Lacase

Abstract

The present invention provides a laccase exhibiting excellent activity in a pH region that includes alkalinity. Both a laccase comprising the amino acid sequence of a laccase derived from Chrysocorona lucknowensis and a laccase having a similar sequence in which said amino acid sequence serves as the base skeleton exhibit excellent activity in a pH region that includes alkalinity.

Description

Technical Field

[0001] The present invention relates to a novel laccase.

Background Art

[0002] Laccase is an enzyme belonging to the multicopper oxidase family, which catalyzes the reaction of oxidizing a substrate and reducing oxygen by four electrons with the remaining electrons to generate water. Typical substrates for laccase oxidation include diphenols, as well as methoxy-substituted phenols and diamines. Such catalytic ability can be applied to various chemical reactions and is expected to be used in many industries. Laccase is known to exist in microorganisms, plants, and animals.

[0003] For example, Patent Document 1 describes a thermostable laccase derived from Trametes versicolor TV-1 strain, which exhibits the best laccase activity at pH 2.

[0004] Patent Document 2 states that the pH range in which laccase derived from Botrytis cinerea, Trametes versicolor or other microbial sources, as well as laccase purchased from commercial sources and / or laccase produced using recombinant technology exhibits activity is pH 3 to pH 7.

[0005] Patent Document 3 describes that the optimum pH of laccase derived from microorganisms belonging to the genus Streptomyces is about 4.5 and the activity disappears at pH 6.5.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

[0007] As mentioned above, laccase is expected to have widespread applications in industry. These applications include catalyzing oxidation reactions under alkaline conditions. However, currently, industrially produced and commercially available laccases operate within the acidic to weakly acidic pH range, and are unusable in alkaline conditions. Thus, the limited pH range in which conventional laccases exhibit activity prevents them from adequately meeting the applications expected by industry.

[0008] Therefore, the present invention aims to provide a laccase that exhibits excellent activity in a pH range including the alkaline range. [Means for solving the problem]

[0009] As a result of diligent research, the inventors of this invention, from a library of more than 10,000 bacterial strains, serendipitously discovered a laccase that exhibits activity in the alkaline range. This invention was completed based on this finding.

[0010] In other words, the present invention provides inventions in the following embodiments. Item 1. Lacase comprising any of the polypeptides shown in (a) to (c) below: (a) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1; (b) A polypeptide having an amino acid sequence in which one or more amino acids are substituted, added, inserted, or deleted in the amino acid sequence shown in Sequence ID No. 1, and having laccase activity equivalent to that of the polypeptide shown in (a) above in a pH range including the alkaline range; (c) A polypeptide having an amino acid sequence with 87% or more sequence identity to the amino acid sequence shown in Sequence ID No. 1, and having laccase activity equivalent to that of the polypeptide shown in (a) above in a pH range including the alkaline range. Item 2. The DNA encoding the laccase described in Item 1. Item 3. An expression cassette or recombinant vector containing the DNA described in Item 2. Item 4. A transformant obtained by transforming a host using the expression cassette or recombinant vector described in Item 3. Item 5. A method for producing laccase, comprising the step of culturing the transformant described in Item 4. Item 6. An enzyme preparation containing the laccase described in Item 1. Item 7. An enzyme preparation as described in Item 6, used as a protein crosslinking agent. Item 8. Enzyme preparations as described in Item 6, used as oxidative modifiers for food and beverages or food and beverage ingredients, industrial materials or industrial waste components, or pharmaceutical or scientific analysis materials. Item 9. A method for producing a cross-linked protein, comprising the step of reacting a protein with the laccase described in Item 1. Item 10. The manufacturing method according to Item 9, wherein the above step is carried out under alkaline conditions. Item 11. The method for producing a product according to item 9 or 10, wherein in the step, a mediator selected from the group consisting of 3-(3,4-dihydroxyphenyl)alanine, catechin, and caffeic acid is used in combination with the laccase. Item 12. A method for producing oxidatively modified food or beverages or food or beverage ingredients, industrial materials or industrial waste components, or pharmaceutical or scientific analysis materials, comprising the step of reacting the food or beverage ingredients or food or beverage ingredients, industrial materials or industrial waste components, or pharmaceutical or scientific analysis materials with the laccase described in Item 1. [Effects of the Invention]

[0011] According to the present invention, a laccase exhibiting excellent activity in a pH range including the alkaline range is provided. [Brief explanation of the drawing]

[0012] [Figure 1] This study investigated the cross-linking activity of laccase derived from Chrysocorona lucknowensis towards pea proteins under various pH conditions, including alkaline pH ranges. [Figure 2] The result of confirming the cross-linking activity of laccase derived from Chrysocorona lucknowensis against soybean protein under various pH conditions including alkaline region pH. [Figure 3] The result of confirming the cross-linking activity of laccase (5537) derived from Chrysocorona lucknowensis against wheat protein under various pH conditions including alkaline region pH. [Figure 4] The result of examining the protein cross-linking activity when various mediators are used in combination with laccase derived from Chrysocorona lucknowensis. [Figure 5] Shows the pH stability of laccase (5537) derived from Chrysocorona lucknowensis. [Figure 6] Shows the temperature stability of laccase (5537) derived from Chrysocorona lucknowensis. [Figure 7] The appearance of a meat-like processed food (derived from strain 5537) produced using laccase derived from Chrysocorona lucknowensis is shown in comparison with the appearances of a meat-like processed food (LC-Y120) produced using a commercially available laccase and a meat-like processed food (without enzyme treatment) produced without using laccase. [Figure 8] The binding property of texturized vegetable protein in a meat-like processed food (derived from strain 5537) produced using laccase derived from Chrysocorona lucknowensis is shown in comparison with that in a meat-like processed food (LC-Y120) produced using a commercially available laccase. [Figure 9] The cooking loss in the production of a meat-like processed food (derived from strain 5537) produced using laccase derived from Chrysocorona lucknowensis is shown in comparison with that in a meat-like processed food (LC-Y120) produced using a commercially available laccase.

Mode for Carrying Out the Invention

[0014] 1. Laccase The laccase of the present invention consists of a polypeptide shown in any of (a) to (c) below.

[0015] (a) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1; (b) A polypeptide having an amino acid sequence in which one or more amino acids are substituted, added, inserted, or deleted in the amino acid sequence shown in Sequence ID No. 1, and having laccase activity equivalent to that of the polypeptide shown in (a) above in a pH range including the alkaline range; (c) A polypeptide having an amino acid sequence with 87% or more sequence identity to the amino acid sequence shown in Sequence ID No. 1, and having laccase activity equivalent to that of the polypeptide shown in (a) above in a pH range including the alkaline range. The following describes in detail the laccase composed of polypeptides (a) to (c).

[0016] Sequence ID 1 is the amino acid sequence of laccase derived from Chrysocorona lucknowensis.

[0017] Polypeptides (b) and (c) are sequence-similar laccases that use the amino acid sequence of polypeptide (a) as their basic backbone.

[0018] The amino acid modifications introduced into the polypeptide of (b) above may include only one type of modification (e.g., substitution) from among substitution, addition, insertion, and deletion, or they may include two or more types of modifications (e.g., substitution and insertion). In the polypeptide of (b) above, there may be one, more, or several amino acids that are substituted, added, inserted, or deleted, for example, 1 to 10, preferably 1 to 8, 1 to 6, 1 to 5, or 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2 or 1.

[0019] Furthermore, in the polypeptide of (c) above, sequence identity should be 87% or more, but preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, even more preferably 99.5% or more, and particularly preferably 99.8% or more.

[0020] Here, in the polypeptide of (c) above, the sequence identity with respect to the amino acid sequence shown in, for example, SEQ ID NO: 1 is the sequence identity calculated by comparing it with the amino acid sequence shown in SEQ ID NO: 1. Furthermore, "sequence identity" refers to the value of amino acid sequence identity obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999) of BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0021] In the polypeptides of (b) and (c) above, the amino acids at positions 136, 138, 181, 183, 474, 477, 479, 545, 546, 547, and 551 in the amino acid sequence shown in SEQ ID NO: 1 are thought to contribute to activity, therefore it is desirable not to introduce substitutions or deletions at these sites.

[0022] When amino acid substitutions are introduced into the polypeptides of (b) and (c) above, one type of amino acid substitution is a conservative substitution. Specifically, in the polypeptides of (b) and (c), examples of amino acid substitutions introduced into the amino acid sequence shown in SEQ ID NO: 1 include substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution with another noncharged amino acid if the amino acid before substitution is an uncharged amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0023] When amino acid addition is introduced to the polypeptides of (b) and (c) above, examples of amino acid addition include the addition of a methionine residue to the N-terminus and the addition of a purification tag (e.g., a binding oligopeptide such as oligohistidine).

[0024] The polypeptides in (b) and (c) above include not only polypeptides obtained by artificial mutation, but also polypeptides resulting from naturally occurring mutations (mutants or variants) based on individual differences or species differences in the organism from which the polypeptide originates.

[0025] With respect to the polypeptides in (b) and (c) above, "alkaline range" refers to a range where the pH is greater than 8.5, preferably pH 9 or higher.

[0026] Furthermore, in this invention, "laccase activity" refers to the activity that catalyzes the reaction that produces cross-linked proteins when a protein is used as a substrate (i.e., protein cross-linking activity). Specifically, protein cross-linking activity is measured as follows.

[0027] The protein material is suspended and mixed in an alkaline pH buffer to a concentration of 15% (w / v), and the supernatant is obtained as a protein solution by centrifugation. 100 μl of this protein solution is mixed with 97 μl of alkaline pH buffer and 3 μl of 200 mM DL-catechin (as a mediator) to obtain a substrate solution. 20 μl of this substrate solution is mixed with 10 μl of enzyme (used so that the laccase concentration is 1.7 μg / ml in protein concentration), and the reaction is carried out at 37°C for 18 hours. The reaction solution is then 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 (i.e., protein polymerized by cross-linking, which appears as a band at the origin) in the SDS-PAGE.

[0028] Furthermore, "having laccase activity equivalent to that of the polypeptide shown in (a)" means that, when a protein is used as a substrate, the amount of cross-linked protein produced by the action of polypeptides (b) and (c) is 80-120% of the amount of cross-linked protein produced by polypeptide (a).

[0029] 2. DNA The 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.

[0030] 2-1. DNA Design Those skilled in the art can appropriately design the base sequence of the DNA encoding the laccase of the present invention according to the amino acid sequence described in the laccase of the present invention.

[0031] Specifically, examples of DNA encoding the laccase of the present invention include any of the following DNAs (i) to (iii).

[0032] (i) DNA consisting of the nucleotide sequence shown in Sequence ID No. 2; (ii) DNA consisting of a DNA sequence that hybridizes under stringent conditions with DNA consisting of a DNA sequence complementary to the DNA sequence shown in Sequence ID No. 2; (iii) DNA consisting of a nucleotide sequence having 87% or more homology to the nucleotide sequence shown in Sequence ID No. 2. The following details the DNA described in (i) to (iii).

[0033] The nucleotide sequence shown in Sequence ID No. 2 is the nucleotide sequence that encodes the amino acid sequence shown in Sequence ID No. 1.

[0034] DNA (ii) and (iii) are sequence-similar DNAs that use the base sequence of DNA (i) as their basic backbone.

[0035] In the DNA described in (ii) above, "stringent conditions" refers to conditions in which 6×SSC (1×SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing 0.5% SDS, 5×Denhartz's [Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficol 400] and 100 μg / ml salmon sperm DNA is incubated at 50°C to 65°C for 4 hours to overnight.

[0036] Hybridization under stringent conditions is specifically performed by the following method: A nylon membrane immobilizing a DNA library or cDNA library is prepared, and the nylon membrane is blocked at 65°C in a pre-hybridization solution containing 6×SSC, 0.5% SDS, 5×Denharts, and 100 μg / ml salmon sperm DNA. Then, 32Each probe labeled with P is added and incubated overnight at 65°C. After washing this nylon membrane in 6×SSC for 10 minutes at room temperature, in 2×SSC containing 0.1% SDS for 10 minutes at room temperature, and in 0.2×SSC containing 0.1% SDS for 30 minutes at 45°C, autoradiography can be performed to detect DNA that specifically hybridizes with the probe.

[0037] In the DNA of (iii) above, the homology should be 87% or more, but preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, even more preferably 99% or more, even more preferably 99.5% or more, and particularly preferably 99.8% or more.

[0038] Here, "homology" of the base sequence refers to the identity value obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol.174, 247-250, 1999) from BLAST PACKAGE [sgi32 bitedition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters should be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

[0039] In the DNA of (ii) and (iii) above, at least one of the base sequences encoding a purification tag (for example, a binding oligopeptide such as oligohistidine) may be further added to the DNA of (i) above.

[0040] 2-2. DNA preparation The DNA of the present invention can be obtained, for example, by using DNA encoding any of the polypeptides (a) to (c) above as a template and obtaining at least the region encoding any of the polypeptides (a) to (c) above by PCR or the like. Furthermore, the DNA encoding the laccase of the present invention can also be artificially synthesized by gene synthesis methods.

[0041] Furthermore, when introducing specific mutations to specific sites in a base sequence, the methods for introducing mutations are publicly known, and for example, site-directed mutagenesis of DNA can be used. As for specific methods for converting bases in DNA, for example, commercially available kits can be used.

[0042] DNA into which mutations have been introduced into the base sequence can be sequenced using a DNA sequencer. Once the base sequence is determined, the DNA encoding the laccase can then be obtained by chemical synthesis, PCR using a cloned probe as a template, or hybridization using a DNA fragment containing the sequence as a probe.

[0043] Furthermore, mutant versions of the DNA encoding the laccase that have the same function as the pre-mutation DNA can be synthesized by site-directed mutagenesis or the like. The mutation in the DNA encoding the laccase can be introduced using known methods such as the Kunkel method, gapped duplex method, or megaprimer PCR method.

[0044] The DNA of the present invention is preferably one whose codon usage frequency is optimized for the host. For example, if E. coli is used as the host, DNA with codon usage frequency optimized for E. coli is preferable.

[0045] 3. Expression cassette or recombinant vector An 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") contains DNA encoding the laccase of the present invention. The expression cassette or recombinant vector of the present invention can be obtained by linking a promoter and a terminator to the DNA of the present invention, or by inserting the expression cassette or DNA of the present invention into an expression vector.

[0046] The expression cassette or recombinant vector of the present invention may, as regulatory factors, include a promoter and a terminator, and optionally further include transcription elements such as enhancers, CCAAT boxes, TATA boxes, and SPI sites. These regulatory factors only need to be operably ligated to the DNA of the present invention. Operable ligation means that the various regulatory factors that regulate the DNA of the present invention and the DNA of the present invention are ligated in a manner that allows them to function in a host cell. Furthermore, the expression cassette or recombinant vector of the present invention may be configured to further include a protease recognition sequence and an N-terminal sequence and / or a C-terminal sequence.

[0047] Preferred expression vectors are those constructed for genetic recombination from phages, plasmids, or viruses that can autonomously proliferate within a host. Such expression vectors are well known, and those skilled in the art can appropriately select and use suitable combinations with host cells. For example, when using microorganisms as hosts, examples include pBluescript (pBS) II SK(-) (Stratagene), pSTV vectors (Takara Bio), pUC vectors (Takara Bio), pET vectors (Merck), pGEX vectors (GE Healthcare), pCold vectors (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Mckenzie, T. et al., 1986, Plasmid 15(2), pp. 93-103), pBR322 (Takara Bio), pRS403 (Stratagene), and pMW218 / 219 (Nippon Gene). Examples of vectors used with algae or microalgae as hosts include pUC19 (Takara Bio Inc.), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 (see Yangmin Gong, et al., Journal of Basic Microbiology, 2011, vol. 51, pp. 666-672), or pJET1 (Cosmo Bio Inc.). Examples of vectors used with plant cells as hosts include pRI vectors (Takara Bio Inc.), pBI vectors (Clontech Inc.), and IN3 vectors (Implanta Innovations Inc.).

[0048] 4. Transformed organism A transformant (hereinafter sometimes referred to as "the transformant of the present invention") can be obtained by transforming a host using the expression cassette or recombinant vector of the present invention.

[0049] The host used to produce the transformant is not particularly limited as long as it is capable of gene introduction, autonomous replication, and expression of the gene traits of the present invention. Suitable examples include bacteria belonging to the Escherichia genus such as Escherichia coli, the Bacillus genus such as Bacillus subtilis, the Pseudomonas genus such as Pseudomonas putida, actinomycetes, yeasts, filamentous fungi, etc. Other examples include animal cells, insect cells, plant cells, etc. Among these, Escherichia coli is particularly preferred.

[0050] The host used for the production of the transformant may be Chrysocorona lucknowensis, which is the bacterium or cell from which the laccase of the present invention originates.

[0051] 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 site on which the DNA of the present invention is introduced is not particularly limited as long as the target gene can be expressed, and may be on a plasmid or on the genome. Specific methods for introducing the expression cassette or recombinant vector of the present invention include, for example, recombinant vectoring and genome editing.

[0052] The conditions for introducing the expression cassette or recombinant vector of the present invention into a host can be appropriately set according to the type of host, etc. If the host is a microorganism, examples include methods using competent cells treated with calcium ions, electroporation, spheroplast, and lithium acetate. If the host is an animal cell, examples include electroporation, calcium phosphate, and lipofection. If the host is an insect cell, examples include calcium phosphate, lipofection, and electroporation. If the host is a plant cell, examples include electroporation, Agrobacterium, particle gun, and PEG.

[0053] 5. Method for producing laccase The laccase of the present invention can be produced by culturing the transformant of the present invention. Alternatively, the laccase of the present invention can also be produced by culturing Chrysocorona lucknowensis itself (untransformed).

[0054] The culture conditions for the transformant, derived bacteria, or derived cells of the present invention may be set appropriately considering the nutritional and physiological properties of the host, derived bacteria, or derived cells, but liquid culture is preferred. Furthermore, in the case of industrial production, aerated and stirred culture is preferred.

[0055] The transformed organism or derived bacteria or cells of the present invention are cultured, and the culture supernatant, cultured cells, or cultured cells are recovered from the culture medium by methods such as centrifugation. If the laccase of the present invention is accumulated in the cultured cells or within the cultured bacteria, the cells or bacteria are treated with mechanical methods such as ultrasound or French press, or with lytic enzymes such as lysozyme, and if necessary, solubilized by using enzymes such as proteases or surfactants such as sodium dodecyl sulfate (SDS) to obtain a water-soluble fraction containing the laccase of the present invention.

[0056] Furthermore, by selecting an appropriate expression vector and host, the expressed laccase of the present invention may be secreted into the culture medium.

[0057] The culture medium, water-soluble fraction, or protease-treated product containing the laccase of the present invention obtained as described above may be subjected to purification treatment as is, or the laccase of the present invention in the culture medium, water-soluble fraction, or protease-treated product may be concentrated before being subjected to purification treatment.

[0058] Concentration can be carried out, for example, by vacuum concentration, membrane concentration, salting-out treatment, or fractional precipitation using hydrophilic organic solvents (e.g., methanol, ethanol, and acetone).

[0059] The purification process of laccase according to the present invention can be carried out by appropriately combining methods such as gel filtration, hydrophobic chromatography, ion exchange chromatography, and affinity chromatography.

[0060] The laccase of the present invention, purified in this manner, may be powdered by freeze-drying, vacuum drying, spray drying, or the like, if necessary.

[0061] 6. Enzyme preparations The laccase of the present invention can be provided in the form of an enzyme preparation. Therefore, the present invention also provides an enzyme preparation containing the laccase of the present invention as described above.

[0062] 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 laccase activity is exhibited.

[0063] The enzyme preparation of the present invention may contain other components in addition to the laccase of the present invention, to the extent that they do not affect the effects of the present invention. Examples of other components include other enzymes other than the laccase of the present invention, mediators, additives, and culture residues generated by the above manufacturing method.

[0064] Other enzymes can be appropriately selected depending on their intended use, but examples include amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), galactosidase (α-galactosidase, β-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 deamide enzyme, pullulanase, etc. These other enzymes may be included individually or in combination.

[0065] Examples of mediators include 3-(3,4-dihydroxyphenyl)alanine (DOPA), catechins, and caffeine. acid These are some examples. These mediators may be included individually or in combination of multiple types. Among these mediators, DOPA and catechins are preferred.

[0066] The additives can be appropriately determined depending on the intended use of the laccase of the present invention and the formulation of the enzyme preparation, but examples include excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, and physiological saline. Examples of excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, and pectin. Examples of buffers include phosphates, citrates, and acetates. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of antiseptics include ethanol, benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. These additives may be included individually or in combination of multiple types.

[0067] Culture residues include components derived from the culture medium, contaminating proteins, bacterial components, and cellular components.

[0068] The formulation form of the enzyme preparation of the present invention is not particularly limited and can be liquid, solid (powder, granules, etc.), etc. Enzyme preparations in these formulation forms can be prepared by generally known methods.

[0069] Specifically, the enzyme preparation of the present invention can be used as a protein crosslinking agent, and can also be used as an oxidative modifier for food and beverages or food and beverage ingredients, industrial materials or industrial waste components, or pharmaceuticals or materials for scientific analysis. The targets of oxidative modification and the details of the oxidative modification are described below in "7. Use of Lacase".

[0070] 7. Use of laccase The laccase of the present invention can be used in any application that utilizes the oxidation reaction of a substrate and / or the various incidental chemical reactions resulting from the radical species of reaction intermediates produced by the oxidation reaction. Examples of such oxidation reactions or incidental chemical reactions include the oxidation of phenolic compounds such as o-,p-diphenols, urushiol, and laccol; the oxidation of aromatic amines such as p-phenylenediamine; the decomposition of lignin; and the crosslinking of proteins having easily oxidizable functional groups such as tyrosine side chains (phenolic hydroxyl groups), cysteine ​​side chains (sulfhydryl groups), lysine side chains (ε-amino groups), and histidine side chains (imidazole groups). In this specification, the chemical modification of a substrate by such oxidation reactions and incidental chemical reactions of laccase is also referred to as "oxidative modification."

[0071] 7-1. Method for producing cross-linked proteins Among the above embodiments of oxidative modification, a particularly preferred method is modification by crosslinking of proteins. Accordingly, the present invention also provides a method for producing crosslinked proteins, which includes the step of reacting a protein with the laccase of the present invention described above.

[0072] In the method for producing crosslinked proteins of the present invention, from the viewpoint of further improving reaction efficiency, 3-(3,4-dihydroxyphenyl)alanine (DOPA), catechin, and caffeine are added in the step. acid It is preferable to use mediators such as these in combination with laccase. These mediators may be included individually or in combination of multiple types. Among these mediators, DOPA and catechins are particularly preferred.

[0073] In the method for producing cross-linked proteins of the present invention, the reaction conditions between laccase and protein (e.g., enzyme amount, reaction time, reaction pH, reaction temperature, etc.) are not particularly limited as long as the desired degree of protein cross-linking is achieved, and can be appropriately set by those skilled in the art depending on the type and / or concentration of laccase of the present invention, the type and / or concentration of protein, and / or the desired degree of protein cross-linking.

[0074] The laccase used in the method for producing cross-linked proteins of the present invention exhibits excellent laccase activity at least in the alkaline range; therefore, the reaction pH is preferably greater than 8.5, more preferably 9 or greater. The upper limit of the reaction pH is, for example, 10.5 or less, preferably 10 or less, more preferably 9.5 or less, and even more preferably 9.3 or less.

[0075] Furthermore, the laccase used in the method for producing cross-linked proteins of the present invention preferably exhibits excellent laccase activity not only in the alkaline range but also in the neutral range, so the reaction pH may be, for example, 5 or higher, 6 or higher, 7 or higher, or 8 or higher. The upper limit of the reaction pH can be, for example, 10.5 or lower, preferably 10 or lower, more preferably 9.5 or lower, and even more preferably 9.3 or lower.

[0076] Furthermore, preferred reaction temperatures include 4 to 55°C, more preferably 20 to 50°C.

[0077] 7-2. Method for producing oxidatively modified food and beverages or food and beverage ingredients, industrial ingredients or industrial waste components, or pharmaceutical or scientific analysis ingredients. Examples of materials to be subjected to the above-mentioned oxidation modification include food and beverages or food and beverage ingredients, industrial materials or industrial waste components, or pharmaceuticals or materials for scientific analysis. Accordingly, the present invention further provides a method for producing oxidation-modified food and beverages or food and beverage ingredients, industrial materials or industrial waste components, or pharmaceuticals or materials for scientific analysis, which includes the step of reacting the above-mentioned laccase of the present invention with food and beverages or food and beverage ingredients, industrial materials or industrial waste components, or pharmaceuticals or materials for scientific analysis.

[0078] Examples of oxidative modification of food and beverages or food and beverage materials include protein cross-linking, improvement of the binding properties of textured plant proteins, thickening or gelling of food, browning treatment of black tea, and removal of bitterness and astringency from food. Examples of oxidative modification of industrial materials or industrial waste components include the manufacture of artificial lacquer, removal of lignin from pulp, detoxification of wastewater containing highly toxic phenolic compounds and / or aromatic amines, synthesis of organic compounds, manufacture of adhesives, and synthesis of concrete admixtures. Examples of oxidative modification of pharmaceutical or chemical analysis materials include conversion of target components into sensable components and cross-linking of analyte proteins.

[0079] In the present invention's method for producing oxidatively modified food and beverages or food and beverage materials, industrial materials or industrial waste components, or pharmaceutical or chemical analysis materials, it is preferable to use a mediator in combination with laccase in the process from the viewpoint of further improving reaction efficiency. Examples of mediators that can be used are as described in "7-1. Method for producing cross-linked proteins" above.

[0080] In the method for producing oxidatively modified food and beverages or food and beverage ingredients, industrial materials or industrial waste components, or pharmaceutical or chemical analysis materials according to the present invention, the reaction conditions (e.g., enzyme amount, reaction time, reaction pH, reaction temperature, etc.) between laccase and the object to be oxidatively modified (i.e., food and beverages or food and beverage ingredients, industrial materials or industrial waste components, or pharmaceutical or chemical analysis materials) are not particularly limited as long as the desired degree of oxidative modification is achieved, and can be appropriately set by those skilled in the art depending on the type and / or concentration of laccase of the present invention, the type and / or concentration of the object to be oxidatively modified, and / or the desired degree of oxidative modification. Furthermore, preferred examples of reaction pH and reaction temperature are as described in "7-1. Method for producing cross-linked proteins" above. [Examples]

[0081] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0082] Test Example 1: Culture and Enzyme Purification (1-1) Culture and filtration A 1 cm square section of a Chrysocorona lucknowensis strain 5537 (NBRC 9681) colony, grown on potato dextrose agar at 30°C for 3 days, was inoculated into a 500 ml shaking flask containing 100 ml of the laccase production medium shown in Table 1. The culture was then incubated at 30°C for 3 days with reciprocating shaking (140 r / min). After separating the solid and liquid from the culture medium by centrifugation, the supernatant was filtered off, and the collected filtrate was used as the crude enzyme solution.

[0083] [Table 1]

[0084] (1-2) Primary purification (anion chromatography) The crude enzyme solution was concentrated by ultrafiltration and dialyzed against 20 mM sodium phosphate buffer (pH 7.0) + 0.1 M NaCl to obtain the dialysate. This dialysate was subjected to a HiTrap Q Fast Flow (ID × L = 1.6 × 2.5 cm; Cytiva) column equilibrated with the above buffer. After washing the column with 50 ml of the same buffer, the laccase adsorbed on the column was eluted using a linear gradient of NaCl concentration (0.1~0.3 M, 50 ml) and fractionated. Laccase activity (oxidation of ABTS and protein crosslinking) was confirmed in a portion of each obtained fraction using the method described below, and the fraction exhibiting both activities was collected to obtain the primary purified solution.

[0085] (1-3) Secondary purification (gel filtration chromatography) The primary purified solution was concentrated by ultrafiltration and then dialyzed against 20 mM sodium phosphate buffer (pH 7.0) + 0.5 M NaCl to obtain the dialysate. This dialysate was subjected to a HiLoad 16 / 600 Superdex 200 pg (ID × L = 1.6 × 60 cm; Cytiva), and the active fraction was combined and dialyzed against 20 mM sodium phosphate buffer (pH 7.0) to obtain the purified laccase solution. Purification was confirmed by SDS-PAGE and Native-PAGE.

[0086] <Confirmation of laccase activity - Oxidation of ABTS> A 50 mM solution of ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) as a substrate, 160 μl of 125 mM sodium phosphate buffer (pH 7.0), and 20 μl of enzyme solution were added to a microplate and mixed. The reaction was carried out 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.

[0087] <Confirmation of laccase activity - protein cross-linking> The following procedures were performed on each of the three plant protein powders listed in Table 2 below. The plant protein powder was suspended in 50 mM sodium phosphate buffer (pH 7.0) to a concentration of 15% (w / v) and thoroughly mixed. Solid-liquid separation was then performed by centrifugation at 12,000 × g for 5 minutes, and the supernatant was obtained as the protein solution. 100 μl of this protein solution was mixed with 97 μl of 50 mM sodium phosphate buffer (pH 7.0) and 3 μl of 200 mM DL-catechin (mediator) to obtain the substrate solution. 20 μl of this substrate solution was mixed with 10 μl of enzyme (used so that the laccase concentration was 1.7 μg / ml, which can be quantified using a DC protein assay (Bio-Rad)), and the mixture was reacted at 37°C for 18 hours. Subsequently, the reaction solution was diluted 10-fold, and the application amounts shown in Table 2 were subjected to SDS-PAGE. Protein cross-linking activity was confirmed by the presence of high-molecular-weight proteins (proteins that have been polymerized by cross-linking, appearing as bands at the origin) in SDS-PAGE.

[0088] Test Example 2: Enzyme Property Evaluation (2-1) Evaluation of laccase activity (protein crosslinking activity) The protein cross-linking activity of purified laccase obtained from Chrysocorona lucknowensis strain 5537 and a comparative laccase, "Laccase Y120" (also referred to as LC-Y120) manufactured by Amano Enzyme Co., Ltd., was evaluated for each of the three plant proteins shown in Table 2 at predetermined pH values ​​(pH 3.0, 5.0, 7.0, and 9.0).

[0089] The following procedures were performed on each of the three plant protein powders listed in Table 2. The plant protein powder was suspended in a buffer of a predetermined pH [50 mM sodium citrate buffer (pH 3.0 or 5.0), 50 mM sodium phosphate buffer (pH 7.0), or 50 mM Tris-HCl buffer (pH 9.0)] to a concentration of 15% (w / v), mixed thoroughly, and then separated into solid and liquid by centrifugation at 12,000 × g for 5 minutes, and the supernatant was obtained as the protein solution. 100 μl of this protein solution was mixed with 97 μl of the buffer of the predetermined pH and 3 μl of 200 mM DL-catechin (mediator) to obtain a substrate solution. 20 μl of this substrate solution was mixed with 10 μl of enzyme (used so that the laccase concentration was 1.7 μg / ml, as quantified using a DC protein assay (Bio-Rad)), and the mixture was reacted at 37°C for 18 hours. Subsequently, the reaction solution was diluted 10-fold, and the applied amounts shown in Table 2 were subjected to SDS-PAGE. Protein cross-linking activity was visually evaluated by assessing the amount of cross-linked protein (protein polymerized by cross-linking, appearing as a band at the origin) in SDS-PAGE, using the amount obtained using a comparative laccase (LC-Y120) as a baseline. If the presence of the cross-linked protein was confirmed, the amount of cross-linked protein was evaluated by the number of "+" signs. A higher number of "+" signs indicates a greater amount of cross-linked protein. The results are shown in Figures 1-3. Note that the amount of the cross-linked protein can also be quantified using image analysis software such as imageJ.

[0090] [Table 2]

[0091] In Figures 1-3, the leftmost lane (lane 1 in Figures 1 and 2, and the "Blanc lane" lane in Figure 3) shows the results of the control group without enzyme addition; the middle lane (lane 2 in Figures 1 and 2, and the "LCY120" lane in Figure 3) shows the results when using a comparative laccase; and the rightmost lane (lane 3 in Figures 1 and 2, and the "5537" lane in Figure 3) shows the results when using purified laccase obtained from Chrysocorona lucknowensis strain 5537.

[0092] As is clear from Figures 1-3, when using the comparative laccase LC-Y120 (lane 2 in Figures 1 and 2, and the "LCY120" lane in Figure 3) for any plant protein, almost no protein cross-linking activity was observed in the alkaline range (pH 9). In contrast, when using the laccase obtained from Chrysocorona lucknowensis strain 5537 (lane 3 in Figures 1 and 2, and the "5537" lane in Figure 3), excellent protein cross-linking activity was observed in the alkaline range (pH 9). Furthermore, it was found that the laccase obtained from Chrysocorona lucknowensis strain 5537 exhibits excellent protein cross-linking activity not only in the alkaline range but also in the pH range of 5-7, demonstrating its high versatility.

[0093] (2-2) Evaluation of the protein cross-linking effect by mediators The protein cross-linking effect of various mediators was evaluated for laccase obtained from Chrysocorona lucknowensis strain 5537, except that only 50 mM Tris-HCl buffer (pH 9.0) was used, the mediators were changed to those listed in Table 3 (however, used to achieve a concentration of 100 mM), the laccase concentration was set to 33 μg / ml in terms of protein concentration, and the reaction time was set to 1 hour, by performing the same procedure as described in "(1-2) Method for Evaluating Laccase Activity (Protein Cross-linking Activity)" above. The results are shown in Figure 4. In Figure 4, the numbers in each lane correspond to the numbers in Table 3.

[0094] [Table 3]

[0095] As is clear from Figure 4, L-DOPA, DL-catechin, and caffeic acid were found to have a protein cross-linking-promoting effect. Among these mediators, L-DOPA and DL-catechin (especially L-DOPA) showed a particularly significant protein cross-linking effect.

[0096] (2-3)pH stability Purified laccase obtained from Chrysocorona lucknowensis strain 5537 and comparative laccase LC-Y120 were dissolved in 50 mM Britton-Robinson buffer (pH 2-12) with different pH values, and the resulting enzyme solutions were pH-treated by incubation at 4°C for 1 hour. For each enzyme solution, the same procedure as in (1-3) "<Confirmation of Laccase Activity - Oxidation of ABTS>" ​​above was performed. Enzyme activity values ​​were measured, with one unit (U) defined as the enzyme activity that catalyzes the oxidation of 1 μmol of ABTS per minute.

[0097] The relative value of the enzyme activity after pH treatment, with the enzyme activity value at the treatment pH showing the highest residual activity set to 100%, was derived as residual activity (%). The results are shown in Figure 5. As is clear from Figure 5, the laccase obtained from Chrysocorona lucknowensis strain 5537 showed significantly improved stability in the alkaline range compared to the commercially available laccase LC-Y120.

[0098] (2-4) Temperature stability Purified laccase obtained from Chrysocorona lucknowensis strain 5537 and comparative laccase LC-Y120 were each dissolved in 50 mM Britton-Robinson buffer (pH 7.0). The resulting enzyme solutions were then incubated at 4°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, or 80°C for 1 hour. The enzyme activity of each enzyme solution before and after temperature treatment was measured using the same method as described in (2-3) above. The relative value of the enzyme activity after temperature treatment, with the enzyme activity before temperature treatment set to 100%, was derived as the residual activity (%). The results are shown in Figure 6. As is clear from Figure 6, the laccase obtained from Chrysocorona lucknowensis strain 5537 showed high stability below 50°C. Furthermore, from the results in (2-3) above, since the stability of this enzyme is almost the same at pH 7 and pH 9, it can be inferred that the temperature stability at pH 9 is similar to that shown above and in Figure 6.

[0099] Test Example 3: Sequence Identification Laccase obtained from Chrysocorona lucknowensis strain 5537 was sequenced. As a result, the gene sequence shown in SEQ ID NO: 3 (2392 bp), the coding region sequence shown in SEQ ID NO: 2 (1845 bp), and the amino acid sequence shown in SEQ ID NO: 1 (614 aa) were determined.

[0100] Test Example 4: Improving the binding properties of tissue-like plant proteins Granular soy protein (manufactured by Marukome Co., Ltd.) was mixed with five times its weight in warm water (40°C) and allowed to stand for 10 minutes to swell. After removing the water, the swollen granular soy protein was weighed in 25g portions. 2.75g of powdered pea protein (NUTRALYS F85M, manufactured by Rocket Co., Ltd.) was mixed with the weighed swollen granular soy protein, and 125U (5U per 1g of swollen granular soy protein) of purified laccase obtained from Chrysocorona lucknowensis strain 5537 or comparative laccase LC-Y120 was added to prepare a soy protein mixture. The soy protein mixture was thoroughly mixed and formed into hamburger patties, which were left 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.

[0101] Figure 7 shows a photograph of the appearance of the obtained meat-like processed food. For comparison, Figure 7 also shows a meat-like processed food obtained in the same manner except that laccase was not used (no enzyme treatment). LC-Y120 can perform oxidative modification that improves the binding properties of textured plant proteins. As shown in Figure 7, no binding occurred at all without enzyme treatment, but binding properties were confirmed when treated with LC-Y120 and when treated with laccase derived from strain 5537.

[0102] Furthermore, to evaluate the degree of binding ability of granular soy protein in the obtained meat-like processed food, the hardness of the meat-like processed food was measured using a rheometer (manufactured by Sun Science Co., Ltd.). A higher hardness measurement indicates a greater degree of binding. The results are shown in Figure 8.

[0103] As shown in Figure 8, it was confirmed that the laccase derived from strain 5537 exhibited superior binding properties compared to LC-Y120.

[0104] Furthermore, to evaluate the degree of cooking loss in the resulting meat-like processed food, the weight was measured before and after the baking process, and the cooking loss (%) was derived based on the following formula. A smaller derived value indicates less cooking loss. The results are shown in Figure 9.

[0105]

number

[0106] As shown in Figure 9, the laccase derived from strain 5537 resulted in less cooking loss than LC-Y120, confirming its usefulness in the production of meat-like processed foods.

Claims

1. Lacase consisting of one of the polypeptides shown in (a) to (c) below: (a) A polypeptide consisting of the amino acid sequence shown in Sequence ID No. 1; (b) A polypeptide having an amino acid sequence in which 1 to 10 amino acids are substituted, added, inserted, or deleted in the amino acid sequence shown in Sequence ID No. 1, and having laccase activity equivalent to that of the polypeptide shown in (a) above in a pH range including the alkaline range; (c) A polypeptide having an amino acid sequence with 90% or more sequence identity to the amino acid sequence shown in Sequence ID No. 1, and having laccase activity equivalent to that of the polypeptide shown in (a) above in a pH range including the alkaline range.

2. DNA encoding the laccase described in claim 1.

3. An expression cassette or recombinant vector comprising the DNA described in claim 2.

4. A transformant obtained by transforming a host using the expression cassette or recombinant vector described in claim 3.

5. A method for producing laccase, comprising the step of culturing the transformant described in claim 4.

6. An enzyme preparation comprising laccase as described in claim 1.

7. An enzyme preparation according to claim 6, which is used as a protein crosslinking agent.

8. The enzyme preparation according to claim 6, which is used as an oxidative modifier for food and beverages or food and beverage ingredients, industrial materials or industrial waste components, or pharmaceuticals or materials for scientific analysis.

9. A method for producing a cross-linked protein, comprising the step of reacting a protein with the laccase described in claim 1.

10. The manufacturing method according to claim 9, wherein the above step is carried out under alkaline conditions.

11. The manufacturing method according to claim 9, wherein in the above step, a mediator selected from the group consisting of 3-(3,4-dihydroxyphenyl)alanine, catechin, and caffeic acid is used in combination with the laccase.

12. A method for producing oxidatively modified food or beverages, food or beverage materials, industrial materials, industrial waste components, or pharmaceutical or scientific analysis materials, comprising the step of reacting food or beverages or food or beverage materials, industrial materials, or industrial waste components, or pharmaceutical or scientific analysis materials, with the laccase described in claim 1.

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