WO2015133146A1 - メタクリル酸エステルの製造方法および新規メタクリル酸エステル合成酵素 - Google Patents
メタクリル酸エステルの製造方法および新規メタクリル酸エステル合成酵素 Download PDFInfo
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- WO2015133146A1 WO2015133146A1 PCT/JP2015/001186 JP2015001186W WO2015133146A1 WO 2015133146 A1 WO2015133146 A1 WO 2015133146A1 JP 2015001186 W JP2015001186 W JP 2015001186W WO 2015133146 A1 WO2015133146 A1 WO 2015133146A1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
Definitions
- the present invention relates to a method for producing an organic acid ester, particularly a methacrylic acid ester, using a biocatalyst. More specifically, the present invention relates to a method for producing a methacrylate ester using an alcohol acyltransferase capable of producing a methacrylate ester, and further relates to these alcohol acyltransferases and methods for using the alcohol acyltransferase.
- Methacrylic acid ester is mainly used as a raw material for acrylic resin, and there is a great demand as a comonomer in fields such as paints, adhesives and resin modifiers.
- ACH acetone cyanohydrin
- the direct acid method using isobutylene and tert-butyl alcohol as raw materials are known. These chemical production methods depend on fossil raw materials and require a lot of energy.
- Patent Documents 1 and 2 For example, a method for producing 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid, which are precursors of methacrylic acid, from natural products such as sugars by using naturally occurring microorganisms has been proposed (Patent Documents 1 and 2). And Non-Patent Document 1). However, these methods still rely on chemical methods for the step of dehydrating the precursor to produce methacrylic acid.
- Patent Documents 3 to 5 exemplifies various biocatalysts (hydrolyzing enzyme, wax ester synthase, alcohol acetyltransferase) having general ester-forming activity, but the exemplified biocatalyst is a synthesis of methacrylate ester. It was unclear whether it had activity.
- Patent Document 6 discloses a method for producing an acrylate ester by allowing a hydrolase to act in the presence of acrylyl-CoA and alcohol. This document suggests that methacrylic acid esters can be produced in the same manner. However, considering the diversity of biocatalysts and substrate specificity, it has only shown that some hydrolases can produce acrylate esters, and methacrylic esters with different structures are also hydrolyzing enzymes. It was unclear whether it could be produced by Furthermore, it was completely unknown whether it could be produced with other types of biocatalysts having different reaction mechanisms. In addition, when an ester is synthesized by the hydrolase described in Patent Document 6, it is expected that the produced ester will be degraded by the hydrolysis activity in the first place, and it is difficult to consider an effective production method.
- Patent Document 7 proposes a method for synthesizing various esters that are fruit flavors by identifying the enzyme gene contained in a specific fruit. However, whether or not methacrylic acid esters can be synthesized by these enzymes has not been reported and it has been completely unknown.
- the present invention was completed by successfully obtaining a novel alcohol acyltransferase from a suspension of a plant body. That is, the present invention is as follows.
- the plant is selected from Osmanthus fragrans, grapes (Vitis vinifera), crepefruit (Citrus x paradisi), durian (Durio zibethinus), Karataneo gamum (Michelia figo) and roman Chamo mel A method for producing a methacrylic acid ester of [1] to [3], which is a plant.
- Methacrylic acid ester comprising a step of synthesizing methacrylic acid ester by reacting methacrylyl-CoA with alcohol or phenol in the presence of alcohol acyltransferase having the following physicochemical properties (1) to (3) Manufacturing method.
- An alcohol acyltransferase having the following physicochemical properties (1) to (5) or an enzyme composition thereof.
- Km value for methacrylyl-CoA is 0.5 mM or less.
- the optimum pH is 8-9 when methacrylyl-CoA and n-butanol are used as substrates.
- [7] The alcohol acyltransferase or the enzyme composition according to [6], which is derived from a plant belonging to the order of Asterales.
- the alcohol acyltransferase or the enzyme composition thereof according to [9] which is derived from Roman chamomile (Chamaemelum nobile).
- the plant belongs to a plant belonging to the family Oleaceae, a plant belonging to the Vitaceae, a plant belonging to the Rutaceae, a plant belonging to the Malvaceae, or a Magnoliaceae Alcohol acyltransferase according to [12], which is a plant and a plant belonging to the family Asteraceae.
- the plant belongs to a plant belonging to the genus Osmanthus, a plant belonging to the genus Grapes (Vitis), a plant belonging to the genus Citrus, a plant belonging to the genus Durian (Magnio), or a genus from Magnolia Alcohol acyltransferase according to [13], which is a plant and a plant belonging to the genus Chamaemelum.
- the plant is selected from Osmanthus fragrans, grapes (Vitis vinifera), crepefruit (Citrus x paradisi), durian (Durio zibethinus), calata neo gamum (Michelia figumol)
- Alcohol acyltransferase of [14] which is a plant.
- the alcohol acyltransferase of [15] having the following physicochemical properties (1) to (6): (1) Acts on methacrylyl-CoA in the presence of alcohols or phenols to produce a methacrylic ester. (2) Activity against methacrylyl-CoA is higher than activity against acetyl CoA.
- the present invention makes it possible to produce a methacrylic acid ester using a biocatalyst.
- fermentation production of methacrylic acid esters can also be achieved.
- a methacrylic acid ester by significantly reducing the load on energy, resources and the environment.
- novel enzyme of this invention to manufacture organic acid esters, such as methacrylic acid ester, more efficiently.
- FIG. 2 is a graph showing purification (elution pattern) using a DEAE-Toyopearl column (second time). It is a graph which shows the refinement
- the methacrylic acid ester is a compound represented by the formula 1.
- R represents a linear or branched hydrocarbon group having 1 to 20 carbon atoms.
- the hydrocarbon group may be saturated or unsaturated acyclic and may be saturated or unsaturated cyclic.
- a straight chain or branched chain unsubstituted alkyl group having 1 to 10 carbon atoms, an aralkyl group or an aryl group is preferred.
- Metal acid (IUPAC name: 2-methyl-2-propenoic acid) means a compound having the following formula, and includes any salt or ionized form thereof.
- methacrylic acid salts include sodium salts, potassium salts, calcium salts, and magnesium salts.
- methacrylyl-CoA is a compound represented by the following structural formula.
- Methacrylyl-CoA is known in vivo as a metabolic intermediate for valine.
- the methacrylyl-CoA used in the present invention may be a known or novel method.
- As the synthesis method a method of organically synthesizing methacrylic anhydride and coenzyme A (Methods in Enzymology. 324, 73-79 (2000)) or a synthesis method using an enzyme reaction is known.
- methacrylyl-CoA or 3-hydroxyisobutyryl converted by the action of acyl CoA dehydrogenase (EC 1.3.99.3) (hereinafter referred to as ACD) using isobutyryl-CoA as a raw material.
- ACD acyl CoA dehydrogenase
- -Methacrylyl-CoA converted from CoA by the action of enoyl CoA hydratase (EC 4.2.1.17) (hereinafter referred to as ECH)
- ECH enoyl CoA hydratase
- the methacrylyl-CoA used in the present invention can also be produced from 2-oxoisovaleric acid via isobutyryl-CoA.
- methacrylyl-CoA produced from isobutyryl-CoA or 3-hydroxyisobutyryl-CoA
- a continuous reaction by an enzyme leads to an improvement in yield and suppression of impurities, and to a living body.
- methacrylic acid esters without passing through or by-product of highly toxic methacrylic acid.
- the alcohol or phenol used as a raw material for producing the methacrylic acid ester in the present invention is a compound represented by the following formula 2. Since the structure of the alcohol or phenol corresponds to a methacrylic ester, the structure has the same definition as R in the formula 1, and represents a linear or branched hydrocarbon group having 1 to 20 carbon atoms.
- the hydrocarbon group may be saturated or unsaturated acyclic and may be saturated or unsaturated cyclic.
- the alcohol acyltransferase (hereinafter referred to as AAT) of the present invention is an enzyme having a catalytic action of synthesizing an ester by transferring an acyl group of acyl-CoA to alcohol or phenol.
- AAT is said to be involved in the formation of esters in various fruits.
- AAT is ginger (banana), rose (strawberry, apple, pear, peach), cucumber (melon), azalea (kiwi), perilla (olive), eggplant (tomato), mugwort (lemon, mango) ) And the like are known to exist in plants.
- AAT used in the present invention is from plants belonging to each order of the order Lamiales, Grapes (Vitales), Mukurodi (Sapindales), Aoi (Malvales), Magnoliaes (Asterales) and Asterales (Asterales).
- AAT derived from a plant selected from the group consisting of methacrylyl-CoA and alcohol or phenol as raw materials, and having the ability to produce methacrylic acid esters, is not particularly limited, the type and origin It doesn't matter.
- AAT suitable for the present invention can be easily obtained from the plant by the following method. Acquired by cutting the appropriate part of the tissue as necessary. A solution containing methacrylyl-CoA and an alcohol or phenol represented by Formula 2 is added to the cutting site, shaken, and allowed to react for a certain time. The synthetic activity can be confirmed by confirming the presence or absence of the methacrylic acid ester in the reaction solution by GC (gas chromatography). Specifically, for example, leaves, flowers, persimmons, flesh or pericarp are cut, and a solution containing 0.01 to 10 mM methacrylyl-CoA and 2 to 50-fold molar amount of n-butanol is added to the mixture at 30 ° C. Shake for 1-10 hours. After completion of the reaction, AAT applicable to the present invention can be obtained by confirming the presence or absence of a methacrylic ester by GC.
- Suitable AAT enzyme sources for the present invention are the group consisting of Lamiales, Grapes (Vitales), Mucarids (Sapindales), Mallows (Malvales), Magnoliaes (Asterales) and Asterales A plant belonging to any eye selected from.
- Lamiaceae As for belonging to Lamiaceae, Acanthaceae, Bignoniaceae, Bybridaceae, Calceolaeaceae, Carlemanniaceae, Carlemanniaaceae, Carlemanniaaceae , Linderinaceae, Lentibulariaceae, Martynaceae, Oleaceae, Obanchaedae, Paulowaceae, Paulowaceae Department (Planta inaceae), plocosperma (Plocospermataceae, Shuregeria family (Schlegeliaceae), Scrophulariaceae (Scrophulariaceae), Sutirube family (Stilbaceae), Tetorakondora family (Tetrachondraceae), plant Tomanderushia family (Thomandersiaceae) and Verbenaceae (Verbenaceae) are preferred.
- Vitaceae plants are preferred as belonging to the order of Grapes.
- the species belonging to the order of the saccharidaceae (Anacardiaceae), Bibelsteiniaceae (Biebersteiniaceae), Orchidaceae (Burseraceae), Kirkiaceae, Melidaceae, Neridaeaceae (Niteraeuceae) ), Plants of the family Sapindaceae and Simarobaceae.
- the family Bixaceae, Cistaceae, Cytinaceae, Dipterocarpacae, Malvaceae, Naceae Plants of the family Sarcolaenaceae, Sphaerosepalaceae and Thymelaeaceae are preferred.
- the order of the magnolia are the Annonaceae, Degeneriaceae, Eupomatiae, Himantaceae, and Magnoceae.
- Asteraceae family Arseosmiaceae
- Argophyllaceae family Asteraceae family
- Caryceraceae family Campanulaceae family
- Cetaceen family family Preference is given to plants of Menyanthaceae, Pentaphragmataceae, Phelinaceae, Rousesaceae and Stylidiaceae.
- plants belonging to the family Asteraceae, vines, citrus, mallow, magnolia or asteraceae are more preferred.
- the vines are the genus Vitis, Ampelopsis, Cayratia, Cissus, Cyphostemma, Leea, Parthenocissus and Parthenocissus Plants of the genus (Tetrastigma) are preferred.
- Citrus genus (Citrus), Aegle genus (Aegle), Salamander genus (Zanthoxylum), Gecki genus (Murraya), Henruda genus (Ruta), Kokusagi genus (Orixa), Miyashikimi genus (Skimmia) In the genus Euodia, Phellodendron, Boronia, Acronymia, Clauena, Correa, Corly, Glycosmis and M Plants belonging to the genus Litchi (Litchi) and those belonging to the family Ursiaceae are preferred.
- a plant belonging to the magnolia family is preferably a plant of the genus Magnolia.
- the genus Chamaemelum, Achillea, Echinacea, Matricaria, Tanacetum, Taramacum, Teramacum, Genus (Petasites), wheat genus (Helichrysum), cotton genus (Santolina), genus Cynara, sylybum, genus Calendula, genus Cichorum Plants belonging to (Chrysanthemum) are preferred.
- plants belonging to the genus Moxae, grape genus, mandarin genus, durian genus, magnolia genus or chamomile genus are particularly preferable.
- citrus are lemon (Citrus limon), sudachi (Citrus sudachi), kabosu (Citrus x paradisi), grapefruit (Citrus x paradisi), citrus (Citrus junarus), Citrus auran (Citrus sinensis);
- Aegle Aegle marmelos (Aegle marmelos);
- the genus Reishi Litchi chinensis;
- Mangifera indica is preferable.
- Dorian As belonging to the genus Dorian (Durio zibethinus, Durio testdinarius, Durio kutejenissis, Duri oxleyanus, Durio graveolens, Durio dulicis); Kara seeds as belonging to Magnolia Ogatama (Magnolia figo), Ogatamanoki (Magnolia compressa), Kinkouboku (Magnolia champaca), magnolia (Magnolia liliiflora), fist (Magnolia kobus), Magnolia obovata (Magnolia obovata and Magnolia laevifolia) are preferred.
- Ogatama Magnnolia figo
- Ogatamanoki Magnnolia compressa
- Kinkouboku Magnnolia champaca
- magnolia Magnnolia liliiflora
- fist Magnnolia kobus
- Magnolia obovata Magnnolia
- Roman chamomile As those belonging to the genus Chamomile, Roman chamomile (Chamaemelum nobile and Chamaemelum fuscatum) is preferred. Among them, mokusei, grape, crepe fruit, durian, carataneotama or roman chamomile are particularly preferable.
- plant classification shall be in accordance with the APG plant classification system 3rd edition (Botanical Journal of the Linnian Society, 2009, 161, 105121).
- a biological tissue containing AAT or a processed product thereof can be used as it is.
- a biological tissue an entire plant body, a plant organ (for example, fruit, leaf, petal, stem, seed, etc.), or a plant tissue (for example, fruit epidermis, pulp, etc.) can be used.
- the processed tissue include AAT crude enzyme solution or purified enzyme extracted from the living tissue.
- the method for purifying AAT is not particularly limited, it is preferably isolated by the following method.
- the plant tissue having AAT activity is disrupted and then suspended in a buffer such as Tris-HCl buffer or phosphate buffer.
- a buffer such as Tris-HCl buffer or phosphate buffer.
- this crude enzyme solution (1) fractionation by precipitation, (2) various chromatographies, (3) methods for removing low-molecular substances by dialysis, ultrafiltration, etc. Or use in combination as appropriate.
- a living tissue is frozen with liquid nitrogen or the like and ground, and then extracted with a Tris-HCl buffer containing 5 times the amount of DTT (dithiothreitol) and glycerol.
- the crude enzyme extract is subjected to ion exchange chromatography, and the non-adsorbed portion is recovered to obtain the enzyme extract.
- this method eliminates the influence of polyphenols contained in plants and can be obtained stably and efficiently. It has also been found that fractionation by precipitation with ammonium sulfate or the like induces inactivation of enzyme activity.
- Enzyme protein can be efficiently purified by using the obtained enzyme extract using ion exchange chromatography, gel filtration column or the like.
- Gene information can be obtained by genetic engineering techniques based on the purified AAT protein.
- the gene information is isolated or totally synthesized by a known method, introduced into a general host vector system, a candidate protein is expressed by a microorganism transformed with the vector system, and the methacrylic acid ester according to the present invention is produced. Can be used.
- the AAT of the present invention catalyzes a reaction that acts on methacrylyl-CoA to produce a methacrylic acid ester in the presence of an alcohol or a phenol.
- AAT is preferably highly reactive when methacrylyl-CoA is used as a substrate. Specifically, it is preferable that the activity against methacrylyl-CoA is higher than the activity against acetyl-CoA and the Km value against methacrylyl-CoA is 0.5 mM or less. According to AAT having such properties, a methacrylic acid ester can be produced with good selectivity.
- Substrate specificity AAT preferred in the present invention is preferably an AAT having a low reactivity to at least acetyl-CoA based on the reactivity to methacrylyl-CoA. More specifically, the preferred AAT of the present invention preferably has a reactivity to acetyl-CoA equal to or less than that when n-butanol is used as a substrate and the reactivity to methacrylyl-CoA is 100%. Is 50% or less, more preferably 40% or less.
- the reactivity with respect to methacrylyl-CoA is 100%
- the reactivity with respect to acetyl-CoA is preferably equal to or less, more preferably 70% or less, and still more preferably. 50% or less.
- the AAT of the present invention preferably has a high affinity for methacrylyl-CoA.
- the affinity for the substrate can be evaluated by the Michaelis constant (Km).
- Km for methacrylyl-CoA is a value obtained by measurement and calculation according to the examples described later.
- the Km value for methacrylyl-CoA of AAT of the present invention is preferably not more than the Km value for acetyl-CoA, preferably 0.5 mM or less, more preferably 0.2 mM or less, and still more preferably 0 0.1 mM or less, particularly preferably 0.05 mM or less. Due to the high affinity, even if the concentration of methacrylyl-CoA as a raw material is low, the above-described catalytic reaction can proceed, and a methacrylic acid ester can be produced more efficiently.
- AAT genes have been published. Based on the information, a DNA probe can be prepared, for example, a primer used for PCR can be prepared, and the gene can be isolated by performing PCR. It is also possible to completely synthesize the base sequence of the AAT gene by a usual method. Whether or not AAT for which genetic information is known has methacrylic acid ester synthesis activity can be confirmed in the same manner as described above. On the other hand, for AAT for which genetic information is unknown, AAT can be purified, and genetic information can be obtained by genetic engineering techniques based on the protein.
- preferred AAT genes belong to the order of the order Lamiales, Grapes (Vitales), Mukuroji (Sapindales), Aoi (Malvales), Magnolia (Asterales) and Asterales (Asterales). It is not particularly limited as long as it is derived from a plant selected from the group consisting of plants and the translation product has the ability to produce a methacrylic acid ester, and is appropriately selected from the AAT enzyme sources.
- the AAT gene includes an amino acid sequence in which one or several amino acids are substituted, deleted, or added in the wild-type amino acid sequence, and has an activity of producing a methacrylate ester from methacrylyl-CoA and alcohol. Also included are genes that encode the proteins they have.
- “several” means 1 to 40, preferably 1 to 20, more preferably 10 or less.
- a mutation introduction kit using site-directed mutagenesis by a known method such as the Kunkel method or the Gapped duplex method, for example, QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene), GeneTailorTM Site-Directed Mutagenesis System (Invitrogen Corporation), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: Takara Bio Inc.) and the like can be used.
- the entire gene having a sequence containing a mutation may be artificially synthesized.
- the DNA base sequence can be confirmed by sequencing by a conventional method. For example, based on the Sanger method, it is also possible to confirm the sequence using an appropriate DNA sequencer.
- the AAT gene has 90% or more, preferably 95% or more, more preferably 99.5% or more, and still more preferably 99.9% or more identity with a protein comprising a wild-type amino acid sequence. Also included are genes that encode proteins that have the activity of producing methacrylic acid esters from methacrylyl-CoA and alcohols.
- the AAT gene is hybridized under stringent conditions to a polynucleotide having a base sequence complementary to the wild-type base sequence to produce a methacrylate ester from methacrylyl-CoA and alcohol.
- a gene encoding a protein having activity is also included.
- the stringent conditions include, for example, a nylon membrane on which DNA is immobilized, 6 ⁇ SSC (1 ⁇ SSC is 8.76 g of sodium chloride, 4.41 g of sodium citrate dissolved in 1 liter of water), Hybridization by incubation with a probe at 65 ° C.
- washing conditions after hybridization for example, “2 ⁇ SSC, 0.1% SDS, 42 ° C.”, “1 ⁇ SSC, 0.1% SDS, 37 ° C.”, and more stringent conditions, for example, Conditions such as “1 ⁇ SSC, 0.1% SDS, 65 ° C.” and “0.5 ⁇ SSC, 0.1% SDS, 50 ° C.” can be mentioned.
- the AAT gene contains 80% or more, more preferably 90% or more, most preferably, when calculated using a wild-type base sequence and BLAST or the like (for example, default or default parameters).
- a gene encoding a protein consisting of a base sequence having an identity of 95% or more and having an activity of forming a methacrylic acid ester from methacrylyl-CoA and an alcohol is also included.
- the codon of the AAT gene may be converted according to the codon usage frequency of the microorganism host used for transformation.
- the “identity” of the sequences means that both base sequences are aligned so that the bases of the two base sequences to be compared match as much as possible. What is divided by the number of bases is expressed as a percentage.
- a gap is appropriately inserted in one or both of the two sequences to be compared as necessary.
- Such alignment of sequences can be performed using a known program such as BLAST, FASTA, CLUSTALW, and the like.
- the total number of bases is the number of bases obtained by counting one gap as one base.
- the identity (%) is calculated by dividing the total number of bases of the longer sequence and dividing the number of matched bases. The same applies to amino acid sequence identity.
- For the methacrylic acid ester synthesis reaction use the culture solution obtained by culturing these recombinant microorganisms as they are, or use the microbial cells obtained from the culture solution by a collection operation such as centrifugation or the processed product thereof.
- treated cells include cells treated with acetone, toluene, etc., freeze-dried cells, disrupted cells, cell-free extracts obtained by disrupting cells, and crude enzymes or purified enzymes extracted from these. Can be mentioned.
- An ACD gene, ECH gene, BCKAD (2-oxoisovalerate dehydrogenase) gene or the like is introduced into a microorganism into which an AAT gene has been introduced as necessary, and isobutyryl-CoA, 3-hydroxyisobutyryl-CoA or 2- It is also possible to synthesize methacrylate esters from precursors such as oxoisovaleric acid.
- Precursor means a compound derivable to methacrylyl-CoA, and indicates a substance derivable to isobutyryl-CoA or 3-hydroxyisobutyryl-CoA, and further to these two compounds.
- Substances derivable into two compounds include, for example, 2-oxoisovaleric acid, isobutyric acid, 3-hydroxyisobutyric acid, acetic acid, pyruvic acid, lactic acid, acetoacetic acid, butyric acid, propionic acid, malic acid, fumaric acid, citric acid and Examples thereof include acids such as succinic acid, amino acids such as valine, alanine, leucine, lysine and glutamic acid, and sugars such as glucose, fructose and xylose.
- various metabolic systems originally possessed by the host microorganism can be used as they are.
- a gene can be introduced or deleted as necessary.
- Step of synthesizing methacrylic acid ester The production of methacrylic acid ester can be carried out by the following method. To the solvent, methacrylyl-CoA and the alcohol or phenol represented by Formula 2 are added and dissolved or suspended. Then, AAT is brought into contact with this solution or suspension to react methacrylyl-CoA with alcohol or phenols while controlling conditions such as temperature. By the reaction, the methacrylyl-CoA methacrylic group is transferred to the alcohol or phenol of formula 2 to produce a methacrylic ester.
- a solution containing methacrylyl-CoA and the alcohol or phenol represented by the formula 2 is usually prepared in an aqueous medium such as a buffer solution.
- an osmotic pressure adjusting agent may be a water-soluble substance added for the purpose of adjusting to be isotonic or hypertonic with respect to the osmotic pressure of the solution inside the cell, for example, a salt or a saccharide, preferably Salt.
- the salt is preferably a metal salt, more preferably an alkali metal salt, more preferably an alkali metal halide, and examples thereof include sodium chloride and potassium chloride.
- the saccharide is preferably a monosaccharide or oligosaccharide, more preferably a monosaccharide or disaccharide, and examples thereof include glucose, sucrose, and mannitol.
- the osmotic pressure adjusting agent is preferably added at a concentration of 1 mM or more, and particularly preferably adjusted to be isotonic or hypertonic as compared with the solution in the living cell to be used.
- organic solvent for example, linear, branched or cyclic, saturated or unsaturated aliphatic hydrocarbon, saturated or unsaturated aromatic hydrocarbon, etc. can be used alone or in admixture of two or more.
- hydrocarbon solvents eg, pentane, hexane, cyclohexane, benzene, toluene, xylene, etc.
- halogenated hydrocarbon solvents eg, methylene chloride, chloroform, etc.
- ether solvents eg, diethyl ether, Dipropyl ether, diisopropyl ether, dibutyl ether, tert-butyl methyl ether, dimethoxyethane and the like
- ester solvents for example, methyl formate, methyl acetate, ethyl acetate, butyl acetate, methyl propionate
- the molar ratio and concentration of methacrylyl-CoA and the alcohol or phenol represented by formula 2 in the reaction solution are arbitrary and are not particularly limited.
- the amount of AAT used or the reaction conditions are appropriately determined according to the raw materials used.
- the concentration of each raw material is set in the range of 0.0000001 to 10% by mass in the case of methacrylyl-CoA, and the alcohol or phenol is 0.1 to 1000 times mol, preferably 0, of methacrylyl-CoA to be used. Add at a concentration of 5-500 moles.
- reaction temperature or reaction time are appropriately determined depending on the raw materials used, the activity of the enzyme, and the like, and are not particularly limited.
- the reaction can be performed at 5 to 80 ° C. for 1 hour to 1 week. That's fine.
- it is 10 to 70 ° C. for 1 to 120 hours, more preferably 1 hour or more, and further preferably 3 hours or more.
- the pH of the reaction solution is not particularly limited as long as the reaction proceeds efficiently, but is, for example, in the range of pH 4 to 10, preferably pH 5.0 to 9.0. It is preferable to select conditions for completing the reaction, such as temperature, time and pH of the reaction solution.
- Suitable conditions are such that the concentration of methacrylyl-CoA is directly or indirectly within the range of 0.000001 to 1% by mass under the condition of pH 5.5 to 9.0, and alcohol or phenols are used. The concentration is adjusted to 1 to 500 times mol with respect to methacrylyl-CoA. Then, the temperature is adjusted to a range of 20 to 40 ° C., and the reaction is performed for 1 hour or more. These raw materials (substrates) can be continuously supplied so as to be in the above-described range, and the accumulated concentration of the product can be improved by doing so.
- methacrylic acid ester using isobutyryl-CoA as a raw material using methacrylyl-CoA converted by the action of ACD or methacrylyl-CoA converted by the action of ECH from 3-hydroxyisobutyryl-CoA It is preferable to adjust the conditions so as to be within the range.
- the methacrylyl-CoA synthesis reaction by ACD or ECH can be carried out by a known method (for example, the conditions described in Microbiology (1999), 145, 2323-2334 as reaction conditions for ACD). Furthermore, by combining with other biological reactions, a continuous reaction (fermentation production) of methacrylic acid esters in vivo becomes possible.
- the methacrylic acid ester produced in the culture medium or in the reaction solution and the amount of the produced methacrylic acid ester can be detected and measured using ordinary methods such as high performance liquid chromatography and LC-MS. Moreover, the methacrylic acid ester volatilized in the gas phase part (head space part) of the culture vessel or the reaction vessel and the amount of production thereof can be detected and measured using a usual method such as gas chromatography.
- Isolation of the methacrylic acid ester from the reaction solution may be carried out by appropriately combining known operations such as filtration, centrifugation, vacuum concentration, ion exchange or adsorption chromatography, solvent extraction, distillation and crystallization as necessary. it can.
- the obtained methacrylic acid ester is polymerized by a usual method, and can be used for conventional applications.
- the methacrylic acid ester and its polymer obtained in this way can significantly reduce the burden on energy, resources and the environment, and are less environmentally friendly than conventional chemical products made from petroleum products. It has a great social value as a load material.
- the AAT of the present invention is an enzyme that catalyzes a reaction that acts on acyl-CoA to produce an organic acid ester in the presence of alcohol or phenols.
- the enzyme composition in the present invention is not particularly limited as long as it contains AAT having a synthetic activity of methacrylic acid ester.
- a culture solution obtained by culturing the above-described genetically modified organism, a microbial cell obtained from the culture solution or a treated product thereof, a crude enzyme or a purified enzyme obtained by extracting an enzyme from these, and the like can be exemplified.
- the AAT of the present invention has high reactivity with methacrylyl-CoA, propionyl-CoA and isobutyryl-CoA as substrates. That is, the activity for methacrylyl-CoA, propionyl-CoA and isobutyryl-CoA is higher than the activity for acetyl-CoA. More specifically, the AAT of the present invention has the same reactivity with propionyl-CoA and isobutyryl-CoA when n-butanol is used as a substrate and the reactivity with methacrylyl-CoA is 100%, and acetyl.
- the reactivity with respect to CoA is less than these, more preferably 50% or less, and still more preferably 40% or less. Furthermore, the reactivity of these three substrates (methacrylyl-CoA, propionyl-CoA and isobutyryl-CoA) to butyryl-CoA and hexanoyl-CoA is as low as acetyl-CoA, which is 50% or less.
- the AAT of the present invention has a high affinity for methacrylyl-CoA.
- the affinity for the substrate can be evaluated by the Michaelis constant (Km).
- Km for methacrylyl-CoA is a value obtained by measurement and calculation according to the examples described later.
- the Km value for methacrylyl-CoA of AAT of the present invention is not more than the Km value for acetyl-CoA, preferably not more than 0.5 mM, more preferably not more than 0.2 mM, still more preferably not more than 0.1 mM. And particularly preferably 0.05 mM or less. Due to the high affinity, even if the concentration of methacrylyl-CoA as a raw material is low, the above-described catalytic reaction can proceed, and a methacrylic acid ester can be produced more efficiently.
- Optimal reaction pH The reactivity of the AAT of the present invention at each pH is recognized in a relatively wide range of pH 6 to 10.5.
- the optimum pH for the reaction is in the range of 7-9. More specifically, it is around 8 to 9, and shows the highest activity particularly when using a tris (trishydroxymethylaminomethane) -hydrochloric acid buffer solution at pH 8.5.
- the AAT of the present invention is a group consisting of the above-mentioned plants belonging to the perilla, the plants belonging to the grapes, the plants belonging to the mucrotids, the plants belonging to the mallow, the plants belonging to the magnolia and the plants belonging to the chrysanthemum It can be isolated from a plant selected from Preferably, it is derived from a plant belonging to the family Asteraceae. More preferably, it is derived from a plant belonging to the genus Chamomile. Particularly preferably, it is derived from Roman chamomile.
- the AAT of the present invention having excellent characteristics is extremely useful as a synthase for a saturated or unsaturated organic acid ester having 3 to 4 carbon atoms. That is, according to the AAT of the present invention, the target organic acid ester can be selectively produced even when acetyl-CoA is present as a contaminant in the raw material. Therefore, this enzyme has an excellent effect for uses (such as fermentative production of esters from biomass raw materials) in which the presence of contaminants is expected or concerned.
- Example 1 Synthesis of butyl methacrylate by Mokusei
- 1 g of chopped leaves of Osmanthus fragrans was weighed into a 20 ml GC headspace vial (23 ⁇ 75 mm, National Scientific).
- 1 ml of a substrate solution 50 mM Tris-HCl (pH 8.5), n-butanol 40 mM, methacrylyl CoA 0.125 mM
- 10 ⁇ l of 10 mM 2-hexanone was added as an internal standard, and the product was analyzed by GC-MS by SPME (solid phase microextraction) method.
- Carboxen / PDMS (75um, Fused Silica, manufactured by Sigma-Aldrich) was used for SPME and adsorbed at 30 ° C. for 10 minutes.
- GC-MS analysis conditions Column: TC-70 (inner diameter 0.25 mm x 60 m, 0.25 ⁇ m, GL Sciences) Column temperature: 50 ° C. ⁇ 5 min ⁇ 7.5 ° C./min ⁇ 200° C. ⁇ 10 min
- Carrier gas Helium Flow rate: 1.13 ml / min Inject: 250 ° C
- Table 2 shows the decomposition activity of butyl methacrylate by cinnamon leaf. A significant decrease in butyl methacrylate was confirmed, suggesting that the production rate of methacrylic acid ester by the action of AAT derived from cinnamon rose exceeds the degradation rate of methacrylic acid ester by esterolytic enzymes contained in the plant tissue. It was.
- Example 2 Synthesis of methyl methacrylate by grape
- Grains red glove: Vitis vinifera
- fruit cut finely with skin were weighed into a vial.
- 0.5 ml of a substrate solution 50 mM Tris-HCl (pH 8.5), methanol 40 mM, methacrylyl CoA 10 mM
- Analysis was carried out in the same manner as in Example 1, and production of 0.8 ⁇ M methyl methacrylate was observed (Table 1).
- Example 3 Synthesis of butyl methacrylate by grape
- n-butanol was used instead of methanol.
- formation of 9.0 ⁇ M butyl methacrylate was observed.
- the hydrolysis reaction of methacrylic acid ester was also confirmed in the same manner as in Example 2.
- Example 4 Synthesis of hexyl methacrylate by crepe fruit
- 2 g of crepefruit (Citrus x paradisi) fruit loosened was poured into a vial.
- a substrate solution 50 mM Tris-HCl (pH 8.5), n-hexanol 40 mM, methacrylyl CoA 10 mM
- Analysis was carried out in the same manner as in Example 1, and formation of 0.3 ⁇ M hexyl methacrylate was observed.
- Example 5 Synthesis of ethyl methacrylate by durian
- a substrate solution 50 mM Tris-HCl (pH 8.5), ethanol 40 mM, methacrylyl CoA 0.125 mM
- Analysis was carried out in the same manner as in Example 1, and production of 6.7 ⁇ M ethyl methacrylate was observed.
- Example 6 Synthesis of butyl methacrylate by durian
- n-butanol was used instead of ethanol.
- formation of 14 ⁇ M butyl methacrylate was observed.
- the hydrolysis reaction of the methacrylic acid ester was also confirmed in the same manner as in Example 5.
- Example 7 Synthesis of butyl methacrylate by Calataneotama] 1 g and 0.5 g of chopped leaves and flower buds of Magnolia figo were weighed into a vial, respectively. 1 ml of a substrate solution (50 mM Tris-HCl (pH 8.5), n-butanol 40 mM, methacrylyl CoA 0.125 mM) was added and sealed, and reacted at 30 ° C. for 12 hours. Analysis was performed in the same manner as in Example 1, and production of 4.4 ⁇ M and 0.4 ⁇ M butyl methacrylate was observed, respectively.
- a substrate solution 50 mM Tris-HCl (pH 8.5), n-butanol 40 mM, methacrylyl CoA 0.125 mM
- Example 8 Synthesis of butyl methacrylate by Roman chamomile
- 0.5 g of chopped leaves of Roman chamomile (Chamaemelum nobile) was weighed into a vial.
- 0.5 ml of a substrate solution 50 mM Tris-HCl (pH 8.5), n-butanol 40 mM, methacrylyl CoA 0.125 mM
- a substrate solution 50 mM Tris-HCl (pH 8.5), n-butanol 40 mM, methacrylyl CoA 0.125 mM
- Example 9 Purification of Roman chamomile-derived AAT
- enzyme purification was performed at a temperature of 4 ° C. or lower.
- the AAT activity of each fraction was measured using GC with n-butanol and methacrylyl-CoA as substrates.
- AAT activity measurement method 500 ⁇ l of a reaction solution 50 mM Tris-HCl (pH 8.0), 40 mM n-butanol, 0.12 mM methacrylyl-CoA was prepared in a 2 ml screw vial (National Scientific autosampler vial). .
- the purified enzymes of the following steps (3) to (6) were added, sealed, and reacted at 30 ° C. for 1 hour.
- the dialysis fraction was again applied to a DEAE-Toyopearl column (10 ml) equilibrated with buffer B. After thoroughly washing with buffer B, the concentration of sodium chloride in buffer B was linearly increased from 0M to 0.3M to perform concentration gradient elution. The eluate was divided into 5.5 ml fractions. The elution pattern is shown in FIG. The obtained AAT active fraction was collected and dialyzed against buffer B. In the figure, “AAT activity” (white circle) is defined as 1 U of the amount of enzyme that gives 1 ⁇ mol of ester per minute. The “protein concentration” (black circle) was measured using a Bio-Rad protein assay Kit (Bio-Rad, USA) with bovine serum albumin as a standard.
- Table 3 shows the yield and activity of the enzyme composition in each purification stage. A total of 5 column separations yielded 201 mU / mg AAT, which was 209-fold purified in activity.
- Example 10 Substrate specificity of Roman chamomile-derived AAT
- the purified substrate fraction of MonoQ 5/50 GL column described in Example 9 was used to evaluate the substrate specificity of AAT.
- reaction solution 50 mM Tris-HCl (pH 8.5), 40 mM alcohol, 0.12 mM acyl-CoA
- 50 mM Tris-HCl (pH 8.5) 40 mM alcohol, 0.12 mM acyl-CoA
- the purified fraction was added to make 500 ⁇ l. Sealed and reacted at 30 ° C. for 1 hour.
- n-butyl propionate For the determination of n-butyl propionate, the separation of the n-butanol peak and the separation was insufficient in this column, so the GC-MS analysis conditions described in Example 1 (however, the temperature conditions were 80 ° C., 1 minute ⁇ 10 ° C. / Min ⁇ 200 ° C., changed to 5 minutes) by the absolute calibration curve method.
- Example 11 Optimal pH of Roman chamomile-derived AAT
- the optimum pH was evaluated using the purified fraction by the MonoQ 5/50 GL column described in Example 9.
- a reaction solution having the composition of butanol and 0.12 mM methacrylyl-CoA was prepared. In any buffer, the buffer concentration was 50 mM. The purified fraction was added to each reaction solution to make 500 ⁇ l, sealed and reacted at 30 ° C. for 1 hour.
- Example 12 Km of AAT derived from Roman chamomile
- Km values for methacrylyl-CoA and acetyl-CoA were measured.
- a purified fraction, 40 mM n-hexanol and each concentration of methacrylyl-CoA or acetyl-CoA are added to a 50 mM Tris-HCl (pH 8.5) buffer to prepare 500 ⁇ l of a reaction solution, which is sealed and sealed at 30 ° C. for 2 hours. Reacted for hours.
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Abstract
Description
[2]モクセイ科(Oleaceae)に属する植物、ブドウ科(Vitaceae)に属する植物、ミカン科(Rutaceae)に属する植物、アオイ科(Malvaceae)に属する植物、モクレン科(Magnoliaceae)に属する植物およびキク科(Asteraceae)に属する植物からなる群から選択される植物由来のアルコールアシルトランスフェラーゼの存在下、メタクリリル-CoAにアルコールまたはフェノール類を作用させて、メタクリル酸エステルを合成する工程を含むメタクリル酸エステルの製造方法。
[3]モクセイ属(Osmanthus)に属する植物、ブドウ属(Vitis)に属する植物、ミカン属(Citrus)に属する植物、ドリアン属(Durio)に属する植物、モクレン属(Magnolia)に属する植物およびカミツレ属(Chamaemelum)に属する植物からなる群から選択される植物由来のアルコールアシルトランスフェラーゼの存在下、メタクリリル-CoAにアルコールまたはフェノール類を作用させて、メタクリル酸エステルを合成する工程を含むメタクリル酸エステルの製造方法。
[4]植物が、モクセイ(Osmanthus fragrans)、ブドウ(Vitis vinifera)、クレープフルーツ(Citrus x paradisi)、ドリアン(Durio zibethinus)、カラタネオガタマ(Michelia figo)およびローマンカモミール(Chamaemelum nobile)から選択される植物である[1]~[3]のメタクリル酸エステルの製造方法。
[5]以下の(1)~(3)の理化学的性質を有するアルコールアシルトランスフェラーゼの存在下、メタクリリル-CoAにアルコールまたはフェノール類を作用させて、メタクリル酸エステルを合成する工程を含むメタクリル酸エステルの製造方法。
(1)アルコールまたはフェノール類の存在下、メタクリリル-CoAに作用してメタクリル酸エステルを生成する。
(2)アセチルCoAに対する活性に対してメタクリリル-CoAに対する活性が高い。
(3)メタクリリル-CoAに対するKm値が0.5mM以下。
(1)アルコールまたはフェノール類の存在下、メタクリリル-CoAに作用してメタクリル酸エステルを生成する。
(2)アセチルCoAに対する活性に対してメタクリリル-CoAに対する活性が高い。
(3)アセチルCoAに対する活性に対してイソブチリル-CoAに対する活性が高い。
(4)メタクリリル-CoAに対するKm値が0.5mM以下。
(5)メタクリリル-CoAおよびn-ブタノールを基質としたときの至適pHが8~9である。
[7]キク目(Asterales)に属する植物由来である[6]のアルコールアシルトランスフェラーゼ又は同酵素組成物。
[8]キク科(Asteraceae)に属する植物由来である[7]のアルコールアシルトランスフェラーゼ又は同酵素組成物。
[9]カミツレ属(Chamaemelum)に属する植物由来である[8]のアルコールアシルトランスフェラーゼ又は同酵素組成物。
[10]ローマンカモミール(Chamaemelum nobile)由来である[9]のアルコールアシルトランスフェラーゼ又は同酵素組成物。
[11][6]~[10]のいずれかのアルコールアシルトランスフェラーゼ又は同酵素組成物を用いた有機酸エステルの製造方法。
[13]前記植物が、モクセイ科(Oleaceae)に属する植物、ブドウ科(Vitaceae)に属する植物、ミカン科(Rutaceae)に属する植物、アオイ科(Malvaceae)に属する植物、モクレン科(Magnoliaceae)に属する植物およびキク科(Asteraceae)に属する植物である[12]のアルコールアシルトランスフェラーゼ。
[14]前記植物が、モクセイ属(Osmanthus)に属する植物、ブドウ属(Vitis)に属する植物、ミカン属(Citrus)に属する植物、ドリアン属(Durio)に属する植物、モクレン属(Magnolia)に属する植物およびカミツレ属(Chamaemelum)に属する植物である[13]のアルコールアシルトランスフェラーゼ。
[15]前記植物が、モクセイ(Osmanthus fragrans)、ブドウ(Vitis vinifera)、クレープフルーツ(Citrus x paradisi)、ドリアン(Durio zibethinus)、カラタネオガタマ(Michelia figo)およびローマンカモミール(Chamaemelum nobile)から選択される植物である[14]のアルコールアシルトランスフェラーゼ。
[16]以下の(1)~(6)の理化学的性質を有する[15]のアルコールアシルトランスフェラーゼ。
(1)アルコールまたはフェノール類の存在下、メタクリリル-CoAに作用してメタクリル酸エステルを生成する。
(2)アセチルCoAに対する活性に対してメタクリリル-CoAに対する活性が高い。
(3)アセチルCoAに対する活性に対してイソブチリリル-CoAに対する活性が高い。
(4)アセチルCoAに対する活性に対してプロピオニル-CoAに対する活性が高い。
(5)メタクリリル-CoAに対するKm値が0.5mM以下。
(6)メタクリリル-CoAおよびn-ブタノールを基質としたときの至適pHが8~9である。
[17]キク科(Asteraceae)に属する植物由来である[16]のアルコールアシルトランスフェラーゼ。
[18]カミツレ属(Chamaemelum)に属する植物由来である[17]のアルコールアシルトランスフェラーゼ。
[19]ローマンカモミール(Chamaemelum nobile)由来である[18]のアルコールアシルトランスフェラーゼ。
[メタクリル酸エステル]
本発明において、メタクリル酸エステルとは式1で示される化合物である。式1において、Rは直鎖あるいは分岐の炭素数1~20の炭化水素基を表す。炭化水素基は、飽和又は不飽和の非環式であってもよく、飽和又は不飽和の環式であってもよい。好ましくは直鎖あるいは分岐鎖の炭素数1~10の無置換のアルキル基、アラルキル基またはアリール基である。特に好ましくはメチル基、エチル基、n-プロピル基、イソプロピル基、n-ブチル基、イソブチル基、sec-ブチル基、tert-ブチル基、n-ペンチル基、イソペンチル基、tert-ペンチル基、n-ヘキシル基、イソヘキシル基、2-ヘキシル基、ジメチルブチル基、エチルブチル基、ヘプチル基、オクチル基、2-エチルヘキシル基の炭素数1~8のアルキル基、ベンジル基またはフェニル基である。
本発明において、メタクリリル-CoAとは、以下の構造式で示される化合物である。メタクリリル-CoAは、生体内ではバリンの代謝中間体として知られている。本発明で使用するメタクリリル-CoAは公知又は新規な方法で製造したものともできる。その合成方法としては無水メタクリル酸と補酵素Aを有機化学的に合成する方法(Methods in Enzymology. 324, 73-79 (2000))あるいは酵素反応を用いた合成方法が知られている。
本発明におけるメタクリル酸エステルの製造の原料となるアルコールまたはフェノール類は以下の式2で示される化合物である。アルコールまたはフェノール類の構造は、メタクリル酸エステルに対応することから、その構造は、前記式1のRと同じ定義であり、直鎖あるいは分岐の炭素数1~20の炭化水素基を表す。炭化水素基は、飽和又は不飽和の非環式であってもよく、飽和又は不飽和の環式であってもよい。好ましくは直鎖あるいは分岐の炭素数1~10の無置換のアルコール、アラルキルアルコールまたはフェノール類であり、より好ましくはメタノール、エタノール、n-プロパノール、イソプロパノール、n-ブタノール、イソブタノール、sec-ブタノール、tert-ブタノール、n-ペンチルアルコール、イソペンチルアルコール、tert-ペンチルアルコール、n-ヘキシルアルコール、イソヘキシルアルコール、2-ヘキシルアルコール、ジメチルブチルアルコール、エチルブチルアルコール、ヘプチルアルコール、オクチルアルコール、2-エチルヘキシルアルコールの炭素数1~8のアルキルアルコール、ベンジルアルコールまたはフェノールである。特に好ましくは、メタノール、エタノール、n-ブタノール、イソブタノール、n-ヘキシルアルコールである。
本発明のアルコールアシルトランスフェラーゼ(以下、AATという)は、アルコールまたはフェノール類にアシル-CoAのアシル基を転移させてエステルを合成する触媒作用を有する酵素である。AATは、種々の果物におけるエステルの生成に関与していると言われている。AATはショウガ目(バナナ)、バラ目(イチゴ、リンゴ、ナシ、モモ)、ウリ目(メロン)、ツツジ目(キウイ)、シソ目(オリーブ)、ナス目(トマト)、ムクロジ目(レモン、マンゴー)等の植物に存在することが知られている。
ブドウ目に属するものとしてはブドウ科(Vitaceae)の植物が好ましい。
ムクロジ目に属するものとしてはウルシ科(Anacardiaceae)、ビーベルステイニア科(Biebersteiniaceae)、カンラン科(Burseraceae)、キルキア科(Kirkiaceae)、センダン科(Meliaceae)、ソウダノキ科(Nitrariaceae)、ミカン科(Rutaceae)、ムクロジ科(Sapindaceae)およびニガキ科(Simaroubaceae)の植物が好ましい。
アオイ目に属するものとしてはベニノキ科(Bixaceae)、ハンニチバナ科(Cistaceae)、キティヌス科(Cytinaceae)、フタバガキ科(Dipterocarpaceae)、アオイ科(Malvaceae)、ナンヨウザクラ科(Muntingiaceae)、ネウラダ科(Neuradaceae)、サルコラエナ科(Sarcolaenaceae)、スファエロセパルム科(Sphaerosepalaceae)およびジンチョウゲ科(Thymelaeaceae)の植物が好ましい。
モクレン目に属するものとしてはバンレイシ科(Annonaceae)、デゲネリア科(Degeneriaceae)、エウポマティア科(Eupomatiaceae)、ヒマンタンドラ科(Himantandraceae)、モクレン科(Magnoliaceae)およびニクズク科(Myristicacea)の植物が好ましい。
キク目に属するものとしてはアルセウオスミア科(Alseuosmiaceae)、アルゴフィルム科(Argophyllaceae)、キク科(Asteraceae)、カリケラ科(Calyceraceae)、キキョウ科(Campanulaceae))、クサトベラ科(Goodeniaceae)、ミツガシワ科(Menyanthaceae)、ユガミウチワ科(Pentaphragmataceae)、フェリネ科(Phellinaceae)、ロウッセア科(Rousseaceae)およびスティリディウム科(Stylidiaceae)の植物が好ましい。
その中でも、モクセイ科、ブドウ科、ミカン科、アオイ科、モクレン科またはキク科に属する植物がより好ましい。
ブドウ科に属するものとしてはブドウ属(Vitis)、ノブドウ属(Ampelopsis)、ヤブガラシ属(Cayratia)、セイシカズラ属(Cissus)、キフォステンマ属(Cyphostemma)、ウドノキ属(Leea)、ツタ属(Parthenocissus)およびミツバカズラ属(Tetrastigma)の植物が好ましい。
ミカン科に属するものとしてはミカン属(Citrus)、アエグレ属(Aegle)、サンショウ属(Zanthoxylum)、ゲッキツ属(Murraya)、ヘンルーダ属(Ruta)、コクサギ属(Orixa)、ミヤマシキミ属(Skimmia) ゴシュユ属(Euodia)、キハダ属(Phellodendron)、ボロニア属(Boronia)、アクロニキア属(Acronychia)、ワンピ属(Clausena)、コレア属(Correa)、ハナシンボウギ属(Glycosmis)およびアワダン属(Melicope);ムクロジ科に属するものとしてはレイシ属(Litchi);ウルシ科に属するものとしてはマンゴー属(Mangifera)の植物が好ましい。
アオイ科に属するものとしてはドリアン属(Durio)、カカオ属(Theobroma)、イチビ属(Abutilon)、トロロアオイ属(Abelmoschus)、ワタ属(Gossypium)、ヤノネボンテンカ属(Pavonia)、フヨウ属(Hibiscus)、キンゴジカ属(Sida)およびアオイ属(Malva)の植物が好ましい。
モクレン科に属するものとしてはモクレン属(Magnolia)の植物が好ましい。
キク科に属するものとしてはカミツレ属(Chamaemelum)、ノコギリソウ属(Achillea)、ムラサキバレンギク属(Echinacea)、シカギク属(Matricaria)、ヨモギギク属(Tanacetum)、タンポポ属(Taraxacum)、ヨモギ属(Artemisia)、フキ属(Petasites)、ムギワラギク属(Helichrysum)、ワタスギギク属(Santolina)、チョウセンアザミ属(Cynara)、オオアザミ属(Silybum)、キンセンカ属(Calendula)、キクニガナ属(Cichorium)、ベニバナ属(Carthamus)およびキク属(Chrysanthemum)に属する植物が好ましい。
その中でも、モクセイ属、ブドウ属、ミカン属、ドリアン属、モクレン属またはカミツレ属に属する植物が特に好ましい。
ブドウ属に属するものとしてはブドウ(Vitis vinifera、Vitis labrusca)、サマーグレープ(Vitis aestivalis)、ヤマブドウ(Vitis coignetiae)およびエビヅル(Vitis ficifolia)が好ましい。
ミカン属に属するものとしてはレモン(Citrus limon)、スダチ(Citrus sudachi)、カボス(Citrus sphaerocarpa)、グレープフルーツ(Citrus x paradisi)、ユズ(Citrus junos)ライム(Citrus aurantifolia)、ウンシュウミカン(Citrusunshiu)およびオレンジ(Citrus sinensis);アエグレ属に属するものとしてはアエグレ・マルメロス(Aegle marmelos);レイシ属に属するものとしてはライチ(Litchi chinensis);マンゴー属に属するものとしてはマンゴー(Mangifera indica)が好ましい。
ドリアン属に属するものとしてはドリアン(Durio zibethinus、Durio testudinarius、Durio kutejensis、Durio oxleyanus、Durio graveolens、Durio dulcis);カカオ属に属するものとしてはカカオ(Theobroma cacao)が好ましい。
モクレン属に属するものとしてはカラタネオガタマ(Magnolia figo)、オガタマノキ(Magnolia compressa)、キンコウボク(Magnolia champaca)、モクレン(Magnolia liliiflora)、コブシ(Magnolia kobus)、ホオノキ(Magnolia obovataおよびMagnolia laevifolia)が好ましい。
カミツレ属に属するものとしてはローマンカモミール(Chamaemelum nobileおよびChamaemelum fuscatum)が好ましい。
その中でも、モクセイ、ブドウ、クレープフルーツ、ドリアン、カラタネオガタマまたはローマンカモミールが特に好ましい。
得られた酵素抽出液をイオン交換クロマトグラフィー、ゲルろ過カラム等を用いることで酵素タンパク質を効率よく精製することができる。
本発明のAATは、アルコールまたはフェノール類の存在下、メタクリリル-CoAに作用してメタクリル酸エステルを生成する反応を触媒する。AATは、メタクリリル-CoAを基質とした場合に高い反応性を有するものが好ましい。具体的には、アセチル-CoAに対する活性に対してメタクリリル-CoAに対する活性が高く、メタクリリル-CoAに対するKm値が0.5mM以下のものが好適である。このような性質を有するAATによれば、選択性良くメタクリル酸エステルを生産できる。
本発明で好ましいAATとしては、メタクリリル-CoAに対する反応性を基準とした場合に、少なくともアセチル-CoAに対する反応性が低いものが良い。より具体的に、本発明の好ましいAATは、n-ブタノールを基質として、メタクリリル-CoAに対する反応性を100%とした場合に、アセチル-CoAに対する反応性が同等以下であることが好ましく、より好ましくは50%以下であり、さらに好ましくは40%以下である。あるいは、n-ヘキサノールを基質として、メタクリリル-CoAに対する反応性を100%とした場合に、アセチル-CoAに対する反応性が同等以下であることが好ましく、より好ましくは70%以下であり、さらに好ましくは50%以下である。
本発明のAATは、メタクリリル-CoAに対する親和性が高いことが好ましい。基質に対する親和性は、ミカエリス定数(Km)によって評価ことができる。メタクリリル-CoAに対するKmは、後述の実施例に従って測定・算出することによって得られる値である。
さらに、AATを反応に供するに際しては、前記AATの遺伝子を単離し、例えば一般的な宿主ベクター系に導入し、該ベクター系で形質転換した微生物を利用することも可能である。宿主としては、細菌では大腸菌、Rhodococcus属、Pseudomonas属、Corynebacterium属、Bacillus属、Streptococcus属、Streptomyces属などが挙げられ、酵母ではSaccharomyces属、Candida属、Shizosaccharomyces属、Pichia属、糸状菌ではAspergillus属などが挙げられる。これらの中で、特に大腸菌を用いることが簡便であり、効率もよく好ましい。
2化合物へ誘導可能な物質とは例えば、2-オキソイソ吉草酸、イソ酪酸、3-ヒドロキシイソ酪酸、酢酸、ピルビン酸、乳酸、アセト酢酸、酪酸、プロピオン酸、リンゴ酸、フマル酸、クエン酸およびコハク酸等の酸、バリン、アラニン、ロイシン、リジンおよびグルタミン酸等のアミノ酸類、グルコース、フルクトースおよびキシロース等の糖類などが挙げられる。
これら前駆体からメタクリル酸エステルを生成させるには宿主微生物が本来有する各種代謝系をそのまま利用することも可能である。必要に応じて遺伝子を導入あるいは欠損させることもできる。
メタクリル酸エステルの製造は、以下の方法で行うことができる。溶媒にメタクリリル-CoA及び式2で表されるアルコールまたはフェノール類を添加して溶解又は懸濁させる。そして、この溶液又は懸濁液に、AATを接触させ、温度等の条件を制御しながらメタクリリル-CoAとアルコールまたはフェノール類とを反応させる。前記反応により、メタクリリル-CoAのメタクリル基を式2のアルコールまたはフェノール類に転移させて、メタクリル酸エステルを生成させる。
培地中又は反応液中に生成したメタクリル酸エステル及びその生成量は、高速液体クロマトグラフィー及びLC-MSなどの通常の方法を用いて検出し、測定することができる。また、培養容器又は反応容器の気相部(ヘッドスペース部)に揮発したメタクリル酸エステル及びその生成量は、ガスクロマトグラフィーなどの通常の方法を用いて検出し、測定することができる。
本発明の一側面である新規AATおよびそれを用いた有機酸エステルの製造について以下に詳述する。
本発明のAATとしては、基質としてメタクリリル-CoA、プロピオニル-CoAおよびイソブチリリル-CoAに高い反応性を有する。すなわち、アセチル-CoAに対する活性に対してメタクリリル-CoA、プロピオニル-CoAおよびイソブチリリル-CoAに対する活性が高い。より具体的に、本発明のAATは、n-ブタノールを基質として、メタクリリル-CoAに対する反応性を100%とした場合に、プロピオニル-CoAおよびイソブチリリル-CoAに対する反応性は同等程度であり、且つアセチル-CoAに対する反応性がそれら以下、より好ましくは50%以下、さらに好ましくは40%以下である。さらに、これら3基質(メタクリリル-CoA、プロピオニル-CoAおよびイソブチリリル-CoA)に対して、ブチリリル-CoAおよびヘキサノイル-CoAへの反応性はアセチル-CoAと同様に低く、50%以下である。
本発明のAATは、メタクリリル-CoAに対する親和性が高い。基質に対する親和性は、ミカエリス定数(Km)によって評価ことができる。メタクリリル-CoAに対するKmは、後述の実施例に従って測定・算出することによって得られる値である。
本発明のAATの各pHにおける反応性は、pH6~10.5の比較的広い範囲で認められる。同反応の至適pHとしては、7~9の範囲にある。より具体的には8~9付近であり、特にpH8.5のトリス(トリスヒドロキシメチルアミノメタン)-塩酸緩衝液を用いたとき最も高い活性を示す。
本発明のAATは、上述したシソ目に属する植物、ブドウ目に属する植物、ムクロジ目に属する植物、アオイ目に属する植物、モクレン目に属する植物およびキク目に属する植物からなる群から選択される植物から単離することができる。好ましくはキク科に属する植物由来である。さらに好ましくは、カミツレ属に属する植物由来である。特に好ましくは、ローマンカモミール由来である。
キンモクセイ(Osmanthus fragrans)の葉を刻んだもの1gを20ml容GCヘッドスペース用バイアル(23×75mm、National Scientific製)にはかり取った。基質溶液(50mM Tris-HCl(pH8.5)、n-ブタノール40mM、メタクリリルCoA 0.125mM)を1ml加え密閉し、30℃で12時間反応させた。反応終了後、内部標準として10mM 2-ヘキサノンを10μl加えた後、SPME(固相マイクロ抽出)法により、生成物をGC-MSで分析した。SPMEにはCarboxen/PDMS(75um、Fused Silica、Sigma-Aldrich製)を使用し、30℃で10分間吸着させた。
GC―MS分析条件
カラム:TC-70(内径0.25mm×60m、0.25μm、GLサイエンス社)
カラム温度:50℃・5min→7.5℃/min→200℃・10min
キャリアガス:ヘリウム
流量:1.13ml/min
Inject:250℃
ブドウ(レッドグローブ:Vitis vinifera)果実を皮付きで細かく裁断したもの1gをバイアルにはかり取った。基質溶液(50mM Tris-HCl(pH8.5)、メタノール40mM、メタクリリルCoA10mM)を0.5ml加え、密閉後、30℃で12時間反応させた。実施例1と同様に分析を実施し、0.8μMのメタクリル酸メチルの生成を認めた(表1)。
メタノールの代わりにn-ブタノールを用いた以外は実施例2と同様に実施した。その結果、9.0μMのメタクリル酸ブチルの生成が認められた。メタクリル酸エステルの加水分解反応も実施例2と同様に確認した。
クレープフルーツ(Citrus x paradisi)果実の砂じょうをほぐしたもの2gをバイアルにはかり取った。基質溶液(50mM Tris-HCl(pH8.5)、n-ヘキサノール40mM、メタクリリルCoA10mM)を0.5ml加え、密閉後、30℃で12時間反応させた。実施例1と同様に分析を実施し、0.3μMのメタクリル酸ヘキシルの生成を認めた。
ドリアン(Durio zibethinus)の果実を細かく裁断したもの1gをバイアルにはかり取った。基質溶液(50mM Tris-HCl(pH8.5)、エタノール40mM、メタクリリルCoA 0.125mM)を0.5ml加え密閉し、30℃で3時間反応させた。実施例1と同様に分析を実施し、6.7μMのメタクリル酸エチルの生成を認めた。
エタノールの代わりにn-ブタノールを用いた以外は実施例5と同様に実施した。その結果、14μMのメタクリル酸ブチルの生成が認められた。メタクリル酸エステルの加水分解反応も実施例5同様に確認した。
カラタネオガタマ(Magnolia figo)の葉および花芽を刻んだものそれぞれ1gおよび0.5gをバイアルにはかり取った。基質溶液(50mM Tris-HCl(pH8.5)、n-ブタノール40mM、メタクリリルCoA 0.125mM)を1ml加え密閉し、30℃で12時間反応させた。実施例1と同様に分析を実施し、それぞれ4.4μMおよび0.4μMのメタクリル酸ブチルの生成を認めた。
ローマンカモミール(Chamaemelum nobile)の葉を刻んだもの0.5gをバイアルにはかり取った。基質溶液(50mM Tris-HCl(pH8.5)、n-ブタノール40mM、メタクリリルCoA 0.125mM)を0.5ml加え密閉し、30℃で12時間反応させた。実施例1と同様に分析を実施し、6.7μMのメタクリル酸ブチルの生成を認めた。
特に明記しない限り、酵素精製は、4℃以下の温度で行った。各画分のAAT活性の測定はn-ブタノールおよびメタクリリル-CoAを基質とし、GCを用いて分析した。
ローマンカモミール(Chamaemelum nobile)の葉38gを液体窒素中で粉末化した。粉末を190mlの抽出用緩衝液(10%グリセロール、5mMジチオスレイトール(DTT)、5%ポリビニルピロリドン、250mMトリス-HCl(pH7.5))に懸濁しし、4層のガーゼを通して濾過した。濾液を15,000gで15分間遠心分離し、粗酵素溶液を得た。
2ml容スクリューバイアル(National Scientific製オートサンプラーバイアル)に、反応液(50mM Tris-HCl(pH8.0)、40mM n-ブタノール、0.12mMメタクリリル-CoA)を500μl調製した。以下の(3)~(6)の各段階の精製酵素を加え密閉し、30℃で1時間反応させた。
GC分析条件
カラム:DB-WAX(内径0.25mm×60m、0.5μm、Agilent Technologies)
カラム温度:115℃・5min→40℃/min→200℃・2min キャリアガス:ヘリウム
検出:FID
Inject温度:230℃
Detect温度:250℃
粗酵素溶液を10%グリセロールおよび2mM DTTを含む250mMトリス-HCl(pH8.0)緩衝液で平衡化したDEAE-トヨパールカラム(20ml)に供した。素通り画分を回収し、10%グリセロールおよび2mM DTTを含む20mMトリス-HCl(pH8.0)緩衝液(以下、緩衝液B)に対して透析した。
得られた透析済みのAAT活性画分を緩衝液Bで平衡化したQセファロースカラム(10ml)に供した。緩衝液Bで十分洗浄した後、緩衝液Bの塩化ナトリウムの濃度を0Mから0.3Mまで直線的に上昇させて濃度勾配溶出を行った。なお、溶出液は5.5mlずつフラクション分けした。溶出パターンを図2に示す。得られたAAT活性画分を回収し、2mM DTTを含む20mMトリス-HCl(pH8.0)緩衝液(以下、緩衝液C)に対して透析した。
Qセファロースカラムにより得られた透析済みのAAT活性画分を、緩衝液Cで平衡化したMonoQ 5/50 GLカラム(1mL)に供した。緩衝液Cの塩化ナトリウムの濃度を0Mから0.5Mまで直線的に上昇させて濃度勾配溶出を行った。溶出パターンを図3に示す。得られたAAT活性画分を回収し、アミコンウルトラ(Amicon Ultra)-0.5mL遠心式フィルターを用いて濃縮を行った。なお、本カラムによる精製はAKTA Explorer 10S(GE Healthcare)を用いて流速0.5ml/分の条件で、0.5mlずつフラクション分けした。
MonoQ 5/50 GLカラムにより得られたAAT活性濃縮画分を、0.3M塩化ナトリウムを含む緩衝液Cで平衡化したSuperdex 200 10/300 GLカラムに供した。本カラムによる精製はAKTA Explorer 10Sを用いて流速0.5ml/分の条件で、0.5mlずつフラクション分けした。溶出パターンを図4に示す。得られたAAT活性画分を回収し、緩衝液Bに対して透析した。
各精製段階における酵素組成物の収量、活性を表3に示す。合計5回のカラム分離により、活性において209倍に精製された、201mU/mgのAATが得られた。
実施例9記載のMonoQ 5/50 GLカラムによる精製画分を用いて、AATの基質特異性を評価した。
実施例9記載のMonoQ 5/50 GLカラムによる精製画分を用いて、至適pHを評価した。
実施例9記載のMonoQ 5/50 GLカラムによる精製画分を用いて、メタクリリル-CoAおよびアセチル-CoAに対するKm値を測定した。50mM Tris-HCl(pH8.5)緩衝液に、精製画分、40mM n-ヘキサノールおよび各濃度のメタクリリル-CoAまたはアセチル-CoAを加えて500μlの反応液を調製し、密閉して30℃で2時間反応させた。
Claims (4)
- モクセイ属(Osmanthus)に属する植物、ブドウ属(Vitis)に属する植物、ミカン属(Citrus)に属する植物、ドリアン属(Durio)に属する植物、モクレン属(Magnolia)に属する植物およびカミツレ属(Chamaemelum)に属する植物からなる群から選択される植物由来のアルコールアシルトランスフェラーゼの存在下、メタクリリル-CoAにアルコールまたはフェノール類を作用させて、メタクリル酸エステルを合成する工程を含むメタクリル酸エステルの製造方法。
- キンモクセイ、ブドウ、クレープフルーツ、ドリアン、カラタネオガタマおよびローマンカモミールからなる群から選択される植物由来のアルコールアシルトランスフェラーゼの存在下、メタクリリル-CoAにアルコールまたはフェノール類を作用させて、メタクリル酸エステルを合成する工程を含むメタクリル酸エステルの製造方法。
- 以下の(1)~(5)の理化学的性質を有する、カミツレ属(Chamaemelum)に属する植物由来のアルコールアシルトランスフェラーゼ又は同酵素組成物。
(1)アルコールまたはフェノール類の存在下、メタクリリル-CoAに作用してメタクリル酸エステルを生成する。
(2)アセチルCoAに対する活性に対してメタクリリル-CoAに対する活性が高い。
(3)アセチルCoAに対する活性に対してイソブチリル-CoAに対する活性が高い。
(4)メタクリリル-CoAに対するKm値が0.05mM以下。
(5)メタクリリル-CoAおよびn-ブタノールを基質としたときの至適pHが8~9である。 - 請求項3記載のアルコールアシルトランスフェラーゼ又は同酵素組成物を用いた有機酸エステルの製造方法。
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WO2018096326A1 (en) * | 2016-11-23 | 2018-05-31 | Lucite International Uk Limited | Process for the production of methyl methacrylate |
WO2019168154A1 (ja) | 2018-03-02 | 2019-09-06 | 三菱ケミカル株式会社 | 3-ヒドロキシイソ酪酸エステルおよびメタクリル酸エステルの製造方法 |
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EP2894224B1 (en) | 2012-09-10 | 2021-09-01 | Mitsubishi Chemical Corporation | Method for producing methacrylic acid and/or ester thereof |
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- 2015-03-05 US US15/124,331 patent/US10570426B2/en active Active
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- 2015-03-05 KR KR1020167022513A patent/KR20160108546A/ko active Search and Examination
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WO2000032789A1 (en) * | 1998-12-02 | 2000-06-08 | Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro-Dlo) | Fruit flavour related genes and use thereof |
WO2007039415A1 (en) * | 2005-09-19 | 2007-04-12 | Ciba Specialty Chemicals Holding Inc. | Biocatalytic manufacturing of (meth)acrylic esters |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018096326A1 (en) * | 2016-11-23 | 2018-05-31 | Lucite International Uk Limited | Process for the production of methyl methacrylate |
WO2019168154A1 (ja) | 2018-03-02 | 2019-09-06 | 三菱ケミカル株式会社 | 3-ヒドロキシイソ酪酸エステルおよびメタクリル酸エステルの製造方法 |
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US20170022525A1 (en) | 2017-01-26 |
AU2015225348A1 (en) | 2016-10-06 |
AU2015225348B2 (en) | 2017-06-08 |
US10570426B2 (en) | 2020-02-25 |
BR112016019997A8 (pt) | 2018-01-02 |
EP3115460A4 (en) | 2017-05-03 |
JPWO2015133146A1 (ja) | 2017-04-06 |
JP6296512B2 (ja) | 2018-03-20 |
CN106062204A (zh) | 2016-10-26 |
BR112016019997A2 (ja) | 2017-08-15 |
EP3115460A1 (en) | 2017-01-11 |
KR20160108546A (ko) | 2016-09-19 |
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