KR102068459B1 - Method for preparation of catechol derivatives by using tyrosinase derived from burkholderia thailandensis and application thereof - Google Patents

Abstract

A method for preparing a catechol-type structural material from a monophenolic structural material using tyrosinase, wherein the tyrosinase is a tyrosinase derived from Burkholderia thailandensis , and the preparation is pH 1.5 to A production method, characterized in that the acidic conditions of pH 6.5.

Description

METHOD FOR PREPARATION OF CATECHOL DERIVATIVES BY USING TYROSINASE DERIVED FROM BURKHOLDERIA THAILANDENSIS AND APPLICATION THEREOF}

The present invention relates to a method for producing a catechol-type structural material using Berkholderia tyrandensis-derived tyrosinase and its application.

Tyrosinase is the primary hydroxylation activity of hydroxylating monophenolic structural materials (eg L-tyrosine) to catecholic structural materials, and melanin pigment precursors which are biotransformed from monophenolic structural materials. The secondary oxidation reaction (dipehnolase activity) to oxidize to the quinonic compounds (quinonic compounds) is carried out continuously. Under basic aerobic conditions, at basic pH conditions above the monophenolic pKa, the catechol-type structural material readily oxidizes to form a quinone-like material. The quinone type material forms radicals to rapidly form melanin, and the resulting melanin pigment has a function of blocking light in the ultraviolet region with a stable structure. Tyrosinase traps two copper ions with six histidine amino acids around the active site, and the distance between the two copper ions is constant within the catechol oxidase and other tyrosinase families containing copper. It is preserved. Tyrosinase has three forms, deoxy-tyrosinase, oxy-tyrosinase and met-tyrosinase, depending on the presence of oxygen and the number of oxygen atoms bonded between the two coppers. Separated by.

The deoxy-tyrosinase is a form that does not contain oxygen between two copper ions, and is an inactive substance that cannot undergo hydroxylation and oxidation without oxygen supply.

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

In the primary hydroxylation, oxygen molecular bonds are broken, one oxygen atom bonds with ortho-positioned carbon, a hydroxyl group is inserted, and only one oxygen atom remains between the two copper tyrosinase. This form is called a mat form. At this time, the catechol-type structural material and meth-tyrosinase are separated, one oxygen atom between the two copper rearranges the angle formed, and prepares for the secondary oxidation reaction. As mentioned above, the metform can oxidize the catechol-type structural material but does not participate in the monophenol-type hydroxide reaction. Therefore, after the oxidation reaction of the monophenolic structural material of oxy-tyrosinase, if the resulting catechol-type structural material is not oxidized by met-tyrosinase, meth-tyrosinase is converted into deoxy-tyrosinase. It can't be converted and will accumulate, and tyrosinase is inactivated.

Usually there is because the reaction rate is the speed (V _{max_di)} of the secondary oxidation than (V _{max_mono)} of the primary hydroxylation size 10 times difficult to accumulate the category kolhyeong structure material intermediate. Therefore, many enzymatic studies have been conducted to control the ratio of the first and second oxidation rates of tyrosinase for the production of catechol-type structural materials which are intermediates of this continuous oxidation reaction (product of the first oxidation reaction). Document 1).

Catechol-type structural substances, which are the primary hydroxylation products of monophenolic structural substances, have been reported to exhibit good physiological activity in antioxidant, anti-cancer, anti-inflammatory or antiviral effects, and are reported to have higher effects than monophenolic structural substances. have. For example, 3-hydroxy phloretin, a type of catechol-type structural material, has a higher antioxidant activity than floretine and inhibits cholesterol accumulation, which may cause atherosclerosis and Parkinson's disease. It is well known to inhibit the lipid peroxidation which exists (nonpatent literature 2). In addition, among polyphenolic phytochemicals, dydzein and genistein are isoflavones present in soybean, and 3'-orthohydroxy didezein and 3'-orthohydroxy zenistein are hydroxylated at specific positions. It is known to exist in soybean fermented foods, and is known to have antioxidant function and anticancer function (Non Patent Literatures 6 and 7). It is known that the glycosylation agents, genistin and dydazine, also improve antioxidative function by site specific hydroxylation (Non Patent Literature 8). In addition, catechol-type substances such as quercetin in various plants, hydroxytyrosol in olive oil, and astringin in grapes are more effective than monophenolic substances. Has strong antioxidant capacity Therefore, catechol-type structural materials are in the spotlight as raw materials for food additives and therapeutic agents, and research on production methods using biological technologies has been continuously conducted (Non-Patent Documents 3 and 4).

The problem with the biosynthesis of the catechol type structural material is that, as mentioned above, most of the catechol type structural materials are unstable, and are easily oxidized at a pH higher than neutral to be modified into a quinone type material which is a radical forming material (Non Patent Literature 5). In addition, since the reported tyrosinase is active at pH 6 to pH 9, when catechol-type structural materials are produced using these tyrosinase, it is easily oxidized by dissolved oxygen regardless of the second oxidation reaction by the enzyme. There is a problem. In addition, since most plants contain a large amount of various types of polyphenols, polyphenol materials can be easily obtained, but location-specific ortho hydroxide reactions in the monophenol structure of the polyphenol materials have a problem in that chemical synthesis is difficult. Therefore, there is a need for a technique that can efficiently produce useful catecholtic structural materials.

Korean Patent Publication No. 10-2013-0092168 Korean Patent Publication No. 10-2015-0001169

 Goldfeder, M., Kanteev, M., Adir, N. and Fishman, A. (2013) Influencing the monophenolase / diphenolase activity ratio in tyrosinase BBA-Proteins and Proteomics. 1834: 629-633.  Mylonas C, Kouretas D, Lipid peroxidation and tissue damage, In Vivo, 1999, 13 (3), 295-309  Tim Ridgway et al., Antioxidant action of novel derivatives of the apple-derived flavonoid phloridzin compared to oestrogen: relevance to potential cardioprotective action, Biochem Soc Trans, 1997, 25, 160S  Regioselective Hydroxylation of trans-Resveratrol via Inhibition of Tyrosinase from Streptomyces avermitilis MA4680 [ACS Chem. Biol., 2012, 7 (10), pp 1687-1692]  Nina Schweigert. Et al., Chemical properties of catechols and their molecular modes of toxic action in cells, from microorganisms to mammals, Environmental Microbiology, 2001, 3 (2), 81  DE Lee. Et al., 7,3 ', 4'-Trihydroxyisoflavone, a Metabolite of the Soy Isoflavone Daidzein, Suppresses Ultraviolet B-induced Skin Cancer by Targeting Cot and MKK4, Journal of Biological Chemistry, 2011, 286, 14246-14256  S.Isonishi. Et al., Differential regulation of the cytotoxicity activity of paclitaxel by orobol and platelet derived growth factor in human ovarian carcinoma cells, Oncology Reports, 2007, 18, 195-201  Chaing CM, Et al., Improving Free Radical Scavenging Activity of Soy Isoflavone Glycosides Daidzin and Genistin by 3'-Hydroxylation Using Recombinant Escherichia coli, Molecules, 2016, 21 (12), E1723

An object of the present invention is to develop a technique capable of producing a catechol type structural material with high productivity and yield in synthesizing a catechol type structural material from a monophenolic structural material. In particular, the inventors have sought to effectively produce functional catechol type structural materials using tyrosinase specialized for acidic conditions.

In order to solve the above problems, the present inventors classify a thousand microorganisms derived from microorganisms from the NCBI database, and many reported S. avermitilis- derived tyrosinase and Bacillus megaterium ( B. megaterium ). Burkholderia thailandensis- derived tyrosinase from the genetically much different from the derived tyrosinase was selected and produced in E. coli (see FIG. 1).

As a result, the Berkholderia tyrandensis-derived tyrosinase (BT_ty) has an optimal pH condition of pH 1.5 to 6.5, which is a perfect acidic condition, and reactivity is completely suppressed at a pH higher than neutral. In addition, the present invention was completed by discovering that under acidic conditions below the pka value of the product, the automatic oxidation of the catechol-type structural material is inhibited, thereby protecting the catechol-type structural material which is the primary hydroxylation product.

Specifically, the present invention

(1) A method for producing a catechol-type structural material from a monophenolic structural material using tyrosinase, wherein the tyrosinase is a Berkholderia tyrandensis-derived tyrosinase, and the preparation is pH 1.5 to 6.5 It relates to a production method characterized in that the acidic conditions of.

(2) The preparation may preferably be made in acidic conditions of pH 3.5 to 6.0.

(3) the ratio (Vmax_mono) of the primary hydroxylation rate (Vmax_mono) in which the catechol-type structural material is formed from the monophenolic structural material and the rate (Vmax_di) of the secondary oxidation reaction in which the quinone type material is produced from the catechol-type structural material (Vmax_mono / Vmax_di) may be greater than or equal to 0.8.

(4) The manufacturing method may further comprise (a) coordinating the resulting catechol-type structural material with transition metal ions or boron ions.

(5) The boron ion may be boric acid.

(6) The preparation method may further comprise one or more of the following steps (b) and (c):

(b) reducing the quinone type structural material produced by the peroxidation of tyrosinase to a catechol type structural material by introducing a reducing force;

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

(7) The reducing power may be provided from one selected from the group consisting of electrochemistry, NADH, NADPH, ascorbic acid, cysteine, p- coumaric acid, hydroxylamine, catechol, pyrogallol and caffeic acid.

(8) The monophenolic structural material is tyrosine amino acid, tyrosol, resveratrol, dydzein, genistein, apigenin, floretine, phenol, para-coumaric acid, para-nitrophenol, para-cresol, para-vinylphenol , Ortho-aminophenol, cepadrox, amocillin and nocardycin.

(9) The catechol-type structural material is luteolin, 3-hydroxyproletin, 3'- ortho hydroxydizezein, aurobol, pisethanol, tyrosol hydroxide, dopa, caffeic acid, 3, 4-dihydroxynitrobenzene, 3,4-dihydroxytoluene and 3,4-dihydroxystyrene.

(10) The monophenolic structural material may be floretine, and the catechol-type structural material may be 3-hydroxy floretine.

(11) The monophenol-type structural material may be a resin, and the catechol-type structural material may be 3-hydroxy resin.

(12) The monophenol-type structural material may be diedzein, phloretin, camphorol, kaempfereol, naringenin, resveratrol, daidzin, genistein ), Glycidin (glycitin), polydatin (polydatin), notofagin (nothofagin), tyrosol (tyrosol) and synephrine (synephrine).

(13) The catechol type structural material is a 3'-hydroxy dideze, 3-hydroxy floretine, quercetin, eriodictyol, piceatannol, 3'-hydroxy da To be selected from the group consisting of idine, 3'-hydroxy genistein, 3'-hydroxy glycidine, astringin, aspalathin, 3-hydroxy tyrosol and epinephrine Can be.

The present invention relates to the production and application of a functional catechol-type structural material using Berkholderia tyrandensis-derived tyrosinase (BT_ty) specialized in acidic conditions, the advantage of producing a catechol-type structural material with high productivity and yield There is this. As a result, it is possible to mass-produce catechol-type structural substances, which have been difficult to extract or organic chemical synthesis, and contribute to the physiological efficacy experiment.

In addition, the present invention can produce a catechol-type structural material having a strong antioxidant ability through the ortho-hydroxyl reaction site-specific to the monophenol structure of the polyphenol material.

Figure 1 is a cladogram based on the sequence of a thousand microorganisms derived from tyrosinase.
Figure 2 is a SDS-PAGE analysis results for identifying the Berkholderia tyrandensis-derived tyrosinase (BT_ty) expressed according to Experimental Example 1.
3 is a diagram confirming the melanin formed from the monophenolic structural material in order to confirm the activity of tyrosinase according to pH in Experimental Example 2.
Figure 4 is a table showing the rate of tyrosinase for L-tyrosine and L-dopa derived in Experimental Example 2.
Figure 5 shows the polyphenol aglycone and polyphenol glycoside (glycone) used in the melanogenesis reaction using Berkholderia tyrandensis-derived tyrosinase (BT_Ty) in Experimental Example 3.
Figure 6 is a result of comparing the melanin formation reaction according to the substrate in Experimental Example 3.
7 is a result of comparing the relative activity of tyrosinase to the saccharification substrate in Experimental Example 3.
8 is a schematic diagram of a reaction for producing 3-hydroxy floretine from floretine using Berkholderia tyrandensis-derived tyrosinase (BT_ty).
9 is a result of qualitative analysis of 3-hydroxy floretine prepared in Experimental Example 4. FIG.
FIG. 10 is a quantitative analysis result using high performance liquid chromatography (HPLC) for confirming separation of floretine and 3-hydroxy floretine in Experimental Example 4. FIG.
FIG. 11 is a view showing a 3-hydroxy floretine production aspect for confirming the conversion rate from a substrate to a reaction product according to reaction conditions in Experimental Example 4. FIG.
12 is a gas chromatograph (GC) / mass spectrometry (MS) result for confirming the production of floretine and 3-hydroxy floretine in Experimental Example 5. FIG.
FIG. 13 is a high performance liquid chromatography (HPLC) result for confirming the production of kaempferol and quercetin in Experimental Example 6. FIG.
FIG. 14 shows the results of conversion to quercetin of camphorol using Burkholdereria tyrandensis-derived tyrosinase (BT_Ty) in Experimental Example 6. FIG.
FIG. 15 is a schematic diagram of a reaction for producing piceatannol from resveratrol (a), a production aspect of piaceanol (b), and gas chromatography for identifying resveratrol and piaceanol in Experimental Example 7; (GC) / mass spectrometry (MS) result ((c)-(f)).
FIG. 16 is a schematic diagram (a) of a reaction for producing eriodictiol from naringenin in Experimental Example (a), a production aspect of eriodicthiol (b), and a gas for identifying naringenin and eriodicthiol Chromatography (GC) / mass spectrometry (MS) results ((c) to (f)).
FIG. 17 is a view showing a schematic diagram of a reaction for generating 3'-hydroxy diadzein (3'-ODI) from diadzein and the generation of 3'-ODI in Experimental Example 9. FIG.
FIG. 18 is a diagram showing a schematic diagram of a reaction for generating 3'-hydroxy diazine from didzin and a production pattern of 3'-hydroxy diazine in Experimental Example 9. FIG.
FIG. 19 is a view showing a schematic diagram of a reaction for generating 3'-hydroxy genistin from genistin and a production pattern of 3'-hydroxy genistin in Experimental Example 9. FIG.
20 is a schematic view of a reaction to generate 3'-hydroxy diazine from didzin in Experimental Example 10 and liquid chromatography (LC) for identifying didazine and 3'-hydroxy diazine / Mass spectrometry (MS) results.
FIG. 21 is a schematic diagram of a reaction for generating 3'-hydroxy genistin from genistin in Experimental Example 10 and liquid chromatography (LC) / mass spectrometry (MS) results for identifying genitin and 3'-hydroxy genistin. to be.
FIG. 22 is a view showing a schematic diagram of a reaction for generating 3'-hydroxyglycitin from glycidin (glycitin) in Experimental Example 11 and a production pattern of 3'-hydroxyglycitin. FIG.
FIG. 23 is a schematic view of a reaction for generating 3'-hydroxyglycitin from glycidin in Experimental Example 12 and liquid chromatography (LC) / mass to identify glycidine and 3'-hydroxyglycithin Analysis (MS) result.
FIG. 24 is a view showing a schematic diagram of a reaction for generating 3-hydroxy polyditatin from polydatin and the production aspect of 3-hydroxy polyd Datin in Experimental Example 13. FIG.
FIG. 25 is a schematic view of a reaction for generating 3-hydroxy polyditatin from polydatidine in Experimental Example 14 and liquid chromatography (LC) / mass spectrometry (MS) results for identifying polydatidine and 3-hydroxy polyditatin. FIG. .
FIG. 26 is a diagram showing a schematic diagram of a reaction for producing 3-hydroxy notopagine from nothofagin and the production of 3-hydroxy notopagine in Experimental Example 15; FIG.
FIG. 27 is a schematic view of a reaction for producing 3-hydroxy notopagine from notopagine and a liquid chromatography (LC) / mass spectrometry (MS) result for confirming notopagine and 3-hydroxy notopagine in Experimental Example 16; FIG. .

Hereinafter, the present invention will be described in detail. However, the present invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein.

The present invention is a method for producing a catechol-type structural material from a monophenolic structural material (substrate) by using a tyrosinase (enzyme), wherein the tyrosinase is tyrosinase derived from Berkholderia tyrandensis (BT_ty) It is characterized in that the preparation is made in an acidic condition of pH 1.5 to pH 6.5.

Berkholderia tyrandensis-derived tyrosinase (BT_ty), unlike other polypropylene oxidase (PPO), has an optimal reaction condition of pH 1.5 to pH 6.5. When Berkholderia tyrandensis-derived tyrosinase (BT_ty) is used as an enzyme, it is possible to express Berkholderia tyrandensis-derived tyrosinase from a monophenolic substrate at low pH conditions. As a result, the catechol-like product generated after the primary hydroxylation reaction in the monophenolic substrate can be stably preserved, thereby making it possible to prepare a catechol-type structural material with high productivity and yield.

Primary hydroxylation by tyrosinase (monooxygenase activity) refers to a tyrosinase enzymatic reaction that catalyzes the introduction of oxygen atoms into carbon-hydrogen bonds and produces hydroxyl moieties. The tyrosinase secondary oxidation reaction (deoxygenase activity) refers to a tyrosinase enzymatic reaction which removes hydrogen and electrons bonded to oxygen in the catechol-type structural material and catalyzes it in the form of benzoquinone.

In this regard, the present invention relates to the ratio of the rate of primary hydroxylation reaction (Vmax_mono) in which the catechol-type structural material is formed from the monophenolic structure material and the rate of the rate of secondary oxidation reaction (Vmax_di) in which the quinone-type material is produced from the catechol-type structural material. (Vmax_mono / Vmax_di) is 0.8 or more. In addition, the present invention enables the industrial use of the catechol-type structural material because the reaction rate is 10 times higher than the previously reported tyrosinase.

As mentioned above, Berkholdereria tyrandensis derived tyrosinase (BT_ty) is active at pH 1.5 to pH 6.5 and inactive at pH above neutral. Therefore, the preparation may be made in an acidic condition of pH 1.5 to pH 6.5, more preferably in an acidic condition of pH 3.5 to pH 6.0. Thus, Berkholderia tyrandensis-derived tyrosinase (BT_ty) forms melanin pigments only under acidic conditions, so when Berkholderia tyrandensis-derived tyrosinase is used together with a substrate, it can be used as a pH indicator. This is possible.

The preparation is carried out in a temperature range of 10 to 70, for 8 to 30 hours It is preferable to react.

In addition, the production method of the present invention may include the step of (a) coordinating the resulting catechol-type structural material with transition metal ions or boron ions. The boron ion may be boric acid. For example, when using boric acid, 3-hydroxy fluoretine is protected from the diphenolase reaction of tyrosinase by boron ester linkage between boric acid and the catechol type material, so that the reaction product remains stable without being oxidized. Thus, the desired catechol type structural material can be obtained in higher yield.

In addition, the process of the present invention may further comprise one or more of the following steps (b) and (c):

(b) reducing the quinone type structural material produced by the peroxidation of tyrosinase to a catechol type structural material by introducing a reducing force;

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

In this case, the reducing power may be one provided from the group consisting of electrochemical, NADH, NADPH, ascorbic acid, cysteine, p- coumaric acid, hydroxylamine, catechol, pyrogallol and caffeic acid.

By further comprising the step of (b) effectively reducing the quinone type material to the catechol type structural material or (c) reactivating tyrosinase, the desired catechol type structural material can be obtained in higher yield. .

In the present invention, the Berkholderia tyrandensis-derived tyrosinase (BT_ty) includes not only tyrosinase itself but also a case in which a tyrosinase-expressing microorganism (whole cell) is used.

In the present invention, as the monophenolic structural material (substrate), tyrosine amino acid (tyrosin) and tyrosol (tyrosol), stilbene series, such as resveratrol (daidzein), genistein (genistein) , Flavonone / isoflavone family such as apigenine, phloretin and phlorezin, as well as phenol, para-coumaric acid, Para-nitrophenol, para-cresol, para-vinylphenol, ortho-aminophenol, and cefadroxil, amocillin phenolic antibiotics such as amoxicillin and nocardicin, and the like, but are not limited thereto.

In addition, the monophenol-type structural material (substrate) in the present invention is a polyphenol material having a monophenol structure, such as didzeze (in), fluorin (phloretin), kaempfereol, naringenin (naringenin), Using resveratrol, daidzin, genistein, glycidin, polydatin, nothofagin, tyrosol and cynephrine May be, but is not limited thereto.

In the present invention, the catechol-type structural material, ortho-specifically hydrated to the monophenolic structural tyrosine material, luteolin (luteolin), 3-hydroxyphloretin (3-hydroxyphloretin, 3'-ODI), orobol flavoneone / isoflavone series such as orobol, stilbene series such as piceatannol, hydroxyltyrosol hydroxide, dopa and caffeic acid acid), 3,4-dihydroxynitrobenzene, 3,4-dihydroxytoluene, 3,4-dihydroxystyrene ). Preferably, the preferred monophenolic structural substances listed above are ortho-specific hydrated forms, in turn, pisethanol, caffeic acid, 3'-ODI, orobol, 3,4-dihydroxynitrobenzene (3, 4-dihydroxynitrobenzene), 3,4-dihydroxytoluene, 3,4-dihydroxystyrene, luteolin, 3-hydroxyproloretin ) And the like, but are not limited thereto.

Also, in the present invention, the catechol-type structural material is ortho-specifically hydrated to the monophenolic structure of the polyphenol, which is a 3'-hydroxy dideze, 3-hydroxy floretine, quercetin, and eryodic. Thiol (eriodictyol), piceatannol, 3'-hydroxy didazine, 3'-hydroxy genistein, 3'-hydroxy glycidine, astringin, aspalathin , 3-hydroxy tyrosol and epinephrine, and the like, but are not limited thereto.

Ortho-hydroxyl reactions specific to the monophenolic structure of the polyphenolic material include sodium citrate buffer, citric phosphate buffer, sodium acetate buffer and acetic acid. It can proceed in ammonium acetate buffer. The concentration of the buffer solution is preferably 10 mM to 100 mM, and the concentration of the polyphenol material (substrate) is preferably 5 mM or less when the concentration of Berkholdereria tyrandensis-derived tyrosinase (BT_ty) is 100 nM.

In addition, the ortho hydroxide reaction may be performed by adding L-ascorbic acid. L-ascorbic acid can turn the quinone-type substance produced by the secondary hydroxylation and natural oxidation of tyrosinase back to the diphenol-type substance, where L-ascorbic acid is an antioxidant (reducing agent) and tyrosine It acts as an inhibitor of activity (secondary reaction inhibitor). In addition, L-ascorbic acid may also act as a reducing agent for reducing the tyrosinase in the form of (met) to deoxy (Non-Patent Document 2). In addition to L-ascorbic acid, hydroxyamine may be added to proceed with ortho hydroxide.

In addition, the ortho hydroxide reaction may be performed in an acidic condition of pH 1.5 to pH 6.5, more preferably in an acidic condition of pH 3.5 to pH 6.0.

On the other hand, the present invention is also significant in that it is possible to convert fluoretine or fluorescein into a useful catechol-type structural material using Berkholderia tyrandensis-derived tyrosinase (BT_ty). Floretine and floresin are a kind of dihydrochalcone that are abundant in the leaves and apple pulp of cheap apple trees and are known for their excellent antioxidant and anti-aging effects (Non-Patent Document 1). Phosphorus 3-hydroxy floretine is known to act as an inhibitor of lipid peroxidation, which may lead to atherosclerosis, Parkinson's disease, etc. because it has better antioxidant efficacy and inhibits cholesterol accumulation than floretine (Non-Patent Literature) 2) In other words, using the present invention, 3-hydroxy floretine and 3-hydroxyflour are difficult to chemically synthesize from inexpensive apples (peel, mouth) under acidic reaction conditions such as apple (peel, leaf) juice. Resin can be produced economically and on an industrial scale.

In addition, the present invention, using the Berkholderia tyrandensis-derived tyrosinase (BT_ty) is selectively selected from a variety of polyphenol non-saccharide material genistein (daidzein), fluoretine (phloretin), camphorol ortho-specific hydroxides for kaempferol and its glycosylation structures: didzin, genistin, glycidin, polydatin, phlorizin, and nothofagin The reaction can proceed. In addition, ortho-specific hydroxylation can be carried out on phenolic structural substances such as tyrosol in olive oil and synephrine, which is rich in citrus peel.

In other words, using the present invention, polyphenol non-saccharides (dyne, floretine, camphorol, naringenin, resveratrol), O-polyphenol glycosides (didazin, genistein, glycidin, polydatin), C- Plant extracts containing polyphenol glycosides (notopagin), phenylethanoids (tyrosol), and alkaloids (cinephrine) are selectively subjected to hydroxylation under acidic reaction conditions without any extraction process, resulting in unstable ortho-hydroxylation in aqueous solution. Only polyphenol materials can be stably produced.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited by the examples.

Experimental Example 1: Expression of Burkholderia tyrandensis-derived tyrosinase (BT_ty)

3 mL of LB liquid medium containing 50 μg / mL Kanamycin was added to colonies obtained by transforming the pET28a vector containing the Berkholderia tyrandensis-derived tyrosinase (BT_ty) gene into E. coli BL21 and applying it to the LB solid medium. It was inoculated to and incubated for 8 hours in 37 shake incubator. For subculture, 50 μg / mL Kanamycin and 500 μL of spawn culture were added to 50 mL of LB liquid medium and cultured in 37 shake incubators.

When D ₆₀₀ (optical density at 600 nm) reached about 0.6, protein overexpression was induced at 18, 30 ° C., and 37 hours after addition of 0.02 mM IPTG or 0.2 mM IPTG and 1 mM CuSO ₄ . Cells were harvested by centrifugation at 4,000 rpm for 10 minutes to obtain protein and cells were washed by adding 5 mL of 50 mM pH 8 Tris-HCl buffer. 5 mL of 50 mM pH 8 Tris-HCl buffer was added thereto, followed by cell disruption using an ultrasonic crusher (Vibra & cell, USA). Cell lysates were dispensed into 1.7 mL tubes and centrifuged at 16,000 rpm for 30 minutes to obtain cell extracts containing proteins.

The result of confirming the protein size and the expression level through the SDS-PAGE analysis of the obtained cell extract is shown in FIG. As can be seen from FIG. 2, it was confirmed that the expression of Berkholdereria tyrandensis-derived tyrosinase (BT_ty) was better at 0.2 mM IPTG and at an expression temperature of 18 ° C. than 0.02 mM IPTG.

Experimental Example 2: Confirmation of optimization conditions for the hydroxylation reaction of monophenol-type structural material using Berkholderia tyrandensis-derived tyrosinase (BT_ty)

Tyrosinase derived from Agaricus bisporus, tyrosinase derived from B. megaterium, tyrosinase derived from S. avermitilis, and the present invention prepared in Experiment 1 Hydroxylation and oxidation of L-tyrosinase, a monophenolic structural material, were carried out using each of Berkholderia tyrlandase-derived tyrosinase (BT_ty). Referring to Figure 3, in the case of the reaction using the Berkholderia tyrandensis-derived tyrosinase (BT_ty) of the present invention, in contrast to other tyrosinase reported previously, L-tyrosinase only at low pH It was confirmed to oxidize to form melanin. In particular, the optimum reaction acidity of Berkholderia tyrandensis-derived tyrosinase (BT_ty) was found to be around pH 5.

In addition, based on the above experiment, Bacillus megaterium (B. megaterium) -derived tyrosinase, S. avermitilis-derived tyrosinase, and the Berkholderia tyrandensis-derived tyrosinase (BT_ty) of the present invention , And the rates for L-tyrosinase and L-dopa, respectively, are shown in FIG. 4. As shown in Fig. 4, when the Berkholderia tyrandensis-derived tyrosinase (BT_ty) of the present invention is used, L-tyrosinase and L-dopa are more effective than the other tyrosinase reported previously. The speed is about 10 times higher. This is because when the catechol-type structural material is prepared from the monophenolic structural material using the Berkholderia tyrandensis-derived tyrosinase (BT_ty) of the present invention as an enzyme, This means that the reaction rate is significantly improved.

In addition, referring to Figure 4, in the case of using the Berkholderia tyrandensis-derived tyrosinase (BT_ty) of the present invention, The ratio (Vmax_mono) of the primary hydroxylation reaction (Vmax_mono) in which the catechol-type structural material is formed from the monophenolic structural material and the rate (Vmax_di) of the secondary oxidation reaction (Vmax_di) in which the quinone type material is produced from the catechol-type structural material is 0.8 It is confirmed that it is abnormal.

Experimental Example 3: Preparation of hydroxy polyphenol through hydroxylation reaction of polyphenol non-saccharide and polyphenol glycoside

Polyphenol non-saccharides using Berkholderia tyrandensis-derived tyrosinase (BT_Ty), Bacillus megaterium-derived tyrosinase (BM_Ty), and Streptomyces avalmitilis-derived tyrosinase (SA_Ty) The substrate specificity of tyrosinase for 7 and 4 polyphenol glycosides was compared. The polyphenol nonsaccharides and polyphenol glycosides used in the reaction are shown in FIG. 5. As the reaction proceeds, the color change due to the production of melanin can be confirmed, and thus the production of hydroxy polyphenol can be confirmed qualitatively, and the result of comparing the melanin formation reaction is shown in FIG. 6. Referring to FIG. 6, it was confirmed that Berkholdereria tyrandensis-derived tyrosinase (BT_ty) has strong activity against the remaining eight substrates except genistein, kaferferol, and quercetin. Could.

In addition, the relative specificity of tyrosinase to the polyphenol glycosides is compared with substrate specificity, and is shown in FIG. 7. Specifically, the relative reactivity to the glycosylation substrate was compared through melanin formation, and as a result, it was confirmed that the selective activity of the polyphenol glycoside of BT_Ty was higher than that of BM_Ty and SA_Ty.

Experimental Example 4: Preparation of 3-hydroxy floretine through the hydroxylation of floretine

3-hydroxy floretine was prepared from floretine present in apples (berries or leaves) using Berkholderia tyrandensis-derived tyrosinase (BT_ty) as an enzyme. A schematic diagram of the reaction is shown in FIG. 8.

At this time, with the reaction (# 3) to which both the enzyme (BT-ty) and the substrate (floletine) were added, the reaction (# 1) and the substrate (floletine) without the addition of the enzyme (BT-ty) as a control Reaction # 2 which was not added was performed together and is shown in FIG. Referring to FIG. 9, the two control groups without the addition of the enzyme or the substrate could not confirm the color change due to melanin production, but the reaction with the enzyme and the substrate added visually confirmed the color change due to the melanin. .

In addition, the results of quantitative analysis of the product 3-hydroxy floretine using HPLC are shown in Figure 10. As can be seen in FIG. 10, fluoretine was eluted at 14.5 minutes and 3-hydroxy fluoretine at 8 minutes, respectively.

Meanwhile, in order to confirm the conversion rate from the substrate to the reaction product according to the reaction conditions, 1 mM of floretine and 1 μM of BT-ty were added to (a) 5 mL of pH 6 50 mM citric acid (Na-citrate) buffer solution, and (b ) After dissolving in 5 mL of each pH 6 500 mM borate buffer, L-ascorbic acid (LAA) was added to the reaction. It is shown in Figure 11 comparing the production of 3-hydroxy floretine over time of the reaction. In the case of the citric acid buffer, it was confirmed that 3-hydroxy floretine produced by the second oxidation reaction of tyrosinase is converted to the quinone form and disappears over time (see FIG. 11 (a)). ). In the case of borate buffer reaction, 3-hydroxy floretine was protected from the diphenolase reaction of tyrosinase by boron ester linkage between boric acid and catechol-type material, so that the reaction product remained stable without oxidation ( See FIG. 11 (b)). As a result, at 9 hours of reaction, 1 mM of floretine was found to be converted to 3-hydroxy floretine in almost 100% yield.

Experimental Example 5: Confirmation of production of 3-hydroxy floretine by reactant analysis

In connection with Experiment 4, a sample was taken immediately after treatment with the enzyme (0 h) and 4 hours after the start of the reaction (4 h), and the reaction product was analyzed by GC / MS. Indicated. In FIG. 12, (a) and (c) show the chromatography of the sample, and (b) and (d) show the mass spectra. In (a) and (c), the peak at 11.81 minutes is the substrate fluoretine and the peak at 13.02 minutes is the 3-hydroxy fluoretine reaction product.

Referring to FIG. 12, after 4 hours of reaction, floretine disappeared and it was confirmed that 3-hydroxy floretine was produced. The two mass peaks of fluoretine are m / z 562.87 and 547.97 (one methyl group removed from TMS ether moiety) and 3-hydroxy fluoretine is 651.01 and 636.15, respectively.

Experimental Example 6 Production of 3′-hydroxy Camperol (Quercetin) through the Hydroxation of Kaempferol

3'-hydroxy camphorol, ie quercetin, was prepared from camphorol using Berkholderia tyrandensis-derived tyrosinase (BT_ty) as an enzyme. HPLC was used to quantitatively analyze the site-specific hydroxylation at the 3 ′ position, which is shown in FIG. 13. Referring to FIG. 13, it was confirmed that the substrate camphorol was eluted at 21 minutes and the product quercetin at 11 minutes.

In addition, in order to determine the conversion yield of the substrate to the product according to the reaction conditions, sodium citrate buffer solution was used, and L-ascorbic acid was added to confirm the production pattern of quercetin over time. The conversion of camphorol to quercetin is shown in FIG. 14, and quercetin was produced with a conversion yield of 4.7%.

Experimental Example 7: Preparation of 3'-hydroxy resveratrol (picetanol) through the hydroxylation of resveratrol

3'-hydroxy resveratrol, i.e., pieatanol, was prepared from resveratrol using Berkholderia tyrandensis-derived tyrosinase (BT_ty) as an enzyme. Specifically, sodium citrate buffer solution was used, and L-ascorbic acid was added to confirm the production of piaceanol over time. The reaction was performed in 100 mL of enzyme and 1 mM of substrate in pH 5.0, 50 mM sodium citrate buffer solution, and the result of the reaction was shown in FIG. 15. Referring to FIG. It was confirmed that the generated. In addition, Figure 15 (b) is a chromatograph of the sample after 5 hours of the reaction can be confirmed that resveratrol disappeared and piceatanole produced. In FIG. 15, (c) shows the chromatography of the (Oh) sample immediately after the enzyme treatment, (d) shows the chromatography of the sample 300 minutes after the start of the reaction (5h), and (e) and (f) the mass. It shows the spectrum. The peak at 10.45 minutes in (c) is the substrate resveratrol and the peak at 12.15 minutes in (d) is the product piaceanol. The mass peak of resveratrol is 444.20 m / z and the mass peak of piaceanol is 532.23 m / z.

Experimental Example 8: Preparation of 3'-hydroxy naringenin (eriodicthiol) through the hydroxylation of naringenin

3'-hydroxy naringenin, ieiodictyol, was prepared from naringenin using Berkholderia tyrdenase derived tyrosinase (BT_ty) as an enzyme. Specifically, sodium citrate buffer solution was used, and L-ascorbic acid was added to confirm a production pattern of eriodic thiol over time. The reaction was carried out in a pH 5.0, 1 mL of 50 Mm sodium citrate buffer solution with enzyme 100 nM, substrate 1 mM is shown in Figure 16. Referring to Figure 16 (b), the conversion yield is about 10% of the product erythroid It was confirmed that thiol was produced.

In addition, Fig. 16 (b) shows the chromatography of the sample, Fig. 16 (c) shows the chromatography of the sample immediately after the enzyme treatment (0h), and (d) shows 300 minutes after the start of the reaction (5h). Chromatography of the samples is shown, and (e) and (f) show the mass spectra. The peak at 12.52 minutes in (c) is the substrate naringenin, and the peak at 13.64 minutes in (d) is the product erythrothiol. The mass peak of resveratrol is 473.15 m / z and the mass peak of piaceanol is 576.22 m / z.

Experimental Example 9 3'-hydroxy didezein (3'-ODI), 3'-hydroxy didazine and the like through the hydroxylation of daidzein, daidzin and genistin Preparation of 3'-hydroxy Geninistine

Location-specific hydroxylation at 3'-position of Dyzezein, an isoflavone substance present in soybeans, Dydazine and Genistin, a large amount of isoflavones present in soybeans, using Berkholderia tyrandensis-derived tyrosinase (BT_ty) as an enzyme This progress was quantitatively analyzed using HPLC, and the results are shown in FIGS. 17 to 19.

To determine the yield of conversion of the substrate to the product according to the reaction conditions, use sodium citrate buffer solution and add L-ascorbic acid to pH 100, 50 Mm sodium citrate buffer solution to 100 nM enzyme, 1 mM substrate. The reaction was progressed, and the production pattern of 3'-hydroxy diazine, 3'-hydroxy diazine, and 3'-hydroxy genistin over time was confirmed.

As can be seen in FIG. 17, the conversion yield of 3'-hydroxy didzezein from dyedzein was no further reaction at 38%, and as shown in FIG. 18, the reaction in the reaction of dydazine was confirmed. After 1 hour all of the substrate dydazine was consumed and 3'-hydroxy didazine was produced with a conversion yield of 87.3%. In addition, as confirmed in FIG. 19, after 1 hour of reaction, the substrate was not consumed, and 3′-hydroxy genistine was produced in 46.5% conversion yield.

Experimental Example 10 Confirmation of Production of 3'-hydroxy Didazine and 3'-hydroxy Zenithin by LC / MS Analysis of Reactions

In relation to Experiment 9, a sample was taken immediately after treatment with enzyme (O h) and 1 hour after the start of the reaction (1 h) in which the reaction solution to which the dydazine was added as a substrate was reacted with LC / MS. The analysis results are shown in FIG. 20. In FIG. 20, (a) shows the chromatography of the sample immediately after the enzyme treatment (Oh), (b) shows the chromatography of the sample (1h) one hour after the start of the reaction, and (c) and (d) show the mass. Represent the mass spectra. The peak at 8.21 minutes in (a) is the substrate didazine and the peak at 8.04 minutes in (b) is 3'-hydroxy diazine, the reaction product.

Referring to FIG. 20, after 1 hour of reaction, the didazine disappeared and the 3'-hydroxy didazine was formed. The mass peak of the didazine was 417.12, and the 3'-hydroxy didazine was present. M / z is 433.08.

In addition, a sample was taken immediately after treatment with the enzyme (O h) and 1 hour after the start of the reaction (1 h), and the reaction product was analyzed by LC / MS. Indicated. In FIG. 21, (a) shows the chromatography of the sample immediately after the enzyme treatment (Oh), (b) shows the chromatography of the sample (1h) one hour after the start of the reaction, and (c) and (d) show the mass. Represent the mass spectra. The peak at 9.4 minutes in (a) is the substrate genistin, and the peak at 8.63 minutes in (b) is 3'-hydroxy geninistin, the reaction product.

Referring to FIG. 21, it can be seen that 1 hour after the reaction, 3'-hydroxygeninistin was formed. The mass peak of genistin was 433.11 m / z and m / z was 449.07.

Experimental Example 11 Preparation of 3'-hydroxyglycitin through Hydroxylation of Glycitin

It was confirmed quantitatively using HPLC that the site-specific hydroxylation reaction at the 3'-position was performed quantitatively in the reaction in which glycidin, a polyphenol glycoside, was added as a substrate, and the results are shown in FIG. 22. In order to determine the yield of conversion of the substrate to the product according to the reaction conditions, sodium citrate buffer solution was used, and L-ascorbic acid was added to confirm the production of 3'-hydroxyglycitin over time. The reaction proceeded with pH 5.0, 1 mL of 50 Mm sodium citrate buffer solution, 100 nM of enzyme and 1 mM of substrate. As can be seen from FIG. 22, after 1 hour of reaction, glycine, a substrate, was consumed. -Hydroxy glycidine was produced with a conversion yield of 80.9%.

Experimental Example 12 Confirmation of Production of 3'-hydroxyglycithin through LC / MS Analysis of Reactions

In connection with Experimental Example 11, after the enzyme was treated to the reaction solution (0 h), and the sample was taken 1 hour after the start of the reaction, the reaction product is analyzed by LC / MS is shown in Figure 23. (a) shows the chromatography of the (Oh) sample immediately after the enzyme treatment, (b) shows the chromatography of the sample (1h) one hour after the start of the reaction, and (c) and (d) the mass spectra (mass) spectra). The peak at 8.33 minutes in (a) is the substrate glycidine. In (b), the peak at 7.84 minutes represents the reaction product 3'-hydroxyglycitin. Referring to FIG. 23, it can be seen that 1 hour after the reaction, 3'-hydroxyglycitin is detected, and the mass peak of glycidine is m / z of 447.12 and the mass peak of 3'-hydroxyglycitin. M / z is 463.10.

Experimental Example 13: Preparation of 3-hydroxy polydatin (astrinzine) through the hydroxylation reaction of polydatin

Polydatin, a polyphenol glycoside, was added as a substrate, and quantitatively using HPLC for the position-specific hydroxylation reaction at the 3-position in the reaction using Berkholderia tyrandensis-derived tyrosinase (BT_ty) as an enzyme. The results are shown in FIG. 24. Specifically, in order to determine the conversion yield of the substrate to the product according to the reaction conditions, sodium citrate buffer solution was used, and L-ascorbic acid was added to confirm the astringin production pattern over time. The reaction proceeded to pH 5.0, 1 mL of 50 Mm sodium citrate buffer solution with enzyme 100 nM and substrate 1 mM. As can be seen from FIG. A yield of 78.6% conversion was produced.

Experimental Example 14 Confirmation of Production of 3-Hydroxy Polydatin (Astringin) by LC / MS Analysis of Reactions

In relation to Experimental Example 13, after the enzyme was treated to the reaction solution (0 h), and 1 hour after the start of the reaction, a sample was taken and the result of analyzing the reaction product by LC / MS is shown in Figure 25. (a) shows the chromatography of the sample immediately after the enzyme treatment (Oh), (b) shows the chromatography of the sample (1h) one hour after the start of the reaction, and (c) and (d) the mass spectrum spectra). The peak at 8.94 minutes in (a) represents the substrate polydatidine and the peak at 8.45 minutes in (b) represents the reaction product astringin. As can be seen from FIG. 25, it can be confirmed that astrine is detected after 1 hour of reaction. The mass peak of polydatin is 390.11 m / z and the mass peak of astringin is 407.09.

Experimental Example 15 Preparation of 3-hydroxy notopagine (aspalatin) through the hydroxylation of nothofagin

Quantitative analysis using the HPLC of site-specific hydroxylation at the 3-position in the reaction using a polyphenol glycoside, notopagine, as a substrate, and using Berkholderia tyrandensis-derived tyrosinase (BT_ty) as an enzyme The results are shown in FIG. 26. Specifically, in order to determine the conversion yield of the substrate to the product according to the reaction conditions, sodium citrate buffer solution was used, and L-ascorbic acid was added to confirm the appearance of aspalathin over time. The reaction proceeded to pH 5.0, 1 mL of 50 Mm sodium citrate buffer solution with 100 nM of enzyme and 1 mM of substrate. As can be seen from FIG. 26, after 60 minutes of reaction, asphaltene was produced with a conversion yield of 75.7%.

Experimental Example 16: Confirmation of production of 3-hydroxy notopagine (aspalatin) by LC / MS analysis of reactants

In connection with Experiment 15, the reaction solution to which notopagin was added as a substrate was treated with Berkholderia tyrandensis-derived tyrosinase (BT_ty) as an enzyme, and after 45 minutes, a sample was taken to obtain a reaction product by LC / MS. Was analyzed and the result is shown in FIG. (a) shows the chromatography of the sample, and (b) and (c) shows the mass spectra. In (a), the peak at 17.84 minutes represents the substrate notopagine and the peak at 13.81 minutes represents the reaction product aspalathin. Asphalt is detected from FIG. 27. The mass peak of notopagine is m / z of 390.11, and the mass peak of asphaltenes of m / z of 407.09.

Claims (13)

As a method for producing a catechol-type structural material from a monophenolic structural material using tyrosinase,
The tyrosinase is a tyrosinase derived from Burkholderia thailandensis ,
The monophenolic structural material is tyrosine amino acid (dyrosine), daidzein (gendstein), genistein (daidzin), genistin (genistin), glycidin (glycitin), resveratrol, poly Selected from the group consisting of polydatin, phloretin, phloridzin, nothofagin, naringenin and kaempferol,
The preparation is characterized in that the production is made under acidic conditions of pH 1.5 to pH 5. The method of claim 1,
The ratio (Vmax_mono) of the primary hydroxylation reaction (Vmax_mono) in which the catechol-type structural material is formed from the monophenolic structural material and the rate (Vmax_di) of the secondary oxidation reaction (Vmax_di) in which the quinone type material is produced from the catechol-type structural material is 0.8 The manufacturing method characterized by the above. The method according to claim 1 or 2,
The preparation method characterized in that the preparation is made in an acidic condition of pH 3.5 to pH 5. The method according to claim 1 or 2,
(a) coordinating the resulting catechol-type structural material with transition metal ions or boron ions. The method of claim 4, wherein
The boron ion is a manufacturing method, characterized in that boric acid. The method according to claim 1 or 2,
Further comprising at least one of the following steps (b) and (c):
(b) reducing the quinone type structural material produced by the peroxidation of tyrosinase to a catechol type structural material by introducing a reducing force;
(c) introducing a reducing force to activate met-tyrosinase. The method of claim 6,
Wherein said reducing power is provided from the group consisting of electrochemistry, NADH, NADPH, ascorbic acid, cysteine, p- coumaric acid, hydroxylamine, catechol, pyrogallol and caffeic acid. delete delete The method according to claim 1 or 2,
Wherein said monophenolic structural material is phloretin and said catechol-type structural material is 3-hydroxy floretine. The method according to claim 1 or 2,
The monophenolic structural material is phloridzin, and the catechol-type structural material is 3-hydroxy floridine. delete The method according to claim 1 or 2,
The catechol-type structural material is dopa (DOPA), 3'- hydroxy didase, orobol, 3'- hydroxy diazine, 3'- hydroxy zenithine, 3'- hydroxy glycine , Group consisting of piceatannol, astringin, 3-hydroxy floretine, 3-hydroxy floridine, aspalathin, eriodictyol and quercetin It is selected from the manufacturing method.

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