄託されて
いる。On May 18, 1989, the H-7746 strain and the H-7467 strain were submitted to the Microorganisms Industrial Technology Research Institute of the Ministry of Industrial Science and Technology, respectively, as Microtechnical Research Institute No. 2427 (FERM BP-2427) and No. 2428 (FERM BP).
BP-2428) under the Budapest Treaty.
KY13184株、Ｈ−7466株およびＨ−7467株を、NaClお
よびKClを含む最少培地寒天平板上で30℃で10日間培養
したときの生育度を第１表および第２表に示す。なお、
浸透圧の測定は、オスモメーター（OSMOMETER）OM−801
（ヴォーゲル社製）を用いておこなった。Tables 1 and 2 show the growth rates when the KY13184, H-7466 and H-7467 strains were cultured at 30 ° C. for 10 days on a minimal medium agar plate containing NaCl and KCl. In addition,
The osmotic pressure is measured using an osmometer (OSMOMETER) OM-801.
(Manufactured by Vogel).
本発明で用いられる微生物の培養に際しては、一般に
核酸の発酵生産に用いられる培地が使用され、微生物が
資化しうる炭素源、窒素源、無機塩類、生育因子などを
含有する培地であれば、合成培地、天然培地などいかな
る培地でも使用できる。 When culturing the microorganism used in the present invention, a medium generally used for fermentative production of nucleic acids is used.If the medium contains a carbon source, a nitrogen source, inorganic salts, growth factors, and the like, which can be assimilated by the microorganism, it is synthesized. Any medium such as a medium and a natural medium can be used.
炭素源としては、グルコース、フラクトース、シュク
ロースあるいは糖蜜、澱粉などの加水分解物のほか、酢
酸、フマール酸、クエン酸などの各種有機酸、エタノー
ル、グリセロールなどのアルコール類などが使用でき
る。Examples of the carbon source include hydrolysates such as glucose, fructose, sucrose, molasses, and starch, various organic acids such as acetic acid, fumaric acid, and citric acid, and alcohols such as ethanol and glycerol.
窒素源としては、アンモニア、塩化アンモニウム、硫
酸アンモニウム、酢酸アンモニウム、燐酸アンモニウム
などの各種無機塩類やフマール酸アンモニウムなどの有
機酸のアンモニウム塩、エチルアミンなどのアミン類、
尿素などの含窒素化合物、ならびにペプトン、肉エキ
ス、酵母エキス、コーン・スティープ・リカー、カゼイ
ン加水分解物、大豆粕またはその加水分解物、アミン酸
発酵、核酸発酵などの各種発酵菌体およびその消化物な
どが用いられる。Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, various inorganic salts such as ammonium phosphate, ammonium salts of organic acids such as ammonium fumarate, amines such as ethylamine,
Nitrogen-containing compounds such as urea, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal or its hydrolyzate, various fermentation cells such as amino acid fermentation, nucleic acid fermentation, and digestion thereof Objects are used.
無機物としては、燐酸第一カリウム、燐酸第二カリウ
ム、燐酸マグネシウム、硫酸マグネシウム、塩化ナトリ
ウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシ
ウムなどが用いられる。As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like are used.
ビオチン、サイアミン、ニコチン酸、β−アナリンな
どのビタミン類やグルタミン酸などのアミノ酸類なども
用いられる。Vitamins such as biotin, thiamine, nicotinic acid and β-analine, and amino acids such as glutamic acid are also used.
培養は、振盪、通気撹拌などの好気的条件下、温度20
〜40℃、好ましくは25〜38℃で、pH5〜９、好ましくは
中性付近に保持しておこなわれ、通常２〜７日間で完了
する。培地のpHは炭酸カルシウム、無機または有機の
酸、アルカリ溶液、アンモニア、pH緩衝液などによって
調整される。Cultivation is performed under aerobic conditions such as shaking and aeration and stirring at a temperature of 20
The reaction is carried out at 4040 ° C., preferably 25-38 ° C., while maintaining the pH at 5-9, preferably near neutrality, and is usually completed in 2-7 days. The pH of the medium is adjusted with calcium carbonate, an inorganic or organic acid, an alkaline solution, ammonia, a pH buffer and the like.
培養終了後、培養液から菌体などの沈殿物を除去し、
イオン交換処理法、吸着法、抽出法、沈澱法などを併用
することにより、培養液から５′−イノシン酸を採取す
ることができる。After completion of the culture, remove precipitates such as cells from the culture solution,
By using an ion exchange treatment, an adsorption method, an extraction method, a precipitation method, or the like, 5′-inosic acid can be collected from the culture solution.
以下に本発明の実施例を示す。 Hereinafter, examples of the present invention will be described.
実施例１ KY13184株、Ｈ−7466株およびＨ−7467株をそれぞれ
２容三角フラスコ中の下記組成からなる種培地300ml
に植菌し、30℃で24時間培養した。Example 1 KY13184 strain, H-7746 strain and H-7467 strain were each placed in a two-volume Erlenmeyer flask in a 300 ml seed medium having the following composition.
And cultured at 30 ° C. for 24 hours.
種培地組成：グルコース20g/、ペプトン10g/、肉
エキス10g/、酵母エキス5g/、NaCl 3g/、ビオチ
ン20μg/、アデニン100mg/、グアニン100mg/、pH
7.2、H2SO4で調整、120℃で10分間加圧蒸煮殺菌。Seed medium composition: glucose 20 g /, peptone 10 g /, meat extract 10 g /, yeast extract 5 g /, NaCl 3 g /, biotin 20 μg /, adenine 100 mg /, guanine 100 mg /, pH
7.2, adjusted with H 2 SO 4 , sterilized by pressure steaming at 120 ° C for 10 minutes.
得られた種培養液300mlを５容培養槽中の下記組成
からなる生産培地３に植菌し、30℃で96時間培養（回
転数600rpm、通気量３/min）した。培養中のpHはアン
モニア水で6.8前後に調整した。300 ml of the obtained seed culture was inoculated into a production medium 3 having the following composition in a 5-volume culture tank, and cultured at 30 ° C. for 96 hours (rotational speed 600 rpm, aeration rate 3 / min). The pH during the culture was adjusted to about 6.8 with aqueous ammonia.
生産培地組成：グルコース150g/、KH2PO4 10g/、
K2HPO4 10g/、MgSO4・7H2O 10g/、CaCl2・2H2O 0.1
g/、ZnSO4・7H2O 5mg/、FeSO4・7H2O 20mg/、MnC
l2・4H2O 5mg/、ビオチン30μg/、パントテン酸カ
ルシウム10mg/、ビタミンB1 5mg/、ニコチン酸5mg/
、アデニン100mg/、グアニン100mg/、コーン・ス
ティープ・リカー20g/、尿素2g/（別途加圧蒸煮殺
菌120℃、５分間）（pH7.2、NaOHまたはH2SO4で調整、1
20℃、10分間加圧蒸煮殺菌。Production medium composition: glucose 150 g /, KH 2 PO 4 10 g /,
K 2 HPO 4 10 g /, MgSO 4・ 7H 2 O 10 g /, CaCl 2・ 2H 2 O 0.1
g /, ZnSO 4 · 7H 2 O 5mg /, FeSO 4 · 7H 2 O 20mg /, MnC
l 2 · 4H 2 O 5mg / , biotin 30 [mu] g /, calcium pantothenate 10 mg /, vitamin B 1 5mg /, nicotinic acid 5mg /
, Adenine 100 mg /, guanine 100 mg /, corn steep liquor 20 g /, urea 2 g / (boiled separately pressurized steam sterilization 120 ° C., 5 minutes) (pH 7.2, adjusted with NaOH or H 2 SO 4, 1
Sterilization by autoclaving at 20 ° C for 10 minutes.
その結果、KY13184株、Ｈ−7466株およびＨ−7467株
の培養液中の５′−イノシン酸生成量はそれぞれ26.5g/
、28.9g/および29.1g/であった。As a result, the amount of 5'-inosic acid produced in the culture of the KY13184, H-7466 and H-7467 strains was 26.5 g /
, 28.9 g / and 29.1 g /.
Ｈ−7466株の培養終了液から菌体を除去して得られた
上清液１を塩酸でpH1.4に調整し、ダイヤイオンSK＃
１（Ｈ型、三菱化成社製）の樹脂塔に通塔後、ただちに
蒸留水を通塔し溶出液を得た。その後樹脂塔を水洗し、
水洗初期の流水液中５′−イノシン酸を含む画分を既に
得られている溶出液と合併し、NaOHでpH7.2に調整し
た。これをロータリーエバポレーターで減圧濃縮するこ
とにより、20.3gの５′−イノシン酸ナトリウム塩の粗
結晶を得た。The supernatant 1 obtained by removing the cells from the culture termination solution of the H-7466 strain was adjusted to pH 1.4 with hydrochloric acid, and Diaion SK # was used.
After passing through a resin tower No. 1 (H type, manufactured by Mitsubishi Kasei Corporation), distilled water was immediately passed through to obtain an eluate. After that, wash the resin tower with water,
The fraction containing 5'-inosic acid in the running water at the initial stage of washing was combined with the eluate already obtained, and adjusted to pH 7.2 with NaOH. This was concentrated under reduced pressure by a rotary evaporator to obtain 20.3 g of a crude crystal of sodium 5'-inosinate.
発明の効果 本発明によれば、５′−イノシン酸を工業的に安価に
効率よく製造することができる。Effects of the Invention According to the present invention, 5'-inosic acid can be produced industrially at low cost and efficiently.
Claims (1)
/KG以上の浸透圧に耐性を有しかつ５′−イノシン酸生
産能を有する微生物を培地に培養し、培養物中に５′−
イノシン酸を生成蓄積させ、該培養物より５′−イノシ
ン酸を採取することを特徴とする発酵法による５′−イ
ノシン酸の製造法。(1) a member belonging to the genus Corynebacterium;
A microorganism which is resistant to osmotic pressure of / KG or more and has 5'-inosinic acid-producing ability is cultured in a medium, and 5'-
A method for producing 5'-inosic acid by fermentation, comprising producing and accumulating inosinic acid and collecting 5'-inosic acid from the culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13383189A JP2886551B2 (en) | 1989-05-26 | 1989-05-26 | Method for producing 5'-inosinic acid by fermentation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP13383189A JP2886551B2 (en) | 1989-05-26 | 1989-05-26 | Method for producing 5'-inosinic acid by fermentation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02312595A JPH02312595A (en) | 1990-12-27 |
| JP2886551B2 true JP2886551B2 (en) | 1999-04-26 |
Family
ID=15114062
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP13383189A Expired - Fee Related JP2886551B2 (en) | 1989-05-26 | 1989-05-26 | Method for producing 5'-inosinic acid by fermentation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2886551B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI222464B (en) * | 1999-02-08 | 2004-10-21 | Kyowa Hakko Kogyo Kk | Process for producing purine nucleotides |
| KR100397321B1 (en) | 2000-12-26 | 2003-09-06 | 씨제이 주식회사 | A microorganism Corynebacterium ammoniagenes CJIP009 being capable of producing 5'-inosinic acid in higher yield and a producing method of 5'-inosinic acid by using the same |
| KR100429925B1 (en) * | 2001-12-28 | 2004-05-03 | 씨제이 주식회사 | Microorganism overproducing 5’-xanthylic acid |
| RU2320717C2 (en) * | 2005-09-14 | 2008-03-27 | Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) | Method for producing purine nucleosides by fermentation method using microorganisms belonging to bacillus genus |
-
1989
- 1989-05-26 JP JP13383189A patent/JP2886551B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02312595A (en) | 1990-12-27 |
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