JP2008054580A - Deferriferrichrysin highly productive variant, liquid medium for producing siderophore and method for producing siderophore - Google Patents
Deferriferrichrysin highly productive variant, liquid medium for producing siderophore and method for producing siderophore Download PDFInfo
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- JP2008054580A JP2008054580A JP2006235307A JP2006235307A JP2008054580A JP 2008054580 A JP2008054580 A JP 2008054580A JP 2006235307 A JP2006235307 A JP 2006235307A JP 2006235307 A JP2006235307 A JP 2006235307A JP 2008054580 A JP2008054580 A JP 2008054580A
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- medium
- siderophore
- aspergillus
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- aspergillus oryzae
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Abstract
Description
本発明は、アスペルギルス・オリゼのデフェリフェリクリシン高生産変異株、及びアスペルギルス属微生物を用いてシデロフォアを生産するのに適した液体培地、及びアスペルギルス属微生物を用いてシデロフォアを製造する方法に関する。 The present invention relates to a high-producing mutant of Aspergillus oryzae, a liquid culture medium suitable for producing siderophores using Aspergillus microorganisms, and a method for producing siderophores using Aspergillus microorganisms.
鉄イオンは一部の乳酸菌を除く、殆ど全ての生物にとって必須の原子である。鉄は、生体内ではFe(II)やFe(III)の形態で利用され、主に酸化還元に関与する酵素群の補欠因子として機能する。しかしながら、一般的に鉄は自然界において鉄鉱石として存在し、可溶化されたイオン状態としてはほとんど存在しない。さらに鉄鉱石から微生物の働きにより可溶化された鉄イオンも、すぐに不溶性の酸化物や水酸化物となるため、生物が利用できる鉄イオンはきわめて微量である。 Iron ions are essential atoms for almost all living organisms except some lactic acid bacteria. Iron is used in the form of Fe (II) or Fe (III) in vivo, and functions as a prosthetic factor for a group of enzymes mainly involved in redox. However, in general, iron exists as iron ore in nature and hardly exists as a solubilized ionic state. Furthermore, since iron ions solubilized from iron ore by the action of microorganisms immediately become insoluble oxides and hydroxides, the amount of iron ions that can be used by organisms is extremely small.
細菌や放線菌、真核微生物はこのような微量な鉄イオンを効率的に獲得するために、シデロフォアと呼ばれる分子量1500以下の低分子の鉄イオンキレート物質を生産する。このシデロフォアに鉄イオンをキレートさせることにより、貴重な鉄イオンの不溶化を防ぎ、鉄イオンを優先的に利用することを図っている。また鉄イオンは生物にとって必須のイオンであるが、過剰に存在すると遊離のラジカルの発生を促し、逆に生体に危害を加える。シデロフォアは鉄イオンの獲得と同時に、このような鉄イオンの無害化にも大きく寄与している。シデロフォアは非キレート状態では無色であるが、鉄イオンをキレートすると、赤色を示し、可視光の吸収を示すことが知られている。 Bacteria, actinomycetes, and eukaryotic microorganisms produce a low-molecular iron ion chelate substance having a molecular weight of 1500 or less called a siderophore in order to efficiently acquire such a small amount of iron ions. By chelating iron ions with this siderophore, insolubilization of valuable iron ions is prevented, and iron ions are used preferentially. Iron ions are essential for living organisms, but if they are present in excess, they promote the generation of free radicals, which in turn harm the living organisms. Siderophores contribute greatly to the detoxification of iron ions as well as the acquisition of iron ions. It is known that siderophores are colorless in an unchelated state, but when chelated with iron ions, they show a red color and absorb visible light.
現在までに様々なシデロフォアが同定されているが、糸状菌が生産するシデロフォアはhydroxamates familyと呼ばれ、一般に構成アミノ酸誘導体としてN−ヒドロキシオルニチンを含む。研究用モデル糸状菌、工業用微生物、病原性糸状菌として幅広く研究が進められているアスペルギルス属糸状菌はhydroxamates familyの中でもフェリクローム類とフザリニン類と呼ばれるシデロフォアを生産する。前者は、N−ヒドロキシオルニチンのトリペプチドにグリシン、セリン、アラニンが環状ペプチドを形成している。一方、後者では一部のN−ヒドロキシオルニチンのN位が無水メバロン酸によってアシル化されている特徴を有する。糸状菌はこのような多種多様なシデロフォアを生産することにより、鉄イオンを優先的に獲得し、自然界での生存競争に活用しているものと考えられる。 Various siderophores have been identified to date, but the siderophores produced by filamentous fungi are called the hydroxamates family and generally contain N-hydroxyornithine as a constituent amino acid derivative. Aspergillus fungi, which are widely studied as research model filamentous fungi, industrial microorganisms, and pathogenic fungi, produce siderophores called ferrichromes and fusarinins in the hydroxamates family. In the former, glycine, serine and alanine form a cyclic peptide on a tripeptide of N-hydroxyornithine. On the other hand, the latter is characterized in that the N-position of some N-hydroxyornithine is acylated with mevalonic anhydride. By producing such a wide variety of siderophores, filamentous fungi preferentially acquire iron ions and use them for survival competition in nature.
ここで、清酒醸造では、アスペルギルス・オリゼを蒸米上に生育させて「麹」を作成し、清酒醸造の原料として利用している。この麹造りにおいてアスペルギルス・オリゼが大量のシデロフォア(主にデフェリフェリクリシン)を生産し、これが酒造用水の鉄イオンをキレートすることでフェリクリシンを形成し、清酒が着色することが知られている。従って、清酒醸造においては、シデロフォアであるフェリクローム類が、品質劣化の原因であり、できるだけフェリクローム類を生産しない菌株の育種が進められてきた。 Here, in sake brewing, Aspergillus oryzae is grown on steamed rice to make “mochi” and used as a raw material for sake brewing. It is known that Aspergillus oryzae produces a large amount of siderophore (mainly deferriferriclysin) in this koji making, and this forms ferriclicin by chelating iron ions in brewing water, and sake is known to be colored. . Therefore, in sake brewing, ferrichromes, which are siderophores, cause quality degradation, and breeding of strains that do not produce ferrichromes as much as possible has been promoted.
一方、シデロフォアは、鉄をキレートする作用により医薬品として使用できる可能性があり、シデロフォアを高生産する方法の開発が求められている。 On the other hand, siderophores may be used as pharmaceuticals by the action of chelating iron, and development of a method for producing siderophores with high production is required.
本発明は、デフェリフェリクリシンを高生産するアスペルギルス・オリゼ変異株を提供することを第1の課題とする。 It is a first object of the present invention to provide an Aspergillus oryzae mutant strain that highly produces deferifericricin.
また本発明は、アスペルギルス属微生物を用いてシデロフォアを効率よく生産することができる方法、及びこの方法に適した培地を提供することを第2の課題とする。 Moreover, this invention makes it a 2nd subject to provide the method which can produce a siderophore efficiently using an Aspergillus microorganism, and the culture medium suitable for this method.
上記課題を解決するために本発明者らは研究を重ね、以下の知見を得た。
(i) アスペルギルス・オリゼO−1013株(FERM P−16528)にN−メチル−N’−ニトロ−N−ニトロソグアニジンによる変異処理を施すことにより得た変異株3129−7株は、デフェリフェリクリシン生産量が親株の3倍以上になっていた。
(ii) 一般に、真菌類の培養には炭素源としてグルコースを含む培地が使用されるが、アスペルギルス属微生物を培養してシデロフォアを生産するに当たり、炭素源としてグリセロールを含む培地を用いることにより生産量が向上する。
(iii) アスペルギルス属微生物を培養してシデロフォアを生産するに当たり、増粘多糖類を含む液体培地を用いることにより、生産量が向上する。
In order to solve the above-mentioned problems, the present inventors have conducted research and obtained the following knowledge.
(i) Mutant strain 3129-7 obtained by subjecting Aspergillus oryzae O-1013 strain (FERM P-16528) to mutation treatment with N-methyl-N′-nitro-N-nitrosoguanidine is Chrysin production was more than three times the parent strain.
(ii) In general, a medium containing glucose as a carbon source is used for culturing fungi, but in producing a siderophore by culturing an Aspergillus microorganism, the production amount is obtained by using a medium containing glycerol as a carbon source. Will improve.
(iii) In producing a siderophore by culturing Aspergillus microorganisms, the production amount is improved by using a liquid medium containing a thickening polysaccharide.
本発明は上記知見に基づき完成されたものであり、下記の変異株などを提供する。 The present invention has been completed based on the above findings, and provides the following mutant strains.
項1. アスペルギルス・オリゼの変異処理により得られ、親株に比べてデフェリフェリクリシン生産量が3倍以上になったデフェリフェリクリシン高生産変異株。 Item 1. A highly deferifericin-producing mutant strain obtained by mutation treatment of Aspergillus oryzae, and the production amount of deferlifericin is more than three times that of the parent strain.
項2. アスペルギルス・オリゼの変異処理により得られ、鉄を含まずグルコースを2%(w/v)含むツァペックドックス培地に胞子を106個/ml接種し30℃で7日間振盪培養した場合に、培養上清中に100ppm以上のデフェリフェリクリシンを生産する、デフェリフェリクリシン高生産変異株。 Item 2. Obtained by mutagenesis of Aspergillus oryzae, 2% glucose free of iron when (w / v) with shaking Czapek-Dox medium spores 10 6 cells / ml inoculated 7 days at 30 ° C. cultures containing culture A deferifericrin high-producing mutant strain that produces 100 ppm or more of deferifericin in the supernatant.
項3. アスペルギルス・オリゼ(Aspergillus oryzae)のデフェリフェリクリシン高生産変異株3129−7株(FERM P-20961)。 Item 3. Aspergillus oryzae deferlifericin high production mutant strain 3129-7 (FERM P-20961).
項4. グリセロールを含む、アスペルギルス属微生物の培養によりシデロフォアを生産するための液体培地。 Item 4. A liquid medium for producing siderophores by culturing Aspergillus microorganisms, which contains glycerol.
項5. グリセロール濃度が3%(v/v)以下である項4に記載の培地。 Item 5. Item 5. The medium according to Item 4, wherein the glycerol concentration is 3% (v / v) or less.
項6. 増粘多糖類を含む項4又は5に記載の培地。 Item 6. Item 6. The medium according to Item 4 or 5, comprising a thickening polysaccharide.
項7. 培地の粘度が3〜30mPa・sである項6に記載の培地。 Item 7. Item 7. The medium according to Item 6, wherein the medium has a viscosity of 3 to 30 mPa · s.
項8. 増粘多糖類を含む、アスペルギルス属微生物の培養によりシデロフォアを生産するための液体培地。 Item 8. A liquid medium for producing a siderophore by culturing an Aspergillus microorganism comprising a thickening polysaccharide.
項9. 粘度が3〜30mPa・sである項8に記載の培地。 Item 9. Item 9. The medium according to Item 8, wherein the viscosity is 3 to 30 mPa · s.
項10. タンパク質分解物を含むものである項4〜9のいずれかに記載の培地。 Item 10. Item 10. The medium according to any one of Items 4 to 9, which comprises a protein degradation product.
項11. 穀類の醸造により得られるもろみまたは粕のプロテアーゼ分解物を含むものである項4〜9のいずれかに記載の培地。 Item 11. Item 10. The medium according to any one of Items 4 to 9, which comprises a mash or koji protease degradation product obtained by brewing cereals.
項12. 項4〜11のいずれかに記載の培地を用いて、アスペルギルス属微生物を培養する工程と、培養物からシデロフォアを回収する工程とを含むシデロフォアの製造方法。 Item 12. Item 12. A method for producing a siderophore comprising a step of culturing an Aspergillus microorganism using the medium according to any one of items 4 to 11, and a step of recovering a siderophore from the culture.
項13. アスペルギルス属微生物が、アスペルギルス・オリゼであり、シデロフォアがデフェリフェリクリシンである項12に記載の方法。 Item 13. Item 13. The method according to Item 12, wherein the Aspergillus microorganism is Aspergillus oryzae and the siderophore is deferifericlysin.
項14. アスペルギルス・オリゼが、アスペルギルス・オリゼのデフェリフェリクリシン高生産変異株3129−7株(FERM P-20961)である項13に記載の方法。 Item 14. Item 14. The method according to Item 13, wherein the Aspergillus oryzae is a highly fertile deferifericin mutant strain 3129-7 (FERM P-20961) of Aspergillus oryzae.
本発明によれば、アスペルギルス・オリゼにN−メチル−N’−ニトロ−N−ニトロソグアニジン処理を施すことにより、デフェリフェリクリシン生産量が親株の3倍以上になった変異株3129−7株が得られた。 According to the present invention, mutant Aspergillus oryzae was treated with N-methyl-N′-nitro-N-nitrosoguanidine, resulting in a mutant 3129-7 strain in which the production amount of deferifericrin was more than three times that of the parent strain. was gotten.
また、アスペルギルス属微生物を鉄欠乏培地で培養してシデロフォアを生産させるに当たり、炭素源としてグリセロールを用いることにより、さらには液体培地中に増粘多糖類を加えることにより、シデロフォアの生産量が向上する。 In addition, when cultivating Aspergillus microorganisms in an iron-deficient medium to produce siderophore, the production of siderophore is improved by using glycerol as a carbon source and further adding a thickening polysaccharide to the liquid medium. .
以下、本発明を詳細に説明する。
(I)デフェリフェリクリシン高生産変異株
本発明の第1のデフェリフェリクリシン高生産変異株は、アスペルギルス・オリゼの変異処理により得られ、親株に比べてデフェリフェリクリシン生産量が3倍以上になった変異株である。
Hereinafter, the present invention will be described in detail.
(I) Deferifericrin high production mutant The first deferifericricin high production mutant of the present invention is obtained by a mutation treatment of Aspergillus oryzae, and the production of deferlifericin is three times that of the parent strain. It is a mutant strain as described above.
変異処理方法は、特に限定されない。代表的には、N−メチル−N’−ニトロ−N−ニトロソグアニジン、エチルメタンスルホン酸(EMS)、メチルメタンスルホン酸、4−ニトロソキノン、亜硝酸、ブロモウラシルのような変異誘発剤による処理、紫外線照射、放射線(X線など)照射のような物理的変異処理などが挙げられる。この他、細胞融合やゲノムシャッフリングなどの原因で、染色体上に変異を有するようになった変異株も本発明の第1の変異株に含まれる。 The mutation treatment method is not particularly limited. Typically, treatment with mutagens such as N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethanesulfonic acid (EMS), methylmethanesulfonic acid, 4-nitrosoquinone, nitrous acid, bromouracil. And physical mutation treatment such as ultraviolet irradiation and radiation (X-ray irradiation). In addition, mutant strains that have mutations on the chromosome due to cell fusion or genome shuffling are also included in the first mutant strain of the present invention.
アスペルギルス・オリゼは、通常、菌体外にデフェリフェリクリシンを分泌生産する。従って、デフェリフェリクリシンの生産量は、液体培地を用いて同一条件で親株と変異株とをそれぞれ培養し、培養上清中のデフェリフェリクリシン濃度を比較する。デフェリフェリクリシン濃度は、例えば、鉄イオンをキレートさせた後に、フェリクリシンの特異的な吸収波長である430nmにおける吸光度を分光光度計で測定して、算出すればよい。デフェリフェリクリシン濃度測定時の培地の組成や培養条件は特に限定されないが、例えば、鉄を含まないツァペックドックス培地に2%(w/v)のグルコースを添加した培地を用いて30℃で7日間培養する条件が挙げられる。 Aspergillus oryzae normally secretes and produces deferifericrin outside the cells. Therefore, as for the production amount of deferifericlysin, the parent strain and the mutant strain are cultured under the same conditions using a liquid medium, and the concentration of deferlifericin is compared in the culture supernatant. For example, the deferriferricin concentration may be calculated by chelating iron ions and then measuring the absorbance at 430 nm, which is a specific absorption wavelength of ferricricin, with a spectrophotometer. The composition of the medium and the culture conditions at the time of measuring the deferifericin concentration are not particularly limited. For example, at 30 ° C. using a medium in which 2% (w / v) glucose is added to a zapek dox medium without iron. The conditions for culturing for 7 days can be mentioned.
本発明の第2のデフェリフェリクリシン高生産変異株は、アスペルギルス・オリゼの変異処理により得られ、鉄を含まずグルコースを2%(w/v)含むツァペックドックス培地に胞子を106個/ml接種し30℃で7日間振盪培養した場合に、培養上清中に100ppm以上のデフェリフェリクリシンを生産する変異株である。 The second deferlifericin high-producing mutant of the present invention is obtained by a mutation treatment of Aspergillus oryzae, and 10 6 spores are present in a Czapedoc medium containing iron and glucose 2% (w / v). Is a mutant that produces 100 ppm or more of deferlifericin in the culture supernatant when inoculated / ml and cultured with shaking at 30 ° C. for 7 days.
変異の原因、及びデフェリフェリクリシン濃度の測定方法は、第1の変異株について述べたとおりである。鉄を含まずグルコースを2%(w/v)含むツァペックドックス培地の組成は実施例に記載している。 The cause of the mutation and the method for measuring the concentration of deferlifericin are as described for the first mutant. The composition of the Czapek Dox medium without iron and 2% glucose (w / v) is described in the examples.
上記の第1及び第2の変異株の具体例として、アスペルギルス・オリゼ(Aspergillus oryzae)のデフェリフェリクリシン高生産変異株3129−7株(平成18年7月14日にFERM P-20961として独立行政法人産業技術総合研究所特許生物寄託センター(日本国茨城県つくば市東1-1-1 つくばセンター 中央第6)に寄託済み)が挙げられる。この株の取得方法、及び菌株の特徴は後述する。
(II)アスペルギルス属微生物の培養によりシデロフォアを生産するための液体培地
本発明のアスペルギルス属微生物の培養によりシデロフォアを生産するための第1の液体培地は、グリセロールを含む液体培地である。炭素源としてグリセロールを含むことにより、その他の汎用の低分子炭素源であるグルコースなどを含む培地に比べて、シデロフォアの生産量が高くなる。
As a specific example of the first and second mutants described above, the Aspergillus oryzae deferifericrisin high-producing mutant 3129-7 (independently named FERM P-20961 on July 14, 2006) National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (deposited at Tsukuba Center, Central 1-1-1 Tsukuba, Ibaraki, Japan). The method for obtaining this strain and the characteristics of the strain will be described later.
(II) Liquid medium for producing siderophore by culturing Aspergillus microorganism The first liquid medium for producing siderophore by culturing Aspergillus microorganism of the present invention is a liquid medium containing glycerol. By including glycerol as a carbon source, the production amount of siderophore is higher than in a medium containing glucose, which is another general-purpose low-molecular carbon source.
液体培地中のグリセロール濃度は、3%(v/v)以下が好ましく、1〜2.5%(v/v)程度がより好ましい。上記グリセロール濃度であれば、シデロフォアの生産を十分に向上させることができる。また、上記グリセロール濃度の範囲であれば菌の代謝系に影響を与えず、シデロフォアを高生産することができる。 The glycerol concentration in the liquid medium is preferably 3% (v / v) or less, and more preferably about 1 to 2.5% (v / v). With the above glycerol concentration, the production of siderophore can be sufficiently improved. In addition, within the above glycerol concentration range, siderophores can be produced at a high rate without affecting the bacterial metabolic system.
培地には炭素源として、グリセロールの他に、本発明の効果に影響を与えない範囲で、トウモロコシ、小麦、米、豆、馬鈴薯、甘藷、キャッサバなどに由来する各種デンプン、デキストリンなどの高分子炭素源;グルコース、シュクロース、フラクトース、マンニトール、ソルビトール、ガラクトース、マルトース、エリスリット、ラクトース、キシロース、イノシット、トレハロースなどの単糖やオリゴ糖(特に二糖類)のような低分子炭素源が含まれていてもよい。但し、グリセロール以外の低分子炭素源が含まれる場合は、それが先に資化されてグリセロール添加によるシデロフォア生産亢進の効果が阻害される可能性があるため、低分子炭素源はグリセロールだけであることが好ましい。 As a carbon source in the medium, in addition to glycerol, polymer carbon such as various starches and dextrins derived from corn, wheat, rice, beans, potatoes, sweet potatoes, cassava, etc., as long as the effects of the present invention are not affected. Sources: Low molecular carbon sources such as monosaccharides and oligosaccharides (especially disaccharides) such as glucose, sucrose, fructose, mannitol, sorbitol, galactose, maltose, erythritol, lactose, xylose, inosit, trehalose May be. However, if a low molecular carbon source other than glycerol is included, glycerol is the only low molecular carbon source because it may be first assimilated to inhibit the effect of increasing siderophore production by adding glycerol. It is preferable.
また、この液体培地は、さらに増粘多糖類を含むことが好ましい。本発明の液体培地では、グリセロールと増粘多糖類とを両方含むことにより、シデロフォアが一層大量に生産されるようになる。増粘多糖類の種類は特に限定されず、公知の増粘多糖類を用いることができる。このような公知の増粘多糖類として、カルボキシメチルセルロースナトリウム(CMC)、アルギン酸、キサンタンガム、アラビアガム、カラギナン、グアーガム、ペクチン、タマリンドシードガム、寒天などが挙げられる。これらは食品添加物としての使用が認められている物質であることから、食品や医薬品用途のシデロフォアの製造に好適である。増粘多糖類は1種を単独で、又は2種以上を組み合わせて使用できる。 The liquid medium preferably further contains a thickening polysaccharide. In the liquid medium of the present invention, siderophores are produced in a larger amount by containing both glycerol and thickening polysaccharide. The kind of thickening polysaccharide is not specifically limited, A well-known thickening polysaccharide can be used. Such known thickening polysaccharides include sodium carboxymethylcellulose (CMC), alginic acid, xanthan gum, gum arabic, carrageenan, guar gum, pectin, tamarind seed gum, agar and the like. Since these are substances that are approved for use as food additives, they are suitable for the production of siderophores for food and pharmaceutical applications. A thickening polysaccharide can be used individually by 1 type or in combination of 2 or more types.
増粘多糖類の含有量は、液体培地の粘度が好ましくは3mPa・s以上、より好ましくは5mPa・s以上、さらにより好ましくは6mPa・s以上、さらにより好ましくは9mPa・s以上となる量であればよい。また、好ましくは30mPa・s以下、より好ましくは15mPa・s以下になる量であればよい。上記増粘多糖類濃度であれば、シデロフォアの生産を十分に向上させることができる。また、上記増粘多糖類濃度の範囲であれば容易に攪拌でき、またシデロフォア生産に必要な溶存酸素を十分に確保することができる。本発明において、培地の粘度は東機産業株式会社製 TVB-10粘度計を用いて測定した値である。例えば、CMCを使用する場合、培地中に0.15〜0.45(w/v)%程度含まれることが好ましい。 The content of the thickening polysaccharide is such that the viscosity of the liquid medium is preferably 3 mPa · s or more, more preferably 5 mPa · s or more, even more preferably 6 mPa · s or more, and even more preferably 9 mPa · s or more. I just need it. Further, the amount is preferably 30 mPa · s or less, more preferably 15 mPa · s or less. If it is the said thickening polysaccharide density | concentration, the production of siderophore can fully be improved. Moreover, if it is the range of the said thickening polysaccharide density | concentration, it can stir easily and can fully ensure the dissolved oxygen required for siderophore production. In the present invention, the viscosity of the medium is a value measured using a TVB-10 viscometer manufactured by Toki Sangyo Co., Ltd. For example, when using CMC, it is preferable that about 0.15-0.45 (w / v)% is contained in a culture medium.
また、本発明のアスペルギルス属微生物の培養によりシデロフォアを生産するための第2の液体培地は、増粘多糖類を含む液体培地である。増粘多糖類の種類、及び含有量は、第1の液体培地について説明した通りである。本発明の第2の液体培地は、増粘多糖類を上記範囲で含むことにより、シデロフォアの生産量が高くなる。 The second liquid medium for producing siderophores by culturing the Aspergillus microorganism of the present invention is a liquid medium containing a thickening polysaccharide. The kind and content of the thickening polysaccharide are as described for the first liquid medium. When the second liquid medium of the present invention contains the thickening polysaccharide in the above range, the production amount of siderophore is increased.
第1及び第2の液体培地には、アスペルギルス属微生物の生育に必要なその他の成分が含まれていればよい。窒素源としては、硝酸塩、アンモニウム塩のような無機窒素化合物、アミノ酸、ペプチドのような有機窒素化合物などが含まれていれば良い。窒素源は1種を単独で、又は2種以上を組み合わせて使用することができる。 The first and second liquid media may contain other components necessary for the growth of Aspergillus microorganisms. The nitrogen source only needs to contain inorganic nitrogen compounds such as nitrates and ammonium salts, and organic nitrogen compounds such as amino acids and peptides. A nitrogen source can be used individually by 1 type or in combination of 2 or more types.
窒素源としては、有機窒素化合物を添加した時の方が微生物はよく生育し、結果としてシデロフォアの生産量が高くなるため、好ましい。但し、鉄はシデロフォアの生産を抑制することから、鉄含有量の少ない有機窒素化合物を使用することが望まれる。 As the nitrogen source, it is preferable to add an organic nitrogen compound because microorganisms grow better and, as a result, the production amount of siderophore increases. However, since iron suppresses the production of siderophore, it is desirable to use an organic nitrogen compound with a low iron content.
本発明の第1及び第2の液体培地には、タンパク質の加水分解物(特にカザミノ酸)が0.05〜5%(w/v)程度含まれることが好ましく、0.5〜2.5%(w/v)程度含まれることがより好ましい。また、タンパク質の加水分解物(特にカザミノ酸)が、それに由来する培地の鉄含有量が1ppm以下となるように添加することが好ましい。上記添加量の範囲であれば、十分に微生物が生育するとともに、タンパク質加水分解物に含まれることがある鉄によりシデロフォアの生産が抑制され難く、その結果、シデロフォアを効率よく生産することができる。 The first and second liquid media of the present invention preferably contain about 0.05 to 5% (w / v) of a protein hydrolyzate (particularly casamino acid), preferably 0.5 to 2.5. % (W / v) is more preferable. Further, it is preferable to add a protein hydrolyzate (particularly casamino acid) so that the iron content of the medium derived therefrom is 1 ppm or less. If it is the range of the said addition amount, while microorganisms will fully grow, production of a siderophore is hard to be suppressed with the iron which may be contained in a protein hydrolyzate, As a result, a siderophore can be produced efficiently.
本発明の第1及び第2の各液体培地には、窒素源として、穀類の醸造により得られるもろみまたは粕のプロテアーゼ分解物が含まれていても良い。 Each of the first and second liquid media of the present invention may contain a mash or koji protease degradation product obtained by brewing cereals as a nitrogen source.
穀類の種類は、特に限定されず、米、麦、そば、あわなど、醸造に用いられる穀類を制限なく使用することができる。例えば、米を用いた醸造により得られるもろみ・粕としては、清酒もろみ・粕(普通もろみ・粕、液化もろみ・粕)、焼酎もろみ・粕、味醂もろみ・粕などが挙げられるが、一般に精白度が高い清酒、味醂のもろみ・粕が好ましい。この他、麦を用いた焼酎やビールの醸造のもろみ・粕を用いることもできる。 The kind of cereal is not particularly limited, and cereals used for brewing such as rice, wheat, buckwheat, and wax can be used without limitation. For example, moromi and koji obtained from brewing using rice include sake moromi and koji (ordinary moromi and koji, liquefied moromi and koji), shochu mash and koji, miso mash and koji. High-quality sake, miso moromi and koji are preferred. In addition, shochu using wheat and beer brewing mash and koji can also be used.
本発明において、もろみは、穀類、水に麹若しくは酵母、又はその両方を混合して発酵させたもの、発酵中のもの、及び仕込んだ直後のものをいう。また、粕は、もろみから液体部(例えば酒類)を搾ったあとに残る固形物をいう。 In the present invention, moromi refers to cereals, water, koji or yeast, or a mixture of both, fermented, fermented, and immediately after charging. Moreover, koji refers to a solid material remaining after squeezing a liquid part (eg, alcoholic beverage) from moromi.
もろみまたは粕のプロテアーゼ分解物は、例えば、以下のようにして得ることができる。粕は、固体であるため、水やバッファーを添加して流動状にしたものにプロテアーゼを作用させればよい。また、もろみは液状または流動状であるため、そのままプロテアーゼを作用させればよい。プロテアーゼの作用温度、時間は、特に限定されず、そのプロテアーゼが活性を示す条件とすればよい。 The mash or koji protease degradation product can be obtained, for example, as follows. Since sputum is a solid, protease should be allowed to act on fluidized water and buffer. Moreover, since the moromi is liquid or fluid, it is sufficient to act on the protease as it is. The action temperature and time of the protease are not particularly limited, and may be any conditions under which the protease exhibits activity.
プロテアーゼの種類は特に限定されず、公知のプロテアーゼを制限なく使用できる。また、プロテイナーゼ(エンドペプチダーゼ)を用いてタンパク質をペプチドにまで分解してもよく、ペプチダーゼ(エキソペプチダーゼ)を用いてタンパク質をアミノ酸にまで分解してもよい。培養する微生物が利用できる程度にタンパク質が分解されていればよい。 The kind of protease is not particularly limited, and a known protease can be used without limitation. In addition, proteinase (endopeptidase) may be used to decompose the protein into peptides, and peptidase (exopeptidase) may be used to decompose the protein into amino acids. It is sufficient that the protein is degraded to such an extent that the microorganism to be cultured can be used.
粕含有液、またはもろみにプロテアーゼを作用させて得られる産物をそのまま培地に添加しても良い。このプロテアーゼ分解物の中には、タンパク質がプロテアーゼにより分解されて生じたアミノ酸やペプチド、水溶性タンパク質の未分解物、少量のアルコールのような水溶性成分と、水不溶性タンパク質、穀類残渣、麹菌や酵母のような水不溶性成分とが含まれる。アルコールは少量しか含まれないため、生物の生育を実質的に阻害しない。 A koji-containing solution or a product obtained by allowing protease to act on moromi may be added to the medium as it is. Among these protease degradation products, amino acids and peptides produced by the degradation of proteins by proteases, undegraded products of water-soluble proteins, water-soluble components such as small amounts of alcohol, water-insoluble proteins, cereal residues, koji molds, And water-insoluble components such as yeast. Since alcohol is contained only in a small amount, it does not substantially inhibit the growth of the organism.
また、粕含有液、またはもろみにプロテアーゼを作用させて得られる産物を濾過または遠心分離して水不溶性成分を除去し、残った水溶性成分を添加しても良い。目的生産物を単離し易くするため、水不溶性成分を除去しておくことが好ましい。 Alternatively, the water-insoluble component may be removed by filtering or centrifuging the product obtained by allowing protease to act on the koji-containing liquid or moromi, and the remaining water-soluble component may be added. In order to facilitate isolation of the target product, it is preferable to remove water-insoluble components.
粕、又はもろみのプロテアーゼ分解物をそのまま培地に添加する場合も、水不溶性成分を除去したものを培地に添加する場合も、さらに乾燥してから添加することができる。これにより、添加量を正確に制御することができる。 When adding the protease decomposition product of koji or moromi to the medium as it is, or when adding the water-insoluble component to the medium, it can be added after further drying. Thereby, the addition amount can be accurately controlled.
培地中のもろみまたは粕のプロテアーゼ分解物の含有量は、プロテアーゼ分解物の乾燥重量に換算して、0.05〜5w/v%程度であることが好ましく、0.2〜1.5w/v%程度であることがより好ましく、0.3〜0.7w/v%程度であることがさらにより好ましい。上記範囲であれば、生物の増殖を十分に促進するとともに、得られる培地中の上記プロテアーゼ分解物に由来する鉄含有量が多くなりすぎることがなく、その結果、シデロフォアを効率よく得ることができる。なお、プロテアーゼ分解物は、それに由来する培地の鉄含有量が1ppm以下となるように含まれることが好ましい。 The content of the mash or koji protease degradation product in the medium is preferably about 0.05 to 5 w / v in terms of the dry weight of the protease degradation product, 0.2 to 1.5 w / v % Is more preferable, and about 0.3 to 0.7 w / v% is even more preferable. Within the above range, the growth of the organism is sufficiently promoted, and the iron content derived from the protease degradation product in the obtained medium is not excessively increased. As a result, the siderophore can be obtained efficiently. . In addition, it is preferable that a protease degradation product is contained so that the iron content of the culture medium derived from it may be 1 ppm or less.
本発明の第1及び第2の液体培地には、アスペルギルス属微生物の生育に必要な、又は生育を促進する、P,K,S,Mg,Zn,Cuのようなミネラルや、ビオチン、チアミンのようなビタミンなどが含まれていても良い。 The first and second liquid media of the present invention include minerals such as P, K, S, Mg, Zn, Cu, biotin, and thiamine that are necessary for or promote the growth of Aspergillus microorganisms. Such vitamins may be included.
培地のpHは、アスペルギルス属微生物が生育する範囲であればよく、3〜6程度であればよい。 The pH of the medium may be in a range where Aspergillus microorganisms grow, and may be about 3 to 6.
本発明の第1及び第2の液体培地を用いれば、アスペルギルス属微生物のシデロフォアの分泌生産量が向上する。従って、本発明の本発明の第1及び第2の液体培地は、アスペルギルス属微生物によるシデロフォアの生産に好適に使用できる。本発明の液体培地は、特にフェリクローム生産に適しており、中でもアスペルギルス・オリゼによるデフェリフェリクリシンの生産に適している。
(III)シデロフォアの製造方法
本発明のシデロフォアの製造方法は、上記説明した本発明の液体培地を用いて、アスペルギルス属微生物を培養する工程と、培養物からシデロフォアを回収する工程とを含む方法である。アスペルギルス属微生物は、シデロフォアを生産する菌種であればよく、特に限定されないが、病原性を有さず、培養に使用し易い菌種として、アスペルギルス・オリゼ、アスペルギルス・アワモリ、アスペルギルス・カワチ、アスペルギルス・ニガー、アスペルギルス・ソヤ、アスペルギルス・ウサミなどが挙げられる。中でも、安全性や性質が良く知られている点でアスペルギルス・オリゼが好ましい。アスペルギルス・オリゼの生産する主なシデロフォアはデフェリフェリクリシンである。中でも、アスペルギルス・オリゼのデフェリフェリクリシン高生産変異株3129―7株(FERM P-20961)を用いることが好ましい。
If the 1st and 2nd liquid culture medium of this invention is used, the secretory production amount of the siderophore of Aspergillus microorganisms will improve. Therefore, the first and second liquid culture media of the present invention can be suitably used for the production of siderophores by Aspergillus microorganisms. The liquid medium of the present invention is particularly suitable for ferrichrome production, and particularly suitable for the production of deferlifericin by Aspergillus oryzae.
(III) Method for Producing Siderophore The method for producing siderophore of the present invention is a method comprising the steps of culturing Aspergillus microorganisms using the liquid medium of the present invention described above and the step of recovering siderophores from the culture. is there. The microorganism belonging to the genus Aspergillus is not particularly limited as long as it is a bacterial species that produces siderophore. -Niger, Aspergillus soya, Aspergillus usami and so on. Of these, Aspergillus oryzae is preferred because of its well-known safety and properties. The main siderophore produced by Aspergillus oryzae is deferlifericin. Among them, it is preferable to use the Aspergillus oryzae deferifericricin high production mutant strain 3129-7 (FERM P-20961).
培養方法は、回分、流加、連続などのいずれの方法であってもよい。培養温度、及び時間は菌種、菌株によって異なるが、概ね25〜35℃程度で3〜7日間程度培養すればよい。
実施例
以下、本発明を実施例を示してより詳細に説明するが、本発明はこれらに限定されるものではない。
The culture method may be any method such as batch, fed-batch or continuous. The culture temperature and time vary depending on the bacterial species and strain, but may be cultured at about 25 to 35 ° C. for about 3 to 7 days.
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
デフェリフェリクリシン(Dfcy)高生産変異株の取得
親株として、麹菌アスペルギルス・オリゼO−1013株(FERM P−16528)を用いた。アスペルギルス・オリゼO−1013株の胞子懸濁液(108個/ml)と飽和N−メチル−N’−ニトロ−N−ニトロソグアニジン(NTG)溶液を等量混合し、30℃で30分間反応させた。遠心分離後の胞子を水で2回洗浄しNTGを除去した後、変異処理した胞子群を回収した。変異処理した胞子をCz―dox,2%(w/v)Glucose,0.25%(v/v)TritonX―100, 2%(w/v) Agarプレートに塗布した後30℃で3日間培養した。
As an acquisition parent strain of a deferifericlysin (Dfcy) high production mutant strain , Aspergillus oryzae O-1013 strain (FERM P-16528) was used. Spore suspension of Aspergillus oryzae O-1013 strain (10 8 cells / ml) and saturated N-methyl-N′-nitro-N-nitrosoguanidine (NTG) solution are mixed in equal amounts and reacted at 30 ° C. for 30 minutes. I let you. The spore after centrifugation was washed twice with water to remove NTG, and then the spore group subjected to the mutation treatment was recovered. The spore after mutation treatment was applied to Cz-dox, 2% (w / v) Glucose, 0.25% (v / v) Triton X-100, 2% (w / v) Agar plate, and then cultured at 30 ° C. for 3 days. did.
生育した株のDfcy生産量を後述する方法で測定し、Dfcy生産量が最も多い株として、3129−7株を選抜した。この株は、平成18年7月14日に産業技術総合研究所特許生物寄託センターにFERM P−20961として寄託済みである。
<培養方法>
親株、及び変異処理した各株を保存スラントからポテトデキストロースアガープレート(ニッスイ社製)に塗布し、胞子を形成させた。各株の胞子を回収し、鉄制限ツァペックドックス培地 (0.2%(w/v)NaNO3,0.1%(w/v)K2HPO4,0.05%(w/v)KCl,0.05%(w/v)MgSO4・7H2O,pH6.0)に2%(w/v)グルコースを添加した液体培地40mlに、それぞれ106個/ml植菌し、30℃で7日間振盪培養した。
<Dfcy生産量の測定方法>
培養上清100μlに10μlの0.2Mクエン酸バッファー(pH4.0)、10μlの3000ppm FeCl3溶液を添加し、デフェリフェリクリシン(Dfcy)に鉄をキレートしフェリクリシン(Fcy)とした。
The Dfcy production amount of the grown strain was measured by the method described later, and 3129-7 strain was selected as the strain having the largest Dfcy production amount. This strain has been deposited as FERM P-20961 at the National Institute of Advanced Industrial Science and Technology on July 14, 2006.
<Culture method>
The parent strain and each mutant-treated strain were applied from a preserved slant to a potato dextrose agar plate (manufactured by Nissui) to form spores. Spores of each strain were collected, and iron-restricted zapek dox medium (0.2% (w / v) NaNO 3 , 0.1% (w / v) K 2 HPO 4 , 0.05% (w / v) 10 6 cells / ml were inoculated into 40 ml of a liquid medium in which 2% (w / v) glucose was added to KCl, 0.05% (w / v) MgSO 4 .7H 2 O, pH 6.0), 30 Cultured with shaking at 7 ° C. for 7 days.
<Dfcy production measurement method>
10 μl of 0.2 M citrate buffer (pH 4.0) and 10 μl of 3000 ppm FeCl 3 solution were added to 100 μl of the culture supernatant, and iron was chelated to deferriferricin (Dfcy) to obtain ferriclysin (Fcy).
これをHPLC(SHIMADZU社製;Prominence)を用い逆相クロマト分析に供し、Fcyに特異的な吸収波長である波長430nmを指標にFcyピークを同定した。そのピーク面積からFcyの定量を行った。定量したFcy量に、分子量比より0.93(744/800)を乗じてDfcy量を算出した。 This was subjected to reverse phase chromatographic analysis using HPLC (manufactured by SHIMADZU; Prominence), and the Fcy peak was identified using the wavelength 430 nm, which is an absorption wavelength specific for Fcy, as an index. Fcy was quantified from the peak area. The Dfcy amount was calculated by multiplying the quantified Fcy amount by 0.93 (744/800) from the molecular weight ratio.
親株、及び変異株の培養上清中のDfcy生産量を以下の表1に示す。 Table 1 below shows the amount of Dfcy produced in the culture supernatant of the parent strain and the mutant strain.
また、この変異株3129−7株は、親株O−1013株に比べて、グルコースプレート上での胞子形成能が低下しており、かつ麹酸生産能が低下している。 In addition, this mutant strain 3129-7 has a reduced spore-forming ability on the glucose plate and a reduced succinic acid-producing ability as compared to the parent strain O-1013.
デフェリフェリクリシンの生産
アスペルギルス・オリゼを実施例1と同じ条件で培養して、デフェリフェリクリシンを生産した。但し、培地としては、以下の4種類の液体培地を用いた。
培地1:
鉄制限ツァペックドックス培地に2.5%(w/v)グルコース(ナカライテスク社)を添加したもの
培地2:
鉄制限ツァペックドックス培地に2.5%(v/v)グリセロール(ナカライテスク社)を添加したもの
培地3:
鉄制限ツァペックドックス培地に2.5%(v/v)グリセロール(ナカライテスク社)、0.3%(w/v)CMC(ナカライテスク社)を添加したもの
培地4:
鉄制限ツァペックドックス培地に2.5%(v/v)グリセロール(ナカライテスク社)、0.3%(w/v)CMC(ナカライテスク社)、1.5%(w/v)カザミノ酸(Difco社)を添加したもの
培地5:
鉄制限ツァペックドックス培地に2.5%(v/v)グリセロール(ナカライテスク社)、0.3%(w/v)CMC(ナカライテスク社)、0.5%(w/v)液化粕プロテアーゼ分解物を添加したもの
培地3〜5の粘度を東機産業株式会社製 TVB-10粘度計を用いて測定したところ、9.0〜9.5mPa・sの範囲であった。
<液化粕分解物作成方法>
液化粕の凍結乾燥品10gに蒸留水50mlを加え均一にし、市販のプロテアーゼであるプロテアーゼSアマノ(アマノエンザイム社製)を0.2g加え50℃で3時間攪拌しながら反応させた。100℃で10分加熱し酵素を失活させた後遠心分離により不溶性の残渣を除去し、上清液を凍結乾燥し液化粕分解物の粉末を得た。
Production of deferlifericin Aspergillus oryzae was cultured under the same conditions as in Example 1 to produce deferifericlysin. However, the following four types of liquid media were used as the media.
Medium 1:
A medium in which 2.5% (w / v) glucose (Nacalai Tesque) is added to an iron-restricted zapek dox medium 2
Iron-restricted zapek dox medium supplemented with 2.5% (v / v) glycerol (Nacalai Tesque) Medium 3:
A medium in which 2.5% (v / v) glycerol (Nacalai Tesque) and 0.3% (w / v) CMC (Nacalai Tesque) are added to an iron-restricted zapek dox medium.
2.5% (v / v) glycerol (Nacalai Tesque), 0.3% (w / v) CMC (Nacalai Tesque), 1.5% (w / v) casamino acid Medium 5 supplemented with (Difco):
2.5% (v / v) glycerol (Nacalai Tesque), 0.3% (w / v) CMC (Nacalai Tesque), 0.5% (w / v) liquefied koji What added protease decomposition product When the viscosity of the culture media 3-5 was measured using the Toki Sangyo Co., Ltd. TVB-10 viscometer, it was the range of 9.0-9.5 mPa * s.
<Liquefied soot decomposition method>
Distilled water (50 ml) was added to 10 g of the lyophilized product of the liquefied koji, homogenized, and 0.2 g of protease S Amano (manufactured by Amano Enzyme), a commercially available protease, was added and reacted at 50 ° C. with stirring for 3 hours. After inactivating the enzyme by heating at 100 ° C. for 10 minutes, the insoluble residue was removed by centrifugation, and the supernatant was lyophilized to obtain a powder of a liquefied soot decomposition product.
変異株3129−7株を培地1〜5を用いて培養した培養上清中のDfcy生産量を以下の表2に示す。 Table 2 below shows the amount of Dfcy produced in the culture supernatant obtained by culturing the mutant strain 3129-7 using the media 1 to 5.
表2から分かるように、炭素源として汎用されているグルコースを用いるより、グリセロールを用いる方がDfcy生産量は高かった。また、増粘剤としてCMCを添加することによりDfcy生産量は一層高くなった。 As can be seen from Table 2, the production amount of Dfcy was higher when glycerol was used than when glucose which is widely used as a carbon source was used. Moreover, the production amount of Dfcy was further increased by adding CMC as a thickener.
また、窒素源として、硝酸ナトリウムに加えて、カザミノ酸、又は液化粕プロテアーゼ分解物を添加することにより、Dfcy生産量は著しく高くなった。特に液化粕酵素分解物を添加すると、カザミノ酸添加の場合と比較してDfcyの生産量が1.5倍以上に増加した。 Moreover, by adding casamino acid or liquefied koji protease degradation product in addition to sodium nitrate as a nitrogen source, the production amount of Dfcy was remarkably increased. In particular, when the liquefied koji enzyme degradation product was added, the production amount of Dfcy increased 1.5 times or more as compared with the case of adding casamino acid.
1.5%(w/v)カザミノ酸を添加した培地4中のカザミノ酸由来の鉄濃度を以下のようにして測定したところ、21ppbであった。また、0.5%(w/v)液化粕分解物を添加した培地5中の液化粕由来の鉄濃度を以下のようにして測定したところ、482ppbであった。いずれも1ppm以下の値であった。
<鉄濃度測定方法>
カザミノ酸、及び液化粕のプロテアーゼ分解物の、それぞれ0.1%(w/v)水溶液を調製し、原子吸光分析装置(Perkin Elmer社製、AAnalyst800)にてFe含量を測定したところ、それぞれ1.4ppb、及び96.4ppbであった。
The iron concentration derived from casamino acid in the medium 4 supplemented with 1.5% (w / v) casamino acid was measured as follows and found to be 21 ppb. Further, the iron concentration derived from the liquefied koji in the medium 5 to which 0.5% (w / v) liquefied koji decomposition product was added was measured as follows and found to be 482 ppb. In all cases, the value was 1 ppm or less.
<Iron concentration measurement method>
0.1% (w / v) aqueous solutions of casamino acid and liquefied koji protease degradation products were prepared, respectively, and the Fe content was measured by an atomic absorption spectrometer (Akinalst 800, manufactured by Perkin Elmer). And 96.4 ppb.
このことから、カザミノ酸を1.5%(w/v)添加した培地4中のカザミノ酸由来の鉄濃度は21ppbであり、液化粕分解物を0.5%(w/v)添加した培地5中の液化粕分解物由来の鉄濃度は482ppbであることが計算された。 Therefore, the concentration of iron derived from casamino acid in the medium 4 supplemented with 1.5% (w / v) casamino acid was 21 ppb, and the liquefaction in the medium 5 supplemented with 0.5% (w / v) liquefied koji decomposition product. It was calculated that the iron concentration derived from soot decomposition product was 482ppb.
増粘多糖類の種類・濃度の検討
2.5%(v/v)グリセロール、1.5%(w/v)カザミノ酸、50ppmクロラムフェニコール、及び増粘多糖類からなる液体培地に、アスペルギルス・オリゼ3129−7株の胞子を106個/ml植菌して、30℃で7日間振盪培養した。
Examination of types and concentrations of thickening polysaccharides In a liquid medium consisting of 2.5% (v / v) glycerol, 1.5% (w / v) casamino acid, 50 ppm chloramphenicol, and thickening polysaccharides, Aspergillus 10 6 cells / ml of the spores of Oryzae 3129-7 were inoculated and cultured at 30 ° C. with shaking for 7 days.
増粘多糖類は、CMC0.15%(w/v)、0.3%(w/v)、0.45%(w/v)、及びアルギン酸ナトリウム0.3%(w/v)、精製寒天末0.15%(w/v)とした。また、増粘多糖類を添加しないコントロール培地も使用した。 The thickening polysaccharides are CMC 0.15% (w / v), 0.3% (w / v), 0.45% (w / v), and sodium alginate 0.3% (w / v), purified agar powder 0.15% (w / v). Moreover, the control medium which does not add thickening polysaccharide was also used.
培養液中のDfcy濃度と、培地の粘度とを以下の表3に示す。 The Dfcy concentration in the culture solution and the viscosity of the medium are shown in Table 3 below.
表3から、添加する増粘剤の種類に関わらず、コントロールと比較して粘度の上昇によりDfcy生産性は向上した。これらのことから、増粘剤の添加により培地の粘度を上げることでDfcy生産性を向上させられることが分かる。 From Table 3, Dfcy productivity was improved by the increase in viscosity as compared with the control, regardless of the type of thickener added. From these facts, it can be seen that Dfcy productivity can be improved by increasing the viscosity of the medium by adding a thickener.
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