JP2008127409A - Polymer bonded with mannosylerythritol lipid, method for producing the same and analyzing instrument using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mannosylerythritol lipid-bonded polymer having a mannosylerythritol lipid (MEL) bonded to the polymer chain at the fatty acid terminal site through a covalent bond without changing the sugar skeleton of MEL having high physiological activity and biofunctionality, and to provide a method for the adsorption and separation of useful proteins such as antibody proteins by using the mannosylerythritol lipid-bonded polymer having high affinity, excellent hydrophilicity, dispersibility and corrosion resistance and exhibiting high physiological activities such as antibacterial property and antiviral activity as an affinity carrier. <P>SOLUTION: The mannosylerythritol lipid-bonded polymer contains a mannosylerythritol lipid compound bonded at the fatty acid terminal through a covalent bond to at least a part of the reactive functional groups of a polymer having reactive functional groups on the main chain or the side chain. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a polymer in which mannosyl erythritol lipid (hereinafter sometimes referred to as MEL), which is a microorganism-produced glycolipid, is introduced into a polymer chain by a covalent bond at a fatty acid terminal site, a method for producing the same, and a utility using the same The present invention relates to technology for recognizing, detecting, separating and purifying proteins and the like.

  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 amphiphilic substances that have both hydrophilic properties derived from sugar properties and lipophilic properties derived from lipid properties. Substances having such properties are called surfactant substances. It is.

  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 groups: glycolipids, acylpeptides, phospholipids, fatty acids, and polymer compounds. For active agents, the most studied and many types of substances produced by bacteria and yeast have been reported.

  These biosurfactants such as glycolipids 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.

  Mannosyl erythritol lipid (MEL) is a kind of glycolipid type biosurfactant, and was discovered from Ustilago nuda (Ustilago Nuda) and Shizonella melanogramma (see Non-Patent Documents 1 and 2). Subsequently, Candida genus yeast (see Patent Literature 1 and Non-Patent Literature 3), Candida antarctica (see Candida Andalucica) (see Non-Patent Literatures 4 and 5), and Kurtzmannomices (Kurzmanomyces) genus (mutants of itaconic acid production) It is reported that yeast such as Non-Patent Document 6) produces. At present, it is possible to produce 300 g / L or more by performing continuous culture and production for a long time.

  MEL generally has a characteristic structure having mannose and erythritol in a sugar skeleton that is a hydrophilic group and one or two fatty acid esters in a lipid domain that is a hydrophobic group. In addition to exhibiting excellent interface physical properties due to this unique structure, it has been reported to have various physiological functions (see Non-Patent Document 7).

  Among them, it has been confirmed that MEL has a specific interaction with antibody protein (IgG), and it has been reported that a resin in which MEL is physically adsorbed on the surface of polymer particles can specifically adsorb and desorb IgG. (See Non-Patent Documents 8 and 9). Therefore, by preparing fine particles whose surface is stably modified with MEL and using this as a carrier, various useful proteins such as antibody proteins can be detected, recognized, adsorbed, separated and collected with high efficiency. It is possible to provide an affinity carrier and a similar structure and material.

  As protein antibody affinity ligands, the most prominent ones are special proteins such as protein A and protein G (see Non-Patent Documents 10 and 11). However, advanced cell culture techniques using genetic recombination techniques are essential for these productions, and the ligand efficiency is low due to the random orientation of the immobilization on the carrier. As a result, currently used affinity carriers are extremely expensive, and the high cost of downstream processes such as separation and purification is a major factor that hinders the commercial use of useful proteins such as antibody proteins. Yes. For these reasons, there is a need for the development of a low molecular weight compound ligand that has a high affinity for the target protein and that can relatively easily control the position and orientation of immobilization, and MEL is extremely promising as a candidate.

  On the other hand, when a resin whose surface is modified with MEL is used as an affinity carrier, it is unstable just by physically adsorbing MEL to the polymer chain, and it cannot prevent the outflow of MEL in the solution, and its use is remarkable. It will be restricted. For use in various applications, it has been desired to bond MEL to a polymer chain with a chemically stable covalent bond. Furthermore, in order to realize high performance, it has been desired that the sugar skeleton showing a high intermolecular interaction with a biomolecule be bonded so as to face the outside of the resin. Therefore, it is strongly possible to synthesize polymers and resins with controlled MEL orientation by introducing a reactive functional group at the fatty acid terminal in MEL and binding it to the polymer chain that is the base of the carrier as a reactive site. It was desired.

Japanese Patent Publication No.57-145896 R. H. RH Haskins, Jay. A. Tone (JA Thorn), B.M. Boothroyd, “Canadean Journal of Chemistry”, Volume 1, p 749-756 (1955). Gee. Dem (G. Deml), Tee. Anke (T. Anke), EF. Overwinker (F. Oberwinkler), Bee. M. Gianetti, W. Steglich, “Phytochemistry”, Vol. 19, p83-87 (1980). Tee. T. Nakahara, H. H. Kawasaki, T. T. Sugisawa, Wy. Y. Takamori, T. T. Tabuchi, “Journal of Fermentation Technology (J. Ferment. Technol.)” (Japan), Japan Fermentation Engineering Society, Vol. 61, p19-23 (1983). Di. Kitamoto, S. et al. Akiba, See. C. Hioki, T. T. Tabuchi, “Agricultural and Biological Chemistry” (Japan), Japan Society for Agricultural Chemistry, Vol. 54. p31-36 (1990). H. S. Kim (H.-S. Kim), Bee. Di. Yoon (B.-D. Yoon), Di. H. Chung (D.-H. Chouung), H. M. Oh (H.-M. Oh), Tee. T. Katsuragi, Wye. Yani, “Applied Microbiology and Biotechnology”, (Germany), Springer-Verlag, Vol. 52, p 713-721 (1999). K. Kakukawa, M. Tamai, I. Mura, K. Miyamoto, S. Miyoshi, Y. Morinaga, Suzuki, O. Suzuki, Miyagawa (T. Miyakawa) “Bioscience, Biotechnology and Biochemistry” (Japan), Japanese Society of Agricultural Chemistry, 66, p188-191 (2002). Dai Kitamoto (Oreoscience), (Japan), Japan Oil Chemists' Society, Volume 3, p663-672 (2003). Jay. H. Im (J.-H. Im), T. T. Ikegami, H. H. Yanagishita, T. T. Nakane, Di. D. Kitamoto “BMC Biotechnology”, (Web Journal), Biomed Central, 1: 5, p1-7 (2001). Jay. H. Im (J.-H. Im), H. H. Yanagishita, T. T. Ikegami, Wy. Y. Takeyama, Wy. Y. Idemoto, N. N. Koura, Di. D. Kitamoto, “Journal of Microbiology and Biotechnology (J. Biomed. Mater. Res.)”, (USA), Wiley, 65, p 379-385 (2003). Gee. G. Kronvall, You. S. Seal (US Seal), Jay. Finstad, J. Finstad. Sea. Williams. Junior (RC Williams, Jr.) “The Journal of Immunology” (USA), 104, p140-147 (1970). El. L. Bjorck, G. G. Kronvall, “The Journal of Immunology (J. Immunol.)” (USA), 133, p969-974 (1984).

  An object of the present invention is to provide a mannosyl erythritol lipid-binding polymer that is covalently bonded to a polymer chain at a fatty acid terminal site without changing the sugar skeleton of mannosyl erythritol lipid having high physiological activity and biofunctionality. . Adsorption of useful proteins such as antibody proteins by using the above mannosyl erythritol lipid binding polymer, which is excellent in hydrophilicity, dispersibility, and corrosion resistance, and exhibits high physiological activities such as antibacterial and antiviral properties, as an affinity carrier It is to provide a separation method.

The present inventors examined the manufacturing method of the MEL compound which has a reactive functional group in the fatty-acid terminal as the 1st for solving the said subject. Although the target MEL compound is extremely difficult to produce by conventional chemical synthesis methods, it focuses on the carbon-carbon double bond introduced into the fatty acid chain of MEL via a biosynthetic pathway by microorganisms, and this is cleaved or metathesized. It has been found that by recombination with other carbon-carbon double bonds by reaction, MEL compounds having a reactive functional group at the fatty acid terminal can be produced efficiently and without complicated operations without changing the sugar skeleton structure. It was.
Furthermore, by reacting the obtained MEL compound with a polymer having a reactive functional group as a novel ligand, it was possible to construct a polymer in which MEL was introduced in a state in which orientation was controlled by a covalent bond. It has been found that by using a polymer as a carrier, interaction with biomolecules such as antibody proteins and glycoproteins can be utilized. The present invention has been made based on these findings.

That is, the present invention is as follows.
[1] A mannosyl erythritol lipid compound is bonded to a part or all of the reactive functional group of a polymer having a reactive functional group in a main chain or a side chain via a covalent bond at a fatty acid terminal. Mannosyl erythritol lipid binding polymer.
[2] The polymer according to [1] above, wherein the polymer forming the main skeleton of the polymer having a reactive functional group in the main chain or side chain is a polysaccharide, a protein, or an organic synthetic polymer. Mannosyl erythritol lipid binding polymer.
[3] The reactive functional group of the polymer having a reactive functional group in the main chain or side chain is an amino group, carboxyl group, hydroxyl group, aldehyde group, thiol group, epoxy group, isocyanate group, ester group, The mannosyl erythritol lipid-bonded polymer according to [1] above, which is a sulfone group, a phenol group, an azide group or a carbon-carbon double bond.
[4] Formation of a Schiff base between the aldehyde group of the fatty acid-terminally aldehyded mannosylerythritol lipid compound represented by the following general formula (1) or (2) and the amino group of a polymer having an amino group in the main chain or side chain Then, a mannosyl erythritol lipid-bonded polymer obtained by covalently bonding the mannosyl erythritol lipid compound to the polymer.

(In the general formula (1), n and m are each a number of 1 to 18 and constitute a saturated aliphatic alkylene group, and ^one of R ¹ and R ² is an aldehyde group and the other is methyl. Or both are aldehyde groups, and R ³ and R ⁴ each represents a hydrogen atom or an acetyl group.
Moreover, in the said General formula (2), l is a number of 1-18, R < ⁵ >, R < ⁶ >, R < ⁷ > represents a hydrogen atom or an acetyl group each independently. )
[5] The polymer having a reactive functional group in the main chain or side chain and the mannosyl erythritol lipid compound having at least one reactive functional group at a fatty acid terminal are covalently bonded by a reaction between the reactive functional groups. The method for producing a mannosyl erythritol lipid-bonded polymer according to [1] above, wherein the polymer is bound via an acid.
[6] The reactive functional group of the mannosylerythritol lipid compound having at least one reactive functional group at the fatty acid terminal is an amino group, a carboxyl group, a hydroxyl group, an aldehyde group, a thiol group, an epoxy group, or an isocyanate group. , An ester group, a sulfone group, a phenol group, an azide group, or a carbon-carbon double bond. The method for producing a mannosylerythritol lipid-bonded polymer according to [5] above.
[7] Using a mannosyl erythritol lipid compound produced by a microorganism as a raw material, introducing a reactive functional group into a fatty acid terminal by an ozone oxidation reaction or a metathesis reaction of a carbon-carbon double bond contained in the fatty acid chain A method for producing a mannosyl erythritol lipid compound having at least one reactive functional group at a fatty acid terminal as described in [5] or [6].
[8] A mannosyl erythritol lipid compound having at least one reactive functional group at a fatty acid terminal represented by any one of the following general formulas (1) to (4).

(In the general formula (1), n and m are each a number of 1 to 18 and constitute a saturated aliphatic alkylene group, and ^one of R ¹ and R ² is an aldehyde group and the other is methyl. Or both are aldehyde groups, and R ³ and R ⁴ each represents a hydrogen atom or an acetyl group.
In formula (2), l is the number of ^{1~18, R 5, R 6,} R 7 each independently represent a hydrogen atom or an acetyl group.
In the general formula (3), p and q each represent a number of 1 to 18 and constitute a saturated aliphatic alkylene group, and one of R ³¹ and R ³² is a 2-allyloxymethyloxirane group. And the other is a methyl group, or both are 2-allyloxymethyloxirane groups. R ³³ and R ³⁴ each represent a hydrogen atom or an acetyl group.
Moreover, in said general formula (4), r is a number of 1-18, and R < ³⁵ >, R < ³⁶ >, R < ³⁷ > represents a hydrogen atom or an acetyl group each independently. )

[9] A method for detecting a biomolecule, comprising using the mannosyl erythritol lipid-binding polymer according to [1].
[10] A method for separating biomolecules, comprising using the mannosyl erythritol lipid-binding polymer according to [1].
[11] A method for collecting biomolecules, comprising using the mannosyl erythritol lipid-binding polymer according to [1].
[12] The method according to any one of [9] to [11], wherein the biomolecule is an antibody protein, a glycoprotein, or a lectin.
[13] A biomolecule adsorbent comprising the mannosyl erythritol lipid-binding polymer according to [1].
[14] Properties of hydrophilicity, high dispersibility, high fluidity, antistatic property or anticorrosion property, antibacterial property, antiviral property, antitumor property, antithrombotic property, inhibition of cell adhesion or inhibition of protein adsorption The biomolecule adsorbent according to [13], wherein the bioactivity is imparted.
[15] An affinity carrier comprising the mannosyl erythritol lipid-binding polymer according to [1].
[16] Hydrophilic property, high dispersibility, high fluidity, antistatic property or anticorrosion property, antibacterial property, antiviral property, antitumor property, antithrombotic property, inhibition of cell adhesion or inhibition of protein adsorption The affinity carrier according to [15] above, which is imparted with physiological activity.
[17] An affinity column using the mannosyl erythritol lipid-binding polymer according to [1].
[18] Hydrophilic property, high dispersibility, high fluidity, antistatic property or anticorrosion property, antibacterial property, antiviral property, antitumor property, antithrombotic property, inhibition of cell adhesion or inhibition of protein adsorption The affinity column according to [17], which is imparted with physiological activity.
[19] A test reagent comprising the mannosyl erythritol lipid-binding polymer according to [1].
[20] Hydrophilic property, high dispersibility, high fluidity, antistatic property or anticorrosion property, antibacterial property, antiviral property, antitumor property, antithrombotic property, inhibition of cell adhesion or inhibition of protein adsorption The test reagent according to [19], wherein physiological activity is imparted.
[21] A sensor using the mannosyl erythritol lipid-binding polymer according to [1].
[22] Hydrophilic property, high dispersibility, high fluidity, antistatic property or anticorrosion property, antibacterial property, antiviral property, antitumor property, antithrombotic property, inhibition of cell adhesion or inhibition of protein adsorption The sensor according to [21], wherein physiological activity is imparted.

[23] A microarray using the mannosyl erythritol lipid-binding polymer according to [1].
[24] Hydrophilic property, high dispersibility, high fluidity, antistatic property or anticorrosion property, antibacterial property, antiviral property, antitumor property, antithrombotic property, inhibition of cell adhesion or inhibition of protein adsorption The microarray according to [23], wherein physiological activity is imparted.
[25] Separation, quantification or inspection using the affinity carrier according to [15], the affinity column according to [17], the sensor according to [21], or the microarray according to [23] Analytical equipment.
In the present specification and claims, the “fatty acid terminal” refers to an aliphatic group of a fatty acid ester group formed by bonding to a hydroxyl group of a mannosyl residue of mannosylerythritol.

In the mannosyl erythritol lipid-bonded polymer of the present invention, MEL, which is a functional glycolipid having excellent interfacial physical properties and various physiological activities, is introduced by a chemically stable covalent bond without significant structural change. . In particular, the sugar (mannosyl erythritol) skeleton, which is the key to its functional expression, is arranged on the polymer surface, which promotes hydrophilicity of the surface and improves the dispersibility of the polymer in aqueous solution. Molecular recognition and adsorption behavior can be controlled. For example, the polymer exhibits high affinity with various biomolecules including antibody proteins, glycoproteins, lectins, etc. due to specific intermolecular interactions of the sugar skeleton arranged on the surface. Thus, it can be used for various purposes such as biological substance analysis / test reagents, adsorbents, separation / purification affinity carriers.
According to the method for producing an MEL-bonded polymer of the present invention, a material comprising the polymer by a combination of an MEL compound having a reactive functional group at a fatty acid terminal and a polymer having a corresponding reactive functional group in the chain, Regardless of the shape, depending on the purpose and application, it is possible to produce a MEL-bonded polymer having an arbitrary structure and bonding mode. Also, the introduction rate can be controlled.
The MEL compound having a reactive functional group at the fatty acid terminal is useful for producing the MEL-binding polymer of the present invention as a novel glycolipid ligand that can be introduced into various carriers.
The method for producing a MEL compound having a reactive functional group at the fatty acid terminal used in the present invention uses an unsaturated group introduced into the fatty acid chain of MEL through a delicate biosynthetic pathway of microorganisms as a reaction point. Arbitrary functional groups can be selectively introduced at the fatty acid terminals without requiring complicated operations.

The method for detecting a biomolecule of the present invention can specifically detect various useful biomolecules such as antibody proteins.
The method for separating biomolecules of the present invention can be separated based on the specific action of useful biomolecules and MEL.
The biomolecule collection method of the present invention can collect useful biomolecules based on specific action with MEL.
The biomolecule adsorbent of the present invention can specifically adsorb useful proteins such as antibody proteins.
As described above, the mannosyl erythritol lipid-bonded polymer of the present invention is expected in the development of materials useful in a wide range of fields closely related to recognition and control of biological functions, including the pharmaceutical, food and cosmetic industries.
In particular, the affinity carrier, the affinity column and the apparatus using the same according to the present invention bring about a major revolution in biomolecular analysis technology. In the separation process of useful proteins, in general, a carrier having a specific protein that specifically interacts with them as a ligand is often used. However, the production of these proteins requires the use of genetic recombination techniques. Since it is expensive (> 1 million yen / kg) and the method of immobilizing the ligand on the carrier is random, the orientation of the site showing affinity cannot be controlled, and the efficiency of the ligand is low (1 to several mol%). The high cost of this downstream process has suppressed the spread to the market.

On the other hand, the affinity carrier using the MEL-binding polymer of the present invention has a high production amount of glycolipid (MEL) as a ligand, a low molecular weight compared to proteins, etc., a simple molecular structure, and an affinity site ( Since the sugar skeleton is arranged on the surface, the ligand efficiency is also high. Compared with a conventional affinity carrier using a protein as a ligand, the cost can be easily reduced to 1/10 to 1/100.
From the above, it can be said that the MEL-bonded polymer of the present invention is excellent as a low-cost and high-efficiency affinity carrier.

Further, the affinity column using the MEL-binding polymer of the present invention can separate other biomolecules such as glycoprotein and lectin without centrifugation.
The analyzer for separation, quantification, or inspection of the present invention is useful as a separation system for any biomolecule such as antibody protein.
The test reagent of the present invention can capture and quantify specific biomolecules present in a sample.
The sensor of the present invention can detect biomolecules such as proteins in a minute sample.
Similarly, the microarray of the present invention can detect biomolecules such as proteins in a minute sample.

First, the mannosyl erythritol lipid-bonded polymer of the present invention (hereinafter sometimes simply referred to as “MEL-bonded polymer”) will be described.
The mannosyl erythritol lipid-bonded polymer of the present invention has a mannosyl erythritol lipid compound in part or all of the reactive functional group of the polymer having a reactive functional group on the polymer chain (main chain) or side chain. It is a polymer formed by bonding at a fatty acid terminal through a covalent bond. The polymer may be in the form of gel, fine particles, plate, film, fiber, etc., and from the viewpoint of controlling the orientation of the MEL to the polymer surface through the covalent bond, the reactive functional group is surfaced. It is preferably in the form of gel, fine particles, plate, film, fiber, etc.
Here, the gel is a porous solid in which a polymer chain forms a three-dimensional network structure by crosslinking, and the polymer constituting the main skeleton may be any of polysaccharides, proteins, or organic synthetic polymers. good.
The polymer having a reactive functional group may be in the form of a resin having a reactive functional group in the side chain. In the same manner as described above, from the viewpoint of binding the MEL to the surface by controlling the orientation, The form of the resin on the surface is preferable.
Glucose, glucosamine, N-acetylglucosamine, galactose, galactosamine, N-acetylgalactosamine, glucuronic acid, etc. bonded to the main chain of the polymer having reactive functional groups by β1,4-bond or α1,4-bond Also included are polysaccharide chains such as sugar chains consisting of repetition of the above.

In the polymer having a reactive functional group, the polymer forming the main skeleton is preferably a polysaccharide, protein, or organic synthetic polymer.
Examples of the polysaccharide that is a polymer that forms the main skeleton include cellulose, chitin, and chitosan. Examples of the protein that is a polymer forming the main skeleton include gelatin and collagen. Examples of the organic synthetic polymer that is a polymer that forms the main skeleton include vinyl polymers, polyethers, polyesters, polyamides, polyurethanes, and copolymers thereof.
Examples of the reactive functional group include amino group, carboxyl group, hydroxyl group, aldehyde group, thiol group, epoxy group, isocyanate group, ester group, sulfone group, phenol group, azide group, carbon-carbon double bond, etc. Is mentioned.
The method of introducing the reactive functional group into a polymer to obtain a polymer having the reactive functional group is a carbodiimide (dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC)). Etc.), and after introduction of epichlorohydrin, it can be carried out by any method such as a method of ring-opening addition of epoxy.

  In the polymer having a reactive functional group in the main chain or side chain, the reactive functional group is preferably present in an amount of 0.1 μmol to 1 mol, preferably 10 μmol to 1 mol, per unit mass (g) of the polymer. More preferred.

Specific examples of the polymer having the reactive functional group in the side chain include cellulose gel having 15 to 20 μmol of 2-hydroxy-3-aminopropyloxy group on the surface per unit gel volume (ml), and formyl group in the side chain. For example, a methacrylic acid polymer having 10 to 30% by mass. For example, amino-cellulofine (trade name, manufactured by Seikagaku Corporation), Toyopearl AF series (trade name, manufactured by Tosoh Corporation) and the like are commercially available.
Specific examples of the polymer having the reactive functional group in the main chain include polyethylene oxide and polyester having a carboxyl group at one end or both ends.

Next, the manufacturing method of the mannosyl erythritol lipid binding polymer of this invention is demonstrated.
The mannosyl erythritol lipid-bonded polymer of the present invention is obtained by reacting the mannosyl erythritol lipid compound having at least one reactive functional group at a fatty acid terminal with the polymer having the reactive functional group in the main chain or side chain described above. It can manufacture by forming a covalent bond by reaction of a functional functional group.
Examples of the reactive functional group that the mannosyl erythritol lipid compound (hereinafter also referred to as “MEL compound”) used in the production method of the present invention has at the fatty acid terminal include an amino group, a carboxyl group, a hydroxyl group, and an aldehyde group. Thiol group, epoxy group, isocyanate group, ester group, sulfone group, phenol group, azide group, carbon-carbon double bond, and the like.
Examples of the covalent bond formed by the reaction between the reactive functional groups include an amide bond consisting of a reaction of an amino group and a carboxyl group, a Schiff base consisting of a reaction of an amino group and an aldehyde group, and reducing it. Examples thereof include an ester bond composed of a reaction between a carboxyl group and a hydroxyl group, a disulfide bond composed of a reaction between thiol groups, an amino bond composed of a reaction between an epoxy group and an amino group, and an ether bond composed of a reaction with a hydroxyl group. These covalent bonds control the orientation of the bound MEL.
Here, “orientation” means that the sugar skeleton (mannosylerythritol skeleton) does not participate in the binding with the polymer and faces the outside (surface) of the polymer. This makes it possible to utilize specific intermolecular interactions of the sugar skeleton without being sterically hindered.

  In the mannosyl erythritol lipid-bonded polymer of the present invention, the mannosyl erythritol lipid compound is preferably present at 0.01 to 30% by mass, more preferably 1% by mass to 30% by mass, based on the mass of the polymer of the present invention.

Subsequently, as one preferred embodiment of the method for producing a mannosyl erythritol lipid-bonded polymer of the present invention, a fatty acid-terminated aldehyde-modified mannosyl erythritol lipid compound (hereinafter simply referred to as a fatty acid) is applied to a cellulose gel having an amino group introduced in the side chain. A production method in which terminal aldehyded MEL compound is also reacted will be described.
Although the structure of the fatty acid-terminated aldehyded MEL compound will be described later in detail, a Schiff base is formed by an arbitrary method with a compound having an amino group, so that it has an amino group in the side chain as shown in the following formula A. The MEL compound can be covalently bonded to the polymer by a carbon-nitrogen bond.

Specifically, an aminated cellulose gel and a fatty acid-terminated aldehyde-modified MEL are dispersed in water, and an arbitrary reducing agent such as sodium cyanoborohydride (NaCNBH ₃ ) is added thereto. Stir for hours. After completion of the reaction, the gel is filtered off and washed thoroughly to recover the MEL-bonded cellulose gel. This allows MEL to be bound in the gel by a carbon-nitrogen bond at the fatty acid end.
Regarding the determination of the MEL introduction rate in the MEL-bonded cellulose gel, the elemental analysis of the MEL-bonded cellulose gel is performed, the carbon / nitrogen weight ratio (C / N ratio) is calculated, and the same is applied to the original cellulose gel. It is possible to determine the number of carbon introductions relative to the amino group. By dividing the obtained value by the number of carbon atoms of the fatty acid-terminated aldehyded MEL estimated at the time of structure determination, the MEL binding ratio relative to the total amount of amino groups can be calculated.

  Furthermore, even when a polymer having an amino group in the side chain other than the cellulose gel is used, the MEL-bonded polymer of the present invention can be produced and the MEL introduction rate can be determined in the same manner as in the above method.

  In addition, for MEL compounds synthesized using an olefin metathesis reaction and having various reactive functional groups (for example, amino group, carboxyl group, hydroxyl group, aldehyde group, thiol group, epoxy group, etc.) at the fatty acid terminal, The MEL-bonded polymer of the present invention can be produced by appropriately reacting with a polymer having a corresponding reactive functional group.

  As described above, according to the method for producing an MEL-bonded polymer of the present invention, the polymer is obtained by combining a MEL compound having a reactive functional group at a fatty acid terminal and a polymer having a corresponding reactive functional group in the chain. Regardless of the material and shape of the structure, it is possible to produce a MEL-bonded polymer of any structure and bonding mode depending on the intended use. Further, the introduction rate can be easily controlled.

The MEL compound having at least one reactive functional group at the fatty acid terminal used in the production method of the present invention will be described.
The MEL compound having a reactive functional group at the fatty acid terminal used in the production method of the present invention is not particularly limited as long as it has at least one reactive functional group described above at the fatty acid terminal. However, since it has a reactive functional group at the end of the fatty acid bound exclusively at the 2′-position or the 3′-position, the fatty acid is from the viewpoint of controlling the orientation of the MEL moiety in the resulting MEL-bonded polymer of the present invention. A terminal aldehyde-modified MEL compound or a fatty acid-terminated epoxidized mannosyl erythritol lipid compound (hereinafter sometimes simply referred to as a fatty acid-terminated epoxidized MEL compound) is preferable.
The fatty acid-terminated aldehyded MEL compound is a novel compound represented by the following general formula (1) or (2) and having an aldehyde group introduced at the fatty acid terminal. In the fatty acid terminal aldehyded MEL compound, the MEL such as MEL-A which is a raw material has an unsaturated fatty acid residue as a substituent, whereby the carbon-carbon double bond is cleaved and an aldehyde group is introduced into the terminal part. It has the structure which becomes.
Ozonide formation by carbon-carbon double bond cleavage and subsequent aldehyde formation by any reductive treatment proceed as follows: For example, it can be performed according to the method described in “Chemical Course of 4th Edition”, Vol. 23, pp. In the formula, R ⁴¹ and R ⁴² are arbitrary groups.

In the general formula (1), n and m each represent a number of 1 to 18 and constitute a saturated aliphatic alkylene group, and ^one of R ¹ and R ² is an aldehyde group and the other is a methyl group. Or both are aldehyde groups. R ³ and R ⁴ each represent a hydrogen atom or an acetyl group.
Moreover, in the said General formula (2), l is a number of 1-18, R < ⁵ >, R < ⁶ >, R < ⁷ > represents a hydrogen atom or an acetyl group each independently.
In the present invention, each of l, m, and n preferably constitutes a saturated aliphatic alkylene group that is 1 to 18, and more preferably constitutes a saturated aliphatic alkylene group that is 1 to 10.
The fatty acid-terminated epoxidized MEL compound is represented by the following general formula (3) or (4), and is a novel compound in which an epoxy group is introduced at the fatty acid terminal. Fatty acid-terminated epoxidized MEL compounds have an unsaturated fatty acid residue in the substituent of MEL such as MEL-A as a raw material, so that an olefin metathesis reaction with allyl glycidyl ether causes a carbon-carbon double bond Recombination takes place and has a structure in which an epoxy group is introduced into the terminal portion. For the olefin metathesis reaction, see J. et al. Am. Chem. Soc. 125, p11360-11370 (2003) and the method described in the cited document.

In the general formula (3), p and q each represent a number of 1 to 18 and constitute a saturated aliphatic alkylene group, and one of R ³¹ and R ³² is a 2-allyloxymethyloxirane group. (

) And the other is a methyl group, or both are 2-allyloxymethyloxirane groups. R ³³ and R ³⁴ each represent a hydrogen atom or an acetyl group.
Moreover, in said general formula (4), r is a number of 1-18, and R < ³⁵ >, R < ³⁶ >, R < ³⁷ > represents a hydrogen atom or an acetyl group each independently.
In the present invention, each of p, q, and r preferably constitutes a saturated aliphatic alkylene group that is 1 to 18, and more preferably constitutes a saturated aliphatic alkylene group that is 1 to 10.

Next, the manufacturing method of the MEL compound which has a reactive functional group in the fatty acid terminal used for manufacture of the mannosyl erythritol lipid binding polymer of this invention is demonstrated.
Mannosyl erythritol lipid (hereinafter sometimes simply referred to as MEL) used as a raw material for the production of an MEL compound having a reactive functional group at the fatty acid terminal is continuously cultured and produced for a long time using a microorganism such as yeast. Therefore, it is preferable that MEL used as a raw material is produced using microorganisms.
In particular, MEL used as production raw materials of MEL compound having a reactive functional group in said fatty acid terminus, Pseudozyma Antakutika (Pseudozyma antarctica T-34 strain), Pseudozyma Rugurosa (Pseudozyma rugulosa Ltd.), Pseudozyma para Anta click Rustica More preferably, it is obtained by culturing MEL-producing bacteria such as ( Pseudozyma parantarctica strain) by any method.
For example, using a Pseudozyma Antakutika (Pseudozyma antarctica T-34 strain), it was separated and purified by any method from the resulting product by culturing soybean oil as a carbon source to obtain a MEL-A as a raw material be able to.

  MEL having a high unsaturated group content is preferable as a raw material, but the composition of the fatty acid chain of MEL varies depending on the type of carbon source contained in the production medium and the utilization mode of the production bacteria. By using fats and oils containing a large amount of linoleic acid, linolenic acid, etc. having a high unsaturated group content as a source, MEL having a high unsaturated group content can be produced. In addition to fats and oils, MEL-producing bacteria can also assimilate compounds such as medium to long chain aliphatic hydrocarbons, medium to long chain aliphatic alcohols, medium to long chain fatty acid esters, etc. By including the above-mentioned compound having a saturated group in the MEL production medium, it is possible to prepare MEL in which a double bond is introduced at a specific position in the fatty acid chain.

  MEL used as a raw material for producing an MEL compound having a reactive functional group at the fatty acid terminal is a compound having a structure represented by the following general formula (5) or (6), and 4-O-β-D-mannopyranosyl -Meso-erythritol is the basic structure.

In the general formulas (5) and (6), the substituents R ¹¹ , R ¹² and R ²² are each independently an unsaturated aliphatic group having at least one alkyl group having 3 to 21 carbon atoms or a double bond. A hydrocarbon group, preferably an alkyl group having 5 to 17 carbon atoms or an unsaturated linear hydrocarbon group having at least one double bond. Further, the MEL used as a raw material for producing the MEL compound having a reactive functional group at the fatty acid terminal is an unsaturated linear hydrocarbon group in which R ¹¹ , R ¹² or R ²² has one double bond. More preferably, an unsaturated straight chain hydrocarbon group having one double bond near the terminal is even more preferable.
Specific examples of the alkyl group representing R ¹¹ , R ¹² or R ²² include a pentyl group, a heptyl group, a nonyl group, an undecyl group, and a tridecyl group.
Specific examples of the unsaturated hydrocarbon group having at least one double bond representing R ¹¹ , R ¹² or R ²² include 2-nonel group, 2-undecel group, 4-undecel group, 6-undecel group, 8 -An undecel group etc. are mentioned.
Said R <^21> , R < ²³ > or R < ²⁴ > represents a hydrogen atom or an acetyl group each independently.
In the general formula (5) or (6), the carbon number of the substituents R ¹¹ , R ¹² , and R ²² in each MEL is the number of carbon atoms of the fatty acid constituting the triglyceride in the fats and oils contained in the MEL production medium. It varies depending on the degree of utilization of fatty acids of MEL-producing bacteria to be used. Further, when the triglyceride has an unsaturated fatty acid residue, the MEL-producing bacterium does not assimilate up to the double bond portion of the unsaturated fatty acid, so that R ¹¹ , R ¹² , and R ²² are an unsaturated fatty acid residue. It is preferable to become.
As is apparent from the above description, the MEL used as a raw material is usually in the form of a mixture of compounds in which the fatty acid residue portions of R ¹¹ , R ¹² and R ²² are different.

As an example of the MEL compound having a reactive functional group at the fatty acid terminal, a method for producing the fatty acid terminal aldehyded MEL compound will be described.
The fatty acid-terminated aldehyded MEL compound can be produced by using MEL produced by a microorganism as described above as a starting material and ozone-oxidizing unsaturated groups contained in the fatty acid chain. This is characterized in that an aldehyde group can be selectively introduced at the fatty acid terminal without requiring a complicated operation.
Specifically, MEL compounds (for example, MEL-A (see the general formula (5))) dissolved in a solvent (for example, methanol) and ozone generated by a general ozone generator at 50 to 75 ° C. Can be selectively oxidatively cleaved at the double bond portion. By allowing the reaction to proceed under any reducing conditions, the double bond is cleaved and an aldehyde is produced. The aldehyde product can be recovered by separating and purifying the product by a known column chromatography method.

The identification of the structure determination and the like of the fatty acid-terminated aldehyded MEL compound is performed as follows.
The molecular structure (sugar skeleton, fatty acid composition, etc.) of MEL such as MEL-A as a raw material and the determination method thereof are known.
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. This glycolipid is subjected to NMR analysis, and the resulting spectrum is compared with the spectrum of MEL-A or the like used as a starting material (see the general formula (5) or (6)) to perform structural analysis. be able to.

Specifically, fatty acid-terminated aldehyded MEL compounds can be identified by the following 1) and 2).
1) Confirmation of aldehyde group introduction
^{In the 1} H-NMR spectrum (DMSO-d ₆ ), protons bonded to the unsaturated group in the MEL fatty acid chain are detected as broad peaks around 5.4 ppm. The heavy bond is cleaved and this signal disappears. In addition, a proton signal derived from an aldehyde group appears newly in the vicinity of 9.8 ppm. Furthermore, the disappearance of the fatty acid terminal reduces or eliminates the signal of protons derived from the terminal methyl group at around 0.9 ppm. From the movement of the above signal, it can be easily confirmed that the aldehyde group has been introduced into the fatty acid terminal.

2) Confirmation of structure and fatty acid chain length of aldehyde compound by molecular weight measurement The obtained glycolipid is recovered as a mixture of components having different fatty acid chain lengths in the lipid portion, but matrix-assisted laser desorption / ionization mass spectrometry (MALDI-) By performing TOF / MS), the molecular weight of the aldehyde and the distribution of the fatty acid chain length are known, and the structure of the main component is determined.

  Furthermore, this can also be easily separated into a compound having a single fatty acid by performing HPLC analysis using a reverse phase column (ODS column).

  Furthermore, other MEL analogs (MEL-B to D (see the general formula (5))) and fatty acid chains other than MEL-A are different from one type of MEL compound (see the general formula (6)). Even when MEL having a different fatty acid composition cultured from a carbon source is used as a starting material, it is possible to produce and identify a fatty acid-terminated aldehyded MEL compound in the same manner as described above.

  As another example of the MEL compound having a reactive functional group at the fatty acid terminal, a method for producing the fatty acid terminal epoxidized MEL compound will be described.

The fatty acid-terminated epoxidized MEL compound can be produced by performing an olefin metathesis reaction with allyl glycidyl ether using a MEL compound produced by a microorganism as described above as a raw material.
Specifically, an MEL compound (for example, MEL-A (see the above general formula (5)) is dissolved in a solvent (for example, dichloromethane), and 1 to 10 mol% of a Grubbs catalyst generally used for an olefin metathesis reaction is used as a catalyst. The fatty acid-terminated epoxidized MEL compound can be synthesized by adding about a degree and stirring at room temperature for 1 to 3 days, and recovering the epoxidized product by separating and purifying the product by a known column chromatography method. Examples of the Grubbs catalyst include a first generation Grubbs catalyst (CAS 172222-30-9) and a second generation Grubbs catalyst (CAS 246047-72-3) represented by the following formulae, respectively.

In the above formula, Cy represents a cyclohexyl group.
The identification of the structure determination and the like of the fatty acid-terminated epoxidized MEL compound is performed as follows in the same manner as in the case of the fatty acid-terminated aldehyded MEL compound.
Specifically, fatty acid-terminated epoxidized MEL can be identified by the following 3) and 4).
3) Confirmation of epoxy group introduction
^{In the 1} H-NMR spectrum (CDCl ₃ ), an epoxy-derived proton signal appears around 2.6 ppm, around 2.8 ppm, and around 3.2 ppm. Furthermore, the disappearance of the fatty acid terminal reduces or eliminates the signal of protons derived from the terminal methyl group at around 0.9 ppm. Further, in the ¹³ C-NMR spectrum (CDCl ₃ ), signals of epoxy-derived carbon appear around 44 ppm and around 51 ppm. From the above signal movement, it can be easily confirmed that the epoxy group has been introduced into the fatty acid terminal.

4) Confirmation of structure and fatty acid chain length of epoxidized product by molecular weight measurement As in the case of the above-mentioned fatty acid-terminated aldehyded MEL compound, by performing matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF / MS), The molecular weight and fatty acid chain length distribution of the epoxidized product is known, and the structure of the main component is determined.

  Furthermore, this can also be easily separated into a compound having a single fatty acid by performing HPLC analysis using a reverse phase column (ODS column).

  Furthermore, other MEL analogs (MEL-B to D (see the general formula (5))) and fatty acid chains other than MEL-A are different from one type of MEL compound (see the general formula (6)). Even when MEL having a different fatty acid composition cultured from a carbon source is used as a starting material, the fatty acid-terminated epoxidized MEL compound can be produced and identified in the same manner as described above.

  In a MEL compound having a complex molecular structure, it is extremely difficult to selectively introduce a functional group to the fatty acid terminal of the MEL compound while maintaining the three-dimensional structure of the glycolipid.

However, as described above, the method for producing a MEL compound having a reactive functional group at the fatty acid terminal used in the present invention uses an unsaturated group introduced into a fatty acid chain of MEL through a biosynthetic pathway of a microorganism as a reactive site. Thus, an arbitrary functional group can be selectively introduced at the fatty acid terminal without requiring a complicated operation.
Moreover, the position of the unsaturated group in the fatty acid chain of the produced MEL can be controlled to some extent by appropriately setting the raw materials and culture conditions using the biosynthetic mechanism of microorganisms. The structure of the MEL compound having a reactive functional group can be designed to some extent.

  As described above, the MEL compound having a reactive functional group at the fatty acid terminal is useful for producing the MEL-binding polymer of the present invention as a novel glycolipid ligand that can be introduced into various carriers.

The MEL compound having a reactive functional group at the end of the fatty acid has an excellent surface activity like the conventional MEL represented by the general formula (5) or (6), and should be used as a biosurfactant. Can do.
Furthermore, in the method for producing an MEL compound having a reactive functional group at the fatty acid terminal, the reactive functional group can be converted into a desired functional group by an olefin metathesis reaction as described above. For example, if an unsaturated hydrocarbon having an amino group at the end (such as allylamine) is used in the reaction, an MEL compound having an amino group at the end of a fatty acid or an unsaturated hydrocarbon having an thiol group at the end (such as allyl mercaptan) will be used in the reaction. If it uses, the MEL compound etc. which have a thiol group at the fatty-acid terminal can be manufactured. Thereby, the modification | reformation of the function as a biosurfactant which MEL originally has can also be performed systematically.

The surface activity can be easily observed by the following method.
That is, an isolated aqueous solution of MEL having a reactive functional group at the terminal of fatty acid (for example, fatty acid-terminated aldehyded MEL) can be spotted on a hydrophobic film, and the change in the surface tension can be observed. Further, as a control, water and an aqueous solution of conventional MEL can be spotted for comparison.

Hereinafter, the use of the MEL binding polymer of the present invention will be described.
The MEL-bonded polymer of the present invention is preferably covalently bonded by arranging MEL on the polymer surface in the following applications.

By using the MEL binding polymer of the present invention, it is possible to specifically adsorb various biomolecules such as antibody proteins, glycoproteins, lectins, etc. to the resin by utilizing the intermolecular interaction of the sugar skeleton of MEL. is there.
Thereby, biomolecules can be detected using the MEL binding polymer of the present invention, biomolecules can be separated, and biomolecules can be collected.
From the specific adsorption action described above, the MEL-binding polymer of the present invention can be used as, for example, a biomolecule adsorbent.
The resin that physically adsorbs MEL has the function of suppressing nonspecific adsorption of proteins (serum albumin, etc.) due to hydrophobic interaction and specifically adsorbing certain useful proteins such as antibody proteins. Examples of reports of expression (J.-H. Im, T. Ikegami, H. Yanagishita, T. Nakane, D. Kitamoto) (D. Kitamoto) “BMC Biotechnology” (Web Journal), Biomed Central, 1: 5, p1-7 (2001). -H. Im), H. Yanagishita (H. Yanagi) Shita, T. Ikegami, Y. Takeyama, Y. Idemoto, N. Koura, D. Kitamoto “Journal of Microbiology and Biotechnology (J. Biomed. Mater. Res.) "(USA), Wiley, Vol. 65, p379-385 (2003). However, the physical adsorption method is concerned about elution of MEL from the resin into the solvent, and is not suitable for long-time use in the solvent or high-purity separation and purification.
On the other hand, the MEL-bonded polymer of the present invention does not cause elution of MEL into the solvent because MEL is firmly bonded to the carrier by covalent bond. Therefore, since it can be handled under various solvent conditions, it is extremely effective as an affinity carrier for biomolecules that involve dramatic changes in solvent conditions such as salt concentration and pH in the separation operation. Moreover, since it is resistant to organic solvents, it can be used for a wide range of applications.

As described above, the MEL-binding polymer of the present invention can be used as an affinity carrier. As a specific example of the affinity carrier using the MEL-binding polymer of the present invention, antibody protein selection using a MEL-bonded cellulose gel is used. The method for selective adsorption and elution is described below.
A predetermined amount of MEL-bonded cellulose gel is weighed in a centrifuge tube, and selective adsorption and elution test of antibody protein can be performed by the following procedure.
(1) Blocking non-specific adsorption surface (blocking treatment with serum albumin)
An aqueous solution of serum albumin (SA) is added to the gel, and the adsorption operation is performed by stirring for 2 hours on a shaker. Thereafter, the gel is precipitated by centrifugation, and the supernatant is recovered.
Subsequently, the gel used for the solution used for adsorption is washed with the same solvent, and after centrifugation, the supernatant is recovered.
(2) Adsorption of antibody protein An adsorption operation is performed by adding an aqueous solution of immunoglobulin (IgG) to the gel and stirring for 2 hours on a shaker. Thereafter, the gel is precipitated by centrifugation, and the supernatant is recovered.
Subsequently, the gel used for the solution used for adsorption is washed with the same solvent, and after centrifugation, the supernatant is recovered.
(3) Elution of antibody protein Add the elution solution to the gel, thoroughly wash it, centrifuge it, and collect the supernatant.
(4) Protein quantification The protein in the solution is quantified by high-performance liquid chromatography (HPLC) analysis using a column for size exclusion chromatography (SEC) or an ion exchange column for all the collected supernatants. . The amount adsorbed on the gel can be determined from the amount eluted with respect to the total amount of protein administered.
Furthermore, the above-described method is not limited to the MEL-bonded cellulose gel but can be applied to all the MEL-bonded polymers of the present invention.

In addition, by using the MEL-binding polymer of the present invention, other biomolecules such as glycoproteins and lectins can be adsorbed and separated by the same operation as that described above.
In addition to the above-described method, the procedure for centrifugation is performed by filling the medium pressure open column and the empty column for HPLC system with the MEL-bonded cellulose gel and continuously flowing the solution used in the above (1) to (3). Can be omitted.
Thereby, the affinity column which uses the MEL binding polymer of this invention can be provided.
In addition, by connecting a differential refractive index (RI) detector and an ultraviolet-visible light absorption (UV) detector to the outlet, a separation system for any biomolecule such as an antibody protein can be created.
Thus, the separation, quantification or test analyzer of the present invention using the affinity column or the affinity carrier can be provided.

  Similarly, the MEL-binding polymer of the present invention can also be used as a test reagent capable of capturing and quantifying specific biomolecules present in a sample, and detects biomolecules such as proteins in a minute sample. It can also be used as a sensor or microarray. Specifically, a test apparatus that can detect, quantify, analyze, etc. by selectively capturing a specific antibody protein from a blood-derived sample such as plasma and combining it with a fluorescently labeled secondary antibody. It is possible to introduce the MEL binding polymer of the present invention into the above.

In addition, the biomolecule adsorbent, the test reagent, the sensor, the microarray, the affinity carrier, or the affinity column are hydrophilic, highly dispersible, highly fluid, by using the MEL binding polymer of the present invention. Physical properties such as antistatic properties and corrosion resistance can be imparted.
Furthermore, the biomolecule adsorbent, the test reagent, the sensor, the microarray, the affinity carrier or the affinity column can be used for antibacterial, antiviral, antitumor, Physiological activities such as antithrombogenicity, inhibition of cell adhesion, and inhibition of protein adsorption can be imparted.

Specifically, the MEL-bonded polymer of the present invention can be imparted with hydrophilicity on the surface of the material by directly introducing MEL showing extremely excellent surface activity, and is expected to improve dispersibility in an aqueous solution. Improvement in operability is expected, such as an antistatic effect appearing due to the smoothness of the material surface.
In order to impart these performances to the material, the surface is generally coated with a surfactant, etc., but the MEL binding polymer of the present invention incorporates a surfactant into the material itself, That process is unnecessary.
In general, the affinity carrier is basically used in an aqueous solution, so that it is necessary to add a preservative or the like. However, glycolipid biosurfactants such as MEL have a solubilizing ability for lipids. High antibacterial and antiviral properties due to effects such as membrane permeability to cell membranes. Therefore, the biomolecule adsorbent, the test reagent, the sensor, the microarray, the affinity carrier, or the affinity column are provided with the antimicrobial action on the material itself by using the MEL binding polymer of the present invention. In addition, it has excellent corrosion resistance and does not require the addition of other components.
Furthermore, as described above, high hydrophilicity is imparted to the material surface, so that not only protein-specific adsorption and cell adhesion due to hydrophobic interaction are suppressed, but also differentiation induction and growth inhibition of tumor cells are suppressed. The biomolecule adsorbent using the MEL-binding polymer of the present invention, the test reagent, the sensor, and the like, such that cell growth on the material surface can be suppressed by arranging MELs having effects and the like on the surface, The microarray, the affinity carrier, or the affinity column is considered to satisfy the performance as a biofunctional material (for example, Oreoscience, Vol. 3, p663-672 (2003), J. Biosci. Bioeng., 94). Vol., P187-201 (2002), and references cited therein).
The above performance is widely required for medical engineering materials, but the MEL-bonded polymer of the present invention has these properties added to the material itself and is expected to be widely used as an extremely useful material.
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Reference example 1
(Production of unsaturated MEL)
In the present invention, since a carbon-carbon double bond contained in the fatty acid chain of MEL is used as a reaction point, a single-structure unsaturated aliphatic alcohol is added to a carbon source as a raw material for microbial production. Production of MEL with controlled content and position of heavy bonds was performed. (See J. Jpn. Oil Chem. Soc., 42, p346-358 (1993)).
a) Pseudozyma antarctica T-34 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), glucose 20 g / L, 1 platinum ear was inoculated into a test tube containing 4 mL of a liquid medium composed 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. Incubate with shaking and then
b) 6% cis-11-tetradecene-1-ol and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0. A Sakaguchi flask containing 20 mL of a liquid medium having a composition of 5 g / L was inoculated and cultured at 30 ° C. with shaking.
After culturing a) for 1 day, the culture solution after culturing b) for 7 days was collected, and an equal amount of ethyl acetate was added and stirred to obtain an ethyl acetate extract. MEL was separated from the obtained ethyl acetate extract containing glycolipid by a known purification method. That is, the ethyl acetate extract was applied to a silica gel column and purified by column chromatography using a mixed solution of chloroform and acetone as a developing solvent. The raw materials and MEL-A, -B, and -C were sequentially separated and recovered by changing the ratio of chloroform and acetone while changing the ratio from 80:20 to 30:70. Components contained in the obtained fraction were confirmed by thin layer chromatography. As the developing solvent, chloroform: methanol: 7N aqueous ammonia = 65: 15: 2 was used, and as the indicator, an anthrone sulfate reagent that colored the glycolipid to blue-green was used.

Reference example 2
In the same manner as in Reference Example 1, MEL was produced using Pseudozyma antarctica JCM 10317 strain, Pseudozyma rugulosa NBRC 10877 strain, and Pseudozyma parantarctica JCM 11752 strain as production strains. It recovered MEL total amount, P.antarctica T-34 strain: 64.4mg, P.antarctica JCM 10317 strain: 111.4mg, P.rugulosa strains: 51.4mg, P.parantarctica strains: a 120.8mg, P The production of MEL was the highest in the .parantarctica strain.
Subsequent reactions, measurements, etc. were performed using MEL-A produced and separated and purified using the P. parantarctica strain.

Reference example 3
The recovered MEL-A is subjected to ¹ H, ¹³ C, ¹ H- ¹ H COSY, HMQC (Heteronical Multiple Quantum Coherence), and HMBC (Heteronomic Multiple Bond Coherence) to perform each NMR spectrum analysis. It was.
FIG. 1 shows the ¹ H NMR spectrum of MEL-A. As is clear from FIG. 1, the proton signal assigned to the carbon-carbon double bond (c in FIG. 1) is 5.5 to 5.0 ppm, and the proton is bonded to the second carbon atom from the end of the fatty acid. Was observed at around 2.1 ppm, and a proton signal of the fatty acid terminal methyl group (a in FIG. 1) was observed at around 0.9 ppm, confirming the structure of MEL-A.
In addition, from the results of the above various NMR measurements, it was confirmed that the obtained MEL-A was MEL-A in which an unsaturated double bond was present between the third and fourth carbons from the fatty acid terminal. It was.
The composition of the lipid part of MEL-A was determined by GC / MS analysis of a fatty acid methyl ester obtained by extraction with hexane from hydrochloric acid methanol hydrolyzate. In this glycolipid, unsaturated fatty acid having 8 carbon atoms (5-octenoic acid) is 33%, unsaturated fatty acid having 10 carbon atoms (7-decenoic acid) is 34%, unsaturated fatty acid having 12 carbon atoms (9-dodecene). Acid) was 13% and 80% unsaturated fatty acids were included as a whole.
It can be seen that MEL was produced by controlling the content and position of the double bond by the production using the above microorganisms.
Note that 5-octenoic acid, 7-decenoic acid, and 9-dodecenoic acid all have an unsaturated double bond between the third and fourth carbons from the fatty acid ω terminal.

Example 1-1
(Ozone oxidation of MEL-A)
0.65 g of MEL-A was dissolved in 10 mL of dehydrated methanol and cooled to -90 ° C. Ozone generated using an ozone generator (trade name ED-OG-R4, manufactured by Ecodesign) was blown into the methanol solution while cooling and stirring. At this time, the generation rate of ozone was quantified in advance with an aqueous potassium iodide solution, and ozone equivalent to MEL-A was blown in. After 20 minutes, trimethyl phosphorous acid was added, and then slowly returned to room temperature (25 ° C.) and stirring was continued for 70 minutes. The mixture was diluted with a mixed solvent of ethyl acetate and methanol (9: 1), washed with water, magnesium sulfate was added to the oil phase and dried, and the solvent was distilled off. After removing impurities by column chromatography using ethyl acetate as a developing solvent, 10% (v / v) ethanol was added to the developing solvent to purify 0.12 g of a colorless viscous liquid.

(Structural analysis of product by NMR measurement)
FIG. 2 is a diagram showing a ¹ H NMR spectrum of the target product, fatty acid-terminated aldehyded MEL. As is clear from FIG. 2b, the 5.4 ppm peak specific to the carbon-carbon double bond disappeared, and as shown in FIG. 2a, a peak derived from an aldehyde group appeared near 9.8 ppm. . In addition, the integral value of protons derived from fatty acid terminal methyl groups was greatly reduced from about 6 to 3.7. From the above spectral change, the production of the target fatty acid-terminated aldehyded MEL was confirmed. The chemical shifts are summarized below.
¹ H NMR (DMSO, δ): 0.87 (t, 3.7H, C H ₃ CH ₂ CH ₂ ), 1.2-1.4 (m, 14H, CH ₂ ), 1.5 (m, 1.2H, -C H ₂ CH ₂ COO), 1.64 (m, 1.5H, -C H ₂ CH ₂ COO), 1.97-2.03 (m, 7H, C H ₂ -CHO, CH ₃ COO), 2.24 (t, 1.2H, CH ₂ COO), 2.30-2.60 (m, 2H, CH ₂ COO) 2.60-2.80 (m, 2H), 3.40-3.55 (m, 3.5H), 3.89 (m, 2H), 4.00 (d, 1H), 4.16 (dd, 2H ), 4.37 (s, 1H), 4.58 (s, 1H), 4.65 (s, 1H), 4.97 (s, 1H), 5.04 (d, 1H), 5.15 (d, 1H), 5.36 (m, 1H) , 9.60-9.70 (m, 0.82H, CHO)

(Molecular weight measurement of fatty acid terminal aldehyde MEL)
FIG. 3 is a diagram showing a spectrum in the vicinity of the molecular weight of the quasi-molecular ion of the main component, which is a MALDI-TOF / MS measurement result of the obtained fatty acid-terminated aldehyded MEL.
As apparent from FIG. 3, pseudomolecular ions [M + Na] ⁺ m / z 630.0 and 601.9 were detected in this glycolipid in MALDI-TOF / MS analysis. Therefore, in the fatty acid terminal aldehyde MEL of the present invention, a C10 linear saturated fatty acid is ester-bonded to either the 2 ′ or 3 ′ hydroxyl group of the mannosylerythritol skeleton represented by the following formula (7). The main component is a compound having a structure in which an ester having an aldehyde group at the end of the ethylene chain is bonded to the other end, and the linear saturated fatty acid represented by the following formula (8) is C8. It turns out that a certain compound is contained a lot.

Example 1-2
(Olefin metathesis reaction of MEL-A)

In the above formula, Cy represents a cyclohexyl group.
MEL-A 68 mg (0.1 mmol), allyl glycidyl ether 57 mg (0.5 mmol), second generation Grubbs catalyst represented by the above formula (C ₄₆ H ₆₅ Cl ₂ N ₂ PRu, FW: 848.98, CAS 246047) -72-3) 4 mg (5 μmol) was dissolved in 2.5 mL of dichloromethane, purged with nitrogen, sealed, and stirred at room temperature for 3 days. A TLC chart of the product after the reaction is shown in FIG.
As apparent from FIG. 4, a spot of fatty acid-terminated epoxidized MEL as a product appeared at an Rf value of 0.65 with respect to an Rf value of 0.71 of MEL-A.
After the reaction, the solvent was removed by an evaporator, and a homo-coupled product of allyl glycidyl ether (chloroform) and unreacted MEL (chloroform / acetone = 70/30) by column chromatography using a mixed solvent of chloroform and acetone as a developing solvent. (Vol / vol)) was removed, and then 16 mg of colorless viscous liquid (chloroform / acetone = 50/50 (vol / vol)) was purified.

(Structural analysis of product by NMR measurement)
As a result of ¹ H NMR measurement of the purified fatty acid-terminated epoxidized MEL, a peak derived from an epoxy group appeared in the vicinity of 2.6 ppm, 2.85 ppm, and 3.2 ppm. In addition, the integral value of protons derived from fatty acid terminal methyl groups was greatly reduced from about 6 to 2.8. From the above spectrum change, it was confirmed that the target product, fatty acid-terminated epoxidized MEL, was produced. The chemical shifts are summarized below.
¹ H NMR (CDCl ₃ , δ): 0.85-1.0 (m, 2.8H, C H ₃ CH ₂ CH ₂ ), 1.2-1.5 (br, 7H, CH ₂ ), 1.55-1.7 (m, 4H, -C H ₂ CH ₂ COO), 1.97-2.1 (m, 7H, C H ₂ -CHO, CH ₃ COO), 2.2-2.3 (t, 2H, CH ₂ COO), 2.4-2.5 (t, 2H, CH ₂ COO ), 2.6-2.7 (br, 1H, epoxy), 2.8-2.9 (br, 1H, epoxy), 3.2-3.4 (br, 1H, epoxy), 3.6-3.85 (br, 6H), 4.0 (dd, 1H) 4.16-4.3 (m, 2H), 4.78 (s, 1H), 5.09 (dd, 1H), 5.25 (t, 1H), 5.3-5.45 (br, 4H), 5.52 (d, 1H).

(Molecular weight measurement of fatty acid-terminated epoxidized MEL)
As a result of MALDI-TOF / MS measurement of the obtained fatty acid-terminated epoxidized MEL, pseudo molecular ion [M + Na] ⁺ m / z 725.7 was detected. Therefore, in the fatty acid-terminated epoxidized MEL of the present invention, a linear unsaturated fatty acid having 8 carbon atoms is present at either the 2 ′ or 3 ′ hydroxyl group of the mannosylerythritol skeleton represented by the following formula (9). It was found that the main component is a compound having a structure in which a linear unsaturated fatty acid ester having 9 carbon atoms having an ester bond and a glycidyl ether group bonded to the other end is bonded to the other end.

(Observation of surface activity)
A dilute aqueous solution of the above-obtained fatty acid-terminated aldehyded MEL was prepared and spotted on a hydrophobic film, and the change in the surface tension was observed. Further, water was compared as a control, and an aqueous solution of MEL-A used as a starting material was spotted for comparison.
As a result, when the diameter of the droplet 3 minutes after the solution spot was measured, the water spot was 5 mm in diameter, the fatty acid-terminated aldehyded MEL spot was 6.5 mm, and the MEL-A spot was 8 mm.
It was shown that fatty acid-terminated aldehyded MEL has a reduced surface tension of aqueous solution and has a function as a surfactant. In addition, the spot spread of the aqueous solution of fatty acid-terminated aldehyded MEL was smaller than that of MEL-A. Fatty acid-terminated aldehyded MEL is presumed to be a more hydrophilic biosurfactant because an aldehyde group is introduced at the end of a fatty acid ester which is a hydrophobic group. Therefore, the affinity with the hydrophobic film was lower than that of the conventional MEL, and the spread of the spot was expected to be slightly smaller, but the result of this observation was in accordance with that.

Example 1-3
(Production of MEL-bonded cellulose gel)
1 g (wet weight) of side chain aminated cellulose gel (trade name Amino-Cellulofine: manufactured by Seikagaku Corporation) was dispersed in 2 mL of 0.1 mol / L phosphate buffer (pH 7) and obtained in Example 1. 60 mg of fatty acid-terminated aldehyded MEL and 31 mg of sodium cyanoborohydride were added and stirred in a 70 ° C. oil bath for 3 days. After completion of the reaction, the product was sufficiently washed with water and ethanol, and dried under reduced pressure to obtain about 100 mg of a white powdered gel.

(Elemental analysis of MEL-bonded cellulose gel)
Elemental analysis of the obtained gel was performed. The results are summarized in Table 1. The C / N ratio (mol / mol) obtained from the result was 143.8 in the gel of the present invention with respect to 117.2 of the original cellulose gel, and 26.6 carbons were introduced into one amino group. Was confirmed. From the results of the molecular weight measurement performed in Example 1-1, the main component of the fatty acid-terminated aldehyded MEL used for the introduction is a compound having 28 carbon atoms having a structure represented by the general formula (7). Therefore, the introduction rate was 26.6 / 28 × 100 = 95 (%), and it was confirmed that MEL could be introduced with high efficiency.

Example 2
(Antibody separation using MEL-conjugated cellulose gel)
Using the gel obtained in Example 1-3, selective adsorption / elution test of antibody protein was performed. 80 mg (dry weight) of the gel was weighed into a centrifuge tube, and an experiment was performed according to the following procedure. As a control experiment, 160 mg (wet weight) of the original gel not adsorbed with MEL was weighed into a centrifuge tube, and the same experiment was performed.

(1) Blocking treatment with serum albumin 1.5 mL of 40 mg / mL human serum albumin (HSA) solution was added and stirred for 2 hours. As the solvent, a 50 mmol / L phosphate buffer solution (pH 4.6) in which sodium sulfate was dissolved so as to be 1 mol / L was used. After stirring, the mixture was centrifuged and the supernatant was recovered.
Subsequently, the solution was washed with 3.3 mL of the same solvent three times (total 10 mL), and the supernatant was recovered after centrifugation.
The collected supernatant was subjected to HPLC analysis, and protein was quantified from the peak area using a UV detector with a wavelength of 280 nm. In both gels, almost all of the added HSA was contained in the supernatant after operation. It was confirmed that From the above, it was confirmed that the gel obtained in Example 1-3 hardly caused non-specific adsorption of HSA due to hydrophobic interaction.

(2) Adsorption of antibody protein Subsequently, (1) 3 mL of a 1 mg / mL human immunoglobulin (IgG) solution was added to the gel after the operation and stirred for 2 hours. As the solvent, the same buffer as in (1) was used. After stirring, the mixture was centrifuged and the supernatant was recovered.
Subsequently, the solution was washed with 3.3 mL of the same solvent three times (total 10 mL), and the supernatant was recovered after centrifugation.

(3) Elution of antibody protein Following the operation of (2) above, washing with 3.3 mL each of 50 mmol / L phosphate buffer solution (pH 7) was performed three times (total 10 mL), and the supernatant was recovered after centrifugation. .
The collected supernatant was subjected to HPLC analysis, and protein was quantified from the peak area using a UV detector with a wavelength of 280 nm. The results are summarized in Table 2. The gel to which MEL was bound significantly increased the amount of recovered antibody by about 7 times compared to the control.

Is a diagram showing the ¹ H NMR spectrum of the MEL-A obtained in Reference Example. 1 is a diagram showing a ¹ H NMR spectrum of fatty acid-terminated aldehyded MEL obtained in Example 1. FIG. It is a figure which shows the mass spectrum of the molecular weight vicinity of the quasi-molecular ion of a main component which is a MALDI-TOF / MS measurement result of the fatty acid terminal aldehyde-ized MEL obtained in Example 1. It is a TLC chart which shows that the fatty acid terminal epoxidized MEL produced | generated in Example 1-2.

Claims (25)

  A mannosyl, wherein a mannosyl erythritol lipid compound is bonded to a part or all of the reactive functional group of a polymer having a reactive functional group in a main chain or a side chain through a covalent bond at a fatty acid terminal. Erythritol lipid binding polymer.   The mannosyl erythritol lipid bond according to claim 1, wherein the polymer forming the main skeleton of the polymer having a reactive functional group in the main chain or side chain is a polysaccharide, a protein, or an organic synthetic polymer. polymer.   The reactive functional group of the polymer having a reactive functional group in the main chain or side chain is an amino group, carboxyl group, hydroxyl group, aldehyde group, thiol group, epoxy group, isocyanate group, ester group, sulfone group, The mannosyl erythritol lipid-bonded polymer according to claim 1, wherein the polymer is a phenol group, an azide group or a carbon-carbon double bond. Through the formation of a Schiff base between the aldehyde group of the fatty acid-terminated aldehyde-modified mannosyl erythritol lipid compound represented by the following general formula (1) or (2) and the amino group of a polymer having an amino group in the main chain or side chain, A mannosyl erythritol lipid-bonded polymer obtained by covalently bonding the mannosyl erythritol lipid compound to the polymer.
(In the general formula (1), n and m are each a number of 1 to 18 and constitute a saturated aliphatic alkylene group, and ^one of R ¹ and R ² is an aldehyde group and the other is methyl. Or both are aldehyde groups, and R ³ and R ⁴ each represents a hydrogen atom or an acetyl group.
Moreover, in the said General formula (2), l is a number of 1-18, R < ⁵ >, R < ⁶ >, R < ⁷ > represents a hydrogen atom or an acetyl group each independently. )   A polymer having a reactive functional group in the main chain or side chain and a mannosyl erythritol lipid compound having at least one reactive functional group at a fatty acid terminal are bonded via a covalent bond by a reaction between the reactive functional groups. The method for producing a mannosyl erythritol lipid-bonded polymer according to claim 1, wherein the polymer is bound.   The reactive functional group of the mannosyl erythritol lipid compound having at least one reactive functional group at the fatty acid terminal is an amino group, a carboxyl group, a hydroxyl group, an aldehyde group, a thiol group, an epoxy group, an isocyanate group, or an ester group. The method for producing a mannosyl erythritol lipid-bonded polymer according to claim 5, which is a sulfone group, a phenol group, an azide group or a carbon-carbon double bond.   Using a mannosyl erythritol lipid compound produced by a microorganism as a raw material, a reactive functional group is introduced into a fatty acid terminal by an ozone oxidation reaction or a metathesis reaction of a carbon-carbon double bond contained in the fatty acid chain. A method for producing a mannosyl erythritol lipid compound having at least one reactive functional group at a fatty acid terminal according to claim 5 or 6. A mannosyl erythritol lipid compound having at least one reactive functional group at a fatty acid terminal represented by any one of the following general formulas (1) to (4).
(In the general formula (1), n and m are each a number of 1 to 18 and constitute a saturated aliphatic alkylene group, and ^one of R ¹ and R ² is an aldehyde group and the other is methyl. Or both are aldehyde groups, and R ³ and R ⁴ each represents a hydrogen atom or an acetyl group.
In formula (2), l is the number of ^{1~18, R 5, R 6,} R 7 each independently represent a hydrogen atom or an acetyl group.
In the general formula (3), p and q each represent a number of 1 to 18 and constitute a saturated aliphatic alkylene group, and one of R ³¹ and R ³² is a 2-allyloxymethyloxirane group. And the other is a methyl group, or both are 2-allyloxymethyloxirane groups. R ³³ and R ³⁴ each represent a hydrogen atom or an acetyl group.
Moreover, in said general formula (4), r is a number of 1-18, and R < ³⁵ >, R < ³⁶ >, R < ³⁷ > represents a hydrogen atom or an acetyl group each independently. )   A method for detecting a biomolecule, comprising using the mannosyl erythritol lipid-binding polymer according to claim 1.   A method for separating a biomolecule, comprising using the mannosyl erythritol lipid-binding polymer according to claim 1.   A method for collecting biomolecules, comprising using the mannosyl erythritol lipid-binding polymer according to claim 1.   The method according to claim 9, wherein the biomolecule is an antibody protein, glycoprotein or lectin.   A biomolecule adsorbent comprising the mannosylerythritol lipid-binding polymer according to claim 1.   Has hydrophilic, highly dispersible, highly fluid, antistatic or anticorrosive properties, or has antibacterial, antiviral, antitumor, antithrombogenic, cell adhesion suppression or protein adsorption suppression 14. The biomolecule adsorbent according to claim 13, which is provided.   An affinity carrier comprising the mannosyl erythritol lipid-binding polymer according to claim 1.   Has hydrophilic, highly dispersible, highly fluid, antistatic or anticorrosive properties, or has antibacterial, antiviral, antitumor, antithrombogenic, cell adhesion suppression or protein adsorption suppression The affinity carrier according to claim 15, which is provided.   An affinity column comprising the mannosyl erythritol lipid-binding polymer according to claim 1.   Has hydrophilic, highly dispersible, highly fluid, antistatic or anticorrosive properties, or has antibacterial, antiviral, antitumor, antithrombogenic, cell adhesion suppression or protein adsorption suppression The affinity column according to claim 17, which is provided.   A test reagent comprising the mannosyl erythritol lipid-binding polymer according to claim 1.   Has hydrophilic, highly dispersible, highly fluid, antistatic or anticorrosive properties, or has antibacterial, antiviral, antitumor, antithrombogenic, cell adhesion suppression or protein adsorption suppression The reagent for a test | inspection of Claim 19 characterized by the above-mentioned.   A sensor comprising the mannosyl erythritol lipid-binding polymer according to claim 1.   Has hydrophilic, highly dispersible, highly fluid, antistatic or anticorrosive properties, or has antibacterial, antiviral, antitumor, antithrombogenic, cell adhesion suppression or protein adsorption suppression The sensor according to claim 21, wherein the sensor is applied.   A microarray using the mannosyl erythritol lipid-binding polymer according to claim 1.   Has hydrophilic, highly dispersible, highly fluid, antistatic or anticorrosive properties, or has antibacterial, antiviral, antitumor, antithrombogenic, cell adhesion suppression or protein adsorption suppression 24. The microarray according to claim 23, wherein the microarray is applied.   An analyzer for separation, quantification, or testing, comprising the affinity carrier according to claim 15, the affinity column according to claim 17, the sensor according to claim 21, or the microarray according to claim 23.