JP3611881B2 - Method for producing vitamin B12 - Google Patents

Description

（Ｃ）生理学的性質
【表２】

（Ｄ）各種糖類の利用性
【表３】

【０００６】
また、上記糖類からのガス及び酸の生成は１４日間培養後認められなかった。
以上に掲げた各性質をバージーズ（Ｂｅｒｇｅｙ’ｓ） マニュアル オブ システマティック バクテリオロジー（Ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ）第１版（１９８４）の記載をもとに分類したところ、これらの菌はリゾビウム属（Ｇｅｎｕｓ Ｒｈｉｚｏｂｉｕｍ）菌であると同定した。この菌について、ＤＮＡのＧＣ含量を求めたところ、６３．４％であった。さらに、キノン類の抽出及び分析を行った結果、すべて、コエンザイムＱ−１０を持っていた。また、菌体脂肪酸組成の結果は、ヒドロキシ脂肪酸として、３−ヒドロキシ脂肪酸１４：０、１６：０、１８：０（炭素数：二重結合数）を有しており、その割合は、６６：１７：７であった。直鎖脂肪酸は、１６：０、１８：０、１８：１を有しており、１８：１が最も多かった。リゾビウム属の多くは上記のような菌体脂肪酸組成を示すことはすでに知られている。しかし直鎖脂肪酸２１：１及び１９：０を同定株は有していないこと、またＬ−アラビノースから酸の生成が認められない点において従来知られているリゾビウム属細菌とは性質が、異なっている。さらに、ＤＮＡホモロジー解析をエザキ（Ｅｚａｋｉ）らの方法〔エザキ エト アール（Ｅｚａｋｉ ｅｔ ａｌ．）， 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），３９，２２４−２２９（１９８９）〕により、従来より知られているリゾビウム属保存株と比較して行った。その結果を以下に示す。
【０００７】
【表４】

２７Ｂ７４株は、いずれの株とも同一種としての相同性を示さなかった。従って本株をリゾビウム属に属する新種菌株であると結論し、該種名をコバラミノゲヌム（ｃｏｂａｌａｍｉｎｏｇｅｎｕｍ）と称することとした。本明細書において、ＩＦＯ番号は、財団法人発酵研究所（ＩＦＯ、大阪府大阪市淀川区十三本町２丁目１７番８５号）への寄託番号、また、ＦＥＲＭ ＢＰ番号は工業技術院生命工学技術研究所（ＦＲＩ 茨城県つくば市東１丁目１番３号）へのブタペスト条約に基づく寄託番号である。
リゾビウム コバラミノゲヌム ２７Ｂ７４は１９９３年９月２日にＩＦＯへ受託番号ＩＦＯ １５５４３、１９９３年９月２９日にＦＲＩへ受託番号ＦＥＲＭ ＢＰ−４４２９としてそれぞれ寄託されている。
【０００８】
上記のようにして得られたビタミンＢ_１２生産菌の培養には、通常の微生物の培養と同様の方法が用いられる。すなわち、培地としては炭素源、窒素源、無機物、金属塩、あるいは、酵母エキス、酵母菌体、リボ核酸などの物質などのほかに、必要に応じてアミノ酸、ビタミン類などの栄養源を含有する培地が用いられる。例えば炭素源としては、グルコース、シュークロス、マルトース、ソルビトール、でんぷん、でんぷん糖化液、糖蜜などの炭水化物、ピルビン酸、フマル酸、リンゴ酸、コハク酸などの各種有機酸、エタノール、メタノールなどのアルコール類、ベタイン、コリン、モノエタノールアミンなどのアミン類などが用いられる。窒素源としては、ペプトン、コーンスチープリカー、大豆粉、尿素などの有機窒素源のほかに、硫酸、硝酸、塩酸、炭酸などのアンモニウム塩や、アンモニアガス、アンモニア水などの無機窒素源が、それぞれ単独もしくは混合して用いられる。その他の栄養源としては、酵母エキス、酵母菌体、リボ核酸などの物質などのほかに、カルシウム塩、マグネシウム塩、カリウム塩、リン酸塩などの無機塩類、アミノ酸類、ビタミン類などが適宜選択の上、それぞれ単独もしくは混合して用いられる。さらに、培地には、必要に応じてシリコンオイルなどの消泡剤、ポリアルキレングリコールエーテルなどの界面活性剤などを添加することができる。また、ビタミンＢ_１２の前駆体としてコバルト化合物、または５，６−ジメチルベンズイミダゾール化合物を加えることが好ましい。これらの化合物により培地中のビタミンＢ_１２の収量が増大する。コバルト化合物は、コバルト源またはその前駆体であればいずれも用いられる。コバルト化合物としては、例えばハロゲン化コバルト（例、塩化コバルト，臭化コバルト等）、硝酸コバルト、硫化コバルト、酢酸コバルト、硫酸アンモニウムコバルト、炭酸コバルト、４−シクロヘキシル酪酸コバルト、２−エチルヘキサン酸コバルト、水酸化コバルト、リン酸コバルト、酸化コバルトまたはチオシアン酸コバルト等が挙げられる。また、５，６−ジメチルベンズイミダゾール化合物は、５，６−ジメチルベンズイミダゾール源またはその前駆体であればいずれも用いられる。５，６−ジメチルベンズイミダゾール化合物としては、例えば５，６−ジメチルベンズイミダゾール、ニコチンアミド、ニコチネートアデニンジヌクレオチド（ＮＡＤ）、ニコチンアミドアデニンジヌクレオチド（ＮａＡＤ）、ニコチネートモノヌクレオチド（ＮａＭＮ）、ニコチンアミドアデニンジヌクレオチドフォスフェート（ＮＡＤＰ）またはニコチン酸等が挙げられる。
【０００９】
培養は通常、好気的条件下に行われる。具体的には、例えば振とうあるいは通気撹はん深部培養等により行われる。培地のｐＨは通常約４ないし９の範囲が好ましい。培養中にｐＨの変動が観察される場合には、これを好ましい範囲に修正するために、酸（例、硫酸，塩酸，酢酸等）、アルカリ（例、炭酸カルシウム，水酸化ナトリウム，アンモニアガス，アンモニア水等）を適宜添加してもよい。培養温度は、通常約２０℃ないし４５℃の範囲から使用される微生物の生育及びビタミンＢ_１２の蓄積に好適な温度が選択される。培養は実質的にビタミンＢ_１２蓄積量が最大になるまで行われるが、通常約２日間ないし１０日間の培養で目的を達することができる。
リゾビウム コバラミノゲヌムに属するビタミンＢ_１２生産菌は、他の細菌の場合と同様に、たとえば紫外線、放射線などの照射、単細胞分離、種々の変異処理、その他の慣用の手段により変異させることができ、このような変異株あるいは自然に得られる突然変異株であっても、上記した分類学的性状との比較において実質的に別種とするに足らず、しかもビタミンＢ_１２を生産する能力を有するものは、すべて本発明方法に利用し得る。
【００１０】
培養物からビタミンＢ_１２及びビタミンＢ_１２各成分を分離、採取するにはすでに公知にされている通常の精製手段、例えば、フェノール、ブタノールなどを用いた溶媒抽出法や、沈殿法、イオン交換樹脂やシリカゲル、活性炭などによるクロマトグラフィー法などの分離精製法が用いられる〔ジェイ フローレント アンド エル ニネット（Ｊ． Ｆｌｏｒｅｎｔ ａｎｄ Ｌ． Ｎｉｎｅｔ），バイタミン（Ｖｉｔａｍｉｎ）Ｂ_１２ミクロービアル テクノロジー（Ｍｉｃｒｏｂｉａｌ Ｔｅｃｈｎｏｌｏｇｙ） エイチ ジェイ ペプラー アンド デー パールマン（Ｈ． Ｊ． Ｐｅｐｐｌｅｒ ａｎｄ Ｄ． Ｐｅｒｌｍａｎ）編，アカデミック プレス エヌ ワイ（Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ， Ｎ． Ｙ．），４９７−５１９頁（１９７９年）〕。
さらに具体的には、例えば、培養物を遠心分離して、菌体を得た後、無機酸（例、塩酸，硫酸等）または有機酸（例、酢酸，酒石酸等）を用いることによりｐＨを約５．０に調整後、シアンイオンを添加して加熱することによりシアノ型として水中へ抽出する。また、ビタミンＢ_１２をメチル型または補酵素型として得る場合には、常法により菌体から暗所でアルコール類（例、エタノール，プロパノール等）、ケトン類（例、アセトン，メチルエチルケトン等）などを用いて抽出する。菌体よりビタミンＢ_１２を抽出するとき、所望により菌体を自体公知の方法により破砕することも有効である。
【００１１】
【実施例】
以下の実施例により、本発明についてさらに具体的に説明する。実施例中の％および比は特にことわりのない限り、容量％および容量比をそれぞれ示す。
実施例１
１％（重量％）ショ糖を含んだ肉汁液体培地５ｍｌを、綿栓付試験管に分注した後、加熱殺菌した。この培地に予め肉汁寒天培地上で３０℃、３日間生育させたリゾビウム コバラミノゲヌム２７Ｂ７４（ＩＦＯ １５５４３、ＦＥＲＭ ＢＰ−４４２９）を一白金耳量接種し、３０℃で２４時間振とう培養した。〔表５〕に示す組成の培地２０ｍｌを２００ｍｌ容フラスコにいれ加熱滅菌した後、これに上記培養物１．０ｍｌを接種し、毎分２３０回転のロータリーシェーカー上で３０℃、６日間培養した。
上記と同様の方法で得られた培養物１リットルから遠心分離により菌体を集めた。得られた菌体を酢酸バッファーに懸濁してｐＨ５．０に調整し、さらに、これにＫＣＮを最終濃度が１００μｇ／ｍｌとなるように添加した後、１００℃、１５分加熱した。冷却後、遠心分離により沈殿物を除き、ビタミンＢ_１２抽出液を得た。抽出液のビタミンＢ_１２量を高速液体クロマトグラフィー法で定量した。カラムはＯＤＰ−５０ ４．６×１５０ｍｍ（旭化成社製）を使用し、カラムからのビタミンＢ_１２の溶出には、４０ｍＭ酢酸アンモニウム：アセトニトリル＝８８：１２を用いた。また溶出液中のビタミンＢ_１２含量は、３６１ｎｍにおける吸光度の測定により求めた。その結果、上記培養物には、２２μｇ／ｍｌのビタミンＢ_１２が生成していたことが認められた。
次に上記のビタミンＢ_１２抽出液を活性炭カラムにかけて、ビタミンＢ_１２を吸着させた。カラムを蒸留水により洗浄した後、吸着物を４０％アセトンで溶出した。ビタミンＢ_１２画分を集めて、減圧下に濃縮した後、ダウエックス５０のカラムにかけた。カラムを水洗した後、５０ｍＭ酢酸ナトリウム緩衝液（ｐＨ６．５）により吸着物を溶出した。ビタミンＢ_１２画分を集めて、中和した後、再度活性炭カラムにかけた。カラムを蒸留水により洗浄した後、吸着物を４０％アセトンで溶出した。ビタミンＢ_１２画分を集めて減圧下に濃縮した後、これにアセトンを加えて、ビタミンＢ_１２を晶出させたところ、１６．５ｍｇのビタミンＢ_１２（シアノ型）結晶が得られた。
【００１２】
【表５】

【００１３】
実施例２
補酵素型でビタミンＢ_１２各成分を得るために、実施例１で得られた培養物１リットルから遠心分離により菌体を集めた。暗所でエタノール（ＣＮ無添加）濃度が８０％となるように調整し、得られた菌体を８０℃で２０分間加熱抽出した。抽出液のエタノールを減圧下に除き、水溶液を得た。得られた水溶液をＳｅｐ−Ｐａｋ Ｃ１８（ウォーターズ社製）を用いて脱塩処理した後、補酵素型ビタミンＢ_１２の同定を高速液体クロマトグラフィー法により行った。カラムはＬｉｃｈｒｏｓｐｈｅｒ ＲＰ １８（５×２５０ｍｍ）（メルク社製）を使用し、カラムからの溶出には、８５ｍＭリン酸（ｐＨ３．０）とアセトニトリルによるグラジェント法を用いた。アセトニトリルの濃度は、０％（溶出開始時）から５０％（溶出開始２５分後）になるようにした。また、溶出液中の補酵素型ビタミンＢ_１２含量は、３６１ｎｍにおける吸光度の測定により求めた。その結果、上記培養物には、アデノシルコバラミン（Ａｄｏ−Ｂ_１２）（１８μｇ／ｍｌ）、メチルコバラミン（ＣＨ_３−Ｂ_１２）（２．５μｇ／ｍｌ）およびヒソロキソコバラミン（ＯＨ−Ｂ_１２）（１．０μｇ／ｍｌ）が生成していたことが認められた。
【００１４】
比較例
実施例１と同様にして、リゾビウム メリロッテイ（Ｒｈｉｚｏｂｉｕｍ ｍｅｌｉｌｏｔｉ）ＩＦＯ １４７８２，リゾビウム ガレガエ（Ｒｈｉｚｏｂｉｕｍ ｇａｌｅｇａｅ）ＩＦＯ １４９６５，リゾビウム フアクイー（Ｒｈｉｚｏｂｉｕｍ ｈｕａｋｕｉｉ）ＩＦＯ １５２４３，リゾビウム レグミノサルム（Ｒｈｉｚｏｂｉｕｍ ｌｅｇｕｍｉｎｏｓａｒｕｍ）ＩＦＯ １３３３７，リゾビウム レグミノサルム（Ｒｈｉｚｏｂｉｕｍ ｌｅｇｕｍｉｎｏｓａｒｕｍ）ＩＦＯ １４７８４，リゾビウム ロッテイ（Ｒｈｉｚｏｂｉｕｍ ｌｏｔｉ）ＩＦＯ １３３３６およびリゾビウム トロピシー（Ｒｈｉｚｏｂｉｕｍ ｔｒｏｐｉｃｉ）ＩＦＯ １５２４７を３０℃、６日間培養した。得られた培養物から実施例１記載の方法を用いて、抽出液のビタミンＢ_１２含量を定量した。その結果を〔表６〕に示す。
【表６】

【００１５】
【発明の効果】
本発明により、工業的に安価に、容易に、効率的にビタミンＢ_１２が製造できる。[0001]
[Industrial application fields]
The present invention, pernicious anemia, nervous diseases, is utilized in such therapeutic agents methylmalonic aciduria, In addition, a process for producing vitamin B _12, which is widely used as a feed additive for poultry and / or livestock.
[0002]
[Prior art]
For the preparation of vitamin B _12, because of the complexity of its structure, the use of chemical synthesis is difficult for industrial fermentation process according Currently microorganisms are used.
With respect to the production of vitamin _{B 12} by microorganisms, Streptomyces (Streptomyc es) the genus Nocardia (Nocardia) sp., Micromonospora (Micromonospora) genus, Aerobacter (Aerobacter) genus, Agrobacterium (Agrobacterium) genus Alcaligenes (Alcaligenes ) genus, Azotobacter (Azotobacter) genus Bacillus (Bacillus) sp., Clostridium (Clostridium) genus Corynebacterium (Corynebacterium) genus Escherichia (Escherichia) sp., Flavobacterium (Flavobacterium) genus Mycobacterium (Mycobacterium) genus The pseudo Monas (Pseudomonas) genus Propionibacterium (Propionibacterium) genus, Proteus (Proteus) genus Serratia (Serratia) genus Streptococcus (Streptococcus) genus Xanthomonas (Xanthomonas) genus, professional data Mino Enterobacter (Protaminobacter) genus, Metanobachirusu (Methanobacillus) genus [E Jay Bandame (E. J. Vandamme), biotechnology of Baitaminzu pig Apartments and growth factors (biotechnology of Vitamins, Pigments and growth factors), Erusebia Science publishers El tee Day (E sevier Science Publisher LTD), pp. 1989,261-263], Arthrobacter (Arthrobacter) belonging to the genus (JP-A-52-94498), Klebsiella (Klebsiella) belonging to the genus (JP-A-50-132186), Rhodopseudomonas (Rhodopseudomonus) genus (JP 60-16597), blanking Chile Agrobacterium (Butyribacterium) genus (JP 62-44197), pseudoephedrine Nocardia (Pseudonocardia) genus (JP 55-96091), Methanosarcina (Methanosarcina) genus (Japanese Unexamined 1- 257490), Yu Mycobacterium (Eubacterium) genus (JP 62-44172), aceto Mycobacterium (Acetoba Cterium ) (Japanese Patent Laid-Open No. 62-122593) has been reported. Further Rhizobium Merirottei (Rhizobium meliloti), Rhizobium Fazeori (Rhizobium phaseoli), Rhizobium japonicum (Rhizobium japonicum), Rhizobium Torifori (Rhizobium trifolli), various Rhizobium leguminosarum (Rhizobium leguminosarum) is reported to accumulate vitamin _{B 12} [Plant Physiology, 38 , 99-104 (1963)].
Among the above Rhizobium species, Rhizobium phaseori and Rhizobium trifoli have been renamed Rhizobium regminosalum [Farming Institute, List of Cultures, 1992 Microorganisms, 9th edition] , Page 169]. Also, a part of Rhizobium japonicum has been renamed Rhizobium leguminosalum, and the rest has been reclassified to Bradyrhizobium japonicum [Food Research Institute, List of Cultures] 1992, Microorganisms, 9th edition, page 169].
[0003]
[Problems to be solved by the invention]
In general, however, the production of vitamin B ₁₂ by microorganisms has become extremely low. In some microorganisms, available carbon sources are limited (such as methanol-utilizing bacteria) or special culture conditions such as anaerobic conditions are required (propionic acid bacteria, acetobacterium, methane) Many production methods are possible only under limited conditions such as methanogenic bacteria, propion bacteria, etc. that require a long incubation time due to low growth rate, Development of improved industrial processes is desired.
[0004]
[Means for Solving the Problems]
The present invention is to provide an industrial production process of an inexpensive and efficient vitamin B _12. The present inventors generally used vitamin B ₁₂ using aeration and agitation culture method, which is a widely used fermentation production condition using glucose, sucrose or the like widely used for fermentation production as a carbon source. In order to develop a method to efficiently fermentatively produce sucrose, a new strain Rhizobium cobalaminogen 27B74 having a high ability to produce vitamin B ₁₂ was isolated from soil in Hyogo Prefecture, and earned research was repeated. Based on the findings, the present invention has been completed.
That is, the present invention
(1) the Rhizobium Kobaraminogenumu having an ability to produce vitamin B ₁₂ were cultured in a medium, yielding accumulate vitamin B _12, the preparation of vitamin B _12, characterized in that collecting the,
(2) The production method according to (1), wherein the medium contains a cobalt compound,
(3) The production method according to the above (2), wherein the cobalt compound is cobalt halide,
(4) The production method according to claim 1, wherein the medium contains a 5,6-dimethylbenzimidazole compound,
(5) The production method according to (1), wherein the Rhizobium cobalaminogenum is Rhizobium cobalaminogenum 27B74 strain (FERM BP-4429),
(6) Rhizobium Kobaraminogenumu having the ability to produce vitamin _{B 12,} and a (7) a microorganism Rhizobium Kobaraminogenumu 27B74 strain (FERM BP-4429).
[0005]
In the present invention, vitamin _{B 12} is coenzyme vitamin _{B 12} [eg, adenosyl cobalamin, methylcobalamin (methyl-type cobalamin)], cyano-type vitamin _{B 12} (cyanocobalamin) or hydroxo-type vitamin _{B 12} of (hydroxocobalamin) Either may be sufficient.
The morphological properties of Rhizobium cobalaminogenum 27B74 (IFO 15543, FERM BP-4429), which have excellent ability to produce vitamin B ₁₂ separated from the soil, the growth state and physiological properties in each medium are described below. Show.
(A) Morphological properties (when grown on a broth agar medium at 28 ° C. for 24 hours)
1. Cell morphology: Neisseria gonorrhoeae Cell size: 0.5-0.8 μm × 1-2.8 μm
3. 3. Presence or absence of spores: None Motility: Existence (meat liquid culture, observation after growth at 28 ° C for 16 hours)
5. Gram stainability: negative 6. Anti-acidity: None (B) Growth condition in each medium [Table 1]

(C) Physiological properties [Table 2]

(D) Usability of various sugars [Table 3]

[0006]
Moreover, the production | generation of the gas and acid from the said saccharide | sugar was not recognized after culture | cultivation for 14 days.
The properties listed above were classified based on the description of Bergey's Manual of Systematic Bacteriology, 1st Edition (1984). These bacteria were classified into Genus Rhizobium . Identified as a fungus. For this bacterium, the GC content of the DNA was determined to be 63.4%. Furthermore, as a result of extracting and analyzing quinones, all had coenzyme Q-10. Moreover, the result of a cell fatty-acid composition has 3-hydroxy fatty acid 14: 0, 16: 0, 18: 0 (carbon number: double bond number) as a hydroxy fatty acid, The ratio is 66: 17: 7. Linear fatty acids had 16: 0, 18: 0, 18: 1, with 18: 1 being the most. It is already known that many Rhizobium genera have the above-mentioned cell fatty acid composition. However, it differs from the conventionally known Rhizobium bacteria in that it does not have strains identified with linear fatty acids 21: 1 and 19: 0 and that no acid is produced from L-arabinose. Yes. Furthermore, DNA homology analysis was performed by the method of Ezaki et al. (Ezaki et al., International Journal of Systematic Bacteriology, 39 , 224-229 (conventional), 39 , 224-229 (conventional)). The comparison was made with a more known Rhizobium stock. The results are shown below.
[0007]
[Table 4]

The 27B74 strain did not show homology as the same species with any strain. Therefore, it was concluded that this strain was a new strain belonging to the genus Rhizobium, and the species name was designated as cobalaminogenum . In this specification, the IFO number is the number assigned to the Fermentation Research Institute (IFO, 2-17-85 Jusanhoncho, Yodogawa-ku, Osaka-shi, Osaka), and the FERM BP number is the Biotechnology of the Industrial Technology Institute. This is a deposit number based on the Budapest Treaty with the Institute (FRI Ibaraki Prefecture Tsukuba City East 1-3-3 East).
Rhizobium cobalaminogenum 27B74 has been deposited with the IFO under the accession number IFO 15543 on September 2, 1993 and under the accession number FERM BP-4429 at the FRI on September 29, 1993.
[0008]
The culture of the vitamin B ₁₂ producing bacterium obtained as described above, the same method as ordinary microbial culture is used. In other words, the medium contains carbon sources, nitrogen sources, inorganic substances, metal salts, or substances such as yeast extract, yeast cells, and ribonucleic acid as well as nutrient sources such as amino acids and vitamins as necessary. A medium is used. For example, carbon sources include glucose, shoecloth, maltose, sorbitol, starch, starch saccharified liquid, carbohydrates such as molasses, various organic acids such as pyruvic acid, fumaric acid, malic acid and succinic acid, and alcohols such as ethanol and methanol. , Amines such as betaine, choline and monoethanolamine are used. Nitrogen sources include organic nitrogen sources such as peptone, corn steep liquor, soy flour, and urea, as well as ammonium salts such as sulfuric acid, nitric acid, hydrochloric acid, and carbonic acid, and inorganic nitrogen sources such as ammonia gas and aqueous ammonia. Used alone or in combination. As other nutrient sources, in addition to substances such as yeast extract, yeast cells, and ribonucleic acid, inorganic salts such as calcium salts, magnesium salts, potassium salts, phosphates, amino acids, vitamins, etc. are selected as appropriate. In addition, each is used alone or in combination. Furthermore, an antifoaming agent such as silicone oil, a surfactant such as polyalkylene glycol ether, and the like can be added to the medium as necessary. Further, it is preferable to add a cobalt compound, or 5,6-dimethyl benzimidazole compounds as precursors of vitamin B _12. The yield of vitamin B ₁₂ in the medium is increased by these compounds. As the cobalt compound, any cobalt source or precursor thereof may be used. Examples of the cobalt compound include cobalt halide (eg, cobalt chloride, cobalt bromide, etc.), cobalt nitrate, cobalt sulfide, cobalt acetate, cobalt sulfate ammonium, cobalt carbonate, cobalt 4-cyclohexylbutyrate, cobalt 2-ethylhexanoate, water Examples thereof include cobalt oxide, cobalt phosphate, cobalt oxide, and cobalt thiocyanate. Any 5,6-dimethylbenzimidazole compound may be used as long as it is a 5,6-dimethylbenzimidazole source or a precursor thereof. Examples of the 5,6-dimethylbenzimidazole compound include 5,6-dimethylbenzimidazole, nicotinamide, nicotinate adenine dinucleotide (NAD), nicotinamide adenine dinucleotide (NaAD), nicotinate mononucleotide (NaMN), and nicotine. Amidoadenine dinucleotide phosphate (NADP), nicotinic acid, etc. are mentioned.
[0009]
Culture is usually performed under aerobic conditions. Specifically, it is carried out by, for example, shaking or aeration stirring deep culture. The pH of the medium is usually preferably in the range of about 4 to 9. If a change in pH is observed during culture, an acid (eg, sulfuric acid, hydrochloric acid, acetic acid, etc.), alkali (eg, calcium carbonate, sodium hydroxide, ammonia gas, Ammonia water or the like) may be added as appropriate. Culture temperature, a temperature suitable for the accumulation of normal growth and vitamin B ₁₂ in the microorganisms used in the range of about 20 ° C. to 45 ° C. is selected. The culture is carried out until the amount of accumulated vitamin B ₁₂ is substantially maximized, but the purpose can be usually achieved by culturing for about 2 to 10 days.
Vitamin B ₁₂ producing bacteria belonging to Rhizobium Kobaraminogenumu, as in the case of other bacteria, such as ultraviolet light, radiation, such as radiation, single cell isolation, various mutagenesis, can be mutated by means of other conventional, such even Do mutant or obtained naturally mutants, not trivial to be substantially different type in comparison to the taxonomic properties described above, moreover those having the ability to produce vitamin B _12, all the It can be used for the inventive method.
[0010]
In order to separate and collect vitamin B ₁₂ and vitamin B ₁₂ components from the culture, conventional known purification means such as solvent extraction using phenol, butanol, precipitation, ion exchange resin, etc. And separation and purification methods such as chromatography using silica gel, activated carbon, etc. [J. Florent and L. Ninet, Vitamin B ₁₂ Microbiology Technology, H. J. Pepper and Day Edited by H. J. Pepper and D. Perlman, Academic Press, NY, 497-519 (1979)].
More specifically, for example, after centrifuging the culture to obtain bacterial cells, the pH is adjusted by using an inorganic acid (eg, hydrochloric acid, sulfuric acid, etc.) or an organic acid (eg, acetic acid, tartaric acid, etc.). After adjusting to about 5.0, cyanide ions are added and heated to extract the cyano form into water. Further, in the case of obtaining the vitamin B ₁₂ as a methyl type or coenzyme-type, alcohol dark from the bacterial cells by conventional methods (e.g., ethanol, propanol, etc.), ketones (e.g., acetone, methyl ethyl ketone), etc. Use to extract. When extracting the vitamin B ₁₂ from the cells, it is effective to crush by a method known per se the cells desired.
[0011]
【Example】
The following examples further illustrate the present invention. Unless otherwise specified,% and ratio in the examples indicate volume% and volume ratio, respectively.
Example 1
5 ml of a broth liquid medium containing 1% (weight%) sucrose was dispensed into a test tube with a cotton plug, and then heat sterilized. This medium was inoculated with one platinum loop of Rhizobium cobalaminogenum 27B74 (IFO 15543, FERM BP-4429) previously grown on a broth agar medium at 30 ° C. for 3 days, and cultured with shaking at 30 ° C. for 24 hours. 20 ml of a medium having the composition shown in [Table 5] was placed in a 200 ml flask and sterilized by heating. Then, 1.0 ml of the above culture was inoculated and cultured on a rotary shaker at 230 rpm for 6 days at 30 ° C.
Bacteria were collected from 1 liter of the culture obtained by the same method as above by centrifugation. The obtained microbial cells were suspended in an acetate buffer to adjust to pH 5.0, and KCN was added thereto so that the final concentration was 100 μg / ml, followed by heating at 100 ° C. for 15 minutes. After cooling, the precipitate was removed by centrifugation to obtain a vitamin B ₁₂ extract. The amount of vitamin B _{12 in the} extract was quantified by high performance liquid chromatography. Column using ODP-50 4.6 × 150mm (manufactured by Asahi Chemical Industry Co., Ltd.), the dissolution of the vitamin _{B 12} from the column, 40 mM ammonium acetate: acetonitrile = 88: 12 was used. The vitamin _{B 12} content in the eluate was determined by measurement of absorbance at 361 nm. As a result, it was confirmed that 22 μg / ml vitamin B ₁₂ was produced in the culture.
Next subjected activated charcoal column of the above vitamin B ₁₂ extract to adsorb the vitamin B _12. After the column was washed with distilled water, the adsorbate was eluted with 40% acetone. Vitamin B ₁₂ fractions were collected and concentrated under reduced pressure and then applied to a Dowex 50 column. After the column was washed with water, the adsorbate was eluted with 50 mM sodium acetate buffer (pH 6.5). The ₁₂ fractions of vitamin B were collected and neutralized, and then applied again to the activated carbon column. After the column was washed with distilled water, the adsorbate was eluted with 40% acetone. Vitamin B ₁₂ fractions were collected and concentrated under reduced pressure, and then acetone was added thereto to crystallize vitamin B _{12. As} a result, 16.5 mg of vitamin B ₁₂ (cyano-type) crystals were obtained.
[0012]
[Table 5]

[0013]
Example 2
In order to obtain each component of vitamin B ₁₂ in a coenzyme type, cells were collected from 1 liter of the culture obtained in Example 1 by centrifugation. In a dark place, the concentration of ethanol (without addition of CN) was adjusted to 80%, and the obtained cells were extracted by heating at 80 ° C. for 20 minutes. Ethanol of the extract was removed under reduced pressure to obtain an aqueous solution. After desalted using the obtained aqueous solution Sep-Pak C18 (Waters), it was identified coenzyme vitamin B ₁₂ by high performance liquid chromatography. The column used was Lichlorosphere RP 18 (5 × 250 mm) (manufactured by Merck), and the gradient method using 85 mM phosphoric acid (pH 3.0) and acetonitrile was used for elution from the column. The concentration of acetonitrile was adjusted from 0% (at the start of elution) to 50% (25 minutes after the start of elution). Further, the content of coenzyme-type vitamin B ₁₂ in the eluate was determined by measuring the absorbance at 361 nm. As a result, adenosylcobalamin (Ado-B ₁₂ ) (18 μg / ml), methylcobalamin (CH ₃ -B ₁₂ ) (2.5 μg / ml) and hisoloxocobalamin (OH-B ₁₂ ) (1.0 μg / ml) was found to be produced.
[0014]
In the same manner as in Comparative Example 1, Rhizobium Merirottei (Rhizobium meliloti) IFO 14782, Rhizobium Garegae (Rhizobium galegae) IFO 14965, Rhizobium Fuakui (Rhizobium huakuii) IFO 15243, Rhizobium leguminosarum (Rhizobium leguminosarum) IFO 13337, Rhizobium leguminosarum (Rhizobium leguminosarum) IFO 14784, Rhizobium Rottei (Rhizobium loti) IFO 13336 and Rhizobium Toropishi (Rhizobium tropici) IFO 15247 and 30 ° C., and cultured for 6 days. The vitamin B ₁₂ content of the extract was quantified from the obtained culture using the method described in Example 1. The results are shown in [Table 6].
[Table 6]

[0015]
【The invention's effect】
According to the present invention, vitamin B ₁₂ can be produced easily and efficiently industrially at low cost.

Claims (7)

Rhizobium Kobaraminogenumu having an ability to produce vitamin B ₁₂ were cultured in a medium, yielding accumulate vitamin B _12, the preparation of vitamin B _12, characterized in that collecting the. The production method according to claim 1, wherein the medium contains a cobalt compound. The process according to claim 2, wherein the cobalt compound is a cobalt halide. The process according to claim 1, wherein the medium contains a 5,6-dimethylbenzimidazole compound. The method according to claim 1, wherein the Rhizobium cobalaminogenum is Rhizobium cobalaminogenum 27B74 strain (FERM BP-4429). Rhizobium Kobaraminogenumu having the ability to produce vitamin B _12. Microorganism Rhizobium Kobaraminogenumu 27B74 strain having an ability to produce vitamin B ₁₂ (FERMBP-4429).