JP2004049014A - Method for producing optically active substance by using enzyme presented on cell surface layer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for more efficiently carrying out a synthetic reaction by using an enzyme presented on the cell surface layer and to find out the field of the synthetic reaction for making the utilization of the enzyme advantageous. <P>SOLUTION: A method for producing an optically active substance comprises a step of bringing a recombinant cell presenting the enzyme on the cell surface layer into contact with a racemate to be a substrate for the enzyme in a reaction liquid and forming an optically active enzymic reaction product. <P>COPYRIGHT: (C)2004,JPO

Description

【００６１】
図２からわかるように、ｓｈｏｒｔ型Ｆｌｏ１リパーゼを表層発現する酵母ＭＴ８−１−ｓｈｏｒｔを用いた反応系では、Ｒ体のみを生成する反応がすみやかに進行し、効率よく生成物を得ることができた。一方、Ｓ体は反応中いずれの時間においても生成量はごく微量であった。これと比較して、粉末酵素Ｆ−ＡＰ１５を用いた場合では、２４時間以降でＲ体生成物の減少、ならびにＳ体の生成が確認された。
【００６２】
反応開始から４８時間後の（Ｒ）−および（Ｓ）−α−メチルベンジルアセテートの生成量、およびエナンチオ選択性を表１に示す。ｓｈｏｒｔ型Ｆｌｏ１リパーゼを表層発現する酵母ＭＴ８−１−ｓｈｏｒｔを用いた反応系では、反応生成物の大部分がＲ体であり、９６．４％というエナンチオ選択性を達成した。一方、粉末酵素Ｆ−ＡＰ１５を用いた系では、Ｒ体およびＳ体とも生成量は低く、エナンチオ選択性も３１．６％にとどまった。
【００６３】
このように、ｓｈｏｒｔ型Ｆｌｏ１リパーゼを発現する酵母ＭＴ８−１−ｓｈｏｒｔを用いた反応系は、生成物収量およびエナンチオ選択性のいずれにおいても遊離型のリパーゼである粉末酵素を用いた場合よりも良好な成績であった。
【００６４】
【発明の効果】
本発明の方法によれば、効率的に光学活性体を得ることができる。特に、リパーゼを細胞表層に提示する酵母を用いる場合、反応液として有機溶媒を使用することができるので、得られた光学活性体の回収や精製に好適である。したがって、本発明の方法は、医薬中間体に代表されるエナンチオ選択的なファインケミカルの製造などの分野で非常に有用である。
【００６５】
【配列表】

【図面の簡単な説明】
【図１】
ＦＬＯ１の５’領域およびプロリパーゼの構造遺伝子をこの順で有するプラスミドの構築の模式図である。
【図２】
エステル合成反応における反応時間と反応生成物の生成量との関係を示すグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing an optically active substance using a cell surface display enzyme.
[0002]
[Prior art]
In recent years, so-called biodiesel fuel, which uses oils and fats produced by naturally occurring plants, animals, fish or microorganisms, has been expected as a fuel to replace fossil fuels such as light oil. Of these fats and oils, fats and oils used for food production are often disposed of in the environment, causing environmental problems. Therefore, biodiesel fuel from waste oils is important in preventing air pollution and effectively using waste oils. Is especially expected.
[0003]
Fatty acid lower alcohol esters are preferably used as biodiesel fuel. In order to produce fatty acid lower alcohol esters from fats and oils, a technique for separating fats and oils into glycerin and fatty acids constituting the same, and then producing an ester from alcohols and fatty acids is required. As one of the methods, various studies on the production of fatty acid esters using lipase have been conducted.
[0004]
However, the method currently used is exclusively a method in which fats and oils are dissolved in a solvent (for example, hexane) and reacted with lipase in the presence of alcohol. In this method, it is necessary to separate the fatty acid ester from the solvent, and an operation for recovering the solvent is required, which complicates the process, increases the cost, and also involves a risk of explosion. . Therefore, development in a solvent-free system is being studied.
[0005]
Experimental examples in a solvent-free system are described in JAOCS 73, 1191-1195 (1996). According to this experiment, when a branched alcohol such as isopropanol, isobutanol, or 2-butanol is used, 90% or more of fatty acid esters can be obtained. However, inexpensive industrially used alcohols such as methanol and ethanol are used. It is described that the fatty acid esterification reaction hardly proceeds. As described above, at present, a method for producing non-energy-consuming biodiesel (fatty acid ester) using an inexpensive alcohol in a solventless system has not yet been established.
[0006]
On the other hand, in a fatty acid ester synthesis reaction using lipase, a method of isolating, immobilizing, and using lipase has been most often studied. However, this method has a problem that it takes time and cost to isolate and immobilize lipase.
[0007]
In addition, when using microbial cells themselves, it is pointed out that it is necessary to consider the permeability of the cell membrane, and treatment of microorganisms with a solvent such as alcohol has been studied (for example, Felix et al., Anal. Biochem., 120: 211). -234 (1982)). This solvent treatment also requires time and cost for the treatment and recovery of the solvent, and also has a problem of explosion.
[0008]
On the other hand, a method has been developed in which a lipase is expressed on the cell surface using the GPI anchoring domain of a protein localized on the cell surface (JP-A-11-290078). That is, a lipase structural gene is arranged upstream of DNA encoding the GPI anchoring domain, and a secretory signal sequence is further arranged upstream, and lipase is expressed on the cell surface so that the N-terminal side is outside the cell. I have.
[0009]
[Problems to be solved by the invention]
The protein expressed in this way can exhibit sufficient activity on the cell surface as long as it has an active center on the N-terminal side. However, when there is an active center on the C-terminal side such as lipase, there is a problem that the active center is too close to the cell surface layer, causing steric hindrance and failing to exhibit sufficient activity. Therefore, there is a need for a means for more efficiently performing a synthesis reaction using an enzyme displayed on the cell surface, and further, a search for a field of a synthesis reaction in which the use of such an enzyme is advantageous.
[0010]
[Means for Solving the Problems]
Therefore, various studies have been made on methods for expressing proteins on the cell surface, and it has been found that the GPI anchor protein is retained on the cell surface even when the function of the GPI anchoring domain, which has been considered essential, is lost. Was found. That is, as long as at least the aggregation functional domain of the GPI anchor protein is contained, the desired protein can be expressed on either the N-terminal side or the C-terminal side, or on both the N-terminal side and the C-terminal side. By constructing a suitable plasmid, it became possible to express a desired protein on the cell surface.
[0011]
According to such a method, lipase was used as an example of an enzyme appropriately presented on the cell surface in accordance with the position of the active center, and various transesterification reactions were repeatedly examined.Unexpectedly, secretory lipase and In comparison, the present inventors have found that an ester can be produced in a high yield and enantioselectively, thereby completing the present invention.
[0012]
That is, the present invention provides a method for producing an optically active substance, which comprises contacting a recombinant cell that presents an enzyme on the cell surface with a racemic substance that can be a substrate of the enzyme in a reaction solution. Forming active enzymatic reaction products.
[0013]
In a preferred embodiment, the enzymatic reaction is a transesterification reaction.
[0014]
In a more preferred embodiment, the enzyme is a lipase, the racemate is an alcohol, and the enzymatic reaction product is a carboxylate.
[0015]
In a further preferred embodiment, the reaction solution is an organic solvent.
[0016]
In another preferred embodiment, the cells are yeast.
[0017]
In a more preferred embodiment, the enzyme is a fusion protein with a carbohydrate binding protein domain.
[0018]
In a further preferred embodiment, the fusion protein is a protein in which the N-terminal side of the enzyme is linked to the C-terminal side of the sugar chain binding protein domain.
[0019]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the enzyme displayed on the cell surface is appropriately selected according to the target synthesis reaction, and is not particularly limited. Secretory proteins are preferably used. A hydrolase is more preferable, and examples of the hydrolase include esterase (including lipase), glycosidase, peptidase and the like. In the method of the present invention, it is preferable to utilize a substrate transfer reaction of these enzymes. Among them, a hydrolysis reaction using an esterase or a transesterification reaction using a lipase is particularly preferable. The origin of these enzymes is not limited, and for example, those derived from microorganisms are preferably used.
[0020]
In the present invention, the cell that expresses the enzyme on the cell surface is not particularly limited as long as it has a cell wall, such as bacteria, fungi, and plant cells. Preferably, yeast is used.
[0021]
A general method for presenting an enzyme on the cell surface will be described. As a method of presenting an enzyme on the cell surface, (a) a method of presenting the enzyme to the cell surface via a GPI anchor of the cell surface localized protein, and (b) a method of binding the sugar chain binding protein domain of the cell surface localized protein to the cell surface There is a method of presenting the enzyme to the cell surface via the interface.
[0022]
Examples of cell surface localization proteins that can be used include α- or a-agglutinin, which is a yeast sex aggregation protein, FLO proteins (eg, FLO1, FLO2, FLO4, FLO5, FLO9, FLO10, and FLO11), alkaline phosphatase, and the like. No.
[0023]
(A) Method using GPI anchor
The gene encoding a protein localized on the cell surface by the GPI anchor encodes, in order from the N-terminal, a secretory signal sequence, a cell surface localized protein (sugar chain binding protein domain), and a GPI anchor adhesion recognition signal sequence. It has a gene. The cell surface localization protein (sugar chain binding protein) expressed from this gene in the cell is led out 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 on the cell surface.
[0024]
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.
[0025]
The GPI anchor attachment recognition signal sequence is a sequence recognized when the GPI anchor binds to a cell surface localization protein, and is usually located at or near the C-terminal of the cell surface localization protein. As the GPI anchor attachment signal sequence, for example, the sequence of the C-terminal part of α-agglutinin of yeast is suitably used. On the C-terminal side of the sequence of 320 amino acids from the C-terminal of the α-agglutinin, a GPI anchor attachment recognition signal sequence is contained. Therefore, the gene used in the above method encodes a sequence of 320 amino acids from the C-terminal. DNA sequences are particularly useful.
[0026]
Therefore, for example, in a sequence having a DNA sequence encoding a secretory signal sequence-a structural gene encoding a cell surface localization protein-a DNA sequence encoding a GPI anchor attachment recognition signal, the structural gene encoding this cell surface localization protein By replacing all or part of the sequence with the sequence of the structural gene of the target enzyme, a recombinant DNA that displays the target enzyme on the cell surface via a GPI anchor can be obtained. When the cell surface localization protein is α-agglutinin, it is preferable to introduce a target enzyme gene so as to leave a sequence encoding a sequence of 320 amino acids from the C-terminal of α-agglutinin.
[0027]
When, for example, a lipase gene is used as the structural gene of the desired enzyme, a recombinant DNA that displays lipase on the cell surface via a GPI anchor can be obtained. The lipase presented on the cell surface in this manner has its C-terminal fixed to the surface.
[0028]
(B) Method using sugar chain binding protein domain
When the cell surface localization protein is a sugar chain binding protein, the sugar chain binding protein domain has a plurality of sugar chains, and the sugar chains interact with or become entangled with the sugar chains in the cell wall. It is possible to stay. For example, sugar chain binding sites such as lectins and lectin-like proteins are exemplified. Typically, the aggregation functional domain of a GPI anchor protein is mentioned. The aggregation functional domain of the GPI anchor protein refers to 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.
[0029]
The enzyme is displayed on the cell surface by binding the protein on the cell surface localization (aggregation functional domain) with the target enzyme. Depending on the type of the target enzyme, (1) the enzyme is bound to the N-terminus of the cell surface localized protein (aggregation functional domain), (2) the enzyme is bound to the C-terminus, and (3) the N-terminus and The same or different enzymes can be bound to both C-terminal sides.
[0030]
So, for example,
(1) DNA encoding a secretory signal sequence-structural gene of an enzyme of interest-structural gene encoding a cell surface localization protein (aggregation functional domain);
(2) DNA encoding a secretory signal sequence-structural gene encoding a cell surface localized protein (aggregation function domain)-structural gene of the enzyme of interest;
(3) DNA sequence such as DNA encoding secretory signal sequence-structural gene of target enzyme-structural gene coding for cell surface localization protein (aggregation function domain)-structural gene of target enzyme is prepared. As a result, a recombinant DNA displaying the target enzyme on the cell surface can be obtained. When the aggregation function domain is used, since the GPI anchor is not involved in the display of the cell surface, the DNA sequence encoding the GPI anchor attachment recognition signal sequence may or may not be present in the recombinant DNA. You may.
[0031]
If, for example, a lipase gene is used as the structural gene of the desired enzyme, a recombinant DNA that presents lipase on the cell surface using a sugar chain binding protein can be obtained. The lipase displayed on the cell surface in this manner has its N-terminal side or C-terminal side fixed to the surface layer.
[0032]
As the secretory signal sequence used in the above-mentioned recombinant DNA, a secretory signal sequence of a protein localized on the cell surface may be used, or another secretory signal sequence capable of guiding the expressed enzyme to the outside of the cell may be used. 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, part or all of the secretory signal sequence and prosequence may remain at the N-terminus after cell surface display.
[0033]
The cells used in the present invention are cells that have been transformed by introducing DNA so that the target enzyme is displayed on the cell surface. The DNA to be introduced contains at least a secretory signal sequence, a sugar chain binding protein domain, and a sequence encoding the enzyme of interest. In addition, it may include a sequence encoding another enzyme or protein.
[0034]
The synthesis and ligation of DNAs containing the various sequences described above can be performed by techniques commonly used by those skilled in the art.
[0035]
Preferably, the DNA is in the form of a plasmid. From the viewpoint of simplification of DNA acquisition, a shuttle vector with Escherichia coli is preferable. The starting material for this DNA has, for example, an origin of replication (Ori) of the yeast 2 μm plasmid and an origin of replication of ColE1, as well as 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). Further, in order to express the structural gene of the enzyme, it is preferable to include a so-called regulatory sequence such as an operator, a promoter, a terminator, and an enhancer that regulates the expression of the gene. Examples include the GAPDH (glyceraldehyde 3'-phosphate dehydrogenase) promoter and GAPDH terminator. Examples of such starting plasmids include plasmids pYGA2270 or pYE22m containing a GAPDH (glyceraldehyde 3'-phosphate dehydrogenase) promoter sequence and a GAPDH terminator sequence, or a UPR-ICL (isocitrate lyase upstream region) sequence. And a plasmid pWI3 containing a Term-ICL (terminator region of isocitrate lyase) sequence.
[0036]
Preferably, a DNA encoding a desired enzyme is inserted between the GAPDH promoter sequence and the GAPDH terminator sequence of the plasmid pYGA2270 or pYE22m, or between the UPR-ICL sequence and the Term-ICL sequence of the plasmid pWI3. For example, a plasmid used for introduction into yeast is produced. In the present invention, a multicopy plasmid pWIFS or pWIFL is preferably used, and for example, pWIFSpmROL or pWIFLpmROL is produced.
[0037]
As the host yeast, any yeast may be used, but flocculent yeast is preferred because it can be separated easily after the reaction, or can be fixed easily, so that a continuous reaction can be performed. Alternatively, when an aggregation function domain is used as the sugar chain binding protein domain, strong aggregation can be imparted to any yeast.
[0038]
The cells used in the method of the present invention can be obtained by introducing the above DNA into the cells. Introduction of DNA means introducing DNA into a cell and expressing it. 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.
[0039]
The DNA to be introduced may be in the form of a plasmid as described above, or may be inserted into a host gene or may be introduced into a chromosome by homologous recombination with the host gene.
[0040]
Cells into which the DNA has been introduced are selected with a selectable marker (eg, TRP) and selected by measuring the activity of the expressed enzyme. The immobilization of the enzyme on the cell surface can be confirmed by an immunoantibody method using an anti-protein antibody against the enzyme and a FITC-labeled anti-IgG antibody.
[0041]
The recombinant cells displaying the enzyme 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 can be cryo-dried or lyophilized. Can be saved.
[0042]
The recombinant cells used in the present invention may be immobilized on a carrier. In the present specification, the carrier means a substance capable of immobilizing cells, and is preferably a substance that is insoluble in water or a specific solvent. As the material of the carrier that can be used in the present invention, for example, a foam or a resin such as a polyvinyl alcohol, a polyurethane foam, a polystyrene foam, a polyacrylamide, a polyvinyl formal resin porous body, a silicon foam, and a cellulose porous body are preferable. A porous carrier is preferable in consideration of the drop of cells having decreased proliferation and activity or dead cells. 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. The thickness is preferably from 50 μm to 1,000 μm, but is not limited thereto.
[0043]
The method for producing an optically active substance of the present invention comprises contacting a cell presenting the above enzyme on the cell surface with a racemic substance which can be a substrate of the enzyme in an appropriate reaction solution to produce an optically active enzyme reaction product. Forming step.
[0044]
In the present invention, the combination of the enzyme and its substrate can be arbitrarily selected depending on the purpose. Hereinafter, the method for producing an optically active substance of the present invention will be specifically described with reference to an example of a transesterification reaction using lipase as an enzyme.
[0045]
First, a cell (eg, yeast) in which lipase is displayed on the cell surface via a GPI anchor or a sugar chain binding protein domain of a cell surface localization protein is obtained. In the case of lipase, it is preferable that the lipase is displayed on the cell surface as a fusion protein in which the C-terminal side of the sugar chain binding protein domain and the N-terminal side of lipase are linked as described above, since high activity can be maintained. Then, for example, the cells are incubated in a reaction solution containing a carboxylic acid ester (RCOOR ') and a racemic alcohol (R "OH) as a substrate, and the alkoxyl groups (R'O- and R" O- The carboxylic acid ester (RCOOR ") can be formed enantioselectively with respect to R" by catalyzing the exchange reaction of (2).
[0046]
The racemic alcohol that is a substrate for the transesterification reaction may be any alcohol as long as it is a racemic alcohol. It may be a monohydric alcohol or a polyhydric alcohol. Further, any of primary alcohol, secondary alcohol, and tertiary alcohol may be used. Most preferably, it is a monohydric secondary alcohol. Examples of the racemic alcohol include (R, S) -1-phenylethanol (α-methylbenzyl alcohol), (R, S) -1-phenyl-1-propanol, and (R, S) -1-phenyl -2-propanol, (R, S) -3-methyl-2-butanol, isohydrobenzoin, (R, S) -2-tetralol and the like.
[0047]
The carboxylic acid ester which is a substrate of the transesterification reaction may be a monobasic acid ester or a dibasic or higher acid ester. In the case of an ester of a dibasic acid or more, it is preferably a neutral ester. Examples of the carboxylate include an acetate, a propionate, a succinate, a maleate, and a benzoate.
[0048]
The reaction solution used in the method of the present invention is not particularly limited as long as the cell that presents the enzyme on the cell surface can exhibit the enzyme activity, and may be a buffer solution or an organic solvent. When the enzyme is lipase as described above, it is preferable to use an organic solvent other than an ester and an alcohol. Examples of such an organic solvent include pentane, hexane, cyclohexane, heptane, octane, nonane, decane, benzene, toluene, diethyl ether, acetone, acetonitrile, chloroform, dichloroethane, tetrahydrofuran, and the like.
[0049]
In the method of the present invention, for example, by mixing the above-mentioned cells and the above-mentioned reaction solution containing the above-mentioned substrate, the enzyme and the substrate are brought into contact with each other to obtain the desired optically active carboxylic acid ester. The reaction conditions in this case are not particularly limited as long as the cells presenting the enzyme on the cell surface can exhibit the enzyme activity, and the substrate concentration, the reaction temperature, the reaction time, and the like are appropriately determined by those skilled in the art. Can decide. After completion of the reaction, the desired product (carboxylate) can be recovered and purified by a method corresponding to the product.
[0050]
The optically active carboxylic acid ester thus obtained can be used as a raw material for pharmaceuticals or the like as it is, or as an optically active alcohol obtained by hydrolysis or further transesterification.
[0051]
【Example】
[Preparation Example 1] Preparation of DNA having FLO1 5 ′ region and prolipase structural gene in this order
A: Acquisition of the gene for the 5 'region (signal sequence and aggregation functional domain) of FLO1
The FLO1 gene was obtained as follows. First, S. Chromosomal DNA was extracted from S. cerevisiae ATCC 60715. Next, this was used as a template, PCR-amplified using the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 as primers, cut with BamHI and BglII, and a BamHI-BglII fragment (BamHI-BglII FLO1) having a length of about 3300 bp. 3300 bp fragment). This 3300 bp fragment seems to have a sequence 5 ′ to FLO1 (secretory signal sequence and FLO1 aggregation functional domain).
[0052]
B: Acquisition of lipase gene
The lipase gene of Rhizopus oryzae was obtained as follows. Briefly, first, R.I. Chromosomal DNA was extracted from oryzae IFO4697. Next, using this as a template, PCR amplification was performed using the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 as primers, digestion with BamHI and SalI, and a BamHI-SalI fragment (BamHI-Sal) of about 1100 bp in length. SalI lipase fragment) was obtained. This lipase fragment had a lipase pro-sequence and a mature protein sequence, and almost matched the sequence described in Beer et al.'S report (Biochim Biophys Acta, 1399: 173-180, 1998).
[0053]
C: Construction of plasmid having FLO1 5 ′ region and prolipase structural gene in this order
The plasmid having the target DNA can be obtained by connecting the 5 ′ region gene of FLO1 obtained in the above A with the prolipase gene obtained in the above B. The following operation was performed to prepare a fusion protein of a FLO1 derivative and a lipase. FIG. 1 shows a schematic diagram of the preparation.
[0054]
First, the multicopy type plasmid pWI3 was digested with BglII, dephosphorylated, and then the BamHI-BglII FLO1 3300 bp fragment obtained in the above A was inserted to obtain a plasmid pWIFS. Next, this plasmid pWIFS was digested with BglII and XhoI, and the BamHI-SalI lipase fragment obtained in B above was inserted to obtain pWIFSpmROL. The protein expressed from the gene inserted into this plasmid pWIFSpmROL was named short-type FLO1 lipase.
[0055]
[Preparation Example 2] Preparation of yeast having lipase on cell surface
Saccharomyces diastaticus ATCC60712 (MATa leu2-3,112 his2 lys2 sta1 FLO8), which is an aggregating yeast, and W303-1B (MATα ura3-52 trp1Δ2 leu2-3,1a1-11a1a1-3a1a1-11a2a1a1-3a1a1a1a1a1a1a1a1a1a1a2a1a1a1a1a1a1a1a1a1a1a1a1a2a1a1a1a1a1a1a1a1a1a1a1a1a1a1a2a1a1a1a1a1a1a2a1a1a1a1a2a1a1a1a1a1a1a1a1a1a1a1a1a1a1a1a1a1a1a1a2a1a1a1-100a2a1) ) Is used. D. According to the method of Rose et al. (Supra), a new agglutinating strain Saccharomyces cerevisiae YF207 (MATa ura3-52 trp1Δ2 his ade2-1 can1-100 sta1 FLO8) was obtained.
[0056]
Plasmid pWIFSpmROL obtained in Preparation Example 1 above was prepared by the lithium acetate method using Yeast Maker (Clontech Laboratories, Inc., Palo Alto, Calif.). cerevisiae MT8-1 (MATa ade his3 leu2 trp1 ura3) (Tajima et al., Yeast, 1: 67-77, 1985) and the flocculent yeast YF207. This was purified using an SD-W agar selection medium supplemented with an appropriate amino acid and base not containing L-tryptophan (6.7% yeast nitrogen base w / o amino acids (Difco Laboratories), 2% glucose, 2% agar powder) ). The grown yeast was selected, and yeasts expressing the short-type FLO1 lipase were named MT8-1-short and YF207-short, respectively.
[0057]
The obtained yeast was cultured in an SDC liquid medium, centrifuged to separate the medium into cells and cells, and the lipase activity of each was measured. As a control, plasmid pWI3 was used for S. cerevisiae respectively. Yeast introduced into S. cerevisiae MT8-1 was used. As a result, no lipase activity was observed in the medium and the cells of the control. Also, almost no lipase activity was found in the culture supernatants of the transformants MT8-1-short and YF207-short, but all of these yeast cells themselves had lipase activity. The lipase activity was measured using a lipase kit S (manufactured by Dainippon Pharmaceutical).
[0058]
[Example 1] Enantioselective transesterification by yeast having lipase on cell surface
The yeast MT8-1-short that expresses the short-type Flo1 lipase obtained in Preparation Example 2 on the surface thereof was subjected to an enantioselective transesterification reaction in an organic solvent as follows. After culture, 30 mg of yeast MT8-1-short collected, washed and freeze-dried was placed in a 10 ml vial, and the reaction solution (heptane 3 ml, (R, S) -1-phenylethanol 30 mg, and vinyl acetate 21.2 mg) was further added. ) And shaken 150 times per minute at 30 ° C. The amount of (R)-and (S) -α-methylbenzyl acetate produced was measured by HPLC, and the reaction rate and enantioselectivity were calculated. The results are shown in FIG.
[0059]
Comparative Example 1 Transesterification with Soluble Lipase
A transesterification reaction was performed in the same manner as in Example 1 except that lipase F-AP15 (derived from Rhizopus oryzae, Amano Enzyme Co., Ltd.), which was a powder enzyme, was used instead of yeast MT8-1-short. The results are shown in FIG.
[0060]
[Table 1]

[0061]
As can be seen from FIG. 2, in the reaction system using yeast MT8-1-short that expresses the short-type Flo1 lipase on the surface, the reaction for producing only the R-form proceeds promptly, and the product can be obtained efficiently. Was. On the other hand, the amount of S-form was very small at any time during the reaction. In comparison, in the case of using the powdered enzyme F-AP15, a decrease in the R-form product and generation of the S-form after 24 hours were confirmed.
[0062]
Table 1 shows the amount of (R)-and (S) -α-methylbenzyl acetate produced 48 hours after the start of the reaction, and the enantioselectivity. In the reaction system using yeast MT8-1-short that expresses the short-type Flo1 lipase on the surface, most of the reaction products were R-forms, and an enantioselectivity of 96.4% was achieved. On the other hand, in the system using the powdered enzyme F-AP15, both the R-isomer and the S-isomer produced low amounts and had an enantioselectivity of 31.6%.
[0063]
As described above, the reaction system using the yeast MT8-1-short expressing the short-type Flo1 lipase is better in both the product yield and the enantioselectivity than when the powder enzyme which is the free lipase is used. It was a good result.
[0064]
【The invention's effect】
According to the method of the present invention, an optically active substance can be obtained efficiently. In particular, when using yeast that displays lipase on the cell surface, an organic solvent can be used as a reaction solution, which is suitable for collecting and purifying the obtained optically active substance. Therefore, the method of the present invention is very useful in fields such as the production of enantioselective fine chemicals represented by pharmaceutical intermediates.
[0065]
[Sequence list]

[Brief description of the drawings]
FIG.
FIG. 3 is a schematic diagram of the construction of a plasmid having the 5 ′ region of FLO1 and the structural gene of prolipase in this order.
FIG. 2
3 is a graph showing a relationship between a reaction time and an amount of a reaction product generated in an ester synthesis reaction.

Claims (7)

A method for producing an optically active substance, comprising a step of contacting a recombinant cell presenting an enzyme on a cell surface with a racemic substance which can be a substrate of the enzyme in a reaction solution to form an optically active enzyme reaction product. The method according to claim 1, wherein the enzymatic reaction is a transesterification reaction. 3. The method of claim 2, wherein the enzyme is lipase, the racemate is an alcohol, and the enzymatic reaction product is a carboxylic acid ester. The method according to claim 2, wherein the reaction solution is an organic solvent. The method according to any one of claims 1 to 4, wherein the cells are yeast. The method according to any one of claims 1 to 5, wherein the enzyme is a fusion protein with a sugar chain binding protein domain. The method according to claim 6, wherein the fusion protein is a protein in which the N-terminal side of the enzyme is linked to the C-terminal side of the sugar chain binding protein domain.

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