WO2022270590A1 - ラッカーゼ - Google Patents

ラッカーゼ Download PDF

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Publication number
WO2022270590A1
WO2022270590A1 PCT/JP2022/025138 JP2022025138W WO2022270590A1 WO 2022270590 A1 WO2022270590 A1 WO 2022270590A1 JP 2022025138 W JP2022025138 W JP 2022025138W WO 2022270590 A1 WO2022270590 A1 WO 2022270590A1
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Prior art keywords
laccase
protein
amino acid
present
food
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English (en)
French (fr)
Japanese (ja)
Inventor
寛之 谷内
杏匠 酒井
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Amano Enzyme Inc
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Amano Enzyme Inc
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Priority to EP22828506.0A priority Critical patent/EP4361275A4/en
Priority to US18/573,368 priority patent/US20240287476A1/en
Priority to CN202280038366.0A priority patent/CN117425729A/zh
Priority to JP2023530123A priority patent/JPWO2022270590A1/ja
Publication of WO2022270590A1 publication Critical patent/WO2022270590A1/ja
Anticipated expiration legal-status Critical
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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
    • A23B2/00—Preservation of foods or foodstuffs, in general
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/16—Vegetable proteins from soybean
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/14—Vegetable proteins
    • A23J3/18—Vegetable proteins from wheat
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/22—Working-up of proteins for foodstuffs by texturising
    • A23J3/225—Texturised simulated foods with high protein content
    • A23J3/227—Meat-like textured foods
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)

Definitions

  • the present invention relates to a novel laccase.
  • Laccase is an enzyme that belongs to multicopper oxidase, and catalyzes a reaction that oxidizes a substrate and uses the surplus electrons to reduce oxygen to four electrons to produce water.
  • Substrates to be oxidized by laccase typically include diphenols, as well as methoxy-substituted phenols and diamines. Since such catalytic ability can be applied to various chemical reactions, it is expected to be used in many industrial fields. Laccase is known to exist in microorganisms, plants and animals.
  • Patent Document 1 describes a thermostable laccase derived from the Trametes versicolor TV-1 strain, which exhibits the best laccase activity at pH 2.
  • WO 2005/010000 discloses the pH range in which laccases derived from Botrytis cinerea, Trametes versicolor, or other microbial sources, as well as laccases that can be purchased from commercial sources and/or produced using recombinant technology, exhibit activity. is described as being between pH 3 and pH 7.
  • Patent Document 3 describes that the optimum pH of laccase derived from microorganisms belonging to the genus Streptomyces is about 4.5, and that the activity is lost at pH 6.5.
  • laccase is expected to be widely applied in industry. Its application mode also includes the case of catalyzing an oxidation reaction under alkaline conditions.
  • laccases produced on an industrial scale and put into practical use have an action pH in the acidic to weakly acidic range, and cannot be used in the alkaline range in particular.
  • conventional laccases are limited in the pH range in which they exhibit activity, and thus cannot sufficiently meet the application conditions expected in the industrial world.
  • an object of the present invention is to provide a laccase that exhibits excellent activity in a pH range including an alkaline range.
  • Section 1 A laccase consisting of a polypeptide shown in any of the following (a) to (c): (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1; (b) in the amino acid sequence shown in SEQ ID NO: 1, consisting of an amino acid sequence in which one or several amino acids are substituted, added, inserted or deleted, and in a pH range including an alkaline range, wherein (a) A polypeptide having laccase activity equivalent to the polypeptide shown in; (c) a polypolypeptide consisting of an amino acid sequence having a sequence identity of 87% or more to the amino acid sequence shown in SEQ ID NO: 1 and having a laccase activity equivalent to that of the polypeptide shown in (a) above in a pH range including an alkaline range; peptide.
  • Section 2. A DNA encoding the laccase according to Item 1.
  • Item 3. An expression cassette or recombinant vector containing the DNA according to item 2.
  • Section 4. A transformant obtained by transforming a host with the expression cassette or recombinant vector according to item 3.
  • Item 5. A method for producing laccase, comprising a step of culturing the transformant according to item 4.
  • Item 6. Item 1. An enzyme preparation containing the laccase according to item 1.
  • Item 7. The enzyme preparation according to Item 6, which is used as a protein cross-linking agent.
  • Item 8. Item 7.
  • Item 9 A method for producing a crosslinked protein, comprising the step of allowing the laccase of Item 1 to act on the protein.
  • Item 10. The production method according to Item 9, wherein the step is performed under alkaline conditions.
  • Item 11. Item 11.
  • Item 12. Oxidatively modified food or food or food material, which comprises the step of allowing the laccase described in Item 1 to act on food or food or food or food material, industrial material or industrial waste component, or pharmaceutical or scientific analysis material. , industrial materials or industrial waste components, or methods of manufacturing pharmaceutical or scientific analytical materials.
  • laccase that exhibits excellent activity in a pH range including an alkaline range is provided.
  • Figure 2 shows pH stability of laccase from Chrysocorona lucknowensis (5537).
  • Figure 2 shows the temperature stability of laccase from Chrysocorona lucknowensis (5537). Appearance of meat-like processed food produced using Chrysocorona lucknowensis-derived laccase (derived from strain 5537) was compared with meat-like processed food produced using commercially available laccase (LC-Y120) and meat-like processed food produced without using laccase. It is shown in comparison with the appearance of each food (no enzyme treatment).
  • nonpolar amino acids include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan.
  • Uncharged amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • Acidic amino acids include aspartic acid and glutamic acid.
  • Basic amino acids include lysine, arginine, and histidine.
  • Laccase The laccase of the present invention comprises a polypeptide shown in any one of (a) to (c) below.
  • SEQ ID NO: 1 is the amino acid sequence of laccase derived from Chrysocorona lucknowensis.
  • polypeptides (b) and (c) are laccases similar in sequence to the amino acid sequence of the polypeptide (a) as a basic skeleton.
  • the amino acid modification introduced into the polypeptide of (b) above may include only one type of modification (e.g., substitution) selected from substitutions, additions, insertions, and deletions. may include modifications (eg, substitutions and insertions) of
  • the number of amino acids to be substituted, added, inserted or deleted may be one or more or several, for example 1 to 10, preferably 1 to 8, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3, particularly preferably 1, 2, or 1.
  • sequence identity may be 87% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more, and still more preferably 99%. Above, more preferably 99.5% or more, particularly preferably 99.8% or more.
  • sequence identity refers to the sequence identity calculated by comparison with the amino acid sequence shown in SEQ ID NO: 1.
  • sequence identity refers to the bl2seq program of BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] Microbiol.Lett., Vol.174, p247-250, 1999).
  • NCBI National Center for Biotechnology Information
  • the parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
  • positions 136, 138, 181, 183, 474, 477, 479, and 136 in the amino acid sequence shown in SEQ ID NO: 1 Since amino acids at positions 545, 546, 547 and 551 are thought to contribute to activity, it is desirable not to introduce substitutions or deletions at these sites.
  • amino acid substitutions introduced into the amino acid sequence shown in SEQ ID NO: 1 include, for example, if the amino acid before substitution is a nonpolar amino acid, other nonpolar amino acids If the amino acid before substitution is a non-charged amino acid, it is substituted with another non-charged amino acid, if the amino acid before substitution is an acidic amino acid, it is substituted with another acidic amino acid, and the amino acid before substitution is If it is a basic amino acid, it may be substituted with other basic amino acids.
  • amino acid addition When amino acid addition is introduced into the polypeptides of (b) and (c) above, examples of aspects of amino acid addition include addition of a methionine residue to the N-terminus, purification tags (e.g., binding properties such as oligohistidine) oligopeptide), and the like.
  • purification tags e.g., binding properties such as oligohistidine
  • polypeptides of (b) and (c) include not only polypeptides obtained by artificial mutation, but also naturally occurring mutations (mutants) based on individual differences in organisms from which the polypeptides are derived or differences in species. or variants) are also included.
  • alkaline range refers to a pH range of more than 8.5, preferably pH 9 or higher.
  • laccase activity refers to at least an activity that catalyzes a reaction that produces a crosslinked protein when a protein is used as a substrate (that is, protein crosslinking activity). Specifically, protein cross-linking activity is measured as follows.
  • a supernatant is obtained as a protein solution by suspending and mixing the protein material in an alkaline pH buffer solution to 15% (w/v) and centrifuging. 100 ⁇ l of this protein solution, 97 ⁇ l of alkaline pH buffer, and 3 ⁇ l of 200 mM DL-catechin (as a mediator) are mixed to obtain a substrate solution. 20 ⁇ l of this substrate solution and 10 ⁇ l of enzyme (used so that the concentration of laccase is 1.7 ⁇ g/ml in protein concentration) are mixed and reacted at 37° C. for 18 hours. Thereafter, the reaction solution is diluted 10-fold and subjected to SDS-PAGE. The degree of protein cross-linking activity can be evaluated by the amount of high-molecular weight protein (that is, protein polymerized by cross-linking, appearing as a band at the origin) in SDS-PAGE.
  • DNA A DNA encoding the laccase of the present invention (hereinafter sometimes referred to as "the DNA of the present invention") can be appropriately prepared and designed by those skilled in the art according to the amino acid sequence of the laccase of the present invention. .
  • examples of the DNA encoding the laccase of the present invention include DNA shown in any of (i) to (iii) below.
  • DNA consisting of the base sequence shown in SEQ ID NO: 2 (i) DNA consisting of the base sequence shown in SEQ ID NO: 2; (ii) a DNA comprising a nucleotide sequence that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2; (iii) A DNA consisting of a nucleotide sequence having 87% or more homology to the nucleotide sequence shown in SEQ ID NO:2.
  • the DNAs (i) to (iii) are described in detail below.
  • the nucleotide sequence shown in SEQ ID NO: 2 is a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO: 1.
  • the DNAs (ii) and (iii) are DNAs similar in sequence to the base sequence of the DNA (i).
  • stringent conditions means 0.5% SDS, 5 ⁇ Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400] and 100 ⁇ g/ml salmon sperm DNA in 6xSSC (1xSSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) at 50-65°C. Refers to conditions for keeping warm for 4 hours to overnight.
  • Hybridization under stringent conditions is specifically performed by the following method. That is, a nylon membrane on which a DNA library or cDNA library was immobilized was prepared and incubated at 65° C. in a prehybridization solution containing 6 ⁇ SSC, 0.5% SDS, 5 ⁇ Denhardts, 100 ⁇ g/ml salmon sperm DNA. Block the nylon membrane. After that, each 32 P-labeled probe is added and incubated overnight at 65°C. This nylon membrane was placed in 6 ⁇ SSC at room temperature for 10 minutes, in 2 ⁇ SSC containing 0.1% SDS at room temperature for 10 minutes, and in 0.2 ⁇ SSC containing 0.1% SDS at 45° C. for 30 minutes. After washing, autoradiography can be performed to detect DNA specifically hybridizing with the probes.
  • the homology may be 87% or higher, preferably 90% or higher, more preferably 95% or higher, even more preferably 98% or higher, still more preferably 99% or higher, and more preferably 99% or higher. More preferably 99.5% or more, particularly preferably 99.8% or more.
  • the "homology" of the nucleotide sequence refers to the BLAST PACKAGE [sgi32 bitedition, Version 2.0.12; Madden, FEMS Microbiol. Lett., Vol. 174, 247-250, 1999).
  • the parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.
  • a base sequence encoding a purification tag for example, a binding oligopeptide such as oligohistidine
  • a purification tag for example, a binding oligopeptide such as oligohistidine
  • the DNA of the present invention can be prepared, for example, by using, as a template, a DNA encoding any of the polypeptides of (a) to (c), and at least one of the polypeptides of (a) to (c). can be obtained by obtaining the region encoding the by PCR or the like.
  • the DNA encoding the laccase of the present invention can be artificially synthesized by a gene synthesis method.
  • a specific mutation is introduced into a specific site of a base sequence
  • the method of introducing mutation is known, and for example, site-specific mutagenesis of DNA can be used.
  • a specific method for converting bases in DNA for example, a commercially available kit can be used.
  • DNA with mutations introduced into the base sequence can be confirmed using a DNA sequencer. Once the nucleotide sequence is determined, then chemical synthesis, PCR using the cloned probe as a template, or hybridization using a DNA fragment having the nucleotide sequence as a probe to obtain the DNA encoding the laccase. can be done.
  • the DNA of the present invention is preferably one whose codon usage frequency is optimized for the host.
  • a DNA whose codon usage frequency is optimized for E. coli is suitable.
  • expression cassette or recombinant vector containing DNA encoding the laccase of the present invention (hereinafter also referred to as "expression cassette of the present invention” or “recombinant vector of the present invention”) is It includes DNA encoding the laccase of the invention.
  • the expression cassette or recombinant vector of the present invention can be obtained by ligating a promoter and a terminator to the DNA of the present invention, or by inserting the expression cassette or the DNA of the present invention into an expression vector.
  • the expression cassette or recombinant vector of the present invention may contain transcription elements such as promoters and terminators, as well as enhancers, CCAAT boxes, TATA boxes, and SPI sites, as required. These regulatory factors need only be operably linked to the DNA of the present invention.
  • operably linked means that various control factors that regulate the DNA of the present invention and the DNA of the present invention are linked in a state in which they are operable in host cells.
  • the expression cassette or recombinant vector of the present invention may be configured to further contain a protease recognition sequence and an N-terminal sequence and/or a C-terminal sequence.
  • expression vector those constructed for genetic recombination from phages, plasmids, or viruses that are capable of autonomous replication within the host are suitable. Such expression vectors are known, and suitable combinations with host cells can be appropriately selected and used by those skilled in the art.
  • pBluescript (pBS) II SK (-) manufactured by Stratagene
  • pSTV-based vector manufactured by Takara Bio
  • pUC-based vector manufactured by Takara Bio
  • pET-based vector Merck
  • pGEX vector GE Healthcare
  • pHY300PLK Tekara Bio
  • pUB110 Mckenzie, T. et al., 1986, Plasmid 15 (2 ), pp.
  • pBR322 manufactured by Takara Bio
  • pRS403 manufactured by Stratagene
  • pMW218/219 manufactured by Nippon Gene.
  • pUC19 manufactured by Takara Bio Inc.
  • P66 Chlamydomonas Center
  • P-322 Cholamydomonas Center
  • pPha-T1 Yangmin Gong, et al., Journal of Basic Microbiology, 2011, vol.51, p.666-672
  • pJET1 manufactured by Cosmo Bio
  • pRI-based vectors manufactured by Takara Bio
  • pBI-based vectors manufactured by Clontech
  • IN3-based vectors manufactured by Implanta Innovations
  • Transformant A transformant (hereinafter sometimes referred to as “the transformant of the present invention") is obtained by transforming a host with the expression cassette or recombination vector of the present invention.
  • the host used for the production of the transformant is not particularly limited as long as it can introduce a gene, can grow autonomously, and can express the trait of the gene of the present invention.
  • Examples include Escherichia coli.
  • Preferable examples include bacteria belonging to the genus Escherichia such as Bacillus subtilis, Pseudomonas such as Pseudomonas putida; , but animal cells, insect cells, plant cells and the like may also be used. Among these, Escherichia coli is particularly preferred.
  • the host used for the production of the transformant may be Chrysocorona lucknowensis, which is the bacterium or cell derived from the laccase of the present invention.
  • the transformant of the present invention can be obtained by introducing the expression cassette or recombinant vector of the present invention into a host.
  • the place where the DNA of the present invention is introduced is not particularly limited as long as the gene of interest can be expressed, and may be on a plasmid or genome.
  • Specific methods for introducing the expression cassette of the present invention or the recombinant vector of the present invention include, for example, recombinant vector methods and genome editing methods.
  • the conditions for introducing the expression cassette or recombinant vector of the present invention into the host may be appropriately set according to the type of host.
  • the host is a microorganism, for example, a method using competent cells by calcium ion treatment, an electroporation method, a spheroplast method, a lithium acetate method and the like can be used.
  • the host is an animal cell, for example, the electroporation method, calcium phosphate method, lipofection method and the like can be used.
  • the host is an insect cell, for example, the calcium phosphate method, lipofection method, electroporation method and the like can be used.
  • the host is a plant cell, for example, the electroporation method, the Agrobacterium method, the particle gun method, the PEG method and the like can be used.
  • the laccase of the present invention can be produced by culturing the transformant of the present invention.
  • the laccase of the present invention can also be produced by culturing Chrysocorona lucknowensis itself (untransformed).
  • the culture conditions for the transformants or derived bacteria or derived cells of the present invention may be appropriately set in consideration of the nutritional and physiological properties of the host or derived bacteria or derived cells, preferably liquid culture. Further, in the case of industrial production, aeration and agitation culture is preferable.
  • the transformant or derived bacteria or derived cells of the present invention are cultured, and the culture solution is collected by a method such as centrifugation to recover the culture supernatant or cultured cells or cultured cells. If the laccase of the present invention is accumulated in cultured bacteria or cultured cells, the bacteria or cells are treated with ultrasonic waves, a mechanical method such as French press, or a lytic enzyme such as lysozyme.
  • the water-soluble fraction containing the laccase of the present invention can be obtained by solubilization by using an enzyme such as protease or a surfactant such as sodium dodecyl sulfate (SDS).
  • the expressed laccase of the present invention may be secreted into the culture medium.
  • the culture medium, the water-soluble fraction, or the protease-treated product containing the laccase of the present invention obtained as described above may be directly subjected to a purification treatment, but the culture medium, the water-soluble fraction, or the protease-treated product may be purified. After concentrating the laccase of the present invention in the product, it may be subjected to a purification treatment.
  • Concentration can be performed, for example, by vacuum concentration, membrane concentration, salting-out treatment, fractional precipitation using a hydrophilic organic solvent (eg, methanol, ethanol, and acetone), or the like.
  • a hydrophilic organic solvent eg, methanol, ethanol, and acetone
  • the purification treatment of the laccase of the present invention can be performed, for example, by appropriately combining methods such as gel filtration, hydrophobic chromatography, ion exchange chromatography, and affinity chromatography.
  • the laccase of the present invention thus purified may be pulverized by freeze-drying, vacuum-drying, spray-drying, etc., if necessary.
  • the laccase of the present invention can be provided in the form of an enzyme preparation. Accordingly, the present invention also provides an enzyme preparation containing the above-described laccase of the present invention.
  • the content of the laccase of the present invention in the enzyme preparation of the present invention is not particularly limited, and can be appropriately set within a range in which the laccase activity is exhibited.
  • the enzyme preparation of the present invention may contain other ingredients in addition to the laccase of the present invention to the extent that the effects of the present invention are not affected.
  • examples of other components include enzymes other than the laccase of the present invention, mediators, additives, and culture residue produced by the above production method.
  • enzymes can be appropriately determined depending on the intended use. galactosidase, ⁇ -galactosidase), protease (acid protease, neutral protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, Dehydrogenase (other than the above active ingredients), glutaminase, pectinase, catalase, dextranase, transglutaminase, protein deamidase, pullulanase and the like. These other enzymes may be contained singly or in combination of multiple types.
  • mediators examples include 3-(3,4-dihydroxyphenyl)alanine (DOPA), catechin, and caffeine. These mediators may be contained singly or in combination of multiple types. Among these mediators, DOPA and catechin are preferred.
  • the additive can be appropriately determined according to the use of the laccase of the present invention and the formulation form of the enzyme preparation. Salt solution etc. are mentioned.
  • Excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, pectin and the like.
  • Buffers include phosphate, citrate, acetate and the like.
  • Stabilizers include propylene glycol, ascorbic acid and the like.
  • Preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like.
  • antiseptics include ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like.
  • the culture residue includes medium-derived components, contaminant proteins, fungal components, cell components, and the like.
  • the formulation form of the enzyme preparation of the present invention is not particularly limited, and examples thereof include liquid and solid forms (powder, granules, etc.). These formulation forms of enzyme preparations can be prepared by generally known methods.
  • the enzyme preparation of the present invention can be used as a cross-linking agent for proteins, and can also be used for the oxidative modification of foods and beverages, materials for foods and beverages, industrial materials, industrial waste components, or materials for pharmaceuticals or scientific analysis. It can also be used as a substance.
  • the target of oxidative modification and the details of oxidative modification are as described in "7. Use of laccase" below.
  • laccases of the present invention can be used in any application that utilizes a variety of incidental chemical reactions resulting from oxidation of a substrate and/or radical species of reaction intermediates produced by the oxidation.
  • oxidation reactions or concomitant chemical reactions are oxidation of phenolic compounds such as o-, p-diphenol, urushiol, laccol; oxidation of aromatic amines such as p-phenylenediamine; decomposition of lignin. ; tyrosine side chain (phenolic hydroxyl group), cysteine side chain (sulfhydryl group), lysine side chain ( ⁇ -amino group), histidine side chain (imidazole group), etc. be done.
  • chemically changing a substrate by such an oxidation reaction of laccase and accompanying chemical reactions is also referred to as "oxidative modification”.
  • the present invention also provides a method for producing a crosslinked protein, comprising the step of allowing the laccase of the present invention to act on the protein.
  • mediators such as 3-(3,4-dihydroxyphenyl)alanine (DOPA), catechin, and caffeine are replaced with laccase in the process. It is preferable to use in combination with These mediators may be contained singly or in combination of multiple types. Among these mediators, DOPA and catechin are preferred.
  • the reaction conditions e.g., amount of enzyme, reaction time, reaction pH, reaction temperature, etc.
  • the reaction conditions e.g., amount of enzyme, reaction time, reaction pH, reaction temperature, etc.
  • the desired degree of protein crosslinking is achieved.
  • those skilled in the art can appropriately set it according to the type and/or concentration of the laccase of the present invention, the type and/or concentration of the protein, and/or the desired degree of protein cross-linking.
  • the reaction pH is preferably above 8.5, more preferably 9 or above.
  • the upper limit of the reaction pH is, for example, 10.5 or less, preferably 10 or less, more preferably 9.5 or less, still more preferably 9.3 or less.
  • the laccase used in the method for producing a crosslinked protein of the present invention preferably exhibits excellent laccase activity not only in the alkaline range but also in the neutral range. , or 8 or more.
  • the upper limit of the reaction pH is, for example, 10.5 or less, preferably 10 or less, more preferably 9.5 or less, still more preferably 9.3 or less.
  • reaction temperature is 4 to 55°C, more preferably 20 to 50°C.
  • Process for producing oxidatively modified foods and beverages, materials for foods, industrial materials, industrial waste components, or materials for pharmaceuticals or scientific analysis examples include industrial materials or industrial waste components, or materials for pharmaceuticals or scientific analysis. Therefore, the present invention further provides an oxidatively modified food or drink material, an industrial material or an industrial waste component, or a pharmaceutical or scientific analysis material, which comprises the step of allowing the laccase of the present invention to act. Also provided is a method for producing food or beverage materials, industrial materials or industrial waste components, or pharmaceutical or scientific analysis materials.
  • Examples of oxidative modification of food or food materials include cross-linking of proteins, improvement of binding properties of textured vegetable proteins, thickening or gelling of foods, browning treatment of black tea, removal of bitterness and astringency of foods. etc.
  • Examples of oxidative modification of industrial materials or components of industrial waste include production of artificial lacquer, removal of lignin from pulp, detoxification of waste liquids containing highly toxic phenolic compounds and/or aromatic amines, and synthesis of organic compounds. , production of adhesives, and synthesis of concrete admixtures.
  • Examples of oxidative modification of materials for pharmaceuticals or scientific analysis include conversion of analyte components into sensing components, cross-linking of analyte proteins, and the like.
  • the step Preferably, the mediator is used in combination with laccase.
  • mediators that can be used are as described above in “7-1. Production method of crosslinked protein”.
  • reaction conditions for example, the amount of enzyme, reaction time, reaction pH, reaction temperature, etc.
  • reaction pH and reaction temperature are as described in the above “7-1. Method for producing crosslinked protein”.
  • Test Example 1 Culture and Enzyme Purification (1-1) Culture and Filtration Chrysocorona lucknowensis 5537 grown on potato dextrose agar medium at 30° C. for 3 days in a 500 ml shake flask containing 100 ml of the laccase production medium shown in Table 1. A 1 cm square section of a strain (NBRC 9681) colony was inoculated and cultured with reciprocating shaking (140 r/min) at 30° C. for 3 days. After solid-liquid separation of the culture solution by centrifugation, the supernatant was filtered off, and the collected filtrate was used as a crude enzyme solution.
  • NBRC 9681 reciprocating shaking
  • ⁇ Confirmation of laccase activity-oxidation of ABTS 20 ⁇ l of a 50 mM solution of ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) as substrate, 160 ⁇ l of 125 mM sodium phosphate buffer (pH 7.0), and the enzyme 20 ⁇ l of the solution was added to a microplate, mixed, reacted at 37°C for 15 minutes, and the absorbance at 405 nm was measured before and after the reaction. The presence of laccase activity was confirmed by confirming the oxidation of ABTS.
  • Test Example 2 Evaluation of enzyme properties (2-1) Evaluation of laccase activity (protein cross-linking activity) Purified laccase obtained from Chrysocorona lucknowensis strain 5537 and a laccase for comparison, "Laccase Y120" manufactured by Amano Enzyme Co., Ltd. ( Also referred to as LC-Y120), the protein cross-linking activity at a predetermined pH (pH 3.0, 5.0, 7.0 and 9.0) for each of the three plant proteins shown in Table 2 was measured. evaluated.
  • Plant protein powder was adjusted to 15% (w/v) with a predetermined pH buffer [50 mM Na citrate buffer (pH 3.0 or 5.0), 50 mM Na phosphate buffer (pH 7.0), 0), or 50 mM Tris-HCl buffer (pH 9.0)] and mixed well, followed by solid-liquid separation by centrifugation at 12,000 x g for 5 minutes to obtain a supernatant as a protein solution. . 100 ⁇ l of this protein solution, 97 ⁇ l of the above-mentioned predetermined pH buffer solution, and 3 ⁇ l of 200 mM DL-catechin (mediator) were mixed to obtain a substrate solution.
  • a predetermined pH buffer 50 mM Na citrate buffer (pH 3.0 or 5.0), 50 mM Na phosphate buffer (pH 7.0), 0), or 50 mM Tris-HCl buffer (pH 9.0)
  • the amount of the crosslinked protein was evaluated by the number of "+". The greater the number of "+”s, the greater the amount of crosslinked protein produced. The results are shown in Figures 1-3.
  • the amount of the crosslinked protein can also be quantified using image analysis software such as imageJ.
  • the leftmost lane (lane 1 in FIGS. 1 and 2, blank lane in FIG. 3) represents the results of a control to which no enzyme was added, and the middle lane (lane 2 in FIGS. 1 and 2) , lane "LCY120” in FIG. 3) represents the results when the laccase for comparison was used, and the rightmost lane (lane 3 in FIGS. 1 and 2, lane “5537” in FIG. 3) represents Chrysocorona Lucknowensis strain 5537 strain purified laccase was used.
  • the relative value of the enzyme activity value after pH treatment was derived as the residual activity (%) when the enzyme activity value at the treatment pH that showed the highest residual activity was taken as 100%.
  • the results are shown in FIG.
  • the laccase obtained from the Chrysocorona lucknowensis strain 5537 has significantly improved stability in the alkaline range compared to the commercially available laccase LC-Y120.
  • Test Example 3 Sequence Identification Laccase obtained from Chrysocorona lucknowensis strain 5537 was sequenced. As a result, the sequence (2392 bp) shown in SEQ ID NO: 3 as the gene sequence, the sequence (1845 bp) shown in SEQ ID NO: 2 as the coding region sequence, and the sequence (614 aa) shown in SEQ ID NO: 1 as the amino acid sequence were determined.
  • Test Example 4 Improving Adhesion of Textured Vegetable Protein Granular soybean protein (manufactured by Marukome Co., Ltd.) was added with warm water (40° C.) of 5 times its weight and allowed to stand for 10 minutes to swell. After removing moisture, 25 g of the swollen granular soybean protein was weighed. 2.75 g of powdered pea protein (NUTRALYS F85M manufactured by Roquette) was mixed with the weighed swollen granular soybean protein, and 125 U of purified laccase obtained from Chrysocorona lucknowensis strain 5537 or comparative laccase LC-Y120 (swollen granular soybean protein) was added.
  • NUTRALYS F85M powdered pea protein
  • soy protein mixture 5 U per gram of protein was added to prepare the soy protein mixture.
  • the soy protein mixture was mixed well and formed into a hamburger shape and allowed to stand at 25°C for 60 minutes. After baking in an oven at 190°C for 15 minutes, a meat-like processed food was obtained.
  • FIG. 7 shows a meat-like processed food (without enzyme treatment) obtained in the same manner except that laccase was not used.
  • LC-Y120 is capable of oxidative modification to improve binding properties to textured vegetable proteins. As shown in FIG. 7, no binding occurred in the case of no enzyme treatment, whereas binding was confirmed in the case of treatment with LC-Y120 and the case of treatment with 5537-derived laccase.
  • the hardness of the meat-like processed food was measured with a rheometer (manufactured by Sun Kagaku Co., Ltd.) in order to evaluate the degree of adhesion of the granular soybean protein in the obtained meat-like processed food. A higher hardness measurement indicates a higher degree of cohesiveness. The results are shown in FIG.
  • the 5537 strain-derived laccase has less cooking loss than LC-Y120, so it was confirmed that it is useful in the production of meat-like processed foods.

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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
WO2024247292A1 (ja) * 2023-05-26 2024-12-05 天野エンザイム株式会社 アルデヒド化合物の製造方法及びその応用

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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
WO2024247292A1 (ja) * 2023-05-26 2024-12-05 天野エンザイム株式会社 アルデヒド化合物の製造方法及びその応用

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