JP4976863B2 - Immobilized phospholipid metabolizing enzyme treated with surfactant and method for producing functional phospholipid using the same - Google Patents

Description

  The present invention relates to an immobilized phospholipid metabolizing enzyme treated with a surfactant and a method for producing a functional phospholipid using the same. More specifically, the immobilized phospholipid metabolizing enzyme can be a cell (particularly yeast) that presents the phospholipid metabolizing enzyme on the cell surface or a phospholipid metabolizing enzyme immobilized on a carrier.

  Phospholipids are one of the important biological components and have been confirmed to exist widely in nature. The phospholipid has in its molecule a hydrophobic site composed of two long-chain fatty acids and a hydrophilic (polar) site composed of a phosphate ester. Examples of the fatty acid include lauric acid, oleic acid and docosahexaenoic acid having different chain lengths and numbers of unsaturated bonds, and examples of the polar group include a choline group, an ethanolamine group, an inositol group, and a serine group. Phospholipids having different structures at each site have specific physiological activities and functions, and can be expected to be applied to various fields such as foods, cosmetics, industrial products, and pharmaceuticals. Therefore, technical development for efficiently producing phospholipids having the target molecular structure is an extremely important issue.

  Currently, soybean is mainly used as a raw material for industrial production of phospholipids. Specifically, low-purity phospholipids are mass-produced by using by-products (oil cake) generated from the refinement process of soybean oil. However, the production of functional phospholipids with the desired structure from naturally derived phospholipids such as soybeans and egg yolk is complicated by the extraction and purification process and the amount recovered is small. Not suitable.

  Conventionally, studies have been conducted to modify an inexpensive phospholipid raw material into a target structure using a group of enzymes (phospholipid metabolizing enzymes) acting on each part of the phospholipid shown in FIG. 1 (Non-patent Document 1). ~ 3). For example, the reaction of substituting the hydrophobic part of phospholipids with other fatty acids is called transesterification, and the reaction of substituting polar parts is called transphosphorylation. These reactions are called phospholipid metabolizing enzymes, phospholipids. It is carried out by dissolving the raw material and the fatty acid to be substituted (or polar group) in a specific organic solvent.

In general, phospholipid-metabolizing enzymes are obtained by purifying, concentrating, immobilizing and recovering enzymes contained in a culture solution of microorganisms. However, since the process is complicated, there is a disadvantage that the equipment cost and the running cost become very high.
JP 2004-49014 A I. Svensson et al., Journal of the American Oil Chemists' Society, 1992, 69, 986-991. CW Park et al., Biotechnology Letter, 2000, 22, pp.147-150 M. Hosokawa et al., Journal of agricultural and food chemistry, 2000, 48, 4550-4554 T. Matsumoto et al., Applied and Environmental Microbiology, 2002, 68, 4517-4522

  In the previous studies, the present inventors have developed a vector system for producing and fixing (presenting) the target enzyme lipase on the cell surface of yeast (Patent Document 1 and Non-Patent Document 4). Accordingly, the object of the present invention is to develop a functional phospholipid production system using yeast that presents phospholipid metabolizing enzymes on the cell surface as a whole cell catalyst.

  In general, microorganisms such as yeast have a high affinity for water, and it is considered difficult to disperse them in a hydrophobic organic solvent such as hexane which is frequently used for ester synthesis. For this reason, contact between the enzyme localized on the cell surface and the substrate in the solvent is hindered, resulting in a decrease in productivity. Therefore, in order to apply the surface-displaying yeast of the phospholipid metabolizing enzyme to the synthesis reaction of functional phospholipid, it is necessary to impart cell affinity to the organic solvent and to efficiently contact the surface lipase with the substrate. .

  The present invention is completed by treating the surface layer of a phospholipid-metabolizing enzyme surface-presenting yeast cell with a surfactant soluble in an organic solvent, and confirming that yeast having high reactivity can be used for the synthesis of functional phospholipids. It was done. It was also clarified that the same treatment can be applied to an immobilized phospholipid metabolizing enzyme using an ion exchange resin as a carrier.

  The present invention provides an immobilized phospholipid metabolizing enzyme treated with a surfactant.

The present invention also includes contacting the immobilized phospholipid metabolizing enzyme with a surfactant; and then washing the immobilized phospholipid metabolizing enzyme with distilled water;
A method for producing an immobilized phospholipid metabolizing enzyme treated with a surfactant is provided.

  In one embodiment, the phospholipid metabolizing enzyme is presented on the cell surface.

  In a preferred embodiment, the cell is a yeast cell.

  In another embodiment, the phospholipid metabolizing enzyme is an isolated or extracted purified or crude enzyme.

  In a further embodiment, the surfactant is a nonionic surfactant.

  In a preferred embodiment, the nonionic surfactant is a Tween surfactant.

  In yet another embodiment, the phospholipid metabolizing enzyme is a lipase.

The present invention further provides a method for producing a functional phospholipid, comprising the step of bringing any of the above immobilized phospholipid metabolizing enzymes into contact with the phospholipid and a substrate; and the step of recovering the obtained functional phospholipid. provide.

  In one embodiment, the phospholipid metabolizing enzyme is a lipase and the substrate is a fatty acid.

  According to the present invention, a highly reactive phospholipid metabolizing enzyme is provided by a simple process. Therefore, this enzyme can be used for the synthesis of functional phospholipids, and the current production process of functional phospholipids can be simplified. Therefore, a significant cost reduction in the production of phospholipid can be achieved.

  In the present invention, the phospholipid metabolizing enzyme refers to an enzyme group that acts on each site of phospholipid. For example, as shown in FIG. 1, an enzyme that acts on a fatty acid ester moiety (for example, lipase, phospholipase A1 (PLA1), phospholipase A2 (PLA2)), an enzyme that acts on a phosphate ester moiety (for example, phospholipase D (PLD)). ) And the like. The phospholipid metabolizing enzyme to be immobilized is appropriately selected according to the synthesis reaction of the target phospholipid, and is not particularly limited. Moreover, the origin of these phospholipid metabolizing enzymes is not limited, For example, the thing derived from microorganisms is used suitably. The microorganism may be a naturally occurring wild type or a transformant.

  In the present invention, the immobilized phospholipid metabolizing enzyme refers to a phospholipid metabolizing enzyme immobilized on an arbitrary carrier. It may be an immobilized enzyme immobilized on a general carrier such as a resin, or may be an enzyme displayed on the surface layer of a cell. As will be described later, a cell in which the phospholipid metabolizing enzyme is immobilized on the cell surface (that is, the phospholipid metabolizing enzyme is presented on the cell surface) may be further immobilized on an arbitrary carrier.

  As the phospholipid-metabolizing enzyme immobilized on a carrier, generally, a purified enzyme or a crudely purified enzyme isolated or extracted from a natural product or a recombinant is used. Examples of the carrier on which the purified enzyme or the crudely purified enzyme is immobilized include a carrier usually used for immobilizing the enzyme. Examples thereof include organic polymer compounds such as various ion exchange resins, inorganic porous materials such as ceramics, and the like. For the immobilization, for example, methods commonly used by those skilled in the art, such as a carrier binding method, a crosslinking method, and an inclusion method, can be applied. The carrier binding method includes a chemical adsorption method or a physical adsorption method for adsorbing to an ion-exchange resin.

  In the present invention, the cell that presents the phospholipid metabolizing enzyme on the cell surface is not particularly limited as long as it has a cell wall, such as a bacterium, a fungus, or a plant cell. Preferably yeast is used.

  A general method for presenting phospholipid metabolizing enzymes on the cell surface will be described. As a method for presenting phospholipid metabolizing enzymes on the cell surface, (a) a method for presenting phospholipid metabolizing enzymes to the cell surface via the GPI anchor of the cell surface localized protein, and (b) cell surface localized protein There is a method for presenting a phospholipid metabolizing enzyme to the cell surface layer via a sugar chain binding protein domain.

  Cell surface localized proteins that can be used include α- or a-agglutinin which is a sex aggregation protein of yeast, FLO proteins (eg, FLO1, FLO2, FLO4, FLO5, FLO9, FLO10, and FLO11), alkaline phosphatase, and the like. Can be mentioned.

(A) Method using GPI anchor A gene encoding a protein localized on the cell surface by the GPI anchor includes, in order from the N-terminal side, a secretory signal sequence, a cell surface localized protein (sugar chain binding protein domain), and GPI. It has a gene encoding each anchor adhesion recognition signal sequence. A cell surface localized protein (glycan binding protein) expressed from this gene in the cell is guided to the outside of the cell membrane by a secretion signal, and the GPI anchor attachment recognition signal sequence is selectively cleaved at the C-terminal. It binds to the GPI anchor of the cell membrane via the portion and is fixed to the cell membrane. Thereafter, the root of the GPI anchor is cut by PI-PLC, incorporated into the cell wall, fixed to the cell surface, and presented to the cell surface.

  Here, the GPI anchor refers to a glycolipid having a basic structure of ethanolamine phosphate-6 mannose α1-2 mannose α1-6 mannose α1-4 glucosamine α1-6 inositol phospholipid, called glycosylphosphatidylinositol (GPI). , PI-PLC refers to phosphatidylinositol-dependent phospholipase C.

  The GPI anchor attachment recognition signal sequence is a sequence recognized when the GPI anchor binds to the cell surface localized protein, and is usually located at or near the C-terminal of the cell surface localized protein. As the GPI anchor attachment signal sequence, for example, the sequence of the C-terminal part of yeast α-agglutinin is preferably used. Since the GPI anchor adhesion recognition signal sequence is included in the C-terminal side of the sequence of 320 amino acids from the C-terminal of α-agglutinin, the gene used in the above method encodes a sequence of 320 amino acids from the C-terminal. DNA sequences are particularly useful.

  Therefore, for example, a DNA encoding a secretory signal sequence-a structural gene encoding a cell surface localized protein-a sequence having a DNA sequence encoding a GPI anchor adhesion recognition signal, and a structural gene encoding this cell surface localized protein By substituting all or part of the sequence with the sequence of the structural gene of the target phospholipid metabolizing enzyme, recombinant DNA presenting the target phospholipid metabolizing enzyme on the cell surface via the GPI anchor can be obtained. When the cell surface localized protein is α-agglutinin, it is preferable to introduce a target phospholipid metabolizing enzyme gene so as to leave a sequence encoding a 320 amino acid sequence from the C-terminal of α-agglutinin. Thus, the C-terminal side of the phospholipid metabolizing enzyme presented on the cell surface is fixed to the surface.

(B) Method using a sugar chain binding protein domain When the cell surface localized protein is a sugar chain binding protein, the sugar chain binding protein domain has a plurality of sugar chains, and these sugar chains are sugars in the cell wall. It is possible to stay on the cell surface by interacting or entanglement with the chains. Examples thereof include sugar chain binding sites such as lectins and lectin-like proteins. Typically, the aggregation functional domain of GPI anchor protein is mentioned. The aggregation functional domain of the GPI anchor protein is a domain that is located on the N-terminal side of the GPI anchoring domain, has a plurality of sugar chains, and is considered to be involved in aggregation.

  By binding this cell surface localized protein (aggregation functional domain) and the target phospholipid metabolizing enzyme, the enzyme is presented on the cell surface. For example, (1) a phospholipid metabolizing enzyme is bound to the N-terminal side of the cell surface localized protein (aggregation functional domain), (2) a phospholipid metabolizing enzyme is bound to the C-terminal side, and (3) the N-terminal side The same or different phospholipid metabolizing enzymes can be bound to both the C-terminal side and the C-terminal side. The N-terminal side or C-terminal side of the phospholipid metabolizing enzyme presented on the cell surface in this way is fixed to the surface layer.

  The secretory signal sequence used for the recombinant DNA may be the secretory signal sequence of the cell surface localized protein, or other secretory signal sequence that can guide the expressed phospholipid metabolizing enzyme to the outside of the cell. Also good. For example, a glucoamylase secretion signal sequence, a yeast α- or a-agglutinin secretion signal sequence, and a lipase secretion signal sequence are preferably used. If the enzyme activity is not affected, a part or all of the secretory signal sequence and pro-sequence may remain at the N-terminus after cell surface display.

  The cell used in the present invention is a cell transformed by introducing DNA so that the target phospholipid metabolizing enzyme is presented on the cell surface. The introduced DNA includes at least a secretory signal sequence, a sugar chain binding protein domain, and a sequence encoding a target phospholipid metabolizing enzyme. Furthermore, it may contain a sequence encoding another same or another phospholipid metabolizing enzyme.

  Synthesis and binding of DNA containing the various sequences described above can be performed by techniques that can be commonly used by those skilled in the art.

  The DNA is preferably in the form of a plasmid. From the viewpoint of simplification of DNA acquisition, a shuttle vector with E. coli is preferable. The starting material for this DNA has, for example, a 2 μm plasmid origin of replication (Ori) and a ColE1 origin of replication, and yeast selectable markers (eg, drug resistance genes, TRP, LEU2 etc.) and E. coli. It is more preferable to have a selection marker (such as a drug resistance gene). In addition, in order to express the structural gene of the enzyme, it is desirable to include so-called regulatory sequences such as an operator, promoter, terminator, enhancer and the like that regulate the expression of this gene. Examples include the GAPDH (glyceraldehyde 3'-phosphate dehydrogenase) promoter and the GAPDH terminator. Examples of such starting material plasmids include GAPDH (glyceraldehyde 3′-phosphate dehydrogenase) promoter sequence and GAPDH terminator sequence plasmid pYGA2270 or pYE22m, or UPR-ICL (isocitrate lyase upstream region) sequence and And a plasmid pWI3 containing Term-ICL (terminator region of isocitrate lyase) sequence.

  Preferably, a DNA encoding a desired phospholipid metabolizing enzyme between the GAPDH promoter sequence and GAPDH terminator sequence of plasmid pYGA2270 or pYE22m, or between the UPR-ICL sequence and Term-ICL sequence of plasmid pWI3. Is used to produce a plasmid used for introduction into yeast. In the present invention, a multi-copy type plasmid pWIFS or pWIFL is preferably used. For example, pWIFS-ProROL (see Non-Patent Document 4), pWIFS-PLA1, or pWIFL-ProROL is produced.

  Any yeast may be used as the host yeast, but aggregating yeast is preferable in that it can be easily separated after the reaction, or can be fixed easily and can perform a continuous reaction. Alternatively, when an aggregation functional domain is used as the sugar chain binding protein domain, strong aggregability can be imparted to any yeast.

  The cells used in the method of the present invention can be obtained by introducing the above DNA into the cells. The introduction of DNA means that DNA is introduced into a cell and expressed. Methods for introducing DNA include methods such as transformation, transduction, transfection, co-transfection, and electroporation. Specific examples include a method using lithium acetate and a protoplast method.

  The DNA to be introduced may be in the form of a plasmid as described above, or may be inserted into a chromosome by being inserted into a host gene or undergoing homologous recombination with a host gene.

  Cells into which DNA has been introduced are selected with a selectable marker (eg, TRP) and selected by measuring the activity of the expressed phospholipid metabolizing enzyme. The fact that the phospholipid metabolizing enzyme is immobilized on the cell surface layer can be confirmed by an immunoantibody method using an anti-protein antibody against this phospholipid metabolizing enzyme and a FITC-labeled anti-IgG antibody.

  Recombinant cells presenting the phospholipid metabolizing enzymes obtained as described above on the cell surface can be cryopreserved or cryopreserved in a suspension containing a medium capable of maintaining the cells, or cryopreserved or It can be stored lyophilized.

  The phospholipid metabolizing enzyme surface-presenting cell used in the present invention may be immobilized on a carrier. As the material of the carrier that can be used in the present invention, for example, a foam or a resin such as polyvinyl alcohol, polyurethane foam, polystyrene foam, polyacrylamide, polyvinyl formal resin porous body, silicon foam, and cellulose porous body is preferable. In consideration of dropping of cells whose growth and activity are reduced or dead cells, a porous carrier is preferable. The size of the opening of the porous body varies depending on the cell, but it is appropriate that the cell can sufficiently enter and grow. Although 50 micrometers-1000 micrometers are suitable, it is not limited to this.

  The shape of the carrier is not limited. Considering the strength of the carrier, culture efficiency, etc., the shape is spherical or cubic, and the size is preferably 2 mm to 50 mm in the case of a sphere, and 2 mm to 50 mm square in the case of a cube.

  In the present specification, the immobilization of yeast means a state in which the yeast is not in a free state, for example, a state in which the yeast is bound to or attached to a carrier or taken into the carrier. For the immobilization of yeast, for example, methods commonly used by those skilled in the art, such as a carrier binding method, a crosslinking method and an inclusion method, can be applied. Among these, the carrier binding method is optimal for immobilizing aggregating yeast. The carrier binding method includes a chemical adsorption method or a physical adsorption method for adsorbing to an ion-exchange resin.

  In the present invention, the immobilized phospholipid metabolizing enzyme is treated with a surfactant. Here, the treatment of the phospholipid metabolizing enzyme with the surfactant refers to bringing the phospholipid metabolizing enzyme into contact with the surfactant, and the phospholipid metabolizing enzyme is coated with the surfactant by this treatment. For example, a surfactant-treated phospholipid-metabolizing enzyme can be obtained by placing the immobilized phospholipid-metabolizing enzyme in an aqueous solution of a surfactant, shaking, and then washing with distilled water.

  The surfactant used in the present invention is not particularly limited, but those generally used in the biochemical region are preferable. Any of ionic surfactants (anionic surfactants, cationic surfactants, and amphoteric surfactants) and nonionic surfactants may be used.

  In the present invention, a nonionic surfactant is particularly preferably used. As a nonionic surfactant, such a nonionic surfactant includes, for example, fatty acid type (nonionic) (for example, sucrose fatty acid ester sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, fatty acid alkanolamide). Higher alcohols (nonionic) (for example, polyoxyethylene alkyl ether), and alkylphenols (for example, polyoxyethylene alkylphenyl ether). More specifically, a Tween surfactant, a Triton surfactant, and a Brij surfactant are included. In the present invention, Tween 20, Tween 40, Tween 60, and Tween 80, and more preferably Tween 20 are used.

  In the present invention, anionic surfactants are preferred among the ionic surfactants. Examples of the anionic surfactant include sodium alkyl sulfate (for example, sodium dodecyl sulfate (SDS)), alkylbenzene sulfonate, sodium cholate and the like.

  In the treatment step of the immobilized phospholipid metabolizing enzyme, the immobilized phospholipid metabolizing enzyme is brought into contact with the surfactant in an aqueous solution. The concentration of the aqueous surfactant solution varies depending on the surfactant used. When a nonionic surfactant is used, it is usually 0.05% (w / v) to 5% (w / v), preferably 0.1% (w / v) to 3% (w / v). More preferably, it is 0.5% (w / v) to 2% (w / v). The contact time varies depending on the concentration, but is usually 1 to 48 hours, preferably 6 to 36 hours, and more preferably 12 to 24 hours. The temperature at the time of contact may be a temperature at which the enzyme is not deactivated, and is usually within a range of 20 to 37 ° C. It is preferable to wash the enzyme with water after the contact between the enzyme and the surfactant.

  A method for producing a functional phospholipid using a phospholipid metabolizing enzyme treated with a surfactant as described above is provided. In the present invention, the functional phospholipid means a phospholipid modified so as to have a target structure, and the structure is not specified. For example, the modified phospholipid obtained by transesterification which substitutes the hydrophobic part of a phospholipid with another fatty acid, or the phosphate group transfer which substitutes the polar part of a phospholipid is mentioned.

  The origin of the phospholipid used in the method of the present invention is not particularly limited, and it may be derived from a natural product (for example, an extract or a concentrate), or may be chemically synthesized. Examples of such phospholipids include phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and the like, and may be a mixture thereof. .

  The constituent fatty acids of these phospholipids are the same or different saturated or unsaturated fatty acids having 8 to 24 carbon atoms. Such fatty acids include caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, arachidic acid, palmitooleic acid, oleic acid, linoleic acid, α- and γ-linolenic acid, erucic acid, Examples include arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and tetracosatetraenoic acid.

  The fatty acid used for the transesterification reaction is appropriately selected according to the purpose. Preferably, it is the same saturated or unsaturated fatty acid having 8 to 24 carbon atoms. Such fatty acids are as described above.

  The ester used for the phosphoric acid group transfer reaction may be a monobasic acid ester or an acid ester of a dibasic acid or higher. In the case of an ester of an acid that is a dibasic acid or more, a neutral ester is preferred. Examples of the carboxylic acid ester include acetate ester, propionate ester, succinate ester, maleate ester, and benzoate ester.

  The reaction solution used in the method for producing a functional phospholipid of the present invention is not particularly limited as long as the phospholipid metabolizing enzyme immobilized on a cell surface layer or a carrier and treated with a surfactant can exhibit enzyme activity. It is not limited, A buffer solution and an organic solvent may be sufficient. As described above, when the phospholipid metabolizing enzyme is lipase, it is preferable to use an organic solvent other than the ester and alcohol. Examples of such an organic solvent include pentane, hexane, cyclohexane, heptane, octane, nonane, decane, benzene, toluene, diethyl ether, acetone, acetonitrile, chloroform, dichloroethane, and tetrahydrofuran.

  In the method of the present invention, for example, by mixing the immobilized phospholipid-metabolizing enzyme and the reaction solution containing the substrate (for example, phospholipid and fatty acid), the enzyme and the substrate are contacted, Phospholipids can be obtained. The reaction conditions in this case are not particularly limited as long as the immobilized enzyme can exhibit enzyme activity, and the substrate concentration, reaction temperature, reaction time, and the like can be appropriately determined by those skilled in the art. . After completion of the reaction, the target product can be recovered and purified by means according to the product (for example, extraction, column chromatography, etc.).

  The functional phospholipid thus obtained can be used as a phospholipid as it is as a raw material for foods and pharmaceuticals.

(Preparation Example 1: Preparation of lipase cell surface display yeast)
As a yeast strain, Saccharomyces cerevisiae MT8-1 (MATa, ade, his3, leu2, trp1, ura3) (Tajima et al., Yeast, 1985, Vol. 1, pages 67-77) was used. The plasmid pWIFS-ProROL constructed in the previous study (Non-patent Document 4) was introduced into the yeast strain by the lithium acetate method. This plasmid is a vector having a gene for producing and fixing a lipase (ROL) derived from the filamentous fungus Rhizopus oryzae on the cell surface of yeast.

The transformed yeast was added to 5 mL of SD (Synthetic Dropout) medium [0.67% (w / v) DIFCO ^™ Yeast nitrogen base w / o amino acid, 2% (w / v) D-glucose, 100 mg / L L-leucine, and 20 mg / L adenine, uracil, and L-histidine, respectively] for 1 day (30 ° C., 150 opm) so that the initial cell density OD (Optical density) at a wavelength of 600 nm is 0.03. Inoculated into SDC medium [SD medium with 2% (w / v) casamino acid added]. The cells were cultured at 30 ° C. for 7 days. The cultured cells were washed with distilled water and lyophilized. This microbial cell was used as lipase cell surface display yeast (hereinafter abbreviated as ROL surface display yeast).

(Example 1: Preparation of lipase cell surface display yeast treated with surfactant)
The powdered ROL surface layer-presenting yeast obtained in Preparation Example 1 was suspended in distilled water so as to be 10 mg-dry cell / mL, and 1% (w / v) of various surfactants were added. As the surfactant, non-ionic surfactants polyoxyethylene sorbitan monolaurate Tween 20, Tween 40, Tween 60, and Tween 80, and anionic surfactant sodium dodecyl sulfate (SDS) were used. This suspension was shaken at 150 ° C. for 16 hours at 30 ° C., and the yeast surface layer was coated with these surfactants. The treated suspension was centrifuged at 8000 rpm for 5 minutes, and the operation of washing the precipitated yeast with distilled water was repeated twice, and then dried under reduced pressure. The white powder thus obtained was used as a surfactant-treated lipase surface-displaying yeast (hereinafter referred to as “surfactant-treated ROL surface-displaying yeast”).

(Reference Example 1: Examination of the effect of the amount of ROL surface-displaying yeast cells on the transesterification reaction)
As a phospholipid raw material, egg yolk-derived phosphatidylcholine (hereinafter abbreviated as PC) purchased from Wako Pure Chemical Industries, Ltd. was used. The transesterification reaction was initiated by dissolving 50 mg of PC and 100 mg of lauric acid (abbreviated as LA) in 5 mL of hexane, and adding various amounts of ROL surface-displaying yeast obtained in Preparation Example 1 above. The reaction temperature was 30 ° C. and the shaking conditions were 150 pm. In order to measure the transesterification rate at each reaction time, about 300 μL of a hexane layer was collected.

  Next, the collected hexane was volatilized, and the remaining lipid component was dissolved in a small amount (about 10 μL) of chloroform. This chloroform was developed by thin layer chromatography (developing solvent: chloroform / methanol / water, 65/35/4, v / v / v) to separate phospholipid, lysophospholipid, fatty acid and the like. Silica gel containing only the separated phospholipid was collected and added to 3 mL of a methanol / toluene solution (4/1, v / v) to extract phospholipid. 200 μL of acetyl chloride was added to this extract and stirred at 50 ° C. for 1 hour to methylate fatty acids contained in phospholipids. The composition of fatty acid methyl was analyzed by gas chromatography to determine the phospholipid fatty acid composition, and the transesterification rate was calculated. The results are shown in FIG.

  ROL surface layer display yeast is known to exhibit high activity in hydrolysis reaction and ester synthesis reaction without using an organic solvent (Non-patent Document 4). However, as shown in FIG. 2, in the transesterification reaction of PC using the untreated ROL surface-displaying yeast, the transesterification rate is hardly improved even if the amount of cells is increased, and at any amount of cells. A value of 10% or less was shown. This is considered to be a problem of the dispersibility of yeast in hexane, which is a hydrophobic organic solvent.

(Example 2: Transesterification of phospholipids using Tween 20-treated lipase surface-displaying yeast)
50 mg of PC and 100 mg of LA were dissolved in 5 mL of hexane, and 50 mg of Tween 20-treated ROL surface-displaying yeast obtained in Example 1 was added to initiate the reaction. The reaction temperature was 30 ° C. and the shaking condition was 150 opm. In addition, reaction was similarly performed about the ROL surface layer display yeast obtained by the said preparation example 1 as control. In order to measure the transesterification rate at each reaction time, about 300 μL of a hexane layer was collected.

  Next, the collected hexane was volatilized, and the remaining lipid component was dissolved in a small amount (about 10 μL) of chloroform. This chloroform was developed by thin layer chromatography (developing solvent: chloroform / methanol / water, 65/35/4, v / v / v) to separate phospholipid, lysophospholipid, fatty acid and the like. The silica gel fraction containing only the separated phospholipid was collected and added to 3 mL of a methanol / toluene solution (4/1, v / v) to extract phospholipid. 200 μL of acetyl chloride was added to this extract and stirred at 50 ° C. for 1 hour to methylate fatty acids contained in phospholipids. The composition of fatty acid methyl was analyzed by gas chromatography to determine the phospholipid fatty acid composition, and the transesterification rate was calculated. The results are shown in FIG.

  As shown in FIG. 3, when Tween 20 treatment was performed, a dramatic improvement in transesterification activity was confirmed. Therefore, it was found that Tween 20 treatment of the cell surface in the lipase surface-displaying yeast is extremely effective in modifying the molecular structure of the phospholipid.

(Example 3: Examination of type of surfactant)
For the ROL surface-displaying yeast treated with various surfactants obtained in Example 1 above, the transesterification rate was measured in the same manner as in Example 2 above. The reaction time was 48 hours. The results are shown in Table 1.

  As shown in Table 1, transesterification activity was improved in all nonionic surfactant (various Tween) -treated ROL surface-displaying yeasts. Among them, Tween 20 was the most effective.

(Example 4: Cell surface hydrophobicity of lipase surface display yeast)
In order to investigate the cause of the increase in transesterification activity due to the surfactant treatment, the hydrophobicity of the yeast cell surface was measured as follows.

First, ROL surface-displaying yeast and various surfactant-treated ROL surface-displaying yeasts are suspended in physiological saline [0.49% (wt) NaCl, 0.49% (wt) KCl)], and bacteria at a wavelength of 600 nm are used. Body concentration (OD ₆₀₀ ) was measured. The value at this time is set as C _1. To 3 mL of the yeast suspension, 1 mL of hexane was added, mixed by vortexing for 10 seconds, and allowed to stand for 10 minutes. The aqueous phase cell concentration of (lower layer) _{(OD 600)} was measured and this value as _{C 2.}

The formula for calculating the hydrophobicity of the cell surface (Hydropathy index: HI) is
(C ₁ -C ₂ ) / C ₂ = HI (1)
Is defined. Also, the equation (1) is
log (C ₁ −C ₂ ) = log C ₂ + log HI (2)
And can be converted. From this equation (2), when logC ₂ = 0, logHI = log (C ₁ -C ₂ ), so log (C ₁ -C ₂ ) is the Y (vertical) axis, and logC ₂ is the X (horizontal) axis. When an approximate straight line is obtained by plotting to, the value of the Y intercept indicates logHI. This log HI value was used as an index representing the hydrophobicity of the cell surface. For example, when cells have strong hydrophobicity, most cells migrate to the upper layer (hexane layer). In this case, since the Y intercept shows a higher value, a high log HI value indicates strong hydrophobicity. The results are shown in Table 2.

  As shown in Table 2, it was found that logHI, which is an index of hydrophobicity, increases by treatment with a nonionic surfactant. In particular, the increase in log HI was remarkable when Tween 20 was used. This was considered to be closely related to the increase in transesterification activity shown in Table 1. From the above results, it was suggested that the change in the hydrophobicity of the yeast cell surface due to the treatment with the nonionic surfactant may be largely involved in the transesterification activity of the phospholipid.

(Preparation Example 2: Preparation of immobilized lipase)
250 mg of R. oryzae-derived lipase powder (product name: Lipase F-AP15, Amano Enzyme Inc.) was dissolved in 5 mL of 100 mM phosphate buffer (pH 7). When performing treatment with Tween 20, 1% (w / v) Tween 20 was added to the buffer. 1 g of DIAION HPA-25 (Mitsubishi Chemical Corporation), which is an anion exchange resin, was added to the above solution and shaken at 20 ° C. and 150 opm to immobilize the lipase. The resin on which the lipase was immobilized was washed twice with distilled water and dried under reduced pressure to obtain an immobilized lipase.

(Example 5: Examination of influence on transesterification activity of Tween20-treated immobilized lipase)
It was verified whether Tween 20 treatment, which was effective for yeast cells, was also effective for immobilized lipases that are commonly used.

  The immobilized lipase obtained in Preparation Example 2 was operated in the same manner as in Example 1 to obtain a Tween 20-treated immobilized lipase. Subsequently, a transesterification reaction of PC was performed in the same manner as in Example 2 except that 50 mg of Tween 20-treated immobilized lipase was used. The reaction time was 48 hours. The results are shown in Table 3.

  As shown in Table 3, the transesterification rate of the Tween 20-treated immobilized lipase was significantly increased. Therefore, it was suggested that the surfactant treatment is effective not only for cells such as yeast but also when immobilized secretory enzymes are immobilized.

  According to the present invention, a highly reactive phospholipid metabolizing enzyme is provided by a simple process, which can be used for the synthesis of a functional phospholipid, and further simplifies the production process of the current functional phospholipid. it can. Therefore, a significant cost reduction in the production of phospholipid can be achieved. Therefore, functional phospholipids can be provided at a lower cost as raw materials for foods and pharmaceuticals.

It is a schematic diagram which shows the basic structure of phospholipid, and the action site | part of an enzyme. It is a graph which shows the relationship between the amount of ROL surface layer presentation yeast added to the transesterification reaction system, and transesterification rate. It is a graph which shows a time-dependent change of the transesterification by Tween20 process ROL surface layer display yeast.

Claims (10)

An immobilized phospholipid metabolizing enzyme treated with a nonionic surfactant ,
An enzyme that is presented on the cell surface . Wherein the cell is a yeast cell, an enzyme of claim 1. The enzyme according to claim 1 or 2 , wherein the nonionic surfactant is a Tween surfactant. The enzyme according to any one of claims 1 to 3 , wherein the phospholipid-metabolizing enzyme is lipase. Contacting the immobilized phospholipid metabolizing enzyme with a nonionic surfactant; and then washing the immobilized phospholipid metabolizing enzyme with distilled water;
A method for producing an immobilized phospholipid metabolizing enzyme treated with a nonionic surfactant, comprising :
A method wherein the phospholipid metabolizing enzyme is presented on the cell surface . 6. The method of claim 5 , wherein the cell is a yeast cell. The method according to claim 5 or 6 , wherein the nonionic surfactant is a Tween surfactant. The method according to any one of claims 5 to 7 , wherein the phospholipid-metabolizing enzyme is lipase. A step of contacting the immobilized phospholipid-metabolizing enzyme according to any one of claims 1 to 4 with a phospholipid and a substrate; and a step of recovering the obtained functional phospholipid. Production method. 10. The method of claim 9 , wherein the phospholipid metabolizing enzyme is a lipase and the substrate is a fatty acid.

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