JP2023182179A - Method for producing 1,5-anhydro-D-mannitol - Google Patents
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Abstract
Description
本発明は、１，５－アンヒドロ－Ｄ－マンニトールの微生物による製造法に関する。 The present invention relates to a method for producing 1,5-anhydro-D-mannitol using microorganisms.
１，５－アンヒドロ－Ｄ－マンニトール（以下１，５－ＡＭ）は、マンノースの１位の水酸基が還元された構造をもつポリオールである。１，５－ＡＭは、動物細胞の培養基材として用いることで、抗体の産生が増強することが知られている。また、１，５－ＡＭに脂肪酸を結合させることで脂質のゲル化剤となることも報告されている。このように１，５－ＡＭは、産業的に価値の高い糖である。 1,5-Anhydro-D-mannitol (hereinafter referred to as 1,5-AM) is a polyol having a structure in which the hydroxyl group at the 1-position of mannose is reduced. 1,5-AM is known to enhance antibody production when used as a culture substrate for animal cells. It has also been reported that 1,5-AM can be used as a lipid gelling agent by binding a fatty acid to it. Thus, 1,5-AM is an industrially valuable sugar.
１，５－ＡＭの製造法としては、化学的に水素付加する方法が報告されている（非特許文献１参照）。しかし、この方法では、１，５－ＡＭの他に１，５－ＡＭの２位のエピマーである、１，５－アンヒドロ－Ｄ－グルシトール（以下１，５－ＡＧ）も生成されてしまうため、１，５－ＡＭの収率が低いという問題があった。例えば、水素化ホウ素ナトリウムを用いた化学的な水素付加においては、副生物の１，５－ＡＧの収率が６０％と多量に生成する。また、パラジウム触媒存在下での水素付加においては１，５－ＡＭが優勢となるものの、１，５－ＡＭの収率は５８％と低収率である。 As a method for producing 1,5-AM, a method of chemical hydrogenation has been reported (see Non-Patent Document 1). However, in this method, in addition to 1,5-AM, 1,5-anhydro-D-glucitol (hereinafter referred to as 1,5-AG), which is an epimer at the 2-position of 1,5-AM, is also produced. , there was a problem that the yield of 1,5-AM was low. For example, in chemical hydrogenation using sodium borohydride, a large amount of the by-product 1,5-AG is produced at a yield of 60%. Furthermore, although 1,5-AM becomes predominant in hydrogenation in the presence of a palladium catalyst, the yield of 1,5-AM is as low as 58%.
１，５－ＡＭを食品素材として使用する場合、摂取しても安全である素材であることが求められる。しかしながら、上記のような化学合成法では、生成物中に化学物質の残留等のおそれがあるため、化学合成法ではなく、より自然な製造法、例えば微生物を用いた１，５－ＡＭの製造法が望ましいといえる。 When using 1,5-AM as a food material, it is required that the material is safe to ingest. However, with the above chemical synthesis method, there is a risk that chemical substances may remain in the product. It can be said that a law is preferable.
微生物を用いた１，５－ＡＭの製造法として、１，５－アンヒドロ－Ｄ－フルクトース（以下１，５－ＡＦ）に、パン酵母を接触させて１，５－ＡＭを製造する方法が報告されている（非特許文献２参照）。しかし、この方法においても１，５－ＡＧが副生物として生成されるという問題があり、１，５－ＡＭの収率は約５０％と低い。 As a method for producing 1,5-AM using microorganisms, a method for producing 1,5-AM by bringing baker's yeast into contact with 1,5-anhydro-D-fructose (hereinafter referred to as 1,5-AF) has been reported. (See Non-Patent Document 2). However, this method also has the problem that 1,5-AG is produced as a by-product, and the yield of 1,5-AM is as low as about 50%.
上記の通り従来の合成方法においては、副生物が多く生成して１，５－ＡＦから１，５－ＡＭへの変換率が低く十分でなかった。従って、副生物を生成せず、１，５－ＡＦから１，５－ＡＭのみを選択的に効率良く製造する方法が望まれている。 As mentioned above, in the conventional synthesis method, many by-products were produced and the conversion rate of 1,5-AF to 1,5-AM was low and insufficient. Therefore, a method for selectively and efficiently producing only 1,5-AM from 1,5-AF without producing by-products is desired.
微生物を用いて１，５－ＡＦから１，５－ＡＭのみを選択的に製造する方法の一つとして、シノリゾビウム・モレレンス種の菌株から、１，５－ＡＦを１，５－ＡＭに変換する還元酵素を分離し、当該還元酵素を用いて１，５－ＡＭを製造する技術が提案されている（非特許文献３参照）。
この製造法においては反応系に１，５－ＡＦから１，５－ＡＭに還元する際に補酵素（ニコチンアミドアデニンジヌクレオチドリン酸；ＮＡＤＰＨ）が必要であり、反応系内にグルコース－６－リン酸とグルコース－６－リン酸脱水素酵素が添加される。ここで、反応においては１モルの１，５－ＡＦの還元に１モルのＮＡＤＰＨが必要であり、１モルのＮＡＤＰＨの発生に、１モルのグルコース－６－リン酸が必要となる。従って、１，５－ＡＦの還元には、１，５－ＡＦと等モルのグルコース６リン酸が必要となる。しかしながら、グルコース－６－リン酸は非常に高価であることから、この反応による製造法では１，５－ＡＭの製造コストが非常に高くなるという問題があるため、より安価な１，５－ＡＭの製造法が求められている。
One method for selectively producing only 1,5-AM from 1,5-AF using microorganisms is to convert 1,5-AF to 1,5-AM from a strain of Sinorhizobium morelens species. A technique for separating a reductase and producing 1,5-AM using the reductase has been proposed (see Non-Patent Document 3).
In this production method, a coenzyme (nicotinamide adenine dinucleotide phosphate; NADPH) is required in the reaction system to reduce 1,5-AF to 1,5-AM, and glucose-6- Phosphoric acid and glucose-6-phosphate dehydrogenase are added. Here, in the reaction, 1 mol of NADPH is required to reduce 1 mol of 1,5-AF, and 1 mol of glucose-6-phosphate is required to generate 1 mol of NADPH. Therefore, reduction of 1,5-AF requires equimolar amount of glucose 6-phosphate to 1,5-AF. However, since glucose-6-phosphate is very expensive, there is a problem in that the production cost of 1,5-AM becomes very high with this reaction-based production method. A manufacturing method is required.
本発明はこのような従来の問題点を解決して、１，５－ＡＦを出発物質とし１，５－ＡＭへの高い変換能を有する微生物を用いた１，５－ＡＭの製造法を提供することにある。 The present invention solves these conventional problems and provides a method for producing 1,5-AM using a microorganism that uses 1,5-AF as a starting material and has a high conversion ability to 1,5-AM. It's about doing.
本発明においては、ウィッカーハモマイセス・アノマルス種の菌株を１，５－アンヒドロ－Ｄ－フルクトースと接触させて１，５－アンヒドロ－Ｄ－マンニトールを生成させ、次いで生成した１，５－アンヒドロ－Ｄ－マンニトールを採取することを特徴とする１，５－アンヒドロ－Ｄ－マンニトールの製造法が提供される。
本発明の１，５－アンヒドロ－Ｄ－マンニトールの製造法においては、前記ウィッカーハモマイセス・アノマルス種の菌株がＮＢＲＣ ０７０７またはＮＢＲＣ １０２１３であることが好ましい。
In the present invention, a strain of Wickerhamomyces anomalus sp. is contacted with 1,5-anhydro-D-fructose to produce 1,5-anhydro-D-mannitol, and then the produced 1,5-anhydro- A method for producing 1,5-anhydro-D-mannitol is provided, which comprises collecting D-mannitol.
In the method for producing 1,5-anhydro-D-mannitol of the present invention, the strain of Wickerhamomyces anomalis species is preferably NBRC 0707 or NBRC 10213.
本発明においては、ウィッカーハモマイセス・アノマルス種の菌株を用いることにより、１，５－ＡＦを１，５－ＡＭのみに選択的に変換し副生物を生成しないため、生産性が向上する。
ウィッカーハモマイセス・アノマルス種の菌株は、従来から食品分野で幅広く使用されてきた菌株であり、例えばワインの製造に用いられる酵母の一種である。従って当該菌株を用いた製造法は、化学的に水素付加する方法等に比べ、安全性が高い。
さらに、ウィッカーハモマイセス・アノマルス種の菌株を用いた１，５－ＡＭの製造は、グルコース－６－リン酸等の高価な添加剤を用いる必要がないため、製造コストが低く安価な１，５－ＡＭの製造法である。
In the present invention, by using a strain of Wickerhamomyces anomalus species, 1,5-AF is selectively converted to 1,5-AM only, and by-products are not produced, thereby improving productivity.
The Wickerhamomyces anomalus strain is a strain that has been widely used in the food field, and is a type of yeast used, for example, in wine production. Therefore, the production method using this strain is safer than methods such as chemical hydrogenation.
Furthermore, the production of 1,5-AM using Wickerhamomyces anomalus strain does not require the use of expensive additives such as glucose-6-phosphate, resulting in low production costs and inexpensive 1,5-AM. This is a method for producing 5-AM.
１，５－ＡＦを１，５－ＡＭに変換する高い能力を持つウィッカーハモマイセス・アノマルス種の菌株としては、具体的にはＮＢＲＣ ０７０７またはＮＢＲＣ １０２１３などが挙げられる。これら菌株は、ブダペスト条約による国際寄託がなされており、独立行政法人製品評価技術基盤機構バイオテクノロジー本部（Ｉｎｃｏｒｐｏｒａｔｅｄ Ａｄｍｉｎｉｓｔｒａｔｉｖｅ Ａｇｅｎｃｙ Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅ ｏｆ Ｔｅｃｈｎｏｌｏｇｙ ａｎｄ Ｅｖａｌｕａｔｉｏｎ）千葉県木更津市かずさ鎌足２－５－８ から入手可能である。但し、本発明に用いられるウィッカーハモマイセス・アノマルス種の菌株は、１，５－ＡＦを１，５－ＡＭに変換する能力を有するウィッカーハモマイセス・アノマルス種の菌株であれば全てが使用可能であり、上記菌株に限定されるものではない。
本発明のウィッカーハモマイセス・アノマルス種の菌株は、前記の菌株より派生する変異株、例えば紫外線照射、Ｎ－メチル－Ｎ－ニトロソグアニジン（ＮＴＧ）処理、エチルメタンスルホネート（ＥＭＳ）処理、亜硝酸処理、アクリジン処理等による変異株、あるいは細胞融合もしくは遺伝子組み換え法などの遺伝学的手法により誘導される遺伝子組み換え株などいずれの株であっても良い。
Specific examples of strains of Wickerhamomyces anomalus that have a high ability to convert 1,5-AF to 1,5-AM include NBRC 0707 and NBRC 10213. These strains have been internationally deposited under the Budapest Treaty and have been deposited with the Incorporated Administrative Agency Department of Biotechnology National Institute of Technology. ogy and Evaluation) 2-5- Kazusa Kamatari, Kisarazu City, Chiba Prefecture Available from 8. However, the strain of Wickerhamomyces anomalus used in the present invention may be any strain of Wickerhamomyces anomalus that has the ability to convert 1,5-AF to 1,5-AM. possible, and is not limited to the above strains.
The Wickerhamomyces anomalis strain of the present invention is a mutant strain derived from the above-mentioned strain, such as ultraviolet irradiation, N-methyl-N-nitrosoguanidine (NTG) treatment, ethyl methanesulfonate (EMS) treatment, nitrous acid treatment, etc. It may be any strain, such as a mutant strain induced by treatment, acridine treatment, etc., or a genetically modified strain induced by genetic techniques such as cell fusion or genetic recombination.
１，５－ＡＦを原料として１，５－ＡＭを製造するには１，５－ＡＦにウィッカーハモマイセス・アノマルス種の菌株に１，５－ＡＦを接触させればよく、具体的には、微生物用の培地中に１，５－ＡＦとウィッカーハモマイセス・アノマルス種の菌株を共存させる方法が好適に採用される。
微生物培養のための１，５－ＡＦを含む培養液は、炭水化物、窒素源、無機イオン、更に必要に応じて有機栄養源を含む培地を用いることができる。炭水化物としては、例えばグルコース、フルクトース、マルトース、スクロース、グリセロール、マンノースなどが挙げられる。グルコースが特に好ましい。有機栄養源としては、例えばビタミン、アミノ酸等を含有する酵母エキス、麦芽エキス、ペプトン、肉エキス、コーンスティープリカー、カゼイン分解物などが適宜使用される。無機イオンとしては、例えばマグネシウムイオン、リン酸イオン、カルシウムイオンなどが適宜使用される。その培地に、別にフィルター滅菌した１，５－ＡＦ水溶液を添加して１，５－ＡＭ採取用の培地とすることができる。
To produce 1,5-AM using 1,5-AF as a raw material, it is sufficient to contact 1,5-AF with a strain of Wickerhamomyces anomalus species, and specifically, A method of coexisting 1,5-AF and a strain of Wickerhamomyces anomalus in a microbial culture medium is preferably employed.
As a culture solution containing 1,5-AF for culturing microorganisms, a medium containing carbohydrates, a nitrogen source, inorganic ions, and, if necessary, an organic nutrient source can be used. Examples of carbohydrates include glucose, fructose, maltose, sucrose, glycerol, and mannose. Glucose is particularly preferred. As the organic nutrient source, for example, yeast extract, malt extract, peptone, meat extract, corn steep liquor, casein decomposition product, etc. containing vitamins, amino acids, etc. are used as appropriate. As the inorganic ion, for example, magnesium ion, phosphate ion, calcium ion, etc. are used as appropriate. A separately filter-sterilized 1,5-AF aqueous solution can be added to the medium to prepare a medium for collecting 1,5-AM.
反応温度は１０℃から４０℃が良く、好ましくは２０℃～３５℃、より好ましくは２５℃から３５℃が良い。ｐＨは７．０以下、好ましくは６．５以下、より好ましくは５．０以下が良い。これらの反応条件下においては、ウィッカーハモマイセス・アノマルス種の菌株の１，５－ＡＦから１，５－ＡＭへの変換能力が適切に作用する。また、微生物の活性化のために反応中にグルコースなどの炭素源を供給しても良い。 The reaction temperature is preferably 10°C to 40°C, preferably 20°C to 35°C, more preferably 25°C to 35°C. The pH is preferably 7.0 or less, preferably 6.5 or less, more preferably 5.0 or less. Under these reaction conditions, the ability of the strain of Wickerhamomyces anomalus species to convert 1,5-AF to 1,5-AM works properly. Furthermore, a carbon source such as glucose may be supplied during the reaction to activate microorganisms.
生成された物質が１，５－ＡＭであることは、ＨＰＬＣにより１，５－ＡＭ標準試料とのピークを比較することで確認できる。培養液中の１，５－ＡＭ生成量も、同様にＨＰＬＣで速やかに測定することができ、１，５－ＡＭ生成量が最高に達した時点で培養を終了する。なお、１，５－ＡＭ生成量は、ＨＰＬＣ測定により検出された１，５－ＡＭのピーク面積によりあらかじめ作成した標準試料の検量線から求めることができる。
ＨＰＬＣ測定の詳細条件を以下に示す。
分離カラム：ＭＩＴＳＵＢＩＳＨＩ ＭＣＩＧＥＬＣＫ０８Ｓの２連結（三菱化学（株）製）、
移動相：水、流速：１．０ｍＬ／分、
カラム温度：６０℃、
検出器：示差屈折率検出器、
サンプル供与量：２０μＬ
It can be confirmed that the produced substance is 1,5-AM by comparing the peak with a 1,5-AM standard sample by HPLC. The amount of 1,5-AM produced in the culture solution can also be rapidly measured by HPLC, and the culture is terminated when the amount of 1,5-AM produced reaches the maximum. Note that the amount of 1,5-AM produced can be determined from a calibration curve of a standard sample prepared in advance based on the peak area of 1,5-AM detected by HPLC measurement.
Detailed conditions for HPLC measurement are shown below.
Separation column: 2 connections of MITSUBISHI MCIGELCK08S (manufactured by Mitsubishi Chemical Corporation),
Mobile phase: water, flow rate: 1.0 mL/min,
Column temperature: 60℃,
Detector: differential refractive index detector,
Sample amount: 20μL
このようにして培養液中に生成した１，５－ＡＭを通常実施される周知の手段で培養物より精製して採取する。具体的には、遠心分離、珪藻土ろ過で菌体および固形物を除去した後、活性炭で脱色、イオン交換樹脂で脱塩し、濃縮後、結晶化させる。結晶化の方法としては、例えば、１，５－ＡＭの水溶液から結晶１，５－ＡＭを析出させる方法等が挙げられるが、特に限定されるものではない。 The 1,5-AM thus produced in the culture solution is purified and collected from the culture using commonly practiced and well-known means. Specifically, after microbial cells and solid matter are removed by centrifugation and diatomaceous earth filtration, the product is decolorized with activated carbon, desalted with an ion exchange resin, concentrated, and then crystallized. Examples of the crystallization method include, but are not particularly limited to, a method of precipitating crystals of 1,5-AM from an aqueous solution of 1,5-AM.
以下、実施例にて本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。以後の説明中に用いる％は、特に断りがない限り容量（ｗ／ｖ）％である。
＜１，５－ＡＭ変換率＞
１，５－ＡＭへ変換された割合（１，５－ＡＭ変換率）は、培地中に添加した１，５－ＡＦが１，５－ＡＭに変換された割合を示し、ＨＰＬＣで検出される１，５－ＡＭと１，５－ＡＧのピーク面積を用いて下記の式に従って求めた。
１，５－ＡＭ変換率（％）＝Ｓ１／（Ｓ１＋Ｓ２）×１００
Ｓ１：１，５－ＡＭのピーク面積の実測値（％）
Ｓ２：１，５－ＡＧのピーク面積の実測値（％）
なお、１，５－ＡＭのピーク面積の実測値が５％以下または９５％以上については、少ない成分のピーク面積が正確に検出できないため、それぞれ０％または１００％と表記した。
また、実施例で用いたＮＢＲＣ ０７０７およびＮＢＲＣ １０２１３の菌株は、独立行政法人製品評価技術基盤機構バイオテクノロジー本部（Ｉｎｃｏｒｐｏｒａｔｅｄ Ａｄｍｉｎｉｓｔｒａｔｉｖｅ Ａｇｅｎｃｙ Ｄｅｐａｒｔｍｅｎｔ ｏｆ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅ ｏｆ Ｔｅｃｈｎｏｌｏｇｙ ａｎｄ Ｅｖａｌｕａｔｉｏｎ）千葉県木更津市かずさ鎌足２－５－８ から入手したものである。
EXAMPLES Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited to these Examples. % used in the following description is capacity (w/v) % unless otherwise specified.
<1,5-AM conversion rate>
The rate of conversion to 1,5-AM (1,5-AM conversion rate) indicates the rate at which 1,5-AF added to the medium was converted to 1,5-AM, and is detected by HPLC. It was calculated according to the following formula using the peak areas of 1,5-AM and 1,5-AG.
1,5-AM conversion rate (%) = S 1 / (S 1 + S 2 ) x 100
S 1 : Actual value of peak area of 1,5-AM (%)
S 2 : Actual value of peak area of 1,5-AG (%)
Note that when the actual value of the peak area of 1,5-AM is 5% or less or 95% or more, it is expressed as 0% or 100%, respectively, because the peak area of a small component cannot be accurately detected.
In addition, the strains NBRC 0707 and NBRC 10213 used in the examples were obtained from the Incorporated Administrative Agency Department of Biotechnology National Institute. e of Technology and Evaluation) 2- Kazusa Kamatari, Kisarazu City, Chiba Prefecture It was obtained from 5-8.
＜実施例１＞（カビおよび酵母の１，５－ＡＭ変換率の比較）
１，５－ＡＦは微生物の増殖を抑制する作用を有するが、その作用は細菌に対しては強いものの、カビや酵母には殆ど作用しない。従って、１，５－ＡＦから１，５－ＡＭを微生物により製造するには、１，５－ＡＦの増殖抑制の影響が少ない酵母やカビが良いと考えられるため、表１に示すカビおよび酵母を準備し、１，５－ＡＭ変換率を調べた。
グルコース１．０％、ペプトン０．５％、酵母エキス０．３％および麦芽エキス０．３％、からなるＹＭ液体培地（ｐＨ６．２）５ｍＬを１５ｍＬ容の遠心チューブに分注し、この遠心チューブ中に寒天培地（グルコース１．０％、ペプトン０．５％、酵母エキス０．３％、麦芽エキス０．３％、寒天粉末１．５％、ｐＨ６．２）で培養した表１に示す菌株をそれぞれ１白金耳接種し、３０℃、１３０ｒｐｍで一晩振盪培養した。この培養液を遠心分離（３０００ｇ、１５分）して菌体を回収した。回収した菌体に、孔径０．４５μｍフィルターで除菌処理した０．１％／２２ｍＭリン酸緩衝液（ｐＨ６．８６）の１，５－ＡＦ溶液を２ｍＬ添加し、３０℃、１３０ｒｐｍで一晩菌体と１，５－ＡＦを接触させた。
この液をＨＰＬＣ分析し、１，５－ＡＭおよび１，５－ＡＧのピーク面積を測定して１，５－ＡＭ変換率を求めた。その結果を表１に示す。カビに比べて、酵母の方が１，５－ＡＭ変換率が高い傾向にあった。
<Example 1> (Comparison of 1,5-AM conversion rates of mold and yeast)
1,5-AF has the effect of inhibiting the growth of microorganisms, and although its effect is strong against bacteria, it has little effect on molds and yeast. Therefore, in order to produce 1,5-AM from 1,5-AF using microorganisms, yeasts and molds that are less affected by the growth inhibition of 1,5-AF are considered to be suitable. was prepared and the 1,5-AM conversion rate was investigated.
Dispense 5 mL of YM liquid medium (pH 6.2) consisting of 1.0% glucose, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract into a 15 mL centrifuge tube, and centrifuge it. The cells shown in Table 1 were cultured on agar medium (glucose 1.0%, peptone 0.5%, yeast extract 0.3%, malt extract 0.3%, agar powder 1.5%, pH 6.2) in tubes. One platinum loop of each strain was inoculated and cultured overnight at 30° C. and shaking at 130 rpm. This culture solution was centrifuged (3000 g, 15 minutes) to collect bacterial cells. 2 mL of 1,5-AF solution of 0.1%/22 mM phosphate buffer (pH 6.86) that had been sterilized using a 0.45 μm pore size filter was added to the collected cells, and the mixture was incubated at 30°C and 130 rpm overnight. The bacterial cells were brought into contact with 1,5-AF.
This liquid was analyzed by HPLC, and the peak areas of 1,5-AM and 1,5-AG were measured to determine the 1,5-AM conversion rate. The results are shown in Table 1. Compared to mold, yeast tended to have a higher 1,5-AM conversion rate.
＜実施例２＞（各酵母の１，５－ＡＭ変換率の比較）
実施例１より、酵母の方が１，５－ＡＭ変換率が高いことが分かったため、表２に示す酵母を準備し、その中で１，５－ＡＭ変換率の高い酵母を調べた。
グルコース１．０％、ペプトン０．５％、酵母エキス０．３％および麦芽エキス０．３％、からなるＹＭ液体培地（ｐＨ６．２）５ｍＬを１５ｍＬ容の遠心チューブに分注し、この遠心チューブ中に寒天培地（グルコース１．０％、ペプトン０．５％、酵母エキス０．３％、麦芽エキス０．３％、寒天粉末１．５％、ｐＨ６．２）で培養した表２に示す菌株をそれぞれ１白金耳接種し、３０℃、１３０ｒｐｍで一晩振盪培養した。この培養液を遠心分離（３０００ｇ、１５分）して菌体を回収した。回収した菌体に、リン酸緩衝生理食塩水（ＰＢＳ）を１ｍＬ添加して混合し、遠心分離（３０００ｇ、１５分）後に上澄みを廃棄し菌体を回収した。これに孔径０．４５μｍフィルターで除菌処理した０．１％の１，５－ＡＦ／２２ｍＭリン酸緩衝液（ｐＨ６．８６）を１ｍＬ添加して、２５℃、１３０ｒｐｍで一晩菌体と１，５－ＡＦを接触させた。
この液をＨＰＬＣ分析し、生成した１，５－ＡＧと１，５－ＡＭのピーク面積を測定し、１，５－ＡＭ変換率を求めた。その結果、評価した殆どの微生物が１，５－ＡＧと１，５－ＡＭの両方を生成したが、ウィッカーハモマイセス・アノマルス種の菌株だけが１，５－ＡＭのみを選択的に生成し、１，５－ＡＭ変換率は１００％であった。
<Example 2> (Comparison of 1,5-AM conversion rates of each yeast)
From Example 1, it was found that yeast had a higher 1,5-AM conversion rate, so yeasts shown in Table 2 were prepared, and yeasts with a high 1,5-AM conversion rate among them were investigated.
Dispense 5 mL of YM liquid medium (pH 6.2) consisting of 1.0% glucose, 0.5% peptone, 0.3% yeast extract, and 0.3% malt extract into a 15 mL centrifuge tube, and centrifuge it. The cells shown in Table 2 were cultured on agar medium (glucose 1.0%, peptone 0.5%, yeast extract 0.3%, malt extract 0.3%, agar powder 1.5%, pH 6.2) in tubes. One platinum loopful of each strain was inoculated and cultured overnight at 30° C. and 130 rpm with shaking. This culture solution was centrifuged (3000 g, 15 minutes) to collect bacterial cells. 1 mL of phosphate buffered saline (PBS) was added to and mixed with the collected bacterial cells, and after centrifugation (3000 g, 15 minutes), the supernatant was discarded and the bacterial cells were collected. To this, 1 mL of 0.1% 1,5-AF/22 mM phosphate buffer (pH 6.86) that had been sterilized using a 0.45 μm pore size filter was added, and the bacterial cells were incubated overnight at 25°C and 130 rpm. , 5-AF was contacted.
This liquid was analyzed by HPLC, the peak areas of the produced 1,5-AG and 1,5-AM were measured, and the 1,5-AM conversion rate was determined. The results showed that most of the microorganisms evaluated produced both 1,5-AG and 1,5-AM, but only one strain of Wickerhamomyces anomalus selectively produced only 1,5-AM. , 1,5-AM conversion rate was 100%.
＜実施例３＞（各ウィッカーハモマイセス属の１，５－ＡＭ変換率の比較）
実施例２から、ウィッカーハモマイセス・アノマルス種の菌株を用いると１，５－ＡＭ変換率が高いことが分かったため、表３に示すウィッカーハモマイセス属の菌株を準備し、その中で１，５－ＡＭ変換率の高い菌株を調べた。
用いる菌体を、表３に示すウィッカーハモマイセス属の菌体にした以外は実施例２と同様にして１，５－ＡＭ変換率を求めた。その結果、ウィッカーハモマイセス属の中でもウィッカーハモマイセス・アノマルス種の菌株のみが１，５－ＡＭ変換率が１００％であった。
<Example 3> (Comparison of 1,5-AM conversion rates of each Wickerhamomyces genus)
From Example 2, it was found that the 1,5-AM conversion rate was high when using a strain of Wickerhamomyces anomalis species. Therefore, the strains of Wickerhamomyces species shown in Table 3 were prepared, and among them, 1,5-AM conversion rate was high. , strains with high 5-AM conversion rates were investigated.
The 1,5-AM conversion rate was determined in the same manner as in Example 2, except that the bacterial cells of the genus Wickerhamomyces shown in Table 3 were used as the bacterial cells. As a result, among the Wickerhamomyces genus, only the Wickerhamomyces anomalus strain had a 1,5-AM conversion rate of 100%.
＜実施例４＞（ウィッカーハモマイセス・アノマルス種の菌株の１，５－ＡＭ資化性）
合成培地（ＹＥＭ培地：Ｙｅａｓｔ Ｎｉｔｒｏｇｅｎ Ｂａｓｅ）５ｍＬを１５ｍＬ容の遠心チューブに分注し、この遠心チューブ中に寒天培地（グルコース１．０％、ペプトン０．５％、酵母エキス０．３％、麦芽エキス０．３％、寒天粉末１．５％、ｐＨ６．２）で培養したウィッカーハモマイセス・アノマルス種の菌株（ＮＢＲＣ０７０７）を１白金耳接種し、３０℃、１３０ｒｐｍで一晩振盪培養した。これを種培養液とした。別に合成培地（ＹＥＭ培地）５ｍＬを試験管に分注し、孔径０．４５μｍフィルターで除菌処理した表４に示す糖類をそれぞれの試験管に１％になるように添加し、種培養液を２０μＬ添加して、２５℃、１３０ｒｐｍで一晩振盪培養した。
この培養液を適量サンプリングし、濁度計（バイオクロム社製、ジーンクオント１００）を用いて６６０ｎｍの波長における培養液の濁度を測定した。この結果を、添加した糖類ごとに表４に示す。
添加した糖類がグルコースの場合は、６６０ｎｍの波長の透過率が減少し濁度が増加したことから、ウィッカーハモマイセス・アノマルス種の菌株（ＮＢＲＣ０７０７）が増殖していることが分かった。これに対して、添加した糖類が１，５－ＡＭの場合は、濁度の増加は認められず、ウィッカーハモマイセス・アノマルス種の菌株（ＮＢＲＣ０７０７）が増殖しないことが分かった。
上記より、ウィッカーハモマイセス・アノマルス種の菌株（ＮＢＲＣ０７０７）は１，５－ＡＭを資化することができず、生成した１，５－ＡＭは培地に蓄積されていくことが分かった。よって、ウィッカーハモマイセス・アノマルス種の菌株（ＮＢＲＣ０７０７）は、１，５－ＡＭの生成に適した菌であることが分かった。
<Example 4> (1,5-AM assimilation ability of Wickerhamomyces anomalus strain)
Dispense 5 mL of synthetic medium (YEM medium: Yeast Nitrogen Base) into a 15 mL centrifuge tube, and add agar medium (glucose 1.0%, peptone 0.5%, yeast extract 0.3%, malt) into the centrifuge tube. One platinum loop of Wickerhamomyces anomalus strain (NBRC0707) cultured in 0.3% extract, 1.5% agar powder, pH 6.2) was inoculated and cultured overnight at 30° C. and 130 rpm with shaking. This was used as a seed culture solution. Separately, 5 mL of synthetic medium (YEM medium) was dispensed into test tubes, and the saccharides shown in Table 4, which had been sterilized using a 0.45 μm pore size filter, were added to each test tube at a concentration of 1%. 20 μL was added and cultured with shaking at 25° C. and 130 rpm overnight.
An appropriate amount of this culture solution was sampled, and the turbidity of the culture solution at a wavelength of 660 nm was measured using a turbidimeter (
When the added saccharide was glucose, the transmittance at a wavelength of 660 nm decreased and the turbidity increased, indicating that the strain of Wickerhamomyces anomalus (NBRC0707) was growing. On the other hand, when the added sugar was 1,5-AM, no increase in turbidity was observed, and it was found that the Wickerhamomyces anomalis strain (NBRC0707) did not grow.
From the above, it was found that the strain of Wickerhamomyces anomalus species (NBRC0707) was unable to assimilate 1,5-AM, and the produced 1,5-AM was accumulated in the medium. Therefore, the strain of Wickerhamomyces anomalus species (NBRC0707) was found to be suitable for producing 1,5-AM.
＜実施例５＞（発酵時間と１，５－ＡＭ変換率の関係）
ＹＭ液体培地を１５ｍＬ容の遠心チューブに５ｍＬ調製し、この遠心チューブ中に寒天培地（グルコース１２．０％、ペプトン０．５１．０％、酵母エキス０．３％、麦芽エキス０．３％、寒天粉末１．５％、ｐＨ６．２）で培養したウィッカーハモマイセス・アノマルス種の菌株（ＮＢＲＣ０７０７）を１白金耳接種し、３０℃、１３０ｒｐｍで一晩振盪培養した。これを種培養液とした。別途、ペプトンを１．０ｇ、酵母エキスを０．６ｇ、麦芽エキスを０．６ｇ、および１，５－ＡＦ１２ｇを１００ｍＬの精製水に溶解した。該水溶液をポアサイズ０．２μＭのメンブレンフィルターでろ過し、次いでろ過後の水溶液に種培養液を５ｍＬ加え、２５℃、１３０ｒｐｍで振盪培養した。培養中に、５％グルコース水溶液を当該培地に連続して添加した。また、ペプトン、酵母エキス、麦芽エキスも当該培地に適時添加した。
この培養中の培地から、２００μＬを随時サンプリングした。サンプリングした培地を精製水で１０倍希釈し、ＨＰＬＣ分析により１，５－ＡＭと１，５－ＡＧのピーク面積を測定して、１，５－ＡＭ変換率を求めた。発酵時間と１，５－ＡＭ変換率（％）の関係を図１に示す。この結果、発酵時間が２６４時間での１，５－ＡＭ変換率は７６．２％に達した。
<Example 5> (Relationship between fermentation time and 1,5-AM conversion rate)
Prepare 5 mL of YM liquid medium in a 15 mL centrifuge tube, and add agar medium (glucose 12.0%, peptone 0.51.0%, yeast extract 0.3%, malt extract 0.3%, One platinum loop of Wickerhamomyces anomalis strain (NBRC0707) cultured in agar powder (1.5%, pH 6.2) was inoculated and cultured overnight at 30° C. and 130 rpm with shaking. This was used as a seed culture solution. Separately, 1.0 g of peptone, 0.6 g of yeast extract, 0.6 g of malt extract, and 12 g of 1,5-AF were dissolved in 100 mL of purified water. The aqueous solution was filtered through a membrane filter with a pore size of 0.2 μM, and then 5 mL of the seed culture solution was added to the filtered aqueous solution, and cultured with shaking at 25° C. and 130 rpm. During the culture, 5% glucose aqueous solution was continuously added to the medium. Furthermore, peptone, yeast extract, and malt extract were also added to the medium at appropriate times.
From the medium during this culture, 200 μL was sampled at any time. The sampled medium was diluted 10 times with purified water, and the peak areas of 1,5-AM and 1,5-AG were measured by HPLC analysis to determine the 1,5-AM conversion rate. The relationship between fermentation time and 1,5-AM conversion rate (%) is shown in Figure 1. As a result, the 1,5-AM conversion rate reached 76.2% after a fermentation time of 264 hours.
Claims (2)
The method for producing 1,5-anhydro-D-mannitol according to claim 1, wherein the strain of Wickerhamomyces anomalus species is NBRC 0707 or NBRC 10213.
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