JP4820277B2 - Method for producing ketone body and / or secondary alcohol - Google Patents
Method for producing ketone body and / or secondary alcohol Download PDFInfo
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Description
本発明は、各種化粧品等のフレーバーとして有用なケトン体及び２級アルコールを、微生物を利用して製造する方法に関する。 The present invention relates to a method for producing ketone bodies and secondary alcohols useful as flavors for various cosmetics and the like using microorganisms.
ケトンや２級アルコールの中には、化粧品のフレーバーとして、また香料や染料の溶剤として有用なものが数多く存在する。これらの化合物の多くは、天然物から抽出する手段の他、アルカンの酸化反応、ケトンやアルコールの酸化還元による相互変換等により製造されている。 Many ketones and secondary alcohols are useful as cosmetic flavors and as perfumes and dye solvents. Many of these compounds are produced not only by means of extraction from natural products, but also by alkane oxidation reaction, interconversion by oxidation and reduction of ketones and alcohols, and the like.
微生物を用いた油脂からメチルケトン及び／又は２級アルコールの製造法は知られており、その反応は油脂の加水分解、脂肪酸のβ位酸化、脱炭酸及びさらに一部還元によって進行するものと考えられており、脂肪酸からメチルケトン及び／又は２級アルコールに関してはフザリウム属等の微生物を用いれば製造できることが知られている（特許文献１及び２）。
しかしながら、アルカン類から直接ケトン体及び／又は２級アルコールを生産する微生物は知られていない。
従って、本発明は、アルカン類からケトン体及び／又は２級アルコールを直接、かつ効率良く生産する方法を提供することにある。
However, microorganisms that produce ketone bodies and / or secondary alcohols directly from alkanes are not known.
Accordingly, an object of the present invention is to provide a method for directly and efficiently producing a ketone body and / or a secondary alcohol from alkanes.
そこで本発明者は、アルカン類からケトン体及び／又は２級アルコールを直接生産する能力を有する微生物を探索したところ、酵母様微細藻であるプロトテカ（Prototheca）に属する微生物の中に、アルカン類の２〜６位を酸化する能力を有するものが存在し、当該微生物をアルカン類の存在下に培養するか、当該微生物とアルカン類を接触させれば、ケトン体及び／又は２級アルコールが効率良く製造できることを見出した。 Therefore, the present inventors searched for microorganisms having the ability to directly produce ketone bodies and / or secondary alcohols from alkanes. Among the microorganisms belonging to Prototheca, a yeast-like microalga, Those having the ability to oxidize 2 to 6 position exist, and if the microorganism is cultured in the presence of alkanes or the microorganism and alkanes are brought into contact with each other, the ketone body and / or the secondary alcohol can be efficiently obtained. We found that it can be manufactured.
すなわち、本発明は、プロトテカ属に属し、次の一般式（１） That is, the present invention belongs to the genus Prototheca and has the following general formula (1)
（式中、Ｒ1は置換基を有していてもよい炭化水素基を示し、Ｒ2は水素原子又は−ＣＯＯＲ3（Ｒ3は水素原子又は炭化水素基を示す）を示し、ｎは１〜６の整数を示す）
で表される化合物を一般式（２）
(Wherein R 1 represents a hydrocarbon group which may have a substituent, R 2 represents a hydrogen atom or —COOR 3 (R 3 represents a hydrogen atom or a hydrocarbon group), and n represents 1 Represents an integer of ~ 6)
The compound represented by general formula (2)
（式中、ｎ１は０〜５の整数、ｎ２は０〜５の整数を示し、ｎ１＋ｎ２は０〜５の整数を示し、Ｒ1及びＲ2は前記と同じ）
で表されるケトン体及び/又は一般式（３）
(Wherein n1 is an integer of 0 to 5, n2 is an integer of 0 to 5, n1 + n2 is an integer of 0 to 5, and R 1 and R 2 are the same as above)
And / or the general formula (3)
（式中、Ｒ1、Ｒ2、ｎ１及びｎ２は前記と同じ）
で表される２級アルコールに変換する能力を有する微生物を、当該一般式（１）で表される化合物の存在下に培養するか、又は当該微生物又はその処理物と当該一般式（１）で表される化合物とを接触させることを特徴とする当該一般式（２）で表されるケトン体及び／又は一般式（３）で表される２級アルコールの製造法を提供するものである。
(Wherein R 1 , R 2 , n1 and n2 are the same as above)
The microorganism having the ability to convert to the secondary alcohol represented by the above is cultured in the presence of the compound represented by the general formula (1), or the microorganism or a treated product thereof and the general formula (1) The present invention provides a method for producing a ketone body represented by the general formula (2) and / or a secondary alcohol represented by the general formula (3), which is characterized by contacting with a compound represented by the formula.
本発明方法によれば、種々のアルカン類から直接、かつ効率良く、ケトン体及び／又は２級アルコールを製造することができる。 According to the method of the present invention, ketone bodies and / or secondary alcohols can be produced directly and efficiently from various alkanes.
本発明に用いられる微生物は、プロトテカ属に属し、一般式（１）の化合物を、一般式（２）のケトン体及び／又は一般式（３）の２級アルコールに変換する能力をするものであれば、特に限定されないが、プロトテカ ゾフィ（Prototheca zopfii）、プロトテカ スタグノラ（Prototheca stagnora）、プロトテカ エリボトリアエ（Prototheca eriobotryae）、プロトテカ サーモデュリカ（Prototheca thermodurica）、プロトテカ トリスポア（Prototheca trispoa）、プロトテカ プロトリセンシス（Prototheca protoricensis）等が挙げられる。具体的な菌株としては、Prototheca stagnora JCM 9642、Prototheca zopfii NBRC 6998、Prototheca zopfii NBRC 7532、Prototheca zopfii NBRC 7533、Prototheca zopfii NBRC 7534、Prototheca zopfii NBRC 7535、Prototheca zopfii NBRC 7536、Prototheca zopfii JCM 9400、Prototheca zopfii JCM 9646、Prototheca eriobotryae NBRC 32449、Prototheca thermodurica JCM 8557、Prototheca trispoa NBRC 6996、Prototheca protoricensis IAM C-177等が挙げられる。これらの菌株は、独立行政法人製品評価技術基盤機構の生物遺伝資源部門（ＮＢＲＣ）、独立行政法人理化学研究所バイオリソースセンター微生物材料開発室（ＲＩＫＥＮＢＲＣ−ＪＣＭ）又は東京大学分子生物学研究所のＩＡＭＣＣから入手可能である。 The microorganism used in the present invention belongs to the genus Prototheca and has the ability to convert a compound of the general formula (1) into a ketone body of the general formula (2) and / or a secondary alcohol of the general formula (3). If there is, but not limited to, Prototheca zopfii, Prototheca stagnora, Prototheca eriobotryae, Prototheca thermodurica, Prototheca thermodurica, Prototheca ) And the like. Specific strains include Prototheca stagnora JCM 9642, Prototheca zopfii NBRC 6998, Prototheca zopfii NBRC 7532, Prototheca zopfii NBRC 7533, Prototheca zopfii NBRC 7534, Prototheca zopfii NBRC 7535, Prototheca zopfii NBRC 7535, Prototheca zopfzo ii 9646, Prototheca eriobotryae NBRC 32449, Prototheca thermodurica JCM 8557, Prototheca trispoa NBRC 6996, Prototheca protoricensis IAM C-177 and the like. These strains can be obtained from the Biogenetic Resource Division (NBRC) of the National Institute for Product Evaluation and Technology, RIKEN BioResource Center, Microbial Materials Development Office (RIKENBRC-JCM), or IAMCC of the University of Tokyo Institute for Molecular Biology. It is available.
本発明におけるケトン体及び／又は２級アルコールの製造は、一般式（１）の化合物の存在下に上記微生物を培養するか、又は上記微生物又はその処理物と一般式（１）の化合物とを接触させることによって行なわれる。 In the present invention, the ketone body and / or the secondary alcohol is produced by culturing the microorganism in the presence of the compound of the general formula (1), or combining the microorganism or a treated product thereof with the compound of the general formula (1). It is done by contacting.
まず、培養法については、通常、一般式（１）の化合物を含有する培地で上記微生物を培養し、当該培養物から目的ケトン体及び／又は２級アルコールを採取することにより行なわれる。 First, the culture method is usually carried out by culturing the microorganism in a medium containing the compound of the general formula (1) and collecting the target ketone body and / or secondary alcohol from the culture.
ここで培地には、上記微生物の生育に必要な栄養源、例えば窒素源、無機物、微量栄養素、一般式（１）の化合物以外の炭素源を含有せしめるのが好ましい。窒素源としては、硫酸アンモニウム、硝酸アンモニウム、酒石酸アンモニウム、硝酸ナトリウム、アスパラギン、ペプトン、コーンスティープリカー等が挙げられる。無機物、微量栄養素としては、硫酸マグネシウム、リン酸二水素カリウム、塩化カルシウム、塩化ナトリウム、硫酸マンガン、硫酸亜鉛、硫酸銅、塩化第二鉄、モリブデン酸アンモニウム、ヨウ化カリウム、ホウ酸、ビタミン類等が用いられる。また、炭素源としては、グルコース、スクロース、グリセリン、デキストリン、カルボキシメチルセルロース等が挙げられる。 Here, the medium preferably contains a nutrient source necessary for the growth of the microorganism, for example, a nitrogen source, an inorganic substance, a micronutrient, and a carbon source other than the compound of the general formula (1). Examples of the nitrogen source include ammonium sulfate, ammonium nitrate, ammonium tartrate, sodium nitrate, asparagine, peptone, corn steep liquor and the like. Inorganic and micronutrients include magnesium sulfate, potassium dihydrogen phosphate, calcium chloride, sodium chloride, manganese sulfate, zinc sulfate, copper sulfate, ferric chloride, ammonium molybdate, potassium iodide, boric acid, vitamins, etc. Is used. Examples of the carbon source include glucose, sucrose, glycerin, dextrin, carboxymethyl cellulose and the like.
培地中における一般式（１）の化合物の濃度は、目的物の生産性及び回収性の点から、０．５〜８質量％、さらに１〜４質量％、特に２〜４質量％が好ましい。 The concentration of the compound of the general formula (1) in the medium is preferably 0.5 to 8% by mass, more preferably 1 to 4% by mass, and particularly preferably 2 to 4% by mass from the viewpoint of productivity and recoverability of the target product.
培養条件は、好気的条件下で液体培養法、例えば振盪培養法、通気撹拌培養法が好ましい。培養温度は２８℃、pH５〜７で３〜７日間行なうのが好ましい。 The culture condition is preferably a liquid culture method under aerobic conditions, for example, a shaking culture method or an aeration stirring culture method. The culture temperature is preferably 28 ° C. and pH 5-7 for 3-7 days.
接触法は、一般式（１）の化合物を添加しない以外は前記条件と同様にして培養した上記微生物の培養上清、培養物から採取した酵素等に、一般式（１）の化合物を接触させることにより行なわれる。接触反応は、水性媒体、水性溶液中で２８℃、pH５〜７の条件で３〜７日間行なうのが好ましい。また接触反応は、上記微生物を固定化した固定化菌体、酵素を固定化した固定化酵素を用いて行なうことも可能である。 In the contact method, the compound of the general formula (1) is brought into contact with the culture supernatant of the microorganisms cultured in the same manner as described above except that the compound of the general formula (1) is not added, the enzyme collected from the culture, and the like. Is done. The contact reaction is preferably performed in an aqueous medium or an aqueous solution at 28 ° C. and pH 5 to 7 for 3 to 7 days. The contact reaction can also be performed using an immobilized microbial cell in which the microorganism is immobilized and an immobilized enzyme in which an enzyme is immobilized.
原料化合物を示す一般式（１）中、Ｒ1で示される炭化水素基としては、炭素数１〜２０の炭化水素基、例えばアルキル基、アルケニル基、アルキニル基、シクロアルキル基、シクロアルケニル基、芳香族炭化水素基等が挙げられる。 In the general formula (1) representing the raw material compound, the hydrocarbon group represented by R 1 is a hydrocarbon group having 1 to 20 carbon atoms such as an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, An aromatic hydrocarbon group etc. are mentioned.
アルキル基としては直鎖又は分岐鎖のアルキル基が好ましく、特に炭素数１〜２０、特に１〜１８のアルキル基が好ましい。アルケニル基としては、炭素数２〜１８のアルケニル基が好ましい。アルキニル基としては炭素数２〜１８のアルキニル基が好ましい。シクロアルキル基としては、炭素数３〜８のシクロアルキル基が好ましい。シクロアルケニル基としては、炭素数４〜８のシクロアルキニル基が好ましい。芳香族炭化水素基としては、炭素数６〜１４の芳香族炭化水素基、例えばフェニル基、ナフチル基等が好ましい。 As the alkyl group, a linear or branched alkyl group is preferable, and an alkyl group having 1 to 20 carbon atoms, particularly 1 to 18 carbon atoms is particularly preferable. As the alkenyl group, an alkenyl group having 2 to 18 carbon atoms is preferable. The alkynyl group is preferably an alkynyl group having 2 to 18 carbon atoms. As the cycloalkyl group, a cycloalkyl group having 3 to 8 carbon atoms is preferable. As a cycloalkenyl group, a C4-C8 cycloalkynyl group is preferable. As an aromatic hydrocarbon group, a C6-C14 aromatic hydrocarbon group, for example, a phenyl group, a naphthyl group, etc. are preferable.
これらの炭化水素基に置換し得る基としては、ハロゲン原子、アルコキシ基、ハロゲノアルキル基、ハロゲノアルコキシ基等が挙げられる。これらの置換基の炭素数は１〜８が好ましい。 Examples of the group that can be substituted with these hydrocarbon groups include a halogen atom, an alkoxy group, a halogenoalkyl group, and a halogenoalkoxy group. These substituents preferably have 1 to 8 carbon atoms.
Ｒ2としては水素原子又は-ＣＯＯＲ3（Ｒ3は水素原子又は炭化水素基を示す）を示すが、水素原子が好ましい。 R 2 represents a hydrogen atom or —COOR 3 (R 3 represents a hydrogen atom or a hydrocarbon group), and is preferably a hydrogen atom.
Ｒ3で示される炭化水素基としては、アルキル基、アルケニル基、芳香族炭化水素基等が挙げられる。特に炭素数１〜８のアルキル基、炭素数２〜８のアルケニル基、炭素数６〜１４の芳香族炭化水素基が好ましい。Ｒ3としては、水素原子が好ましい。 Examples of the hydrocarbon group represented by R 3 include an alkyl group, an alkenyl group, and an aromatic hydrocarbon group. In particular, an alkyl group having 1 to 8 carbon atoms, an alkenyl group having 2 to 8 carbon atoms, and an aromatic hydrocarbon group having 6 to 14 carbon atoms are preferable. R 3 is preferably a hydrogen atom.
本発明方法によれば、アルカン類の末端から２〜６位のメチレン基がケトン化及び／又はヒドロキシル化され、そのうち、３〜５位のメチレン基、特に４〜５位のメチレン基が主にケトン化及び／又はヒドロキシル化される。従って、目的物である一般式（２）及び（３）中、ｎ１が０〜５であり、ｎ２が１〜４であるのがより好ましい。さらに、ｎ１が０〜３であり、ｎ２が３又は４であるのが特に好ましい。 According to the method of the present invention, the methylene group at the 2-6 position from the end of the alkane is ketonated and / or hydroxylated, of which the methylene group at the 3-5 position, particularly the methylene group at the 4-5 position, is mainly used. Ketonation and / or hydroxylation. Therefore, it is more preferable that n1 is 0-5 and n2 is 1-4 in the general formulas (2) and (3), which are target products. Furthermore, it is particularly preferable that n1 is 0 to 3 and n2 is 3 or 4.
培養物又は接触反応混合物から、目的化合物を分離するには、各種溶媒による抽出、クロマトグラフィー等に付すことにより行なわれる。 In order to separate the target compound from the culture or the contact reaction mixture, extraction with various solvents, chromatography, and the like are performed.
実施例１ ケトン体蓄積型プロトテカ属微生物のスクリーニング
プロトテカ属の各菌株を、試験管中の５mLのＧＹ培地（２．０％酵母エキス，１．０％グルコース）に４％ｎ−ヘキサデカンを添加した液体培地に接種し、２８℃で５日間往復振盪培養（１４０rpm）した。培養終了液に１mLのクロロホルム及び２mLのメタノールを添加し激しく撹拌後１０分間放置した。その後さらに１mLのクロロホルム及び１mLの１．５％ＫＣｌを添加し攪拌後、遠心分離を行い、クロロホルム層（下層）を回収した。クロロホルムで抽出した脂溶性画分を遠心エバポレーターで濃縮しガスクロマトグラフィー（ＧＣ）に供し、保持時間８．８分に出現するヘキサデカノンの有無を確認した。その結果を表１に示した菌株で、ケトン体の生成が認められた。なおＧＣ分析は、カラム：ＨＲ−５２（信和化工）、分析装置：ＧＣ−１７Ａ（Ｓｈｉｍａｄｚｕ）、カラム温度：１８０〜２４０℃、２℃／分昇温、注入口検出器温度：２５０℃、検出器：ＦＩＤ、キャリアガス：Ｈｅ、メイクアップガス：Ｎ2の条件で行なった。
Example 1 Screening of ketone body-accumulating prototheca microorganisms Each prototheca strain was added with 4% n-hexadecane in 5 mL of GY medium (2.0% yeast extract, 1.0% glucose) in a test tube. The medium was inoculated and reciprocally shaken (140 rpm) at 28 ° C. for 5 days. 1 mL of chloroform and 2 mL of methanol were added to the culture end solution, and the mixture was vigorously stirred and allowed to stand for 10 minutes. Thereafter, 1 mL of chloroform and 1 mL of 1.5% KCl were further added and stirred, followed by centrifugation to recover a chloroform layer (lower layer). The fat-soluble fraction extracted with chloroform was concentrated with a centrifugal evaporator and subjected to gas chromatography (GC) to confirm the presence or absence of hexadecanone appearing at a retention time of 8.8 minutes. With the strains whose results are shown in Table 1, formation of ketone bodies was observed. In addition, GC analysis is as follows: column: HR-52 (Shinwa Chemical), analyzer: GC-17A (Shimadzu), column temperature: 180-240 ° C., 2 ° C./minute temperature increase, inlet detector temperature: 250 ° C., detection Apparatus: FID, carrier gas: He, makeup gas: N 2
実施例２ ケトン体高蓄積型菌株の選抜
実施例１によりケトン体の蓄積が確認された菌株を用い、２０mL容三角フラスコ中の４mLのＧＹ培地（０．５％酵母エキス，０．５％グルコース）に４％ｎ−ヘキサデカンを添加した液体培地に接種し、２８℃で５日間往復振盪培養（１４０rpm）した。培養終了後、培養液に内部標準として７−ペンタデカノンを２５０μｇ添加し、実施例１と同様に脂溶性画分をクロロホルムにて抽出し、ＧＣ分析に供した。内部標準のピーク面積を元に算出したケトン体の生産量を表２に示した。
Example 2 Selection of Highly Accumulated Ketone Body Strains Using strains confirmed to accumulate ketone bodies in Example 1, 4 mL of GY medium (0.5% yeast extract, 0.5% glucose) in a 20 mL Erlenmeyer flask Was inoculated into a liquid medium supplemented with 4% n-hexadecane, followed by reciprocal shaking culture (140 rpm) at 28 ° C. for 5 days. After completion of the culture, 250 μg of 7-pentadecanone was added to the culture medium as an internal standard, and the fat-soluble fraction was extracted with chloroform in the same manner as in Example 1 and subjected to GC analysis. Table 2 shows the production amount of ketone bodies calculated based on the peak area of the internal standard.
実施例３ ケトン体生産量
実施例２において最大の生産量が認められたプロトテカ ゾフィ ＪＣＭ９４００株（Ｐｒｏｔｏｔｈｅｃａ ｚｏｐｆｉｉ ＪＣＭ９４００）を用い、２０mL容三角フラスコ中のＹｅａｓｔ培地（０．５％酵母エキス）又はＧｌｙ培地（０．５％グリシン、０．０７％リン酸１カリウム、０．０３％リン酸２カリウム、０．０３％硫酸マグネシウム・７水和物、０．０３％硫酸鉄・７水和物、１０ppmチアミン塩酸塩）に４％から８％のｎ−ヘキサデカンを添加した液体培地に接種し、２８℃で５日間から８日間往復振盪培養（１４０rpm）した。培養終了後、実施例２と同様に７−ペンタデカノンを内部標準として添加し、クロロホルムにて抽出した脂溶性画分をＧＣ分析に供した。内部標準のピーク面積を元に算出したケトン体の生産量を表３に示した。
Example 3 Production of Ketone Body Using Prototheca zofi JCM9400 strain (Prototheca zopfii JCM9400) in which the maximum production amount was recognized in Example 2, Yeast medium (0.5% yeast extract) or Gly medium in a 20 mL Erlenmeyer flask (0.5% glycine, 0.07% monopotassium phosphate, 0.03% dipotassium phosphate, 0.03% magnesium sulfate heptahydrate, 0.03% iron sulfate heptahydrate, 10ppm Thiamine hydrochloride) was inoculated into a liquid medium supplemented with 4% to 8% n-hexadecane, followed by reciprocal shaking culture (140 rpm) at 28 ° C. for 5 to 8 days. After completion of the culture, 7-pentadecanone was added as an internal standard in the same manner as in Example 2, and the fat-soluble fraction extracted with chloroform was subjected to GC analysis. Table 3 shows the production amount of ketone bodies calculated based on the peak area of the internal standard.
実施例４ ケトン体の構成比（経時変化）
ｎ−ヘキサデカンを資化させた際に蓄積するヘキサデカノンのケトン基の位置を確認するために、高極性カラムを用いたＧＣ分析を行なった。使用したカラムはＴＣ−ＷＡＸ（ＧＬサイエンス社製）、分析は、カラム温度：１２０〜１８０℃まで２℃／分昇温、１８０〜２２０℃まで１０℃／分昇温、注入口検出器温度：２５０℃、検出器：ＦＩＤ、キャリアガス：Ｈｅ、メイクアップガス：Ｎ2の条件で行った。プロトテカ ゾフィ ＪＣＭ９４００株を用い、実施例２と同様の条件で培養を行ない、３日目から１２日目までの培養液からそれぞれ抽出した脂溶性画分を上記ＧＣ分析条件により解析し、ヘキサデカノン構成比率を算出した。結果を表４に示した。
Example 4 Composition ratio of ketone body (change over time)
In order to confirm the position of the ketone group of hexadecanone accumulated when n-hexadecane was assimilated, GC analysis using a high polarity column was performed. The column used was TC-WAX (manufactured by GL Science Co., Ltd.), and the analysis was performed at a column temperature of 120 ° C. to 180 ° C./2° C./min. The measurement was performed under the conditions of 250 ° C., detector: FID, carrier gas: He, makeup gas: N 2 . Prototheca zophyll JCM9400 strain was cultured under the same conditions as in Example 2, and the fat-soluble fractions extracted from the culture solution from the 3rd day to the 12th day were analyzed under the above GC analysis conditions. Was calculated. The results are shown in Table 4.
実施例５ ケトン体の構成比（基質による変化）
炭素鎖長１２から１７までのｎ−アルカンをそれぞれ２０mL容三角フラスコ中の４mLのＧＹ培地に４％添加し、プロトテカ ゾフィ ＪＣＭ９４００株を接種した。２８℃で７日間往復振盪培養（１４０rpm）後、培養液から抽出した脂溶性画分を実施例３と同様の条件でＧＣ分析に供し、ケトン体構成の９５％以上を占める４位体、５位体の比率を算出した。結果を表５に示した。
Example 5 Composition ratio of ketone body (change by substrate)
N-alkanes having a carbon chain length of 12 to 17 were each added to 4 mL of GY medium in a 20 mL Erlenmeyer flask and inoculated with Prototecazofi JCM9400 strain. After reciprocating shaking culture (140 rpm) at 28 ° C. for 7 days, the fat-soluble fraction extracted from the culture solution was subjected to GC analysis under the same conditions as in Example 3, and the 4-position, occupying 95% or more of the ketone body composition, 5 The ratio of the position was calculated. The results are shown in Table 5.
実施例６ アルコール体生産量
実施例２に用いた菌株を、２０mL容三角フラスコ中の４mLのＧＹ培地（０．５％酵母エキス，０．５％グルコース）に４％ｎ−ヘキサデカンを添加した液体培地に接種し、２８℃で５日間往復振盪培養（１４０rpm）した。培養終了後、培養液に内部標準として７−ペンタデカノンを２５０μｇ添加し、実施例２と同様に脂溶性画分をクロロホルムにて抽出し、ＧＣ分析に供した。保持時間９．０分に出現する内部標準のピーク面積を元に算出したアルコール体の生産量を表６に示した。
Example 6 Alcohol Production A liquid obtained by adding 4% n-hexadecane to 4 mL of GY medium (0.5% yeast extract, 0.5% glucose) in a 20 mL Erlenmeyer flask. The medium was inoculated and reciprocally shaken (140 rpm) at 28 ° C. for 5 days. After completion of the culture, 250 μg of 7-pentadecanone was added to the culture medium as an internal standard, and the fat-soluble fraction was extracted with chloroform in the same manner as in Example 2 and subjected to GC analysis. Table 6 shows the amount of alcohol produced based on the peak area of the internal standard appearing at a retention time of 9.0 minutes.
実施例７ ケトン体及びアルコール体の構造解析
４％のヘキサデカンを含むＹｅａｓｔ培地（０．５％酵母エキス）５００mLにて培養したプロトテカ ゾフィ ＪＣＭ９４００（Ｐｒｏｔｏｔｈｅｃａ ｚｏｐｆｉｉ ＪＣＭ９４００）より実施例１と同様に脂溶成分をクロロホルムにて抽出し、薄層クロマトグラフィーに供した。シリカゲルでコートされたＭＥＲＣＫ社製１．０５７２１（２００×２００×０．２５mm）を用い、ヘキサン／ジエチルエーテル／酢酸（８０：２０：１）にて展開した。展開後０．０１％プリムリン溶液を噴霧し、紫外線照射にて発色するＲｆ値０．５以下のスポットを掻き取り、ヘキサン／水（４：１）を加えて混合し、脂溶成分であるヘキサン層を回収し減圧エバポレーター（ＥＹＥＲＡ社）によりヘキサンを除いた。
取得した脂質成分をアセトニトリル／水（８０：２０）に溶解しＨＰＬＣに供した。ＨＰＬＣはＳＩＭＡＤＺＵ社ＬＣ−１０Ａシステムを用い、カラム：Ｓｈｉｍ−ｐａｃｋ ＣＬＣ−ＯＤＳ（Ｍ）、溶媒：アセトニトリル／水（８０：２０）、流量：１mL／min、検出器：ＲＩにて行なった。本条件下でＲ．Ｔ．３０．０分付近、３３．１分付近の化合物を1Ｈ−ＮＭＲ解析に供したところ、それぞれ５−ヘキサデカノール、５−ヘキサデカノンと同定された。
Example 7 Structural analysis of ketone bodies and alcohol bodies From Prototheca Zofi JCM9400 (Prototheca zopfii JCM9400) cultured in Yeast medium (0.5% yeast extract) containing 4% hexadecane in the same manner as in Example 1, fat-soluble components Was extracted with chloroform and subjected to thin layer chromatography. Development was performed with hexane / diethyl ether / acetic acid (80: 20: 1) using 1.05721 (200 × 200 × 0.25 mm) manufactured by MERCK and coated with silica gel. After development, spray with 0.01% primulin solution, scrape off spots with an Rf value of 0.5 or less that develop color by UV irradiation, add hexane / water (4: 1), mix, and mix with hexane, a fat-soluble component. The layer was collected and hexane was removed with a vacuum evaporator (EYERA).
The obtained lipid component was dissolved in acetonitrile / water (80:20) and subjected to HPLC. HPLC was carried out using a SIMADZU LC-10A system with a column: Shim-pack CLC-ODS (M), a solvent: acetonitrile / water (80:20), a flow rate: 1 mL / min, and a detector: RI. Under these conditions, R.I. T.A. When the compounds at around 30.0 minutes and around 33.1 minutes were subjected to 1 H-NMR analysis, they were identified as 5-hexadecanol and 5-hexadecanone, respectively.
なお、市販の試薬５−ヘキサデカノール（ＳＩＧＭＡ―ＡＬＤＲＩＣＨ）及び５−ヘキサデカノン（Ａｌｆａ Ａｅｓａｒ）と培養サンプルのＧＣ解析結果の比較により、各々の生産物の特定を行なった。また、ＧＣ−ＭＳ解析にても各々のフラグメントパターンが一致することを確認した。ＧＣ−ＭＳの解析条件は、カラム：ＨＲ−ＳＳ−１０（信和化工）、分析装置：ＧＣＭＳ−ＱＰ５０５０（Ｓｈｉｍａｄｚｕ）、カラム温度：１６０〜２２０℃、２℃／分昇温、注入口検出器温度：２５０℃、検出器：ＦＩＤ、キャリアガス：Ｈｅ、メイクアップガス：Ｎ2で行なった。 Each product was identified by comparing the GC analysis results of commercially available reagents 5-hexadecanol (SIGMA-ALDRICH) and 5-hexadecanone (Alfa Aesar) with the culture sample. Further, it was confirmed by GC-MS analysis that the respective fragment patterns were identical. GC-MS analysis conditions are as follows: Column: HR-SS-10 (Shinwa Chemical), Analytical device: GCMS-QP5050 (Shimadzu), Column temperature: 160-220 ° C., 2 ° C./min temperature rise, inlet detector temperature : 250 ° C., detector: FID, carrier gas: He, makeup gas: N 2
実施例８
ヘキサデカンに代えて、１−トリデセン、１−ヘキサデセン、フェニルノナンを用いて、実施例３と同様に培養し、その培養液をＧＣ分析したところ、１−トリデセンからは１−トリデセン−９−オンの生成を、１−ヘキサデセンからは１−ヘキサデセン−１２−オンの生成を、フェニルノナンからは５−フェニルノナノンの生成を確認した。
Example 8
In place of hexadecane, 1-tridecene, 1-hexadecene, and phenylnonane were used in the same manner as in Example 3, and the culture was analyzed by GC. From 1-tridecene, 1-tridecene-9-one was obtained. Production of 1-hexadecene-12-one was confirmed from 1-hexadecene, and formation of 5-phenylnonanone was confirmed from phenylnonane.
Claims (6)
で表される化合物を一般式（２）
で表されるケトン体及び／又は一般式（３）
（式中、Ｒ１、Ｒ２、ｎ１及びｎ２は前記と同じ）
で表される２級アルコールに変換する能力を有する微生物を、当該一般式（１）で表される化合物の存在下に培養するか、又は当該微生物又はその処理物と当該一般式（１）で表される化合物とを接触させることを特徴とする当該一般式（２）で表されるケトン体及び／又は一般式（３）で表される２級アルコールの製造法。 It belongs to the genus Prototheca and has the following general formula (1)
The compound represented by general formula (2)
And / or the general formula (3)
(Wherein R 1 , R 2 , n1 and n2 are the same as above)
The microorganism having the ability to convert to the secondary alcohol represented by the above is cultured in the presence of the compound represented by the general formula (1), or the microorganism or a treated product thereof and the general formula (1) The manufacturing method of the secondary alcohol represented by the ketone body represented by the said general formula (2) and / or general formula (3) characterized by making the compound represented contact.
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