JP4122184B2 - Method of performing catalytic reaction in organic solvent using yeast - Google Patents

Description

【００３６】
実施例２：長鎖脂肪酸の有機溶媒中でのエステル化反応
実施例１で作成した酵母MT8-1/pWRSL17の凍結乾燥粉末を用いヘプタン中でのパルミチン酸とn-ペンタノールとのエステル化反応について評価した。100 mMのパルミチン酸及び100 mMのn-ペンタノールを含むヘプタン溶液2 mL中に、前記実施例１により得られた酵母（MT8-1/pWRLS17）6 mgを添加し、30℃で振とうしながら反応させた。ヘプタンはモレキュラーシーブ3Aにより脱水したものを用いた。また、この反応系に水 0.2, 0.5, 1.0%を添加し、同様の反応を行った。一定時間ごとに反応液を取り出しテトラメチルシリルジアゾメタンのヘキサン溶液（東京化成）を添加して残存するカルボン酸をメチルエステル化した後、ガスクロマトグラフィーにより各時点での反応量を測定した（カラム；J&W Scientific DB-５（Length：１５m、内径 : 0.25mm）、カラム温度：100℃→ 昇温：５℃/分 → 250℃、気化室温度：280℃、検出器温度：300℃）。
【００３７】
実施例１と同様にして、Ａｍａｎｏ Ｆ−ＡＰ１５についても同様の対照実験を行った。結果を表２に示す。酵母（MT8-1/pWRLS17）及びＡｍａｎｏ Ｆ−ＡＰ１５とも脱水ヘプタン中での酵素活性はともに0.03 mM/hrと同じであった。微量の水の添加により酵素活性はいずれも高まったが、Ａｍａｎｏ Ｆ−ＡＰ１５が最大約５倍（0.5%添加時）であるのに対し、酵母（MT8-1/pWRLS17）では約２１倍（0.2%添加時）にまで高まった。これはＡｍａｎｏ Ｆ−ＡＰ１５中のリパーゼは微量の水が存在しても変性失活し易いのに対し、酵母表層に発現したリパーゼは活性構造維持に必要な水和水が細胞壁成分中に安定に保持されるため、高い酵素活性を発現したものと考えられる。
【００３８】
【表２】

【００３９】
実施例３：長鎖脂肪酸の有機溶媒中でのエステル化反応
実施例１で作成した酵母MT8-1/pWRSL17の凍結乾燥粉末を用いヘプタン中での硬化油の有機溶媒中でのエステル化活性について評価した。100 mMの硬化油加水分解物（日本化成、C₁₇H₃₅CO₂H ; 18.4wt%, C₁₉H₃₉CO₂H ; 8.5wt%, C₂₁H₄₃CO₂H ; 73.1wt%）及び100mMのn-ペンタノールを含むn-オクタン溶液3 mL中に、前記実施例１により得られた酵母（MT8-1/pWRLS17）10 mgを添加し、45℃で振とう（160 rpm）しながら反応させた。一定時間ごとに反応液を取り出しテトラメチルシリルジアゾメタンのヘキサン溶液（東京化成）により残存するカルボン酸をメチルエステル化後、ガスクロマトグラフィーにより各時点での反応量を測定した（カラム；J&W Scientific DB-17（Length：30 m、内径 : 0.25 mm）、カラム温度：100℃（5分）→ 昇温：10℃/分 → 250℃、気化室温度：280℃、検出器温度：300℃）。結果を図１に示す。対照実験としてリパーゼＡｍａｎｏ ＡＫ２０について同様の実験を行い、その結果を図２に示した。図１及び２から明らかな様に、酵素表層にリパーゼを発現させた酵母MT8-1 /pWRSL17の方がエステル化反応速度が速いことがわかった。
【００４０】
【発明の効果】
本発明の方法によれば、触媒反応のための酵素をアグルチニンの全長又はその一部の配列とともに融合タンパク質として細胞表層に発現させた酵母を用いて、有機溶媒中又は有機溶媒と水との二層系中で触媒反応を行うことができる。例えば２５〜４０℃及び中性などの温和な条件下で効率的に触媒反応を行うことが可能であり、有機溶媒との接触による酵素の失活もないという特徴がある。
【図面の簡単な説明】
【図１】 本発明の酵母を用いてn-オクタン中で硬化油のエステル化を行った結果（実施例３）を示した図である。
【図２】 比較例として市販のリパーゼを用いてn-オクタン中で硬化油のエステル化を行った結果を示した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to yeast in which an enzyme is expressed in a cell surface layer as a fusion protein with a DNA containing a gene encoding the full length or a part of agglutinin and a gene encoding an enzyme for catalytic reaction in an organic solvent or organically. The present invention relates to a method for use as a catalyst in a two-layer system of a solvent and water.
[0002]
[Prior art]
In recent years, a technique using an enzyme, a microorganism, or the like as a catalyst for synthesizing an organic compound has attracted attention. These biocatalysts are usually capable of reacting under mild conditions of 25-40 ° C. and neutral pH conditions. Moreover, since it is excellent in the regioselectivity and stereospecificity of the reaction product, it has already been used for the production of drug substances. The enzyme reaction is usually carried out in an aqueous solution system in order to prevent enzyme denaturation. If the reaction raw material is an organic solvent-soluble substance that is insoluble in water, suspend the raw material in an aqueous solution containing the biocatalyst, or allow the reaction solution to coexist with the biocatalyst. However, in this method, the reaction efficiency is generally low because the reaction raw material concentration in the aqueous solution is low.
[0003]
For this reason, a water-insoluble reaction raw material is dissolved in an organic solvent, and the reaction is carried out using the enzyme or microorganism in the organic solvent as a biocatalyst, or the organic solvent and a reaction aqueous solution containing the enzyme or microorganism are mixed. Thus, a method for carrying out the reaction has been proposed. However, in these methods, the enzyme is often deactivated or microorganisms are killed by contact with an organic solvent. In particular, when a microorganism is used as a biocatalyst, when the microorganism is killed, only a reaction using dead cells is performed. Therefore, this method cannot be used for reactions that require regeneration of the coenzyme system.
[0004]
In the case of using an enzyme as a biocatalyst, means for immobilizing the enzyme on various carriers or modifying it with a polymer such as polyethylene glycol to keep its activity in an organic solvent has been studied. However, a new process is required for the modification reaction, which leaves a problem for practical use in which the production cost increases. A method using microorganisms themselves as a biocatalyst has also been proposed. However, in this method, it is generally necessary for the reaction raw material to pass through the cell membrane of the microorganisms and reach the place where the target enzyme exists. Since the product needs to pass through the cell membrane and be released to the outside of the cell, the reaction efficiency may not reach the reaction with the enzyme alone. In particular, in the case of a substance that cannot permeate the cell membrane, there is a problem that the reaction cannot occur. On the other hand, when the reaction is carried out with an enzyme alone, there is no such problem of permeation of the cell membrane. However, in order to obtain a large amount of enzyme, a plurality of complicated purification steps are necessary, and there is a problem in practical use.
[0005]
There has also been proposed a method of performing an enzyme reaction using a microorganism in which an enzyme is expressed on the cell surface. The use of prokaryotic gram-positive bacteria, gram-negative bacteria, and eukaryotes such as yeast, animal cells, and plant cells has been proposed as microorganisms capable of expressing enzymes on the cell surface. Yeast is the best because it is easy, safe, and allows biosynthesis of glycoproteins. For example, Ueda et al. Succeeded in immobilizing glucoamylase on the yeast surface and growing starch as the sole carbon source to produce ethanol (Ueda et al, Appl. Environ. Microbiol., 63, 1332). -1336 (1997)). Regarding a technique for expressing an enzyme on a cell surface of a microorganism and using it as a biocatalyst, an enzyme such as lipase is expressed on a yeast surface in JP 7-508652 and JP 11-290078 A, and in an aqueous solution. A method for use as a biocatalyst is disclosed. However, any of these methods in which an enzyme reaction is performed using a microorganism in which an enzyme is expressed on the cell surface layer is performed in an aqueous solution. In the above publication, the use of an organic solvent as a reaction solvent, or There is no suggestion or teaching regarding the reaction in a two-layer system of an organic solvent and water.
[0006]
As for the technique of using an enzyme or microorganism as a catalyst in an organic solvent, for example, JP-A-6-269285 discloses a method of using an enzyme modified with an isoprenoid in an organic solvent, JP-A-6-000090 discloses a hydrophilic method. Methods for immobilizing microorganisms on a functional carrier and using them as a catalyst in an organic solvent have been disclosed, but these methods do not use those in which enzymes are expressed on the cell surface of microorganisms as catalysts.
[0007]
SUMMARY OF THE INVENTION Problems to be Solved by the Invention and Means for Solving the Problems
An object of the present invention is to provide a method for efficiently using an enzyme as a catalyst in an organic solvent or in a two-layer system of an organic solvent and water. As a result of intensive studies to solve the above problems, the present inventors have used a DNA containing a gene encoding the full length of agglutinin or a part thereof and a gene encoding an enzyme for catalytic reaction, The enzyme is expressed as a fusion protein on the cell surface of the yeast, and the yeast is used as a catalyst in a two-layer system of an organic solvent or an organic solvent and water, thereby reducing inactivation of the enzyme due to contact with the organic solvent. It was found that various catalytic reactions can be performed very efficiently. Further, in the above fusion protein, it can be expressed in a form further including a peptide or protein having an action of enhancing the enzyme activity of the enzyme in an organic solvent, or co-expressed as another fusion protein. It has been found that high catalytic activity can be achieved. The present invention has been completed based on the above findings.
[0008]
That is, according to the present invention, a cell comprising a DNA comprising a gene encoding the full length or a part of agglutinin and a gene encoding an enzyme for performing a catalytic reaction as a fusion protein containing the full length or a part of agglutinin. Provided is a method for performing the catalytic reaction in an organic solvent or a two-layer system of an organic solvent and water using yeast expressed on the surface layer. Further, according to a preferred embodiment, the yeast substantially enhances the catalytic reaction activity and / or stereoselectivity and / or regioselectivity of the enzyme encoding the full-length or part of agglutinin and the enzyme, and / or The above-described method, which is a yeast in which the peptide and / or protein is coexpressed on the cell surface as a fusion protein containing the full length of agglutinin or a part thereof, is provided by DNA containing a gene encoding the protein.
[0009]
Further, the gene encoding the full length of agglutinin or a part thereof, the gene encoding the enzyme for performing the catalytic reaction, and the catalytic reaction activity and / or stereoselectivity and / or regioselectivity of the enzyme are substantially increased. Using a yeast containing a DNA containing a gene encoding the peptide and / or protein and expressing the enzyme on the cell surface as a full-length or part of agglutinin and a fusion protein containing the peptide and / or protein, an organic solvent A method for carrying out the catalytic reaction in or in a two-layer system of an organic solvent and water is provided by the present invention.
[0010]
From another point of view, according to the present invention, an enzyme for performing a catalytic reaction in an organic solvent or a two-layer system of an organic solvent and water, the gene encoding the full length of agglutinin or a part thereof and the catalyst An enzyme expressed on the surface of a yeast cell as a fusion protein containing the full length of agglutinin or a part thereof is provided by a DNA containing a gene encoding an enzyme for carrying out the reaction. According to a preferred embodiment, the yeast substantially enhances the catalytic activity and / or stereoselectivity and / or regioselectivity of the enzyme encoding the full-length or part of agglutinin and the enzyme and / or The above enzyme, which is a yeast in which the peptide and / or protein is coexpressed on the cell surface as a fusion protein containing the full length of agglutinin or a part thereof, is provided by a DNA containing a gene encoding a protein.
[0011]
An enzyme for performing a catalytic reaction in an organic solvent or in a two-layer system of an organic solvent and water, the gene encoding the full length of agglutinin or a part thereof, and an enzyme for performing the catalytic reaction A full-length agglutinin or a part thereof, and a DNA containing a gene encoding the peptide and / or protein that substantially enhances the catalytic reaction activity and / or stereoselectivity and / or regioselectivity of the enzyme An enzyme expressed on the surface of yeast cells as a fusion protein containing the peptide and / or protein is provided. Also provided by the present invention is a catalyst containing these enzymes for performing a catalytic reaction in an organic solvent or in a two-layer system of an organic solvent and water.
[0012]
From yet another aspect, a yeast for performing a catalytic reaction in an organic solvent or a two-layer system of an organic solvent and water, wherein the catalytic reaction is performed with a gene encoding the full length of agglutinin or a part thereof. A yeast comprising the enzyme expressed on the cell surface as a fusion protein comprising the full length of agglutinin or a part thereof by a DNA comprising a gene encoding an enzyme for encoding the full length of agglutinin or a part thereof The peptide and / or protein is agglutinin by a DNA containing the gene encoding the peptide and / or protein that substantially enhances the catalytic reaction activity and / or stereoselectivity and / or regioselectivity of the enzyme A yeast co-expressed on the cell surface as a fusion protein comprising the full length or a part thereof; and an organic solvent or an organic solvent A yeast for performing a catalytic reaction in a two-layer system of water and water, a gene encoding the full length of agglutinin or a part thereof, a gene encoding an enzyme for performing the catalytic reaction, and a catalyst for the enzyme DNA comprising a gene encoding the peptide and / or protein that substantially enhances reaction activity and / or stereoselectivity and / or regioselectivity, and includes the full length of agglutinin or a part thereof and the peptide and / or protein Yeast containing the enzyme expressed on the cell surface as a fusion protein is also provided by the present invention.
[0013]
According to preferred embodiments of these inventions, the gene sequence encoding a peptide and / or protein that substantially increases the catalytic reaction activity and / or stereoselectivity and / or regioselectivity of the enzyme is, for example, 100 bp or more. It is a sequence selected from a DNA fragment library derived from a microorganism, and yeast having enhanced organic solvent resistance can be used as the host yeast. When a yeast coenzyme system is used in the catalytic reaction with the enzyme, it is preferable to use a yeast having resistance to organic solvents as the yeast. The enzyme can be selected from the group consisting of, for example, oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The method of the present invention uses a DNA containing a gene encoding the full length or a part of agglutinin and a gene encoding an enzyme for performing a catalytic reaction, and a fusion containing the full length of agglutinin or a part thereof and the enzyme Protein is expressed on the cell surface layer of yeast, and the catalytic reaction is carried out in an organic solvent or a two-layer system of an organic solvent and water using the yeast. According to a preferred embodiment, the yeast comprises the peptide and / or which substantially increases the catalytic activity and / or stereoselectivity and / or regioselectivity of the enzyme encoding the full length of agglutinin or a part thereof and the enzyme. A yeast in which the peptide and / or protein is co-expressed on the cell surface as a fusion protein containing the full length of agglutinin or a part thereof by DNA containing a gene encoding a protein. Also, a fusion comprising an enzyme for performing a catalytic reaction, a peptide and / or protein that substantially increases the catalytic reaction activity and / or stereoselectivity and / or regioselectivity of the enzyme, and the full length of agglutinin or a part thereof. It is also possible to express the protein on the cell surface of yeast and use the yeast for the catalytic reaction.
[0015]
The kind of the enzyme for performing the catalytic reaction is not particularly limited, and an enzyme that catalyzes any chemical reaction can be used. Examples of the chemical reaction may include, but are not limited to, esterification reaction, hydrolysis reaction, acylation reaction, oxidation reaction such as hydroxylation and dehydrogenation reaction, reduction reaction such as hydrogenation, and the like. Rather, it is possible to use enzymes that catalyze various chemical reactions. There may be two or more chemical reactions catalyzed by one enzyme. In the method of the present invention, two or more kinds of enzymes that catalyze different reactions can be expressed on the cell surface of the same yeast. Specific examples of the enzyme include, but are not limited to, oxidoreductase, transferase, hydrolase, lyase, isomerase, esterase, and ligase. The enzyme may be naturally derived, but may be a modified form in which substitution, insertion, and / or deletion with another amino acid is introduced into the amino acid sequence of the naturally occurring enzyme.
[0016]
The kind of the organic solvent for performing the reaction is not particularly limited, and an organic solvent that does not substantially mix with water or an organic solvent that has a property of mixing with water may be used. For example, hydrocarbon solvents such as pentane and hexane, alcohol solvents such as methanol and ethanol, ester solvents such as ethyl acetate, ether solvents such as diethyl ether, dioxane and tetrahydrofuran, ketone solvents such as acetone and methyl ethyl ketone, Halogenated hydrocarbon solvents such as chloroform and dichloroethane, aromatic hydrocarbon solvents such as toluene and xylene, nitrogen-containing aromatic solvents such as pyridine, amide solvents such as acetamide and N, N-dimethylformamide, N- Examples thereof include, but are not limited to, methyl pyrrolidone, dimethyl sulfoxide, and sulfolane.
[0017]
The organic solvent may be used as a substantially completely anhydrous solvent, but it can also be used as an appropriate water-containing solvent as necessary. Although content of water is not specifically limited, it is about 0.01-40 mass% (the numerical range represented by "-" in this specification includes a numerical value of an upper limit and a minimum). When using an organic solvent that is substantially immiscible with water, the reaction can be carried out in a two-layer system.
[0018]
Means for expressing an enzyme as a fusion protein on the cell surface of yeast are known. For example, production of yeast MT8-1 / pWRSL17 containing plasmid pWRSL17 for expressing lipase derived from Rhizopus oryzae on the cell surface The method is known (Appl. Microbiol. Biotechnol., 56, 681-686 (2001)). The yeast used in the method of the present invention can be easily produced by those skilled in the art by adding appropriate modifications or alterations as necessary according to the method described in the above publication.
[0019]
The type of yeast used as a host is not particularly limited. However, when the yeast coenzyme system is involved in the catalytic reaction, it is preferable to use a yeast having resistance to organic solvents. On the other hand, when it is not necessary to participate in the coenzyme system possessed by the yeast in the catalytic reaction, it is not necessary to use yeast having resistance to organic solvents because the yeast may be killed in the organic solvent. . For example, yeast belonging to the genus Saccharomyces, Dizosaccharomyces, and Candida can be preferably used, but yeast belonging to the genus Saccharomyces is more preferably used, and specifically, Saccharomyces cerevisiae MT8-1. (Tajima, M., Yeast, 1, 67-77 (1985)).
[0020]
In general, as methods for obtaining microorganisms resistant to organic solvents, methods using screening from soil or the like and mutation using nitrosating agents are known (for example, Aono et al, Biosci. Biotechnol). Biochem., 56, 145-146 (1992)), organic solvent resistant yeast can be obtained using this method. However, microorganisms obtained by screening from soil or the like may not be used for genetic engineering expression of foreign enzymes without genetic manipulation. As a method alternative to such a method, a method for producing a microorganism that has acquired resistance to an organic solvent by introducing a gene encoding a foreign peptide or protein has been proposed. According to this method, a microorganism that has acquired high organic solvent resistance by introducing a gene encoding a foreign peptide or protein is used, and the microorganism is directly used as a host for expressing any enzyme on the cell surface. Can do. For example, Ueda et al. Reported that a yeast-derived DNA fragment library was expressed on the surface of yeast as a fusion protein with a-agglutinin to produce yeast resistant to organic solvents (Nonane) (Udea et al. , Appl. Microbiol. Biotechnol., 58, 806-812, 2002).
[0021]
The type of peptide and / or protein that substantially enhances catalytic reaction activity and / or stereoselectivity and / or regioselectivity is not particularly limited, depending on the type of enzyme, the type of substrate, the conditions of the catalytic reaction, etc. It can be selected appropriately. Such peptides and / or proteins include peptides and / or proteins for increasing resistance to organic solvents. For example, a gene sequence encoding a peptide and / or protein that substantially enhances the catalytic activity and / or stereoselectivity and / or regioselectivity of the enzyme is obtained from a DNA fragment library derived from a microorganism of, for example, 100 bp or more. It can be selected but is not limited to such sequences.
[0022]
The yeast provided by the present invention expresses an enzyme for catalytic reaction on its cell surface, and the enzyme is expressed as a fusion protein with a sequence encoding the full length or a part of agglutinin. In addition, according to a preferred embodiment, the yeast substantially enhances the catalytic reaction activity and / or stereoselectivity and / or regioselectivity of the gene encoding the full length or a part of agglutinin and the enzyme, and Yeast in which the peptide and / or protein is co-expressed on the cell surface as a fusion protein containing the full length of agglutinin or a part thereof by DNA containing a gene encoding a protein. Furthermore, the gene encoding the full length of agglutinin or a part thereof, the gene encoding the enzyme for performing the catalytic reaction, and the catalytic reaction activity and / or stereoselectivity and / or regioselectivity of the enzyme are substantially increased. It may be a yeast in which the enzyme is expressed on the cell surface as a fusion protein containing the full length or a part of agglutinin and the peptide and / or protein by DNA containing the gene encoding the peptide and / or protein.
[0023]
Preferred DNAs for expressing the enzyme as a fusion protein containing agglutinin or a partial sequence thereof on the cell surface of yeast include, for example, a promoter, a secretion signal sequence, a gene sequence of the enzyme, a sequence encoding the full length or a part of agglutinin, and Has a GPI anchor attachment signal sequence. A preferable DNA for expressing the peptide and / or protein that substantially enhances the catalytic activity and / or stereoselectivity and / or regioselectivity of the enzyme as a fusion protein containing agglutinin or a partial sequence thereof is as described above. It has a gene encoding the peptide or protein instead of the gene sequence of the enzyme in DNA. A preferable DNA for expressing an enzyme and the peptide or protein as a fusion protein containing agglutinin or a partial sequence thereof contains a gene encoding the peptide or protein together with the gene sequence of the enzyme in the above DNA. Hereinafter, although the preferable aspect of the said DNA is demonstrated, this invention is not limited to the case where this preferable DNA is used.
[0024]
The promoter is not particularly limited as long as it is a promoter that is usually used for protein expression in yeast. T. et al, Biosci. Biotechnol. Biochem., 59, 1221-1228 (1995)), and the promoter of Galactokinase (GAL1) (Davis, Mol. Cell Biol., 4, 1440-1448 (1984) ), Candida tropicalis isocitrate lyase (ICL) promoter (UPR-ICL) (Kanai et al, Appl. Microbiol. Biotechnol., 44, 759-765 (1996)) .
[0025]
The secretion signal is not particularly limited as long as it is a signal region normally used for expression and secretion in yeast. For example, the secretion signal region of a glucoamylase gene derived from Rhizopus oryzae, the yeast α-factor Prepro sequences (Walker, GM, in Yeast Physiology and Biotechnology, p265, John Wiley & Sons Lyd., Chichester (1998)) can be used. These promoters and secretion signals can be obtained by a known method, for example, a PCR method using a primer prepared on the basis of a known sequence and a genomic DNA of the microorganism as a template, or Appl. Microbiol. Biotechnol., 51, 65 The plasmids pGA11 and pGPM6 described in -70 (1995) and Appl. Microbiol. Biotechnol., 57, 697-701 (2001) may be excised by an appropriate restriction enzyme reaction.
[0026]
The agglutinin gene or a fragment thereof is obtained from yeast. Among them, yeast belonging to the genus Saccharomyces is preferable, and examples thereof include Saccharomyces cerevisiae MT8-1 (Tajima, M., Yeast, 1, 67-). 77 (1985)). Agglutinin may be either a-arginine or α-agglutinin, and the fragment thereof may have any sequence as long as it does not adversely affect the enzyme activity expressed on the surface layer. Preferably, a sequence encoding the AGA2 sequence of a-agglutinin derived from Saccharomyces is used. The AGA2 is immobilized on the cell surface layer by binding to AGA1 immobilized on the cell wall via a GPI anchor with two disulfide bonds. In addition, a sequence encoding a sequence of 320 amino acids from the C-terminal of α-agglutinin derived from the genus Saccharomyces is preferably used. There are four sugar chain binding sites in this amino acid sequence. After the glycosyl phosphatidylinositol (GPI) anchor is cleaved with phosphatidylinositol-dependent phospholipase (PI-PLC), Are covalently bonded to each other, whereby the C-terminal sequence portion of α-agglutinin is held in association with the cell wall.
[0027]
When the a-arginine is used, the target enzyme sequence can be fused to the 3 ′ end of the AGA1 sequence, and the N-terminus of the enzyme is fused to a-arginine. On the other hand, when α-agglutinin is used, the target enzyme sequence can be fused to the 5 ′ end of α-agglutinin, and the C-terminus of the enzyme is fused to α-arginine. . In general, when an enzyme is expressed as a fusion protein, it is preferably fused at a site as far as possible from the active site of the enzyme, and the a-arginine and α-agglutinin can be selectively used depending on the target enzyme.
[0028]
The above-mentioned promoter, secretory signal sequence, enzyme gene sequence, sequence encoding the full length or part of agglutinin, and DNA containing the GPI anchor attachment signal sequence may be in the form of a plasmid or incorporated into a chromosome. From the viewpoint of simplification of DNA acquisition, a shuttle vector with E. coli is preferable. The starting material for preparing the DNA is not particularly limited. For example, it has a replication origin (2 μm) of yeast 2 μm plasmid and a replication origin (ori) of ColE1, and a yeast selection marker (for example, TRP, URA3 etc.) and E. coli selectable markers (drug resistance genes etc.) are preferred. Further, in order to express 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. Transformation of yeast with a vector for expressing the enzyme on the cell surface can be performed according to a standard method. For example, a transformant can be obtained by a lithium acetate method, an electric pulse method, a protoplast method, or the like. Methods for culturing transformants are also known, and for example, the methods described in MD Rose et al, Method In Yeast Genetics, Gold Spring Harbar Laboratory Press (1990) can be used as appropriate.
[0029]
The method for performing the catalytic reaction in the organic solvent or in the two-layer system of the organic solvent and water using the yeast is not particularly limited, and the yeast is in the organic solvent or in the two-layer system of the organic solvent and water. And the reaction may be performed by adding a starting material as a substrate or an organic compound as a reaction species at an appropriate concentration. The order of adding yeast or substrate is not particularly limited. In addition, the concentration of the yeast or the substrate concentration to be used can be appropriately selected depending on conditions such as the type of reaction and reaction temperature. The reaction can usually be carried out in the range from room temperature to 50 ° C, but when using a heat-resistant enzyme, for example, a temperature range of 100 ° C or lower may be selected. The pH of the reaction solution is not particularly limited, and can generally be selected near neutral. However, when an acid-resistant or alkali-resistant enzyme is used, the reaction should be performed on the acid side or alkaline side, respectively. Is also possible.
[0030]
When the reaction is carried out in a two-layer system of an organic solvent and water, vigorous stirring is performed as in a normal two-layer system, and the yeast present mainly in the aqueous layer and the substrate mainly present in the organic solvent, etc. It is necessary to sufficiently contact the organic compound. When the reaction is carried out in a two-layer system, components necessary for yeast growth can be added to the aqueous layer. Moreover, when reacting in a two-layer system, you may react by mixing a phase transfer catalyst as needed. Examples of the phase transfer catalyst include, but are not limited to, tetrabutylammonium chloride, benzyltributylammonium chloride, tetrabutylammonium hydrogen sulfate, and the like.
[0031]
【Example】
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to these examples.
Example 1: Hydrolysis of long-chain fatty acid esters in organic solvents
A: Preparation of MT8-1 / pWRSL17 powder
Plasmid pWRSL17, which contains a sequence encoding the C-terminal 320 amino acids of the agglutinin gene and expresses a lipase derived from Rhizopus oryzae described in Appl. Microbiol. Biotechnol., 56, 681-686 (2001) on the cell surface Yeast MT8-1 / pWRSL17 containing yeast was cultured in SD medium containing 0.67% Yeast nitrogen base w / o amino acids and 2% glucose for 24 hours, and the resulting yeast was separated by centrifugation (3,000 rpm, 5 minutes) And washed twice with distilled water. The obtained yeast was freeze-dried overnight to obtain MT8-1 / pGPMROL powder. About 0.7 mg of MT8-1 / pWRSL17 powder was obtained per 1.0 and 1.0 mL of absorbance at 600 nm of the culture solution.
[0032]
B: Measurement of enzyme activity in organic solvent
According to the method described in JC Baratti et al, Enzyme Microbiol. Technol., 18, 417-422 (1996), the hydrolysis activity of long-chain fatty acid esters in heptane in an organic solvent was evaluated. To 2 mL of heptane solution of 50 mM palmitic acid p-nitrophenyl ester (Sigma), 0.5, 1, and 2 mg of the yeast obtained in the above step A were added and reacted while shaking at 30 ° C. . The reaction solution was taken out at regular intervals and mixed with a 0.1N aqueous sodium hydroxide solution, and the absorbance at 410 nm of p-nitrophenol produced by hydrolysis present in the aqueous layer was measured. Using the molar extinction coefficient: 17950, the reaction amount at each time point was measured, and the initial reaction rate was calculated. The enzyme activity was calculated from the slope of the graph in which this initial reaction rate was plotted against the yeast concentration. Enzyme activity was expressed in U / g, and 1 U was defined as the enzyme activity that hydrolyzes 1 micromole of substrate per minute.
[0033]
As a control experiment, the same experiment was performed on the enzyme Amano F-AP15 (Amanoenzyme Co., Ltd.), which is a dry powder of cell extract of Rhizopus oryzae. The enzyme is a powder containing the same lipase as that expressed on the surface of the yeast. The results are shown in Table 1. The hydrolysis activity of yeast (MT8-1 / pWRSL17) in which the lipase was expressed in an aqueous solution was about 1/5 that of Amano F-AP15, but the hydrolysis activity in heptane was that of Amano F-AP15. It was about 15 times. This is probably because lipase present as an enzyme alone in Amano F-AP15 is denatured and inactivated in heptane, whereas lipase expressed on the surface of yeast is not denatured and deactivated due to the protective action of surrounding cell wall components. Is done.
[0034]
C: Measurement of enzyme activity in aqueous solution
As a comparative experiment, hydrolysis activity in an aqueous solution was evaluated according to the method described in the literature described in B above. A 2-propanol solution of 16.5 mM palmitic acid p-nitrophenyl ester was prepared and diluted 10-fold with Tris / HCl (pH 8.0) containing 0.4% Triton X and 0.1% gum arabic. To 0.9 mL of this solution, 0.1 mL of a Tris / HCl (pH 8.0) solution to which 2, 4, 6 mg of the yeast obtained in Step A was added, respectively, was allowed to react while shaking at 30 ° C. The reaction solution was taken out at regular intervals, the yeast was removed by centrifugation (15,000 rpm, 1 minute), and the absorbance at 410 nm of p-nitrophenol present in the supernatant was measured. Enzyme activity was calculated in the same manner as B using a molar extinction coefficient of 12750.
[0035]
[Table 1]

[0036]
Example 2: Esterification reaction of long chain fatty acid in organic solvent
The lyophilized powder of yeast MT8-1 / pWRSL17 prepared in Example 1 was used to evaluate the esterification reaction between palmitic acid and n-pentanol in heptane. To 2 mL of a heptane solution containing 100 mM palmitic acid and 100 mM n-pentanol, 6 mg of the yeast (MT8-1 / pWRLS17) obtained in Example 1 was added and shaken at 30 ° C. It was made to react. Heptane was dehydrated with molecular sieve 3A. Further, 0.2, 0.5, 1.0% of water was added to this reaction system, and the same reaction was performed. After taking out the reaction solution at regular intervals and adding a hexane solution (Tokyo Kasei) of tetramethylsilyldiazomethane to methyl esterify the remaining carboxylic acid, the reaction amount at each time point was measured by gas chromatography (column; J & W Scientific DB-5 (Length: 15 m, ID: 0.25 mm), column temperature: 100 ° C. → temperature increase: 5 ° C./min→250° C., vaporization chamber temperature: 280 ° C., detector temperature: 300 ° C.).
[0037]
In the same manner as in Example 1, a similar control experiment was performed for Amano F-AP15. The results are shown in Table 2. Both yeast (MT8-1 / pWRLS17) and Amano F-AP15 had the same enzyme activity in dehydrated heptane as 0.03 mM / hr. Enzymatic activity increased with the addition of a small amount of water, but Amano F-AP15 was up to about 5 times (when 0.5% was added), whereas yeast (MT8-1 / pWRLS17) was about 21 times (0.2%). %). This is because the lipase in Amano F-AP15 tends to be denatured and inactivated even in the presence of a small amount of water, whereas the lipase expressed on the surface of yeast has stable hydration water necessary for maintaining the active structure in the cell wall components. Since it is retained, it is considered that high enzyme activity was expressed.
[0038]
[Table 2]

[0039]
Example 3: Esterification reaction of long chain fatty acid in organic solvent
The freeze-dried powder of yeast MT8-1 / pWRSL17 prepared in Example 1 was used to evaluate the esterification activity of hardened oil in heptane in an organic solvent. 100 mM hydrogenated oil hydrolyzate (Nippon Kasei, C ₁₇ H ₃₅ CO ₂ H; 18.4wt%, C ₁₉ H ₃₉ CO ₂ H; 8.5wt%, C _{twenty one} H ₄₃ CO ₂ H; 73.1 wt%) and 10 mg of the yeast (MT8-1 / pWRLS17) obtained in Example 1 above was added to 3 mL of n-octane solution containing 100 mM n-pentanol and shaken at 45 ° C. The reaction was carried out while continuing (160 rpm). The reaction solution was taken out at regular intervals, and the remaining carboxylic acid was methyl esterified with a hexane solution of tetramethylsilyldiazomethane (Tokyo Kasei), and the reaction amount at each time point was measured by gas chromatography (column; J & W Scientific DB- 17 (Length: 30 m, ID: 0.25 mm), column temperature: 100 ° C (5 minutes) → temperature rise: 10 ° C / min → 250 ° C, vaporization chamber temperature: 280 ° C, detector temperature: 300 ° C). The results are shown in FIG. As a control experiment, a similar experiment was performed for lipase Amano AK20, and the results are shown in FIG. As apparent from FIGS. 1 and 2, it was found that the yeast MT8-1 / pWRSL17 in which lipase was expressed on the enzyme surface layer had a faster esterification reaction rate.
[0040]
【The invention's effect】
According to the method of the present invention, a yeast in which an enzyme for catalytic reaction is expressed on a cell surface as a fusion protein together with the full length of agglutinin or a partial sequence thereof is used in an organic solvent or an organic solvent and water. Catalytic reactions can be carried out in the layer system. For example, the catalytic reaction can be efficiently performed under mild conditions such as 25 to 40 ° C. and neutrality, and the enzyme is not deactivated by contact with an organic solvent.
[Brief description of the drawings]
FIG. 1 is a diagram showing the results (Example 3) of esterification of hydrogenated oil in n-octane using the yeast of the present invention.
FIG. 2 is a graph showing the results of esterification of hydrogenated oil in n-octane using a commercially available lipase as a comparative example.

Claims (7)

An organic solvent using yeast in which the lipase is expressed on the cell surface as a fusion protein containing the full length of agglutinin or a part thereof using a DNA containing a gene encoding the full length of agglutinin or a part thereof and a gene encoding a lipase In which the catalytic reaction with the lipase is carried out. The method according to claim 1, wherein the organic solvent is a hydrocarbon solvent. The process according to claim 1, wherein the organic solvent is heptane. The method according to claim 1, wherein the organic solvent is heptane containing 0.2 to 1.0% by mass of water. The method according to claim 1, wherein the catalytic reaction is a hydrolysis reaction. The method according to claim 5, wherein the organic solvent is a hydrocarbon solvent. The process according to claim 5, wherein the organic solvent is heptane.

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