WO2023032952A1 - リパーゼを有効成分として含有するエステル交換用酵素剤 - Google Patents

リパーゼを有効成分として含有するエステル交換用酵素剤 Download PDF

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WO2023032952A1
WO2023032952A1 PCT/JP2022/032516 JP2022032516W WO2023032952A1 WO 2023032952 A1 WO2023032952 A1 WO 2023032952A1 JP 2022032516 W JP2022032516 W JP 2022032516W WO 2023032952 A1 WO2023032952 A1 WO 2023032952A1
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Prior art keywords
lipase
amino acid
transesterification
seq
acid sequence
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English (en)
French (fr)
Japanese (ja)
Inventor
貫暉 松田
明紅 楊
侑朔 児玉
志帆 田伏
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Amano Enzyme Inc
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Amano Enzyme Inc
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Priority to US18/687,533 priority Critical patent/US20240425829A1/en
Priority to EP22864534.7A priority patent/EP4397754A4/en
Priority to JP2023545585A priority patent/JPWO2023032952A1/ja
Priority to CN202280058405.3A priority patent/CN117916366A/zh
Publication of WO2023032952A1 publication Critical patent/WO2023032952A1/ja
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
    • C11C3/10—Ester interchange
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a transesterification enzymatic agent containing lipase as an active ingredient and its use.
  • Lipases derived from Candida, Alcaligenes, Pseudomonas, Humicola (trade name: Novozyme, Lipozyme TL IM) can be used for enzymatic transesterification of fats and oils. However, there are few enzymes that are practical for food applications.
  • Patent Document 1 describes a lipase derived from Talaromyces thermophilus, which is used as a catalyst for biofuel synthesis (butyl oleate synthesis) and an esterification reaction.
  • Non-Patent Document 1 describes a lipase derived from Talaromyces thermophilus, and describes its use as a catalyst for a biofuel synthesis reaction (fatty acid methyl ester synthesis).
  • oils and fats containing stearic acid and palmitic acid as constituent fatty acids are solid at room temperature, it is necessary to maintain the temperature of the oils and fats at the melting point or higher during the reaction. Since lipases generally have low thermostability, development of lipases with better thermostability is desired. Furthermore, development of a lipase with high 1- and 3-position specificity is also required.
  • An object of the present invention is to provide a transesterification enzymatic agent containing, as an active ingredient, a lipase with excellent heat resistance and high 1,3-position specificity.
  • a further object of the present invention is to provide a method for producing modified fats and oils using the above transesterification enzymatic agent.
  • thermostable lipase from a strain library of more than 10,000 strains
  • Talaromyces thermophilus NBRC31798T strain sometimes referred to as Thermomyces dupontii NBRC31798T strain
  • the present invention was completed by discovering two types of thermostable lipases. According to the present invention, the following inventions are provided.
  • a lipase consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
  • ⁇ 3> The enzymatic agent according to ⁇ 1> or ⁇ 2>, wherein the lipase is derived from Talaromyces thermophilus (also referred to as Thermomyces duponti).
  • ⁇ 4> A method for producing transesterified fats and oils, comprising allowing the enzymatic agent according to any one of ⁇ 1> to ⁇ 3> to act on the fats and oils.
  • ⁇ 5> The method according to ⁇ 4>, wherein the enzymatic agent acts on fats and oils at a temperature of 60°C or higher.
  • ⁇ 6> A lipase comprising an amino acid sequence having 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, after treatment at 70 ° C. for 30 minutes in 50 mM PIPES buffer (pH 7.0) lipase, which still retains enzymatic activity.
  • ⁇ 7> A method for producing a lipase comprising the following steps (1) and (2): (1) culturing the microorganism having the ability to produce lipase according to ⁇ 6>; (2) recovering lipase from the culture medium and/or cells after culturing; ⁇ 8> The production method according to ⁇ 7>, wherein the microorganism is Talaromyces thermophilus (also referred to as Thermomyces duponti) strain NBRC31798T.
  • Talaromyces thermophilus also referred to as Thermomyces duponti
  • the lipase in the transesterification enzymatic agent of the present invention has high heat resistance and high 1- and 3-position specificity.
  • FIG. 1 shows the results of heat resistance evaluation.
  • the transesterification enzymatic agent of the present invention contains a lipase consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 as an active ingredient.
  • the lipase in the present invention is preferably a lipase derived from bacteria of the genus Talaromyces or Thermomyces, more preferably derived from Talaromyces thermophilus (also referred to as Thermomyces dupontii).
  • Lipase. “Derived from Talaromyces thermophilus” means a lipase produced by a microorganism classified as Talaromyces thermophilus (whether it is a wild strain or a mutant strain), or a lipase produced by Talaromyces thermophilus It means that it is a lipase obtained by a genetic engineering method using the lipase gene of Talaromyces thermophilus (or a modified gene).
  • a gene or a recombinant produced by a host microorganism into which a gene having a nucleotide sequence equivalent thereto has been introduced is also a lipase derived from Talaromyces thermophilus. is called the originating bacterium of the present enzyme, and the microorganism (Talaromyces thermophilus, host microorganism) used to produce the present enzyme is called the producing bacterium.
  • NBRC31798T strain A specific example of Talaromyces thermophilus is the Talaromyces thermophilus NBRC31798T strain.
  • the NBRC31798T strain is a strain (published as NBRC31798T in the NBRC Culture catalog) stored in the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture), and the prescribed procedure is performed. You can get it over time.
  • the amino acid sequences (SEQ ID NO: 2 and SEQ ID NO: 4) of the lipase produced by the NBRC31798T strain were determined.
  • One aspect of the lipase in the present invention consists of a protein having the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4.
  • the protein after modification may have the same function as the protein before modification.
  • modification of the amino acid sequence does not substantially affect the function of the protein, and the function of the protein may be maintained before and after modification.
  • a protein having an amino acid sequence equivalent to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and having transesterification activity hereinafter also referred to as "equivalent protein" may be used.
  • the present invention relates to an enzymatic agent useful for transesterification of fats and oils (enzymatic agent for transesterification).
  • the enzyme agent of the present invention (hereinafter also referred to as “this enzyme agent”) contains transesterified lipase (hereinafter also referred to as “this enzyme”) as an active ingredient.
  • the active ingredient transesterification lipase that is, the present enzyme, consists of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 or an amino acid sequence equivalent thereto.
  • the “equivalent amino acid sequence” is partially different from the reference amino acid sequence (amino acid sequence of SEQ ID NO: 2 or amino acid sequence of SEQ ID NO: 4), but the difference is related to protein function (here, transesterification ability).
  • the degree of activity is not particularly limited as long as it can exhibit its function as a transesterified lipase. However, it is preferably the same as or higher than the enzyme consisting of the reference amino acid sequence.
  • Partial difference in amino acid sequence includes, for example, deletion or substitution of one or more amino acids in the amino acids constituting the amino acid sequence, addition or insertion of one or more amino acids to the amino acid sequence, or caused by any combination of
  • a partial difference in the amino acid sequence is permissible as long as the transesterification activity is maintained (there may be some variation in the activity).
  • the positions where the amino acid sequences differ are not particularly limited.
  • differences in amino acid sequences may occur at multiple sites (sites).
  • the number of amino acids that cause partial differences in the amino acid sequence is, for example, a number corresponding to less than about 30%, preferably less than about 20%, more preferably a number corresponding to all amino acids constituting the amino acid sequence.
  • the equivalent protein is, for example, about 70% or more, preferably about 80% or more, more preferably about 85% or more, even more preferably about 90% or more, even more preferably about 93% or more, and further Even more preferably, they have an identity of about 95% or greater, more preferably about 97% or greater, even more preferably about 98% or greater, and most preferably about 99% or greater.
  • partial difference in amino acid sequence is 1 to 40 (preferably 1 to 30, more preferably 1 to 10, more preferably 1 1 to 40 (preferably 1 to 30, more preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, still more preferably 1 to 3) amino acid addition, insertion, or a combination thereof to mutate (change) the amino acid sequence is occurring.
  • equivalent amino acid sequences are obtained by conservative amino acid substitutions at amino acid residues that are not essential for transesterification.
  • conservative amino acid substitution refers to substitution of an amino acid residue with an amino acid residue having a side chain with similar properties.
  • Amino acid residues can have, depending on their side chain, a basic side chain (e.g. lysine, arginine, histidine), an acidic side chain (e.g. aspartic acid, glutamic acid), an uncharged polar side chain (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine). , cysteine), nonpolar side chains (e.g.
  • alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine), aromatic side chains (e.g. tyrosine, phenylalanine, Tryptophan, histidine) are classified into several families. Conservative amino acid substitutions are preferably between amino acid residues within the same family.
  • the identity (%) of two amino acid sequences can be determined, for example, by the following procedure. First, the two sequences are aligned for optimal comparison (eg, gaps may be introduced in the first sequence to optimize alignment with the second sequence). When the molecule (amino acid residue) at a particular position in the first sequence is the same as the molecule at the corresponding position in the second sequence, the molecules at that position are said to be identical.
  • the transesterified lipase which is the active ingredient of the enzymatic agent, that is, the enzyme may be part of a larger protein (eg, fusion protein).
  • Additional sequences in the fusion protein include, for example, sequences that aid in purification such as multiple histidine residues, additional sequences that ensure stability during recombinant production, and the like.
  • the transesterification reaction refers to a reaction in which an acid group (carboxylic acid group) in an ester compound is exchanged with another acid (carboxylic acid). distinguished.
  • the transesterification reaction in the present invention refers to fatty acid residues constituting an acceptor substrate (oil (triglyceride), glycerin fatty acid ester (diglyceride, monoglyceride)) and a donor substrate (fatty acid, ester compound (fatty acid ester, etc.) or fat ( It refers to the reaction that exchanges the fatty acid of (which may be the same as the acceptor substrate)).
  • this enzymatic agent is used to modify and improve the physical properties of fats and oils and fat processed products, it will be possible to transesterify fats and oils with high melting points, resulting in a different fatty acid composition (especially the 2nd fatty acid) than before treatment. Improvement in quality (acquisition of desired physical properties) can be expected by becoming oils and fats with different compositions.
  • the content of the active ingredient (the present enzyme) in the enzymatic preparation is not particularly limited, but for example, the content of the active ingredient is set so that the enzymatic activity per gram of the enzymatic preparation is 1 U to 100000 U, preferably 10 U to 30000 U. or can be adjusted.
  • the enzymatic agent of the present invention is usually solid (for example, granules, powder, silica, diatomaceous earth, porous polymer, etc.). ) or in liquid form.
  • the enzyme preparation may contain excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, and the like.
  • excipients include lactose, sorbitol, D-mannitol, maltodextrin, and sucrose.
  • Phosphate, citrate, acetate and the like can be used as buffers.
  • Propylene glycol, ascorbic acid and the like can be used as stabilizers.
  • Preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like. As antiseptics, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.
  • the present invention further provides a lipase consisting of an amino acid sequence having a sequence identity of 99% or more with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein It relates to a lipase that retains enzymatic activity even after treatment.
  • the invention further relates to a lipase consisting of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
  • the present enzyme can be obtained by culturing a microorganism that produces transesterified lipase (transesterified lipase-producing strain).
  • the method for producing a lipase of the present invention may include the following steps (1) and (2).
  • the transesterified lipase-producing strain may be a wild strain or a mutant strain (for example, a mutant strain can be obtained by ultraviolet irradiation).
  • a specific example of the strain producing this enzyme is Talaromyces thermophilus NBRC31798T strain. Mutants can be obtained by irradiation with ultraviolet rays, X-rays, gamma rays, or the like, treatment with nitrous acid, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine, or the like. Mutants are not limited as long as they produce the present enzyme. Mutant strains include strains with improved productivity of the present enzyme, strains with reduced productivity of contaminants, strains with easier culture, and strains with easier recovery from the culture medium.
  • a step of culturing a microorganism that produces this enzyme (step (1)) and a step of recovering lipase from the culture solution and/or bacterial cells after culturing (step (2)) are performed. can be done.
  • the culture conditions and culture method are not particularly limited as long as the enzyme is produced. That is, on the condition that the present enzyme is produced, a method and culture conditions suitable for culturing the microorganism to be used can be appropriately set.
  • a culture method either liquid culture or solid culture may be used, but liquid culture is preferably used. Taking liquid culture as an example, the culture conditions will be described.
  • the medium is not particularly limited as long as it allows the microorganisms to be used to grow.
  • glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses carbon sources such as organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein.
  • Nitrogen sources such as hydrolysates, bran and meat extracts, and inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts and zinc salts can be used. Vitamins, amino acids, etc.
  • the pH of the medium is adjusted to, for example, about 3 to 8, preferably about 4 to 7, and the culture temperature is usually about 20 to 40°C, preferably about 25 to 35°C, for 1 to 20 days, preferably 3 to 3. Incubate under aerobic conditions for about 10 days.
  • a shake culture method and an aerobic submerged culture method using a jar fermenter can be used.
  • the target enzyme is recovered from the culture medium or the cells (step (2)).
  • the culture solution for example, after removing insoluble matter by filtering or centrifuging the culture supernatant, concentration with an ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, ion exchange resin, etc.
  • the present enzyme can be obtained by separating and purifying by appropriately combining various chromatographic techniques.
  • the present enzyme when it is recovered from the cells, can be obtained by, for example, crushing the cells by pressure treatment, ultrasonic treatment, etc., followed by separation and purification in the same manner as described above.
  • the series of steps may be performed after collecting the bacterial cells from the culture solution in advance by filtration, centrifugation, or the like.
  • This enzyme can be easily prepared by genetic engineering techniques.
  • DNA encoding the present enzyme for example, the DNA sequence encoding the present enzyme consisting of the amino acid sequence of SEQ ID NO: 2 is sequenced as SEQ ID NO: 1, and the DNA sequence encoding the present enzyme consisting of the amino acid sequence of SEQ ID NO: 4 is sequenced. 3, respectively
  • transforming an appropriate host cell eg, E. coli
  • recovering the protein expressed in the transformant eg, E. coli
  • the recovered protein is appropriately purified depending on the purpose. If the present enzyme is obtained as a recombinant protein in this way, various modifications are possible.
  • the recombinant protein consists of any peptide or protein linked thereto.
  • This enzyme can be obtained.
  • addition of sugar chains and/or lipids, or modification that causes N-terminal or C-terminal processing may be performed. Such modifications enable extraction of recombinant proteins, simplification of purification, addition of biological functions, and the like.
  • an appropriate host-vector system is usually used to express the gene and recover the expression product (this enzyme), but a cell-free synthesis system may also be used.
  • the "cell-free synthesis system does not use living cells, but ribosomes derived from living cells (or obtained by genetic engineering techniques), It refers to the in vitro synthesis of mRNA or protein encoded by a template nucleic acid (DNA or mRNA) using transcription/translation factors.
  • a cell-free synthesis system generally uses a cell extract obtained by purifying a cell lysate as necessary.
  • Cell extracts generally contain various factors such as ribosomes and initiation factors necessary for protein synthesis, and various enzymes such as tRNA. When synthesizing proteins, other substances necessary for protein synthesis such as various amino acids, energy sources such as ATP and GTP, and creatine phosphate are added to the cell extract. Of course, ribosomes, various factors, and/or various enzymes prepared separately may be supplemented as necessary during protein synthesis.
  • cell-free transcription/translation system is used interchangeably with cell-free protein synthesis system, in vitro translation system or in vitro transcription/translation system.
  • In vitro translation systems use RNA as a template to synthesize proteins. Total RNA, mRNA, in vitro transcripts and the like are used as template RNA.
  • the other in vitro transcription/translation system uses DNA as a template.
  • the template DNA should contain a ribosome binding region and preferably contains a suitable terminator sequence.
  • conditions are set such that factors necessary for each reaction are added so that the transcription reaction and the translation reaction proceed continuously.
  • the purified enzyme obtained as described above can be powdered by, for example, freeze-drying, vacuum-drying, or spray-drying. At that time, the purified enzyme may be dissolved in advance in acetate buffer, phosphate buffer, triethanolamine buffer, Tris-HCl buffer, or GOOD buffer. Preferably, acetate buffers, phosphate buffers and triethanolamine buffers can be used.
  • the GOOD buffer includes PIPES, MES or MOPS.
  • the degree of purification of the enzyme is not particularly limited, for example, it can be purified to have a specific hydrolytic activity of 1 to 100,000 (U/ ⁇ g), preferably 10 to 20,000 (U/ ⁇ g).
  • the final form may be liquid or solid (including powder).
  • transesterification method for oils and fats there is provided a method for producing transesterified fats and oils, which comprises allowing the present enzymatic agent to act on fats and oils. That is, according to the present invention, a method for transesterification of fats and oils using the present enzymatic agent is provided.
  • a step of acting the present enzymatic agent or the present enzyme on fats and oils is performed, and by this step, transesterification of fats and oils, i.e., triacylglycerol (abbreviated as TG or TAG) in fats and oils. are rearranged (rearranged).
  • Oils and fats that can be processed by the transesterification method of the present invention include soybean oil, rapeseed oil, rice oil, corn oil, sunflower oil, cottonseed oil, peanut oil, safflower oil, palm oil, soft palm oil, fractionated palm oil, and palm kernel.
  • Vegetable oils and fats such as oil, coconut oil and cacao butter, animal oils and fats such as fish oil, lard, beef tallow and milk fat, fractionated oils thereof, hydrogenated oils, and synthetic oils and fats such as trilaurin, triolein and tripalmitin can be exemplified.
  • the transesterification method of the present invention can be used for transesterification between fats and oils, as well as for transesterification between fats and oils and fatty acids or fatty acid esters.
  • fatty acids are stearic acid, palmitic acid, lauric acid, arachidic acid, behenic acid, oleic acid, linoleic acid
  • fatty acid esters are ethyl stearate, ethyl palmitate, ethyl oleate, ethyl linoleate.
  • the present enzymatic agent or the present enzyme is added to oils and fats, and the is 60° C. to 100° C., more preferably 70° C. to 100° C., more preferably 80° C. to 100° C., for a predetermined time (for example, 10 minutes to 72 hours, 1 hour to 48 hours, 2 hours to 24 hours). react. In order to promote the reaction, it is preferable to stir during the reaction.
  • the enzymatic agent or the enzyme may be subjected to an immobilization treatment, and the reaction with the immobilized enzyme may be performed.
  • an immobilization treatment for reactions with immobilized enzymes, a batch stirred tank reactor, a flow stirred tank reactor, a packed bed reactor, a fluidized bed reactor, or the like can be used.
  • the transesterification method of the present invention is useful for modifying and improving the physical properties of fats and oils or fat processed products (eg, shortening, margarine, cocoa butter alternatives). For example, improvement of spreadability, improvement of emulsion stability, optimization of solid fat content (SFC), improvement of solidification property, selective concentration of specific fatty acids, production of low trans acid fat or low trans acid fat processed products For such purposes, the transesterification method of the present invention can be applied.
  • the oil or fat obtained by applying the transesterification method of the present invention or the oil-and-fat processed product containing the same has improved physical properties compared to before the treatment, and has high industrial utility value.
  • transesterified fats and oils can be produced. That is, the present invention also provides a method for producing a transesterified fat.
  • an acceptor substrate fat (triglyceride), glycerin fatty acid ester (diglyceride, monoglyceride)
  • a donor substrate fatty acid, ester compound (fatty acid ester, etc.) or fat (which may be the same as the acceptor substrate)
  • the present enzymatic agent or the present enzyme is allowed to act (that is, the enzymatic reaction is performed in the presence of the acceptor substrate and the donor substrate).
  • the fats, oils, fatty acids, and fatty acid esters used for the acceptor substrate and donor substrate are those described above.
  • the enzyme reaction conditions are the above conditions (for example, 30 to 100°C, preferably 40 to 100°C, more preferably 60 to 100°C, more preferably 70 to 90°C, for a predetermined time (for example, 1 hour to 48°C). time) to react) can be employed.
  • OEO triolein
  • SOS fat S: A method for producing stearic acid, O: oleic acid
  • SOS fat is a triglyceride in which stearic acid (S) and oleic acid (O) are bound to glycerin in the order of SOS.
  • Example 1 Determination of sequence ⁇ Preparation of TDL2 gene probe for Southern blotting> Using the genomic DNA of Talaromyces thermophilus NBRC31798T as a template, the primers shown in Table 1 were designed, and a TDL2 gene probe was prepared by PCR labeling using a PCR DIG Synthesis kit (manufactured by Roche).
  • a sample obtained by treating the genomic DNA of Talaromyces thermophilus NBRC31798T with BamHI overnight at 37°C and a TDL2 gene probe were used to perform Southern blotting, and two bands were detected. It was presumed that the band of about 3.0 kbp was a fragment containing the entire TDL2 gene, and the band of about 5.0 kbp was a fragment containing the entire length of TDL1. About 5.0 kbp and about 3.0 kbp fragments thought to contain the TDL1 gene and TDL2 gene, respectively, were used to carry out a ligation reaction to self-circularize the DNA.
  • PCR was performed using primer F2 and primer R2 in Table 2 using the self-circularized DNA (about 5.0 kbp) as a template.
  • self-circularized DNA approximately 3.0 kbp
  • PCR was performed with primer F3 and primer R3 of . The amplified DNA fragment was purified and subjected to sequence analysis.
  • TDL1 and TDL2 gene sequences are listed in Table 3, and the amino acid sequences translated based on the determined TDL1 and TDL2 gene sequences are listed in Table 4.
  • thermostable lipase gene Based on the identified thermostable lipase gene, the cDNA sequence was estimated by FGENESH (FGENESH - HMM-based gene structure prediction (softberry.com)).
  • Example 2 Evaluation of heat resistance ⁇ Preparation of purified enzyme>
  • Talaromyces thermophilus NBRC31798T was solid-cultured (cultivated at 40°C for 4 days) in an autoclaved wheat bran medium and filtered.
  • anion exchange chromatography HiPrep DEAE FF manufactured by GE Healthcare
  • 20 mM phosphate buffer pH 7.0
  • 20 mM phosphate buffer pH 7.0
  • a purified enzyme was obtained by hydrophobic chromatography (Hitrap Butyl FF manufactured by GE Healthcare) under linear gradient conditions of a liquid (pH 7.0).
  • TDL2 showed high heat resistance even after treatment at 70°C and 100°C for 30 minutes, as reaction products (diolein and oleic acid) were observed even in samples treated at 70°C and 100°C. I have confirmed that I have it.
  • TDL1 gene SEQ ID NO: 5
  • TDL2 gene SEQ ID NO: 6
  • the amplified DNA was ligated to a filamentous fungal expression vector (pUC19 containing an Aspergillus oryzae-derived ⁇ -amylase-modified promoter, an Aspergillus oryzae-derived FAD-GDH terminator and a pyrG gene) and introduced into A. oryzae by the protoplast-PEG method.
  • a filamentous fungal expression vector pUC19 containing an Aspergillus oryzae-derived ⁇ -amylase-modified promoter, an Aspergillus oryzae-derived FAD-GDH terminator and a pyrG gene
  • ⁇ Method for obtaining recombinant enzyme The resulting transformant was inoculated into a liquid medium (pH 6.0) containing starch, yeast extract and inorganic salts, and cultured at 30° C. for 90 hours with aeration. The culture supernatant was filtered through diatomaceous earth to remove producing cells, concentrated with an ultrafiltration membrane, and freeze-dried to obtain a dry powder of the enzyme.
  • a liquid medium pH 6.0
  • the culture supernatant was filtered through diatomaceous earth to remove producing cells, concentrated with an ultrafiltration membrane, and freeze-dried to obtain a dry powder of the enzyme.
  • Diatomaceous earth was used as an immobilization support.
  • the amount of protein in the dry enzyme powder was measured using the Bradford method kit (BIO-RAD), and based on the measured value, the dry enzyme powder was dissolved in 1 mL of purified water so that the protein amount was 0.1 g, and the enzyme was dissolved. liquid. According to a conventional method, it was immobilized on an immobilization carrier (Celite No. 535 (manufactured by Celite)), and immobilized enzymes (TDL1-IM, TDL2-IM) were prepared.
  • an immobilization carrier Celite No. 535 (manufactured by Celite)
  • immobilized enzymes TDL1-IM, TDL2-IM
  • ⁇ Evaluation method> 3.4 g of triolein (olein rich, manufactured by Showa Sangyo) and 5 g of methyl palmitate (manufactured by Wako) were added to a 50 mL tube, and the mixture was kept at the reaction temperature (40° C., 80° C.) for 30 minutes.
  • Lipozyme TL-IM (Novozymes), TDL1-IM and TDL2-IM were used as immobilized enzymes. After adding 0.05 g of each immobilized enzyme, the mixture was shaken to react. After 20 hours, the supernatant was collected and used as a reaction sample.
  • POP palmitic acid at the 1st and 3rd positions of glycerol, oleic acid at the 2nd position of glycerin
  • PPO palmitic acid at the 1st and 2nd positions of glycerin, oleic acid at the 3rd position
  • the acid-ester bond) ratio was analyzed by HPLC. After adding 0.9 mL hexane and 0.1 mL GC analysis sample to the vial and stirring well using a vortex mixer, HPLC analysis was performed under the following conditions. From the areas of POP and PPO, the POP ratio (%) was calculated by POP/(POP+PPO).
  • TDL1-IM and TDL2-IM showed a high POP ratio at 80°C. It was suggested that the 1,3-position specificity in the high-temperature reaction is higher than that of Lipozyme TL-IM.
  • the POP ratio was evaluated after reaction with each lipase so that the amount of OAME produced was about 20%.
  • the reaction temperature was 80° C. and the reaction was carried out according to the evaluation method described above. The amount of enzyme added and the reaction time were appropriately adjusted, and the supernatant was collected when the amount of OAME produced reached about 20%.
  • Table 2 shows the POP ratio (%) when using each lipase.
  • TDL1-IM and TDL2-IM showed a higher POP ratio than Lipozyme TL-IM, even when the amount of OAME produced was the same.
  • the enzymatic agent of the present invention is suitable for use in the fields of food and medical applications, and has high industrial utility value.
  • the enzymatic agent for transesterification of the present invention which contains a lipase with high heat resistance and high 1,3-position specificity, is useful for producing cocoa butter substitute fat.

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LIAN WEISHUAI; WANG WEIFEI; TAN CHIN PING; WANG JIANRONG; WANG YONGHUA: "ImmobilizedTalaromyces thermophiluslipase as an efficient catalyst for the production of LML-type structured lipids", BIOPROCESS AND BIOSYSTEMS ENGINEERING, vol. 42, no. 2, 12 November 2018 (2018-11-12), DE , pages 321 - 329, XP036690455, ISSN: 1615-7591, DOI: 10.1007/s00449-018-2036-7 *
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WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

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