CN105555776B - Catechol-type structural material, its preparation method and its application prepared using tyrosinase - Google Patents
Catechol-type structural material, its preparation method and its application prepared using tyrosinase Download PDFInfo
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Abstract
The present invention is the relevant technology of biosynthesis reaction with the catechol-type structural material using tyrosinase, is the technology for effectively inhibiting second of oxidation reaction of tyrosinase to be optionally only catalyzed the first time hydroxylating of single phenol type structural material.The present invention provides the technology using the several functions catechol-type material of tyrosinase with high productivity and produced in yields.
Description
Technical field
The present invention relates to the use of the tyrosinase (tyrosianse) with wide substrate specificity with high productivity and
Yield sieve selectively only generates catechol-type works from a variety of single phenol type structural materials (monophenolic compounds)
The method of matter.Thus, the batch production of the feature catechol-type structural material using enzyme reaction can be achieved in the present invention, and can fit
For raw material and medical production.
Background technology
It was reported that many catechol-type structural materials (catechol derivatives) are in anti-oxidant, anticancer, anti-inflammatory
Or it is antiviral and other effects in the physiological activity that has presented, with single phenol type structural material (monophenolic compounds) phase
Than its effect higher.Especially, catechol-type structural material is utilized as the inhibitor of tyrosinase (tyrosinase), and is had
It is reduced the effect of the production of the melanin (melanin) based on tyrosinase.It is also known that in daidzein
In (daidzein, from the isoflavones of soybean), adjacent hydroxylating (ortho-hydroxylation) is specifically realized
A part for 3 '-neighbour hydroxyl daidzein (3 '-ODI, 3 '-orthohydorxydaidzein) is present in the salty sauce of maturation,
Prevention is suppressed and relevant activity (JBC, 2010, vol.285, pp.21458 of metastasis of cancer by UV-induced cutaneum carcinoma；
JBC, 2011, vol.286, pp.14246).It is hydroxylated as the catechol-type material from apiolin (apigenin)
Cyanidenon (luteolin) can extract very small amount in the arithoke (arichoke) of good in oxidation resistance, can be according to newest report
(Food Chemistry, 2013, " Luteolin sensitises drug-resistant are used in breast cancer treatment
human breast cancer cells to tamoxifen via the inhibition of cyclin E2
expression").In addition, it is well known that in the hydroxytyrosol (hydroxytyrosol), grape wine in olive oil
The catechol-type material of piceatannol (piceatannol) etc. also serves as the antioxidant of strength.Catechu on polymer substance
Phenolic structural material can imitate the function of mussel protein matter, its application field is just wide from medical treatment material to surface-modified nano engineering
(Annu Rev Mater Res.2011 1 41 are studied generally：99-132 Biofouling 2012,28：8,865-
877)。
In the case where producing catechol-type structural material using conventional biocatalyst, exist using including P450 enzymes
Interior a variety of monooxygenases (mono-oxigenase).But, it is necessary to coenzyme in the case of most monooxygenase, and
Need double reductases (reductase) for electron transmission.This can influence the bioconversion reaction speed of matrix, and by low
Yield and productivity cause.On the contrary, the single phenol structural material oxidation reaction of tyrosinase is relatively fast (as reference, from mushroom
The kcat/Km values of the tyrosinase of mushroom class are substantially 1000mM-1s-1), it is not accompanied by the double of electron transfer reaction for coenzyme
Enzyme.
But the problem of tyrosinase, is as follows：Make single phenol type structural material hydroxylating, in production catechol-type works
After the first time hydroxylating (monophenolase activity) of matter, the bioconversion in single phenol type structural material
Catechol-type structural material is biologically converted into by second of oxidation reaction (dipehnolase activity) of tyrosinase
Quinoid material (quinonic compounds).In general, quinoid material forms free radical (radical) quickly to form melanin
(melanin), the light that the melanin of generation has the function of to obstruct ultraviolet range with stable structure.
Tyrosinase fixes two copper ions by 6, active site periphery histidine (Histidine), above-mentioned two copper from
The distance between son provides ground in the catechol-oxydase (catechol oxidase) including copper and other tyrosinase groups
Keep.According to the presence or absence of oxygen, and according to the quantity of the oxygen atom combined between two copper, tyrosinase divides into three kinds
Form, i.e. deoxidation form (deoxidation-tyrosinase (deoxy-tyrosinase)), oxygen form (oxygen-tyrosinase (oxy-
)) and methionine form (methionine-tyrosinase (met-tyrosinase)) tyrosinase.
Deoxidation form (deoxidation-tyrosinase (deoxy-tyrosinase)) does not include the shape of oxygen between two copper ions
State, if not supplying oxygen supply, becomes the inactive form that can not carry out hydroxylating and oxidation reaction.
If in atmosphere, oxygen dissociates or supply in a solvent hydrogen peroxide, then between two copper ions of deoxidation form
Fix an oxygen (O2) molecule, and this form is known as oxygen form (oxygen-tyrosinase (oxy-tyrosinase)).This
In oxygen form, if the oxygen atom of single phenol is combined with a copper ion, start the first time hydroxylating of tyrosinase.
As above-mentioned first time hydroxylating (monophenolase activity), the fracture of oxygen sub-key, an oxygen
Atom is combined with ortho position carbon phase, is inserted into hydroxyl, an oxygen atom is only remained between two copper of tyrosinase.By this shape
State is known as methionine form (methionine-tyrosinase (met-tyrosinase)).At this time, catechol-type structural material and
Methionine-tyrosinase separates, and an oxygen atom between two copper rearranges established angle, and prepares second
Oxidation reaction (diphenolase activity).
Methionine form can aoxidize catechol-type structural material, but be not involved in the hydroxylating of single phenol type.
After hydroxylating, hydrogen ion is removed from the residue of two hydroxyls on aromatic rings, so as to recur and remain
An oxygen atom be combined to generate the oxidation reaction of water.If using two oxygen atoms of the conjunction that finishes, two copper ions it
Between vacated, and come back to deoxidation form.In single phenol type initial substrate, by bioconversion twice (hydroxylating and
Oxidation reaction) generation product be o-quinone form structural material.
, can be from two hydroxyls of catechol-type structural material in second of oxidation reaction (diphenolase activity)
Dehydrogenation is gone in base, to be combined with two copper, an oxygen atom receives two electronics to be reduced to water from catechol-type material
(diphenolase activity).As described above, methionine form can aoxidize catechol-type structural material, but it is not involved in
The hydroxylating of single phenol type.Thus, after the oxidation reaction of the single phenol type structural material of oxygen-tyrosinase, the youngster of generation
In the case that tea phenolic structural material can not be by methionine-tyrosinase oxidation, methionine-tyrosinase is because that can not change
Accumulated for deoxidation-tyrosinase, tyrosinase is deactivated.
Compared with the rate constant (k1) of first time hydroxylating (monophenolase activity), second
The velocity constant (k2) of secondary oxidation reaction (diphenolase activity) is substantially 10 times or so big, thus in accumulation is used as
Between product catechol-type structural material in terms of have difficulties.The research focus of tyrosinase are concentrated mainly on the shape of melanin
Into reaction and the effect of its inhibitor, thus its a variety of catechol-type structural materials form reaction Techniques of Optimum in application
In, this research can be considered initial technology.
Prior art literature
Patent document
Patent document 1：South Korea Patent Publication 10-2012-0114072
Patent document 2：South Korea's special permission logs in 10-0878394
Non-patent literature
Non-patent literature 1：The influence that L-AA produces the single phenol enzymatic activity of tyrosinase, Biochem
J.1993 October 1；295(Pt 1)：309–312
Non-patent literature 2：Direction for the tyrosinase of monophenolase/bis-phenol enzyme activition ratio is evolved, Enzyme and
Microbial Technology, Volume 47, Issue 7,8 December 2010, Pages 372-376
The content of the invention
Technical problem
Two kinds of situations can be divided into by being applicable in the representative production method of the catechol-type structural material of biological method, i.e. youngster
The hydroxylating of the associated methods and single phenol type structural material of tea phenol and other structures.
Representative reactions equivalent to the first situation are Japanese aginomoto (Ajinomoto) company's DOPA (DOPA) production
Method.Catechol, acetic acid and ammonia are biologically converted into using tyrosine-phenol lyases (tyrosin-phenol liase) more
Bar (DOPA), and highest can produce 100g/L or so.In order to using lyases produce catechol-type structural material, it is necessary to screen with
Catechol can form the specific matrix of carbon-carbon bond and corresponding lyases.Thus, in the life of a variety of catechol-type structural materials
There is limitation in production.
Have equivalent to the benzene class formation material of the second situation or the hydroxylating of single phenol type structural material and added using single
The method of oxygenase (monooxygenase).Monooxygenase as oxidoreducing enzyme one kind, it is necessary to by electron transport system
The supply of electronics.Representational monooxygenase reaction enzymes are Cytochrome P450 (cytochrome P450), from positioned at enzyme
The redox induced reaction of the iron ion of ferroheme (heme) structure of the heart.Make the substrate specificity of Cytochrome P450
Conversion, so that the production of the catechol-type structural material suitable for various structures.But in the situation of most monooxygenase
It is lower, it is necessary to coenzyme, and need double reductases (reductase) for electron transmission.The bioconversion that this can influence matrix is anti-
Speed is answered, and is caused by low yield and productivity.
There is the application of tyrosinase as other biological catalytic hydroxylation reaction in addition.The active portion of tyrosinase
Position is primarily present in protein surface, and in the production of a variety of catechol-type structural materials, advantage is to single phenol type structural material
Tyrosinase substrate specificity it is wide.Also, the single phenol structural material oxidation reaction of tyrosinase is relatively fast (as reference,
Kcat/Km values from the tyrosinase of mushroom class are substantially 1000mM-1s-1), it is not accompanied by anti-for the electron transmission of coenzyme
The double enzymes answered.But in the case of tyrosinase, a variety of single phenol type structural materials are used as matrix, thus with it is mentioned
Above-mentioned two situations are compared, the advantages of being quickly converted to catechol-type structural material, but also quickly make catechol-type at the same time
Structural material aoxidizes, so as to form quinone form.The quinoid material of generation easily forms free radical, to form the high score of melanin etc.
Sub- material, or make periphery material oxidation.
In order to effectively accumulate the catechol-type structural material as intermediate, it is necessary to study the reduction based on quinoid material
Catechol-type material accumulation, the oxidation of the catechol-type material accumulated prevent, with catechol-type material accumulate and lose
Tyrosinase living reactivates.
It is an object of the present invention to pass through three kinds as described above researchs, there is provided using tyrosinase with high productivity
With the production method of produced in yields feature catechol-type structural material.
The means solved the problems, such as
In order to suppress second of oxidation reaction of tyrosinase, and effectively produce catechol-type structural material, this technology
Strategy be roughly divided into three parts, i.e. the accumulation of the catechol-type material of the reduction based on quinoid material, the catechu accumulated
The tyrosinase that the oxidation of phenolic material prevents, inactivates reactivates.
Specifically, first, the present invention provides following method, i.e. makes to produce by second of oxidation reaction of tyrosinase
Quinoid material be effectively reduced to catechol-type structural material again.
Second, the present invention provides following method, i.e. the youngster generated for the first time hydroxylating by tyrosinase
Tea phenolic structural material, induction and the coordinate bond of other elements, to be protected from second of oxidation reaction of tyrosinase.
3rd, the present invention provides and effectively adjusts the technology that three kinds of activity morphologies of tyrosinase optimize enzymatic activity.
When catechol-type structural material does not aoxidize, it is not easy to remove an oxygen atom of methionine-tyrosinase, thus tyrosine
Enzyme is in inactivated state.The tyrosinase in inactivated state can be reactivated by reducing power like this.
Specifically, the present invention provides as follows.
(1) method for being prepared catechol-type structural material from single phenol type structural material using tyrosinase is characterized in that,
It comprises the following steps a step in (a) to step (c), and step (a), imports reducing power to make the mistake by tyrosinase
The quinoid structure material of oxidation reaction generation is reduced to catechol-type structural material；Step (b), utilizes transition metal ions or boron
Ion makes the catechol-type structural material of generation form coordinate bond；And step (c), reducing power is imported to make methionine-junket
Propylhomoserin enzyme activition.
(2) side for preparing catechol-type structural material from single phenol type structural material using tyrosinase according to (1)
Method, it is characterised in that the reducing power used in step (a) or step (c) is from selected from fast by electrochemistry, reduced form nicotinoyl amine gland
Nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), ascorbic acid, cysteine, to perfume (or spice)
Material in the group that beans acid, azanol, catechol, pyrogallol and caffeic acid form provides.
(3) side for preparing catechol-type structural material from single phenol type structural material using tyrosinase according to (1)
Method, it is characterised in that single phenol type structural material is selected from by Tyrosine Amino acid, tyrosol, resveratrol, daidzein, dyewood
Element, apiolin, phloretin, phenol, p-Coumaric Acid, p-nitrophenol, paracresol, 4-Vinyl phenol and o-aminophenol composition
Group in.
(4) side for preparing catechol-type structural material from single phenol type structural material using tyrosinase according to (1)
Method, it is characterised in that catechol-type structural material be selected from by cyanidenon, 3- hydroxyls phloretin, 3 '-adjacent hydroxyl daidzein,
Orobol, piceatannol, hydroxytyrosol, DOPA (dopa), caffeic acid, 3,4- dihydroxy nitrobenzene, 3,4- dihydroxy
In the group of toluene and 3,4- dihydroxy benzenes ethene composition.
(5) side for preparing catechol-type structural material from single phenol type structural material using tyrosinase according to (1)
Method, it is characterised in that the step of isolating and purifying catechol-type structural material (d) is further included, in above-mentioned steps (d), organic
The product comprising catechol-type structural material is dissolved in solvent to obtain water layer, after the pH for the water layer that reduction obtains, is also existed
Dissolved in organic solvent to obtain organic layer, and organic layer evaporation is obtained catechol-type structural material.
(6) side for preparing catechol-type structural material from single phenol type structural material using tyrosinase according to (1)
Method, it is characterised in that single phenol type structural material is the tyrosine residue on macromolecule.
(7) high molecular crosslink reaction method is characterized in that, using by utilizing tyrosinase from single phenol type described in (6)
The catechol-type structural material that structural material prepares the method for catechol-type structural material to prepare.
Invention effect
The preparation and its application of the feature catechol-type structural material of tyrosinase are the present invention relates to the use of, is applicable to
The tyrosinase in a variety of sources.This is to be summarised according to tyrosinase substrate specificity in a variety of single phenol type structural materials to function
Property catechol-type material hydroxylating, it can be achieved that existing difficult extraction or being difficult to the catechol of organic chemical synthesis
The production of type structural material, so as to contribute to its physiology Efficacy experiments.Recently, have much to flavones/isoflavones series
The cancer resistance correlative study of hydroxide.However, it is very difficult to realize the organic synthesis method of location specific, and in extraction nature pole
There is also limitation in terms of few existing flavonol/isoflavones.By this research, feature catechol-type structural material can be made
Production becomes easy, and can carry out more Physiologic Studieses related to this and new material/functional raw material exploitation.
Also, the tyrosine (tyrosine) as the single phenol type amino acid residue on protein can be made by the use of tyrosinase
Hydroxyl turns to DOPA, this improvability matter becomes the protein for the attachment function that can imitate mussel protein matter.Like this, ameliorative
The protein of matter can develop into the stent (scaffolds) of organizational project, further can be developed into functional protein administration system
Unite (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).
Brief description of the drawings
Fig. 1 is the schematic diagram for generating effective catechol-type structural material by tyrosinase, and a of Fig. 1 is single phenol type
Structural material, the b of Fig. 1 is catechol-type structural material, and the c of Fig. 1 is quinoid structure material, and the d of Fig. 1 is and catechol-type structure
The other elements of material form the structure of coordinate bond.Also, the first time hydroxylating that the 1 of Fig. 1 is tyrosinase, Fig. 1
2 be tyrosinase second of oxidation reaction, an oxygen molecule is put into for deoxidation-tyrosinase and is present in activity by the 3 of Fig. 1
Between two copper in position, and it is by making methionine-tyrosinase as the reaction of oxidation-tyrosinase, the 4 of Fig. 1
An oxygen atom reduction reducing agent reaction, Fig. 15 for hydroxylated catechol-type structural material formed coordinate bond it is anti-
Should, the 6 of Fig. 1 is the reaction for making the also original accumulation again of quinoid structure material be catechol-type structural material, and the 7 of Fig. 1 is peroxide
The quinoid material producing high-molecular of change is the reaction of melanin.
Fig. 2 is adding each reducing agent (also as the table for enumerating the reducing agent added in the full cell effect of tyrosinase
Prototype nicotinamide adenine dinucleotide, L-AA, glutathione, cysteine, hydroxyquinone, 1- naphthols, p-Coumaric Acid,
Curcumin, catechol, pyrogallol or forulic acid) in the case of, hydroxyl is presented in resveratrol (trans-resveratrol)
The opposite output of the piceatannol (piceatannol) of base.
Fig. 3 is the tyrosinase (BMT_tyr in relation to three kinds of separate sources：Bacillus megaterium tyrosinase, ABS_tys：
Agaricus bisporus tyrosinase, SAV_tyr：Deinsectization streptomycete tyrosinase) Tyrosine Amino acid (L-tyrosine) and DOPA
(L-dopa) monooxygenase reaction speed and the comparison sheet of dioxygenase reaction speed.
Fig. 4 is the multiple catechol-type knots for representing to generate by the hydroxylating of tyrosinase from single phenol type structural material
The reaction equation of structure material.
Fig. 5 be adjusted by pH confirm hydroxylated 3 '-adjacent hydroxyl daidzein (3 '-ODI, 3 '-
Orthohydorxydaidzein the quantitative analysis of high-performance-liquid chromatography of coordinate bond) whether is formed.
Fig. 6 is to adjust to confirm that hydroxylated orobol (orobol) and cyanidenon (luteolin) are by pH
The quantitative analysis of the no high-performance-liquid chromatography for forming coordinate bond.
Fig. 7 a are the junket ammonia of the daidzein comprising 1mM and 100nM in the borate buffer for observed in units of the time pH9
The curve map of the productivity of 3 '-neighbour hydroxyl daidzein in the 5mL of sour enzyme external (in vitro) reaction.
Fig. 7 b are comprising 300 μM in three (methylol) aminomethanes (tris-HCl) for observed in units of the time pH9
The curve map of the productivity of 3 '-neighbour hydroxyl daidzein in the 5mL vitro reactions of the tyrosinase of daidzein and 100nM.
Fig. 8 is the hydroxylating of the genistein of the 1mM that observation with the addition of azanol or ascorbic acid in units of the time
Curve map.
Fig. 9 is the hydroxylating of the resveratrol of the 1mM that observation with the addition of azanol or ascorbic acid in units of the time
Curve map.
Figure 10 is the schematic diagram of the technology of the catechol-type structural material for separating/purifying generation, in embodiment 5 in detail
It is thin and explicitly show its method.
Figure 11 a represent what is generated using gas chromatograph-mass spectrometer (GC-MS) (GC/MS) analysis from daidzein (daidzein)
3 '-adjacent hydroxyl daidzein (3 '-ODI) as a result, being logical by 1 18.18 points of the retention time (retention time) represented
Gas chromatography (GC) separated 3 '-neighbour's hydroxyl daidzein is crossed, by the 486.48 of a expressions, m/ that m/z is molecular weight (TMS)
Z, by b represent 471.50, m/z be molecular weight (TMS) -15 m/z, by c represent 383.39, m/z for molecular weight (TMS) -
103 m/z.
Figure 11 b represent to generate from resveratrol (trans-resveratrol) using gas chromatograph-mass spectrometer (GC-MS) analysis
It is piceatannol (piceatannol) as a result, by 1 14.82 points of the retention time (retention time) represented to pass through
The separated piceatannol of gas chromatography, by the 532.53 of a expressions, m/z that m/z is molecular weight (TMS), is represented by b
517.45th, m/z is the m/z of molecular weight (TMS) -15.
Figure 12 shows to form the state of coordinate bond with iron ion according to the catechol-type structural material of pH.
Embodiment
Hereinafter, the present invention will be described in more detail.
As long as no other definition, all technical terms and scientific terms being used in the present invention have and the present invention
The identical implication of term that person of an ordinary skill in the technical field is generally understood.In general, use in the present specification
Nomenclature and the experimental method of following description is in the art it is known that and commonly used approaches.
The present invention is provided using the microorganism (full cell) of tyrosinase or expression tyrosinase to produce catechol-type knot
The method of structure material.
Also, provide in order to suppress the peroxidization based on tyrosinase catechol-type structural material bioconversion
The method reacted and form coordinate bond to accumulate.
Also, the present invention, which provides, to be made effectively to be reduced to catechol-type structural material by the quinoid material that peroxidating generates
Method.
Specifically, the present invention can pass through the accumulation of the catechol-type material of reduction i) based on quinoid material, ii) accumulate
Catechol-type material oxidation prevent, iii) tyrosinase that accumulates and inactivate with catechol-type material reactivates,
Effectively accumulate catechol-type structural material.
First, the present invention provides the quinoid material weight that produce effectively second of oxidation reaction by tyrosinase
The new method for being reduced to catechol-type structural material.Thus it is melanin to be possible to delay quinoid structure material producing high-molecular.This
It is because multiple free radicals are stabilized by reducing power, and receive electronics because of quinoid material, so as to be converted to again
Tea phenolic structural material.Bad influence is not produced to enzymatic activity and workable effective reducing power has electrochemical method and Fig. 2's
The reducing agent of the compound being explicitly illustrated in reducing agent catalogue etc.If adding the reducing agent in catalogue in the oxidation reaction,
It can confirm that the formation of dark melanin is delayed by.At this time, although the formation of delay melanin, can confirm as initial substrate
Single phenol type material persistently reduce, be thus judged as that reducing power as described above only influences the formation of melanin, and and tyrosine
The activity of enzyme reduces unrelated.Above-mentioned reducing power can be from selected from by electrochemistry, reduced nicotinamide adenine dinucleotide, reduced form
Nicotinamide-adenine dinucleotide phosphate, ascorbic acid, cysteine, p-Coumaric Acid, azanol, catechol, pyrogallol and coffee
Material in the group of coffee acid composition provides.Preferably, above-mentioned reducing power can utilize glucose dehydrogenase (glucose
) etc. dehydrogenase reduced nicotinamide adenine dinucleotide (phosphoric acid) (NAD (P) H) regeneration producing enzyme is come in the reaction
Lasting to provide, this can more effectively be applied when (in vivo) produces catechol-type material in vivo.
Second, the present invention provides following method, i.e. the youngster generated for the first time hydroxylating by tyrosinase
Tea phenolic structural material, induction and the coordinate bond of other elements, to be protected from second of oxidation reaction of tyrosinase.
It is well known that catechol-type structural material forms coordinate bond (Proc Natl Acad Sci USA.2011 according to pH and iron ion
15,108 (7), 2651-2655).This reason is incorporated into the metal ion in organic matter to have cohesive force with mussel protein matter
Reason it is identical (Annu Rev Mater Res.2011 41,99-132).Based on this, in addition to iron ion, to tyrosine
Enzyme it is active harmless, and screened with catechol-type structural material formed coordinate bond material, pass through the implementation for this research
The experiment of example carries out.
Boron (B) has a sp2 function of hybridized orbitals, but when there are when lewis base (Lewis base), becoming sp3.According to report
Accuse, catechol-type structural material plays the role of lewis base, and according to pH, coordinate bond is formed with one or two catechol material
(BBA, 2002,1569,35-44).Increase with the hydrophily for the complex that boric acid is combined, due to this mechanism, have when stablizing
When having the sugar of glycol (diol), using borate ion, borate ion is also used when from coffee bean or Extraction of Caffeine From Tea.
The hydroxyl for forming the catechol-type material of coordinate bond all forms coordinate bond with boric acid, because without being aoxidized by tyrosinase.Thus,
Also utilized when excluding second of oxidation reaction, and only observing first time hydroxylating (JACS, 2003, vol.125,43,
13034-13035)。
3rd, the present invention provides three kinds of activity morphologies (deoxidation form, oxygen form and the first sulphur ammonia for effectively adjusting tyrosinase
Sour form) by enzymatic activity optimize technology.When catechol-type structural material does not aoxidize, methionine-tyrosinase
One oxygen atom is not easily removed, thus tyrosinase is in inactivated state.Like this in inactivated state methionine-
One oxygen atom of tyrosinase is reduced by other reducing agents or electrochemical reduction power, it is possible to being gone as hydrone
Remove, tyrosinase is converted to again can receive oxygen (O2) molecule deoxidation form.As this reducing agent, such as the reducing agent of Fig. 2
It is explicitly illustrated in list, in addition to catechol-type material, there is azanol (NH2OH, hydroxylamine), reduced form niacinamide
Adenine-dinucleotide (phosphoric acid), ascorbic acid (ascorbic acid) etc..
As shown in the reaction 4 of Fig. 1, the oxygen that azanol (hydroxylamine) makes to remain between the copper in active site is former
Son reduction generates water, and make to be used as matrix reception single phenol type material tyrosinase be converted to deoxidation form (JACS,
2003, vol.125,43,13034-13035).Deoxidation-tyrosinase and an oxygen molecule re-form copper-oxygen complex (μ-η
²：η 2 peroxo binuclear copper complex) become oxygen-tyrosinase.The reaction 4 of Fig. 1 is not only by azanol
To realize, but also can by can close to active site reducing agent, i.e. reduced nicotinamide adenine dinucleotide (phosphorus
Acid), ascorbic acid, other catechol-type materials etc. realize.
On the other hand, the term being used in the present invention is term usually used in the art, as long as this
The those of ordinary skill of technical field that the present invention belongs to, is just appreciated that its implication, but in the present specification, carries out briefly
It is bright as follows.
(1) tyrosinase refers to the enzyme for being catalyzed the production of melanin as the protein comprising copper.
(2) single phenol type structural material refers to the material with the structure identical with a of Fig. 1.Above-mentioned single phenol type structural material
There is 1, the 2- talan of Tyrosine Amino acid (tyrosin), tyrosol (tyrosol), resveratrol (resveratrol) etc.
(stilbene) series, daidzein (daidzein), genistein (genistein), apiolin (apigenine), root skin
The flavones (flavonone) of plain (phloretin) etc./isoflavones (isoflavone) series, in addition, also has phenol
(phenol), p-Coumaric Acid (para-coumaric acid), p-nitrophenol (para-nitrophenol), paracresol
(para-cresol), 4-Vinyl phenol (para-vinylphenol) and o-aminophenol (ortho-aminophenol).
Preferably, can be used resveratrol, p-Coumaric Acid, daidzein, genistein, p-nitrophenol, paracresol, to vinyl benzene
Phenol, apiolin or phloretin etc..
Also, single phenol type structural material can be the tyrosine residue on polymer substance, and above-mentioned polymer substance can be with
For protein (polypeptide).
(3) catechol-type structural material refers to the material with the structure identical with the b of Fig. 1.Above-mentioned catechol-type structure
Material has cyanidenon as adjacent hydroxylated material is specifically carried out to single phenol type structure tyrosine material
(luteolin), the hydroxyl of 3- hydroxyls phloretin (3-hydroxyphloretin, 3 '-ODI), orobol (orobol) etc.
The hydroxyl 1 of base flavones (flavonone)/isoflavones (isoflavone) series, piceatannol (piceatannol) etc., 2- bis-
Styrene (stilbene) series, hydroxytyrosol (hydroxyltyrosol), DOPA (dopa), caffeic acid (caffeic
Acid), 3,4- dihydroxy nitrobenzene (3,4-dihydroxynitrobenzene), 3,4- orcins (3,4-
Dihydroxytoluene), 3,4- dihydroxy benzenes ethene (3,4-dihydroxystyrene) etc..Preferably, enumerate above
Preferable multiple single phenol type structural materials as adjacent hydroxylated form is specifically carried out, have successively piceatannol, caffeic acid,
3 '-neighbour hydroxyl daidzein, orobol, 3,4- dihydroxy nitrobenzene (3,4-dihydroxynitrobenzene), 3,
4- orcins (3,4-dihydroxytoluene), 3,4- dihydroxy benzenes ethene (3,4-dihydroxystyrene), wood
Rhinoceros grass element, 3- hydroxyls phloretin (3-hydroxyphloretin) etc..
(4) quinoid structure material refers to the material with the structure identical with the c of Fig. 1.
(5) coordinate bond compound substance refers to be formed and single complex of the d or Figure 12 of Fig. 1 (mono-complex), double multiple
The complex of the identical structure of fit (bis-complex) or trishydroxymethylaminomethane complex (tris-complex).Can
Use transition metal ions (transition metal ions) or borate ion (boric acid, borate ion).
(6) cell extract refers to the microorganism extracts of the invention for expressing auxilin and tyrosinase.
(7) full cell effect refers to the broken cell comprising certain enzyme to utilize cellular content, or enzyme is not divided
The reaction of complete whole cell is utilized from purifying.
(8) tyrosinase first time hydroxylating (monooxygenase activity) refers to importing oxygen atom into carbon-hydrogen link
The tyrosinase enzyme reaction of Shi Jinhang catalysis, generates the residue of hydroxyl.
(9) second of oxidation reaction of tyrosinase (dioxygenase activity) refer in catechol-type structural material remove with
The hydrogen and electronics that oxygen is combined, the tyrosinase enzyme reaction being catalyzed in the form of benzoquinones.
(10) PCR is to instigate deoxyribose core as PCR (Polymerase Chain Reaction)
The method expanded one regiospecificity of acid.
(11) carrier refers to be formed by single-stranded, double-strand, circle or supercoil DNA or ribonucleotide more
Nucleotide.Carrier can be included in the structural element that appropriately distance connects in the operative mode, for that can produce recombinant protein.
This structural element can include replication orgin, promoter, enhancer, 5 ' messenger RNA (5 ' mRNA) targeting sequencing, ribose
Body binding site, nucleic acid cassette, termination and site of polyadenylation and the tag format that can screen etc., multiple said structure key elements can
One or more structural elements are reduced according to specific purposes.Nucleic acid cassette, which can include, to be used to be inserted into restructuring egg to be expressed
The restriction enzyme sites of white matter.In functional vector, nucleic acid cassette contain comprising translation start and termination site it is to be expressed
Nucleotide sequence.As needed, the carrier that can be inserted into two kinds of boxes in carrier is also used, multiple functions as described above can be additionally
Serialized.Expression coli strain BL21 (DE3) etc. can be used in the gene for being inserted in recombinant vector, but can basis
The species of the carrier of insertion and it is different.As long as general technical staff of the technical field of the invention, it is possible to easily selection
This carrier and expression bacterial strain.
(12) it is melanin as delay quinoid structure material producing high-molecular, and quinoid material is effectively reduced to
The reducing agent of tea phenolic structural material, can be used reduced nicotinamide adenine dinucleotide (phosphoric acid), L-AA, paddy Guang
Sweet peptide (glutathione), cysteine (cystein), xenogenesis catechol-type structural material, benzene or single phenol type structural material
Deng, as xenogenesis catechol-type structural material, usable catechol, 3- methyl catechols, 4- methyl catechols, caffeic acid
(caffeic acid), pyrogallol or catechol violet etc..Also, as single phenol type structural material, phenol, daidzin can be used
Member, genistein, p-Coumaric Acid etc..
(13) as it is making that tyrosinase reactivates, azanol, can also can be used close to the reducing agent of active site
Prototype nicotinamide adenine dinucleotide (phosphoric acid), ascorbic acid or other catechol-type materials etc..
Representation 1：The expression of tyrosinase
Make to derive from bacillus megaterium (BMT, Bacillus megaterium) and source by PCR
Expand in the tyrosinase DNA sequence of deinsectization streptomycete (SAV, actinomyces, Streptomyces avermitilis)
After increasing, it is respectively put into after pet28a or petDuet carriers, in this regard, in Escherichia coli (E.coli), such as histidine mark
(histag) expressed respectively shown in (6 histidines).Expression is as follows.To BL21 competent cells
After plasmid of (competent cell) conversion inserted with tyrosinase base sequence, the antibiosis of suitable plasmid is being included respectively
In the LB solid mediums of element, cultivated in 37 DEG C of culture mediums.In the LB fluid nutrient mediums comprising antibiotic of 5mL
Be inoculated with a bacterium colony, and in 37 DEG C of incubators, with 200rmp speed culture 8 it is small when.By it again in the antibiosis comprising 50mL
1v% (500uL) is vaccinated with the new fluid nutrient medium of element.If cell optical density 600nM reaches 0.6, the different of 2mM is put into
The CuSO of propyl group-β-D- Thiogalactopyranosides (IPTG) and 1mM4, and in 18 DEG C of incubators, with the speed of 200rpm,
By protein expression induction of 17 it is small when.17 it is small when after, cell is centrifuged with the speed of 4000rpm, and utilizes
The phosphate buffer (PBS) of 5mL is cleaned to prepare.After cleaning, delay in three (methylol) aminomethane buffer solutions or boric acid
Full cell effect has been carried out in fliud flushing, and the cell extract except having been prepared in a manner of ultrasonication.(In in vitro
Vitro) full cell effect directly uses the water-soluble body portion in cell extract, or make use of to commonly use histidine mark (6-
Histag) the protein of method of purification purifying, and show specifically and clearly in various embodiments.
Reaction method 1：Utilize the production method of tyrosinase and the catechol-type structural material of reducing substances
Make the tyrosinase of the mentioned microorganism of acquisition or the tyrosinase extract of production or purifying and such as a of Fig. 1
Single phenol type structural material is reacted, to produce the catechol-type structural material of the b such as Fig. 1.In the reaction, reducing agent is added
To suppress the production process after the c materials of Fig. 1.
Relative to the concentration of single phenol type structural material, the concentration of the reducing agent of addition is preferably 1 to 100 times, and more preferably 5
To 30 times, especially preferably 7 to 13 times.In the reaction, the material of coordinate bond can be formed with the catechol of generation by adding, from junket
Second of oxidation reaction of propylhomoserin enzyme is protected.
Relative to the concentration of single phenol type structural material, the concentration of the coordinate bond precursor of addition is preferably 1 to 10000 times, more
Preferably 50 to 600 times, especially preferably 100 to 500 times.
The reaction temperature of the present invention is preferably 2 to 80 DEG C, more preferably 20 to 70 DEG C, especially preferably 20 to 40 DEG C.
The pH of reaction solution is preferably 5 to 11, more preferably 5 to 11, especially preferably 7 to 9.As shown in figure 3, each junket ammonia
The reaction speed of sour enzyme is different, according to its substrate specificity, has used tyrosinase.
The specific method of the present invention is described in detail with embodiment, but the technical scope of the present invention is not limited to reality
Apply example.In the examples below, if not referring to particularly, all percentages are with 100 percentage by weights of final composition
On the basis of.
Embodiment 1
According to the changes in solubility of the initial substrate (single phenol type structural material) of pH
Daidzein (daidzein) as isoflavones series has the solubility low to water, thus under normal temperature condition
Neutral water or buffer solution in, there are 0.1% dimethyl sulfoxide (DMSO) (DMSO) in the case of, can dissolve 300uM or so.But
It is that can confirm as follows：In the case of using the borate buffer of high concentration, as pH becomes higher, solubility increase, in pH9,
Horizontal with 5mM, solubility increases by more than ten times, and horizontal with 30mM in pH10, solubility increases by 100 times.Tyrosinase and its
His monooxygenase is different, active in wide pH, so as to improve reaction productivity using the initial substrate of high concentration.
In the pH9 as tyrosinase activity scope, 5mL, daidzein comprising 300 μM and 100nM junket ammonia
External (in vitro) reaction of sour enzyme produces the 3 '-adjacent hydroxyl daidzein of 15mg per hour, on the contrary, 5mL, include pH9
Borate buffer in 1 μM of daidzein and 100nM tyrosinase external (in vitro) reaction produce per hour
The 3 ' of 45mg-neighbour's hydroxyl daidzein, its productivity can increase by three or so.It is shown explicitly in Fig. 7 a and Fig. 7 b.
Embodiment 2
According to the catechol-type structural material yield comparative experiments of reducing agent
It is not broken to express the tyrosinase from deinsectization streptomycete cultivated according to the above-mentioned representation 1 being explicitly illustrated
Cell, and only clean cell, and add a variety of reducing agents (reduced form nicotinamide adenine of the resveratrol of 0.1mM, 1mM
Dinucleotides, L-AA, glutathione, cysteine, hydroxyquinone, 1- naphthols, p-Coumaric Acid, curcumin, catechol, neighbour
Benzenetriol or forulic acid) produce piceatannol.After reaction, using same amount ethyl acetate (EA, ethylacetate) or
Nonpolar solvent is come after extracting, with high-performance-liquid chromatography quantitative analysis, to compare the piceatannol based on reducing agent addition
Production efficiency, and be shown explicitly in Fig. 2.
Embodiment 3
The coordinate bond of catechol-type structural material
Such as reaction method 1, using the tyrosinase from bone-marrow transplantation (BMT) of the purifying of 200nM, to make 500uM's
Daidzein carries out hydroxylating, at this time, is put into boric acid to induce coordinate bond.As shown in (a) part of Fig. 5, pass through high property
Energy-liquid chromatography, which confirms the 3 '-adjacent hydroxyl daidzein that coordinate bond is formed in alkaline reaction liquid, has hydrophily, because without
It is extracted as organic solvent.Also, when the HCl of processing 2M is confirmed in (b) part of Fig. 5, coordinate bond is released from, and is detected
The 3 ' of 200uM-neighbour's hydroxyl daidzein.
Also, the bone-marrow transplantation tyrosinase of the 300nM using purifying, the genistein and apiolin for also making 1mM carry out
Hydroxylating, as shown in fig. 6, confirming hydroxylated orobol and cyanidenon by high-performance-liquid chromatography
Also coordinate bond is formed in alkaline reaction liquid, not to be extracted in organic solvent, if releasing coordinate bond in acid reaction liquid,
Then it is extracted in organic solvent.
Embodiment 4
The inactivation of tyrosinase prevents
Histidine mark purifying is carried out to the tyrosinase cultivated in representation 1, and with the addition of 100nM's in the reaction
The inactivation of tyrosinase, in order to prevent tyrosinase, is put into azanol or ascorbic acid into line activating.At this time, relative to matrix, add
The azanol or the concentration of ascorbic acid added is 1 to 100 times, more preferably 3 times to 50 times, especially preferably 10 times to 20 times.It is logical
This composition is crossed, according to reaction method 1, is reacted the daidzein of 1mM, and temporally utilizes 4 times of ethyl acetate
(ethylacetate, EA) or nonpolar solvent after extracting the reactant of 100uL, utilize high-performance-liquid chromatography to carry out
Quantitative analysis.According to quantitative analysis results, when reacting 6 hours, the 3 ' of 1mM-neighbour's hydroxyl daidzin with 100% produced in yields
Member, and be shown in Fig. 7 a.
In the same way, it is special induction of the position of genistein (genistein) and resveratrol (resveratrol)
Different in nature hydroxylating, single phenol type material as described above can be biologically converted into by the first time hydroxylating of tyrosinase
Feature catechol-type structural material.Genistein is biologically converted into 95% orobol when reacting 1 hour
(orobol), resveratrol is biologically converted into 100% piceatannol (piceatannol) when reacting 1 hour.Shown
In Fig. 8 and Fig. 9.
Embodiment 5
The catechol-type structural material of generation isolates and purifies method
In order to separate the catechol-type structural material generated in example 4, the solubility of product and reactant is utilized.
In alkaline buffer, the catechol-type material for being reacted and being generated forms coordinate bond, so as to improve hydrophily (the 1 of Figure 10
Part).Do not formed the single phenol type structural material of coordinate bond body 4 multiple of reaction volume ethyl acetate (ethylacetate,
EA it is extracted in), as shown in 2 parts of Figure 10, organic solvent and water layer distinguish layer because of difference of specific gravity.Using buret to upper
State organic solvent and water layer is separated, to separate the 3-1 of Figure 10 (water layer) and 3-2 (organic layer), and make separated organic layer
Evaporation, the single phenol type structural material to make to remain in it can be reused in reaction.Water layer is reduced using the HCl of 1M
PH, and catechol-type structural material (the 4 of Figure 10 are extracted again using the ethyl acetate (ethylacetate, EA) of 4 multiples
Part).After extraction, acid reaction liquid layer has been separated using buret.With regard to ethyl acetate (ethylacetate, EA) layer
Speech, ethyl acetate (ethylacetate, EA) is removed using cold boiler (vacuum evaporator), and can be obtained pure
The catechol-type structural material of the powder form of change.
Embodiment 6
Utilize the quality analysis of the hydroxylating product of tyrosinase
Using gas chromatograph-mass spectrometer (GC-MS) (GC-MS) to multiple feature catechol-type knots for being generated in embodiment 5
Structure material carries out qualitative analysis, its result is shown explicitly in Figure 11.
Specifically, Figure 11 a represent to analyze from daidzein using gas chromatograph-mass spectrometer (GC-MS) (GC/MS)
(daidzein) the 3 ' of generation-adjacent hydroxyl daidzein (3 '-ODI) as a result, by the 1 retention time (retention represented
Time) 18.18 points are by gas chromatography (GC) separated 3 '-neighbour's hydroxyl daidzein, are by the 486.48 of a expressions, m/z
The m/z of molecular weight (TMS), by the 471.50 of the b expressions, m/z that m/z is molecular weight (TMS) -15, by the 383.39 of c expressions, m/z
For the m/z of molecular weight (TMS) -103, the above results can refer to existing 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) confirm as 3 '-neighbour hydroxyl daidzein.
Figure 11 b represent to generate from resveratrol (trans-resveratrol) using gas chromatograph-mass spectrometer (GC-MS) analysis
It is piceatannol (piceatannol) as a result, by 1 14.82 points of the retention time (retention time) represented to pass through
The separated piceatannol of gas chromatography, by the 532.53 of a expressions, m/z that m/z is molecular weight (TMS), is represented by b
517.45th, m/z is the m/z of molecular weight (TMS) -15, and the above results can refer to existing paper (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) piceatannol is confirmed as.
Embodiment 7
Bioconversion of the single phenol type material to catechol-type structural material on macromolecule：To imitating for mussel protein matter
In order to which the tyrosine residue on macromolecule is effectively biologically converted into DOPA residue, deinsectization streptomycete junket ammonia is utilized
Sour enzyme.Elastin like polypeptides (ELP, the elastin of totally 180 amino acid sequences with (VPGYG) 12 (VPGVG) 24
Like-polypeptide) by following Elastin like polypeptides Y with the three of the 50mM of pH9 of 0.5 percentage by weight in 300uL
After being dissolved in (methylol) aminomethane buffer solution, the FeCl of 2mM is put into3With ascorbic acid, the deinsectization streptomycete junket of 500nM
Propylhomoserin enzyme, and react two hours after, change pH using the HCl of the NaOH and 1M of 1M.The elastin-like dissolved in pH5
Polypeptide _ Y is precipitated in pH7 in the form of powder.Confirm in the case where pH is adjusted to 11, Elastin like polypeptides _ Y into
Row gelation (gelation).Confirm in the case where reducing pH again, hydrogel (hydrogel) dissolving of formation, so that
It is that there is reversible reaction.This be withImitateConsistent result (the Proc of the pervious paper of the function of mussel protein matter
Natl Acad Sci USA.2011 15,108 (7), 2651-2655).
Industrial applicability
The present invention can realize the batch production of feature catechol-type structural material using enzyme reaction, and be applicable to new
The production of material/functional raw material and medical product.Specifically, the present invention is according to tyrosinase substrate specificity, summarize from
A variety of single phenol type structural materials are to the hydroxylating of feature catechol-type material, it can be achieved that existing be difficult to extract or be difficult to
The production of the catechol-type structural material of organic chemical synthesis is carried out, so as to contribute to its physiology Efficacy experiments.
Claims (2)
- A kind of 1. method for preparing adjacent hydroxylating material from single phenol type structural material using tyrosinase, it is characterised in that described Adjacent hydroxylating material is cyanidenon, 3- hydroxyls phloretin, 3 '-adjacent hydroxyl daidzein or orobol, the method Comprise the following steps (a), (b) and (c)：Step (a), imports ascorbic acid to remove the hydroxyl between the two of de-methionine-tyrosinase copper ions, thus swashs Tyrosinase living；The quinoid structure material that the peroxidization by tyrosinase generates is set to be reduced to adjacent hydroxylate at the same time Matter；Step (b), the adjacent hydroxylating material formation for making generation using transition metal ions or boron ion in alkaline reaction liquid are matched somebody with somebody Position key；WithStep (c), dissolves the product comprising the adjacent hydroxylating material to obtain water layer in organic solvent, what reduction obtained After the pH of water layer, product is dissolved in organic solvent to obtain organic layer, and make the organic layer evaporation described to obtain Adjacent hydroxylating material.
- 2. according to the method described in claim 1, it is characterized in that, single phenol type structural material is selected from by daidzein, dyewood The group of element, apiolin and phloretin composition.
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| KR10-2013-0073957 | 2013-06-26 | ||
| PCT/KR2013/011733 WO2014208847A1 (en) | 2013-06-26 | 2013-12-17 | Catechol derivative prepared using tyrosinase, method for preparing same, and application of same |
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| KR102068459B1 (en) | 2016-12-30 | 2020-01-21 | 서울대학교산학협력단 | Method for preparation of catechol derivatives by using tyrosinase derived from burkholderia thailandensis and application thereof |
| KR101992247B1 (en) * | 2017-09-27 | 2019-06-24 | 전남대학교산학협력단 | Composition for 3-hydroxyl phloretin production |
| KR101983570B1 (en) | 2018-01-31 | 2019-05-29 | 서울대학교 산학협력단 | Lipid nano particle composition for improvement of orobol stability and method for production of it |
| KR102273628B1 (en) * | 2018-10-08 | 2021-07-06 | 포항공과대학교 산학협력단 | Development of Adhesive Mussel Protein Hydrogel Formulation by Introducing Histidine-based Protein Domain from Mussel Byssus |
| CN109825518B (en) * | 2019-02-25 | 2020-07-28 | 佐登妮丝（广州）美容化妆品有限公司 | Preparation method and application of 3' -hydroxy genistein |
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| Stabilization of Tyrosinase for Catechol Production;Jong Hyeon Park，等;《Korean J. Biotechnol. Bioeng.》;19941231;第9卷(第5期);第525页、第528页 * |
| 组氨酸残基的化学修饰对酪氨酸酶活性和构象的影响;芶琳;《四川农业大学硕士学位论文》;20091231;第2-3页 * |
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