KR101364994B1 - Expression of tyrosinase by gene sequence optimization of helpful protein and production methods of catechol-like construction material using tyrosinase and its inhibitor - Google Patents

Abstract

The present invention includes the gene sequence of a helper protein that can help the expression of tyrosinase, and is applicable to the production of catechol-type structures using enzyme catalysis.
In addition, the present invention proposes a method capable of producing a large amount of catechol-type structural substances corresponding to the target substance without side reactions by adding heterogeneous catechol-type structural substances.

Description

Expression of tyrosinase by optimizing gene sequence of help protein and production method of catechol-type structural material using tyrosinase and inhibitor ITS INHIBITOR}

The present invention uses a tyrosinase for melanin synthesis, a helper protein that helps the tyrosinase to have an active structure, and recombinant strains containing respective genes as biocatalysts to produce chemicals having a catechol-like structure. It is about how to.

Catechol-type structures exhibit enhanced physiological activity in antioxidant, anticancer, anti-inflammatory or antiviral effects, and are reported to be more effective than monophenolic structures. In particular, catechol-type structural materials are used as inhibitors of tyrosinase, and have an effect of reducing melanin production by tyrosinase.

When producing a catechol-type structural material by utilizing a conventional biocatalyst, various monooxygenases including P450 monooxygenase are used. Most monooxygenases, however, require coenzymes and require electron transfer. In addition, it is reported that not only affects the rate of biotransformation reaction of the substrate, but also the efficiency of electron transfer reaction between coenzyme and protein is low.

On the other hand, tyrosinase is an enzyme that can produce hydroxylated products without requiring the use of coenzymes. It is mainly applied to the production of dopa (DOPA) from tyrosine using tyrosinase. Tyrosinase is reported to have a function of blocking light in the ultraviolet region by producing melanin after oxidizing the monophenol structure and converting it into a catechol-type structure.

In addition, in the case of tyrosinase which catalyzes the oxidation reaction and the oxidation reaction at the same time, it is mostly utilized as an enzyme related to the production of melanin. In general, since the rate constant (k2) of the secondary oxidation reaction is about 10 times larger than the reaction rate constant (k1) of the primary hydroxide reaction, it is difficult to accumulate an intermediate catechol-type structure.

In addition, when tyrosinase is expressed by introducing genetic engineering techniques in heterologous hosts, it is difficult to have a structure having full activity. The reason for this is that not only are the translation systems present in each microorganism different, but there is also no helper protein that can help to achieve an active structure.

In the current technical context, expression systems are used to apply expression between microorganisms of similar dependencies, or to produce melanin by expressing tyrosinase and helper proteins together in heterologous hosts. However, there have been no reports on the production of catechol-like structures by applying tyrosinase expressed from recombinant strains incorporating tyrosinase and help proteins in heterologous hosts.

On the other hand, the typical production method of the catechol-type structural material applying the biological method can be divided into first, the method of combining catechol and other structures, and second, the hydroxylation reaction of the benzene-type or monophenolic structural material.

A representative example of the first example is a method for producing dopa (DOPA) in Ajinomoto, Japan, which is based on catechol, acetic acid, and ammonia, and produces dopa (DOPA) using tyrosine-phenolase. Reaction. It can be produced at the maximum level of 100 g / L and is a representative example of mass production of catechol-type structural materials. The production of catechol-like structures using lyases requires the screening of lyases corresponding to specific substrates that can form carbon-carbon bonds with catechol.

Second, an example of the hydroxylation reaction of a benzene structure or a monophenolic structure is a method using a monooxygenase. Monooxygenase is a type of oxidoreductase that requires the supply of electrons by an electron transport system. A representative monooxygenase reactive enzyme is cytochrome P450 and the reaction is derived from the redox action of iron ions of the heme structure located at the center of the enzyme. By converting the substrate specificity of cytochrome P450, it is applied to the production of catechol-type structural substances of various structures.

Another hydroxylation reaction is the application of tyrosinase. In the case of tyrosinase, there is an advantage that can be converted to catechol-type structural material using a variety of monophenolic structural material as a substrate. However, in the case of tyrosinase, the catalyzed reaction to hydrate the monophenolic structural material to form a catechol-type structural material, and at the same time, oxidize the catechol-type structural material to form a diquinone form. As a method for accumulating the catechol-type structural material, which is an intermediate of the tyrosinase reaction, treatment with a reducing agent prevents peroxidation, reduction of deoxygenase activity and increase of monooxygenase activity through improvement of tyrosinase. It is applied.

The tyrosinase is a redox catalytic enzyme containing copper and contains oxygen molecules between two copper ions. The tyrosinase catalyzes the monooxygenase action, and the monooxygenase action inserts the oxygen atom of one of the oxygen molecules contained in the tyrosinase into the substrate. An oxidation reaction is generated in which hydrogen ions are removed from two hydroxide residues generated after the hydroxylation, and water is combined with one oxygen ion remaining to generate water. The resulting product produces a structural material in the form of ortho-benzoquinone.

Tyrosinase has the property that the reaction specificity of the substrate is universal. In the case of a structural material containing a monophenolic structure, there is an advantage in that the conversion to a catechol-type structural material as a whole.

As such, tyrosinase having various substrate specificities may be extracted from microorganisms, fungi, and mushrooms, used to separate and purify, or may be produced as recombinant strains using genetic information. After PCR amplification of the gene of tyrosinase, it is inserted into a recombinant plasmid and cultured by inserting it into a strain capable of inducing expression. After treating the strain to induce the expression of the protein in the cultured strains, the expressed tyrosinase is isolated and purified or used as a whole-cell reaction.

As described above, in the method for producing a biological catechol-type structure, it is inevitable to screen for enzymes or to improve enzymes for a desired product because of the high substrate specificity when using lyase or monooxygenase. Production efficiency cannot be guaranteed even if the enzymes that can be produced as catechol-type structures by hydroxylating specific monophenol-type structures are screened or improved.

On the other hand, the method of producing a catechol-type structural material by catalyzing tyrosinase has the advantage that it can be commonly used in the substrate of various monophenolic structural materials. Reaction rates of enzymes also reach industrially applicable levels. Tyrosinase derived from mushrooms has a value of k _cat / K _m of 1000 mM ⁻¹ s ⁻¹ or more, and considering the productivity, it has the most advantageous advantage over other enzymes.

However, catalysis using tyrosinase has a disadvantage in that it is difficult to accumulate a catechol-type structural material, which is regarded as a target material because an additional oxidation reaction proceeds. Since the catechol type structure is oxidized at a very high rate and converted into the ortho-benzoquinone form, the production yield of the catechol type structure that can be actually produced is remarkably low.

In order to solve this drawback, many researchers have made efforts to reduce the rate of oxidation by treating the reducing agent or providing reducing power by an electrochemical method. However, in the case of the reducing agent used, there is a disadvantage in that the price is expensive, and in the case of providing the reducing power by an electric method, there is a disadvantage in that it is limited to tyrosinase immobilized on the electrode surface.

There are also restrictions on the production of units for industrial application of tyrosinase. Since tyrosinase itself has a property of inhibiting reaction by a catechol-type structure, an excess amount of tyrosinase is required, but it is produced in nature in order to be applied to the production of a catechol-type structure using tyrosinase. There is a limit to extracting tyrosinase. Therefore, it is realistically required to produce, amplify, and produce a recombinant microbial strain using genetic information.

Accordingly, attempts have been made to produce recombinant plasmids from the genetic information of tyrosinase and introduce them into heterologous hosts of E. coli. The most problematic process for expression in heterologous hosts is the part that does not achieve normal protein folding of tyrosinase. Since the information of the gene encoding tyrosinase is originally optimized for the expression system of the host, it is difficult to achieve normal expression and protein structure in the foreign environment of the heterologous host. For the same reason, there is a limitation in mass production of catechol-type structural materials using tyrosinase.

On the other hand, for some microorganisms, helper protein genes coexist in the same open reading frame (ORF) as tyrosinase. The function of the helper protein is to put copper ions into the catalytic activity of tyrosinase so that it has a normal folding structure. However, since the help protein is usually present in the cell membrane of the microorganism and consists of a rare codon sequence, the expression itself is difficult in heterologous hosts including Escherichia coli.

KR 1048475 B1 KR 0739871 B1

 Effect of L-ascorbic acid on the monophenolase activity of tyrosinase (L-ascorbic acid on the monophenolase activity of tyrosinase). Biochem J. 1993 October 1; 295 (Pt 1): 309-312.  Directed evolution of tyrosinase for enhanced monophenolase / diphenolase activity ratio (aromatic evolution of tyrosinase for monophenolase / diphenolase activation ratio). Enzyme and Microbial Technology, Volume 47, Issue 7, 8 December 2010, Pages 372-376

The present invention relates to a method for effectively producing a catechol-type structural material having a hydroxyl functional group introduced at the ortho position of a monophenolic chemical using the catalytic activity of tyrosinase present in a microorganism. It is an object to increase the production yield of catechol-type structural material by adding an additional oxidation inhibitor after the hydroxylation reaction of tyrosinase.

In addition, the present invention relates to an E. coli strain overexpressed by introducing a tyrosinase of a heterologous strain, and includes a sequence of genes of a helper protein that may help the expression of tyrosinase.

The present invention uses recombinant genetic information of tyrosinase and helper protein from actinomycetes and other microorganisms to provide a method for producing a recombinant strain and maximizing expression of the protein.

In addition, the present invention synthesized the gene of the help protein into a gene sequence suitable for the expression system of E. coli, a method in which the same amino acid is expressed in heterologous host by replacing the expression codon of the help protein with a sequence rich in E. coli To provide.

In other words, by utilizing the genetic information introduced the improved help protein and tyrosinase sequence information, the expression of the protein is maximized and applied to the catalysis of the catechol-type structural material.

The present invention also provides a method for producing a catechol-type structural material using overexpressed tyrosinase or whole cells expressed with tyrosinase.

The present invention also provides a method of additionally adding a catechol or a hetero catechol type structural material to suppress side reactions of the catechol type structural material due to peroxidation.

The present invention also provides a helper protein gene comprising the DNA sequences of SEQ ID NOs: 2 and 4, a recombinant DNA vector comprising the same, and a host cell transformed with the recombinant DNA vector.

Specifically, the present invention provides the following.

(1) DNA encoding a tyrosinase helper protein, the DNA having the DNA sequence of SEQ ID NO: 2.

(2) DNA encoding a tyrosinase helper protein, the DNA having at least 78% homology with the DNA sequence of SEQ ID NO: 2.

(3) DNA encoding a tyrosinase helper protein, the DNA having the DNA sequence of SEQ ID NO: 4.

(4) DNA encoding a tyrosinase helper protein, the DNA having at least 78% homology with the DNA sequence of SEQ ID NO: 4.

(5) A recombinant DNA vector comprising the DNA of any one of (1) to (4).

(6) A host cell transformed with the recombinant DNA vector of (5).

(7) A tyrosinase helper protein expressed by the DNA of any one of (1) to (4).

(8) A tyrosinase converted into an active structure by the tyrosinase helper protein of (7).

(9) A cell extract comprising the tyrosinase helper protein of (7).

(10) A host cell comprising the tyrosinase of (8).

(11) A cell extract comprising the tyrosinase of (8).

(12) A vector in which the DNA encoding any one of (1) to (4) and a DNA encoding tyrosinase are constructed together.

(13) A host cell transformed with the DNA vector of (12).

(14) An extract of a host cell transformed with the DNA vector of (12).

(15) Using a host cell transformed with a vector constructed with the DNA of any one of (1) to (4) together with a DNA encoding tyrosinase as a biocatalyst, the monophenolic structural substance is converted into a cate Method of producing colloidal structures.

(16) Using the extract of the host cell transformed with the vector constructed with the DNA of any one of (1) to (4) together with the DNA encoding tyrosinase as a biocatalyst, and converts the monophenol-type structural material To produce a catechol type structural material.

(17) The method according to (15), wherein the reaction is carried out at a temperature of 20 to 70 ° C.

(18) The method according to (16), wherein the reaction is carried out at a temperature of 20 to 70 ° C.

(19) The method according to (15), wherein the reaction is performed at pH 5 to 10.

(20) The method according to (16), wherein the reaction is performed at pH 5 to 10.

(21) The monophenolic structural substance used in the reaction according to (15) is phenol, resveratrol, para-coumaric acid, diadzeze, genistein, para-nitrophenol, para-cresol, para-vinylphenol, ortho -Aminophenol, curcumin, apigenin, floretine and chrysin.

(22) The method of (16), wherein the monophenolic structural material used in the reaction is phenol, resveratrol, para-coumaric acid, dyedze, genistein, para-nitrophenol, para-cresol, para-vinylphenol, ortho -Aminophenol, curcumin, apigenin, floretine and chrysin.

(23) The composition according to (15), further comprising a heterologous catechol-type construct which can be used as an inhibitor of tyrosinase, wherein the heterologous catechol-type construct is catechol, 3-methylcatechol, 4-methylcatechol, fatigue Galol and catechol violet.

(24) The method according to (16), further comprising a heterologous catechol-type construct which can be used as an inhibitor of tyrosinase, wherein the heterologous catechol-type construct is catechol, 3-methylcatechol, 4-methylcatechol, fatigue Galol and catechol violet.

(25) The method according to (15), further comprising a benzene and a monophenolic structural substance which can be used as an inhibitor of tyrosinase.

(26) The method according to (16), further comprising a benzene and monophenolic structural substance which can be used as an inhibitor of tyrosinase.

Tyrosinase can be produced in large quantities through expression in heterologous hosts through gene sequence optimization of helper proteins and stabilizing protein structure of tyrosinase. The produced tyrosinase can convert monophenolic structures into catechol type structures through whole cell reactions or cell extract reactions. Catalytic reactions to universal substrates are possible in the reaction of general monooxygenase without the need for custom enzyme improvement and screening.

In addition, when the inhibitor addition method is applied, the monophenolic structural material that is considered as the target substance can be accumulated as a catechol-type structural material without side reaction. High value-added catechol-type structural materials can be used as bioactive substances, and can be used as whitening agents, antioxidants, anticancer substances or antiviral substances.

In addition, it is possible to produce melanin in various kinds and in a large amount using the prepared recombinant strain, and may be applied to polymer synthetic additives, colorants, adhesives, sunscreens and general industrial chemistry.

1 is a schematic diagram of a structural formula of a compound used in the present invention and a method of accumulating catechol type structural substances by preventing peroxidation, wherein (a) is a monophenolic structural material, (b) is a catechol type structural material, and (c) ) Represents an ortho-benzoquinone structure and (d) represents a heterologous catechol type structure.
Figure 2 is a Streptomyces Arbor US subtilis MA4680 (Streptomyces avermitilis MA4680) showing the DNA sequence or amino acid sequence of the help protein derived from (a) is a DNA sequence of SAV1136 (SEQ ID NO: 1), (b) is a sequence transformed to optimize the DNA of SAV1136 (SEQ ID NO: 2), (c) is the DNA sequence of SAV5361 (SEQ ID NO: 3), and (d) is the sequence which converted the DNA of SAV5361 to be optimized (SEQ ID NO: 4).
3 is an electrophoretic gel photograph of the help protein MelC1 * (SAV1136 transformation) and thereby tyrosinase MelC2 with normal structure.
4 shows tyrosinase expressed by the help protein melC1 (SAV1136: SEQ ID NO: 1), melC1 * (SAV1136 conversion: SEQ ID NO: 2), melD1 (SAV5361: SEQ ID NO: 3) or melD1 * (SAV5361 conversion: SEQ ID NO: 4). It is the result of melanin production experiment by aze melC2 (SAV1137).
FIG. 5 is a graph related to the nature of the amino acid residues translated from the DNA sequence of the deleted DNA variant (SEQ ID NO: 5) and the removed sequence of DNA positions 1 to 78 of the help protein (SAV1136 Transformation: SEQ ID NO: 2).
FIG. 6 shows a total of 19 mutations and SEQ ID NO: 2 (WT) in which the TAC sequence encoding tyrosine at positions 274-276 of the helper protein (SAV1136 transformation: SEQ ID NO: 2) was substituted with a gene encoding another other amino acid. Experimental results of the production of melanin by the expressed tyrosinase.
7 is a graph of piacetanol productivity of tyrosinase expressing whole cell response following treatment with resveratrol and catechol.
FIG. 8 is a productivity graph of various catechol-like structures (initial concentration: 1 mM) using tyrosinase-expressing cell extracts, (a) physethanol production (b) 3'-dyezein production (c) caffeic acid Graph about production.
FIG. 9 shows the results of high performance liquid chromatography quantitative analysis for the production of piaceanol using tyrosinase expressing cell extracts.

The terms used in the present invention are commonly used in the art and those skilled in the art will understand the meaning, but briefly described in the present specification are as follows:

(1) Tyrosinase refers to an enzyme that catalyzes the production of melanin as a copper-containing protein.

(2) The monophenolic structural material means a material having a structure as shown in FIG.

(3) The catechol-type structural material means a material having a structure as shown in FIG.

(4) Heterologous catechol-type structural material means a material having a structure as shown in FIG.

(5) Cell extract means a microbial extract of the present invention in which helper protein and tyrosinase are expressed.

(6) Whole-cell reaction refers to a reaction using whole cell cells without breaking down the cell containing the specific enzyme and using the cell contents or separating and purifying the enzyme.

(7) Monooxygenase means an enzyme that catalyzes the introduction of an oxygen atom into a carbon-hydrogen bond and produces a hydroxyl moiety.

(8) Deoxygenase means an enzyme that catalyzes the benzoquinone form by removing hydrogen and electrons bound to oxygen in the catechol-type structural material.

(9) ORF means Open Reading Frame.

(10) PCR is a polymerase chain reaction, and means a method of specifically amplifying a certain region of DNA.

(11) A vector refers to a polynucleotide consisting of single-stranded, double-stranded, circular or ultra-stranded DNA or RNA, and may include components that are operably linked at a suitable distance to produce a recombinant protein.

Such components may include replication origins, promoters, enhancers, 5'mRNA leader sequences, ribosomal binding sites, nucleic acid cassettes, termination and polyadenylation sites, or selectable label formats, and such components may be used for specific applications. So one or more may be missing. The nucleic acid cassette may comprise restriction enzyme sites for insertion of the recombinant protein to be expressed. In a functional vector, the nucleic acid cassette contains a nucleic acid sequence to be expressed that includes a translation initiation and termination site, and may use a vector capable of inserting two kinds of cassettes into the vector, if necessary, and the functions mentioned above may be added. Can be sequenced.

The gene inserted into the recombinant vector may use E. coli strain BL21 (DE3) for expression, but may vary depending on the type of the inserted vector. Such vectors and expression strains can be readily selected by those skilled in the art.

Codon Sequence Changes and Expression of Tyrosinase in Helper Proteins

The sequence of the helper protein of the present invention is the locus number SAV1136 transform (SEQ ID NO: 2) and SAV5361 transform (SEQ ID NO: 4) derived from Streptomyces avermitilis MA4680. and b) and (d) sequences. Previously known Escherichia coli BL21 ( Escherichia coli Genes synthesized with the new sequences were used based on the codon usage table optimized for BL21 (DE3).

Gene synthesized in the present invention was adjusted so that the content of guanine and cytosine within 60% for ease of PCR. Genes synthesized in the present invention were excluded from the gene sequence corresponding to the sequence of NdeI, BamHI, EcoRI, HindIII and XhoI in order to utilize the restriction enzyme in the vector.

The optimized DNA encoding the helper protein in the present invention is not only SEQ ID NO: 2 and 4, but also DNA having at least 78% homology with the DNA sequence of SEQ ID NO: 2 and 4.

For example, a DNA sequence having at least 78% homology may be a DNA variant in which the codon sequence at DNA positions 1 to 78 of SEQ ID NO: 2 is inserted, deleted, substituted, or translocated, or a codon having a different codon corresponding to DNA positions 274 to 276. Substituted DNA variants are possible.

In certain embodiments of the DNA variants, the codon corresponding to DNA positions 274 to 276 in the codon sequence (SEQ ID NO: 2) of the help protein (SAV1136 transformation) is substituted with another codon, and the amino acid sequence translated from the codon sequence is 91 Burn tyrosine is substituted with other amino acids. Substituted amino acids constitute a helper protein of appropriate structure and help tyrosinase to have a normal folding structure.

In another specific embodiment, a primer is designed that can amplify the 79th to the last sequence (SEQ ID NO: 5) of the codon sequence (SEQ ID NO: 2) of the helper protein (SAV1136 transformation), and PCR amplification, restriction enzyme reaction, expression It was expressed together with tyrosinase through a series of processes that were conjugated to a dragon vector and introduced into an expression strain. In this case, the 26 amino acids at the N-terminal translated from the codons at positions 1 to 78 of SEQ ID NO: 2, which are immobilized on the membrane of the cell, are irrelevant to the property of forming the helper protein. Accordingly, DNA variants in which codon sequences are inserted, deleted, substituted or translocated at DNA positions 1 to 78 of SEQ ID NO: 2 may also help tyrosinase to have a normal folding structure, and the expressed tyrosinase may normally synthesize melanin. Could.

The help protein gene and tyrosinase gene synthesized in the present invention were inserted into one or more vectors in the form of a cassette using restriction enzymes and then injected into an expression host. After incubation with the addition of an enhancer capable of inducing the expression of tyrosinase and copper ions, a tyrosinase expressing whole cell or cell extract was produced. 4-40 degreeC is preferable, 4-30 degreeC is more preferable, and 10-20 degreeC is still more preferable. 16 to 24 hours is the most appropriate time for expression.

Tyrosinase Expressing Cell Extracts and Heterologous Catechol type  Using structural materials Catechol type  Production method of structural material

The obtained tyrogenase-expressing whole cell or normally expressed tyrogenase-expressing cell extract is reacted with the monophenol-type structural material as shown in (a) of FIG. 1 to produce a catechol-type structural material as shown in (b). In the reaction, a heterologous catechol-type structural material is added to inhibit the production process after the material (c). The concentration of the added inhibitor is preferably 1 to 100 times, more preferably 5 to 30 times, and more preferably 7 to 13 times the concentration of the monophenolic structural substance. 2-80 degreeC is preferable, 20-70 degreeC is more preferable, and 20-40 degreeC of the reaction temperature of this invention is more preferable. 5-11 are preferable, as for pH of a reaction liquid, 5-11 are more preferable, and 6-8 are more preferable.

Examples of the monophenolic structural material include phenol, resveratrol, para-coumaric acid, dydzein, genistein, para-nitrophenol, para-cresol, para-vinylphenol, ortho-aminophenol, curcumin, apigenin and flore. Tin or chrysin can be used.

As the inhibitor, a heterologous catechol-type structural material, a benzene or a monophenolic structural material, or the like can be used, and as the heterologous catechol-type structural material, catechol, 3-methylcatechol, 4-methylcatechol, pyrogallol or cate Cole violet, etc. can be used. As the monophenolic structural substance, phenol, diindase, genistein, p-coumaric acid, or the like can be used.

Although the specific method of this invention is demonstrated in detail as an Example, the technical scope of this invention is not limited to these Examples. Unless stated otherwise in the examples below, all percentages are based on 100% by weight of the final composition.

[ Manufacturing example  One]: Production of Tyrosinase Extract

Cells cultured in a medium consisting of tryptone (10 g / L), yeast extract (5 g / L) and sodium chloride (10 g / L) were washed three times with buffer (pH 8) to recover the extracellular medium. The component was removed. The recovered cells were suspended in Tris-HCl buffer at a volume ratio of 3 to 10 times and then disrupted with a sonicator. After the crushed extract was centrifuged at 13,000 rpm for 30 minutes, the supernatant was recovered and filtered to produce the final tyrosinase extract. It was possible to produce 12-26 mg / L tyrosinase in the medium.

[ Manufacturing example  2]: Expression of helper protein gene and tyrosinase expression with codon sequence control

Streptomyces Arbor US subtilis MA4680 (Streptomyces avermitilis MA4680) codon sequence (SEQ ID NO: 2), tyrosinase (Locus number of codon sequence (SEQ ID NO: 1), help the protein of the present invention (SAV1136 conversion) optimized for E. coli from the origin help protein (SAV1136): SAV1137) Expressed together. Expression of 1 mM CuSO ₄ and 0.2 mM enhancer (Isopropyl β-D-1-thiogalactopyranoside) was confirmed by electrophoresis that tyrosinase had a normal folding structure, which is shown in FIG. 3. In FIG. 3, MelC1 * is a helper protein (SAV1136 transformation), and MelC2 is a tyrosinase having a normal structure.

In addition, in the case of the experimental group expressing the helper protein of the present invention optimized for E. coli (SAV1136 conversion: SEQ ID NO: 2) and tyrosinase (SAV1137) (Fig. 4 (c)), dark brown from monophenolic structure Or black melanin, while the comparative group, the expression of tyrosinase alone (Fig. 4 (a)), the unconverted helper protein (SAV1136: SEQ ID NO: 1) and the expression of tyrosinase ( (B) of FIG. 4 and the optimized helper protein (SAV5361 transform: SEQ ID NO: 4) and the unconverted helper protein (SAV1136: SEQ ID NO: 1) are transformed together (FIG. It was confirmed that the cells and the medium color, which is shown in Figure 4.

[ Manufacturing example  3]: sequence number 2 of  5 'terminal position 1 to 78 of DNA  Expression of help protein gene and sequence of tyrosinase after removing sequence

It was expressed together with tyrosinase through a series of processes of primer design, PCR amplification, restriction enzyme reaction, conjugation to an expression vector, and introduction into an expression strain. In this case, tyrosinase had a normal structure, and it was confirmed that melanin was produced.

The sequence from which the sequence of DNA positions 1 to 78 of SEQ ID NO: 2 is removed is shown in FIG. 5 (A), and the N-terminal 26 amino acids translated from the DNA of the positions 1 to 78 of SEQ ID NO: 2 are hydrophobic. Consisting of the residues can be predicted through a commercialized prediction program (using Web-based SignalP), the results are shown in Figure 5 (B).

[ Manufacturing example  4]: SEQ ID NO 2 of  Position 274 through Of 276 DNA  Expression of Help Protein Gene Substituted Sequence and Tyrosinase Expression

The entire vector was obtained by using a primer to which a NNK sequence (N is A, C, G or T, and K is G or T), which randomly substituted the TAC sequence corresponding to positions 274 to 276 of SEQ ID NO: 2. The library was constructed by PCR and designed to encode 19 different amino acids via sequencing. If the sequence of the first methionine is omitted and the amino acid sequence is not known, the amino acid sequence corresponding to position 91 is tyrosine.

A total of 19 mutations in which the DNA sequence encoding tyrosine at positions 274 to 276 of SEQ ID NO: 2 with genes encoding other amino acids and the help protein activity of SEQ ID NO 2 (WT) were compared. The expression level of was confirmed, which is shown in FIG.

[ Example  One]: Tyrosinase expression Whole cells  Used Resveratrol hydroxylation of resveratrol and Piaceanol  ( piceatannol ) Production of

Tyrosinase-expressing cells cultured in Preparation Example 2 were washed without disrupting the cells, and 0.1 mM resveratrol and 1 mM catechol were added to produce 70% of piaceanol within 3 hours, as shown in FIG. 7. It was. After the reaction was extracted using the same amount of ethyl acetate or non-polar solvent and quantitatively analyzed by high performance liquid chromatography.

[ Example  2]: Using Tyrosinase-expressing Cell Extracts Resveratrol  hydroxylation of resveratrol and Piaceanol ( piceatannol ) Production of

Using tyrosinase-expressing cell extract obtained in Preparation Example 2, 0.1% resveratrol and 1 mM catechol were added to produce 50% of piaceanol within 10 minutes, which is shown in (a) of FIG. 8. It was. After the reaction was extracted using the same amount of ethyl acetate or non-polar solvent and quantitatively analyzed by high performance liquid chromatography. The high performance liquid chromatography quantitative analysis results are shown in FIG. 9.

[ Example  3]: Tyrosinase expression Whole cells  Used Dyed Jane  ( daidzein Hydroxylation of 3'- Hydroxide Jane  (3'- hydroxydaidzein Production of

Only the cells were washed without disrupting the tyrosinase-expressing cells cultured in Preparation Example 2 to produce 40% of 3'-hydroxydide zein within 3 hours by adding 1 mM catechol, 0.1 mM dyzezein. It was. After the reaction was extracted using the same amount of ethyl acetate or a non-polar solvent and quantitatively analyzed by high performance liquid chromatography.

[ Example  4]: Using Tyrosinase-expressing Cell Extracts Dyed Jane  hydroxylation of (daidzein) and 3'- Hydroxide Jane  (3'- hydroxydaidzein Production of

Using the tyrosinase-expressing cell extract obtained in Preparation Example 2, 40% of 3'-hydroxydidezein was produced within 10 minutes by adding 0.1 mM dyzezein and 1 mM catechol. It is shown to 8 (b). After the reaction was extracted using the same amount of ethyl acetate or a non-polar solvent and quantitatively analyzed by high performance liquid chromatography.

[ Example  5]: Tyrosinase expression Whole cells  Para- Kumaric acid  (p- coumaric  hydroxylation of acid) and Cafe Iksan  ( caffeic acid Production of

Tyrosinase-expressing cells cultured in Preparation Example 2 were washed without breaking the cells, and 0.1 mM para-coumaric acid and 1 mM catechol were added to produce 40% equivalent caffeic acid within 3 hours. After the reaction was extracted using the same amount of ethyl acetate or a non-polar solvent and quantitatively analyzed by high performance liquid chromatography.

[ Example  6]: Para- Using Tyrosinase Expressing Cell Extracts Kumaric acid  (p-coumaric acid Hydroxide reaction and Cafe Iksan  ( caffeic acid Production of

Using tyrosinase-expressing cell extracts obtained in Preparation Example 2, 0.1 mM para-coumaric acid and 1 mM catechol were added to produce 40% of caffeic acid within 10 minutes. Shown in After the reaction was extracted using the same amount of ethyl acetate or a non-polar solvent and quantitatively analyzed by high performance liquid chromatography.

[ Example  7]: Tyrosinase expression Whole cells  Used Resveratrol  hydroxylation of resveratrol and Of piceatannol  Production: As inhibitor, 4-meth Tilcatechol Use of

Tyrosinase-expressing cells cultured in Preparation Example 2 were washed without disrupting cells, and 0.1 mM resveratrol and 1 mM 4-methylcatechol were added to produce 70% of piaceanol within 3 hours. After the reaction was extracted using the same amount of ethyl acetate or a non-polar solvent and quantitatively analyzed by high performance liquid chromatography.

[ Example  8]: Using Tyrosinase-expressing Cell Extracts Resveratrol  hydroxylation of resveratrol and Of piceatannol  Production: As inhibitor, 4-meth Tilcatechol Use of

Using tyrosinase-expressing cell extracts obtained in Preparation Example 2, 0.1% resveratrol and 1 mM 4-methylcatechol were added to produce 50% of piaceanol within 10 minutes. After the reaction was extracted using the same amount of ethyl acetate or non-polar solvent and quantitatively analyzed by high-performance liquid chromatography.

Tyrosinase can be produced in large quantities through expression in heterologous hosts through gene sequence optimization of the aforementioned helper proteins and stabilizing protein structure of tyrosinase. The produced tyrosinase can convert monophenolic structures into catechol type structures through whole cell reactions or cell extract reactions. Catalytic reactions to universal substrates are possible in the reaction of general monooxygenase without the need for custom enzyme improvement and screening.

In addition, when the above-mentioned inhibitor addition method is applied, a monophenolic structural material that is considered as a target substance can be accumulated as a catechol-type structural material without side reactions. High value-added catechol-type structural materials can be used as bioactive substances, and can be used as whitening agents, antioxidants, anticancer or antiviral substances.

In addition, it is possible to produce melanin in various kinds and in a large amount using the prepared recombinant strain, and may be applied to polymer synthetic additives, colorants, adhesives, sunscreens and general industrial chemistry.

Attach an electronic file to a sequence list

Claims (26)

DNA encoding a tyrosinase helper protein, the DNA having the DNA sequence of SEQ ID NO: 2. A DNA in which the TAC sequence corresponding to positions 274 to 276 of SEQ ID NO: 2 is substituted with an NNK sequence, wherein N of the NNK sequence is A, C, G or T, and K is G or T; DNA coding. A DNA encoding a tyrosinase helper protein, the DNA having a DNA sequence of SEQ ID NO. delete A recombinant DNA vector comprising the DNA of any one of claims 1 to 3. A host cell transformed with the recombinant DNA vector of claim 5. A tyrosinase helper protein expressed by the DNA of any one of claims 1 to 3. A tyrosinase converted into an active structure by the tyrosinase helper protein of claim 7. An extract of a host cell comprising the tyrosinase helper protein of claim 7. A host cell comprising the tyrosinase of claim 8. Extract of a host cell comprising the tyrosinase of claim 8. A vector in which the DNA of any one of claims 1 to 3 and a DNA encoding tyrosinase are constructed together. A host cell transformed with the DNA vector of claim 12. Extract of the host cell transformed with the DNA vector of claim 12. Claims 1 to 3 of the host cell transformed with a vector constructed with the DNA and the tyrosinase-encoding DNA as a biocatalyst, converting the monophenolic structural material to the catechol-type structural material How to produce. The extract of a host cell transformed with a vector constructed with the DNA of any one of claims 1 to 3 and the DNA encoding tyrosinase as a biocatalyst, and the monophenolic structural material is converted into a catechol type How to Produce Structural Materials. 16. The process of claim 15, wherein the reaction is carried out at a temperature of 20 to 70 ° C. 17. The process of claim 16, wherein the reaction is carried out at a temperature of 20 to 70 ° C. The method of claim 15, wherein the reaction is carried out at pH 5-10. The method of claim 16, wherein the reaction is carried out at pH 5-10. 16. The monophenolic structural material used for the reaction according to claim 15, wherein the monophenolic structural material used in the reaction is phenol, resveratrol, para-coumaric acid, diadzeze, genistein, para-nitrophenol, para-cresol, para-vinylphenol, ortho-aminophenol , Curcumin, apigenin, floretin and chrysin. 17. The monophenolic structural material used for the reaction according to claim 16, wherein the monophenolic structural material used in the reaction is phenol, resveratrol, para-coumaric acid, didzeze, genistein, para-nitrophenol, para-cresol, para-vinylphenol, ortho-aminophenol , Curcumin, apigenin, floretin and chrysin. The method of claim 15, further comprising a heterologous catechol-like structure that can be used as an inhibitor of tyrosinase, wherein the heterologous catechol-like structure is catechol, 3-methylcatechol, 4-methylcatechol, pyrogallol and catechol. The method is characterized in that it is selected from the group consisting of kohl violet. 17. The method of claim 16, further comprising a heterologous catecholtic construct that can be used as an inhibitor of tyrosinase, wherein the heterologous catecholtic construct is catechol, 3-methylcatechol, 4-methylcatechol, pyrogallol and catechol. The method is characterized in that it is selected from the group consisting of kohl violet. 16. The method of claim 15, further comprising a benzene or monophenolic structure that can be used as an inhibitor of tyrosinase. 17. The method of claim 16, further comprising a benzene or monophenolic structure that can be used as an inhibitor of tyrosinase.

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