KR101566672B1 - Catechol derivatives prepared by using tyrosinase, method for preparation thereof, and application thereof - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a biosynthesis reaction of a catechol-type structural substance using tyrosinase, and effectively suppresses the secondary oxidation reaction of tyrosinase, thereby selectively catalyzing only the primary hydroxylation reaction of the monophenol-type structural substance. The present invention provides a technique for producing various functional catechol type materials using the same at high productivity and yield.

Description

[0001] CATECHOL DERIVATIVES PREPARED BY USING TYROSINASE, METHOD FOR PREPARATION THEREOF, AND APPLICATION THEREOF [0002] The present invention relates to a catechol-type structural substance prepared using tyrosinase,

The present invention relates to a method for producing selectively from various monophenolic compounds to catechol type structural materials with high productivity and yield by using tyrosinase having a wide substrate specificity. Accordingly, the present invention can mass-produce functional catheter-type structural materials using an enzyme reaction, and can be applied to raw materials and production of medical products.

Many catechol derivatives have been reported to exhibit good physiological activity in antioxidant, anti-cancer, anti-inflammatory or antiviral effects and have been reported to have a higher efficacy than monophenolic compounds . In particular, the catechol-type structural material has been utilized as an inhibitor of tyrosinase and has an effect of reducing the production of melanin by tyrosinase. In addition, the ortho-hydroxylation 3'-ODI, 3'-orthohydoroxydidzein, in daidzein (soy isoflavone) is a fermented soybean paste (JBC, 2010, vol. 285, pp. 21458; JBC, 2011, vol. 286, pp. 14246). As a catechol-type substance derived from apigenin, hydroxylated luteolin has been reported to be able to be extracted extensively in arichoke, which has good antioxidative properties, and recently can be used for the treatment of breast cancer (Food Chemistry , 2013, "Luteolin sensitizing drug-resistant human breast cancer cells to tamoxifen via the inhibition of cyclin E2 expression"). In addition, catechol-type substances such as hydroxytyrosol and piceatannol in olive oil are also known as powerful antioxidants. Catechol-based structures on polymeric materials can mimic the function of mussel proteins, and their applications range from medical materials to surface modified nanotechnology (Annu Rev Mater Res. 2011 1; 41: 99-132; Biofouling , 2012, 28: 8, 865-877).

When producing catechol-type structural materials using conventional biocatalysts, various mono-oxigenases including P450 enzymes are used. However, most monooxygenases require coenzyme and require a reductase for electron transfer. This affects the rate of bio-conversion of the substrate and is due to its low yield and productivity. On the other hand, mono-phenolic structure material oxidation of tyrosinase is relatively fast (For reference, kcat / Km value of the mushroom-derived tyrosinase is about 1000 mM ^-1 s ^- a ^1), a pair for the electron transfer reaction of the coenzyme It does not carry enzymes.

However, the problem with tyrosinase is that after the monophenolase activity which hydroxylates the monophenol-type structural material to produce the catechol-type structural material, the catechol-type structural material converted from the monophenol- And is biotransformed into quinonic compounds by diphenolase activity. Usually, the quinone-type material forms a radical to rapidly form melanin, and the resulting melanin dye is a stable structure and has a function of blocking light in the ultraviolet ray region.

Tyrosinase captures two copper ions with six histidine amino acids around the active site, and the distance between the two copper ions is controlled by the catechol oxidase containing copper and the constant . Tyrosinase has three forms of deoxy-tyrosinase, oxy-tyrosinase and met-tyrosinase depending on the presence or absence of oxygen and depending on the number of oxygen atoms bonded between the two copper atoms Respectively.

Deoxy-tyrosinase is a form that does not contain oxygen between the two copper ions. If oxygen is not supplied, the deoxy-tyrosinase becomes an inactivated product that can not undergo hydroxylation and oxidation reactions.

When oxygen in the air dissociates to solvent or hydrogen peroxide is supplied, one oxygen molecule (O ₂ ) is caught between two copper ions of the deoxy form, and the form is called oxy-tyrosinase. When the oxygen atom of the monophenol binds to one copper ion in this oxy form, the primary hydroxylation reaction of tyrosinase begins.

In the monophenolase activity, the oxygen molecule bonds are broken, one oxygen atom bonds with the ortho-position carbon, the hydroxyl group is inserted, and only one oxygen atom remains between the two tyrosinase groups. This form is called met-tyrosinase. At this time, the catechol-type structural material and met-tyrosinase are separated and rearranged at the angle formed by one oxygen atom between the two copper, and prepare for diphenolase activity.

The met form can oxidize the catechol type structure material, but does not participate in the monophenol type hydroxylation reaction. After the primary hydroxylation reaction, an oxidation reaction occurs in which the hydrogen ions are removed from the two hydroxyl moieties on the aromatic ring to combine with the remaining oxygen atom to form water. After using the two oxygen atoms to be bonded, the space between the two copper ions becomes empty and returns to the deoxy form. The product produced by the two biotransformation (hydroxylation and oxidation reactions) in the monophenol-type initial substrate is the ortho-benzoquinone type structure.

In the diphenolase activity, hydrogen is released from two hydroxyl groups of the catechol-type structure and can bond with two copper atoms. One oxygen atom receives two electrons from the catechol-type substance and is reduced to water (diphenolase activity). As mentioned above, the met form can oxidize the catechol type structure material, but does not participate in the monophenol type hydroxylation reaction. Therefore, when the resulting catechol-type structural material can not be oxidized by the met-tyrosinase after the oxidation reaction of the monophenol-type structural material of oxy-tyrosinase, the met-tyrosinase is oxidized by deoxy- tyrosinase It is not converted and is accumulated, and tyrosinase is inactivated.

It is difficult to accumulate the intermediate product catechol type structure material because the rate constant (k2) of the diphenolase activity is about 10 times larger than the kinetics constant k1 of the monophenolase activity. The research of tyrosinase mainly focuses on the role of melanogenesis reaction and its inhibitor. Therefore, this study is the first time to develop various catechol-type structure formation reaction optimization technique.

Korean Patent Publication No. 10-2012-0114072 Korean Patent Registration 10-0878394

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

Typical production methods of catechol-type structural materials employing biological methods can be classified into a combination of catechol and other structures, and a hydroxylation reaction of a monophenol-type structural material.

A representative reaction in the first case is a method of producing DOPA from Ajinomoto Japan. Catechol, acetic acid and ammonia are converted into DOPA using tyrosine-phenol lase and can be produced up to 100 g / L. Screening of the lyase corresponding to the specific substrate capable of forming a carbon-carbon bond with the catechol is required to produce a catechol-type structural substance using lyase. Accordingly, there is a limit to the production of various catechol-type structural materials.

In the second case, there is a method of using monooxygenase for the hydroxylation reaction of the benzene type structural material or the monophenol type structural material. Monooxygenase is a type of oxidoreductase that requires the supply of electrons by an electron transport system. The typical monooxygenase-reactive enzyme is cytochrome P450 (cytochrome P450), and the reaction is induced from the redox action of the heme structure of the iron ion 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 materials of various structures. However, most monooxygenases require coenzyme and require a reductase for electron transfer. This affects the rate of bio-conversion of the substrate and is due to its low yield and productivity.

Another biocatalytic hydroxylation reaction is the application of tyrosinase. The active site of tyrosinase is mainly present on the surface of the protein, and the substrate specificity of tyrosinase for the monophenol type structural material is wide, which is an advantage in the production of various catechol type structural materials. In addition, mono-phenolic structure material oxidation of tyrosinase is relatively fast (For reference, kcat / Km value of the mushroom-derived tyrosinase is about 1000 mM ^-1 s ^- a ¹⁾ mate an enzyme for the electron transfer reaction of the coenzyme . However, in the case of tyrosinase, various types of monophenol-type structural materials can be used as a substrate to convert into a catechol-type structure material faster than the above-mentioned two cases. At the same time, the catechol- To form a quinone form. The resulting quinone-type material readily forms radicals, which form a polymeric material such as melanin or oxidize the surrounding material.

In order to effectively accumulate the catechol-type structural material as an intermediate, first, the catechol-type material accumulation by the reduction of the quinone type material, the second, the oxidation prevention of the accumulated catechol type material, and the third, the inactivation by the accumulation of the catechol type material The reactivation of synapses should be studied.

It is an object of the present invention to provide a method for producing a functional catheter type structure material with high productivity and yield by using tyrosinase through the above three studies.

In order to inhibit the secondary oxidation of tyrosinase and effectively produce the catechol-type structural material, the strategy of the present technology is largely divided into three parts: first, catechol-type material accumulation by reduction of the quinone type material; Second, prevention of oxidation of the accumulated catechol-type material; And third, reactivation of inactivated tyrosinase.

Specifically, first, the present invention provides a method for effectively reducing a quinone-type material produced by a secondary oxidation reaction of tyrosinase to a catechol-type structure material.

Second, the present invention provides a method for protecting a catechol-type structural material produced by a primary hydroxylation reaction of tyrosinase from secondary oxidation of tyrosinase by inducing a coordination bond with another element.

Third, the present invention provides a technique for optimizing enzyme activity by effectively controlling three active forms of tyrosinase. When the catechol-type structural material is not oxidized, tyrosinase is inactivated because one oxygen atom of the meth-tyrosinase is not easily removed. This inactivated tyrosinase can be reactivated by reducing power.

Specifically, the present invention provides the following.

(1) A method for producing a catechol-type structural material from a monophenol type structural material using tyrosinase, which comprises any one of the following steps (a) to (c):

(a) reducing a quinone type structural material produced by a peroxidation reaction of tyrosinase with a catechol type structure material by introducing a reducing power;

(b) coordinating the resulting catechol-type structural material with transition metal ions or boron ions;

(c) introducing a reducing power to activate the met-tyrosinase.

(2) The method according to (1), wherein the reducing power used in step (a) or (c) is selected from the group consisting of electrochemistry, NADH, NADPH, ascorbic acid, cysteine, p -coumaric acid, hydroxylamine, catechol, pyrogallol, ≪ / RTI > phosphoric acid.

(3) The pharmaceutical composition according to item (1), wherein the monophenol type structural substance is selected from the group consisting of tyrosine amino acid, tyrosol, resveratrol, daidzein, genistein, apigenin, floretine, phenol, para- , Para-vinylphenol, and ortho-aminophenol.

(4) The pharmaceutical composition according to item (1), wherein the catechol-type structure substance is luteolin, 3-hydroxyproline, 3'-orthohydroxydizzean, orlovole, Dihydroxynitrobenzene, 3,4-dihydroxytoluene, 3,4-dihydroxystyrene, and 3,4-dihydroxystyrene.

(5) The method according to (1), further comprising the step (d) of separating and refining the catechol structure material,

(d) dissolving the product containing the catechol-type structural material in an organic solvent to obtain an aqueous layer, lowering the pH of the obtained water layer, further dissolving the organic layer in an organic solvent to obtain an organic layer, and evaporating the obtained organic layer to obtain a catechol- step.

(6) The method according to (1), wherein the mono phenol type structural material is a tyrosine residue on the polymer.

(7) A polymer crosslinking reaction method using a catechol-type structural material prepared by the method according to (6).

The present invention relates to the preparation of a functional catheter type structural material using tyrosinase and its application, and it can be applied to tyrosinase derived from various sources. This is because it regulates the hydroxylation reaction from various monophenol type structural materials to functional catechol type substances according to the tyrosinase substrate specificity and enables the production of the catechol type structural material which was difficult to extract or organic chemical synthesis in the past, Can contribute to the experiment of efficacy. Recently, studies on anticancer activity of hydroxides of flavonoids / isoflavonoids have been reported. However, the site-specific organic synthesis method is difficult, and there is a limit in extracting naturally occurring hydroxylated flavonol / isoflavone. Through this study, the production of functional catheter structure materials becomes easy, and physiological studies and new materials / functional materials can be developed.

In addition, tyrosinase can be used to hydrolyze tyrosine, which is a monophenol-type amino acid residue on a protein, and can be improved as a protein capable of simulating the adhesion function of a mussel protein. This property-improving protein can be developed into scaffolds of tissue engineering and further developed into a functional protein drug delivery system (Annu Rev Mater Res. 2011 1; 41: 99-132; Biofouling, 2012, 28: 8, 865-877; Current Opinion in Biotechnology 2013, 24: 1-7).

FIG. 1 is a schematic diagram for producing an efficient catechol-type structural material with tyrosinase, wherein (a) is a monophenol type structural material, (b) is a catechol type structure material, (c) is a quinone type structure material, (d) is a structure that forms a coordination bond with another element of the catechol-type structural material. (1) is a primary hydroxylation reaction of tyrosinase, (2) is a secondary oxidation reaction of tyrosinase, (3) is a reaction of deoxy-tyrosinase with two oxygen molecules present in the active site (4) is a reaction with a reducing agent that reduces one oxygen atom of met-tyrosinase, and (5) is a reaction in which the hydroxylated catechol-type structural material is coordinated (6) reduces the quinone type structural material and accumulates it again in the catechol type structure material, and (7) is a reaction in which the peroxidized quinone type material is polymerized into melanin.
FIG. 2 is a table listing the reducing agents added during the whole cell reaction of tyrosinase. Each of the reducing agents (NADH, L-ascorbic acid, glutathione, cysteine, hydroquinone, 1-naphthol, p -coumaric acid, curcumin, Pyrogallol, or ferulic acid), the relative yield of the hydrolyzed piceatannol in trans-resveratrol.
Figure 3 shows the tyrosine amino acid (L) of three different derived tyrosinase (BMT_tyr: Bacillus megaterium tyrosinase, ABS_tys: Agaricus bisphosphorus tyrosinase, and SAV_tyr: Streptomyces avermitilis tyrosinase) -tyrosine and L-dopa, respectively. The results are shown in Fig.
Fig. 4 is a reaction formula showing catechol-type structural substances produced by the hydroxylation reaction of tyrosinase from a monophenol type structural substance.
FIG. 5 is a quantitative analysis of high performance liquid chromatography to confirm the coordination of hydroxylated 3'-orthohydroxyzase (3'-ODI, 3'-orthohydoroxydidzein) through pH control.
FIG. 6 is a quantitative analysis of high performance-liquid chromatography confirming the coordination bond between hydroxylated orobol and luteolin through pH control.
FIG. 7A is a graph showing the productivity of 3'-ODI in a 5 mL in vitro reaction including 1 mM dideoxyne and 100 nM tyrosinase in a pH 9 boronate buffer in hours.
FIG. 7b is a graph showing the productivity of 3'-ODI in a 5-mL in vitro reaction, including 300 μM daidzein and 100 nM tyrosinase, in hours in a pH 9 tris-HCl buffer.
8 is a graph showing the hydroxylation reaction of 1 mM genistein added with hydroxylamine or ascorbic acid in time units.
9 is a graph showing the hydroxylation reaction of 1 mM resveratrol added with hydroxylamine or ascorbic acid in time units.
FIG. 10 is a schematic diagram of a technique for separating / purifying the resulting catechol blood structure material, and the method thereof is described in detail in Example 5.
11A shows a result of GC / MS analysis of 3'-orthohydroxydizzean (3'-ODI) generated from daidzein. The retention time (1) 18.18 min is the 3'-ODI separated by GC, m / z of 486.48, m / z of TMS and 471.50 of MW (TMS) 15, m / z of 383.39, and m / z of (Mw (TMS) -103)
FIG. 11B shows the results of analysis of piceatannol produced from trans-resveratrol by GC / MS. The retention time of 14.82 minutes represented by (1) And m / z of MW (TMS) is 517.45, and m / z of MW (TMS) -15 is 517.45, which is represented by (b).
FIG. 12 shows a state in which a catechol-type structural material according to pH is coordinated with iron ions. FIG.

Hereinafter, the present invention will be described in more detail.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

The present invention provides a method for producing a catechol-type structural substance using tyrosinase or tyrosinase-expressing microorganism (whole cell).

In addition, a method of coordinating and accumulating catechol-type structural substances in order to inhibit the biotransformation reaction of the catechol-type structural material by the peroxidation reaction of tyrosinase is provided.

The present invention also provides a method for effectively reducing a quinone-type material produced by peroxidation to a catechol-type structure material.

Specifically, the present invention provides a method for inhibiting the oxidation of a catechol-type substance by i) reducing catechol-type substances by reduction of a quinone type substance, ii) preventing oxidation of an accumulated catechol-type substance, and iii) , It is possible to effectively accumulate the catechol-type structural material.

First, the present invention provides a method for effectively reducing a quinone-type material produced by a secondary oxidation reaction of tyrosinase to a catechol-type structure material. This makes it possible to delay the polymerization of the quinone type structural material into melanin. This is because the radicals are stabilized by the reducing power, and the quinone type material can be converted into a catechol type structure material by receiving electrons. As effective reducing power that can be used without adversely affecting enzyme activity, there are electrochemical methods and reducing agents such as the compounds listed in the reducing agent list of FIG. It was confirmed that dark melanin formation was delayed when the reducing agent in the list was added to the oxidation reaction. Although melanin formation is delayed at this time, it was confirmed that the reduction of the monophenol-like substance as the initial substrate is continuously performed. Thus, the above-mentioned reducing power affects only melanin formation and is independent of the inhibition of tyrosinase activity Respectively. The reducing power may be selected from the group consisting of electrochemistry, NADH, NADPH, ascorbic acid, cysteine, p -coumaric acid and hydroxylamine, catechol, pyrogallol and caffeic acid. Preferably, the reducing power can be continuously provided during the reaction using the NAD (P) H regeneration enzyme such as glucose dehydrogenase, which can be more efficiently applied in the production of catechol-type substances in vivo have.

Second, the present invention provides a method for protecting a catechol-type structural material produced by a primary hydroxylation reaction of tyrosinase from secondary oxidation of tyrosinase by inducing a coordination bond with another element. It is known that catechol-type structure materials are coordinated with iron ions depending on pH (Proc Natl Acad Sci USA. 2011, 15 (2), 108 (7), 2651-2655). This is the same as mussel protein binding to metal ions in organic matter (Annu Rev Mater Res. 2011 41, 99-132). Based on this, materials other than iron ions, which are harmless to the activity of tyrosinase and which are coordinated with the catechol-type structural material, were selected and the experiment was conducted for the examples of this study.

Boron (B) has an sp2 hybrid orbital, but becomes sp3 in the presence of a Lewis base. It has been reported that catechol-type structural materials act as Lewis bases and coordinate bond with one or two catechol substances depending on pH (BBA, 2002, 1569, 35-44). The complex bound to the boronic acid increases the hydrophilicity. Due to this mechanism, the boronic acid ion is used to stabilize the sugar having a diol, and also to extract caffeine from coffee beans or tea leaves. The catechol-type material in which coordination bonds are formed is not oxidized by tyrosinase since all of the hydroxyl groups are coordinately bound to the boronic acid. Therefore, it is also used to exclude the secondary oxidation reaction and to observe only the primary hydroxylation reaction (JACS, 2003, vol. 125, 43, 13034-13035).

Third, the present invention provides a technique for optimizing enzyme activity by effectively controlling three active forms of tyrosinase (dioxy form, oxy form and met form). When the catechol-type structural material is not oxidized, tyrosinase is inactivated because one oxygen atom of the meth-tyrosinase is not easily removed. One of the oxygen atoms of the inactivated met-tyrosinase can be reduced by another reducing agent or an electrochemical reductive force and fall into a water molecule. The tyrosinase is again capable of accepting an oxygen molecule (O ₂ ) . Examples of such reducing agents include hydroxylamine (NH ₂ OH, hydroxylamine), NAD (P) H, ascorbic acid, and the like, in addition to catechol-type substances,

As shown in Reaction 4 of FIG. 1, hydroxylamine reduces tyrosinase, which can not accept a monophenol-like substance as a substrate, by reducing oxygen atoms remaining in the copper in the active site to water (JACS, 2003, vol. 125, 43, 13034-13035). Deoxy-tyrosinase forms an oxy-tyrosinase by forming an oxygen molecule and a copper-oxygen complex (μ-η ² : η ² peroxo binuclear copper complex). Reaction 4 of FIG. 1 can be performed not only with hydroxylamine but also with a reducing agent accessible to the active site, that is, NAD (P) H, ascorbic acid, another catechol-type substance or the like.

The term used in the present invention is commonly used in the art and any person skilled in the art can understand the meaning thereof.

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

(2) Monophenol type structural material - This refers to a material having a structure as shown in FIG. 1 (a). Examples of the monophenol type structural material include tyrosine, tyrosol, stilbene such as resveratrol, daidzein, genistein, apigenin, flavonone / isoflavone series such as apigenine and phloretin as well as phenol, para-coumaric acid, para-nitrophenol, Para-cresol, para-vinylphenol, and ortho-aminophenol. Preferably, resveratrol, para-coumaric acid, daidzein, genistein, para-nitrophenol, para-cresol, para-vinylphenol, apigenin or floretine and the like can be used.

In addition, the monophenol-type structural substance may be a tyrosine residue on the polymer substance, and the polymer substance may be a protein (polypeptide).

(3) Catechol-type structural material - This refers to a material having a structure as shown in FIG. 1 (b). The catechol-type structural material is ortho-specifically hydroxylated to a monophenol-type structural tyrosine substance, and is selected from the group consisting of luteolin, 3-hydroxyphoretin, 3'-ODI, orobol, A stilbene series such as the flavonone / isoflavone series and piceatannol, such as hydroxyltyrosol, dopa, caffeic acid, 3,4-dihydroxynthrobene, 3,4-dihydroxytoluene, 3,4-dihydroxystyrene, and the like can be used. have. Preferably, the preferred monophenol-type structural materials listed above are ortho-specific hydroxylated forms, which in turn are selected from the group consisting of: Pisatanol, caffeic acid, 3'-ODI, Orobor, 3,4-dihydroxynitrobenzene (3, 4-dihydroxynthrobenzene, 3,4-dihydroxytoluene, 3,4-dihydroxystyrene, luteolin, 3-hydroxyphloretin ).

(4) Quinone type structure material - This refers to a material having a structure as shown in FIG. 1 (c).

(5) Coordination complex material - A complex forming a structure such as the mono-complex, bis-complex or tris-complex of FIG. 1 (d) . Transition metal ions or boric acid ions can be used.

(6) cell extract-assistant protein and tyrosinase-expressing microbial extract of the present invention.

(7) Whole-cell reaction - refers to a reaction using the whole cell without disrupting or using the cell contents by disrupting cells containing a specific enzyme.

(8) Tyrosinase A primary hydroxylation reaction (monooxygenase activity) - a tyrosinase enzyme reaction that catalyzes the introduction of oxygen atoms into a carbon-hydrogen bond and produces hydroxyl moieties.

(9) Tyrosinase secondary oxidation reaction (dioxygenase activity) - refers to a tyrosinase enzyme reaction catalyzing the removal of hydrogen and electrons bound to oxygen in a catechol-type structural material in the form of benzoquinone.

(10) PCR-Polymerase Chain Reaction (PCR) is a method of specifically amplifying a certain region of DNA.

(11) vector - means a polynucleotide consisting of single strand, double strand, circular or super stranded DNA or RNA. The vector may comprise components operatively linked at appropriate distances so as to produce the recombinant protein. These components may include a replication origin, a promoter, an enhancer, a 5'mRNA leader sequence, a ribosome binding site, a nucleic acid cassette, a termination and a polyadenylation site, and a selectable marker form, One or more may be missed. The nucleic acid cassette may contain a restriction enzyme site for insertion of a recombinant protein to be expressed. In a functional vector, the nucleic acid cassette contains the nucleic acid sequence to be expressed, including the translation initiation and termination sites. If necessary, a vector capable of inserting two types of cassettes into a vector may be used, and the above-mentioned functions may additionally be sequenced. The gene inserted into the recombinant vector may be E. coli strain BL21 (DE3) for expression, but it 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.

(12) a reducing agent capable of retarding the quinone type structural material from being polymerized with melanin and effectively reducing the quinone type material to a catechol type structure material, wherein NAD (P) H, L-ascorbic acid, glutathione, Cysteine, a heterogeneous catechol structure material, a benzene or a monophenol type structure material, and the heterogeneous catechol structure materials include catechol, 3-methylcatechol, 4-methylcatechol, caffeic acid, pyrogallol or catechol beollet. As the monophenol type structure material, phenol, didezane, genistein, p -coumaric acid and the like can be used.

(13) Hydroxylamine, NAD (P) H, ascorbic acid or other catechol-type substances can be used as reductants accessible to active sites for reactivation of tyrosinase.

Expression method 1: Expression of tyrosinase

A tyrosinase DNA sequence derived from Bacillus megaterium (BMT) and Streptomyces avermitilis (SAV) is amplified by PCR and inserted into pet28a or petDuet vector, respectively. And E. coli, respectively, as histag (6 histidine). The expression method is as follows. The plasmid in which the tyrosinase base sequence was inserted was transformed into BL21 competent cells and then cultured in an LB solid medium containing an antibiotic suitable for each plasmid in a 37 ° C incubator. One colony was inoculated into 5 mL LB liquid medium containing antibiotics and cultured at 37 ° C incubator for 8 hours at 200 rpm. This was again inoculated with 1v% (500uL) in a fresh liquid medium containing 50mL of antibiotics. When the cell OD 600nm reached 0.6, 2mM IPTG and 1mM CuSO ₄ were added and the protein expression was induced for 17 hours at 200 rpm in an incubator at 18 ° C. After 17 hours, the cells were centrifuged at 4000 rpm and washed by 5 mL PBS. After washing, the whole cell reaction proceeded in tris-HCl buffer or borate buffer, and the other cell extracts were prepared by ultrasonication. The in vitro whole cell reaction was carried out by using the receptor portion of the cell extract or a protein purified by a conventional 6-histag purification method, and specifically described in each Example.

Reaction method 1: Synthesis of tyrosinase and Reducing substance  Used Catechol type  Method of production of structural material

The obtained microorganism or the produced tyrosinase extract or the purified tyrosinase is reacted with the monophenol type structural material as shown in FIG. 1 (a) to produce a catechol type structure material as shown in (b). (C) the production process after the substance is inhibited by adding a reducing agent to the reaction.

The concentration of the added reducing agent is preferably 1 to 100 times, more preferably 5 to 30 times, and even more preferably 7 to 13 times, the concentration of the monophenol type structural material. The reaction is protected from the secondary oxidation of tyrosinase by adding a substance capable of coordinating with the generated catechol.

The concentration of the coordination bond precursor added is preferably 1 to 10,000 times, more preferably 50 to 600 times, and even more preferably 100 to 500 times, the concentration of the monophenol type structural material.

The reaction temperature of the present invention is preferably 2 to 80 占 폚, more preferably 20 to 70 占 폚, and still more preferably 20 to 40 占 폚.

The pH of the reaction solution is preferably 5 to 11, more preferably 5 to 11, and even more preferably 7 to 9. As shown in Fig. 3, the rate of reaction for tyrosinase was different, and tyrosinase was used depending on the substrate specificity.

The specific method of the present invention will be described in detail by way of examples, but the technical scope of the present invention is not limited to the examples. In the following examples, unless otherwise specified, all percentages are based on 100% by weight of the final composition.

pH  The initial substrate ( Monophenol type  Change in solubility of the structural material)

The isoflavone-based daidzein has low solubility in water, so it can be dissolved in neutral water or buffer at room temperature in the presence of 0.1% DMSO to about 300 μM. However, when using a high-concentration boronic acid buffer solution, the solubility increases with increasing pH, and the solubility increases at a level of 5 mM at a pH of 9 to a level of 10 times, and the solubility increases to a level of 30 mM at a pH of 10 there was. Unlike other monooxygenases, tyrosinase has activity at a wide range of pH, enabling it to increase reaction productivity using a high initial substrate.

At pH 9, the tyrosinase activity range, a 5 mL in vitro reaction involving 300 μM daidzein and 100 nM tyrosinase produced 15 mg of 3'-ODI per hour, whereas 1 mM diiodes in pH 9 borate buffer A 5 mL in vitro reaction involving 50 μM zein and 100 nM tyrosinase produced a 3'-ODI of 45 mg per hour and was able to increase its productivity by three. This is illustrated in Figures 7A and 7B.

Depending on the reducing agent Catechol type  Comparison of yield of structural materials.

Cells expressing SAV-derived tyrosinase, which were cultured according to the above-described expression method 1, were washed without destroying the cells, and the cells were washed with 0.1 mM resveratrol, 1 mM various reducing agents (NADH, L-ascorbic acid, glutathione, cysteine, hydroquinone , 1-naphthol, p -coumaric acid, curcumin, catechol, pyrogallol or ferulic acid) was added to produce fichethanol. After the reaction, the mixture was extracted with the same amount of ethylacetate (EA) or nonpolar solvent, and quantitatively analyzed by high performance liquid chromatography to compare the production efficiency of physisathenol by adding the reducing agent.

Catechol type  Of structural material Coordination  Combination.

500 uM of dideoxyne was reacted with 200 nM of purified BMT-derived tyrosinase in the same manner as in Reaction Scheme 1. At this time, boronic acid was added to induce coordination. As shown in Fig. 5 (a), the 3'-ODI coordinated in the alkaline reaction solution was confirmed by high-performance liquid chromatography because it was hydrophilic and was not extracted with an organic solvent. Also, in FIG. 5 (b), when 2 M of HCl was treated, it was confirmed that 3'-ODI of 200 uM was detected when the coordination bond was released.

Also, 1 mM of genistin and apigenin were subjected to a hydroxylation reaction using purified 300 nM BMT tyrosinase. As shown in FIG. 6, hydroxylated orbital and ruteolin were also co-ordinated in an alkaline reaction solution and extracted into an organic solvent , And when the coordination bond is released from the acidic reaction solution, it is confirmed by high performance liquid chromatography that the compound is extracted into an organic solvent.

Prevention of inactivation of tyrosinase

Tyrosinase cultured in Expression Method 1 was subjected to the hysteresis purification and 100 nM was added to the reaction. To prevent inactivation of tyrosinase, hydroxylamine or ascorbic acid was added to activate the tyrosinase. At this time, the concentration of the hydroxylamine or ascorbic acid added is 1 to 100 times as much as the substrate, more preferably 3 to 50 times, and even more preferably 10 to 20 times. Through the above composition, 1 mM daidzein was reacted according to Reaction Scheme 1 and 100 μL of the reaction product was extracted with 4 times of ethylacetate (EA) or nonpolar solvent, followed by high performance liquid chromatography And analyzed quantitatively. As a result of quantitative analysis, 1 mM 3'-ODI was produced at 100% yield in 6 hours of reaction, as shown in FIG. 7A.

In the same way, the site-specific hydroxylation of genistein and resveratrol was induced, and these monophenol-type substances were converted to functional catechol-type structural materials through primary hydroxylation of tyrosinase there was. Genistein was biotransformed to 95% orobol in response to time, and resveratrol was biotransformed to 100% piceatannol within the time of the reaction. This is shown in Figures 8 and 9.

Separation and purification of the resulting catechol-type structural material

In order to separate the catechol-type structural material produced in Example 4, the solubility of the product and of the reactants was used. The reaction was carried out in an alkaline buffer solution, and the resulting catechol-type material was subjected to coordination bonding to increase the hydrophilicity (1 in FIG. 10). The monophenol type structural material which does not form a coordination complex is extracted into ethylacetate (EA) having four times the reaction volume. The organic solvent and the water layer are divided into layers as shown in FIG. 10 due to the difference in specific gravity. Separately, 3-1 (water layer) and 3-2 (organic layer) in Fig. 10 are separated by buret, and the separated organic layer is evaporated to prepare the remaining monophenol type structural material Respectively. The water layer was lowered in pH using 1M HCl and the catechol type structure material was extracted again with 4 times ethylacetate (EA) (FIG. 10, 4). After extraction, the acidic reaction liquid layer was separated into buret. The ethylacetate (EA) layer was removed from ethylacetate (EA) through a vacuum evaporator to obtain a purified powdery catecholic structure material.

Mass spectrometry of hydroxylation products using tyrosinase

The functional catheter structure materials produced in Example 5 were qualitatively analyzed by gas chromatography-mass spectrometry (GC-MS), and the results are shown in FIG.

Specifically, FIG. 11A shows the result of GC / MS analysis of 3'-orthohydroxydizze (3'-ODI) generated from daidzein, showing the storage time ( retention time 18.18 min is the 3'-ODI separated by GC, 486.48 as shown in (a), m / z as m / z of MW (TMS), 471.50 as shown in (b) TMS) -15, m / z of 383.39 and m / z of MW (TMS) -103, respectively. The results are shown in the preceding paper (Hydroxylation of daidzein by CYP107H1 from Baciilus subtilis 168, Journal of Molecular Catalysis B: Enzymatic, Vol. 59, issue 4, August 2009, Pages 248-253).

FIG. 11B shows the result of analysis of piceatannol produced from trans-resveratrol by GC / MS. The retention time of 14.82 minutes represented by (1) (M / z) of MW (TMS) is 517.45, and m / z is m / z of MW (TMS) -15. The cancer preventative agent resveratrol is converted to the anticancer agent piceatannol by the cytochrome P450 enzyme CYP1B1, British Journal of Cancer (2002) 86, 774-778.

Polymeric stomach Monophenol type  Of matter Catechol type  As a structural material Raw conversion : As a mussel protein  copy

SAV tyrosinase was used to effectively convert the tyrosine residue on the polymer to a doping residue. ELP_Y, which has a total of 180 amino acid sequences of VPGYG 12 (VPGVG) 24, was dissolved in 300 uL of pH 9 50 mM tris-HCl buffer at a concentration of 0.5 w%, followed by addition of 2 mM FeCl ₃ and ascorbic acid, After incubation for 2 hours with 500 nM of SAV tyrosinease, the pH was changed using 1 M NaOH and 1 M HCl. At pH 5, dissolved ELP_Y precipitated in powder form at pH 7. When pH was adjusted to 11, it was confirmed that ELP_Y gelated. When the pH was lowered again, the formed hydrogel was dissolved and confirmed to be a reversible reaction. This is consistent with previous reports that simulate the function of mussel proteins (Proc Natl Acad Sci USA. 2011 15, 108 (7), 2651-2655).

Claims (7)

A method for producing an ortho-hydroxide reaction material from a monophenol-type structural material using tyrosinase,
Wherein the ortho-hydroxylating agent is luteolin, 3-hydroxyproline, 3'-orthohydroxydizane or ourobol,
A method of manufacturing comprising the steps of: (a)
(a) reacting tyrosinase by removing ascorbic acid between the met-tyrosinase dodecyl ion by introducing ascorbic acid; And
(b) dissolving the reaction product containing the generated catechol-type structural material in an organic solvent to separate the substrate that dissolves in the organic solvent and the ortho-hydroxide reaction material remaining in the water layer, and lower the pH of the obtained water layer to remove the boron ion Or a transition metal ion is removed, further dissolved in an organic solvent to obtain an organic layer, and the obtained organic layer is evaporated to obtain an ortho-hydroxide reaction material. 2. The method according to claim 1, wherein the monophenolic structure material is selected from the group consisting of daidze, genistein, apigenin and floretine. delete delete delete delete delete

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