JP5438259B2 - Yeast presenting Candida antarctica-derived lipase B on the cell surface - Google Patents

Description

  The present invention relates to a yeast that presents Candida antarctica-derived lipase B on the cell surface.

  The basidiomycetous yeast Candida antarctica produces two different lipases, which are distinguished by the names A and B and are called CALA and CALB in abbreviated form, respectively. . These enzymes are originated from bacteria isolated in the Antarctic with the aim of discovering new enzymes with extreme characteristics from microorganisms that grow in extreme conditions. In fact, both CALA and CALB exhibit extremely unique properties. (Non-Patent Document 1).

  The crystal structure of CALB was clarified by Uppenberg et al. In 1994 (Non-patent Document 2). Although less homologous to other lipases or esterases, it belongs to the same family as typical hydrolases and forms a Ser-His-Asp catalytic triad in the active site. CALB is composed of 317 amino acid residues, has a molecular weight of 33 kDa, and is a relatively compact protein among lipases. The prepro region, which is important in the process of protein processing, is presumed to consist of a sequence containing an 18 amino acid signal peptide and a 7 amino acid pro peptide (Non-patent Document 2).

  Most lipases exhibit a unique phenomenon called “interfacial activation” during substrate recognition. This activation phenomenon is a movement of opening and closing by a specific part called lid because it resembles a pot lid, and is often essential for lipase. CALA shows a similar phenomenon although it is small, CALB does not have a lid site and hardly shows such movement (Non-Patent Document 1). Instead, a flexible site similar to Lido exists on the surface of the protein, and it is speculated that it moves slightly during substrate recognition.

  In many lipases derived from microorganisms and animals, the common sequence GXSSXX is retained around the catalytic residue serine. In CALB, GXSSXX is It is not preserved, and the first glycine is replaced by threonine, which is TWSGQ (Non-patent Document 2).

  Thus, CALB shows a very unusual structure and property among general lipases.

  CALB is not yet used industrially as compared with other lipases, but its useful value is rapidly expanding because recent studies have shown that it can be applied to various chemical reactions. For example, there are many reports using CALB regarding ester bond decomposition reaction, ester synthesis reaction, and transesterification reaction. For example, in the decomposition reaction of an ester bond, it is used for synthesizing a raw material for pharmaceuticals using the high enantion selectivity of lipase (Non-patent Document 3). In the ester synthesis reaction, an immobilized enzyme with extremely high stability is used for the reaction in an organic solvent or under high temperature conditions (Non-Patent Documents 4 to 6). Furthermore, as an acyl group donor, not only alcohol but also sugar or the like is used to develop functional foods and pharmaceuticals (Non-patent Documents 4 and 7 to 9). The transesterification reaction is also applied to various reactions utilizing the high substrate specificity of CALB for secondary alcohols (Non-patent Documents 10 and 11). Alternatively, it has been reported that a CALB mutant also exhibits a catalytic action in addition reactions such as Michael addition and Aldol addition (Non-patent Documents 12 and 13). Furthermore, it has been reported that mutants for oxyanion holes exhibit aldolase activity.

  In most of the reports to date concerning the catalytic reaction of CALB, an immobilized lipase represented by Novozym435 (Novo-Nordisk) is used (Non-patent Documents 3 to 9 and 14 to 16). Immobilized enzymes have great utility value due to their very high stability, but are produced by special chemical modification, and therefore have a problem of high cost.

  Regarding the CALB expression system, extracellular secretion systems using Aspergillus oryzae, methanol-utilizing yeast Pichia pastoris, or yeast Saccharomyces cerevisiae have been reported, and thermostability and oxidation are reported. Mutants with modified stability or substrate specificity under conditions have been obtained (Non-Patent Documents 11, 17 to 23).

  In recent years, a display method using various biological species such as Escherichia coli and phage as a support has attracted attention as a novel protein expression system. Unlike the conventional intracellular expression and extracellular secretion system, the display method that presents the target protein in a stable form on the surface layer or carrier of individual cells requires complicated operations such as extraction, purification, and concentration. In addition, the function of the target protein can be evaluated by a simple operation of collecting cells and carriers.

For various proteins (including enzymes), yeasts that present the proteins on the cell surface have been produced (for example, Patent Documents 1 to 12). By displaying the target protein on the surface of the yeast cell, the protein can be handled easily. For example, in Patent Document 1, a lipase gene is cloned into a yeast surface display vector, mutagenesis PCR is performed, lipase gene mutation is induced, and the activity of mutant lipase expressed on the surface of a transformant is measured. It is described that a CALB mutant having high lipase activity was produced by a method for screening active mutant lipase quickly and easily. The present inventors have also succeeded in displaying the cell surface of lipase using a cell surface engineering technique (Patent Documents 2 and 7, and Non-Patent Document 24).
JP 2005-538711 A JP-A-11-290078 JP 2002-17368 A Japanese Patent Application Laid-Open No. 2002-176979 JP 2002-253267 A JP 2003-235579 A JP 2004-49014 A JP 2004-305096 A JP 2004-305097 A JP 2005-58010 A JP 2005-176605 A JP 2005-245335 A O. Kirk et al., Organic Process Research & Development, 2002, 6, 446-451 J. Uppenberg et al., Structure, 1994, 15, 293-308 MJ Homann et al., Advanced Synthesis and Catalysis, 2001, 343, 744-749 Y. Shimada et al., Journal of Bioscience and Bioengineering, 2001, 92, 19-23 F. Secundo et al., Biotechnology and Bioengineering, 2001, 73, 157-163 Y. Mei et al., Biomacromolecules, 2003, 4, 70-74 MP Bousquet et al., Biotechnology and Bioengineering, 1999, 62, 225-234 S. Adachi et al., Journal of Bioscience and Bioengineering, 2005, 99, 87-94 G. Afach et al., Bioscience Biotechnology Biochemistry, 2005, 69, 833-835 D. Rotticci et al., Journal of Molecular Catalysis B: Enzymatic, 1998, 5, 267-272 AO Magnusson et al., ChemBioChem, 2005, 6, 1-6 P. Carlqvist et al., ChemBioChem, 2005, 6, 331-336 M. Svedendahl et al., Journal of American Chemical Society, 2005, 127, 17988-17989 L. Araceil et al., Applied Microbiology Biotechnology, 2004, 65, 373-376 N. Weber et al., Journal of Agricultural and Food Chemistry, 2004, 52, 5347-5353 W. Du et al., Biotechnology Applied Biochemistry, 2004, 40, 187-190 JC Rotticci-Mulder et al., Protein Expression & Purification, 2001, 21, 386-392 SA Patkar et al., Journal of Molecular Catalysis B: Enzymatic, 1997, 3, 51-54 SA Patkar et al., Chemistry and Physics of Lipids, 1998, 93, 95-101 N. Zhang et al., Protein Engineering, 2003, 16, 599-605 WC Suen et al., Protein Engineering, Design & Selection, 2004, 17, 133-140 D. Rotticci et al., ChemBioChem, 2001, 2, 766-770 Z. Qian et al., Journal of American Chemical Society, 2005, 127, 13466-13467 S. Shiraga et al., Appl. Environ. Microbiol., 2005, 71, 4335-4338

  An object of the present invention is to provide a highly active and inexpensive CALB as a lipase which is a useful catalyst for ester bond decomposition reaction, ester synthesis reaction, transesterification reaction, asymmetric synthesis of various chiral compounds, and the like. To do.

  The present invention provides Candida antarctica-derived lipase B (CALB) consisting of an amino acid sequence from an alanine residue at position -7 to a proline residue at position 317 of SEQ ID NO: 2 in the sequence listing on the cell surface. Provide yeast to be presented.

In one embodiment, the mutant is
In the amino acid sequence from the alanine residue at position -7 to the proline residue at position 317 in SEQ ID NO: 2 in the sequence listing,
(1) a polypeptide comprising an amino acid sequence in which the lysine residue at the -2 position and / or the arginine residue at the -1 position is substituted with another amino acid;
(2) a polypeptide having an amino acid sequence in which the lysine residue at position 308 and / or the arginine residue at position 309 is substituted with another amino acid, or (3) the lysine residue at position -2 and / or the position -1 Is a polypeptide comprising an amino acid sequence in which the arginine residue is substituted with another amino acid and the lysine residue at position 308 and / or the arginine residue at position 309 is substituted with another amino acid.

In a further embodiment, the variant is selected from the group consisting of:
In the amino acid sequence from the alanine residue at position -7 to the proline residue at position 317 in SEQ ID NO: 2 in the sequence listing,
(1) a polypeptide comprising an amino acid sequence in which positions -2 and -1 are lysine residues, respectively;
(2) a polypeptide comprising an amino acid sequence in which positions -2 and -1 are each an alanine residue;
(3) a polypeptide having an amino acid sequence in which positions 308 and 309 are lysine residues;
(4) a polypeptide comprising an amino acid sequence in which positions 308 and 309 are each an alanine residue;
(5) a polypeptide comprising an amino acid sequence in which positions -2 and -1 are lysine residues, respectively, and positions 308 and 309 are lysine residues;
(6) a polypeptide comprising an amino acid sequence in which positions -2 and -1 are each an alanine residue and positions 308 and 309 are lysine residues;
(7) a polypeptide comprising an amino acid sequence in which positions -2 and -1 are lysine residues, and positions 308 and 309 are alanine residues; and (8) positions -2 and -1 are A polypeptide comprising an amino acid sequence each of which is an alanine residue and each of positions 308 and 309 is an alanine residue.

The present invention further provides a method for producing a lactate ester, the method comprising:
A step of reacting alcohol and lactic acid in the presence of the yeast.

  In one embodiment, the alcohol is ethanol.

  In another embodiment, the reaction step is performed at 30 to 60 ° C.

  ADVANTAGE OF THE INVENTION According to this invention, the yeast which displays CALB with high activity as a lipase on a cell surface layer is provided. The cell surface CALB-presenting yeast of the present invention has high thermal stability.

(Construction of DNA capable of expressing Candida antarctica-derived lipase B (CALB) on the cell surface)
Candida antarctica-derived lipase B (CALB) according to the present invention can be, for example, an enzyme having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing or a variant thereof. This mature CALB consists of, for example, a 317 amino acid sequence from leucine at position 1 to proline residue at position 317 in SEQ ID NO: 2 in the sequence listing, and the propeptide consists of an alanine residue at position -7. It consists of an amino acid sequence of 7 amino acids up to -1 position arginine residue. For display of the cell surface layer, it is desirable to include in a plasmid so that a base sequence encoding a propeptide and a mature form can be expressed.

  In the CALB according to the present invention, when referring to a polypeptide consisting of an amino acid sequence from the alanine residue at position -7 to the proline residue at position 317 in SEQ ID NO: 2, this polypeptide is also referred to as “wild type CALB”. .

  The detailed properties of CALB are described in Non-Patent Document 1. The enzyme having the amino acid sequence described in SEQ ID NO: 2 may be of any origin as long as it has the characteristics described in Non-Patent Document 1, particularly lipase activity. The amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and the base sequence shown in SEQ ID NO: 1 are CALB sequences derived from Candida antarctica LF 058 strain. Based on this sequence information, the target gene is Can be obtained from organisms having CALB. For obtaining the gene, PCR or hybridization screening usually used by those skilled in the art is used. It is also possible to chemically synthesize the full length of a gene by DNA synthesis. For example, by designing a probe using the above base sequence or a part of the base sequence and performing hybridization on DNA prepared from another organism, base sequences encoding CALB derived from various organisms are isolated. can do. Based on the above base sequence information, PCR primers should be designed from a high homology region using sequence information registered in DNA databases such as DNA Databank of JAPAN (DDBJ), EMBL, Gene-Bank, etc. You can also. By using such primers and PCR using chromosomal DNA or cDNA as a template, the base sequence encoding CALB can be isolated from various organisms.

  Unless the lipase activity is greatly reduced, this enzyme may be modified or mutated so that one or more amino acids differ from the above amino acid sequence by substitution, deletion, addition or the like. Such modifications or mutations can occur naturally or can be artificial (eg, site-directed mutagenesis). Modifications that can affect lipase activity include modification of the site of action (eg, lysine-arginine site) by yeast endogenous protease (eg, KEX2 protease). It is preferable that the C-terminal part of CALB is not deleted. The nucleic acid used for substitution of amino acid residues as described above can be designed according to the codon usage of the host for expressing CALB.

  As CALB displayed on the cell surface, a mutant of wild-type CALB is also used. Examples of such a mutant include a lysine residue at position -2 (position 6 of the propeptide) and / or position -1 (propeptide) of SEQ ID NO: 2 in the sequence listing with respect to the amino acid sequence of wild-type CALB. And a polypeptide in which the arginine residue at position 7) is substituted. It is preferable that the amino acid after substitution at this lysine-arginine site suppresses recognition by KEX protease to a low level and does not greatly change the properties of the amino acid. For example, the lysine residue at position -2 (position 6 of the propeptide) is not changed, and the arginine residue at position -1 (position 7 of the propeptide) can be replaced with a lysine residue. Also, the lysine residue at the -2 position (position 6 of the propeptide) and the arginine residue at the position -1 (position 7 of the propeptide) can be substituted with alanine residues.

  In addition, the lysine residue at position 308 and / or the arginine residue at position 309 of SEQ ID NO: 2 in the sequence listing may be substituted for the amino acid sequence of wild-type CALB. The lysine residue at position 308 and / or the arginine residue at position 309 is a lysine-arginine site located at the C-terminal part of CALB. Similarly to the above, it is preferable that the amino acid after substitution suppresses recognition by KEX protease to be low and the properties of the amino acid do not change greatly. For example, an arginine residue at position 309 can be replaced with a lysine residue. In addition, the lysine residue at position 308 and the arginine residue at position 309 can each be substituted with an alanine residue. This lysine-arginine site located at the C-terminal can be modified in combination with the lysine-arginine site of the propeptide.

  In the present invention, in order to guide the expressed CALB to the cell membrane, CALB is expressed in a state where a secretion signal is bound to the N-terminus. A secretory signal sequence is an amino acid sequence containing many amino acids rich in hydrophobicity, which is generally bound to the N-terminus of a protein (secretory protein) secreted extracellularly (including the periplasm) and is usually secretory. Proteins are removed as they pass through the cell membrane and are secreted out of the cell. Any secretory signal sequence can be used as long as it is a secretory signal sequence capable of directing the expressed CALB to the cell membrane, regardless of the origin. For example, a secretion signal sequence of glucoamylase, a signal sequence of yeast α- or a-agglutinin, a secretion signal sequence of CALB itself (for example, -8 from methionine residue at position −25 of SEQ ID NO: 2) A polypeptide having an amino acid sequence consisting of 18 amino acids up to the alanine residue at the position) is preferably used. If the activity of other proteins fused to the cell surface binding protein is not affected, a part or all of the secretory signal sequence and the pro sequence may remain at the N-terminus.

  The elements for presenting CALB on the cell surface will be described. Elements for presenting CALB on the cell surface include (a) a GPI anchor adhesion recognition signal sequence of the cell surface localized protein, and (b) a sugar chain binding protein domain of the cell surface localized protein. As described above, it is desirable to fix to the cell surface in the form of CALB containing a propeptide.

  Examples of cell surface localized proteins that can be used include α- or a-agglutinin which is a sex aggregation protein of yeast (used as a GPI anchor), FLO1 protein (FLO1 protein has various amino acid lengths on the N-terminal side, Can be used as a GPI anchor: for example, FLO42, FLO102, FLO146, FLO318, FLO428, etc .; Appl. Microbiol. Biotech., 60, 469-474, 2002: where FLO1326 represents the full-length FLO1 protein) , FLO protein (FLOshort or FLOlong which does not have GPI anchor function and utilizes aggregation property; Appl. Environ. Microbiol., Pages 4517-4522, 2002), invertase which is a periplasmic localized protein (does not use GPI anchor) ) And the like.

  First, (a) will be described. A gene encoding a protein localized on the cell surface by a GPI anchor encodes a secretory signal sequence, a cell surface localized protein (sugar chain binding protein domain), and a GPI anchor attachment recognition signal sequence in this order from the N-terminal side. Have a gene. 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. Then, it is cut by the PI-PLC near the root of the GPI anchor, incorporated into the cell wall, fixed to the cell surface, and presented on 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 recognition signal sequence, for example, the sequence of the C-terminal portion of yeast α-agglutinin is preferably used. On the C-terminal side of the sequence of 320 amino acids from the C-terminus of α-agglutinin, a GPI anchor attachment recognition signal sequence is included. Therefore, a DNA sequence encoding a sequence of 320 amino acids from the C terminus is 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 a part of the sequence with a DNA sequence encoding CALB, recombinant DNA for displaying CALB on the cell surface via a GPI anchor can be obtained. When the cell surface localized protein is α-agglutinin, it is preferable to introduce a CALB-encoding DNA so as to leave a sequence encoding a 320 amino acid sequence from the C-terminal of the α-agglutinin. The target protein presented on the cell surface by introducing such DNA into yeast and expressing it has its C-terminal side immobilized on the surface.

  Next, (b) will be described. The sugar chain-binding protein domain refers to a domain that has a plurality of sugar chains and can remain on the cell surface layer by interacting or entanglement with sugar chains in the cell wall. Examples thereof include sugar chain binding sites such as lectins and lectin-like proteins. Typically, an aggregation functional domain of GPI anchor protein and an aggregation functional domain of FLO protein can be 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 expression protein CALB, the target expression protein CALB is presented on the cell surface. The target expressed protein can be bound to both the N-terminal side and the C-terminal side of the cell surface localized protein (aggregation functional domain). In the present invention, (1) a DNA encoding a secretory signal sequence-a gene encoding CALB-a structural gene encoding a cell surface localized protein (aggregation functional domain); or (2) a DNA encoding a secretory signal sequence- By producing a structural gene encoding a cell surface localized protein (aggregation function domain)-a gene encoding CALB, recombinant DNA for presenting CALB on the cell surface can be obtained. When the aggregation functional domain is used, since the GPI anchor is not involved in the cell surface presentation, only a part of the DNA sequence encoding the GPI anchor attachment recognition signal sequence may be present in the recombinant DNA. It does not have to exist. In addition, when the aggregation functional domain is used, the length of the domain can be easily adjusted (for example, either FLOShort or FLOLong can be selected), so that CALB can be presented on the cell surface with a more appropriate length, and This is very useful in that it can be bound on either the N-terminus or C-terminus of CALB.

  In both (a) and (b), CALB may be directly bonded to the above element or may be bonded via a linker. Such linkers can be designed and produced by those skilled in the art as appropriate.

  Synthesis and binding of DNAs containing the various base sequences described above can be performed by techniques that can be commonly used by those skilled in the art. The binding can be performed using an appropriate restriction enzyme, a linker or the like.

  The DNA capable of expressing CALB on the cell surface is preferably in the form of a plasmid. From the viewpoint of simplification of DNA acquisition, a shuttle vector with E. coli is preferable. As a starting material for DNA in the form of a plasmid, for example, it has a replication origin (Ori) of 2 μm plasmid of yeast and a replication origin of ColE1, and a yeast selectable marker (for example, drug resistance gene, TRP, LEU2 Etc.) and E. coli selectable markers (drug resistance genes etc.) are more preferred. In addition, in order to express the CALB structural gene, it is desirable to include so-called regulatory sequences such as an operator, a promoter, a terminator, and an enhancer that regulate the expression of this gene. For example, the promoter and terminator of glyceraldehyde 3'-phosphate dehydrogenase (GAPDH) are mentioned. 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 plasmid pWI3 containing Term-ICL (terminator region of isocitrate lyase) sequence.

  For example, if DNA encoding CALB is inserted 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, The plasmid vector used for the introduction is produced.

  Vector pGA11 (Ueda et al., Ann. NY. Acad. Sci., 1998, 864, 528-537) and vector pICAS1 (Murai et al., Appl. Environ. Microbiol., 1998, 64, 4857-4861 Page) fragment (this fragment encodes a sequence encoding the secretion signal of the glucoamylase gene, a multiple cloning site capable of recognizing restriction enzymes SacII, BglII, NcoI, and XhoI, and further coding 320 amino acids from the C-terminal of α-agglutinin) The plasmid vector pCAS obtained by ligation) is suitably used as a yeast cell surface display cassette vector.

  In a host cell into which the plasmid has been introduced, a tag (for example, a FLAG tag) can be expressed in order to confirm that CALB is immobilized on the cell surface. Such a tag can be connected to the downstream of the base sequence of the CALB structural gene using a linker. Such a linker can be designed based on a procedure commonly used by those skilled in the art.

  An example of a plasmid prepared based on the method described above is shown in FIG. The plasmid pCAS-FLAGR-CALB shown in FIG. 1 has a base sequence encoding a secretory signal, a base sequence encoding CALB including a propeptide, a base sequence encoding a FLAG tag, between the GAPDH promoter and the GAPDH terminator, And a base sequence encoding 320 amino acids from the C-terminal of α-agglutinin was inserted in this order.

(Production of yeast displaying CALB on the cell surface)
Yeast that presents CALB on the cell surface (hereinafter, also simply referred to as “surface layer display yeast”) can be obtained by introducing the DNA into yeast cells. “Introduction of DNA” means that DNA is introduced into a cell and expressed. Examples of DNA introduction include transformation, transduction, transfection, cotransfection, and electroporation. Specific examples of the introduction into yeast cells include a method using lithium acetate and a protoplast method.

  The introduced DNA may be incorporated into the chromosome in the form of a plasmid, inserted into a host gene, or subjected to homologous recombination with the host gene.

  The host yeast is not particularly limited as long as it has a marker for plasmid expression and can display CALB on the cell surface. In the following Examples, Saccharomyces cerevisiae MT8-1 strain is used, but it is not limited to this strain.

  Yeast into which DNA has been introduced can be selected with a selectable marker (eg, drug resistance gene, TRP, LEU2, etc.), and is selected by measuring CALB activity. In order to confirm that CALB is immobilized on the cell surface, as described above, a sequence encoding a tag (for example, FLAG tag) is inserted in the plasmid in advance, and an anti-tag antibody (and fluorescent if necessary) is inserted. An immunoantibody method using a labeled antibody can be used.

  The yeast introduced with the DNA has CALB displayed on the cell surface and has lipase activity. This yeast can be used without further purification of the enzyme because CALB is presented in an active form on the cell surface.

  The yeast that presents CALB on the cell surface may be immobilized on a carrier. This is because it can be used repeatedly in continuous reaction or batch culture.

  In terms of immobilization and continuous reaction, aggregating yeast is preferred as the host yeast. Examples of the aggregating yeast include Saccharomyces diastaticus ATCC60715, ATCC60712, Saccharomyces cerevisiae IFO1953, CG1945, and HF7C.

  The carrier means a substance capable of immobilizing yeast, and is preferably a substance that is insoluble in water or a specific solvent. As the material of the carrier 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, cellulose porous body and the like are preferable. In consideration of detachment of yeast whose growth and activity are reduced or dead, a porous carrier is preferable. Although the size of the opening of the porous body varies depending on the cells, a size that allows yeast to sufficiently enter and grow is suitable, and is preferably 50 μm to 1,000 μm, but is not limited thereto.

  Moreover, the shape of a support | carrier is not ask | required. Considering the strength of the carrier, the 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.

  Immobilization 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 the carrier or taken into the carrier. For immobilization of yeast, for example, known methods 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.

  The aggregating yeast presenting CALB on the cell surface is capable of growing despite being immobilized on the carrier, and has the property that it naturally falls off when the activity decreases. The combined yeast is characterized in that the number of viable bacteria is kept almost constant and the activity is high. Considering this feature, physical adsorption is most preferable for binding to the support. Special adsorption is not necessary for physical adsorption. By simply mixing and culturing the aggregating or adherent cells and the porous carrier, the cells enter the opening of the porous body and adhere to the carrier.

  Cohesiveness means the property that yeasts that are floating or dispersed in a liquid gather together to form a lump (aggregate). Adhesiveness means that yeasts adhere or bind to each other. It means the property to form.

  The decreased activity means that the yeast itself has not died, but the activity of the whole cell is weakened, or the activity is reduced at the level of DNA encoding the enzyme related to aggregation, for example, the activity related to aggregation is reduced. It means a state where it cannot aggregate.

  In the present invention, the aggregating or adhesive yeast may be a yeast that has been imparted with the aggregating or adhering property by introduction of a gene related to aggregation or adhesion.

  Examples of the gene relating to aggregation or adhesion include structural genes encoding substances involved in aggregation or adhesion, such as chitin and lectin in yeast. Examples of genes relating to aggregation are FLO1 [J. Watari et al., Agric. Biol. Chem., 55: 1547 (1991), GG Stewart et al., Can. J. Microbiol., 23: 441 (1977), I. Russell et al., J. Inst. Brew., 86: 120 (1980), CW Lewis. J. Inst. Brew., 82: 158 (1976)], FLO5 [I. Russell et al., J. Inst. Brew., 85:95 (1979)], FLO8 [I. Yamashita et al., Agric. Biol. Chem., 48: 131 (1984)].

  These genes for aggregation or adhesion can be incorporated into the starting material plasmids described above and introduced into yeast.

  The immobilized yeast obtained in this way can be used as a so-called bioreactor by being cultured in a floating state while being attached to a carrier, or packed in a column or the like. Even if the cells are cultured continuously or repeatedly in batches, the cells whose activity has been reduced or killed are detached, so that the activity does not decrease and can be used effectively.

(Use of yeast to present CALB on the cell surface)
Candida antarctica-derived lipase B (CALB) maintains high activity and stability in both aqueous and organic solvent systems, and has high stereoselectivity and enantioselectivity as its substrate specificity. Therefore, the yeast of the present invention can be applied as a catalyst for asymmetric synthesis of various chiral compounds, decomposition and synthesis reactions of ester compounds, transesterification reactions, polymerization reactions and the like.

  The CALB surface layer display yeast can be suitably used for the reaction with fats and oils, for example, as described in Patent Document 2, in the production of fatty acids and fatty acid esters, and the transesterification of fats and oils.

  CALB surface layer display yeast can be used suitably for manufacture of lactate ester. Lactic acid ester is obtained by reacting alcohol and lactic acid in the presence of the surface layer-displayed CALB of yeast. The lactic acid to be reacted may be D-form or L-form, or a mixture thereof. As the alcohol, lower alcohols such as methanol, ethanol, propanol and butanol are preferred. The amount of alcohol used in the production may be equal to or less than the number of moles of lactic acid. By this reaction, a type of lactic acid ester depending on the alcohol used is obtained. Lactic acid esters can be used as industrial solvents and cleaning agents.

  In one embodiment, the alcohol is ethanol. In this case, the surface layer-displayed CALB of yeast can mediate a reaction between lactic acid and ethanol to produce ethyl lactate. Ethyl lactate can be used as a raw material for biodegradable plastics.

  CALB expressed by the surface of the yeast of the present invention can retain activity even at around 60 ° C. Therefore, it can be preferably used in the reaction at about 20 ° C to about 60 ° C. Reactions at relatively high temperatures such as about 30 ° C to about 60 ° C are also possible. For example, as shown in the following examples, it can be used for a reaction at a high temperature such as about 50 ° C. for a long time such as about 100 hours. In the production of lactic acid esters, the reaction of lactic acid and surface-presented CALB under alcohol conditions can be carried out at a relatively high temperature, such as from about 30 ° C to about 60 ° C.

  The pH of the reaction can be determined taking into account the nature of the CALB and the substrate to be reacted. For the production of lactic acid esters, about 4.0 to about 8.0 is preferred. Since the pH decreases as the reaction proceeds, it is preferable to adjust the pH to a certain value or a specific range.

  The solvent used for the reaction may be either an aqueous or non-aqueous organic solvent. It can be determined depending on the reaction used.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this Example.

  The procedure of the method used in this example is described below, but is not limited thereto.

(1. Used strain)
The following strains were used in this study:
Escherichia coli DH5α [F-, ψ80dlac ZΔM15, Δ (lac ZYA-argF) U169, deoR, recA1, endA1, hsdR17 (rk-mk +), phoA, supE44, λ-, thi-1, gyrA96, relA1
Yeast Saccharomyces cerevisiae MT8-1 (MATa, ade, his3, leu2, trp1, ura3).

(2. Medium)
The following media, except for some parts, were dissolved in distilled water and then autoclaved (121 ° C., 15 minutes). For preparation of the agar medium, 2% (w / v) agar powder (Nacalai special grade) was used.

<LB (Luria-Bertani) medium>
1% (w / v) bactotryptone (DIFCO); 0.5% (w / v) yeast extract (DIFCO); 1% (w / v) NaCl (Nacalai special grade). After autoclaving and cooling to 60 ° C., 50 mg / l ampicillin sodium (Meiji Seika) was added. Hereinafter, the LB medium supplemented with ampicillin is also referred to as “LBA”.

<YPD (yeast extract peptone dextrose) medium>
1% (w / v) yeast extract; 1% (w / v) bactopeptone (DIFCO); 2% (w / v) D-(+)-glucose (Nacalai special grade).

<SD (Synthetic Dropout) Medium>
It has 0.67% (w / v) DIFCO ^™ Yeast nitrogen base w / o amino acids (DIFCO) and 2% (w / v) glucose. To prepare plates containing tributyrin and soybean oil, add SDC medium (SD medium containing 2% (w / v) casamino acid (DIFCO)) and 1% (w / v) bile powder as an emulsifier. And then autoclaved. After cooling to 60 ° C, mix 0.2% tributyrin (Wako Pure Chemical) or 0.2% soybean oil (Wako Pure Chemical) and 50mg / l ampicillin sodium, dissolve well by sonication, stir, and evenly in a Petri dish Dispensed into

Depending on the auxotrophy of the strain to be grown, bases and amino acids were added as follows:
20mg / l Adenine sulfate dehydrate (Wako Pure Chemical Industries)
20mg / l uracil (Wako Pure Chemical Industries)
100mg / l L-leucine (Wako Pure Chemical Industries)
20mg / l L-histidine monohydrochloride monohydrate (Nacalai)
20mg / l L-tryptophan (Nacalai).

<SOC medium>
2% (w / v) tryptone; 0.5% (w / v) yeast extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgSO ₄ ; 10 mM MgCl ₂ ; and 20 mM glucose.

(3. Gene amplification by PCR)
In this example, PCR was performed using the following polymerases according to the purpose.

(3-1. PCR using KOD Dash (registered trademark) (TOYOBO))
Reaction solution composition (per 50 μl) was: 37 μl sterile water; 5 μl 10 × buffer (according to manufacturer's instructions); 5 μl 2 mM dNTPs; 1 μl primer 1 (100 pmol / μl); 1 μl primer 2 (100 pmol / μl); and KOD 1 μl of Dash®.

  The reaction cycle is 94 ° C 2 minutes; 94 ° C 30 seconds, (Tm-5) ° C 2 seconds, and 74 ° C 30 seconds / kb 30 cycles; 74 ° C 7 minutes; and 4 ° C ∞.

  Since KOD Dash (registered trademark) has high amplification efficiency, it was mainly used for direct colony PCR. Direct colony PCR was performed to confirm the insertion of the insert DNA into the vector. First, a reaction solution excluding the polymerase having the above reaction solution composition was prepared, and 50 μl was dispensed into a PCR tube. Here, a sterilized toothpick with a colony was dipped and gently stirred. Thereafter, polymerase was added to each PCR tube, and PCR was performed in the above reaction cycle. After the reaction, the band of the target amplification product was confirmed using agarose electrophoresis.

(3-2. PCR using KOD Plus (registered trademark) (TOYOBO))
Reaction solution composition (per 50 μl) was: sterile water 37 μl; 10 × buffer (according to manufacturer's instructions) 5 μl; 2 mM dNTPs 5 μl; 25 mM MgSO ₄ 2 μl; primer 1 (100 pmol / μl) 1 μl; primer 2 (100 pmol / μl) ) 1 μl; template DNA 1 μl (1-10 ng); and KOD Plus® 1 μl.

  The reaction cycle is 94 ° C for 2 minutes; 94 ° C for 15 seconds, (Tm-5) ° C for 30 seconds, and 68 ° C for 1 minute / kb for 25 cycles; 68 ° C for 7 minutes; and 4 ° C ∞.

Since KOD Plus (registered trademark) has high accuracy, it was used when preparing insert DNA. The solution was advanced by increasing the concentration of MgSO ₄ when the desired amplification product by PCR was not confirmed, and decreasing the concentration of MgSO ₄ when smear was confirmed in the PCR product.

(4. Purification of PCR products)
When preparing an insert DNA or megaprimer, the amplification product band was confirmed by electrophoresis after the PCR reaction, and then purified using a Quantum Prep (registered trademark) PCR Kleen Spin Column (BIO-RAD). Purification followed the protocol.

(5. Restriction enzyme treatment)
The restriction enzyme treatment of DNA was performed in the following reaction solution. The reaction solution composition (per 20 μl) was DNA 1-2 μg; 10 × buffer 2 μl; restriction enzyme 1 μl; sterilized water (20 μl in total). The reaction buffer and reaction temperature were selected and processed optimally for each restriction enzyme. The reaction time was 2 hours or longer. The reaction buffer is as follows: 10 × High buffer (TOYOBO) [10 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM DTT]; 10 × Low buffer (TOYOBO) [50 mM Tris-HCl (pH 7) _{.5), 10mM MgCl 2, 100mM} NaCl, 1mM DTT].

(6. Mutation introduction by quick change method)
The quick change method is a very simple mutagenesis method that does not require a ligation operation. This is also made into a kit by Stratagene, such as QuikChange (registered trademark) II Site-Directed Mutagenesis Kit. The method consists of three stages. The first step is a polymerase reaction using the whole plasmid as a template, the second step is removal of the template plasmid by DpnI, and the third step is transformation of E. coli.

In this example, all of the site-specific mutations were introduced using this method except for a part. As the polymerase, Pfu Ultra ^™ High-Fidelity DNA polymerase (Stratagene) excellent in accuracy and amplification of long DNA was used. Reaction solution composition (per 50 μl) is: 36 μl sterile water; 5 μl buffer (according to manufacturer's instructions) 5 μl; 2 mM dNTPs 5 μl; Primer 1 (100 pmol / μl) 1 μl; Primer 2 (100 pmol / μl) 1 μl; Template DNA 1 μl (100 ng); and 1 μl of polymerase. The reaction cycle was 95 ° C. for 2 minutes; 95 ° C. for 50 seconds, 55 ° C. for 50 seconds, and 72 ° C. for 2 minutes / kb 18 cycles; 72 ° C. for 7 minutes; The primers used for mutagenesis were designed with the following points in mind: (1) Make the two primers the same length and make them completely complementary; (2) Both ends of the mutagenesis site are 15 Use at least bases to match the template sequence exactly (3) End the primer with G or C.

The procedure is as follows: polymerase reaction using whole plasmid as template; 1 μl of DpnI (20 units) is added directly to the reaction product (50 μl); incubation overnight at 37 ° C .; confirmation of plasmid size band by electrophoresis; 2 μl directly introduced into Competent high DH5α (7. see); after about 16 hours, a single colony inoculated into LBA liquid medium 5 ml; (see 8) miniprep; overnight incubation 160 min ^-1, at 37 ° C. determination of nucleotide sequence Confirm the introduction of the mutation by (see 13.) and obtain the plasmid with the desired mutation introduced.

(7. Transformation of E. coli)
E. coli was transformed by the calcium chloride method. The procedure is as follows: Add 50 μl of Competent high DH5α (TOYOBO) to 2 μl of plasmid solution (10 ng as standard); leave on ice for 30 minutes; heat shock at 42 ° C. for 1 minute; leave on ice for 3 minutes; SOC Add to 300 μl of medium; leave at 37 ° C. for 1 hour; inoculate appropriate amount on LBA plate; leave at 37 ° C. for 16 hours; obtain transformed E. coli colonies.

(8. Plasmid extraction from E. coli)
Plasmid extraction (miniprep) from E. coli was performed using Quantum Prep® Plasmid Miniprep Kit (Bio-Rad) according to the manufacturer's protocol. The following shows the procedure: adding 100 mg / ml ampicillin sodium solution 7μl LB liquid medium; inoculate a single colony of LBA plates of E. coli; 160 min ^-1, incubated overnight at 37 ° C.; at room temperature 3000 rpm 10 Centrifugation for min; Vortex the pellet in 200 μl of resuspension solution; Transfer suspension to 1.5 ml Eppendorf tube with Pasteur pipette; Invert suspension in 250 μl of lysis solution; Add 250 μl of neutralization solution, Invert suspension; centrifuge at 14000 rpm for 5 minutes at room temperature; inject supernatant into spin filter; add 200 μl Quantum Matrix and mix by pipetting; centrifuge at 14000 rpm for 1 minute at room temperature; add 500 μl wash buffer Centrifuge at 14000 rpm for 1 minute at room temperature; add 500 μl of wash buffer; centrifuge at 14000 rpm for 2 minutes at room temperature; move spin filter to new 1.5 ml Eppendorf tube; TE (pH 8. 0) Add 100 μl; centrifuge at 14000 rpm for 1 minute at room temperature; obtain plasmid DNA.

(9. Transformation of yeast)
Yeast transformation was performed according to the manufacturer's protocol using EZ-Yeast Transformation Kit (Q-BIO gene). The procedure is as follows: Inoculate yeast in 10 ml of YPD; incubate 300 min ^-1 overnight at 30 ° C; transfer 500 μl each into a 1.5 ml Eppendorf tube; centrifuge at 3000 rpm for 3 minutes at room temperature; pellet the EZ Transformation solution Mix by pipetting into 125 μl; add 2 μg of plasmid DNA, mix by pipetting; add 5 μl of carrier DNA, mix by pipetting; vortex; leave at 42 ° C. for 30 minutes; plant appropriate amount on SD agar medium Inoculate and leave at 30 ° C. for 2-3 days; obtain transformed yeast colonies.

(10. Agarose electrophoresis of DNA)
Put 2g of agarose in 200ml of TAE solution (Tris 48g; EDTA · 2Na 174mg; acetic acid 1.14ml; distilled water (with a total of 1000ml)), boil and dissolve in microwave oven, then pour into mold, cool and 1% An agarose gel was made. An appropriate amount of 6 × Loading Dye (TOYOBO) was added to the DNA solution. This was applied to an agarose gel and electrophoresed at 100 V using a mini-gel electrophoresis tank i-Mupid (Cosmo Bio). The gel after electrophoresis was stained with ethidium bromide (EtBr) solution for 10 minutes. Thereafter, it was transferred to deionized water and decolorized for 10 minutes. Using a UV sample image server system KYT-101 (TOYOBO), DNA in the gel was irradiated with UV to develop DNA. Photographs were taken as needed.

(11. Purification of DNA using agarose electrophoresis)
A region containing the target DNA in the agarose gel was cut out using a razor. Then we made it as small as possible. After setting the column GenElute ^™ MINUS EtBr SPIN COLUMNS (SIGMA) in the tube, 100 μl of distilled water was added, centrifuged at 15000 rpm for 5 seconds, and prewashed. The column was transferred to a new tube and the agarose gel fragment was placed there. Centrifugation was performed at 15000 rpm for 10 seconds to obtain about 50 μl of DNA solution.

(12. Ligation reaction)
The insert DNA prepared by treating the PCR product with a restriction enzyme was ligated with vector DNA treated with the same restriction enzyme by Ligation high (TOYOBO). The reaction was carried out at 16 ° C. for 2 hours or longer in the reaction solution shown below. This was introduced into DH5α by the calcium chloride method.

  The composition of the reaction solution (per 20 μl) was vector DNA 50 ng; insert DNA 50 ng; Ligation high DNA solution equivalent amount; sterilized water (20 μl in total).

(13. Determination of DNA base sequence)
The base sequence of DNA was determined as follows. First, a terminator reaction was caused using Big Dye (registered trademark) Terminator v3.1 Cycle Sequencing Standard Kit (Applied Biosystems). The composition of the reaction solution was sterile water (20 μl total); 2 μl of 5 × buffer (according to manufacturer's instructions); 3 μl of Big Dye® Terminator; 3.2 μl of primer (100 pmol / μl); and template DNA ( 200-500 ng). The reaction cycle was 96 ° C. 2 minutes; 96 ° C. 10 seconds, 50 ° C. 5 seconds, and 60 ° C. 4 minutes 18 cycles; 60 ° C. 2 minutes; and 4 ° C. ∞. After termination of the terminator reaction, analysis was performed using ABI PRISM ^™ 310 Genetic Analyzer (Applied Biosystems).

(14. Fluorescent antibody staining)
A small amount of culture solution (OD ₆₀₀ = 2.5-3.0 corresponds to 1 ml) was centrifuged at 3000 rpm for 5 minutes to collect cells. The supernatant was discarded, washed with 1 ml of PBS buffer (10 × PBS buffer (Nippon Gene)), and centrifuged again under the same conditions. After discarding the supernatant, it was suspended in 1 ml of 3.7% (w / v) formaldehyde / PBS buffer and allowed to stand at room temperature for 1.5 hours. The cells were collected again by centrifugation at 3000 rpm for 5 minutes. Then, washing with 1 ml of PBS buffer was performed three times. The cells were resuspended in 300 μl of 1% (w / v) calf serum albumin (Nacalai) / PBS buffer and allowed to stand at room temperature for 30 minutes. 1 μl of the primary antibody FLAG antibody (SIGMA) was added to 300 μl of the solution after completion of blocking. After lightly mixing, the mixture was allowed to stand at room temperature for 1.5 hours. The cells were collected again by centrifugation at 3000 rpm for 5 minutes. Then, after washing with 1 ml of PBS buffer, it was resuspended in 300 μl of PBS buffer. 1 μl of secondary antibody Alexa Fluor ™ 488-conjugated goat anti-mouse IgG (Molecular Probes) was added to the solution. After lightly mixing, the mixture was allowed to stand at room temperature for 1.5 hours. The cells were collected again by centrifugation at 3000 rpm for 5 minutes. Then, after washing with 1 ml of PBS buffer, it was resuspended in 30 μl of PBS buffer. The yeast cells in this suspension were observed with a microscope IX71 (Olympus) equipped with a fluorescent filter U-MNIBA2 (Olympus).

(15. Activity measurement)
The lipase activity and esterase activity of lipase expressed in yeast were measured using several methods. The lipase-presenting yeast used for activity measurement was cultured in 10 ml SDC-W liquid medium for 2 days, washed three times with 100 mM Tris-HCl buffer (pH 7.4), and then washed with 100 mM Tris-HCl buffer (pH 7.4). ) And adjusted to OD ₆₀₀ = 3.0.

(15-1. Confirmation of activity by plate assay)
By adding colonies directly to a plate assay containing tributyrin or soybean oil, halos generated around the yeast colonies were confirmed. If active lipase is expressed on the cell surface, the substrate in the medium is decomposed, and the turbid color plate changes clearly and transparently. The presence or absence of enzyme activity was confirmed by observing the color change around the colonies on the plate.

(15-2. Activity measurement using p-nitrophenyl ester)
The esterase activity of the surface lipase was measured by the following procedure: Prepared 2.5 mM p-nitrophenyl ester; added 0.05 M potassium phosphate (pH 6.5) containing 0.5% Triton X-100; complete by sonication Pre-incubation for 3 minutes at 30 ° C; start reaction by adding an equal volume of enzyme solution (yeast) to 0.5 mL of substrate solution; incubate for 10 minutes at 30 ° C; stop reaction by adding 1 mL of acetone; 5 minutes at 12000 g Centrifugation; 200 μl of the supernatant was collected in a 96-well plate; the absorbance at λ405 nm was measured, and the difference from the background was taken as the activity value.

(15-3. Activity measurement using fluorescent substrate fluorescein diester)
Measure the fluorescent substrates fluorescein diacetate and fluorescein dibutyrate in 2 mg screw tubes, add 500 μl of N, N-dimethylformamide, dissolve by pipetting, and suspend in 9.5 ml of 100 mM Tris-HCl buffer (pH 7.4). It became cloudy. The substrate solution and the bacterial cell solution were mixed in 100 μl each in a 96-well plate, and the fluorescence of free fluorescein was measured with a Fluoroscan Ascent Fluorometer (Labsystems OY, Helsinki).

(16. Polyacrylamide gel electrophoresis)
16 μl of sample and SDS-PAGE sample buffer (60 mM Tris-HCl (pH 6.8); 25% (w / v) glycerol; 2% (w / v) SDS; 14.4 mM mercaptoethanol; 0.1% bromophenol blue; dH ₂ O) 4 μl was mixed, boiled for 3 minutes, and then applied to a 10% polyacrylamide gel. Electrophoresis was performed at 150 V for 70 minutes using an electrophoresis buffer (25 mM Tris; 192 mM glycine; 0.1% (w / v) SDS; distilled water). The gel after electrophoresis was immersed in distilled water, and after 5 minutes, water was exchanged three times for desalting. The gel was then immersed in SimplyBlue ^™ SafeStrain (Invitrogen) and stained with shaking for 1 hour at room temperature. Finally, the gel was washed by immersing in 100 ml of distilled water for about 1 hour.

In order to prepare a 10% polyacrylamide gel, the following solutions were mixed to prepare a 10% running gel and a 4% stacking gel. The composition of 10% running gel (12 ml) is H ₂ O 4.8 ml; Solution I (1.5 M Tris-HCl (pH 8.8); 0.4% (w / v) SDS; dH ₂ O) 3.0 ml; 30% screw Acrylamide 4.0 ml; 10% SDS 120 μl; 10% ammonium persulfate 60 μl; and N, N, N ′, N′-tetramethylethylenediamine (TEMED) 5 μl, the composition of 4% stacking gel (10 ml) is H ₂ O 6.5 ml; Solution II (0.5 M Tris-HCl (pH 6.8); 0.4% (w / v) SDS; dH ₂ O) 2.5 ml; 30% bisacrylamide 1.0 ml; 10% SDS 100 μl; 10% ammonium persulfate 75 μl And N, N, N ′, N′-tetramethylethylenediamine (TEMED) 8 μl.

(Example 1: Construction of a CALB surface display plasmid containing a propeptide)
A gene encoding CALB derived from Candida antarctica CBS 6678 strain was obtained from the Graduate School of Natural Sciences, Kobe University in the form of a plasmid pWIFS-pmCALB for cell surface display by Flo1p. In addition to the UPR-ICL promoter, the N-terminal region of Flo1p, the multiple cloning site, and UPR-term, this plasmid has TRP1, yeast replication origin 2 μm, E. coli replication origin ColE1, and ampicillin resistant Amp ^r as auxotrophic markers. The gene is generated by inserting a CALB-encoding gene containing a propeptide derived from Candida antarctica CBS 6678 strain into the cassette vector pWIFS.

(1-1. Introduction of CALB gene into plasmid for surface display)
Primers CALB-BglII-f1 (SEQ ID NO: 3) and CALB-XhoI-r1 (SEQ ID NO: 4) were designed to clone the CALB gene containing the propeptide. The CALB gene derived from Candida antarctica CBS 6678 strain was cloned by PCR using pWIFS-pmCALB as template DNA and CALB-BglII-f1 and CALB-XhoI-r1 as primers. The product was purified using PCR Kleen Column, and it was confirmed by agarose electrophoresis that a band was observed at the target position.

  The vector pICAS1 (Murai et al., Appl. Environ. Microbiol., 1998, 64, 4857-4861) was digested with restriction enzymes EcoRI and KpnI to obtain an EcoRI-KpnI fragment. This EcoRI-KpnI fragment has a sequence encoding a secretion signal of a glucoamylase gene, a multicloning site capable of recognizing restriction enzymes SacII, BglII, NcoI, and XhoI, and a sequence encoding 320 amino acids from the C-terminal of α-agglutinin. included. This 320 amino acid coding sequence includes a base sequence encoding a GPI anchor attachment recognition signal. This fragment was ligated to the vector pGA11 (Ueda et al., Ann. NY. Acad. Sci., 1998, 864, 528-537) digested with EcoRI and KpnI. Thereby, a cassette vector pCAS for surface display of yeast cells was obtained.

  pCAS was treated with BglII and XhoI and ligated with the CALB gene cloning product also treated with BglII and XhoI. The completed plasmid was named pCAS-CALB.

(1-2. Confirmation of CALB gene insertion)
Using a DNA sequencer, the nucleotide sequence near the CALB gene of the completed plasmid DNA was read. As a result, it was found that the base sequence of the region amplified by PCR was correctly inserted.

(1-3. Insertion of linker)
Synthetic oligonucleotides Linker18-F (SEQ ID NO: 5) and Linker18-R (SEQ ID NO: 6) were designed as genes encoding a linker sequence composed of glycine and serine. pCAS-CALB was cleaved using restriction enzyme XhoI and 10 × High buffer. This vector and a synthetic oligonucleotide encoding a linker sequence were ligated at the XhoI site, and the completed plasmid was named pCAS-R-CALB.

(1-4. Confirmation of linker sequence insertion)
The DNA base sequence of the C-terminal of CALB of pCAS-R-CALB and the linker sequence (amino acid sequence shown in SEQ ID NO: 7) was read using a DNA sequencer to confirm the insertion of the linker sequence.

(1-5. FLAG sequence insertion)
For confirmation when CALB was presented on the surface layer, a FLAG sequence generally used as a tag was inserted by the quick change method. Primers CALB-FLAG-f1 (SEQ ID NO: 8) and CALB-FLAG-r1 (SEQ ID NO: 9) were designed for FLAG sequence insertion. Quick change was performed by PCR using pCAS-R-CALB as template DNA and CALB-FLAG-f1 and CALB-FLAG-r1 as primers, and a FLAG sequence was inserted. The completed plasmid was named pCAS-FLAGR-CALB. A schematic diagram of this plasmid is shown in FIG. As shown in FIG. 1, a gene encoding CALB containing a propeptide could be included in the surface layer display plasmid.

(1-6. Confirmation of FLAG sequence insertion)
The CLB terminal of CALB of pCAS-FLAGR-CALB, the linker sequence, and the DNA base sequence of the FLAG sequence were read using a DNA sequencer to confirm the insertion of the FLAG sequence. As a result, it was found that CALB was inserted correctly in the order of C-terminal, FLAG, and linker sequence.

(1-7. Correction of CALB sequence)
The CALB sequence derived from Candida antarctica CBS 6678 strain is slightly different in gene sequence from the CALB sequence derived from the Candida antarctica LF 058 strain disclosed on the database. Seven mutations were introduced: A25T, A28T, S31T, G46Q, A89T, N97R, V286I. The lipase activity was not high in yeast introduced with a plasmid containing the CALB sequence in which these mutations were introduced.

  Therefore, mutations were introduced into these mutation sites and corrected to CALB sequences derived from Candida antarctica LF 058 strain. For mutagenesis, primers CAL B-muta-F1 (SEQ ID NO: 10), CAL B-muta-F2 (SEQ ID NO: 11), CAL B-muta-F3 (SEQ ID NO: 12), and CALB-G46Q-f ( SEQ ID NO: 13) was designed. Using the above four primers, a quick change method was performed in which mutations were simultaneously introduced into a plurality of sites. This introduced mutations for a total of seven amino acids.

(1-8. Confirmation of CALB sequence correction)
The base sequence of CALB mutated by the quick change method was determined using a DNA sequencer. This base sequence is as described in SEQ ID NO: 1 in the sequence listing. Thereby, it was confirmed that the amino acids at all sites were correctly corrected to the CALB sequence derived from Candida antarctica LF 058 strain. The amino acid sequence of this corrected CALB is as shown in SEQ ID NO: 2 (amino acid sequence from alanine at position -7 to proline at position 317). Hereinafter, CALB containing a propeptide having the modified amino acid sequence may be referred to as “wild type CALB”.

(Example 2: Production of surface layer CALB mutant)
(2-1. Modification of C-terminal lysine-arginine site of wild-type CALB)
Near the C-terminus of CALB is a site followed by lysine-arginine that can be recognized by KEX2 protease, the yeast endogenous protease. In the process until CALB is expressed on the cell surface in the yeast Saccharomyces cerevisiae, it is considered that CALB is not correctly presented when it is cleaved by KEX2 protease.

  Therefore, mutations were introduced into lysine at position 308 and arginine at position 309 of wild type CALB. At this time, a mutant (“R309K”) in which arginine at position 309 was substituted with lysine was prepared in order to keep the recognition by KEX2 protease low and not to greatly change the properties of the amino acid. In order to completely suppress recognition by KEX2 protease, mutants in which lysine at position 308 and arginine at position 309 were substituted with alanine (“K308A” and “R309A”, respectively) were also prepared.

The following primers were designed for mutagenesis. As primers for preparing the “R309K” mutant, CALB-kex2K-f (SEQ ID NO: 14) and CALB-kex2K-r (SEQ ID NO: 15) were used as primers for preparing the “K308A” mutant, CALB-K308A. -F (SEQ ID NO: 16) and CALB-K308A-r (SEQ ID NO: 17), and CALB-R309A-f (SEQ ID NO: 18) and CALB-R309A-r (primers for generating the "R309A" mutant) SEQ ID NO: 19) was prepared. Using each primer of the above combination, each mutant R309K, K308A, R309A was prepared by a quick change method using pCAS-FLAGR-CALB as template DNA and Pfu Ultra ^™ as polymerase.

(2-2. Modification of wild-type CALB propeptide)
CALB is presumed that a propeptide composed of 7 amino acids is related to maturation. It is thought that CALB is correctly folded when the C-terminal lysine-arginine residue of the propeptide is cleaved by a KEX2 type protease. Actually, from the result of X-ray crystal structure analysis, it can be seen that the mature CALB is deficient in propeptide. However, it is unclear how a very short peptide consisting of 7 amino acids is involved in protein activation. During the expression of CALB in yeast Saccharomyces cerevisiae, it is not clear whether the yeast endogenous protease acts on the residue of the C-terminal lysine-arginine site of the propeptide. It is not clear how it will affect activation.

  In order to elucidate the effect of this propeptide on CALB activation, lysine at position 6 (-2 position of SEQ ID NO: 2) and 7 position (-1 position of SEQ ID NO: 2) of the wild type CALB propeptide An attempt was made to modify arginine. For this purpose, a mutant in which arginine at position 7 was replaced with lysine ("proR7K") and a mutant in which lysine at position 6 was replaced with alanine and arginine at position 7 was replaced with alanine ("proK6A / R7A") were prepared. A mutant lacking the propeptide (“Δpro”) was also produced. Furthermore, a mutant (“pro → Linker”) in which the propeptide was replaced with a linker peptide was prepared.

The following primers were designed for mutagenesis. ProCALB-R7K-f (SEQ ID NO: 20) and proCALB-R7K-r (SEQ ID NO: 21) are used as primers for producing the “proR7K” mutant, and proCALB-pro7 is used as a primer for producing the “proK6A / R7A” mutant. -K6A / R7A-f (SEQ ID NO: 22) and proCALB-K6A / R7A-r (SEQ ID NO: 23) were used as primers for the production of a “pro → Linker” mutant, and proCALB-Linker-F (SEQ ID NO: 24) And mCALB-f1 (SEQ ID NO: 26) and mCALB-r1 (SEQ ID NO: 27) were prepared as primers for the production of “Δpro” mutant and proCALB-Linker-R (SEQ ID NO: 25). Using the above combinations of primers, the mutants proR7K, proK6A / R7A, pro → Linker, and Δpro were transformed by the quick change method using wild-type pCAS-FLAGR-CALB as the template DNA and Pfu Ultra ^TM as the polymerase. Produced. In addition, by using the R309K mutant as the CALB gene of pCAS-FLAGR-CALB as the template DNA, proR7K / R309K, proK6A / R7A / R309K, and Δpro / R309K into which double mutations were introduced were prepared.

(2-3. Deletion of C-terminal region of wild-type CALB)
A recognition site by KEX2 protease followed by lysine-arginine exists near the C-terminus of CALB. In Candida antarctica, this site is thought to be expressed without cleavage during maturation. Therefore, the effect of the deletion of 8 residues after the C-terminal position 310 of CALB by yeast endogenous protease was examined.

  A mutant lacking the C-terminal 8 residues of wild-type CALB (mutant consisting of 309 amino acids: “ΔC-term”) was prepared. Primers delCtermini-R309A-F1 (SEQ ID NO: 28) and delCtermini-R309A-R1 (SEQ ID NO: 29) were designed for mutagenesis. Using the above primers, a C-terminal deletion mutant ΔC-term was prepared by the quick change method using wild-type pCAS-FLAGR-CALB as the template DNA and Pfu Ultra (registered trademark) as the polymerase. In addition, by using proR7K, proK6A / R7A, or Δpro as template DNA, proR7K / ΔC-term, proK6A / R7A / ΔC-term, and Δpro / ΔC-term into which double mutations were introduced were prepared.

(2-4. Confirmation of gene sequence of CALB mutant)
About all the produced CALB variants, the base sequence of the peripheral region of the mutation introduction site was read by a DNA sequencer, and it was confirmed that the mutation was correctly introduced.

(Comparative Example 1: Preparation of Rhizopus oryzae-derived lipase (ROL) cell surface display plasmid)
As a control, a Rhizopus oryzae-derived lipase (ROL) cell surface display plasmid was prepared based on the description in Non-Patent Document 24. This plasmid can display wild-type ROL (hereinafter, also referred to as “wtROL”; sometimes simply expressed as “ROL”) on the surface of yeast cells.

(Comparative Example 2: Preparation of control cell surface display plasmid)
As another control plasmid, a cell surface display plasmid pCAS-C containing no lipase was prepared. Even if the cell surface display cassette vector pCAS is introduced into yeast, α-agglutinin is not displayed on the cell surface due to frame shift. Therefore, to introduce cytosine into the pCAS multicloning site with a single nucleotide and to match the frame, using pCAS as a template and primers (SEQ ID NO: 30 and SEQ ID NO: 31), KOD Dash (registered trademark) (TOYOBO) PCR was performed. The obtained plasmid pCAS-C has a sequence encoding the secretion signal of the glucoamylase gene, a multicloning site consisting of 21 base pairs that can recognize restriction enzymes SacII, BglII, NcoI, and XhoI, and 320 from the C-terminus of α-agglutinin. Includes sequences encoding amino acids. This plasmid can display the C-terminal region of α-agglutinin to which a polypeptide consisting of 7 amino acids is added at the N-terminus on the surface of yeast cells.

(Comparative Example 3: Preparation of non-surface display plasmid)
As another control plasmid, a non-surface display plasmid pMW1 was prepared. pMW1 was prepared based on the description of T. Kanai et al., Appl. Microbiol. Biotechnol., 1996, 44, 759-765. Briefly, the 2.14 kbp EcoRI-EcoRI fragment of pMT34 (+3) (M. Tajima et al., Yeast, 1985, Vol. 1, pages 67-77) containing 2 μm DNA is inserted into the AatII site of pRS404 (Stratagene). PMW1 was obtained.

(Example 3: Transformation into yeast)
All of the prepared CALB surface expression plasmid DNAs were transformed into the yeast Saccharomyces cerevisiae MT8-1 strain to create CALB surface display yeast. Similarly, the plasmids prepared in Comparative Examples 1 to 3 were transformed into Saccharomyces cerevisiae MT8-1 strain, respectively, and wild-type ROL (wtROL) surface display yeast and pCAS-C recombinant yeast (surface display not containing lipase), respectively. Yeast introduced with a plasmid), and pMW1 recombinant yeast (yeast introduced with a non-surface display plasmid).

(Example 4: Confirmation of CALB cell surface display by immunoantibody staining)
In order to confirm that CALB was expressed and presented on the cell surface of the yeast cell into which the CALB surface expression plasmid was introduced, immunofluorescence microscopy was performed. A fluorescence micrograph of the yeast Saccharomyces cerevisiae MT8-1 strain transformed with pCAS-FLAGR-CALB containing wild-type CALB, created in Example 3 above, is shown in the lower side of FIG. The corresponding phase difference image is shown on the upper side of FIG. When these photographs were examined together, fluorescence was observed in the surface layer of the cells in which the plasmid for expression of the CALB surface layer was introduced. Therefore, it was shown that when the CALB surface expression plasmid is introduced into yeast, the expressed CALB is presented on the cell surface.

(Example 5: Confirmation of lipase activity of surface layer-presented CALB)
(5-1. Confirmation of lipase activity by halo assay)
In order to confirm the presence or absence of the activity of the surface layer-displayed CALB, evaluation by a halo assay using a plate was performed. First, SDC medium supplemented with 0.2% tributyrin for wild-type CALB surface-displaying yeast (wtCALB), wild-type ROL surface-displaying yeast (wtROL), pCAS-C recombinant yeast, and pMW1 recombinant yeast (FIG. 3) And SDC medium supplemented with 0.2% soybean oil (results not shown). A halo was formed in the wild-type CALB surface-displaying yeast and the wild-type ROL surface-displaying yeast, but there was no halo formation in the pCAS-C recombinant yeast and the pMW1 recombinant yeast. Subsequently, wild-type CALB surface display yeast, CALB mutants proR7K, proR7K / R309K, proK6A / R7A, proK6A / R7A / R309K surface display yeast, and pCAS-C recombinant yeast were added with 0.2% tributyrin The SDC medium was tested (FIG. 4). Detection of halo was confirmed in the surface-displaying yeast of wild-type CALB (wt) and all CALB variants. In pCAS-C, no halo was detected.

(5-2. Change in lipase activity related to the growth stage of yeast)
In order to examine the change in the expression level of CALB presented on the surface of the yeast cell depending on the growth stage of the yeast, the activity was measured at each growth period. The growth was confirmed by growing the yeast in SDC-W liquid medium at 30 ° C. and measuring the cell density at OD ₆₀₀ . As a substrate used for activity measurement, p-nitrophenyl butyrate, which is a p-nitrophenyl ester having an acyl group having 4 carbon atoms, was selected. FIG. 5 shows the time course of cell growth during the culture of wild-type CALB surface-displayed yeast and pCAS-C, and FIG. 6 shows the time course of the lipase activity during the culture of wild-type CALB surface-displayed yeast and pCAS-C. (Both diamonds represent wild-type CALB and squares represent pCAS-C). As a result, it was found that the CALB surface-displaying yeast entered the stationary phase from the logarithmic phase around 40 hours (FIG. 5). 5 and 6 were examined together, it was found that the lipase activity value of the CALB surface-displaying yeast hardly changed from the latter half of the log phase to the stationary phase.

(5-3. Change in activity by CALB mutant)
The activity of the various CALB variants constructed was measured using p-nitrophenyl butyrate and fluorescein diester. FIG. 7 shows the lipase activity of wild type CALB and CALB variants measured using p-nitrophenyl butyrate, and FIG. 8 shows the lipase activity of wild type CALB and CALB variants measured using fluorescein diester. Indicates. The mutant lacking the CALB propeptide or the mutant substituted with the linker peptide was less active than the wild type. On the other hand, the activity of the mutant of the residue at the C-terminal lysine-arginine site of the propeptide slightly increased. Thus, it was found that the propeptide is very important for the activity of CALB.

(Example 6: Evaluation of substrate specificity of surface-presented CALB)
In order to examine the substrate specificity of the surface layer-displayed CALB, activity was measured using p-nitrophenyl ester having an acyl group having 2 to 18 carbon atoms as a substrate. It was compared with surface layer-displayed ROL, which is known to have high specificity for medium chain length substrates. In the substrate solution, an enzyme surface layer-displaying yeast having an OD _{600 of} 5 was reacted at 30 ° C. for 5 minutes, and then the absorbance at 405 nm was measured to determine the lipase activity. The result is shown in FIG. In FIG. 9, in each acyl chain length, the left bar represents wild-type CALB, and the right bar represents wild-type ROL. From FIG. 9, it was found that wild-type CALB has a higher reactivity to a substrate having a shorter chain length (C2 to C6) than wild-type ROL.

(Example 7: Evaluation of thermal stability of surface layer presentation CALB)
CALB is known to have higher stability than general lipases. Novozyme435, an immobilized enzyme of CALB, is extremely stable and exhibits high activity even in an organic solvent. When used in a catalytic reaction (for example, ester synthesis reaction) in an organic solvent or under high temperature conditions, the stability of the lipase is extremely important.

  Therefore, in order to examine the thermal stability of the surface layer presented CALB, the residual activity after the heat treatment was measured. The thermal stability of the surface layer presentation CALB was evaluated by comparison with the surface layer presentation ROL. For the measurement of residual activity, p-nitrophenyl butyrate was used as a substrate.

  First, in the reaction of the substrate solution and the enzyme solution (yeast) based on “Activity measurement using p-nitrophenyl ester” in the procedure 15-2 of the above method, at a set temperature from 0 ° C. to 100 ° C. Enzyme activity was measured immediately after each 30 minute yeast treatment. Both CALB and ROL are relative values based on the value at the time of 30 ° C. treatment (activity 100%), and are shown as residual activity (FIG. 10: in the figure, diamonds represent surface layer-presented wild-type CALB and squares represent surface layer) Represents wild type ROL). As a result, the activity of the surface-displayed wild-type ROL rapidly decreased when the temperature exceeded 40 ° C., and almost no activity was observed near 50 ° C. On the other hand, the surface layer-displayed wild-type CALB did not lose its activity as rapidly as the surface layer-displayed wild-type ROL even when the temperature exceeded 40 ° C., and maintained almost half of the activity even near 60 ° C. Thus, it was found that the surface layer-displayed wild-type CALB has higher thermal stability than normal lipase.

  Next, the residual activity for each treatment time at 60 ° C. was measured to evaluate the stability. During each set time, the residual activity after treatment at 60 ° C. was expressed as a relative value with reference to the respective values of wild-type CALB and wild-type ROL at the time of 0 minute measurement (activity 100%) (FIG. 11). : In the figure, diamonds represent surface-presented wild-type CALB, and squares represent surface-presented wild-type ROL). Wild-type ROL lost activity in a few minutes, whereas wild-type CALB retained more than half the activity until nearly 20 minutes.

(Example 8: Ethyl lactate synthesis using surface-presented CALB)
10 mg of freeze-dried wild-type CALB surface-displayed yeast was added to 2 ml of a reaction solution containing 0.1 M ethanol and 0.3 M lactic acid in heptane saturated with water, and reacted at 50 ° C. for 100 hours while shaking at 250 rpm. It was. The reaction solution was subjected to molecular sieves 3A (Wako Pure Chemical Industries, Ltd.) 72 hours after the start of the reaction, and the reaction was further continued. As a control, pCAS-C recombinant yeast, which is a yeast introduced with a surface layer display plasmid not containing lipase, was used.

After completion of the reaction, the reaction solution was analyzed by high performance liquid chromatography (HPLC) under the following conditions:
Column: ODS-W 5C ₁₈ -MS-II (Nacalai Tesque)
Eluent A: 0.1% (v / v) trifluoroacetic acid aqueous solution
B: Methanol A: B = 8: 2 (v / v) used Flow rate 1.0 ml / min Temperature 40 ° C.
Detector UV 210nm
Injection volume: 10 μl.

  FIG. 12 is a chromatogram showing the results of the above HPLC analysis, and the horizontal axis represents the retention time (minutes). FIG. 12 shows the results of HPLC analysis in a reaction solution of lactic acid and ethanol to which wild type CALB surface display yeast or pCAS-C recombinant yeast (“control yeast” in the figure) was added, together with the peak of the ethyl lactate preparation. Show. In the reaction solution to which the wild-type CALB surface-displaying yeast was added (“CALB-presenting yeast” in the figure), a peak similar to that of the ethyl lactate preparation was observed, but in the reaction solution to which the pCAS-C recombinant yeast was added, Such a peak was not obtained.

  As calculated from the calibration curve of the standard product, the amount of ethyl lactate synthesized under the above conditions using the wild-type CALB surface display yeast was 43.5 mM, and the yield was 43.5%.

  The surface layer-displayed CALB of the yeast of the present invention has high thermal stability and high lipase activity. Moreover, the surface layer presentation CALB does not require special chemical modification. Therefore, it can be efficiently and inexpensively used for ester bond decomposition reactions, ester synthesis reactions, transesterification reactions, and asymmetric synthesis of various chiral compounds, which are important catalytic reactions in which general lipases are used. Furthermore, according to the present invention, ethyl lactate can be efficiently produced using the CALB surface-displaying yeast, and an important process can be performed with a biocatalyst during the production of biodegradable plastic polylactic acid.

It is a schematic diagram of plasmid pCAS-FLAGR-CALB. It is the phase-contrast micrograph and immunofluorescence micrograph which show the cell form of wild type CALB surface layer display yeast MT8-1 / pCAS-FLAGR-CALB. It is a photograph which shows the halo formation in the culture medium by the wild-type CALB surface layer display yeast and ROL surface layer display yeast. It is a photograph which shows the halo formation in the culture medium by the wild-type CALB surface display yeast and the CALB mutant surface display yeast. It is a graph which shows a time-dependent change of the cell growth during culture | cultivation of wild type CALB surface layer display yeast and pCAS-C recombinant yeast. It is a graph which shows a time-dependent change of the lipase activity during culture | cultivation of wild type CALB surface layer display yeast and pCAS-C recombinant yeast. It is a graph which shows the lipase activity of the wild-type CALB surface display yeast and CALB mutant surface display yeast which were measured using p-nitrophenyl butyrate as a substrate. It is a graph which shows the lipase activity of the wild-type CALB surface layer display yeast and CALB mutant surface layer display yeast which were measured using fluorescein diester as a substrate. It is a graph which shows the substrate specificity of surface layer presentation wild type CALB and surface layer presentation wild type ROL. It is a graph which shows the residual activity after heating and inactivating the surface layer presentation wild type CALB and the surface layer presentation wild type ROL at various temperatures for 30 minutes. It is a graph which shows the residual enzyme activity after heat-processing a surface layer presentation wild type CALB and a surface layer presentation wild type ROL at 60 degreeC for various time. It is a chromatogram which shows the result of the HPLC analysis in the reaction liquid of lactic acid and ethanol which added wild type CALB surface layer display yeast or pCAS-C recombinant yeast (control yeast).

Claims (3)

A yeast that presents a mutant of Candida antarctica-derived lipase B (CALB) on the cell surface,
The mutant is
In the amino acid sequence from the alanine residue at position -7 to the proline residue at position 317 in SEQ ID NO: 2 in the sequence listing,
(1) a polypeptide comprising an amino acid sequence in which positions -2 and -1 are lysine residues, respectively;
(2) a polypeptide comprising an amino acid sequence in which positions -2 and -1 are lysine residues, respectively, and positions 308 and 309 are lysine residues; and (3) positions -2 and -1 are A yeast selected from the group consisting of a polypeptide consisting of an amino acid sequence each of which is an alanine residue and each of positions 308 and 309 is a lysine residue. A method for producing a lactate ester, the method comprising:
Reacting alcohol and lactic acid in the presence of yeast,
The yeast
A yeast that presents a mutant of Candida antarctica-derived lipase B (CALB ) consisting of an amino acid sequence from an alanine residue at position -7 to a proline residue at position 317 in SEQ ID NO: 2 in the sequence listing on the cell surface. And
The mutant is
In the amino acid sequence from the alanine residue at position -7 to the proline residue at position 317 in SEQ ID NO: 2 in the sequence listing,
(1) a polypeptide comprising an amino acid sequence in which positions -2 and -1 are lysine residues, respectively;
(2) a polypeptide comprising an amino acid sequence in which positions -2 and -1 are lysine residues, respectively, and positions 308 and 309 are lysine residues; and (3) positions -2 and -1 are A method selected from the group consisting of polypeptides consisting of amino acid sequences each of which is an alanine residue and each of positions 308 and 309 is a lysine residue.   The method of claim 2, wherein the alcohol is ethanol.

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