JP5622190B2 - Novel microorganism and method for producing sugar-type biosurfactant using the same - Google Patents
Novel microorganism and method for producing sugar-type biosurfactant using the same Download PDFInfo
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- JP5622190B2 JP5622190B2 JP2010048481A JP2010048481A JP5622190B2 JP 5622190 B2 JP5622190 B2 JP 5622190B2 JP 2010048481 A JP2010048481 A JP 2010048481A JP 2010048481 A JP2010048481 A JP 2010048481A JP 5622190 B2 JP5622190 B2 JP 5622190B2
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- Prior art keywords
- mel
- pseudozyma
- lipid
- microorganism
- mannosyl erythritol
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- 238000004519 manufacturing process Methods 0.000 title claims description 26
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- -1 mannosylerythritol Lipid Chemical class 0.000 claims description 45
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 19
- 229910052799 carbon Inorganic materials 0.000 claims description 17
- 241001210929 Dirkmeia churashimaensis Species 0.000 claims description 12
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Description
本発明は、糖型バイオサーファクタントを生産する能力を有するサトウキビから単離した微生物、ならびにその利用に関する。 The present invention relates to a microorganism isolated from sugarcane having the ability to produce a sugar-type biosurfactant, and its use.
糖脂質は、脂質に１〜１０数個の単糖が結合した物質であり、生体内において細胞間の情報伝達に関与し、神経系・免疫系の機能維持にも重要な役割を果たしていること等が明らかにされつつある。一方で、糖脂質は、糖の性質に由来する親水性と脂質の性質に由来する親油性の二つの性質を合わせ持つ両親媒性物質であり、このような性質を有する物質は界面活性物質と呼ばれている。 Glycolipids are substances in which 1 to 10 monosaccharides are bound to lipids, are involved in the transmission of information between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. On the other hand, glycolipids are amphipathic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. being called.
一方、一部の微生物はこれらの界面活性物質を効率良く生産することが知られており、この生物由来界面活性剤（バイオサーファクタント）は、安全性が高く、環境に対する負荷が少ない生分解性に優れた環境先進型界面活性剤として研究が進められている。現在、微生物が生産する界面活性物質としては、糖型、アシルペプチド型、リン脂質型、脂肪酸型及び高分子化合物型の５つに分類されているが、特にこの内の糖型の界面活性剤については、最もよく研究され、細菌及び酵母によって生産された多くの種類の物質が報告されている。 On the other hand, some microorganisms are known to produce these surfactants efficiently, and this biological surfactant (biosurfactant) is highly safe and biodegradable with low environmental impact. Research is progressing as an excellent environmentally advanced surfactant. Currently, the surface active substances produced by microorganisms are classified into five types: sugar type, acyl peptide type, phospholipid type, fatty acid type, and polymer compound type. Among these, surfactants of the sugar type in particular are included. There are many types of substances that have been best studied and produced by bacteria and yeast.
これらのバイオサーファクタントは、生分解性が高く、低毒性で環境に優しく、新規な生理機能を持つといわれている。このことから、食品工業、化粧品工業、医薬品工業、化学工業、環境分野等にこれらのバイオサーファクタントを幅広く適用することは、持続可能社会の実現と高機能製品の提供という、両面を兼ね備えており極めて有意義である。 These biosurfactants are said to have high biodegradability, low toxicity, environmental friendliness, and novel physiological functions. For this reason, the wide application of these biosurfactants in the food industry, cosmetic industry, pharmaceutical industry, chemical industry, environmental field, etc. has both the realization of a sustainable society and the provision of high-performance products. Meaningful.
糖型バイオサーファクタントには、次のようなものが例として挙げられる。 Examples of sugar-type biosurfactants include the following.
ラムノリピド（Rhamnolipid；以下、ＲＬと省略する。）は、結核菌の抗生物質としてシュードモナス アエルジノーサ（Pseudomonas aeruginosa）（緑膿菌）の培養液から最初に発見されている（非特許文献１参照）。
また、これまでにシュードモナス（Pseudomonas）属の細菌から４種類の同族体が報告されており、当初は数ｇ／Ｌ程度の生産量であったが、現在では１００ｇ／Ｌ以上の生産を可能にしている（非特許文献２参照）。
Rhamnolipid (hereinafter abbreviated as RL) was first discovered from the culture solution of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) as an antibiotic of Mycobacterium tuberculosis (see Non-Patent Document 1).
In addition, four kinds of homologues have been reported so far from bacteria of the genus Pseudomonas , and the initial production was about several g / L, but now it is possible to produce over 100 g / L. (See Non-Patent Document 2).
トレハロースリピド（Trehalose lipid；以下、ＴＬと省略する。）は、コリネバクテリウム（Corynebacterium）等の細胞表層物質として発見された。また、類似の物質が、マイコバクテリウム（Mycobacterium）、ノカルディア（Nocardia）、ロドコッカス（Rodococcus）属細菌からも報告されている（非特許文献３参照）。
一般的に、細胞壁に結合しているために生産量は低いが、ロドコッカス エリスロポリス（Rodococcus erythropolis）を窒素制限下で培養を行うとサクシノイルトレハロースリピッドを３２ｇ／Ｌ生産することが報告されている（非特許文献４参照）。
Trehalose lipid (hereinafter abbreviated as TL) has been discovered as a cell surface material such as Corynebacterium . Similar substances have also been reported from Mycobacterium , Nocardia and Rodococcus bacteria (see Non-Patent Document 3).
In general, production is low due to binding to the cell wall, but it has been reported that succinoyl trehalose lipid is produced at a rate of 32 g / L when cultivating Rhodococcus erythropolis under nitrogen limitation. (Refer nonpatent literature 4).
ソホロースリピド（Sophorose lipids；「ソホロリピッド」とも言われる；以下、ＳＬと省略する。）は、Ｐ．Ａ．Ｇｏｒｉｎらによってスターメレラ（キャンディダ）・ボンビコーラ（Starmerella（Candida）bombicola）の培養液から発見されている（非特許文献５参照）。
その後、その他の酵母菌、例えば、キャンディダ・ボゴリエンシス（Candida bogoriensis）、キャンディダ・マグノリエ（Candida magnoliae）、キャンディダ・グロペンギッセリ（Candida gropengisseri）、キャンディダ・アピコーラ（Candida apicola）によっても、その培養液中に比較的多量に生産されることが報告されている（非特許文献６参照）。
さらに、現在では、３００ｇ／Ｌ以上の生産を可能にしている（非特許文献７参照）。
Sophorose lipids (also referred to as “sophorolipids”; hereinafter abbreviated as SL) A. It has been discovered by Gorin et al. In a culture solution of Starmerella ( Candida ) bombicola (see Non-Patent Document 5).
Later, other yeasts such as Candida bogoriensis , Candida magnoliae , Candida gropengisseri , and Candida apicola were also used in the culture. It has been reported that it is produced in a relatively large amount (see Non-Patent Document 6).
Furthermore, production of 300 g / L or more is now possible (see Non-Patent Document 7).
セロビオースリピド（Cellobiose lipid；以下、ＣＬと省略する。）は、ウスチラジン酸（ustilagic acid）、フロキュロシン（flocculosin）とも呼ばれる抗微生物活性の高い糖脂質である。
また、ＣＬはウスチラゴ マイディス（Ustilago maydis）により１５ｇ／Ｌ（非特許文献８参照）以上生産されるほか、クリプトコッカス・フミコーラ（Cryptococcus humicola）（非特許文献９参照）、シュードザイマ・フロキュローサ（Pseudozyma flocculosa）（非特許文献１０参照）、シュードザイマ・フジフォルメータ（Pseudozyma fusiformata）（非特許文献１１参照）などからも生産されることが報告されている。
Cellobiose lipid (hereinafter abbreviated as CL) is a glycolipid having high antimicrobial activity, also called ustilagic acid or flocculosin.
In addition, CL is produced by Ustilago maydis over 15 g / L (see Non-Patent Document 8), Cryptococcus humicola (see Non-Patent Document 9), Pseudozyma flocculosa ( Pseudozyma flocculosa ) ( Pseudozyma flocculosa ) Non-patent document 10), Pseudozyma fusiformata (see non-patent document 11) and the like have been reported.
マンノシルエリスリトールリピド（ＭＥＬ）は、高い界面活性作用を有し、界面活性剤又はファインケミカルの種々の触媒として用いられる。ヒト急性前骨髄性白血病細胞性ＨＬ６０株にマンノシルエリスリトールリピッドを作用させると顆粒系を分化させる白血病細胞分化誘導作用があり、また、ラット副腎髄質褐色細胞腫由来のＰＣ１２細胞にマンノシルエリスリトールリピドを作用させると神経突起の伸長が生ずる神経系細胞株分化誘導作用等の生理活性作用を有する。更に、微生物産生の糖脂質として初めて、メラノーマ細胞のアポトーシスを誘導することが可能となり、癌細胞増殖抑制作用がある。これらの生理作用から見て、マンノシルエリスリトールリピドには抗ガン剤等の医薬としての用途が期待される。また、マンノシルエリスリトールリピド（ＭＥＬ）には生分解性があり、高い安全性を有すると考えられる（非特許文献１２等参照）。 Mannosyl erythritol lipid (MEL) has a high surface activity and is used as a surfactant or as a catalyst for fine chemicals. When mannosyl erythritol lipid is allowed to act on human acute promyelocytic leukemia cell line HL60, it has a leukemia cell differentiation inducing action that differentiates the granule system, and mannosyl erythritol lipid acts on PC12 cells derived from rat adrenal medullary pheochromocytoma And has a physiologically active action such as a neural cell line differentiation-inducing action that causes neurite outgrowth. Furthermore, for the first time as a glycolipid produced by microorganisms, it becomes possible to induce apoptosis of melanoma cells, and has an effect of suppressing cancer cell growth. In view of these physiological actions, mannosyl erythritol lipid is expected to be used as a medicine such as an anticancer agent. In addition, mannosyl erythritol lipid (MEL) is biodegradable and is considered to have high safety (see Non-Patent Document 12, etc.).
ＭＥＬは、マンノースあるいはヒドロキシル基が一部アセチル化したマンノースと、エリスリトールを糖骨格（親水基）として、１〜３本の脂肪酸を親水基として有する糖脂質である。ＭＥＬの一般式１を化１に示す。
この式において、Ｒ３およびＲ４がアセチル基である構造物はＭＥＬ-Ａ、Ｒ４がアセチル基でありＲ３が水素である構造物はＭＥＬ-Ｂ、Ｒ３がアセチル基でありＲ４が水素である構造物はＭＥＬ-Ｃ、Ｒ３およびＲ４が水素である構造物はＭＥＬ-Ｄと定義されている。
MEL is a phospholipid having mannose or mannose partially hydroxylated and erythritol as a sugar skeleton (hydrophilic group) and 1 to 3 fatty acids as hydrophilic groups. Formula 1 of MEL is shown in Chemical Formula 1.
In this formula, a structure in which R 3 and R 4 are acetyl groups is MEL-A, and a structure in which R 4 is an acetyl group and R 3 is hydrogen is MEL-B, a structure in which R 3 is an acetyl group, and R 4 A structure in which is hydrogen is defined as MEL-C, and a structure in which R 3 and R 4 are hydrogen is defined as MEL-D.
現在ＭＥＬは、洗剤、化粧品等幅広い分野で工業利用が進められており、ＭＥＬの遺伝子導入剤としての利用（特許文献１参照）およびリポソーム形成剤としての利用（特許文献２参照）、ＭＥＬのスキンケアおよびヘアケア剤としての利用（特許文献３参照）、乳化剤・可溶化剤（特許文献４参照）、タンパク質分離用担体（特許文献５参照）などの報告がある。 Currently, MEL is being used industrially in a wide range of fields such as detergents and cosmetics. Use of MEL as a gene introduction agent (see Patent Document 1) and use as a liposome forming agent (see Patent Document 2), MEL skin care In addition, there are reports on utilization as hair care agents (see Patent Document 3), emulsifiers / solubilizers (see Patent Document 4), protein separation carriers (see Patent Document 5) and the like.
マンノシルエリスリトールリピド（ＭＥＬ）については、Ｃａｎｄｉｄａ ｓｐ．ＳＹ１６株を用いて２１１ｇ／Ｌの大豆油から２００時間で９５ｇ／Ｌ（生産速度：０．４７５ｇ／Ｌ／ｈ、原料収率：４５％）のＭＥＬの生産が可能であることが報告されている（非特許文献１３参照）。また、Ｃａｎｄｉｄａ ａｎｔａｒｃｔｉｃａ Ｔ−３４株を用いて大豆油から６日間で４７ｇ／Ｌ（生産速度：０．３２ｇ／Ｌ／ｈ）のＭＥＬの生産が可能であることが報告されている（非特許文献１４参照）。Pseudozyma aphidis株を用いて８０質量％の植物油脂から流加培養法により２４時間で１３．９ｇ／Ｌ（生産速度：０．５７ｇ／Ｌ／ｈ、原料収率：９２質量％）のＭＥＬの生産が可能であることが報告されている（非特許文献１５参照）。 For mannosyl erythritol lipid (MEL), Candida sp. It is reported that 95 g / L (production rate: 0.475 g / L / h, raw material yield: 45%) of MEL can be produced from 211 g / L soybean oil in 200 hours using SY16 strain. (See Non-Patent Document 13). In addition, it has been reported that 47 g / L (production rate: 0.32 g / L / h) of MEL can be produced from soybean oil in 6 days using Candida antarctica T-34 strain (non-patent literature). 14). Using Pseudozyma aphidi s strain, 80% by mass of vegetable oils and fats by the fed-batch method for 13.9 g / L (production rate: 0.57 g / L / h, raw material yield: 92% by mass) in 24 hours It has been reported that production is possible (see Non-Patent Document 15).
バイオサーファクタントには汎用の界面活性剤と比較して環境適合性、生体適合性に優れ、様々な生理活性が認められているが、既存の界面活性剤と同様の幅広い用途に利用されているとはいえず。例えば食品用途等に使用するには制約があった。これは、多くの場合生産微生物に病原性は知られていないものの、自然界からの単離源が明確でないため、安全性に対する知見に乏しいことによる。特に微生物生産物を食品用途等に展開するためには、使用微生物の単離源が重要視される。例えば、酒類やパンの製造に利用される出芽酵母、乳酸菌や枯草菌（納豆菌）等は、食品分野で活躍する微生物として一般的に良く知られている。これらの微生物は全て食品中から容易に単離可能な微生物である。 Biosurfactants are superior in environmental compatibility and biocompatibility compared to general-purpose surfactants, and various physiological activities are recognized, but they are used in a wide range of applications similar to existing surfactants. No. For example, there are restrictions on use for food applications. This is due to the fact that although the pathogenicity of the produced microorganism is not known in many cases, the source of isolation from the natural world is not clear, and thus knowledge about safety is poor. In particular, in order to develop a microbial product for food use, an isolation source of microorganisms to be used is regarded as important. For example, budding yeast, lactic acid bacteria, Bacillus subtilis (natto), and the like used for the production of alcoholic beverages and bread are generally well known as microorganisms that play an active role in the food field. These microorganisms are all microorganisms that can be easily isolated from food.
さらに、バイオサーファクタントの生産効率を高め、生産コストの低減を図ることや、新しい機能性を備えた同族体の生産技術を開発して、幅広い用途へ展開するために、新たなバイオサーファクタント生産菌の探索・取得が強く望まれているのが現状である。 Furthermore, in order to increase the production efficiency of biosurfactants, reduce production costs, and develop homologue production technologies with new functionality and deploy them to a wide range of applications, new biosurfactant producing bacteria The current situation is that search / acquisition is strongly desired.
そこで、食用されている植物から新たに、バイオサーファクタント生産菌を単離し、当該微生物を用いたバイオサーファクタンント製造技術を開発することができれば、得られるバイオサーファクタントは安全性の面で大きな利点を獲得することとなり、生産微生物も含めた生産物全体の適用用途が大きく拡充されるものと期待される。 Therefore, if a biosurfactant-producing bacterium can be newly isolated from an edible plant and a biosurfactant production technology using the microorganism can be developed, the resulting biosurfactant has a great advantage in terms of safety. It is expected that the application of the entire product including the production microorganism will be greatly expanded.
以上事情を鑑みて、本発明では、化粧品、食品、食品添加物、医薬、農畜水産業関連品（飼料、飼料添加物、肥料、農薬、魚餌）、洗剤など、生体に対する安全性がより求められる用途に対して、従来よりも適応性に優れたバイオサーファクタントを提供するべく、安全な環境から単離した微生物、ならびにそれを用いるバイオサーファクタントの発酵製造方法の提供を目的とする。 In view of the above circumstances, in the present invention, safety to living bodies such as cosmetics, foods, food additives, medicines, agricultural and livestock industry-related products (feeds, feed additives, fertilizers, agricultural chemicals, fish foods), detergents, and the like are further required. The purpose of the present invention is to provide a microorganism isolated from a safe environment and a method for fermenting a biosurfactant using the same, in order to provide a biosurfactant that is more adaptable than before.
本発明者は、上記課題を解決するため上記バイオサーファクタント生産菌を探索した結果、日常的に食用されている砂糖の原料となる「サトウキビ」から、新種の酵母であるシュードザイマ チュラシマエンシス（Pseudozyma churashimaensis）(Ustilago esculenta)を単離することに成功した。そして、当該酵母がバイオサーファクタントの１種であるマンノシルエリスリトールリピド（ＭＥＬ）を生産すること、及び既存のＭＥＬだけでなく、既存の生産菌ではほとんど生産できないトリアセチルモノアシル型マンノシルエリスリトールリピドを生産できることを見出し、本発明を完成するに至った。 As a result of searching for the biosurfactant-producing bacteria to solve the above-mentioned problems, the present inventor has developed a new yeast, Pseudozyma churashimaensis, from sugarcane, which is a raw material for sugar that is used daily. ) ( Ustilago esculenta ) was successfully isolated. The yeast can produce mannosyl erythritol lipid (MEL) which is a kind of biosurfactant, and can produce not only existing MEL but also triacetyl monoacyl mannosyl erythritol lipid which can hardly be produced by existing producing bacteria. As a result, the present invention has been completed.
すなわち、本発明は、以下のとおりである。
〔１〕トリアセチルモノアシル型マンノシルエリスリトールリピドを生産する能力を有する、シュードザイマ チュラシマエンシス（Pseudozyma churashimaensis）に属する微生物であって、下記（ａ）及び（ｂ）で示される菌学的性質を有する微生物。
（ａ）形態学的性質
〔３〕シュードザイマ チュラシマエンシス ＯＫ−９６（受託番号 FERM P-２１８９６）である上記〔１〕に記載の微生物。
〔４〕上記〔１〕〜〔３〕のいずれかに記載の微生物を培養し、得られた培養物あるいは分離菌体からマンノシルエリスリトールリピド−Ａを採取することを特徴とする、マンノシルエリスリトールリピドの製造方法。
〔５〕上記〔１〕〜〔３〕のいずれかに記載の微生物を、炭素源として糖類を含有する培地に培養し、得られた培養物からトリアセチルモノアシル型マンノシルエリスリトールリピドを採取することを特徴とする、トリアセチルモノアシル型マンノシルエリスリトールリピドの製造方法。
That is, the present invention is as follows.
[1] A microorganism belonging to Pseudozyma churashimaensis having the ability to produce a triacetyl monoacyl-type mannosyl erythritol lipid, having the bacteriological properties shown in the following (a) and (b) microbial you.
(A) Morphological properties
[3] The microorganism according to the above [1 ], which is Pseudozyma Chulashima Ensis OK-96 (Accession No. FERM P-21896).
[4] Mannosylerythritol lipid, wherein the microorganism according to any one of [1] to [3] above is cultured, and mannosylerythritol lipid-A is collected from the obtained culture or isolated cells. Production method.
[5] Culturing the microorganism according to any one of [1] to [3] above in a medium containing saccharides as a carbon source, and collecting triacetyl monoacyl mannosyl erythritol lipid from the obtained culture A process for producing a triacetyl monoacyl mannosyl erythritol lipid, characterized in that
本発明によれば、古くから広く食される植物であるサトウキビに常在する微生物、より具体的にはシュードザイマ チュラシマエンシス(Ｐｓｅｕｄｏｚｙｍａ ｃｈｕｒａｓｈｉｍａｅｎｓｉｓ)を提供することができる。
当該微生物を生産微生物として用いることにより、得られたバイオサーファクタントの安全性を確保するとともに、これまで利用に制限があった食品用途や家畜飼料、魚餌、農業用途に直接使用できる界面活性剤としての利用が見込まれ、バイオサーファクタントの普及の拡大に著しく貢献できるものと期待される。
According to the present invention, it is possible to provide a microorganism that is resident in sugarcane, which is a widely eaten plant for a long time, more specifically, Pseudozyma churashimaensis .
By using the microorganism as a production microorganism, the safety of the obtained biosurfactant is ensured, and as a surfactant that can be directly used for food use, livestock feed, fish food, and agricultural use that have been limited in use so far. Expected to be used, it is expected to contribute significantly to the spread of biosurfactants.
さらに本発明に係る製造方法によれば、副生物を生じることなく選択的にＭＥＬ−Ａを生産することができる。すなわち、従来の方法では、精製過程を必要とするＭＥＬ−ＢとＭＥＬ−Ｃを含まないＭＥＬ−Ａを、微生物の培養のみで得ることが可能となる。特に、油脂等の疎水性基質を用いず、糖質(グルコースやショ糖)のみを炭素源として含有する培地を用いても、ＭＥＬを生産できるため、ＭＥＬの単離も簡便となる。さらに、本発明の上記微生物は、トリアセチル型ＭＥＬを生産する能力を有する。このトリアセチルモノアシル型ＭＥＬは、ＭＥＬ−Ａに上記定義上属し、その界面活性作用自体は、従来のＭＥＬ−Ａ（ジアセチルジアシル型）と大差がないが、特に、トリアセチルモノアシル型ＭＥＬは脂肪酸が一本であるため、脂肪酸を二本もつ従来のＭＥＬ−Ａと比べて水分子に対する親和性が向上している。また、分子構造の違いに起因する分子間相互作用の点で差違があり、従来のＭＥＬ−Ａとは異なるナノ材料としての応用が期待されるものである。 Furthermore, according to the manufacturing method which concerns on this invention, MEL-A can be selectively produced, without producing a by-product. That is, in the conventional method, it is possible to obtain MEL-B that does not contain MEL-B and MEL-C, which require a purification process, only by culturing microorganisms. In particular, since MEL can be produced even if a medium containing only a carbohydrate (glucose or sucrose) as a carbon source is used without using a hydrophobic substrate such as fat or oil, isolation of MEL becomes simple. Furthermore, the microorganism of the present invention has an ability to produce triacetyl type MEL. This triacetyl monoacyl type MEL belongs to MEL-A as defined above, and its surface activity itself is not much different from conventional MEL-A (diacetyl diacyl type). Since the number of fatty acids is one, the affinity for water molecules is improved as compared with the conventional MEL-A having two fatty acids. Further, there is a difference in the interaction between molecules due to the difference in molecular structure, and application as a nanomaterial different from the conventional MEL-A is expected.
以上のように、本発明によれば、ＭＥＬ−Ａのみを生産可能になり、例えば界面活性剤として使用する場合には精製ステップの簡略化が可能であることから、ＭＥＬ生産コストの低減が可能となる。また、特に上記したトリアセチルモノアシル型ＭＥＬは、水分子に対する親和性が向上している点、また従来のＭＥＬ−Ａとは異なるナノ材料としての利用が期待できる点で特筆すべきものである。 As described above, according to the present invention, only MEL-A can be produced. For example, when used as a surfactant, the purification step can be simplified, so that the MEL production cost can be reduced. It becomes. In particular, the triacetyl monoacyl type MEL described above is notable because it has an improved affinity for water molecules and can be expected to be used as a nanomaterial different from the conventional MEL-A.
以下、本発明につきさらに詳しく説明する。
〈本発明の微生物〉
本発明に係る微生物は、ＭＥＬを生産する能力を有するＰｓｅｕｄｏｚｙｍａ属に属する新種の酵母である。該酵母はマンノシルエリスリトールリピドを生産し、該マンノシルエリスリトールリピドは生理活性が高く実用特性も優れていることから望ましい。また、該酵母は特にトリアセチルモノアシル型ＭＥＬを産生する点で特徴的な性質を有する。該当する微生物としては、シュードザイマ(Ｐｓｅｕｄｏｚｙｍａ)属に属するＯＫ−９６株を挙げることができる。本菌株は、本発明者等が日本国内で採取した植物（サトウキビ）から分離した菌株であり、本菌株は、YM寒天培地、PDB寒天培地および5%麦芽エキス培地上にて25℃4日間培養で、全縁、円錐状、平滑、輝光、湿性でクリーム色から淡桃色のコロニーを形成する。YM液体培地およびPD液体培地、25℃下において、培養３日目に、細胞は楕円形から円筒形であり、極出芽によって増殖する。また、隔壁のある菌糸ならびに偽菌糸の形成が認められる。また、培養一カ月を経過した時点でも、明らかな有性生殖器の形成は認められない。これらの形態学的性質はPseudozyma属の一般的な形態学的特徴と一致し、加えて、Ｏｋ−９６株は糖発酵性を示さず、窒素源として硝酸塩を資化する点でもPseudozyma属の特徴と一致するので、本菌株はPseudozyma属に帰属する菌株といえる。
Hereinafter, the present invention will be described in more detail.
<Microorganism of the present invention>
The microorganism according to the present invention is a new yeast belonging to the genus Pseudozyma having the ability to produce MEL. The yeast produces mannosyl erythritol lipid, and the mannosyl erythritol lipid is desirable because of its high physiological activity and excellent practical properties. In addition, the yeast has a characteristic property particularly in that it produces a triacetyl monoacyl MEL. Examples of such microorganisms include the OK-96 strain belonging to the genus Pseudozyma . This strain is a strain isolated from a plant (sugar cane) collected by the present inventors in Japan. This strain is cultured on a YM agar medium, a PDB agar medium and a 5% malt extract medium at 25 ° C. for 4 days. And forms a cream to light pink colony with full edge, conical, smooth, bright and moist. On the third day of culture in YM liquid medium and PD liquid medium at 25 ° C., the cells are elliptical to cylindrical and proliferate by extreme budding. In addition, formation of hyphae with septa and pseudohyphae is observed. In addition, no apparent sexual genital formation is observed even after one month of culture. These morphological properties are consistent with the general morphological characteristics of the genus Pseudozyma. In addition, the strain of Ok-96 does not exhibit sugar fermentability, and is also characterized by the utilization of nitrate as a nitrogen source. Therefore, this strain can be said to be a strain belonging to the genus Pseudozyma.
また、この分離されたＯＫ−９６株について、リボゾームRNA遺伝子の26SrDNA−D1/D2領域の塩基配列（rDNA配列）を決定し（配列番号１）、DNAデータベース（DDBJ）にアクセスし、FASTAプログラム（http://www.ddbj.nig.ac.jp/search/fasta-j.html）を用いて相同性検索を行ったところ、シュードザイマ アフィディス（Pseudozyma aphidis）CBS517.83TのrDNA配列と最も高い相同性（９９．５％）を示した。しかし、ＯＫ−９６株のITS1-５，８SrDNA−ITS2領域の塩基配列（配列番号２）については、データベース上において９９％以上の相同性を示したものはなく、Ustilago trichophora１３３４株の８SrDNA−ITS2と９４．１％の相同性を示したのが最高であった。各種ジュードザイマ属酵母に対しては、９２．６％の相同性（Pseudozyma hubeiensis CBS1007７）が最高で、上記シュードザイマ アフィディス（Pseudozyma aphidis）CBS517.83T配列とは９０．１％の相同性しか示さなかった。また、ＯＫ−９６株とシュードザイマ アフィディス（Pseudozyma aphidis）CBS517.83Tとは、その生理性状試験尾の結果、本菌株がエリスリトールを資化せず、エタノールを資化し、ビタミン欠乏培地で生育を示した点で明らかに異なる。これらの点から、本菌株は担子菌門の一種であるシュードザイマ属の新たな種であることが分かり、シュードザイマ チュラシマエンシス(Ｐｓｅｕｄｏｚｙｍａ ｃｈｕｒａｓｈｉｍａｅｎｓｉｓ)ＯＫ−９６株と命名した。 For the isolated OK-96 strain, the nucleotide sequence (rDNA sequence) of the 26S rDNA-D1 / D2 region of the ribosomal RNA gene was determined (SEQ ID NO: 1), the DNA database (DDBJ) was accessed, and the FASTA program ( The homology search using http://www.ddbj.nig.ac.jp/search/fasta-j.html) revealed the highest homology with the rDNA sequence of Pseudozyma aphidis CBS517.83T. (99.5%). However, the nucleotide sequence (SEQ ID NO: 2) of the ITS1-5, 8SrDNA-ITS2 region of the OK-96 strain does not show a homology of 99% or more on the database, and the 8SrDNA-ITS2 of Ustilago trichophora 1334 strain The best was 94.1% homology. 92.6% homology (Pseudozyma hubeiensis CBS10077) was the highest for various yeasts of the genus Judezaima, and only 90.1% homology with the Pseudozyma aphidis CBS517.83T sequence. . In addition, as a result of the physiological property test tail, OK-96 strain and Pseudozyma aphidis (Pseudozyma aphidis) CBS517.83T showed that this strain did not assimilate erythritol, assimilated ethanol, and showed growth in a vitamin-deficient medium. Clearly different in respect. From these points, this strain was found to be a new species of the genus Pseudozyma, a kind of basidiomycete, and was designated as Pseudozyma churashimaensis OK-96 strain.
本菌株の形態学的観察結果及び生理性状試験の結果は、以下の表１、表２にまとめて示す。
本菌株は、平成２２年１月２６日付で独立行政法人産業技術総合研究所特許微生物寄託センター（IPOD）（茨城県つくば市東１−１−３）に受託番号FERM P-21896として寄託されている。
The morphological observation results and physiological property test results of this strain are summarized in Tables 1 and 2 below.
This strain was deposited on January 26, 2010 at the National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary Center (IPOD) (Higashi 1-1-3 Tsukuba, Ibaraki) under the accession number FERM P-21896. .
（ａ）形態学的性質
（ｂ）生理学的性質
（産生バイオサーファクタント）
本発明の微生物が産生するバイオサーファクタントは、マンノシルエリスリトール（ＭＥＬ）であり、具体的には、4-O-β-D-mannopyranosyl-erythritolを糖骨格とするＭＥＬ−Ａであり、ＭＥＬ−Ａとしては、以下の一般式において、Ｒ３およびＲ４がアセチル基であって、Ｒ１及びＲ２が炭素数４以上の飽和または不飽和の脂肪族アシル基である化合物と、Ｒ１、Ｒ３およびＲ４がともにアセチル基であって、Ｒ２が炭素数３以上の脂肪族アシル基である化合物が挙げられる。
本明細書において、ジアセチルジアシル型マンノシルエリスリトールリピド（ＭＥＬ）とは前者の化合物を意味し、トリアセチルモノアシル型マンノシルエリスリトールリピド（ＭＥＬ）とは後者の化合物を意味する。
(Production biosurfactant)
The biosurfactant produced by the microorganism of the present invention is mannosylerythritol (MEL), specifically, MEL-A having 4-O-β-D-mannopyranosyl-erythritol as a sugar skeleton, as MEL-A Is a compound in which R 3 and R 4 are acetyl groups and R 1 and R 2 are saturated or unsaturated aliphatic acyl groups having 4 or more carbon atoms, and R 1, R 3 And R 4 are both acetyl groups, and R 2 is an aliphatic acyl group having 3 or more carbon atoms.
In the present specification, the diacetyl diacyl type mannosyl erythritol lipid (MEL) means the former compound, and the triacetyl monoacyl type mannosyl erythritol lipid (MEL) means the latter compound.
本発明の微生物を炭素源として糖類を使用して培養した場合には、培養液中にジアセチルジアシル型マンノシルエリスリトールリピド（ＭＥＬ）及びトリアセチルモノアシル型マンノシルエリスリトールリピド（ＭＥＬ）との混合物として蓄積される。両者は界面活性剤の性質には大差はないので、混合物のまま界面活性剤として使用してもよく、また、トリアセチルモノアシル型マンノシルエリスリトールリピド（ＭＥＬ）の水分子に対する親和性および／または分子間相互作用に着目すれば、さらに、ジアセチルジアシル型マンノシルエリスリトールリピド（ＭＥＬ）及びトリアセチルモノアシル型マンノシルエリスリトールリピド（ＭＥＬ）とを分離しても良い。
一方、本発明の微生物を炭素源として油脂類、脂肪酸あるいは脂肪酸エステルを炭素源として培養する場合には、ジアセチルジアシル型マンノシルエリスリトールリピド（ＭＥＬ）のみが得られる。上記培地の炭素源によって、蓄積されるジアセチルジアシル型マンノシルエリスリトールリピド（ＭＥＬ）は、そのマンノシル２位の脂肪族アシル基（上記一般式中、R1）の炭素数が若干異なる。
When the microorganism of the present invention is cultured using a saccharide as a carbon source, it accumulates in the culture solution as a mixture of diacetyl diacyl mannosyl erythritol lipid (MEL) and triacetyl monoacyl mannosyl erythritol lipid (MEL). The Since there is no great difference between the properties of the surfactants, they may be used as a surfactant in the form of a mixture. Also, the affinity of triacetyl monoacyl mannosyl erythritol lipid (MEL) for water molecules and / or molecules If attention is paid to the interaction, diacetyldiacyl-type mannosylerythritol lipid (MEL) and triacetylmonoacyl-type mannosylerythritol lipid (MEL) may be further separated.
On the other hand, when the microorganism of the present invention is used as a carbon source for culturing oils and fats, fatty acids or fatty acid esters as a carbon source, only diacetyldiacyl mannosylerythritol lipid (MEL) is obtained. The diacetyldiacyl-type mannosylerythritol lipid (MEL) accumulated depending on the carbon source of the medium has a slightly different carbon number of the aliphatic acyl group at the 2-position of mannosyl (in the above general formula, R 1 ).
〈培地・培養条件〉
本発明の微生物の培養形態は液体培地を用いた回分培養あるいは培養系に炭素源および/または有機窒素源を連続添加する流加培養であり、通気攪拌することが望ましい。培地の初期ｐHは酸性から中性の範囲であれば特に問題は無いが、３．０−９．０に調整することが好ましく、６．０−８．０に調整することがより望ましい。培養に適した温度範囲は２０−３０℃、より好ましくは２２−２７℃である。
<Medium and culture conditions>
The culture form of the microorganism of the present invention is batch culture using a liquid medium or fed-batch culture in which a carbon source and / or an organic nitrogen source are continuously added to a culture system, and it is desirable to aeration and agitate. The initial pH of the medium is not particularly limited as long as it is in the acidic to neutral range, but it is preferably adjusted to 3.0-9.0, more preferably 6.0-8.0. The temperature range suitable for the culture is 20-30 ° C, more preferably 22-27 ° C.
本発明において、培地は一般的な半合成培地を用いればよいが、ジアセチルジアシル型マンノシルエリスリトールリピド（ＭＥＬ）及びトリアセチルモノアシル型マンノシルエリスリトールリピド（ＭＥＬ）の混合物を培養液中に蓄積される場合には、炭素源として糖類を用いる。
一方、ジアセチルジアシル型マンノシルエリスリトールリピド（ＭＥＬ）のみを培養液中に蓄積させるには、炭素源として油脂類を用いる。
窒素源としては、有機窒素源と無機窒素源を組み合わせて用いることが望ましい。
糖類としては、本願発明の微生物が資化できるものあれば特に限定されるものではないが、例えば、グルコース、ガラクトース、フルクトースなどの単糖類、ショ糖、マルトースなどの二糖類が用いられるが、好ましくはグルコースである。培養初発濃度は１０−２００ｇ／Ｌ、好ましくは５０−１５０ｇ／Ｌで用いられる。
In the present invention, a general semi-synthetic medium may be used as the medium, but when a mixture of diacetyl diacyl mannosyl erythritol lipid (MEL) and triacetyl monoacyl mannosyl erythritol lipid (MEL) is accumulated in the culture solution. Uses sugars as a carbon source.
On the other hand, in order to accumulate only diacetyldiacyl mannosylerythritol lipid (MEL) in the culture solution, fats and oils are used as a carbon source.
As the nitrogen source, it is desirable to use a combination of an organic nitrogen source and an inorganic nitrogen source.
The saccharide is not particularly limited as long as it can assimilate the microorganism of the present invention. For example, monosaccharides such as glucose, galactose and fructose, and disaccharides such as sucrose and maltose are preferably used. Is glucose. The initial culture concentration is 10-200 g / L, preferably 50-150 g / L.
用いる油脂類としては、特に限定されるものではないが、例えば、植物油、脂肪酸またはそのエステル類を用いても良い。
使用する植物油としては、例えばダイズ油、ナタネ油、コーン油、綿実油、ヒマワリ油、カポック油、ゴマ油、コメ油、落花生油、ベニバナ油、オリーブ油、アマニ油、キリ油、ヒマシ油、パーム油、パーム核油、ヤシ油もしくはこれらの混合物等が挙げられる。
使用する脂肪酸および脂肪酸エステルには、炭素鎖が１０−２４のもの、好ましくは炭素鎖が１６−１８の脂肪酸または脂肪酸エステルを用いることができる。これらの脂肪酸または脂肪酸エステルは分子内に１−３個の不飽和結合を含んでいてもよい。例えば、デカン酸、ウンデカン酸、ラウリン酸、トリデカン酸、ミリスチン酸、ペンタデカン酸、パルミチン酸、マルガリン酸、ステアリン酸、ノナデカン酸、アラキジン酸、ベヘン酸、リグノセリン酸などの飽和脂肪酸またはそのエステル、あるいはトウハク酸、リンデル酸、ツズ酸、ミリストレイン酸、パルミトレイン酸、ペトロセリン酸、オレイン酸、エライジン酸、バクセン酸、エルカ酸、ソルビン酸、リノール酸、リノエライジン酸、γ―リノレン酸、リノレン酸、アラキドン酸などの不飽和脂肪酸またはそのエステルが挙げられる。
培地に添加する上記油脂類の濃度は、２０−１００ｇ／Ｌ、好ましくは５０−８０ｇ／Ｌの範囲である。流加培養の様式をとる場合には、培地中濃度が上記の範囲に収まるように培養期間中に連続的もしくは断続的に添加する。
The fats and oils to be used are not particularly limited, and for example, vegetable oils, fatty acids or esters thereof may be used.
Examples of vegetable oils to be used include soybean oil, rapeseed oil, corn oil, cottonseed oil, sunflower oil, kapok oil, sesame oil, rice oil, peanut oil, safflower oil, olive oil, flaxseed oil, gill oil, castor oil, palm oil, palm Examples thereof include nuclear oil, coconut oil, and mixtures thereof.
As the fatty acid and fatty acid ester to be used, a fatty acid or fatty acid ester having a carbon chain of 10-24, preferably 16-18 carbon chains can be used. These fatty acids or fatty acid esters may contain 1-3 unsaturated bonds in the molecule. For example, saturated fatty acids such as decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, behenic acid, lignoceric acid, etc. Acids, Linderic acid, Tuzic acid, Myristoleic acid, Palmitoleic acid, Petroselinic acid, Oleic acid, Elaidic acid, Vaccenoic acid, Erucic acid, Sorbic acid, Linoleic acid, Linoleic acid, Gamma-linolenic acid, Linolenic acid, Arachidone Examples thereof include unsaturated fatty acids such as acids or esters thereof.
The concentration of the fats and oils added to the medium is in the range of 20-100 g / L, preferably 50-80 g / L. When adopting a fed-batch culture mode, it is added continuously or intermittently during the culture period so that the concentration in the medium falls within the above range.
使用する有機窒素源としては、酵母エキス、麦芽エキス、ペプトン、ポリペプトン、コーンスティープリカー、カザミノ酸、尿素などの内、一種類もしくは二種類以上を組み合わせて用いることができる。好ましくは酵母エキスを１−８ｇ／Ｌの濃度範囲で用いるとよい。
無機窒素源としては、硝酸ナトリウム、硝酸カリウム、硝酸アンモニウム、硫酸アンモニウム、アンモニアなどの内、一種類もしくは二種類以上を組み合わせて用いることができる。好ましくは硝酸ナトリウムを用いるとよい。
As the organic nitrogen source to be used, one or a combination of two or more of yeast extract, malt extract, peptone, polypeptone, corn steep liquor, casamino acid, urea and the like can be used. Yeast extract is preferably used in a concentration range of 1-8 g / L.
As the inorganic nitrogen source, one or a combination of two or more of sodium nitrate, potassium nitrate, ammonium nitrate, ammonium sulfate, and ammonia can be used. Sodium nitrate is preferably used.
（マンノシルエリスリトールリピド（ＭＥＬ）の分離）
培養液中に蓄積されたＭＥＬ−Ａは、本菌株培養後、培養液を抽出することにより培養液から分離できる取得できる。抽出は、酢酸エチル、クロロホルム、メタノール、エタノール、ヘキサン、プロパノール等の有機溶媒を用い当技術分野において通常行われる方法にしたがって行えばよい。
有機溶媒によって抽出されたＭＥＬ−Ａが、ジアセチルジアシル型マンノシルエリスリトールリピド（ＭＥＬ）及びトリアセチルモノアシル型マンノシルエリスリトールリピド（ＭＥＬ）の混合物である場合、ジアセチルジアシル型マンノシルエリスリトールリピド（ＭＥＬ）とトリアセチルモノアシル型マンノシルエリスリトールリピド（ＭＥＬ）は、シリカゲルカラムクロマトグラフィー等で分離すればよい。
(Separation of mannosyl erythritol lipid (MEL))
MEL-A accumulated in the culture solution can be obtained by being separated from the culture solution by extracting the culture solution after culturing the present strain. Extraction may be performed according to a method commonly used in the art using an organic solvent such as ethyl acetate, chloroform, methanol, ethanol, hexane, propanol or the like.
When MEL-A extracted with an organic solvent is a mixture of diacetyl diacyl mannosyl erythritol lipid (MEL) and triacetyl monoacyl mannosyl erythritol lipid (MEL), diacetyl diacyl mannosyl erythritol lipid (MEL) and triacetyl Monoacyl type mannosyl erythritol lipid (MEL) may be separated by silica gel column chromatography or the like.
〈ＭＥＬの構造決定〉
上記により得られる糖脂質成分の構造決定は、以下のようにして行うシュードザイマ チュラシマエンシス(Ｐｓｅｕｄｏｚｙｍａ ｃｈｕｒａｓｈｉｍａｅｎｓｉｓ)ＯＫ−９６株を用いてグルコースあるいは大豆油を炭素源として培養して得られたＭＥＬの構造決定手法を例にして以下説明する）。
単離した糖脂質成分は、ＴＬＣプレート上で、アンスロン硫酸試薬で青緑色に呈色することにより糖脂質成分であると判断できる。この糖脂質について、1Ｈ、13Ｃ―ＮＭＲ解析を行い、得られたスペクトルと、構造既知であるＭＥＬのデータとを比較することで、構造解析を行う。
<Determining the structure of MEL>
The structure of the glycolipid component obtained as described above is determined as follows. The structure of MEL obtained by culturing glucose or soybean oil as a carbon source using Pseudozyma churashimaensis OK-96 strain This will be explained below using the determination method as an example).
The isolated glycolipid component can be judged to be a glycolipid component by being colored blue-green with an anthrone sulfate reagent on a TLC plate. The glycolipid is subjected to 1 H, 13 C-NMR analysis, and the obtained spectrum is compared with MEL data having a known structure to perform structural analysis.
〈ＭＥＬの定量分析方法〉
全糖脂質量はアンスロン硫酸法を用いることで測定できる。ＭＥＬ同属体の含有量と存在比は薄相クロマトグラフィー（ＴＬＣ）法および高速液体クロマトグラフィー（ＨＰＬＣ）を使用することで測定できる。
<Quantitative analysis method of MEL>
The total glycolipid amount can be measured by using the anthrone sulfate method. The content and abundance ratio of MEL congeners can be measured by using thin phase chromatography (TLC) method and high performance liquid chromatography (HPLC).
以下に、本発明について実施例によりさらに詳細に説明するが、本発明はこれにより限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
実施例１
（シュードザイマ チュラシマエンシス(Ｐｓｅｕｄｏｚｙｍａ ｃｈｕｒａｓｈｉｍａｅｎｓｉｓ)を用いた、ＭＥＬの生産；使用する炭素源の影響)
〈種培養〉
ディープフリーザー（-８０℃）で冷凍保存されたシュードザイマ チュラシマエンシス(Ｐｓｅｕｄｏｚｙｍａ ｃｈｕｒａｓｈｉｍａｅｎｓｉｓ)ＯＫ−９６株を３０ｍＬのＹＭ培地（グルコース１０ｇ／Ｌ、酵母エキス３ｇ／Ｌ、麦芽エキス３ｇ／Ｌ、ペプトン５ｇ／Ｌ）に０．５ ｍＬ播種し、２８℃で１日間、振蕩培養した。
〈本培養〉
３００ｍＬ容量の三角フラスコに３０ｍＬの含有する炭素源を変えたＭＥＬ生産用培地を表３および４（ＭＥＬ生産培地組成）のごとく調製し、１２１℃、２０分間オートクレーブで滅菌した培地を用意した。上述の種培養液を１．５ｍＬを播種し、２５℃で５日間、振蕩培養を行った。培養終了後の培養液からＭＥＬ標品を酢酸エチルにて抽出した。抽出液中のＭＥＬ濃度はシリカゲルカラム（イナートシルＳｉｌ−１００Ａ）を接続したＨＰＬＣを用い、定量分析を行った。溶離液にはクロロホルム／メタノール混合溶媒を用い、流速１ｍＬ／ｍｉｎで混合比が１０：０から０：１０まで変化するように設定したグラジエントシステムにより溶出した。検出は蒸発光散乱検出器（ＥＬＳＤ−ＬＴ、島津製作所製）を用いた。精製したＭＥＬを用いて検量線を作成し、ピークエリアからＭＥＬ濃度を算出した。
Example 1
(Production of MEL using Pseudozyma churashimaensis ; Influence of carbon source used)
<Seed culture>
30 mL of YM medium (glucose 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, malt extract 3 g / L, peptone 5 g / L) was stored frozen in a deep freezer (−80 ° C.) in a pseudo- Zima Churashima Ensis L) was inoculated with 0.5 mL and cultured with shaking at 28 ° C. for 1 day.
<Main culture>
MEL production media were prepared as shown in Tables 3 and 4 (composition of MEL production medium) in a 300 mL Erlenmeyer flask with the carbon source contained in 30 mL, and a medium sterilized by autoclaving at 121 ° C. for 20 minutes was prepared. 1.5 mL of the above seed culture solution was inoculated, and shaking culture was performed at 25 ° C. for 5 days. The MEL preparation was extracted with ethyl acetate from the culture broth after completion of the culture. The MEL concentration in the extract was quantitatively analyzed using HPLC connected to a silica gel column (Inertosyl Sil-100A). The eluent was a chloroform / methanol mixed solvent and eluted with a gradient system set so that the mixing ratio was changed from 10: 0 to 0:10 at a flow rate of 1 mL / min. For detection, an evaporative light scattering detector (ELSD-LT, manufactured by Shimadzu Corporation) was used. A calibration curve was created using the purified MEL, and the MEL concentration was calculated from the peak area.
〈薄相クロマトグラフィー分析〉
図１に示すように、グルコース添加した培地で、糖脂質の生産を示す２つのスポットが得られた。すなわち、移動度が０．６６のＧＬ−Ａ、移動度が０．６２のＧＬ−Ｂが得られた。一方、大豆油を添加した培地では、１つのスポットが得られた。すなわち、移動度が０．６６のＧＬ−Ｃが得られた。炭素源として添加する糖の種類を変えても、植物油の種類を変えても、ＴＬＣの結果は同様になった。また、図１の結果から、スタンダードに用いた従来型のＭＥＬ−Ａの移動度が０．６６であるのに対して、ＧＬ−Ｂの移動度は０．６２であり、ＧＬ−Ｂの移動度が、明らかに移動度が小さい（極性が高い）ことが分かる。すなわち、ＧＬ−Ｂの水分子に対する親和性は従来型のＭＥＬ−Ａより大きいことが分かる。ＭＥＬの生産量を測定した結果は図２に示した。
<Thin phase chromatography analysis>
As shown in FIG. 1, two spots showing glycolipid production were obtained in the medium supplemented with glucose. That is, GL-A with a mobility of 0.66 and GL-B with a mobility of 0.62 were obtained. On the other hand, one spot was obtained in the medium supplemented with soybean oil. That is, GL-C having a mobility of 0.66 was obtained. The TLC results were the same whether the type of sugar added as the carbon source or the type of vegetable oil was changed. Further, from the result of FIG. 1, the mobility of the conventional MEL-A used for the standard is 0.66, whereas the mobility of GL-B is 0.62, and the mobility of GL-B It can be seen that the mobility is clearly low (high polarity). That is, it can be seen that the affinity of GL-B for water molecules is larger than that of conventional MEL-A. The result of measuring the production amount of MEL is shown in FIG.
実施例２
シュードザイマ チュラシマエンシス(Ｐｓｅｕｄｏｚｙｍａ ｃｈｕｒａｓｈｉｍａｅｎｓｉｓ)ＯＫ−９６株が生産する糖脂質の構造解析
（糖脂質（ＭＥＬ）の精製）
実施例１と同様に培養した培養液から、酢酸エチルを用いて糖脂質を抽出・回収し、シリカゲルカラムを用いて、各成分（ＧＬ−Ａ，ＧＬ−Ｂ，ＧＬ−Ｃ）を精製した。精製された各糖脂質成分をＴＬＣ分析により分析した結果を図３に示す。
（糖脂質成分のＮＭＲ解析）
上記で得られた各成分（ＧＬ−Ａ，ＧＬ−Ｂ，ＧＬ−Ｃ）について、重水素化メタノール（CD3OD）を溶媒とする1H-ＮＭＲ解析により構造の同定を行った。その結果、シュードザイマ チュラシマエンシス(Ｐｓｅｕｄｏｚｙｍａ ｃｈｕｒａｓｈｉｍａｅｎｓｉｓ)ＯＫ−９６株が生産する糖脂質は全て、マンノースの４位と６位の位置にアセチル基を有するＭＥＬ−Ａであることがわかった（図４中のピークｄおよびｅ）。すなわち、ＧＬ−Ａ，ＧＬ−Ｂ，ＧＬ−Ｃは既知のＭＥＬとほぼ同じ化学シフトを示した。さらに、ＧＬ−ＡとＧＬ−Ｂには、マンノースの２位の位置にアセチル基が存在することを示すピークが得られたことから、マンノースの２位の位置の脂肪酸が極端に短くなり、アセチル基となった構造物が主成分の一つとして含まれていることが分かった（図４中のピークｃ）。また、ＧＬ−Ｂにおいて、マンノースの２位の位置の脂肪酸鎖（アシル基）を示すピークが見られなかったことから、ＧＬ−Ｂに含まれるＭＥＬ−Ａのほとんどは、マンノースの２位の位置がアセチル基となった構造物であるといえる（図４中のピークａ）。それぞれの糖脂質に関して、1H-ＮＭＲ解析において帰属された化学シフトをまとめて表５に示す。
Example 2
Structural analysis of glycolipid produced by Pseudozyma churashimaensis OK-96 strain (Purification of glycolipid (MEL))
Glycolipids were extracted and collected from the culture broth cultured in the same manner as in Example 1 using ethyl acetate, and each component (GL-A, GL-B, GL-C) was purified using a silica gel column. The result of analyzing each purified glycolipid component by TLC analysis is shown in FIG.
(NMR analysis of glycolipid components)
About each component (GL-A, GL-B, GL-C) obtained above, the structure was identified by 1 H-NMR analysis using deuterated methanol (CD 3 OD) as a solvent. As a result, it was found that all glycolipids produced by Pseudozyma churashimaensis OK-96 strain were MEL-A having acetyl groups at positions 4 and 6 of mannose (in FIG. 4). Peaks d and e). That is, GL-A, GL-B, and GL-C showed almost the same chemical shift as known MEL. Furthermore, in GL-A and GL-B, a peak indicating the presence of an acetyl group at the 2-position of mannose was obtained, so that the fatty acid at the 2-position of mannose became extremely short, and acetyl It was found that the base structure was included as one of the main components (peak c in FIG. 4). Moreover, in GL-B, since the peak which shows the fatty acid chain (acyl group) of the 2nd position of mannose was not seen, most of MEL-A contained in GL-B is the 2nd position of mannose. Can be said to be a structure having an acetyl group (peak a in FIG. 4). Table 5 summarizes the chemical shifts assigned in 1 H-NMR analysis for each glycolipid.
（糖脂質成分のＭＡＬＤＩ−ＴＯＦ／ＭＳ解析）
上記で得られた各成分（ＧＬ−Ａ，ＧＬ−Ｂ，ＧＬ−Ｃ）の分子量を、ＭＡＬＤＩ−ＴＯＦ／ＭＳによって解析した結果、ＧＬ−Ａの主成分の分子量は７０４．８、６７６．８、６４８．８、ＧＬ−Ｂの主成分の分子量は６４８．１、ＧＬ−Ｃの主成分の分子量は７０４．１、６７６．１であった。図５に、ＭＡＬＤＩ−ＴＯＦ／ＭＳで得られた分子量のピークを示す。これらの結果から、ＧＬ−Ａは、4-O-[(6',4'-di-O-acetyl-,3'-O-alka(e)noyl-2'-O-caproyl)- β-D-mannopyranosyl]-D-erythritol（一般式３）、4-O-[(6',4'-di-O-acetyl-,3'-O-alka(e)noyl-2'- O-butanoyl)-β-D-mannopyranosyl]-D-erythritol（一般式４）、4-O-[(6',4',2'-tri-O-acetyl-,3'-di-O-alka(e)noyl)-β-D-mannopyranosyl]-D-erythritol（一般式５）を主成分とするＭＥＬ−Ａ混合物の画分、ＧＬ−Ｂは、4-O-[(6',4',2'-tri-O-acetyl-,3'-di-O-alka(e)noyl)-β-D-mannopyranosyl]-D-erythritol（一般式５）が主成分のＭＥＬ-Ａ画分、ＧＬ−Ｃは4-O-[(6',4'-di-O-acetyl-,3'-O-alka(e)noyl-2'-O-caproyl)-β-D-mannopyranosyl]-D-erythritol（一般式３）、4-O-[(6',4'-di-O-acetyl-,3'-O-alka(e)noyl-2'- O-butanoyl)-β-D-mannopyranosyl]-D-erythritol（一般式４）を主成分とするＭＥＬ−Ａの混合物で あることが分かった。化３に一般式３を示す。化４に一般式４を示す。化５に一般式５を示す。
(MALDI-TOF / MS analysis of glycolipid components)
As a result of analyzing the molecular weight of each component (GL-A, GL-B, GL-C) obtained above by MALDI-TOF / MS, the molecular weight of the main component of GL-A is 704.8, 676.8. The molecular weight of the main component of GL-B was 648.1, and the molecular weight of the main component of GL-C was 704.1 and 676.1. FIG. 5 shows the molecular weight peak obtained by MALDI-TOF / MS. From these results, GL-A is 4-O-[(6 ′, 4′-di-O-acetyl-, 3′-O-alka (e) noyl-2′-O-caproyl) -β- D-mannopyranosyl] -D-erythritol (general formula 3), 4-O-[(6 ', 4'-di-O-acetyl-, 3'-O-alka (e) noyl-2'- O-butanoyl ) -β-D-mannopyranosyl] -D-erythritol (general formula 4), 4-O-[(6 ', 4', 2'-tri-O-acetyl-, 3'-di-O-alka (e ) noyl) -β-D-mannopyranosyl] -D-erythritol (general formula 5), a fraction of a MEL-A mixture, GL-B is 4-O-[(6 ′, 4 ′, 2 '-tri-O-acetyl-, 3'-di-O-alka (e) noyl) -β-D-mannopyranosyl] -D-erythritol (general formula 5) as a main component, MEL-A fraction, GL- C is 4-O-[(6 ′, 4′-di-O-acetyl-, 3′-O-alka (e) noyl-2′-O-caproyl) -β-D-mannopyranosyl] -D-erythritol (General formula 3), 4-O-[(6 ', 4'-di-O-acetyl-, 3'-O-alka (e) noyl-2'-O-butanoyl) -β-D-mannopyranosyl] It was found to be a mixture of MEL-A mainly composed of -D-erythritol (general formula 4). Formula 3 shows the general formula 3. Formula 4 shows the general formula 4. Formula 5 shows the general formula 5.
実施例３
（ＭＥＬの界面活性測定）
〈表面張力測定〉
実施例２において、精製した各成分（ＧＬ−Ａ，ＧＬ−Ｂ，ＧＬ−Ｃ）の表面張力低下能を調べた。種々の濃度の各成分の水溶液を作成し、その表面張力をウイルヘルミー方式の表面張力計（ＣＢＶＰ−Ａ３，協和界面科学製）を用いて測定した。
Example 3
(Measurement of MEL surface activity)
<Surface tension measurement>
In Example 2, the surface tension reducing ability of each purified component (GL-A, GL-B, GL-C) was examined. Aqueous solutions of various components at various concentrations were prepared, and the surface tension was measured using a Wilhelmy type surface tension meter (CBVP-A3, manufactured by Kyowa Interface Science).
各成分（ＧＬ−Ａ，ＧＬ−Ｂ，ＧＬ−Ｃ）について、上記の方法を用いて表面張力低下能を測定した。各濃度の各成分に対する表面張力は図６に示す値を示した。図６の曲線からＧＬ−Ａの臨界ミセル濃度（ｃｍｃ）は４．８６× １０−６ Ｍであり、その時の表面張力値（γｃｍｃ値）は２９．２ｍＮ／ｍであった。ＧＬ−Ｂの臨界ミセル濃度（ｃｍｃ）は１．３６× １０−６ Ｍであり、その時の表面張力値（γｃｍｃ値）は２９．３ｍＮ／ｍであった。ＧＬ−Ｃの臨界ミセル濃度（ｃｍｃ）は２．０８× １０−６ Ｍであり、その時の表面張力値（γｃｍｃ値）は２８．７ｍＮ／ｍであった。既存の合成界面活性剤とくらべて、十分小さいｃｍｃ値ならびにγｃｍｃ値を示したことから、本発明で生産されるＭＥＬは界面活性剤として十分な機能を発揮できることが推察できる。 About each component (GL-A, GL-B, GL-C), surface tension reduction ability was measured using said method. The surface tension for each component at each concentration showed the values shown in FIG. From the curve of FIG. 6, the critical micelle concentration (cmc) of GL-A was 4.86 × 10 −6 M, and the surface tension value (γcmc value) at that time was 29.2 mN / m. The critical micelle concentration (cmc) of GL-B was 1.36 × 10 −6 M, and the surface tension value (γcmc value) at that time was 29.3 mN / m. The critical micelle concentration (cmc) of GL-C was 2.08 × 10 −6 M, and the surface tension value (γcmc value) at that time was 28.7 mN / m. Compared with existing synthetic surfactants, they showed sufficiently small cmc values and γcmc values. Therefore, it can be inferred that the MEL produced in the present invention can exhibit a sufficient function as a surfactant.
実施例４
（ＭＥＬの自己集合体形成能）
実施例２において、精製した各成分（ＧＬ−Ａ，ＧＬ−Ｂ，ＧＬ−Ｃ）の自己集合体形成能を調べた。各成分をガラスプレート上に塗りつけ、カバーガラスをかぶせた後、ガラスプレートとカバーガラスの間から水を侵入させ、各成分と水との境界を光学顕微鏡および偏光顕微鏡で観察した。その結果を、図７に示した。比較対象として、従来型ＭＥＬ−Ａの結果も示した。図７によると、従来型ＭＥＬ-Ａの液晶形成パターンと、ＧＬ−Ａ，ＧＬ−Ｂ，ＧＬ−Ｃの液晶形成パターンは、全く異なっており、本発明で生産されるＭＥＬ−Ａは、従来型ＭＥＬ−Ａと自己集合体形成能が異なることが分かる。
Example 4
(MEL self-assembly ability)
In Example 2, the ability of the purified components (GL-A, GL-B, GL-C) to form self-assemblies was examined. Each component was applied onto a glass plate and covered with a cover glass, water was then allowed to enter between the glass plate and the cover glass, and the boundary between each component and water was observed with an optical microscope and a polarizing microscope. The results are shown in FIG. As a comparison object, the result of the conventional MEL-A is also shown. According to FIG. 7, the liquid crystal formation pattern of the conventional MEL-A and the liquid crystal formation patterns of GL-A, GL-B, and GL-C are completely different, and the MEL-A produced in the present invention is the conventional one. It can be seen that the self-assembly forming ability is different from that of the type MEL-A.
Claims (5)
（ａ）形態学的性質
(A) Morphological properties
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