JP4535812B2 - Phenol oxidase gene and method for producing phenol oxidase - Google Patents

Description

  The present invention relates to a nucleic acid encoding phenol oxidase, a recombinant phenol oxidase, an expression vector, a transformant, a method for producing a recombinant phenol oxidase, and an antibody against the recombinant phenol oxidase. More specifically, the present invention relates to a nucleic acid encoding a phenol oxidase exhibiting high enzyme activity near neutrality, a recombinant phenol oxidase, an expression vector, a transformant, a method for producing a recombinant phenol oxidase, and an antibody against the recombinant phenol oxidase.

  Phenol oxidase is an oxidase having an oxidizing action on various substrates such as phenol compounds and polyphenol compounds. Since the phenol oxidase can catalyze the oxidation of various substrates in the presence of oxygen in the atmosphere, coloration, decolorization, polymerization, decomposition, etc. caused by the generation of radical species as intermediates in the presence of oxygen. Suitable for various chemical reactions.

  Examples of the phenol oxidase include phenol oxidase, laccase, tyrosinase, polyphenol oxidase (catechol oxidase), ascorbate oxidase, bilirubin oxidase and the like.

  Therefore, the phenol oxidase is clinical analysis, biosensor, pulp and fiber bleaching, synthetic board production, wood quality improvement, phenol resin production, artificial lacquer coating production, adhesive production, organic compound synthesis, dyeing And discharge, prevention of color transfer during washing, removal of phenol and aniline compounds in the waste liquid, detoxification of toxic compounds, decomposition of endocrine disrupting substances, prevention of turbidity of juice, removal of bitter taste of food, promotion of in vivo digestion of livestock feed In addition, it is expected to have a wide range of applications such as the control of malodor, desulfurization of fossil fuels, and decomposition treatment of harmful environmental substances.

The phenol oxidase, various plants traditionally have been found in bacteria and fungi such as, e.g., Schizophyllum commune (Schizophyllum commune), oyster mushroom (Pleurotus octreatus), Coriolus versicolor (Coriolus versicolor), shiitake mushroom (Lentinula edodes), Irupekkusu-Rakuteusu (Irpex lacteus), Wang Li lenticular rear Poritorika (Auricularia polytricha), Ganoderumu-lucidum (Ganoderm lucidum), Coprinus Mikaseusu (Coprinus micaceus), Daedareopushisu-Suchirashina (Daedaleopsis styracina), Coriolus Konsorusu (Co Iolus consors), the basidiomycetes such Furamurina Berutipesu (Flammulina Veltipes), phenol oxidases have been found (for example, refer to Patent Document 1, etc.).

However, the phenol oxidase has an optimum pH of enzyme activity for various substrates on the acidic side, the usage is limited, the optimum pH varies greatly depending on the substrate used, and various substrates near neutrality. However, it does not work efficiently. Moreover, the productivity of the said phenol oxidase in a basidiomycete is low, and there exists a fault that the purity of the produced phenol oxidase is also low. In addition, the phenol oxidase described in Patent Document 1 is an enzyme found by culturing under acidic conditions.
JP 60-156385 A

  The present invention relates to providing means that can be used for dyeing fibers and hair under conditions mild to the environment and the human body. One aspect of the present invention provides a nucleic acid encoding phenol oxidase that can stably supply phenol oxidase exhibiting high enzyme activity in a high yield, inexpensively and in large quantities in the vicinity of neutrality. About that. Another aspect of the present invention is that it acts on various compounds in the vicinity of neutrality, exhibits high stability in a wide range from neutral pH to weakly alkaline pH, and has an optimum pH depending on the substrate. The present invention relates to providing a recombinant phenol oxidase having characteristics such as small fluctuation, low-priced supply in large quantities, and high purity. Still another aspect of the present invention relates to providing an expression vector capable of efficiently and efficiently expressing a large amount of phenol oxidase exhibiting high enzyme activity in the vicinity of neutrality. Further, another aspect of the present invention is to stably and efficiently express a large amount of phenol oxidase exhibiting high enzyme activity in the vicinity of neutrality, and to express the phenol oxidase in a state suitable for recovery, purification, etc. The present invention relates to providing a transformant that enables at least one of causing the above. Still another aspect of the present invention is a recombinant phenol oxidase which can obtain a phenol oxidase exhibiting high enzyme activity stably in the vicinity of neutrality in a high yield, at a low cost, in a large amount and with a high purity. It is related with providing the manufacturing method of. Still another aspect of the present invention relates to an antibody that enables recovery, purification, screening, and the like of phenol oxidase exhibiting a high enzyme activity stably in the vicinity of neutrality. Further problems of the present invention are apparent from the description of the present specification.

That is, the gist of the present invention is as follows.
[1] (a) a base sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2,
(B) In SEQ ID NO: 2, encodes an amino acid sequence having substitution, deletion, addition or insertion of one or several amino acid residues, and the encoded polypeptide exhibits phenol oxidase activity. nucleotide sequence,及 beauty <br/> (c) SEQ ID NO: 2 sequence identity may encode an amino acid sequence that is at least 85%, and the encoded polypeptide shows a phenol oxidase activity Base sequence,
A nucleic acid encoding phenol oxidase, comprising a base sequence selected from the group consisting of:
[2] The nucleic acid according to [1] above, wherein the encoded polypeptide has phenol oxidase activity at least at pH 5.0 to 8.0,
[3] An expression vector comprising the nucleic acid according to [1] or [2],
[4] The expression vector according to [3] above, wherein a nucleic acid encoding a signal sequence is operably linked upstream of the nucleic acid according to [1] or [2].
[5] A transformant comprising the expression vector according to [3] or [4],
[6] The transformant according to [5], which is derived from a yeast cell or a filamentous fungus, and [7] the transformant according to [5] or [6] are cultured, and the recombinant phenol is cultured in the culture. The present invention relates to a method for producing a recombinant phenol oxidase encoded by the nucleic acid according to [1] or [2], wherein oxidase is generated and recovered.

  According to the nucleic acid encoding the phenol oxidase of the present invention, a phenol oxidase exhibiting a high enzyme activity can be stably expressed in the vicinity of neutrality, and can be supplied in a large amount at a low yield with a high yield. Excellent effect. The recombinant phenol oxidase of the present invention acts on various compounds in the vicinity of neutrality, exhibits high stability in a wide range from neutral pH to weakly alkaline pH, and changes in optimum pH depending on the substrate. Excellent effect of small. Furthermore, the recombinant phenol oxidase of the present invention can be provided at low cost, in large quantities, and with high purity. The expression vector of the present invention exhibits an excellent effect that the phenol oxidase can be efficiently expressed in large quantities. Moreover, according to the transformant of the present invention, there is an excellent effect that the phenol oxidase can be efficiently expressed in a large amount in a state suitable for recovery, purification and the like. Furthermore, according to the method for producing a recombinant phenol oxidase of the present invention, the phenol oxidase can be obtained in a high yield, at a low cost, in a large amount and with a high purity. Moreover, according to the antibody of the present invention, the phenol oxidase can be recovered, purified, screened, etc. easily and with excellent sensitivity.

  In one aspect, the present invention is a phenol oxidase that stably exhibits high enzyme activity in the vicinity of neutrality, that is, a nucleic acid encoding neutral phenol oxidase.

  In the present invention, the phenol oxidase activity refers to an activity evaluated by measuring an oxidative activity on a substrate, a decolorizing activity on a dye, and the like.

  The “oxidation activity on a substrate” refers to a direct oxidation reaction of a substrate that reduces oxygen molecules using a phenol compound, aminophenol compound, diamine compound or the like as a hydrogen donor in the presence of the polypeptide to be measured. It can be determined by measuring. Examples of the hydrogen donor include 2,6-dimethoxyphenol, ortho-aminophenol, para-phenylenediamine, and the like. The oxidation activity is measured, for example, by changes in absorbance in syringaldazine (530 nm), 2,6-dimethoxyphenol and para-phenylenediamine (470 nm), and ortho-aminophenol (420 nm). Specifically, 0.1M of 0.05M 2,6-dimethoxyphenol aqueous solution is added to 0.896ml of 0.2M sodium phosphate buffer (pH 7.0) to obtain a substrate solution. This substrate solution and 0.004 ml of a solution containing the polypeptide to be measured are mixed to initiate the oxidation reaction of 2,6-dimethoxyphenol. Then, the oxidation activity can be determined by measuring the oxidation reaction of 2,6-dimethoxyphenol by the change in absorbance at 470 nm. The oxidation activity is defined as 1 unit (U: unit), which is the amount of enzyme that increases the absorbance at a wavelength corresponding to each substrate by 1 per minute.

Specifically, the nucleic acid of the present invention includes (a) a base sequence encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2,
(B) a base which encodes an amino acid sequence having substitution, deletion, addition or insertion of at least one amino acid residue in SEQ ID NO: 2, and the encoded polypeptide exhibits phenol oxidase activity Array,
(C) a nucleotide sequence of a nucleic acid that hybridizes with an antisense strand of a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and the encoded polypeptide exhibits phenol oxidase activity. And (d) a base sequence that encodes an amino acid sequence having a sequence identity with SEQ ID NO: 2 of at least 65%, and wherein the encoded polypeptide exhibits phenol oxidase activity,
A nucleic acid containing a base sequence selected from the group consisting of:

In the (a), SEQ ID NO: 2 amino acid sequence shown in the Furamurina Berutipesu (Flammulina velutipes) the PH6.0～12.0, preferably, PH7.0～11.0, more preferably pH8.0~ It is an amino acid sequence of neutral phenol oxidase which is detected as an enzyme exhibiting high activity at neutral pH by culturing at 10.0, more preferably around pH 9.0.

  The nucleic acid of the present invention shows that the encoded polypeptide has an optimum pH of 5.0 to 8.0, preferably an optimum pH of 5.0 to 7.5, by evaluating the oxidative activity against the substrate. As long as it is a polypeptide, it may be a variant of the nucleic acid. The variant may be a naturally occurring variant or an artificially created variant. Although it does not specifically limit as said variant, For example, the nucleic acid containing the base sequence in any one of said (c)-(d) is mentioned.

  In the above (b), “at least one” means that the encoded polypeptide has an optimal pH of 5.0 to 8.0, preferably by evaluation of oxidation activity against the substrate and / or evaluation of decolorization activity against dye. May be in a range that is a polypeptide having an optimum pH of 5.0 to 7.5, for example, one or more, preferably one or several. The base sequence (b) preferably encodes an amino acid sequence having a sequence identity with SEQ ID NO: 2 of at least 65%.

As used herein, “substitution, deletion, addition or insertion of amino acid residues”
1) In SEQ ID NO: 2, substitution, deletion, addition or insertion (mutation) of amino acid residues unrelated to the expression of phenol oxidase activity and / or the maintenance of the three-dimensional structure for expressing the phenol oxidase activity,
2) In SEQ ID NO: 2, conservative substitution of amino acid residues essential for expression of phenol oxidase activity and / or maintenance of conformation for expressing the phenol oxidase activity,
Etc.

Examples of the conservative substitution include:
-Substitution with an amino acid residue (hereinafter also referred to as a similar amino acid residue) that exhibits a function similar to hydrophobicity, charge, pK, steric features, etc.,
-Substitution with an amino acid residue capable of changing the three-dimensional structure or folding structure of the polypeptide only to such an extent that the physiological activity of the original polypeptide is maintained. More specific examples of the conservative substitution include the following 1. ~ 6. :
1. Glycine, alanine;
2. Valine, isoleucine, leucine;
3. Aspartic acid, glutamic acid, asparagine, glutamine;
4). Serine, threonine;
5. Lysine, arginine;
6). Phenylalanine, tyrosine;
Substitution of amino acid residues within any group of similar amino acid residues.

  In the above (c), “stringent conditions” refers to moderately stringent conditions, preferably highly stringent conditions. More specifically, the “stringent conditions” are, for example, 6 × SSC (composition of 1 × SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0) and 0.5% by weight. Incubate at room temperature, more stringent at 42 ° C., and even more stringent at 60 ° C. for 10 hours in a solution containing SDS, 5 × Denhardt, 100 μg / ml denatured salmon sperm DNA and 50% by volume formamide. Ionic strength, for example, 2 × SSC, more stringent, conditions such as 0.1 × SSC and / or higher temperature, room temperature, 42 ° C. or higher, more stringent, 55 ° C., more stringent, The conditions for performing cleaning under conditions such as 60 ° C. can be mentioned. In the present invention, the “stringent conditions” are preferably such that specific hybridization with a nucleic acid encoding an amino acid sequence having a sequence identity with SEQ ID NO: 2 of at least 65% can be achieved. It is desirable that the conditions are satisfied.

  In this specification, sequence identity is calculated by appropriately aligning a query sequence (evaluation target sequence) with a BLAST algorithm against a reference sequence (for example, the amino acid sequence shown in SEQ ID NO: 2). sell. In the present specification, examples of setting conditions in the multiple alignment include conditions (cost to open gap 11, Cost to extended gap 1, expect value 10, and wordsize 3) (default values).

  In addition, the sequence identity in this specification is the default algorithm other than the BLAST algorithm, for example, an algorithm that can be used in multiple alignment by, for example, sequence analysis software, product name: GENETYX-MAC (Software Development Co., Ltd.). It may be a value calculated using the value condition.

  In the present invention, the sequence identity is at least 65%, preferably 70% or more, more preferably 75% or more, still more preferably 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 2. More preferably, it is desirable that it is 85% or more.

The nucleic acid containing the base sequence (a) is synthesized by, for example, conventional chemical synthesis, a nucleic acid having a base sequence different from that of SEQ ID NO: 1 through the base sequence shown in SEQ ID NO: 1 or degeneracy. PCR, RT-PCR, Southern blot high, using a primer pair and / or probe designed based on a nucleotide sequence different from that of SEQ ID NO: 1 through the nucleotide sequence shown in SEQ ID NO: 1 or degeneracy. hybridization, colony hybridization, by conventional techniques such as plaque hybridization, from Furamurina Berutipesu (Flammulina velutipes) derived from nucleic acid may be obtained such as by acquiring. In addition, it is confirmed that the obtained nucleic acid contains the base sequence of (a) by sequencing by dideoxy method, Maxam-Gilbert method, improved methods based on these methods, etc. Can be evaluated.

The nucleic acid containing the base sequence (a) is not specifically limited, but examples thereof include the following steps (1) to (5):
(1) Furamurina Berutipesu by culturing (Flammulina velutipes) under conditions suitable for expression of the phenol oxidase, the step of increasing the expression level of mRNA encoding a phenol oxidase in cells,
(2) obtaining mRNA from the cells after the step (1),
(3) Designed on the basis of a base sequence different from SEQ ID NO: 1 through oligo dT primer, the base sequence shown in SEQ ID NO: 1 or degeneracy using the mRNA obtained in the step (2) as a template A step of performing RT-PCR using a primer pair, etc.,
(4) linking the product obtained in the step (3) to an appropriate vector, and transforming an appropriate host cell using the obtained vector;
(5) About the transformant obtained in the step (4), SEQ ID NO: 1 or a base sequence different from SEQ ID NO: 1 or a partial sequence thereof is retained through degeneracy to express phenol oxidase activity A step of selecting a transformant and, when a clone containing a partial sequence is obtained, optionally, (6) a step of obtaining the full length of a nucleic acid encoding phenol oxidase by 5′RACE, nested PCR,
Or the like. In addition, after the step (2), (3 ′) a step of preparing a cDNA library from the mRNA obtained in the step (2),
(4 ′) Southern blot hybridization, colony hybridization, plaque hybridization using a probe designed based on the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence different from SEQ ID NO: 1 through degeneracy Screening a cDNA library to obtain a clone containing a nucleic acid encoding phenol oxidase or a part thereof;
(5 ′) The transformant obtained in the step (4 ′) retains a nucleotide sequence different from that of SEQ ID NO: 1 through the nucleotide sequence shown in SEQ ID NO: 1 or degeneracy, and has a phenol oxidase activity. When a clone containing a part of a nucleic acid encoding phenol oxidase is obtained, a phenol oxidase is optionally encoded by (6 ′) 5 ′ RACE, nested PCR or the like. Obtaining the full length of the nucleic acid to be
May be performed.

Incidentally, the aforementioned "conditions suitable for expression of the phenol oxidase", for example, Furamurina Berutipesu (Flammulina velutipes) mycelium small pieces, liquid culture medium 1 {composition of: 2.4 wt% potato dextrose broth [Difiko ( Difco)], remainder water (pH 5.2); sterilized at 121 ° C. for 15 minutes], reciprocally shaken culture (150 reciprocations / minute) at 25 ° C. for 7 days, In a 2 L Erlenmeyer flask, 500 ml of the liquid culture medium 1 was added, and reciprocal shaking culture (100 reciprocations / min) was performed at 25 ° C. for 3 weeks. The obtained mycelium was added to the liquid culture medium 2 [ composition: 1.0 wt% of glucose, 0.1 wt% yeast extract, 0.14 _{wt% (NH 4) 2 SO 4} , 0.36 wt% K ₂ HPO _4, 0.0 _{Wt% MgSO 4 · 7H 2 O,} 0.10 wt% mineral mixture (composition: 1.0 _{wt% CuSO 4 · 5H 2 O,} 1.0 wt% ZnCl _2, 0.7 wt% FeCl ₃ · 6H ₂ O, 0.5 wt% CoSO ₄ .7H ₂ O, 0.5 wt% MnCl ₂ .4H ₂ O), pH 9.2; sterilized at 121 ° C. for 15 minutes] and cultured at 25 ° C. for 3 days Etc. Culture of mycelium under such conditions is advantageous in that the abundance ratio of mRNA corresponding to the phenol oxidase in the cells is improved and the nucleic acid of the present invention can be obtained.

  Each of the primer pairs is desirably at least 9 nucleotides in length, preferably 9 to 40 nucleotides in length, more preferably 12 to 30 nucleotides in length. In addition, the primer pair is based on the base sequence shown in SEQ ID NO: 1 or a base sequence different from SEQ ID NO: 1 through degeneracy, taking into account the formation of secondary structure, Tm value, etc. 2 types having a sequence suitable for amplifying a nucleic acid containing a base sequence different from SEQ ID NO: 1 or a part thereof by means of primer design software or the like through the base sequence shown in SEQ ID NO: 1 or degeneracy Can be designed as a primer.

  Examples of the probe include all or part of the nucleic acid of the present invention or a complementary strand thereof. Examples thereof include a nucleic acid encoding the amino acid sequence of SEQ ID NO: 2, the complementary strand of the nucleic acid, and a part thereof. . The probe is preferably at least 12 nucleotides in length from the viewpoint of sufficiently obtaining specificity with a nucleic acid encoding phenol oxidase. The part of the pre-nucleic acid or complementary strand may be a fragment consisting of a base sequence characteristic of the nucleic acid of the present invention or its complementary strand. The “base sequence characteristic of the nucleic acid of the present invention or its complementary strand” is not particularly limited. For example, the sequence identity with respect to the database sequence is preferably 15% or less, more preferably 10%. % Or less, particularly preferably 0% base sequence.

  The design of the probe or primer pair and conventional genetic manipulation (for example, hybridization, PCR, etc.) can be performed by, for example, Molecular Cloning Laboratory Manual Second Edition [J. Sambrook et al. Reference may be made to EDITION, 1989].

  The variant nucleic acid can be obtained by screening from a living organism in the natural world; artificially introducing a desired mutation into the nucleic acid containing the base sequence (a).

  When screening from a living organism in nature, for example, screening a nucleic acid extracted from an organism to be evaluated by hybridization under the “stringent conditions” using the probe; The presence or absence of an amplification product by PCR using a primer pair, RT-PCR, or the like can be performed. On the other hand, when artificially creating a variant of the nucleic acid, the variant can be prepared by, for example, a conventional site-directed mutagenesis method.

  Since the nucleic acid of the present invention contains any one of the base sequences (a) to (d), it is stably expressed in the vicinity of neutrality when expressed in an appropriate host, and exhibits high enzyme activity. An excellent effect that phenol oxidase can be supplied in a large amount at a high yield at a low cost is exhibited.

  In another aspect, the invention relates to a recombinant phenol oxidase encoded by a nucleic acid of the invention.

Since the recombinant phenol oxidase of the present invention is encoded by the nucleic acid of the present invention,
-Acting near neutral to various compounds-Showing high stability in a wide range from neutral pH to weak alkaline pH-Various substrates, especially phenolic compounds, aminophenolic compounds and diamine compounds For any of these, it exhibits excellent properties such as small fluctuations in the optimum pH. Therefore, according to the recombinant phenol oxidase of the present invention, fibers and hair can be dyed under conditions that are mild to the environment and the human body.

  Moreover, since the recombinant phenol oxidase of the present invention is encoded by the nucleic acid of the present invention and obtained by expressing the nucleic acid, it can be supplied in a large amount at a low cost. Moreover, since the recombinant phenol oxidase of the present invention is encoded by the nucleic acid of the present invention and obtained by expressing the nucleic acid, the recombinant phenol oxidase has an excellent property of high purity. Therefore, it is suitable for uses requiring a strict catalytic reaction, for example, synthesis of compounds.

  The recombinant phenol oxidase of the present invention has a molecular weight of 28 kDa when calculated by SDS-PAGE. The molecular weight is calculated by, for example, subjecting the target protein to SDS-PAGE, measuring mobility, and comparing the obtained measurement value with the mobility of a molecular weight marker having a known molecular weight. sell.

  Moreover, the recombinant phenol oxidase of the present invention has an optimum pH of 5.0 to 8.0, preferably an optimum pH of 5.0 to 7.5. Since the recombinant phenol oxidase of the present invention exhibits excellent enzyme activity in the above pH range, a water-based reaction solution can be used, and an excellent pH adjustment is not required for efficient enzyme reaction. Show the effect. In addition, the recombinant phenol oxidase of the present invention can use a water-based reaction solution. Specifically, for example, the reaction can be efficiently performed even with an aqueous solution of a substrate and an enzyme not subjected to special pH adjustment. it can.

  Furthermore, the recombinant phenol oxidase of the present invention is specifically neutral in the oxidation reaction using a phenol compound, an aminophenol compound and a diamine compound as substrates, specifically pH 5.0 to 8.0, preferably Has an optimum pH between pH 5.0 and 7.5. More specifically, the phenol oxidase of the present invention, when 2,6-dimethoxyphenol is used as a substrate, has an optimum pH of about 5.0 to about 7.5, more preferably about 5 When using orthoaminophenol as a substrate, the optimum pH is about 5.0 to 8.0, and more preferably about 6.0 to 7.5. When paraphenylenediamine is used as a substrate, the optimum pH is about 5.0 to 6.5, more preferably about 5.0 to 5.5.

  The recombinant phenol oxidase of the present invention has an excellent property of exhibiting high pH stability over a wide range from neutral pH to weak alkaline pH. Specifically, the recombinant phenol oxidase of the present invention has a relative residual activity of at least 25%, preferably at pH 7.5-9. 5, at least 50% relative residual activity under incubation conditions at 25 ° C. for 20 hours, more preferably at 8.0 to 9.0, at least 80% relative residual activity under incubation conditions at 25 ° C. for 20 hours. Maintain activity.

  The recombinant phenol oxidase of the present invention exhibits a thermal stability of maintaining at least 60% relative residual activity with respect to the activity before incubation under incubation conditions of 1 hour at pH 8.5 at 40 ° C. That is, since the recombinant phenol oxidase of the present invention exhibits excellent enzyme activity in the above temperature range, it is highly enzyme at daily living temperatures (room temperature, water temperature, body temperature, temperature, etc.) without adjusting to special temperature conditions. Shows activity. Therefore, according to the recombinant phenol oxidase of the present invention, staining, waste liquid treatment, polymer compound synthesis, and the like can be easily performed.

  The recombinant phenol oxidase of the present invention is expressed by, for example, incorporating the nucleic acid of the present invention into an appropriate vector, transforming an appropriate host using the obtained expression vector, and culturing the resulting transformant. It can be made. Such expression vectors and transformants are also included in the present invention.

  In still another aspect, the present invention relates to an expression vector containing the nucleic acid of the present invention.

  Since the expression vector of the present invention contains the nucleic acid of the present invention, a large amount of phenol oxidase exhibiting high enzyme activity can be stably and efficiently expressed in the vicinity of neutrality.

  The vector that can be used as the expression vector of the present invention can be appropriately selected according to the type of host for expressing the recombinant phenol oxidase, the use of the recombinant phenol oxidase, and the like. Examples include vectors and virus vectors.

  Although it does not specifically limit as said vector, For example, pYES2, pYEUra3, pAcSGHisNT-A, pKCR, these derivatives etc. are mentioned. Such a vector may appropriately include an inducible promoter, a selection marker gene, an element such as a terminator, a nucleic acid encoding a secretory signal sequence, and the like. When a vector containing an inducible promoter is used, the nucleic acid of the present invention is operably linked to the promoter.

  Further, from the viewpoint of easily and mass-producing the recombinant phenol oxidase of the present invention, a vector capable of secreting the recombinant phenol oxidase extracellularly; a vector capable of inducing and expressing the recombinant phenol oxidase; Alternatively, a vector that can be expressed as a fusion protein with a conventional fusion partner (His tag or the like) may be used.

  The expression vector of the present invention is preferably a vector in which a nucleic acid encoding a signal sequence is operably linked upstream of the nucleic acid of the present invention from the viewpoint of easily and mass-producing the recombinant phenol oxidase of the present invention.

  In another aspect, the present invention relates to a transformant containing the expression vector of the present invention.

  Since the transformant of the present invention contains the nucleic acid of the present invention, an excellent effect of being able to efficiently and efficiently express a large amount of phenol oxidase exhibiting high enzyme activity in the vicinity of neutrality. To demonstrate,

  According to the transformant of the present invention, an excellent effect that recombinant phenol oxidase can be expressed in a state suitable for recovery, purification and the like is exhibited. Therefore, according to the present invention, recombinant phenol oxidase can be supplied at low cost.

  The transformant of the present invention can be prepared by introducing the expression vector of the present invention into an appropriate host.

  The host is not particularly limited as long as it is suitable for introducing the nucleic acid of the present invention and expressing recombinant phenol oxidase. Examples thereof include bacterial cells, yeast cells, plant cells, and the like. Among these, yeast cells and filamentous fungi are preferable from the viewpoint of sufficiently expressing the properties of phenol oxidase (also referred to as natural phenol oxidase) isolated from the organism that is the source of the nucleic acid of the present invention.

Examples of the yeast cell include Pichia pastoris GS115 strain, Pichia pastoris KM71 strain, Pichia pastoris SMD1168 strain, Saccharomyces cerevisiae and the like.

  As a method for introducing an expression vector into a host, a known introduction method can be used, and examples thereof include a calcium phosphate method, a DEAE-dextran method, and an electroporation method.

From the viewpoint of expressing the recombinant phenol oxidase in a more suitable state for recovery, purification, etc., the transformant of the present invention is preferably upstream of the nucleic acid of the present invention, preferably a signal sequence, specifically a pre-urotus sager. -A vector (for example, pPIC-F1 (FIG. 2) and the like in Examples described later) to which a nucleic acid encoding a secretion signal of pheasant (Pleurotus sajor-caju) is operably linked is transformed into a yeast cell, for example, Pichia pastoris GS115. A transformant obtained by introduction into a strain is desirable.

  In still another aspect, the present invention relates to a method for producing a recombinant phenol oxidase, comprising culturing the transformant of the present invention, generating and recovering a recombinant phenol oxidase in the culture.

  Since the transformant of the present invention is used for the transformant of the present invention, phenol oxidase exhibiting high enzyme activity stably in the vicinity of neutrality, in a high yield, at low cost, in large quantities, It exhibits an excellent effect that it can be obtained with high purity.

  Further, according to the production method of the present invention, the optimum conditions for the expression of the recombinant phenol oxidase are determined with respect to the medium composition, the pH of the medium, the culture temperature, the culture time, the use amount of the inducer, the use time, and the like. By this, recombinant phenol oxidase can be produced more efficiently.

For example, in the case of the Pichia pastoris GS115 strain, the transformant is cultured using the Pichia pastoris GS115 strain in a liquid culture medium 3 {composition: 1% by weight yeast extract (manufactured by Difco)], 2% by weight polypeptone [ [Nippon Pharmaceutical Co., Ltd.] 2% by weight dextrose (manufactured by Nacalai Tesque), remaining water; sterilized at 121 ° C. for 15 minutes} overnight at 30 ° C., reciprocal shaking culture (120 reciprocations / min. ), And the resulting transformant cells were cultured in a liquid culture medium 4 {Composition: 100 mM potassium phosphate buffer (pH 6.0), 1.34% by weight. Product name: yeast nitrogen base (Invitrogen) 4 × 10-5 wt% D-biotin (manufactured by Nacalai Tesque), In a volume% methanol, remaining water; sterilized at 121 ° C. for 15 minutes}, add 0.5% by volume methanol every 24 hours, and incubate at 20 ° C. for 6 days with reciprocal shaking (120 rounds / min) It is desirable.

  Conventional protein purification methods can be used for the purification of recombinant phenol oxidase from transformant cultures.

  Specifically, when the transformant accumulates recombinant phenol oxidase in the cell, the cell is collected by centrifugation after completion of the culture, and the obtained cell is disrupted by sonication or the like. The cell-free extract is obtained by centrifugation, etc., and the cell-free extract is subjected to salting-out, ion exchange (cation exchange or anion exchange) chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc. The protein can be purified by a general protein purification method. In such a transformant, since recombinant phenol oxidase accumulates in the cells, the proportion of recombinant phenol oxidase increases compared to other contaminating proteins, and thus purification can be performed easily. On the other hand, when the transformant secretes recombinant phenol oxidase outside the cell, it may be purified from the culture supernatant in the same manner. When such a transformant is used, a large amount of recombinant phenol oxidase is accumulated in the culture supernatant, so that purification can be performed more easily, inexpensively and rapidly.

  In addition, when recombinant phenol oxidase is obtained as an inclusion body, recombinant phenol oxidase can be obtained as a polypeptide exhibiting activity by a conventional protein regeneration method. As a method for regenerating the protein, for example, after culturing the transformant, the cells of the transformant are collected by centrifugation, disrupted by sonication or the like, and the inclusion body is recovered by centrifugation or the like. The inclusion body may be solubilized with, for example, urea, guanidine hydrochloride, etc., purified by the protein purification method as necessary, and subjected to a refolding operation using a dialysis method, a dilution method, or the like. .

  Confirmation of the expression of the recombinant phenol oxidase can be performed by measuring the phenol oxidase activity as described above.

  In still another aspect, the present invention relates to an antibody against the recombinant phenol oxidase of the present invention. According to the antibody of the present invention, since it binds to the recombinant phenol oxidase of the present invention, phenol oxidase exhibiting high enzyme activity can be recovered, purified and screened stably in the vicinity of neutrality.

The antibody of the present invention may be a monoclonal antibody or a polyclonal antibody. In addition, the antibody of the present invention includes an antibody fragment as long as it exhibits binding to the recombinant phenol oxidase of the present invention. Examples of the antibody fragment include a single chain antibody, a Fab fragment, and an F (ab ′) ₂ fragment.

  The antibody of the present invention can be obtained by immunizing an animal such as a rabbit using the recombinant phenol oxidase of the present invention, collecting blood from the animal, and purifying it by a conventional technique. As such a technique, reference is made, for example, to John E. Coligan, Current Protocols in Immunology (1992).

The Fab fragment can be obtained by digesting a monoclonal antibody with papain. The F (ab ′) ₂ fragment can be obtained by digesting a monoclonal antibody with pepsin.

  Further, the specificity of the antibody of the present invention for the recombinant phenol oxidase of the present invention can be evaluated by examining the cross-reaction between the recombinant phenol oxidase of the present invention and other phenol oxidases.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention in detail, this invention is not limited by this Example. Unless otherwise specified, the following operations such as seeding, mycelium separation, and medium addition were performed in a clean bench.

1 platinum ear equivalent amount of Flammulina veltipes (Flamulina velutipes ), agar medium for solid culture {composition: 2.4 wt% potato dextrose broth (Difco)}, 2.0 wt% agar, balance water; 121 Sterilized at 15 ° C. for 15 minutes] The cells were inoculated in a sterile petri dish containing 10 ml and cultured at 25 ° C. for 10 days. Thereafter, the mycelium grown on the entire agar medium was cut into 5 mm squares with a sterilized platinum loop to obtain small pieces. 10 pieces of the above-mentioned pieces are seeded on a medium for liquid culture 1 {composition: 2.4% by weight potato dextrose broth (manufactured by Difco), remaining water; sterilized at 121 ° C. for 15 minutes} at 25 ° C. Then, reciprocal shaking culture (150 reciprocations / min) was performed. As the liquid culture medium 1, each medium whose pH was arbitrarily adjusted in the range of 4.0 to 9.0 was used. About the obtained culture, the activity of phenol oxidase was investigated.

Enzyme activity (oxidation activity) was measured by a change in absorbance at 470 nm using sodium acetate buffer (pH 5.0) or Tris- HCl buffer (pH 8.0) as a buffer and para-phenylenediamine as a substrate. . Specifically, in a well of a 96-well microplate (Corning (registered trademark) 3368, manufactured by Corning), 0.025 M para-phenylenediamine aqueous solution was added to 0.1 ml of 0.2 M buffer solution. 0.08 ml was added to prepare a substrate solution. About this substrate solution, the enzyme activity was calculated | required by measuring a light absorbency using Multi Spectrometer (Dainippon Pharmaceutical Co., Ltd. make, brand name: Viento). In addition, the enzyme activity defined the amount of enzyme that increases the absorbance at 470 nm by 1 in 1 minute as 1 unit (U: unit).

  As a result of culturing for 7 days under each culture condition, enzyme activity measurement at each reaction pH of pH 5.0 and pH 8.0 was the highest in enzyme activity on the 7th culture day. Thus, the enzyme activity increased with the passage of the culture days.

  As a result of measuring the enzyme activity at pH 5.0, no clear enzyme activity was observed in all the cultures cultured in the pH range of pH 4.0 to 9.0. Specifically, the highest enzyme activity was observed in a culture solution obtained by culturing for 7 days under a culture condition of pH 9.0, but the change in absorbance at 470 nm 17 hours after the start of the reaction. Was only 0.12. Moreover, about the culture solution obtained by culture | cultivating on culture | cultivation pH conditions of pH 6.0 for 7 days, the change of the light absorbency in 470 nm after 17 hours was 0.06.

  On the other hand, as a result of measuring the enzyme activity at pH 8.0, all of the culture solutions cultured in the pH range of pH 5.0 to 9.0 showed clear enzyme activity. In addition, although the culture solution obtained by culture | cultivating by pH 4.0 did not show clear enzyme activity, the increase in enzyme activity by a culture | cultivation day was seen.

  In addition, the culture solution having a higher culture pH showed higher enzyme activity. The culture solution showing the highest enzyme activity was a culture solution obtained by culturing for 7 days under a culture pH condition of pH 9.0, and the change in absorbance at 470 nm after 17 hours was 1.27. Moreover, about the culture solution obtained by culturing for 7 days on the culture pH conditions of pH 6.0, the change of the light absorbency in 470 nm after 17 hours was 0.78.

From the above results, phenoloxidase induced in Furamurina Berutipesu (Flammulina velutipes) is a neutral phenol oxidase, also acidic phenol oxidase or acidic phenol oxidase having the optimum pH in an acidic side was found not induced . In addition, in Flammulina vertipes, the enzyme activity at pH 8.0 is 7 days under the culture pH condition of pH 6.0. The enzyme activity of the culture solution obtained by culturing for 7 days under the culture pH condition of pH 9.0 is 7 days. It was found to be about 1.6 times higher than the enzyme activity of the culture solution obtained by culturing.

These results, in Furamurina Berutipesu (Flammulina velutipes) in, it was found that the presence of phenol oxidase showing strong activity at neutral pH.

  Agar medium for solid culture [Composition: 2.4% by weight potato dextrose broth (manufactured by Difco)], 2.0% by weight agar, remaining water; sterilized at 121 ° C. for 15 minutes] In a sterile petri dish containing 10 ml Enokidake (Flamulina velutipes) was inoculated in an amount equivalent to one platinum loop and cultured at 25 ° C. for 10 days. Thereafter, the mycelium grown on the entire agar medium was cut into 5 mm squares with a sterilized platinum loop. 10 pieces of the above-mentioned pieces are seeded in a medium for liquid culture 1 [composition: 2.4% by weight potato dextrose broth (manufactured by Difco)], remaining water (pH 5.2); sterilized at 121 ° C. for 15 minutes. Then, reciprocal shaking culture (150 reciprocations / min) was performed at 25 ° C. for 7 days. The total amount of the obtained culture solution was added to 500 ml of the liquid culture medium 1 in a 2 L Erlenmeyer flask, and reciprocal shaking culture (100 reciprocations / min) was performed at 25 ° C. for 3 weeks.

Thereafter, the grown mycelium in the form of pellets is allowed to settle, the culture solution is removed, and the remaining mycelium is added to the liquid culture medium 2 [composition: 1.0 wt% glucose, 0.1 wt% yeast extract, 0 .14 wt% (NH ₄ ) ₂ SO ₄ , 0.36 wt% K ₂ HPO ₄ , 0.02 wt% MgSO ₄ .7H ₂ O, 0.10 wt% Mineral mixture (composition: 1.0 wt% CuSO ₄ .5H ₂ O, 1.0 wt% ZnCl ₂ , 0.7 wt% FeCl ₃ .6H ₂ O, 0.5 wt% CoSO ₄ .7H ₂ O, 0.5 wt% MnCl ₂ .4H ₂ O ), PH 9.2; sterilized at 121 ° C. for 15 minutes] 500 ml was added, and further cultured at 25 ° C. for 3 days.

  Thereafter, the grown pellet-like mycelium was allowed to settle, and the culture solution was collected by decantation.

  The collected culture solution was a pale yellow or tan clear or turbid liquid. The collected culture medium had a total volume of 3060 ml, a total titer of 13100 U, a total protein amount of 778 mg, and a specific activity of 16.8 U / mg protein.

  The culture solution was freeze-dried to obtain a freeze-dried product. The obtained lyophilized product was dissolved in 1M Tris-HCl buffer (pH 8.0) to obtain an enzyme solution. 50 μl of the enzyme solution was subjected to native PAGE using 12.5 wt% polyacrylamide gel. The gel after native PAGE was shaken and stirred in 0.1 M sodium phosphate buffer (pH 7.0), and then in 0.1 M sodium phosphate buffer (pH 7.0) containing 1 mM orthoaminophenol. Active staining was carried out by shaking and agitating the gel for 5 minutes. Then, the band which showed activity was cut out, and it wash | cleaned by stirring for 30 minutes in a 25 mM Tris-192 mM glycine buffer solution (pH 8.2). The gel after washing was subjected to SDS-PAGE using 12.5 wt% polyacrylamide gel.

The gel after SDS-PAGE is set in a transfer device (KS-8460, manufactured by Marisol), and 1 mA per 1 cm ^{2 of} gel using a HIFRED CONSTANT POWER SUPPLY (manufactured by Marysol, MP-7352). For 1.5 hours, and transferred to a brand name: Problot ^™ membrane (Applied Biosystems). The membrane after transfer was shaken and stirred for 3 minutes in a staining solution (composition: 0.1% by weight Coomassie Brilliant Blue R-250, 40% by volume methanol, remaining water), and a decolorizing solution (composition: 50% by volume methanol, Staining was performed by shaking and stirring until the protein band was visible in the remaining water.

  The stained band was excised and subjected to a protein sequencer (Applied Biosystems, Procise ™ 492 HT) to determine the N-terminal amino acid sequence.

As a result, phenol oxidase Furamurina Berutipesu (Flammulina velutipes) as the amino-terminal amino acid sequence, SEQ ID NO: was found to have the amino acid sequence shown in 3.

  In the same manner as in Example 2, Flammulina vertipes was cultured in an agar medium for solid culture, and the obtained mycelium was cultured in medium 1 for liquid culture, and then cultured in medium 2 for liquid culture. As a result, phenol oxidase exhibiting strong activity at neutral pH was produced and the abundance ratio of mRNA corresponding to phenol oxidase in the cells was improved.

The Furamurina Berutipesu of phenol oxidase production in production (Flammulina velutipes), Advantech Toyo Co., Ltd., trade name: qualitative filter paper No. It was recovered using 1 etc., quenched with liquid nitrogen and frozen.

The obtained Flammulina vertipes (Flamulina velutipes ) was ground to a powder using a mortar and pestle in the presence of liquid nitrogen to obtain a frozen cell powder. From about 100 mg of frozen bacterial powder, 20 μl of total mRNA extraction solution (equivalent to 50 μg) was prepared according to the manufacturer's instructions using the trade name: Dynabeads mRNA DIRECT Kit manufactured by Dynal.

  The obtained mRNA was subjected to a reverse transcription reaction using a product name: First strand cDNA synthesis Kit manufactured by Amersham Pharmacia according to the manufacturer's instructions to synthesize single-stranded cDNA.

Based on the N-terminal amino acid sequence (SEQ ID NO: 3) obtained in Example 3, a 5 ′ primer (SEQ ID NO: 5) was designed and synthesized. Further, in the known amino acid sequences of phenol oxidases of fungi other than the genus Flammulina , a 3 ′ primer (SEQ ID NO: 6) is based on a consensus sequence having high identity: WFLHCH (SEQ ID NO: 4) and a DNA sequence. ) Was synthesized.

And the primer (each 20 pmol), using a cDNA 5 ng and TaKaRa Ex Taq ^TM (manufactured by Takara Bio Inc.), and the entire cDNA of Furamurina Berutipesu (Flammulina velutipes) as a template, perform the polymerase chain reaction (PCR), phenol oxidase gene The DNA fragment was amplified.

  The PCR conditions were as follows: after incubation at 95 ° C. for 5 minutes, 35 cycles of reaction were performed with 95 ° C. for 1 minute, 50-60 ° C. for 1 minute, and 72 ° C. for 5 minutes. The conditions were for a minute incubation.

  The obtained PCR product was mixed with Tris / acetate / EDTA buffer [composition: 0.484 wt% tris, 0.14 vol% acetic acid, 0.07885 wt% ethylenediamine-N, N, N ′, N′-tetraacetate triacetate] Sodium salt, the remaining water] was electrophoresed on a 2.0 wt% agarose gel, and a band corresponding to a DNA fragment of about 1.3 kbp was cut out to obtain a gel fragment. Subsequently, using the Prep-A-Gene (registered trademark) DNA Purification System (manufactured by Bio-Rad), the DNA fragment in the gel fragment was purified according to the manufacturer's instructions.

  The obtained DNA fragment was cloned into pGEM (registered trademark) -T Easy Vector using pGEM (registered trademark) -T Easy Vector Systems (manufactured by Promega) according to the manufacturer's instructions. A plasmid containing 1.3 kb DNA is referred to as pGEM-F1-N.

The obtained vector was purified, and as a template, using Thermo Sequenase Primer ^™ Cycle Sequencing Kit (manufactured by Amersham Biosciences) according to the manufacturer's instructions, Hitachi SQ5500E type DNA Sequence (manufactured by Hitachi, Ltd.) The nucleotide sequence was determined by analysis.

  Based on the sequence upstream of the SacI recognition site in the determined base sequence, a 5 'primer (SEQ ID NO: 7) was synthesized. Further, PCR was performed using the oligonucleotide (SEQ ID NO: 8) contained in the product name: First strand cDNA synthesis kit manufactured by Amersham Pharmacia as a primer and cDNA as a template in the same manner as described above. As a result, a DNA fragment of about 700 bp was obtained. Thereafter, the obtained DNA fragment was cloned into pGEM (registered trademark) -T Easy Vector in the same manner as described above. Using the obtained vector as a template, the base sequence was determined in the same manner as described above. A plasmid containing 700 bp DNA is referred to as pGEM-F1-C.

  The determined base sequence is shown in SEQ ID NO: 1. The amino acid sequence deduced from the determined base sequence is shown in SEQ ID NO: 2.

  As a result, it was estimated that phenol oxidase consists of an amino acid sequence of 496 amino acid residues, and it was confirmed that the predicted amino acid sequence completely preserves 12 copper binding sites found in known phenol oxidases. It was done.

  Thereafter, the two plasmids obtained by the above method were digested with the restriction enzyme SacI and ligated to obtain a plasmid (pGEM-F1) containing the phenol oxidase full-length gene. The plasmid map of this pGEM-F1 is shown in FIG.

(1) Construction of heterologous expression system by Pichia pastoris of phenol oxidase 5 ′ primer for PCR synthesized based on the 5 ′ base sequence of the phenol oxidase gene and the 3 ′ base sequence of the signal sequence ( PCR was performed using SEQ ID NO: 11) and a 3 ′ primer (SEQ ID NO: 12) synthesized based on the base sequence downstream of the EcoRI recognition site in the phenol oxidase gene, using the EcoRI digested product of pGEM-F1 as a template. A partial DNA fragment of the phenol oxidase gene was obtained. PCR conditions are as follows: incubation at 94 ° C. for 5 minutes, followed by 25 cycles of reaction at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 30 seconds, and incubation at 7 ° C. for 10 minutes Condition.

  In order to add a nucleic acid corresponding to the signal region to the obtained PCR product (partial DNA modified with the phenol oxidase gene 5 ′), a PCR 5 ′ primer (SEQ ID NO: 9) and a PCR 3 ′ primer (SEQ ID NO: 9) 10 and 12), PCR was performed using the above PCR product as a template to obtain a partial DNA fragment of the phenol oxidase gene to which a signal region was added. PCR conditions are as follows: incubation at 94 ° C for 5 minutes, 30 cycles of reaction at 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 30 seconds, followed by incubation at 72 ° C for 16 minutes It was.

  Both the obtained PCR product (a partial DNA fragment of a phenol oxidase gene containing a nucleic acid corresponding to the signal region) and pGEM-F1 were digested with EcoRI and NotI, respectively.

  The obtained product was subjected to agarose gel electrophoresis. Corresponding bands were cut out from the gel, and DNA fragments were extracted from the obtained gel fragments using Prep-A-Gene (registered trademark) DNA Purification System (manufactured by Bio-Rad).

  The obtained DNA fragment was ligated to EcoRI-NotI digested plasmid pPIC3.5k (manufactured by Invitrogen) to obtain a plasmid for yeast transformation (pPIC-F1). The plasmid map of pPIC-F1 is shown in FIG.

Moreover, according to the manual of the brand name: Multi-Copy Pichia Expression Kit (manufactured by Invitrogen), the pPIC-F1 was opened with the restriction enzyme MssI, and the yeast Pichia pastoris GS115 strain was transformed by electroporation. The resulting transformed yeast is His ⁺ (no histidine requirement).

(2) Culture of transformed bacteria and screening of transformed bacteria An appropriate amount of the transformed yeast (His ⁺ ) obtained in (1) above was added to an MD agar plate medium [composition: 1.34% by weight YNB [Invitrogen (Invitrogen)]. ), 4 × 10 ⁻⁵ wt% biotin, 2 wt% D-glucose], cultured at 30 ° C. overnight and fully grown. Sterile water was added to the resulting plate to suspend the transformed yeast. YPD agar plate medium (1% by weight yeast extract (manufactured by Nacalai Tesque)), 2% by weight peptone (manufactured by Nippon Pharmaceutical Co., Ltd.) containing an appropriate amount of G418 (manufactured by Sigma), 2 wt% D-glucose, 1.5 wt% agar] so that 10 ⁵ cells per cell were applied, and cultured at 30 ° C.

  Transformed yeast grown on a YPD agar plate medium containing 0.25 mg / ml G418 was transformed again with pPIC-F1 opened with the restriction enzyme MssI, and 3.0 mg / ml was obtained in the same manner. Colonies showing resistance to G418 (G418 highly resistant strain) were screened.

(3) Expression of recombinant phenol oxidase (rF1) G418 highly resistant strain obtained in (2) above was added to 4 ml of YPD liquid medium [composition: 1 wt% yeast extract (manufactured by Nacalai Tesque), 2 wt% Peptone, 2% by weight D-glucose], and cultured with shaking in a test tube at 30 ° C. for 24 hours. 4 ml of the obtained culture was inoculated into a 100 ml YPD liquid medium in a 500 ml Sakaguchi flask and cultured with shaking at 30 ° C. for 24 hours. The obtained culture is subjected to centrifugation to recover the yeast, and an appropriate amount of BMM minimal medium [composition: 100 mM potassium phosphate buffer, 1.34 wt% YNB (manufactured by Invitrogen)], 4 × 10 ^-5 wt% biotin, 0.5 vol% methanol). The entire amount of the obtained suspension was inoculated into a 400 ml BMM minimal medium (2 L Erlenmeyer flask) containing 0.2 mM CuCl ₂ .

  Every 24 hours, the cells were cultured with shaking at 20 ° C. for 4 days while adding 0.5% by volume of methanol as an expression induction substrate. By induction with methanol, recombinant phenol oxidase (rF1) was induced and expressed and released into the culture medium. Yeast cells were removed from the total 7.2L culture solution by centrifugation to obtain a crude enzyme solution as a culture supernatant.

  0.1 ml of orthoaminophenol aqueous solution containing 10% dimethyl sulfoxide is added to 800 μl of 0.5 M sodium phosphate (pH 7.0) to obtain a substrate solution. The obtained substrate solution and 0.1 ml of the crude enzyme solution are mixed with each other. About the obtained mixture, the enzyme activity at the time of using orthoaminophenol as a substrate was measured by measuring the light absorbency in 420 nm. The amount of enzyme that increases the absorbance at 420 nm by 1 per minute was defined as 1 unit (U: unit). As a result, the obtained crude enzyme solution had a total volume of 7.07 L, a total titer of 18,600 U, a total protein of 5600 mg, and a specific activity of 3.3 U / mg protein.

  To the crude enzyme solution, ammonium sulfate (manufactured by Nacalai Tesque, special grade reagent) was gradually added, and the mixture was stirred to adjust to 25% saturation. This was equilibrated with a 50 mM potassium phosphate buffer solution (pH 6.0) containing 25% saturated ammonium sulfate, and a column (4.8 × 20 cm) packed with a trade name: TOYOPEARL Butyl-650M (manufactured by Tosoh Corporation). Adsorbed. After adsorption, the column was washed with 2 L of 50 mM potassium phosphate buffer (pH 6.0) containing 25% saturated ammonium sulfate. Thereafter, the recombinant phenol oxidase adsorbed on the column was eluted by a gradient elution method using ammonium sulfate in a concentration range of 25 to 0% saturation. The fraction of recombinant phenol oxidase was determined by measuring the enzyme activity for paraphenylenediamine in the obtained eluted fraction.

  The recombinant phenol oxidase fraction was dialyzed against 50 mM Tris sulfate buffer (pH 8.5) using a trade name: Dialysis membrane, Size 36 (manufactured by Wako Pure Chemical Industries, Ltd.) An oxidase solution was obtained.

  The recombinant phenol oxidase solution was adsorbed on a column (2.4 × 40 cm) packed with trade name: TSK gel DEAE-TOYOPARRL 650M (manufactured by Tosoh Corporation) equilibrated with 50 mM Tris sulfate buffer (pH 8.5). I let you. After adsorption, the column was washed with 50 mM Tris sulfate buffer (pH 8.5) containing 0.1 M sodium sulfate until the absorbance of the eluate at 280 nm was 0.1 or less. Thereafter, the recombinant phenol oxidase adsorbed on the column was eluted by a gradient elution method using sodium sulfate in a concentration range of 0.1 to 0.35M. The fraction of recombinant phenol oxidase was determined by subjecting the obtained eluted fraction to enzyme activity against paraphenylenediamine and activity staining with paraphenylenediamine after SDS-PAGE. An example of purification of recombinant phenol oxidase is shown in Table 1.

  The fraction of the recombinant phenol oxidase was subjected to SDS-PAGE using 10% by weight polyacrylamide gel. The SDS-PAGE gel was equilibrated in 0.1 M potassium phosphate buffer (pH 6.0), and then the gel was added in 0.1 M potassium phosphate buffer (pH 6.0) containing 1 mM paraphenylenediamine. Activity staining was performed by shaking the gel.

  In addition, the fraction of the recombinant phenol oxidase was concentrated using a trade name: Centricon YM-10 (manufactured by Millipore) so as to be about 10 times the amount. The obtained concentrated solution was used as a sample and subjected to SDS-PAGE using a 10% by weight polyacrylamide gel. Electrophoresis was performed according to the method of Laemmli et al. Except that no reducing agent was added to the sample and no heat treatment was performed. The gel after electrophoresis is shaken in a staining solution [composition: 0.05% by weight Coomassie Brilliant Blue R250, 50% by volume methanol, 10% by volume acetic acid, the remaining water], and then a decoloring solution [composition: 25% by volume. % Methanol, 7% by volume acetic acid, the balance water] to perform protein staining. A molecular weight marker (manufactured by Amersham Biosciences, trade name: LMW calibration kit) was simultaneously used, and the molecular weight of the recombinant phenol oxidase was measured by comparing the mobility. FIG. 3 shows the results of activity staining and protein staining after SDS-PAGE.

  As shown in FIG. 3, the fraction of recombinant phenol oxidase shows a single band in activity staining and protein staining, and it can be seen that the molecular weight is 28 kDa.

(4) Optimum pH of recombinant phenol oxidase
A 50 mM paraphenylenediamine aqueous solution, a 50 mM 2,6-dimethoxyphenol aqueous solution, and a 10 volume% dimethyl sulfoxide solution of 50 mM orthoaminophenol were prepared, and each was used as a substrate solution. Mixed buffer solution with pH adjusted to pH 4.5, pH 5.3, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5 and pH 9.0, respectively [Composition : 0.2 M sodium acetate, 0.2 M 2- (N-morpholino) ethanesulfonic acid (MES), 0.2 M Tris aqueous solution is adjusted to the target pH using hydrochloric acid or sodium hydroxide] 895 μl and 50 mM substrate 100 μl of the solution and 5 μl of recombinant phenol oxidase solution (0.125 U, 1.6 μg) were mixed. Changes in absorbance at 470 nm were measured for paraphenylenediamine and 2,6-dimethoxyphenol, and absorbance at 420 nm was measured for orthoaminophenol. The amount of enzyme that increases the absorbance by 1 per minute was defined as 1 unit.

  As a result, the recombinant phenol oxidase showed high activity in the vicinity of neutrality against three types of substrates (paraphenylenediamine, 2,6-dimethoxyphenol, and orthoaminophenol). Recombinant phenol oxidase exhibits an activity of 60% or more with respect to paraphenylenediamine at pH 5.0 to 6.5, compared with the activity at pH 5.3, which exhibits the maximum activity, and pH 5.0 to 5. 5 showed an activity of 80% or more as compared with the activity at pH 5.3 showing the maximum activity. In addition, the recombinant phenol oxidase exhibits an activity of 60% or more with respect to 2,6-dimethoxyphenol at pH 5.0 to 7.5, compared with the activity at pH 6.5 showing the maximum activity, pH 5 In 0.0-7.0, the activity of 80% or more was shown compared with the activity in pH 6.5 which shows maximum activity. Furthermore, the recombinant phenol oxidase exhibits an activity of 60% or more with respect to orthoaminophenol at pH 5.0 to 8.0, compared with the activity at pH 7.0, which exhibits the maximum activity, and pH 6.0. In ˜7.5, the activity was 80% or more as compared with the activity of pH 7.0 showing the maximum activity.

(5) pH stability of recombinant phenol oxidase 22 μl (0.53 U, 0.17 μg) of recombinant phenol oxidase was adjusted to pH 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11. 5, mixed buffer adjusted to 12.0 [Composition: 0.2M acetic acid, 0.2M phosphoric acid, 0.2M boric acid aqueous solution adjusted to the desired pH with sodium hydroxide solution] mixed with 88 μl, 25 ° C. Incubated with After 1 hour and 20 hours from the start of incubation, 50 μl of the obtained mixed solution, 850 μl of 0.5 M potassium phosphate buffer (pH 7.0), and 100 μl of 10% dimethyl sulfoxide solution of 50 mM orthoaminophenol were mixed. The change in absorbance at a wavelength of 420 nm was measured. The pH stability after 1 hour of the recombinant phenol oxidase is shown in FIG. 4 panel (A), and the pH stability after 20 hours is shown in FIG. 4 panel (B).

  Recombinant phenol oxidase has a relative residual activity of at least 25% compared to the activity at pH 9.0, which showed maximum stability under incubation conditions at 25 ° C. for 1 hour at pH 3.5-11.0. Indicated. In addition, at pH 4.0 to 10.5, the relative residual activity was at least 50% as compared with the activity at pH 9.0, which showed the maximum stability under the incubation condition at 25 ° C. for 1 hour. In addition, at pH 7.0-10.0, the relative residual activity was at least 80% compared to the activity at pH 9.0, which showed the maximum stability under the incubation condition at 25 ° C. for 1 hour.

  In addition, the recombinant phenol oxidase has a relative residual activity of at least 25% compared to the activity at pH 9.0, which showed the maximum stability under incubation conditions of 25 ° C. and 20 hours at pH 7.5 to 10.0. showed that. In addition, at pH 7.5-10.0, the relative residual activity was at least 50% as compared with the activity at pH 9.0, which showed the maximum stability under the incubation condition at 25 ° C. for 20 hours. In addition, at pH 8.0 to 9.0, the relative residual activity was at least 80% as compared with the activity at pH 9.0 which showed the maximum stability under the incubation condition at 25 ° C. for 20 hours.

(6) Thermal stability of recombinant phenol oxidase An elution fraction of recombinant phenol oxidase [50 mM Tris sulfate buffer (pH 8.5)] was incubated at 40 ° C. and 50 ° C. Sampling was performed after 5, 10, 15, 20, 30, and 60 minutes, respectively. 50 μl of the solution after incubation, 850 μl of 0.5 M potassium phosphate buffer (pH 7.0), and 100 μl of a 10% dimethyl sulfoxide solution of 50 mM orthoaminophenol were mixed, and the change in absorbance at a wavelength of 420 nm was measured. The results are shown in FIG. In the figure, the case of incubation at 40 ° C. is indicated by a diamond, and the case of incubation at 50 ° C. is indicated by a square.

  As a result, the recombinant phenol oxidase showed a relative residual activity of 25% or more compared to the pre-incubation activity under the incubation condition at 50 ° C. for 10 minutes, and before the incubation under the incubation condition at 50 ° C. for 5 minutes. The relative residual activity of 50% or more was shown in comparison with the activity of. In addition, it shows a relative residual activity of 80% or more compared to the activity before incubation under the incubation condition at 40 ° C. for 20 minutes, and compared with the activity before incubation under the incubation condition at 40 ° C. for 60 minutes. A relative residual activity of 60% or more was exhibited.

  According to the present invention, fibers and hair can be dyed under conditions that are mild to the environment and the human body.

FIG. 1 shows a schematic diagram of pGEM-F1. In the figure, “F1” indicates a phenol oxidase gene.

FIG. 2 shows a schematic diagram of the plasmid pPIC-F1 for yeast expression. In the figure, “F1” indicates a phenol oxidase gene, and “signal” indicates a signal sequence.

FIG. 3 shows the results of activity staining and protein staining. In FIG. 3, lane 1 shows the result of protein staining, lane 2 shows the result of activity staining, and lane M shows the molecular weight marker.

FIG. 4 shows a graph of the results of examining the pH stability of recombinant phenol oxidase. In FIG. 4, panel (A) shows pH stability after 1 hour incubation, and panel (B) shows pH stability after 20 hours incubation.

FIG. 5 shows a graph of the results of examining the thermal stability of recombinant phenol oxidase. In the figure, the case of incubation at 40 ° C. is indicated by a diamond, and the case of incubation at 50 ° C. is indicated by a square.

  SEQ ID NO: 3 is the amino terminal amino acid sequence of phenol oxidase.

  SEQ ID NO: 4 is a consensus sequence for various phenol oxidases.

  SEQ ID NO: 5 is the sequence of the primer.

  SEQ ID NO: 6 is the sequence of the primer.

  SEQ ID NO: 7 is the primer sequence.

  SEQ ID NO: 8 is the sequence of a primer.

  SEQ ID NO: 9 is the sequence of a primer.

  SEQ ID NO: 10 is a primer sequence.

  SEQ ID NO: 11 is the sequence of a primer.

  SEQ ID NO: 12 is the sequence of a primer.

Claims (7)

(A) a base sequence encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2;
(B) In SEQ ID NO: 2, encodes an amino acid sequence having substitution, deletion, addition or insertion of one or several amino acid residues, and the encoded polypeptide exhibits phenol oxidase activity. nucleotide sequence,及 beauty <br/> (c) SEQ ID NO: 2 sequence identity may encode an amino acid sequence that is at least 85%, and the encoded polypeptide shows a phenol oxidase activity Base sequence,
A nucleic acid encoding a phenol oxidase, comprising a base sequence selected from the group consisting of:   The nucleic acid according to claim 1, wherein the encoded polypeptide has phenol oxidase activity at least at a pH of 5.0 to 8.0.   An expression vector comprising the nucleic acid according to claim 1 or 2. The expression vector according to claim 3 , wherein a nucleic acid encoding a signal sequence is operably linked upstream of the nucleic acid according to claim 1 or 2. A transformant comprising the expression vector according to claim 3 or 4 . The transformant according to claim 5 , which is derived from a yeast cell or a filamentous fungus. The recombinant phenol encoded by the nucleic acid according to claim 1 or 2 , wherein the transformant according to claim 5 or 6 is cultured, and a recombinant phenol oxidase is produced in the culture and recovered. Method for producing oxidase.

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