JP2007209332A - Method for producing biosurfactant - Google Patents
Method for producing biosurfactant Download PDFInfo
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- JP2007209332A JP2007209332A JP2006339426A JP2006339426A JP2007209332A JP 2007209332 A JP2007209332 A JP 2007209332A JP 2006339426 A JP2006339426 A JP 2006339426A JP 2006339426 A JP2006339426 A JP 2006339426A JP 2007209332 A JP2007209332 A JP 2007209332A
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- mannosyl erythritol
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 59
- 239000003876 biosurfactant Substances 0.000 title description 12
- -1 mannosyl erythritol lipid Chemical class 0.000 claims abstract description 69
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- 239000008103 glucose Substances 0.000 claims abstract description 60
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 25
- 239000000194 fatty acid Substances 0.000 claims abstract description 25
- 229930195729 fatty acid Natural products 0.000 claims abstract description 25
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 19
- 241000893045 Pseudozyma Species 0.000 claims abstract description 9
- 239000000758 substrate Substances 0.000 claims abstract description 9
- 241001661345 Moesziomyces antarcticus Species 0.000 claims description 34
- 244000005700 microbiome Species 0.000 claims description 24
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 18
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 9
- 235000010344 sodium nitrate Nutrition 0.000 claims description 9
- 239000004317 sodium nitrate Substances 0.000 claims description 9
- UJEADPSEBDCWPS-SGJODSJKSA-N (2R,3R)-1-[(3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]butane-1,2,3,4-tetrol Chemical class C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)C([C@H](O)[C@H](O)CO)O UJEADPSEBDCWPS-SGJODSJKSA-N 0.000 claims description 7
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- HIWPGCMGAMJNRG-UHFFFAOYSA-N beta-sophorose Natural products OC1C(O)C(CO)OC(O)C1OC1C(O)C(O)C(O)C(CO)O1 HIWPGCMGAMJNRG-UHFFFAOYSA-N 0.000 description 3
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
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- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
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- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
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- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
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- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
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- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
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- FCBUKWWQSZQDDI-UHFFFAOYSA-N rhamnolipid Chemical compound CCCCCCCC(CC(O)=O)OC(=O)CC(CCCCCCC)OC1OC(C)C(O)C(O)C1OC1C(O)C(O)C(O)C(C)O1 FCBUKWWQSZQDDI-UHFFFAOYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、バイオサーファクタントの一種であるマンノシルエリスリトールリピッドをグルコースから生産する方法に関する。
The present invention relates to a method for producing mannosylerythritol lipid, which is a kind of biosurfactant, from glucose.
糖脂質は、脂質に１〜数十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、神経系及び免疫系の機能維持にも重要な役割を果たしていることなどが明らかにされつつある。また、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両親媒性物質は界面活性物質と呼ばれている。石油化学工業が隆盛となるまでは、レシチン、サポニン等の生体成分由来の界面活性剤（バイオサーファクタント）が利用されていた。近年、石油化学工業の発展により合成界面活性剤が開発され、その生産量が飛躍的に増加し、日常生活には無くてはならない物質となったが、この合成界面活性剤の使用量の拡大に伴って環境汚染が広がり、社会問題が生じている。このため、安全性が高く、環境に対する負荷を低減できる生分解性の高い界面活性物質の開発が望まれている。 Glycolipids are substances in which one to several tens of monosaccharides are bound to lipids, are involved in information transmission between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. In addition, glycolipids are amphiphilic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. It is called a substance. Until the petrochemical industry prospered, surfactants (biosurfactants) derived from biological components such as lecithin and saponin were used. Synthetic surfactants have been developed in recent years due to the development of the petrochemical industry, and their production has dramatically increased, making it an indispensable substance for daily life. As a result, environmental pollution has spread and social problems have arisen. For this reason, it is desired to develop a highly biodegradable surfactant that is highly safe and can reduce the burden on the environment.
従来より、微生物が生産する界面活性物質としては、糖脂質系、アシルペプタイド系、リン脂質系、脂肪酸系及び高分子系の界面活性物質の５つに分類されている。これらの中でも、糖脂質系の界面活性剤が最もよく研究されており、細菌及び酵母による多くの種類の界面活性物質が報告されている。 Conventionally, the surface active substances produced by microorganisms are classified into five types: surface active substances of glycolipid type, acyl peptide type, phospholipid type, fatty acid type and polymer type. Among these, glycolipid-based surfactants are the most studied, and many types of surfactants from bacteria and yeast have been reported.
前記細菌としては、Pseudomonas属によるラムノリピッド（非特許文献１及び２参照）とユスチラジン酸（非特許文献３参照）、Rhodococcus属によるトレハロースリピッド（非特許文献４参照） などが知られている。しかし、いずれも生産量は１５ｇ／Ｌ以下である。 As the bacterium, rhamnolipid (see Non-Patent Documents 1 and 2) and Pseudomonas genus, ustyrazine acid (see Non-Patent Document 3), trehalose lipid (see Non-Patent Document 4) and Rhodococcus genus, and the like are known. However, in all cases, the production amount is 15 g / L or less.
前記酵母としては、Candida属によるソホロースリピッドとマンノシルエリスリトールリピッド（特許 文献１参照）などが知られている。 As the yeast, a sophorose lipid and a mannosyl erythritol lipid (refer to Patent Document 1) by the genus Candida are known.
前記ソホロースリピッドについては、Candida bombicolaを用いてグルコースとオレイン酸の流 加培養法により２００時間で１８０ｇ／Ｌの効率的なソホロースリピッドの生産が可能であることが報告されている（非特許文献５参照）。 As for the sophorose lipid, it has been reported that an efficient sophorose lipid production of 180 g / L can be produced in 200 hours by the fed -batch culture method of glucose and oleic acid using Candida bombicola (non-patent document). Reference 5).
前記マンノシルエリスリトールリピッド（ＭＥＬ）については、Candida sp. B-7株を用いて５質量％の大豆油から５日間で３５ｇ／Ｌ（生産速度：０．３ｇ／Ｌ／ｈ、原料収率：７０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６及び７参照）。また、Candida antarctica T-34株を用いて８質量％の大豆油から８日間で３８ｇ／Ｌ（生産速度：０．２ｇ／Ｌ／ｈ、原料収率： ４８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８及び９参照）。同じく、 Candida antarctica T-34株を用いて６日間隔で計３回の逐次流加により２４日後に２５質量％ のピーナッツ油から１１０ｇ／Ｌ（生産速度：０．２ｇ／Ｌ／ｈ、原料収率：４４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１０参照）。 For the mannosyl erythritol lipid (MEL), 35 g / L (production rate: 0.3 g / L / h, raw material yield: 70) from 5% by weight soybean oil using Candida sp. Mass%) MEL is reported to be possible (see Non-Patent Documents 6 and 7). Candida antarctica T-34 strain can be used to produce 38g / L (production rate: 0.2g / L / h, raw material yield: 48% by mass) of 8% by weight soybean oil in 8 days. (See Non-Patent Documents 8 and 9). Similarly, 110 g / L (production rate: 0.2 g / L / h, raw material yield) from 25% by mass of peanut oil after 24 days by Caddida antarctica T-34 strain using a total of 3 batches fed every 6 days. It has been reported that production of MEL at a rate of 44 mass% is possible (see Non-Patent Document 10).
Candida sp. SY-16株を用いて１０質量％の植物油脂から回分培養法により２００時間で５０ ｇ／Ｌ（生産速度：０．２５ｇ／Ｌ／ｈ、原料収率：５０質量％）のＭＥＬの生産が可能であると共に、流加培養法により２０質量％の植物油から２００時間で１２０ｇ／Ｌ（生産速度：０．６ｇ／Ｌ／ｈ、原料収率：５０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１１参照）。 MEL of 50 g / L (production rate: 0.25 g / L / h, raw material yield: 50% by mass) in 200 hours from 10% by mass of vegetable oil using Candida sp. SY-16 strain by batch culture method Production of 120 g / L (production rate: 0.6 g / L / h, raw material yield: 50 mass%) from 20 mass% vegetable oil in 200 hours by fed-batch culture method. It has been reported that this is possible (see Non-Patent Document 11).
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１２参照）。 Production of 13.9 g / L (production rate: 0.57 g / L / h, raw material yield: 92% by mass) of MEL from 80% by mass of vegetable oil using Pseudozyma aphidis strain by fed-batch culture for 24 hours Has been reported to be possible (see Non-Patent Document 12).
また、醤油醸造工程において副産物として生産されるしょうゆ油（あぶら）を原料としてCandida antarctica T-34株を用いて７日間で８質量％のしょうゆ油から１７ｇ／Ｌ（生産速度：０．１ｇ／Ｌ ／ｈ、原料収率：２１質量％）のＭＥＬの生産が可能であることが提案されている（特許文献２参照）。 17g / L (production rate: 0.1g / L) from 8% by weight of soy sauce oil in 7 days using soy sauce oil (oil) produced as a by-product in the soy sauce brewing process using Candida antarctica T-34 strain / H, raw material yield: 21% by mass) has been proposed (see Patent Document 2).
一方、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るマンノシルエリスリトールリピッドなどのバイオサーファクタントを食品工業、医薬品工業、化学工業などで広く普及させていくためには、マンノシルエリスリトールリピッドの生産効率を高め、生産コストの低減を図ることが必要である。 On the other hand, in order to widely disseminate biosurfactants such as mannosyl erythritol lipid, which is said to have high biodegradability, low toxicity, environmental friendliness and novel physiological functions, in the food industry, pharmaceutical industry, chemical industry, etc. It is necessary to increase the production efficiency of mannosyl erythritol lipid and to reduce the production cost.
現在までのところ、マンノシルエリスリトールリピッドの生産方法において、生産条件の最適化による生産効率（生産速度、対原料収率、及び収率）の向上が試みられてきた。しかしながら、いずれの方法も、脂肪酸あるいは脂肪酸トリグリセリド、若しくはこれらを含有する原料を培地に添加して、マンノシルエリスリトールリピッド生産菌を培養するものであり、脂肪酸給源を培地に添加して、マンノシルエリスリトールリピッドを生産するものであった。 So far, in the production method of mannosyl erythritol lipid, attempts have been made to improve production efficiency (production rate, yield relative to raw material, and yield) by optimizing production conditions. However, in any of these methods, fatty acid or fatty acid triglyceride, or a raw material containing these is added to a medium to culture a mannosyl erythritol lipid-producing bacterium, and a fatty acid source is added to the medium to add mannosyl erythritol lipid. It was something to produce.
一方、近年物質生産において、石油プロセスからバイオプロセスへの変換が、環境負荷低減の観点から 重要視されてきている。特に、再生可能な重要バイオマス資源として木質系バイオマスが挙げられる。木質系バイオマスからはグルコースが生産され、様々な物質生産の原料としての利用が期待されている。したがって、グルコースからマンノシルエリスリトールリピッドが製造できれば、木質系バイオマスからグルコースの生産を経て、バイオサーファクタントとして有用なマンノシルエリスリトールリピッドを生産するための一貫したバイオプロセスの確立に貢献できる。 On the other hand, in material production, the conversion from petroleum process to bioprocess has been regarded as important from the viewpoint of reducing environmental impact. In particular, woody biomass can be cited as an important renewable biomass resource. Glucose is produced from woody biomass and is expected to be used as a raw material for various substance production. Therefore, if mannosyl erythritol lipid can be produced from glucose, it can contribute to establishment of a consistent bioprocess for producing mannosyl erythritol lipid useful as a biosurfactant through production of glucose from woody biomass.
本発明は、このような要望に応え、従来における前記諸問題を解決することを課題とするものである。
An object of the present invention is to meet these demands and to solve the above-mentioned problems.
前記課題を解決するため、本発明者は鋭意検討を重ねた結果、シュードザイマ属に属する特定のマンノシルエリスリトールリピッド生産菌が、脂肪酸又は脂肪酸トリグリセリド等の脂肪酸エステルを培地に含有させなくとも、グルコース含有培地（グルコース１２０ｇ／Ｌ、酵母エキス１ｇ／Ｌ、硝酸ナトリウム０．５ｇ／Ｌ、リン酸２水素カリウム０．４ｇ／Ｌ、及び硫酸マグネシウム０．２ｇ／Ｌ）で培養することにより、マンノシルエリスリトールリピドを生産し得ることを見いだし、本発明を完成させた。 In order to solve the above problems, the present inventor has made extensive studies, and as a result, the specific mannosylerythritol lipid-producing bacterium belonging to the genus Pseudozyma does not contain a fatty acid or a fatty acid ester such as a fatty acid triglyceride in the medium. By culturing with (glucose 120 g / L, yeast extract 1 g / L, sodium nitrate 0.5 g / L, potassium dihydrogen phosphate 0.4 g / L, and magnesium sulfate 0.2 g / L), mannosyl erythritol lipid is obtained. It was found that it could be produced, and the present invention was completed.
すなわち、本発明は、以下（１）〜（５）に示されるとおりである。
（１） シュードザイマ属に属し、グルコースを基質としてマンノシルエリスリトールリピッドを生成する能力を有する微生物を、グルコースを含有し、脂肪酸あるいは脂肪酸エステル不含培地で培養し、培養物からマンノシルエリスリトールリピッド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ッドの生産方法。
（２） 上記マンノシルエリスリトールリピッドを生産する能力を有する微生物が、シュードザイマ・アンタクチカ（Pseudozyma antarctica）に属する微生物であることを特徴とする、上記（１）に記載の生産方法。
（３） 上記マンノシルエリスリトールリピッドを生産する能力を有する微生物が、シュードザイマ・アンタクチカ（Pseudozyma antarctica）ＫＭ-34（ＦＥＲＭＰ−２０７３０）であることを特徴とする上記（１）または（２）に記載の生産方法。
（４） 培地の初発グルコース濃度が５〜２０重量％の範囲で培養を行うことを特徴とする、上記（１）〜（３）のいずれかに記載の生産方法。
（５） 培地組成及び培養条件が、以下に示されるものであることを特徴とする、上記（１）〜（４）のいずれかに記載の生産方法。
酵母エキス：０．１〜２ｇ／Ｌ
硝酸ナトリウム：０．１〜１ｇ／Ｌ
リン酸２水素カリウム：０．１〜２ｇ／Ｌ
硫酸マグネシウム：０．１〜１ｇ／Ｌ
グルコース：１００〜２００ｇ／Ｌ
培養温度：２６〜３２℃
That is, the present invention is as shown in the following (1) to (5).
(1) A microorganism that belongs to the genus Pseudozyma and has the ability to produce mannosylerythritol lipid using glucose as a substrate is cultured in a medium containing glucose and not containing fatty acid or fatty acid ester, and mannosylerythritol lipid is collected from the culture. A production method of mannosyl erythritol lipid, characterized by
(2) The production method according to (1) above, wherein the microorganism having the ability to produce the mannosyl erythritol lipid is a microorganism belonging to Pseudozyma antarctica .
(3) The production according to (1) or (2) above, wherein the microorganism capable of producing the mannosyl erythritol lipid is Pseudozyma antarctica KM-34 (FERMP-20730) Method.
(4) The production method according to any one of (1) to (3) above, wherein the culture is performed in an initial glucose concentration of 5 to 20% by weight.
(5) The production method according to any one of (1) to (4) above, wherein the medium composition and the culture conditions are as follows.
Yeast extract: 0.1-2 g / L
Sodium nitrate: 0.1-1 g / L
Potassium dihydrogen phosphate: 0.1-2 g / L
Magnesium sulfate: 0.1-1 g / L
Glucose: 100-200 g / L
Culture temperature: 26-32 ° C
本発明の使用微生物は、シュードザイマ属に属し、グルコースを基質としてマンノシルエリスリトールリピッドを生成する能力を有する微生物であり、当該微生物は、従来技術のように脂肪酸あるいは脂肪酸トリグリセリド等の脂肪酸エステル、あるいは植物油等の油脂類を培地に含有させなくとも、グルコースを栄養源として培地に含有させることにより、マンノシルエリスリトールリピッドを生産することができる点で画期的なものである。また、木質系バイオマスからのグルコース生産技術と組み合わせることにより、木質系バイオマスからマンノシルエリスリトールリピッドの一貫生産も可能となる。 The microorganism used in the present invention belongs to the genus Pseudozyma and is a microorganism having the ability to produce mannosylerythritol lipid using glucose as a substrate. The microorganism is a fatty acid ester such as fatty acid or fatty acid triglyceride, vegetable oil or the like as in the prior art. It is a revolutionary point that mannosyl erythritol lipid can be produced by adding glucose to the medium as a nutrient source without including the above oils and fats. In addition, by combining with technology for producing glucose from woody biomass, integrated production of mannosylerythritol lipid from woody biomass becomes possible.
一方、本発明においては、植物油等の油脂類を使用しないので、これら由来の油分が生成物に混入せず、マンノシルエリスリトールリピッドの分離精製においても有利である。 On the other hand, since oils and fats such as vegetable oils are not used in the present invention, oils derived from these oils are not mixed into the product, which is advantageous in the separation and purification of mannosyl erythritol lipids.
さらに、現段階では、本発明によるマンノシルエリスリトールリピッドの収量はそれほど高くはないものの、遺伝子工学的手法等による微生物の改良技術により生産性の向上が期待でき、上記本発明の油脂類を使用しない利点を併せて考察すれば、本発明は極めて有用なものである。 Furthermore, at the present stage, although the yield of mannosyl erythritol lipid according to the present invention is not so high, the improvement in productivity can be expected by the improvement technology of microorganisms by genetic engineering techniques, etc. In addition, the present invention is extremely useful.
したがって、本発明は、医薬等種々用途への使用が期待されるバイオサーファクタント生産技術の発展に大いに貢献するものである。
Therefore, the present invention greatly contributes to the development of biosurfactant production technology expected to be used for various uses such as medicine.
（目的生産物）
本発明の目的生産物であるマンノシルエリスリトールリピッド（ＭＥＬ）は、下記構造式（１）で表される化合物である。
Mannosyl erythritol lipid (MEL), which is a target product of the present invention, is a compound represented by the following structural formula (1).
前記構造式（１）において、Ｒ１〜Ｒ４は、互いに同一であっても異なっていてもよく、水素原子、アセチル基、又は炭素原子数１〜１４、好ましくは３〜１２の飽和若しくは不飽和の脂肪酸残基を表す。 In the structural formula (1), R 1 to R 4 may be the same or different from each other, and may be a hydrogen atom, an acetyl group, or a saturated or unsaturated group having 1 to 14 carbon atoms, preferably 3 to 12 carbon atoms. Represents a saturated fatty acid residue.
前記マンノシルエリスリトールリピッド（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の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ッ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（Ｘ． Ｚｈａｏ ｅｔ． ａｌ．， Ｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ，５９， ４８２−４８６（１９９９））、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ッ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ッド（ＭＥＬ）には生分解性があり、高い安全性を有すると考えられているものである。
The mannosyl erythritol lipid (MEL) has a high surface activity and is used as various surfactants or fine chemical catalysts. When mannosyl erythritol lipid is allowed to act on human acute promyelocytic leukemia cell line HL60, it has the effect of inducing cell differentiation of leukemia cells that differentiate the granule system. It has a physiological activity such as an action of inducing differentiation of a nervous system cell line that causes neurite outgrowth when applied. Furthermore, for the first time as a glycolipid produced by microorganisms, it becomes possible to induce apoptosis of melanoma cells (X. Zhao et. Al., Cancer Research, 59 , 482-486 (1999)) 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.
（使用微生物）
本発明の使用微生物は、シュードザイマ属に属し、グルコースからマンノシルエリスリトールリピッドを生産する能力を有する微生物であり、具体的には、シュードザイム・アンタクチカに属する微生物であり、特に、Pseudozyma antarcticaＫＭ-34株（ＦＥＲＭＰ−２０７３０）がグルコースを基質としてマンノシルエリスリトール リピッドを生産する効率が高い点で好ましい。 なお、Pseudozyma antarcticaＫＭ-34株の場合、最適生育温度は２８℃であり、 生育可能な温度範囲は２４〜３６℃である。
(Used microorganism)
The microorganism used in the present invention belongs to the genus Pseudozyma and is a microorganism having the ability to produce mannosylerythritol lipid from glucose. Specifically, it is a microorganism belonging to Pseudozyme antactica, particularly Pseudozyma antarctica KM-34 strain ( FERMP-20730) is preferred because of its high efficiency in producing mannosylerythritol lipids using glucose as a substrate. In the case of the Pseudozyma antarctica KM-34 strain, the optimum growth temperature is 28 ° C, and the temperature range in which it can grow is 24 to 36 ° C.
マンノシルエリスリトールリピッド生産菌としては、上記したようにシュードザイマ（Pseudozyma）属の微生物を含め、種々の微生物が知られているが、これらは全てマンノシルエリスリトールリピッド生産のために、脂肪酸、脂肪酸トリグリセリド等の脂肪酸エステル、あるいはこれらを含有する植物油等の脂肪酸給源を培地に含有させており、これらを含有させずに、グルコースを基質として、マンノシルエリスリトールリピッドを生産できる能力を有する微生物が存在することは全く知られておらず、このことは、本発明者の新知見にかかるものである。 As described above, various microorganisms are known as mannosyl erythritol lipid-producing bacteria , including microorganisms belonging to the genus Pseudozyma . These are all fatty acids such as fatty acids and fatty acid triglycerides for the production of mannosyl erythritol lipids. It is completely known that there are microorganisms having the ability to produce mannosyl erythritol lipids using glucose as a substrate without the inclusion of fatty acids such as esters or vegetable oils containing them in the medium. This is a new finding of the present inventors.
（マンノシルエリスリトールリピッドの生産）
本発明における使用微生物の培養においては、培地に、脂肪酸、脂肪酸トリグリセリド等の脂肪酸エステル類、あるいは植物油等の油脂類を含有させず、グルコースを微生物の炭素源及び基質として含有させるが、このほかの条件については、特に制限はなく、適宜選定することができる。例えば、酵母に対して一般に用いられる培地を使用でき、このような培地として、例えば、ＹＰＤ培地（イーストイクストラクト１０ｇ、 ポリペプトン２０ｇ、及びグルコース１００ｇ）を挙げることができる。
(Mannosyl erythritol lipid production)
In the culture of the microorganism used in the present invention, the medium does not contain fatty acid esters such as fatty acids, fatty acid triglycerides, or fats and oils such as vegetable oil, but glucose is contained as a carbon source and substrate of the microorganism. The conditions are not particularly limited and can be appropriately selected. For example, a medium generally used for yeast can be used, and examples of such a medium include YPD medium (yeast extract 10 g, polypeptone 20 g, and glucose 100 g).
ＭＥＬの生産量を増加させるためには原料であるグルコースの供給量を増加させることが好ましく、本発明者等は、培養開始時のグルコース濃度（初発グルコース濃度）を変化させて培養を行った結果、培養液中の初発グルコース濃度が少なくとも５％以上、好ましくは１０〜２０重量％の濃度の場合に、良好なＭＥＬの生産速度、生産量、及び収率が得られるという知見を得ている。これは、原料であるグルコースを酵母が基礎代謝のために炭素源として利用するため、基礎代謝に必要な炭素源の供給を十分行う必要があるためと考えられる。 In order to increase the production amount of MEL, it is preferable to increase the supply amount of glucose as a raw material, and the present inventors have performed the culture by changing the glucose concentration at the start of culture (initial glucose concentration). It has been found that when the initial glucose concentration in the culture solution is at least 5% or more, preferably 10 to 20% by weight, a good MEL production rate, production amount, and yield can be obtained. This is considered to be because it is necessary to sufficiently supply the carbon source necessary for basal metabolism because yeast, which is a raw material, uses glucose as a carbon source for basal metabolism.
本発明の使用微生物、特に前記Pseudozyma antarcticaＫＭ-34株を用いてグルコースからマンノシルエリスリトールリピッドを生産する場合の好適な培地組成及び培養条件は、以下のとおりである。
酵母エキスは、０．１〜２ｇ／Ｌが好ましく、１ｇ／Ｌが特に好ましい
硝酸ナトリウムは、０．１〜１ｇ／Ｌが好ましく、０．５ｇ／Ｌが特に好ましい。
リン酸２水素カリウムは、０．１〜２ｇ／Ｌが好ましく、０．４ｇ／Ｌが特に好ましい。
硫酸マグネシウムは、０．１〜１ｇ／Ｌが好ましく、０．２ｇ／Ｌが特に好ましい。
グルコースは、１００ｇ／Ｌ以上が好ましく、１００ｇ／Ｌが特に好ましい。
培養温度は、２６〜３２℃が好ましく、３０℃が特に好ましい
The preferred medium composition and culture conditions for producing mannosylerythritol lipid from glucose using the microorganisms of the present invention, particularly the aforementioned Pseudozyma antarctica KM-34 strain, are as follows.
The yeast extract is preferably 0.1 to 2 g / L, particularly preferably 1 g / L. Sodium nitrate is preferably 0.1 to 1 g / L, particularly preferably 0.5 g / L.
The potassium dihydrogen phosphate is preferably 0.1 to 2 g / L, particularly preferably 0.4 g / L.
Magnesium sulfate is preferably 0.1 to 1 g / L, particularly preferably 0.2 g / L.
The glucose is preferably 100 g / L or more, particularly preferably 100 g / L.
The culture temperature is preferably 26 to 32 ° C, particularly preferably 30 ° C.
本発明のマンノシルエリスリトールリピッドの製造方法は、特に制限はなく、目的に応じて適宜選定。
することができるが、例えば、種培養、本培養及びマンノシルエリスリトールリピッド生産培養の順にスケールアップしていくことが望ましい。
There is no restriction | limiting in particular in the manufacturing method of the mannosyl erythritol lipid of this invention, According to the objective, it selects suitably.
For example, it is desirable to scale up in order of seed culture, main culture, and mannosyl erythritol lipid production culture.
これらの培養における、培地、培養条件を例示すると以下のとおりである。
ａ）種培養；グルコース２０ｇ／Ｌ、酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液体培地４ｍＬが入った試験管に１白金耳接種し、３０℃で１日間振とう培養を行う。
ｂ）本培養；上記種培養と同じ組成の培地１００ｍＬの入った坂口フラスコに接種して、３０℃で２日間培養を行う。
ｃ）マンノシルエリスリトールリピッド生産培養；所定量のグルコースと酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液体培地１．４Ｌが入ったジャーファメンターに接種して、３０℃で８００ｒｐｍの撹拌速度で培養を行う。この培養においては、培養途中からグルコースを培養容器中に流下させて、培地中のグルコース濃度を５０〜２００ｇ／Ｌに保持することが望ましい。
Examples of culture media and culture conditions in these cultures are as follows.
a) Seed culture: 20 ml / L of glucose, 4 g of liquid medium having a composition of 1 g / L of yeast extract, 1 g / L of sodium nitrate, 0.5 g / L of potassium dihydrogen phosphate, and 0.5 g / L of magnesium sulfate A platinum loop is inoculated into a test tube and cultured with shaking at 30 ° C. for 1 day.
b) Main culture: Inoculate into a Sakaguchi flask containing 100 mL of the medium having the same composition as the seed culture and culture at 30 ° C. for 2 days.
c) Mannosylerythritol lipid production culture ; liquid medium 1 having a composition of a predetermined amount of glucose and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L 4. Inoculate a jar fermenter containing 4 L and culture at 30 ° C. with a stirring speed of 800 rpm. In this culturing, it is desirable that glucose is allowed to flow down into the culture vessel during the culturing to maintain the glucose concentration in the medium at 50 to 200 g / L.
生成したマンノシルエリスリトールリピドは、培養液中に不溶性物質として蓄積される。したがって、遠心分離することにより、マンノシルエリスリトールリピドを分離、採取する。 The produced mannosyl erythritol lipid is accumulated as an insoluble substance in the culture solution. Therefore, mannosyl erythritol lipid is separated and collected by centrifugation.
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.
（Pseudozyma antarcticaＫＭ-34の培養）
ａ）保存培地（麦芽エキス３ｇ／Ｌ、酵母エキス３ｇ／Ｌ、ペプトン５ｇ／Ｌグルコース１０ｇ／Ｌ、寒天３０ｇ／Ｌ）に保存しておいたPseudozyma antarcticaＫＭ-34（ＦＥＲＭＰ−２０７３０）株を、 グルコース２０ｇ／Ｌ、酵母エキス１ｇ／Ｌ、硝酸ナトリウムム１ｇ／Ｌ、リン酸２水素カリウム０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液体培地４ｍＬが入った試験管に１白金耳接種し、３０℃で振とう培養を行い、次いで、
ｂ）得られた菌体培養液を同じ組成の培地２０ｍＬの入った坂口フラスコに接種して、３０℃で振とう培養を行い、さらに
ｃ）これを所定量のグルコースと酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム ０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液体培地１．４Ｌが入ったジャーファメンターに接種して、３０℃で８００ｒｐｍの撹拌速度で培養を行った。
上記ａ）〜ｃ）の各培養により得られた菌体培養液を使用して、以下の（１）〜（５）に示される試験を行った。
( Pseudozyma antarctica KM-34 culture)
a) Pseudozyma antarctica KM-34 (FERMP-20730) strain stored in a storage medium (malt extract 3 g / L, yeast extract 3 g / L, peptone 5 g / L glucose 10 g / L, agar 30 g / L) 1 platinum in a test tube containing 4 mL of liquid medium with a composition of 20 g / L, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L Inoculate by ear, perform shaking culture at 30 ° C, then
b) The obtained bacterial cell culture solution is inoculated into a Sakaguchi flask containing 20 mL of the medium having the same composition and cultured at 30 ° C., and c) a predetermined amount of glucose and yeast extract 1 g / L, Inoculate a jar fermenter containing 1.4 L of liquid medium composed of 1 g / L sodium nitrate, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate, and stir at 30 ° C. at 800 rpm The culture was performed at a rate.
The tests shown in the following (1) to (5) were performed using the bacterial cell culture solutions obtained by the above cultures a) to c).
（試験手法、結果）
（１）バイオサーファクタントの生産の確認
上記ａ）の培養を１０日間行い、得られたPseudozyma antarcticaＫＭ-34株（ＦＥＲＭＰ−２０７３０）の菌体培養液を疎水性のフィルム上にスポットしてその表面張力の変化を観察した。また、コントロールとして培養前の培養液、及び比較例としてPseudozyma aphidis ATCC 23657株を同様にして培養して得られた培養液をそれぞれスポットして比較した。これらの結果を図１に示す。
なお、図１においては、Pseudozyma antarcticaＫＭ-34株（ＦＥＲＭＰ−２０７３０）及びPseudozyma aphidis ATCC23657株をそれぞれ大豆油含有培地で培養して得られた培養液を使用した結果も併せて示す。
これによれば、Pseudozyma antarcticaＫＭ-34株（ＦＥＲＭＰ−２０７３０）の菌体培養液のスポットにおいては、コントロールと比べ培地の表面張力が低下しており、このことはグルコースからバイオサーファクタントが生産可能であることを示している。一方、比較の対象としたPseudozyma aphidis ATCC23657株はグルコースからバイオサーファクタントを生産できないことがわかる。なお、両菌株とも大豆油で培養すると表面張力が低下しており、バイオサーファクタントを生産していることが確認できる。
(Test method, results)
(1) Confirmation of biosurfactant production The culture of a) above is carried out for 10 days, and the obtained Pseudozyma antarctica KM-34 strain (FERMP-20730) cell culture is spotted on a hydrophobic film and its surface tension is determined. The change of was observed. Moreover, the culture solution before culture | cultivation as a control, and the culture solution obtained by culture | cultivating Pseudozyma aphidis ATCC 23657 strain | stump | stock similarly as a comparative example were spotted and compared, respectively. These results are shown in FIG.
In addition, in FIG. 1, the result using the culture solution obtained by culture | cultivating Pseudozyma antarcticaKM-34 strain | stump | stock ( FERMP-20730) and Pseudozyma aphidis ATCC23657 strain | stump | stock respectively in a soybean oil containing medium is also shown.
According to this, the surface tension of the medium in the Pseudozyma antarctica KM-34 strain ( FERMP-20730) cell culture broth is lower than that in the control, which means that biosurfactant can be produced from glucose. It is shown that. On the other hand, it can be seen that the Pseudozyma aphidis ATCC23657 strain used as a comparison target cannot produce biosurfactant from glucose. In addition, when both strains are cultured with soybean oil, the surface tension is reduced, and it can be confirmed that biosurfactant is produced.
（２）Pseudozyma antarcticaＫＭ-34株のマンノシルエリスリトールリピッド（ＭＥＬ）生産能の確認
ａ）の培養を１日間行った後、ｂ）の培養を１０日間行った。１日毎に培養液を採取し、これを用いてPseudozyma antarcticaＫＭ-34株の培養時間に伴うバイオサーファクタントの生産性を薄層クロマトグラフィーで確認した。一方、比較例としてPseudozyma aphidis ATCC23657株を上記と同じ条件で培養し、同様にして薄層クロマトグラフィーを行った。また、これら両菌株を、それぞれ大豆油含有培地で培養し、得られた培養液を用いて同様にして薄層クロマトグラフィー（ＴＬＣ）を行った。
結果を図２に示す。なお、図中、左端はマンノシルエリスリトールリピッドの標準である。
これによれば、両株とも大豆油含有培地ではマンノシルエリスリトールリピッド（MEL）を生産しているが、代表的なMEL生産菌であるPseudozyma aphidis ATCC23657株は、大豆油不含のグルコース含有培地ではMEL を全く生産できなかった。他方、Pseudozyma antarcticaＫＭ-34株はグルコースからマンノシルエリスリトールリピッドを生産していることが明らかである。また、マンノシルエリスリトールリピッドの標準と対比して、Pseudozyma antarcticaＫＭ-34株が生産してい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リピッドであることがわかる。
(2) Confirmation of production ability of Pseudozyma antarctica KM-34 strain for mannosylerythritol lipid (MEL) a) was cultured for 1 day, and then b) was cultured for 10 days. A culture solution was collected every day, and the biosurfactant productivity associated with the culture time of Pseudozyma antarctica KM-34 strain was confirmed by thin layer chromatography. On the other hand, as a comparative example, Pseudozyma aphidis ATCC23657 strain was cultured under the same conditions as described above, and thin layer chromatography was performed in the same manner. In addition, both these strains were cultured in a soybean oil-containing medium, and thin layer chromatography (TLC) was performed in the same manner using the obtained culture solution.
The results are shown in FIG. In the figure, the left end is a standard of mannosyl erythritol lipid.
According to this, both strains produced mannosyl erythritol lipid (MEL) in soybean oil-containing medium, but Pseudozyma aphidis ATCC23657 , a representative MEL-producing bacterium, was produced in MEL in a soybean-free glucose-containing medium. Could not be produced at all. On the other hand, it is clear that the Pseudozyma antarctica KM-34 strain produces mannosylerythritol lipid from glucose. Moreover, it can be seen that the biosurfactant produced by the Pseudozyma antarctica KM-34 strain is mannosyl erythritol lipid, in contrast to the mannosyl erythritol lipid standard.
（３）マンノシルエリスリトールリピッド（ＭＥＬ）生産用培地で同リピッドの生産
Pseudozyma antarcticaＫＭ-34株を用い、ａ）の培養を１日間行った後、ｂ）の培養を１０日間行った。一方、大豆油含有培地を用いて同株を１０日間培養した。 これらの培養においては、一日毎に培養液を採取し、そのＭＥＬ生産量を高速液体クロマトグラフィーで検出した。結果を図３に示す。なお、図３は、培養液中の酢酸エチル可溶分を高速液体クロマトグラフィーで検出した結果であり、既知のマンノシルエリスリトールリピッドのものと一致する。図３によれば、大豆油含有培地と比較しその生産量は低いものの、グルコースを基質として培養時間の経過に伴ってMELを確実に生産していることが分かる。この実験条件でグルコースからのMEL生産量は２．６６ｇ／Ｌであった。
(3) Mannosyl erythritol lipid (MEL) production in the same medium
Using Pseudozyma antarctica KM-34 strain , a) was cultured for 1 day, and then b) was cultured for 10 days. On the other hand, the same strain was cultured for 10 days using soybean oil-containing medium. In these cultures, the culture solution was collected every day, and the production amount of MEL was detected by high performance liquid chromatography. The results are shown in FIG. FIG. 3 shows the result of detection of ethyl acetate-soluble components in the culture solution by high performance liquid chromatography, which is consistent with that of known mannosyl erythritol lipids. According to FIG. 3, although the production amount is low compared with a soybean oil containing medium, it turns out that MEL is reliably produced with progress of culture | cultivation time using glucose as a substrate. Under these experimental conditions, the production of MEL from glucose was 2.66 g / L.
（４）マンノシルエリスリトールリピッド（ＭＥＬ）生産に対するグルコース濃度の影響
ａ）に使用した培地においてグルコース濃度を種々変更し、Pseudozyma antarcticaＫＭ-34株を接種しａ）の培養を１０日間行った後、培養液を酢酸エチルで抽出し、該抽出液について高速液体クロマトグラフィーで分析してＭＥＬ生産量を測定した。結果を図４に示す。これによれば培地中のグルコース濃度が１００ｇ／Ｌのとき、ＭＥＬ生産量が最大であった（１０ｇ／Ｌ）。また、１００ｇ／Ｌ以上のグルコース添加においても低濃度の場合（５０ｇ／Ｌ）に比し、生産量の上昇がみられた。
(4) Effect of glucose concentration on mannosylerythritol lipid (MEL) production The glucose concentration was variously changed in the medium used in a), Pseudozyma antarctica KM-34 strain was inoculated, and a) was cultured for 10 days. Was extracted with ethyl acetate, and the extract was analyzed by high performance liquid chromatography to determine the amount of MEL production. The results are shown in FIG. According to this, when the glucose concentration in the medium was 100 g / L, the MEL production amount was the maximum (10 g / L). Moreover, even when glucose of 100 g / L or more was added, an increase in production amount was observed as compared to the case of low concentration (50 g / L).
（５）Pseudozyma antarcticaＫＭ-34株を用い、ａ）の培養を１０日間行った後、培養液を酢酸エチルで抽出し、該抽出液について薄層クロマトグラフィー（ＴＬＣ）を行った後、アンスロン試薬を噴霧し、スポットを検出した。一方、大豆油含有培地を用いる他は、同様にしてPseudozyma antarcticaＫＭ-34株を培養し、同様にスポットの検出を行った。結果を図５に示す。なお図５中、レーン１はＭＥＬ標準を示し、ＭＥＬ−Ａ，ＭＥＬ−Ｂ及びＭＥＬ−Ｃはそれぞれ順に一般式中（Ｒ１、２＝炭素原子数１〜１４の脂肪酸残基、Ｒ３、４=アセチル基）、同（Ｒ１、２＝炭素原子数１〜１４の脂肪酸残基、Ｒ３=水素原子、Ｒ４=アセチル基）及び同（Ｒ１、２＝炭素原子数１〜１４の脂肪酸残基、Ｒ３=アセチル基、Ｒ４=水素原子）で表される化合物を示す。
図５の結果によれば、レーン３，４（大豆油含有培地での培養したもの）では、青色と茶色に呈色するスポットが生じたが、レーン２（大豆油不含、グルコース含有培地で培養したものは）、青色に呈色したスポットのみが生じている。茶色の呈色は、植物油等の脂質成分を表し、青色の呈色は、糖骨格を有する糖脂質、すなわちＭＥＬを表す。この結果は、大豆油含有培地で培養したものは、糖脂質以外の油の混入を示し、他方、本願発明の大豆油不含グルコース含有培地で培養したものは他の脂質の混入がないことを示す。したがって、グルコースを基質として、ＭＥＬを生産すれば、その分離精製において有利である。
(5) After culturing a) for 10 days using Pseudozyma antarctica KM-34 strain , the culture solution was extracted with ethyl acetate, and the extract was subjected to thin layer chromatography (TLC). Sprayed and spot detected. On the other hand, except that the soybean oil-containing medium was used, the Pseudozyma antarctica KM-34 strain was cultured in the same manner, and spots were similarly detected. The results are shown in FIG. In FIG. 5, lane 1 shows the MEL standard, and MEL-A, MEL-B, and MEL-C are in the general formula (R 1 , 2 = fatty acid residue having 1 to 14 carbon atoms, R 3 , 4 = acetyl group), the same (R 1 , 2 = fatty acid residue having 1 to 14 carbon atoms, R 3 = hydrogen atom, R 4 = acetyl group) and the same (R 1 , 2 = carbon atom number 1 to 14) A fatty acid residue, R 3 = acetyl group, R 4 = hydrogen atom).
According to the results of FIG. 5, in lanes 3 and 4 (cultured in a soybean oil-containing medium), spots that were colored blue and brown were produced, but in lane 2 (soy oil-free and glucose-containing medium). In the case of the cultured one), only the spots colored blue are generated. The brown color represents a lipid component such as vegetable oil, and the blue color represents a glycolipid having a sugar skeleton, that is, MEL. This result shows that those cultured in soybean oil-containing medium showed contamination with oils other than glycolipids, while those cultured in soybean oil-free glucose-containing medium of the present invention were free from other lipids. Show. Therefore, producing MEL using glucose as a substrate is advantageous in the separation and purification.
（６）Pseudozyma antarctica JCM ３９４１株とPseudozyma antarctica JCM １０３１７株によるマンノシルエリスリトールリピッド（ＭＥＬ）の生産
Pseudozyma antarctica JCM ３９４１株とPseudozyma antarctica JCM １０３１７株を用い、ａ）の培養を１０日間行った後、培養液を酢酸エチルで抽出し、該抽出液について薄層クロマトグラフィー（ＴＬＣ）を行った後、アンスロン試薬を噴霧し、スポットを検出した。一方、大豆油含有培地を用いる他は、同様にしてPseudozyma antarctica JCM ３９４１株（レーンA）とPseudozyma antarctica JCM １０３１７株（レーンB）を培養し、同様にスポットの検出を行った。結果を図６に示す。なお図５中、レーンSはＭＥＬ標準を示す。
図６の結果によれば、大豆油含有培地での培養したもの（右図、４％大豆油）では、青色と茶色に呈色するスポットが生じ、大豆油不含、グルコース含有培地（左図、１２％グルコース）で培養したものは、青色に呈色したスポットのみが生じている。この結果は、この二種類の株を用いて、Pseudozyma antarcticaＫＭ-34株と同様に、大豆油不含グルコース含有培地でＭＥＬを生産可能であることを示すと共に、生産物に油が混入しないことを示している。
(6) Mannosylerythritol lipid (MEL) production by Pseudozyma antarctica JCM3941 and Pseudozyma antarctica JCM10317
Pseudozyma antarctica JCM3941 strain and Pseudozyma antarctica JCM10317 strain were used and a) was cultured for 10 days. The culture was extracted with ethyl acetate, and the extract was subjected to thin layer chromatography (TLC). Anthrone reagent was sprayed to detect spots. On the other hand, except that a soybean oil-containing medium was used, Pseudozyma antarctica JCM3941 strain (lane A) and Pseudozyma antarctica JCM10317 strain (lane B) were cultured in the same manner, and spots were similarly detected. The results are shown in FIG. In FIG. 5, lane S indicates the MEL standard.
According to the results shown in FIG. 6, spots cultured in a soybean oil-containing medium (right figure, 4% soybean oil) produce blue and brown spots, soy oil-free, glucose-containing medium (left figure). , 12% glucose) has only blue spots. This result shows that MEL can be produced on a soybean-free glucose-containing medium using these two strains, as well as Pseudozyma antarctica KM-34 strain, and that the product does not contain oil. Show.
（7）ジャーファメンターを用いたマンノシルエリスリトールリピッド（ＭＥＬ）の生産
Pseudozyma antarcticaＫＭ-34株を用い、ａ）の培養を１日間行った後、ｂ）の培養を１０日間行い、さらに、ｃ）の培養を、グルコース１００ｇ／Ｌ、酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム ０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液体培地１．４Ｌが入ったジャーファメンターに接種して、３０℃で８００ｒｐｍの撹拌速度で、1週間培養を行った。1週間培養後、グルコース１００ｇ／Ｌを流下して、さらに、３０℃で８００ｒｐｍの撹拌速度で、1週間培養した。その後、培養液を酢酸エチルで抽出し、該抽出液をエバポレーターに供し、該抽出液中のMEL量を計測した。その結果、ジャーファメンターを用いて、６ｇのMELを生産できた。
(7) Mannosyl erythritol lipid (MEL) production using jar fermenters
Using Pseudozyma antarctica KM-34 , a) was cultured for 1 day, b) was cultured for 10 days, and c) was further cultured with glucose 100 g / L, yeast extract 1 g / L, sodium nitrate 1 g. / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L in a jar fermenter containing 1.4 L of liquid medium, and at a stirring speed of 800 rpm at 30 ° C. Culture was performed for 1 week. After culturing for 1 week, glucose 100 g / L was flowed down, and further cultured at 30 ° C. at a stirring speed of 800 rpm for 1 week. Thereafter, the culture broth was extracted with ethyl acetate, the extract was used in an evaporator, and the amount of MEL in the extract was measured. As a result, 6 g of MEL could be produced using a jar fermenter.
Claims (5)
A microorganism that belongs to the genus Pseudozyma and has the ability to produce mannosyl erythritol lipids using glucose as a substrate, is cultured in a medium containing glucose and is free of fatty acids and fatty acid esters, and mannosyl erythritol lipids are collected from the culture. The production method of mannosyl erythritol lipid.
The production method according to claim 1, wherein the microorganism capable of producing the mannosylerythritol lipid is a microorganism belonging to Pseudozyma antarctica .
The production method according to claim 1 or 2, wherein the microorganism capable of producing the mannosyl erythritol lipid is Pseudozyma antarctica KM-34 (FERMP-20730).
The production method according to any one of claims 1 to 3, wherein the culture is performed in an initial glucose concentration of 5 to 20 wt% in the medium.
酵母エキス：０．１〜２ｇ／Ｌ
硝酸ナトリウム：０．１〜１ｇ／Ｌ
リン酸２水素カリウム：０．１〜２ｇ／Ｌ
硫酸マグネシウム：０．１〜１ｇ／Ｌ
グルコース：１００〜２００ｇ／Ｌ
培養温度：２６〜３２℃ The production method according to any one of claims 1 to 4, wherein the medium composition and the culture conditions are as follows.
Yeast extract: 0.1-2 g / L
Sodium nitrate: 0.1-1 g / L
Potassium dihydrogen phosphate: 0.1-2 g / L
Magnesium sulfate: 0.1-1 g / L
Glucose: 100-200 g / L
Culture temperature: 26-32 ° C
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