CN105555776A - Catechol derivative prepared using tyrosinase, method for preparing same, and application of same - Google Patents

Catechol derivative prepared using tyrosinase, method for preparing same, and application of same Download PDF

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CN105555776A
CN105555776A CN201380077815.3A CN201380077815A CN105555776A CN 105555776 A CN105555776 A CN 105555776A CN 201380077815 A CN201380077815 A CN 201380077815A CN 105555776 A CN105555776 A CN 105555776A
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catechol
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tyrosine oxidase
tyrosine
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金秉祺
李相赫
白基宪
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Seoul National University Industry Foundation
SNU R&DB Foundation
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Abstract

The present invention relates to a technology related to the biosynthetic reaction of a catechol derivative using tyrosinase, for effectively preventing the diphenolase activity of tyrosinase and thereby selectively catalyzing only the monophenolase activity of a monophenolic compound. The present invention provides a technology for producing various functional catechol materials with high productivity and yield using same.

Description

The catechol-type structural material utilizing tyrosine oxidase to prepare, its preparation method and application thereof
Technical field
The present invention relates to the tyrosine oxidase (tyrosianse) utilizing and have wide substrate specificity only generates catechol-type structural material with screening property from multiple single phenolic structural material (monophenoliccompounds) method with high productivity and yield.Thus, the present invention can realize the batch production of the functional catechol-type structural material utilizing enzyme reaction, and is applicable to raw material and medical products production.
Background technology
It was reported, the physiologically active that a lot of catechol-type structural material (catecholderivatives) has presented in anti-oxidant, anticancer, anti-inflammatory or the effect such as antiviral, compared with single phenolic structural material (monophenoliccompounds), its effect is higher.Especially, catechol-type structural material utilizes as the inhibitor of tyrosine oxidase (tyrosinase), and has the effect reduced based on the production of the melanochrome (melanin) of tyrosine oxidase.And, as everyone knows, in daidzein (daidzein derives from the isoflavones of soybean), realize 3 '-adjacent hydroxyl daidzein (3 '-ODI of adjacent hydroxylation (ortho-hydroxylation) specifically, 3 '-orthohydorxydaidzein) a part be present in the salty sauce of maturation, prevention by UV-induced skin carcinoma, and suppresses activity (JBC, 2010 relevant to metastasis of cancer, vol.285, pp.21458; JBC, 2011, vol.286, pp.14246).As the catechol-type material deriving from apigenin (apigenin), hydroxylated luteolin (luteolin) can extract minute quantity in the arithoke (arichoke) of good in oxidation resistance, according to up-to-date report, (FoodChemistry can be used in breast cancer treatment, 2013, " Luteolinsensitisesdrug-resistanthumanbreastcancercellsto tamoxifenviatheinhibitionofcyclinE2expression ").In addition, as everyone knows, the catechol-type material of the Hydroxytyrosol (hydroxytyrosol) in sweet oil, the piceatannol (piceatannol) in grape wine etc. is also used as powerful antioxidant.Catechol-type structural material on polymer substance can imitate the function of mussel protein matter, its Application Areas carries out studying (AnnuRevMaterRes.2011141:99-132Biofouling2012 from medical treatment material to surface-modified nano engineering just widely, 28:8,865-877).
When utilizing biological catalyst in the past to produce catechol-type structural material, utilize the multiple monooxygenase (mono-oxigenase) comprising P450 enzyme.But, when most monooxygenase, need coenzyme, and need the two reductase enzymes (reductase) for electron transmission.This can affect the bioconversion reaction speed of matrix, and is caused by low yield and productivity.On the contrary, relatively fast (the kcat/Km value as a reference, deriving from the tyrosine oxidase of mushroom class is roughly 1000mM to single phenol structural material oxidizing reaction of tyrosine oxidase -1s- 1), not with the two enzymes for the electrotransfer reaction of coenzyme.
But, the problem of tyrosine oxidase is as follows: make single phenolic structural material hydroxylation, come after the first time hydroxylating (monophenolaseactivity) producing catechol-type structural material, the catechol-type structural material of bio-transformation in single phenolic structural material is quinoid material (quinoniccompounds) by second time oxidizing reaction (dipehnolaseactivity) bio-transformation of tyrosine oxidase.Usually, quinoid material forms free radical (radical) and forms melanochrome (melanin) fast, and the melanochrome of generation has the function of the light intercepting ultra-violet region with stable structure body.
Tyrosine oxidase fixes two cupric ions by reactive site periphery 6 Histidines (Histidine), and the distance between above-mentioned two cupric ions specifies that in the catechol-oxydase (catecholoxidase) comprising copper and other tyrosine oxidase groups ground keeps.According to the existence of oxygen whether, and according to the quantity of the Sauerstoffatom combined between two copper, tyrosine oxidase divides into three kinds of forms, that is, deoxidation form (deoxidation-tyrosine oxidase (deoxy-tyrosinase)), oxygen form (oxygen-tyrosine oxidase (oxy-tyrosinase)) and methionine(Met) form (methionine(Met)-tyrosine oxidase (met-tyrosinase)).
Deoxidation form (deoxidation-tyrosine oxidase (deoxy-tyrosinase)) is do not wrap oxygen containing form between two cupric ions, if do not supply oxygen supply, then becomes the inactive form that cannot carry out hydroxylation and oxidizing reaction.
If in atmosphere, oxygen dissociates in a solvent or supplies hydrogen peroxide, then between two cupric ions of deoxidation form, fix an oxygen (O 2) molecule, and this form is called oxygen form (oxygen-tyrosine oxidase (oxy-tyrosinase)).In this oxygen form, if the Sauerstoffatom of single phenol combines with a cupric ion, then start the first time hydroxylating of tyrosine oxidase.
As above-mentioned first time hydroxylating (monophenolaseactivity), oxygen molecule bond rupture, a Sauerstoffatom combines with ortho position carbon, inserts hydroxyl, between two copper of tyrosine oxidase, only remains a Sauerstoffatom.This form is called methionine(Met) form (methionine(Met)-tyrosine oxidase (met-tyrosinase)).Now, catechol-type structural material is separated with methionine(Met)-tyrosine oxidase, and a Sauerstoffatom between two copper rearranges established angle, and prepares second time oxidizing reaction (diphenolaseactivity).
Methionine(Met) form can make catechol-type structural material be oxidized, but does not participate in the hydroxylating of single phenolic.After first time hydroxylating, from the residue of the hydroxyl of two aromatic nucleus, remove hydrogen ion, thus recur to combine with a residual Sauerstoffatom and generate the oxidizing reaction of water.If use two Sauerstoffatoms of combination, then vacated between two cupric ions, and come back to deoxidation form.In single phenolic initial substrate, the product generated by twice bio-transformation (hydroxylating and oxidizing reaction) is the structural material of o-quinone form.
In second time oxidizing reaction (diphenolaseactivity), hydrogen can be removed from two of a catechol-type structural material hydroxyl, combine with two copper, a Sauerstoffatom receives two electronics and is reduced to water (diphenolaseactivity) from catechol-type material.As mentioned above, methionine(Met) form can make catechol-type structural material be oxidized, but does not participate in the hydroxylating of single phenolic.Thus, after the oxidizing reaction of single phenolic structural material of oxygen-tyrosine oxidase, when the catechol-type structural material generated cannot be oxidized by methionine(Met)-tyrosine oxidase, methionine(Met)-tyrosine oxidase accumulates because being converted to deoxidation-tyrosine oxidase, and tyrosine oxidase is by inactivation.
Compared with the reaction velocity constant (k1) of first time hydroxylating (monophenolaseactivity), large about roughly 10 times of the velocity constant (k2) of second time oxidizing reaction (diphenolaseactivity), thus has difficulties as in the catechol-type structural material of intermediate product in accumulation.The research focus of tyrosine oxidase mainly concentrates on the effect of melanic forming reactions and its inhibitor, and thus in the multiple catechol-type structural material forming reactions optimization technique applying it, this research can be considered initial technology.
Prior art document
Patent documentation
Patent documentation 1: the open 10-2012-0114072 of Korea S's special permission
Patent documentation 2: Korea S's special permission logs in 10-0878394
Non-patent literature
The impact that the single phenolase activity of non-patent literature 1:L-xitix on tyrosine oxidase produces, BiochemJ.1993October1; 295 (Pt1): 309 – 312
Non-patent literature 2: evolve in the direction for the tyrosine oxidase of single phenolase/bis-phenol enzyme activition ratio, EnzymeandMicrobialTechnology, Volume47, Issue7,8December2010, Pages372-376
Summary of the invention
Technical problem
The representative production method being suitable for the catechol-type structural material of biological method can be divided into two kinds of situations, that is, catechol and the combining method of other structures and the hydroxylating of single phenolic structural material.
The representative reactions being equivalent to the first situation is Japanese aginomoto (Ajinomoto) company DOPA (DOPA) production method.Utilize tyrosine-phenol lyase (tyrosin-phenolliase) to make catechol, acetic acid and ammonia bio-transformation be DOPA (DOPA), and the highlyest produce about 100g/L.In order to utilize lyase to produce catechol-type structural material, need screening can form the specific matrix of C-C and corresponding lyase to catechol.Thus, there is limitation in the production of multiple catechol-type structural material.
The hydroxylating of the benzene class formation material or single phenolic structural material that are equivalent to the second situation has the method utilizing monooxygenase (monooxygenase).Monooxygenase, as the one of oxydo-reductase, needs the supply of the electronics by electron transfer system.Representational monooxygenase reaction enzymes is Cytochrome P450 (cytochromeP450), from the redoxomorphism induced reaction of iron ion of protoheme (heme) structure at center being positioned at enzyme.The substrate specificity of Cytochrome P450 is changed, thus is applicable to the production of the catechol-type structural material of various structures.But, when most monooxygenase, need coenzyme, and need the two reductase enzymes (reductase) for electron transmission.This can affect the bioconversion reaction speed of matrix, and is caused by low yield and productivity.
The application of tyrosine oxidase is had as other biological catalytic hydroxylation reaction in addition.The reactive site of tyrosine oxidase is mainly present in protein surface, and in the production of multiple catechol-type structural material, advantage is wide to the substrate specificity of the tyrosine oxidase of single phenolic structural material.Further, relatively fast (the kcat/Km value as a reference, deriving from the tyrosine oxidase of mushroom class is roughly 1000mM to single phenol structural material oxidizing reaction of tyrosine oxidase -1s -1), not with the two enzymes for the electrotransfer reaction of coenzyme.But when tyrosine oxidase, multiple single phenolic structural material is used as matrix, thus has compared with mentioned above-mentioned two situations, the advantage of catechol-type structural material can be converted to quickly, but also make catechol-type structural material be oxidized simultaneously fast, thus form quinone form.The quinoid material generated easily forms free radical, forms the polymer substance of melanochrome etc., or makes periphery substance oxidation.
In order to effectively accumulate the catechol-type structural material as intermediate, need research based on the catechol-type material of the reduction of quinoid material accumulation, accumulate catechol-type material oxidation prevents, along with catechol-type substance accumulation, the tyrosine oxidase of inactivation reactivates.
The object of the invention is to, by three kinds of researchs as above, provide and utilize tyrosine oxidase with the production method of high productivity and the functional catechol-type structural material of produced in yields.
The means of dealing with problems
In order to the second time oxidizing reaction of restraint of tyrosinase, and effectively produce catechol-type structural material, the strategy of this technology is roughly divided into three parts, that is, prevent based on the accumulation of the catechol-type material of the reduction of quinoid material, the oxidation of catechol-type material that accumulates, the reactivating of the tyrosine oxidase of inactivation.
Particularly, the first, the invention provides following method, that is, make the quinoid material produced by the second time oxidizing reaction of tyrosine oxidase effectively again be reduced to catechol-type structural material.
The second, the invention provides following method, that is, for the catechol-type structural material that the first time hydroxylating by tyrosine oxidase generates, induction and the coordinate bond of other elements, protected from the second time oxidizing reaction of tyrosine oxidase.
3rd, the invention provides and effectively regulate three kinds of activity morphologies of tyrosine oxidase by optimized for enzymic activity technology.When catechol-type structural material is not oxidized, be not easy the Sauerstoffatom removing methionine(Met)-tyrosine oxidase, thus tyrosine oxidase is in inactivated state.The tyrosine oxidase being in inactivated state like this can reactivate by reducing power.
Particularly, the invention provides as follows.
(1) feature utilizing tyrosine oxidase to prepare the method for catechol-type structural material from single phenolic structural material is, it comprises the following steps (a) to the step of in step (c), step (a), is imported reducing power and makes the quinonoid structure material generated by the peroxidation of tyrosine oxidase be reduced to catechol-type structural material; Step (b), utilizes transition metal ion or boron ion to make the catechol-type structural material of generation form coordinate bond; And step (c), import reducing power and methionine(Met)-tyrosine oxidase is activated.
(2) tyrosine oxidase that utilizes Gen Ju (1) prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, the reducing power used in step (a) or step (c) provides from the material be selected from the group that is made up of electrochemistry, nicotinamide adenine dinucleotide reduced (NADH), NADPH (NADPH), xitix, halfcystine, P-coumaric acid, azanol, catechol, pyrogallol and coffic acid.
(3) tyrosine oxidase that utilizes Gen Ju (1) prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, single phenolic structural material is selected from the group be made up of Tyrosine Amino acid, tyrosol, trans-resveratrol, daidzein, genistein, apigenin, Phloretin, phenol, P-coumaric acid, p-NP, p-cresol, 4-Vinyl phenol and Ortho-Aminophenol.
(4) tyrosine oxidase that utilizes Gen Ju (1) prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, catechol-type structural material is selected from by luteolin, 3-hydroxyl Phloretin, 3 '-adjacent hydroxyl daidzein, orobol, piceatannol, Hydroxytyrosol, DOPA (dopa), coffic acid, 3,4-dihydroxyl oil of mirbane, 3, in the group of 4-orcin and 3,4-dihydroxy-benzene ethene composition.
(5) tyrosine oxidase that utilizes Gen Ju (1) prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, also comprise the step (d) of separation and purification catechol-type structural material, in above-mentioned steps (d), dissolving comprises the resultant of catechol-type structural material to obtain water layer in organic solvent, after the pH of the water layer that reduction obtains, also dissolve in organic solvent and obtain organic layer, and make organic layer evaporation obtain catechol-type structural material.
(6) tyrosine oxidase that utilizes Gen Ju (1) prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, single phenolic structural material is the tyrosine residues on polymer.
(7) feature of high molecular crosslink reaction method is, utilizes by the catechol-type structural material utilizing tyrosine oxidase to prepare from the method that single phenolic structural material prepares catechol-type structural material described in (6).
Invention effect
The present invention relates to preparation and the application thereof of the functional catechol-type structural material utilizing tyrosine oxidase, be applicable to the tyrosine oxidase in multiple source.This is summarised in the hydroxylating of functional catechol-type material in multiple single phenolic structural material according to tyrosine oxidase substrate specificity, existing difficulty can be realized extract or be difficult to carry out the production of catechol-type structural material of organic chemical synthesis, thus its physiology Efficacy experiments can be contributed to.Recently, a lot of cancer resistance correlative study to the oxyhydroxide of flavones/isoflavones series is had.But, be difficult to the organic synthesis method realizing location specific, and also there is limitation extracting in flavonol/isoflavones of seldom existing of nature.By this research, the production of functional catechol-type structural material can be made to become easy, and Physiologic Studies more related to this and novel material/functional raw material exploitation can be carried out.
Further, utilize tyrosine oxidase can make to turn to DOPA as tyrosine (tyrosine) hydroxyl of the single phenolic amino-acid residue on protein, this improvability matter becomes the protein of the attachment function can imitating mussel protein matter.Like this, the protein of improved properties can develop into the support (scaffolds) of organizational project, can be developed as functional protein drug delivery system (drugdeliverysystem) (AnnuRevMaterRes.20111 further; 41:99-132; Biofouling, 2012,28:8,865-877; CurrentOpinioninBiotechnology2013,24:1 – 7).
Accompanying drawing explanation
Fig. 1 is the schematic diagram for being generated effective catechol-type structural material by tyrosine oxidase, the a of Fig. 1 is single phenolic structural material, the b of Fig. 1 is catechol-type structural material, and the c of Fig. 1 is quinonoid structure material, and the d of Fig. 1 is the structure forming coordinate bond with other elements of catechol-type structural material.And, 1 of Fig. 1 is the first time hydroxylating of tyrosine oxidase, 2 of Fig. 1 is the second time oxidizing reaction of tyrosine oxidase, 3 of Fig. 1 be deoxidation-tyrosine oxidase an oxygen molecule is put into be present in reactive site two copper between, and become the reaction of oxidation-tyrosine oxidase, 4 of Fig. 1 is the reaction of the reductive agent by the Sauerstoffatom reduction making methionine(Met)-tyrosine oxidase, 5 of Fig. 1 is the reaction that hydroxylated catechol-type structural material forms coordinate bond, Fig. 16 for making the also original accumulation again reaction that is catechol-type structural material of quinonoid structure material, 7 of Fig. 1 is snperoxiaized quinoid material high molecular is melanic reaction.
Fig. 2 is as the table enumerating the reductive agent added in the full cell response of tyrosine oxidase, when adding each reductive agent (nicotinamide adenine dinucleotide reduced, L-AA, gsh, halfcystine, hydroxyquinone, 1-naphthols, P-coumaric acid, curcumine, catechol, pyrogallol or forulic acid), in trans-resveratrol (trans-resveratrol), present the relative turnout of hydroxylated piceatannol (piceatannol).
Fig. 3 is about the Tyrosine Amino acid (L-tyrosine) of the tyrosine oxidase (BMT_tyr: bacillus megaterium tyrosine oxidase, ABS_tys: Twospore Mushroom tyrosine oxidase, SAV_tyr: deinsectization streptomycete tyrosine oxidase) of three kinds of different sourcess and the monooxygenase speed of response of DOPA (L-dopa) and the comparison sheet of dioxygenase speed of response.
Fig. 4 is the reaction formula representing the multiple catechol-type structural materials generated by the hydroxylating of tyrosine oxidase from single phenolic structural material.
By pH regulator, Fig. 5 is for confirm whether hydroxylated 3 '-adjacent hydroxyl daidzein (3 '-ODI, 3 '-orthohydorxydaidzein) forms the quantitative analysis of the high-performance-liquid chromatography (LC) of coordinate bond.
By pH regulator, Fig. 6 is for confirm whether hydroxylated orobol (orobol) and luteolin (luteolin) form the quantitative analysis of the high-performance-liquid chromatography (LC) of coordinate bond.
Fig. 7 a is the graphic representation of the productivity of the 3 '-adjacent hydroxyl daidzein comprised in the borate buffer observing pH9 in units of the time in 5mL external (invitro) reaction of the daidzein of 1mM and the tyrosine oxidase of 100nM.
Fig. 7 b is the graphic representation comprising the productivity of 3 ' in the 5mL vitro reactions of the daidzein of 300 μMs and the tyrosine oxidase of 100nM-adjacent hydroxyl daidzein in three (methylol) aminomethane (tris-HCl) observing pH9 in units of the time.
Fig. 8 observes the graphic representation that with the addition of the hydroxylating of the genistein of the 1mM of azanol or xitix in units of the time.
Fig. 9 observes the graphic representation that with the addition of the hydroxylating of the trans-resveratrol of the 1mM of azanol or xitix in units of the time.
Figure 10 is the schematic diagram of the technology of the catechol-type structural material generated for separating of/purifying, in embodiment 5 in detail and explicitly show its method.
Figure 11 a represents the result utilizing gas chromatograph-mass spectrometer (GC/MS) to analyze the 3 '-adjacent hydroxyl daidzein (3 '-ODI) generated from daidzein (daidzein), the retention time (retentiontime) 18.18 represented by 1 is divided into the 3 '-adjacent hydroxyl daidzein be separated by vapor-phase chromatography (GC), represented by a 486.48, m/z is the m/z of molecular weight (TMS), represented by b 471.50, m/z is the m/z of molecular weight (TMS)-15, represented by c 383.39, m/z is the m/z of molecular weight (TMS)-103.
Figure 11 b represents the result of the piceatannol (piceatannol) utilizing gas chromatograph-mass spectrometer analysis to generate from trans-resveratrol (trans-resveratrol), the retention time (retentiontime) 14.82 represented by 1 is divided into the piceatannol be separated by vapor-phase chromatography, represented by a 532.53, m/z is the m/z of molecular weight (TMS), represented by b 517.45, m/z is the m/z of molecular weight (TMS)-15.
Figure 12 illustrates the state forming coordinate bond according to the catechol-type structural material of pH and iron ion.
Embodiment
Below, the present invention will be described in more detail.
As long as without other definition, all technical term used in the present invention and scientific terms have the identical implication of understood term usual with general technical staff of the technical field of the invention.Usually, the experimental technique of the nomenclature used in this manual and following description is well-known in the art and normally used method.
The invention provides the method that the microorganism (full cell) utilizing tyrosine oxidase or express tyrosine oxidase produces catechol-type structural material.
Further, the method in order to suppress the bioconversion reaction of the catechol-type structural material of the peroxidation based on tyrosine oxidase to form coordinate bond to accumulate is provided.
Further, the invention provides the method that the quinoid material making to be generated by peroxidation is reduced to catechol-type structural material effectively.
Particularly, the present invention is by accumulation, ii i) based on the catechol-type material of the reduction of quinoid material) oxidation of catechol-type material that accumulated prevents, iii) the reactivating of the tyrosine oxidase of inactivation along with catechol-type substance accumulation, effectively accumulate catechol-type structural material.
The first, the invention provides the method that can effectively make the quinoid material produced by the second time oxidizing reaction of tyrosine oxidase again be reduced to catechol-type structural material.Likely postponing quinonoid structure material high molecular is thus melanochrome.This is because multiple free radical by reducing power stabilization, and because quinoid material receives electronics, thus can again be converted to catechol-type structural material.To enzymic activity generation bad influence, spendable effective reducing power does not have the reductive agent of the compound clearly illustrated in the reductive agent catalogue of electrochemical method and Fig. 2 and so on.If add the reductive agent in catalogue in the oxidation reaction, then can confirm that dark melanic formation is delayed by.Now, although postpone melanic formation, can confirm to continue to reduce as single phenolic material of initial substrate, be judged as that reducing power as above only affects melanic formation thus, and reduce irrelevant with the activity of tyrosine oxidase.Above-mentioned reducing power can provide from the material be selected from the group that is made up of electrochemistry, nicotinamide adenine dinucleotide reduced, NADPH, xitix, halfcystine, P-coumaric acid, azanol, catechol, pyrogallol and coffic acid.Preferably, above-mentioned reducing power can utilize nicotinamide adenine dinucleotide reduced (phosphoric acid) (NAD (P) H) the regenerative ratio enzyme of Hexose phosphate dehydrogenase (glucosedehydrogenase) etc. to continue in the reaction to provide, this can more effectively be applied when (invivo) produces catechol-type material in vivo.
The second, the invention provides following method, that is, for the catechol-type structural material that the first time hydroxylating by tyrosine oxidase generates, induction and the coordinate bond of other elements, protected from the second time oxidizing reaction of tyrosine oxidase.As everyone knows, catechol-type structural material forms coordinate bond (ProcNatlAcadSciUSA.201115,108 (7), 2651-2655) according to pH and iron ion.This reason and mussel protein matter are incorporated into metal ion in organism to the reason with cohesive force identical (AnnuRevMaterRes.201141,99-132).Based on this, except iron ion, harmless to the activity of tyrosine oxidase, and screened the material forming coordinate bond with catechol-type structural material, undertaken by the experiment of the embodiment for this research.
Boron (B) has sp2 function of hybridized orbital, but when there is Lewis base (Lewisbase), becomes sp3.It was reported, catechol-type structural material plays the effect of Lewis base, according to pH, forms coordinate bond (BBA, 2002,1569,35-44) with one or two catechol material.The wetting ability of the complex body combined with boric acid increases, due to this mechanism, when stable there is glycol (diol) sugared time, use borate ion, when also using borate ion from when coffee berry or Extraction of Caffeine From Tea.The hydroxyl forming the catechol-type material of coordinate bond all forms coordinate bond with boric acid, is not thus oxidized by tyrosine oxidase.Thus, when eliminating second time oxidizing reaction, and also utilize (JACS, 2003, vol.125,43,13034-13035) when only observing first time hydroxylating.
3rd, the invention provides and effectively regulate three kinds of activity morphologies (deoxidation form, oxygen form and methionine(Met) form) of tyrosine oxidase by optimized for enzymic activity technology.When catechol-type structural material is not oxidized, a Sauerstoffatom of methionine(Met)-tyrosine oxidase is not easy to be removed, and thus tyrosine oxidase is in inactivated state.Be in a Sauerstoffatom of the methionine(Met)-tyrosine oxidase of inactivated state like this by other reductive agents or the reduction of electrochemical reduction power, thus be likely removed as water molecules, tyrosine oxidase is again converted to and can receives oxygen (O 2) the deoxidation form of molecule.As this reductive agent, as clearly illustrated in the reductive agent list of Fig. 2, except catechol-type material, there is azanol (NH 2oH, hydroxylamine), nicotinamide adenine dinucleotide reduced (phosphoric acid), xitix (ascorbicacid) etc.
As shown in the reaction 4 of Fig. 1, azanol (hydroxylamine) makes the Sauerstoffatom reduction remained between the copper in reactive site generate water, and make the tyrosine oxidase that cannot receive single phenolic material as matrix be converted to deoxidation form (JACS, 2003, vol.125,43,13034-13035).Deoxidation-tyrosine oxidase and an oxygen molecule form copper-oxygen complex body (μ-η again 2: η 2peroxobinuclearcoppercomplex) oxygen-tyrosine oxidase is become.The reaction 4 of Fig. 1 not only realizes by azanol, but also can by can near the reductive agent of reactive site, that is, nicotinamide adenine dinucleotide reduced (phosphoric acid), xitix, other catechol-type materials etc. realize.
On the other hand, the term used in the present invention is normally used term in the art, as long as general technical staff of the technical field of the invention, just can understand its implication, but in this manual, simply be described as follows.
(1) tyrosine oxidase is as the protein comprising copper, refers to the enzyme of the melanic production of catalysis.
(2) single phenolic structural material refers to the material with the structure identical with a of Fig. 1.Above-mentioned single phenolic structural material has Tyrosine Amino acid (tyrosin), tyrosol (tyrosol), 1 of trans-resveratrol (resveratrol) etc., 2-toluylene (stilbene) series, daidzein (daidzein), genistein (genistein), apigenin (apigenine), flavones (flavonone)/isoflavones (isoflavone) series of Phloretin (phloretin) etc., in addition, also has phenol (phenol), P-coumaric acid (para-coumaricacid), p-NP (para-nitrophenol), p-cresol (para-cresol), 4-Vinyl phenol (para-vinylphenol) and Ortho-Aminophenol (ortho-aminophenol).Preferably, trans-resveratrol, P-coumaric acid, daidzein, genistein, p-NP, p-cresol, 4-Vinyl phenol, apigenin or Phloretin etc. can be used.
Further, single phenolic structural material can be the tyrosine residues on polymer substance, and above-mentioned polymer substance can be 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 structural material carries out adjacent hydroxylated material specifically as to single phenolic structure tyrosine material, there is luteolin (luteolin), 3-hydroxyl Phloretin (3-hydroxyphloretin, 3 '-ODI), flavonol (flavonone)/isoflavones (isoflavone) series of orobol (orobol) etc., the hydroxyl 1 of piceatannol (piceatannol) etc., 2-toluylene (stilbene) series, Hydroxytyrosol (hydroxyltyrosol), DOPA (dopa), coffic acid (caffeicacid), 3, 4-dihydroxyl oil of mirbane (3, 4-dihydroxynitrobenzene), 3, 4-orcin (3, 4-dihydroxytoluene), 3, 4-dihydroxy-benzene ethene (3, 4-dihydroxystyrene) etc.Preferably, the preferred multiple single phenolic structural material more than enumerated is as carrying out adjacent hydroxylated form specifically, have successively piceatannol, coffic acid, 3 '-adjacent hydroxyl daidzein, orobol, 3,4-dihydroxyl oil of mirbane (3,4-dihydroxynitrobenzene), 3,4-orcin (3,4-dihydroxytoluene), 3,4-dihydroxy-benzene ethene (3,4-dihydroxystyrene), luteolin, 3-hydroxyl Phloretin (3-hydroxyphloretin) etc.
(4) quinonoid structure material refers to the material with the structure identical with the c of Fig. 1.
(5) coordinate bond compound substance refers to the complex body forming the structure identical with single complex body (mono-complex) of d or Figure 12 of Fig. 1, two complex body (bis-complex) or Tutofusin tris complex body (tris-complex).Transition metal ion (transitionmetalions) or borate ion (boricacid, borateion) can be used.
(6) cell extract refers to the microorganism extracts of the present invention of expressing accessory protein and tyrosine oxidase.
(7) full cell response refers to that fragmentation comprises the cell of certain enzyme to utilize entocyte, or does not carry out separation and purification to enzyme and utilize the reaction of complete whole cell.
(8) tyrosine oxidase first time hydroxylating (monooxygenase activity) refers to the tyrosine oxidase enzyme reaction carrying out catalysis when importing Sauerstoffatom in C-H, generates the residue of hydroxyl.
(9) tyrosine oxidase second time oxidizing reaction (dioxygenase activity) refers to the hydrogen and electronics that removal combines with oxygen in catechol-type structural material, comes with the tyrosine oxidase enzyme reaction of benzoquinones form catalysis.
(10) PCR is as polymerase chain reaction (PolymeraseChainReaction), is the method increased with instigating thymus nucleic acid regiospecificity.
(11) carrier refers to the polynucleotide formed by strand, double-strand, circle or superhelix thymus nucleic acid or ribonucleotide.Carrier can be included in the textural element that suitable distance connects in the operative mode, for can Restruction protein.The tag format etc. that this textural element can comprise replication orgin, promotor, enhanser, 5 ' messenger RNA(mRNA) (5 ' mRNA) leader sequence, ribosome bind site, nucleic acid cassette, termination and site of polyadenylation and can screen, multiple said structure key element can reduce one or more textural elements according to specificity purposes.Nucleic acid cassette can comprise the restriction enzyme sites for inserting recombinant protein to be expressed.In functional vector, nucleic acid cassette contains the nucleotide sequence to be expressed comprising translation beginning and termination site.As required, be also used in the carrier that can insert two kinds of boxes in carrier, multiple function as above additionally can carry out serializing.The gene being inserted in recombinant vectors can use expression coli strain BL21 (DE3) etc., but can be different according to the kind of the carrier inserted.As long as general technical staff of the technical field of the invention, just can easily select this carrier and expression strain.
(12) be melanochrome as delay quinonoid structure material high molecular, and make quinoid material effectively can be reduced to the reductive agent of catechol-type structural material, nicotinamide adenine dinucleotide reduced (phosphoric acid) can be used, L-AA, gsh (glutathione), halfcystine (cystein), xenogenesis catechol-type structural material, benzene or single phenolic structural material etc., as xenogenesis catechol-type structural material, catechol can be used, 3-methyl catechol, 4-methyl catechol, coffic acid (caffeicacid), pyrogallol or catechol violet etc.Further, as single phenolic structural material, phenol, daidzein, genistein, P-coumaric acid etc. can be used.
(13) as make that tyrosine oxidase reactivates, near the reductive agent of reactive site, can use azanol, nicotinamide adenine dinucleotide reduced (phosphoric acid), xitix or other catechol-type materials etc.
representation 1: the expression of tyrosine oxidase
Make to derive from bacillus megaterium (BMT by polymerase chain reaction, and derive from deinsectization streptomycete (SAV Bacillusmegaterium), actinomycetes, Streptomycesavermitilis) after tyrosine oxidase DNA sequence amplification, after putting into pet28a or petDuet carrier respectively, to this, in intestinal bacteria (E.coli), express respectively as Suo Shi histidine mark (histag) (6 Histidines).Expression method is as follows.After BL21 competent cell (competentcell) conversion is inserted with the plasmid of tyrosine oxidase base sequence, respectively in the antibiotic LB solid medium comprising applicable plasmid, cultivate in 37 DEG C of substratum.Inoculate a bacterium colony in comprising in antibiotic LB liquid nutrient medium of 5mL, and in 37 DEG C of incubators, cultivate 8 hours with 200rmp speed.It is vaccinated with 1v% (500uL) again in the antibiotic new liquid nutrient medium comprising 50mL.If cell optical density(OD) 600nM reaches 0.6, then put into the isopropyl-beta D-thio galactopyranoside (IPTG) of 2mM and the CuSO of 1mM 4, and in 18 DEG C of incubators, with the speed of 200rpm, by protein expression induction of 17 hours.After 17 hours, with the speed of 4000rpm, centrifugation is carried out to cell, and utilize the phosphate buffered saline buffer of 5mL (PBS) cleaning to prepare.After cleaning, in three (methylol) aminomethane buffer solutions or borate buffer, carry out full cell response, and the cell extract except having prepared in the mode of ultrasonication.External (Invitro) full cell response directly uses the hydrosol part in cell extract, or make use of with the protein of conventional histidine mark (6-histag) method of purification purifying, and in various embodiments specifically and illustrate clearly.
reaction method 1: the production method utilizing the catechol-type structural material of tyrosine oxidase and reducing substance
The tyrosine oxidase extract of the mentioned microorganism of acquisition or production or the tyrosine oxidase of purifying and the single phenolic structural material as a of Fig. 1 are reacted, produces the catechol-type structural material of the b as Fig. 1.In the reaction, interpolation reductive agent suppresses the production process after the c material of Fig. 1.
Relative to the concentration of single phenolic structural material, the concentration of the reductive agent of interpolation is preferably 1 to 100 times, is more preferably 5 to 30 times, is especially preferably 7 to 13 times.In the reaction, add the material that can form coordinate bond with the catechol generated, protected from the second time oxidizing reaction of tyrosine oxidase.
Relative to the concentration of single phenolic structural material, the concentration of the coordinate bond precursor of interpolation is preferably 1 to 10000 times, is more preferably 50 to 600 times, is especially preferably 100 to 500 times.
Temperature of reaction of the present invention is preferably 2 to 80 DEG C, is more preferably 20 to 70 DEG C, is especially preferably 20 to 40 DEG C.
The pH of reaction solution is preferably 5 to 11, is more preferably 5 to 11, is especially preferably 7 to 9.As shown in Figure 3, the speed of response of each tyrosine oxidase is different, according to its substrate specificity, employs tyrosine oxidase.
With embodiment, concrete grammar of the present invention is described in detail, but technical scope of the present invention is not limited to embodiment.In the examples below, if do not mention especially, then all per-cent with 100 weight percents of final composition for benchmark.
Embodiment 1
according to the changes in solubility of the initial substrate (single phenolic structural material) of pH
Daidzein (daidzein) as isoflavones series has the solubleness low to water, in the water of the neutrality thus under normal temperature condition or damping fluid, when there is dimethyl sulfoxide (DMSO) (DMSO) of 0.1%, about solubilized 300uM.But can confirm as follows: when utilizing the borate buffer of high density, along with pH uprises, solubleness increases, and in pH9, with 5mM level, solubleness increases by more than ten times, and in pH10, with 30mM level, solubleness increases by 100 times.Tyrosine oxidase is different from other monooxygenases, in wide pH, have activity, thus the initial substrate of high density can be used to improve reaction productivity.
In the pH9 as tyrosinase activity scope, 5mL, external (invitro) that comprise the daidzein of 300 μMs and the tyrosine oxidase of 100nM react the 3 '-adjacent hydroxyl daidzein of production 15mg per hour, on the contrary, 5mL, external (invitro) of the daidzein of 1 μM in the borate buffer that comprises pH9 and the tyrosine oxidase of 100nM react the 3 '-adjacent hydroxyl daidzein of production 45mg per hour, its productivity can increase about three.It is clearly shown in Fig. 7 a and Fig. 7 b.
Embodiment 2
according to the catechol-type structural material yield comparative experiments of reductive agent
The not broken cell deriving from the tyrosine oxidase of deinsectization streptomycete that have expressed according to above-mentioned representation 1 cultivation clearly illustrated, and only clean cell, and add the trans-resveratrol of 0.1mM, the multiple reductive agent (nicotinamide adenine dinucleotide reduced, L-AA, gsh, halfcystine, hydroxyquinone, 1-naphthols, P-coumaric acid, curcumine, catechol, pyrogallol or forulic acid) of 1mM produces piceatannol.After reaction, after utilization is extracted with the ethyl acetate (EA, ethylacetate) measured or non-polar solvent, with high-performance-liquid chromatography (LC) quantitative analysis, compare the piceatannol production efficiency of adding based on reductive agent, and it is clearly shown in Figure 2.
Embodiment 3
the coordinate bond of catechol-type structural material
As reaction method 1, utilize the tyrosine oxidase deriving from bone marrow transplantation (BMT) of the purifying of 200nM, make the daidzein of 500uM carry out hydroxylating, now, put into boric acid to induce coordinate bond.As shown in (a) part of Fig. 5, confirm by high-performance-liquid chromatography (LC) the 3 '-adjacent hydroxyl daidzein forming coordinate bond in alkali reaction liquid and there is wetting ability, be not thus extracted as organic solvent.Further, when confirming the HCl of process 2M in (b) part of Fig. 5, coordinate bond is removed, and the 3 '-adjacent hydroxyl daidzein of 200uM detected.
And, utilize the bone marrow transplantation tyrosine oxidase of the 300nM of purifying, the genistein of 1mM and apigenin is also made to carry out hydroxylating, as shown in Figure 6, hydroxylated orobol is confirmed and luteolin also forms coordinate bond in alkali reaction liquid by high-performance-liquid chromatography (LC), come not to be extracted in organic solvent, if remove coordinate bond in acid-reaction liquid, be then extracted in organic solvent.
Embodiment 4
the inactivation of tyrosine oxidase prevents
Histidine mark purifying being carried out to the tyrosine oxidase cultivated in representation 1, and with the addition of the tyrosine oxidase of 100nM in the reaction, in order to prevent the inactivation of tyrosine oxidase, putting into azanol or xitix activates.Now, relative to matrix, the azanol of interpolation or the concentration of xitix are 1 to 100 times, are more preferably 3 times to 50 times, are especially preferably 10 times to 20 times.By this composition, according to reaction method 1, the daidzein of 1mM is reacted, and temporally utilize the ethyl acetate (ethylacetate of 4 times, EA) or after non-polar solvent extracts the reactant of 100uL, high-performance-liquid chromatography (LC) is utilized to carry out quantitative analysis.According to quantitative analysis results, constantly little in reaction 6,3 ' of 1mM-adjacent hydroxyl daidzein with 100% produced in yields, and be shown in Fig. 7 a.
In the same way, induction of the location specific hydroxylating of genistein (genistein) and trans-resveratrol (resveratrol), single phenolic material as above is functional catechol-type structural material by the first time hydroxylating bio-transformation of tyrosine oxidase.Genistein is constantly little in reaction 1, and bio-transformation is the orobol (orobol) of 95%, and trans-resveratrol is constantly little in reaction 1, and bio-transformation is the piceatannol (piceatannol) of 100%.Be shown in Fig. 8 and Fig. 9.
Embodiment 5
the separation and purification method of the catechol-type structural material generated
In order to be separated the catechol-type structural material generated in example 4, utilize the solubleness of resultant and reactant.In ealkaline buffer, carry out reacting and the catechol-type material that generates forms coordinate bond, thus improve wetting ability (1 part of Figure 10).The single phenolic structural material not forming coordinate bond body is extracted in the ethyl acetate (ethylacetate, EA) of reaction volume 4 multiple, and as shown in 2 parts of Figure 10, organic solvent and water layer be differentiate layers because of difference of specific gravity.Drop-burette is utilized to be separated with water layer above-mentioned organic solvent, be separated 3-1 (water layer) and the 3-2 (organic layer) of Figure 10, and the organic layer of separation is evaporated, residual single phenolic structural material within it can be reused in reaction.Water layer utilizes the HCl of 1M to reduce pH, and utilizes the ethyl acetate (ethylacetate, EA) of 4 multiples to be again extracted catechol-type structural material (4 parts of Figure 10).After extraction, the acid-reaction liquid layer that utilized drop-burette to be separated.With regard to ethyl acetate (ethylacetate, EA) layer, utilize vacuum-evaporator (vacuumevaporator) to remove ethyl acetate (ethylacetate, EA), and the catechol-type structural material of the powder form of purifying can be obtained.
Embodiment 6
utilize the mass analysis of the hydroxylating resultant of tyrosine oxidase
Utilize gas chromatograph-mass spectrometer (GC-MS) to carry out qualitative analysis to the multiple functional catechol-type structural material generated in embodiment 5, its result is clearly shown in Figure 11.
Particularly, Figure 11 a represents the result utilizing gas chromatograph-mass spectrometer (GC/MS) to analyze the 3 '-adjacent hydroxyl daidzein (3 '-ODI) generated from daidzein (daidzein), the retention time (retentiontime) 18.18 represented by 1 is divided into the 3 '-adjacent hydroxyl daidzein be separated by vapor-phase chromatography (GC), represented by a 486.48, m/z is the m/z of molecular weight (TMS), represented by b 471.50, m/z is the m/z of molecular weight (TMS)-15, represented by c 383.39, m/z is the m/z of molecular weight (TMS)-103, the above results can refer to existing paper (HydroxylationofdaidzeinbyCYP107H1fromBaciilussubtilis168, JournalofMolecularCatalysisB:Enzymatic, Vol59, issue4, August2009, Pages248-253) 3 '-adjacent hydroxyl daidzein is confirmed as.
Figure 11 b represents the result of the piceatannol (piceatannol) utilizing gas chromatograph-mass spectrometer analysis to generate from trans-resveratrol (trans-resveratrol), the retention time (retentiontime) 14.82 represented by 1 is divided into the piceatannol be separated by vapor-phase chromatography, represented by a 532.53, m/z is the m/z of molecular weight (TMS), represented by b 517.45, m/z is the m/z of molecular weight (TMS)-15, the above results can refer to existing paper (Thecancerpreventativeagentresveratrolisconvertedtotheant icanceragentpiceatannolbythecytochromeP450enzymeCYP1B1, BritishJournalofCancer (2002) 86, 774-778) confirm as piceatannol.
Embodiment 7
on polymer, single phenolic material is to the bio-transformation of catechol-type structural material: to imitating of mussel protein matter
In order to by the tyrosine residues on polymer effectively bio-transformation be DOPA residue, utilize deinsectization streptomycete tyrosine oxidase.There is (VPGYG) 12 Elastin like polypeptides (ELP of totally 180 aminoacid sequences of (VPGVG) 24, elastinlike-polypeptide), after being dissolved in three (methylol) aminomethane buffer solution of the 50mM of the pH9 of 300uL with 0.5 weight percent by following Elastin like polypeptides Y, the FeCl of 2mM is put into 3with the deinsectization streptomycete tyrosine oxidase of xitix, 500nM, and after reacting two hours, utilize the HCl of NaOH and 1M of 1M to change pH.Elastin like polypeptides _ the Y dissolved in pH5, in pH7, precipitates with the form of powder.Confirm when being 11 by pH regulator, Elastin like polypeptides _ Y carries out gelation (gelation).Confirm when again reducing pH, the hydrogel (hydrogel) of formation dissolves, from but there is reversible reaction.This is the result (ProcNatlAcadSciUSA.201115,108 (7), 2651-2655) consistent with the former paper of the function of imitating mussel protein matter.
Utilizability in industry
The present invention can utilize enzyme reaction to carry out the batch production of practical function catechol-type structural material, and is applicable to the production of novel material/functional raw material and medical product.Particularly, the present invention is according to tyrosine oxidase substrate specificity, summarize the hydroxylating from multiple single phenolic structural material to functional catechol-type material, the existing production being difficult to the catechol-type structural material extracting or be difficult to carry out organic chemical synthesis can be realized, thus its physiology Efficacy experiments can be contributed to.
Claims (amendment according to treaty the 19th article)
1. utilize tyrosine oxidase to prepare a method for catechol-type structural material from single phenolic structural material, it is characterized in that, comprise the following steps (a) to step (c):
Step (a), is imported reducing power and makes the quinonoid structure material generated by the peroxidation of tyrosine oxidase be reduced to catechol-type structural material;
Step (b), utilizes transition metal ion or boron ion to make the catechol-type structural material of generation form coordinate bond; And
Step (c), imports reducing power and methionine(Met)-tyrosine oxidase is activated.
2. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, the reducing power used in step (a) or step (c) provides from the material be selected from the group that is made up of electrochemistry, nicotinamide adenine dinucleotide reduced, NADPH, xitix, halfcystine, P-coumaric acid, azanol, catechol, pyrogallol and coffic acid.
3. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, single phenolic structural material is selected from the group be made up of Tyrosine Amino acid, tyrosol, trans-resveratrol, daidzein, genistein, apigenin, Phloretin, phenol, P-coumaric acid, p-NP, p-cresol, 4-Vinyl phenol and Ortho-Aminophenol.
4. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, catechol-type structural material be selected from by luteolin, 3-hydroxyl Phloretin, 3 '-adjacent hydroxyl daidzein, orobol, piceatannol, Hydroxytyrosol, DOPA, coffic acid, 3,4-dihydroxyl oil of mirbane, 3, in the group of 4-orcin and 3,4-dihydroxy-benzene ethene composition.
5. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, also comprise the step (d) of separation and purification catechol-type structural material, in above-mentioned steps (d), dissolving comprises the resultant of catechol-type structural material to obtain water layer in organic solvent, after the pH of the water layer that reduction obtains, also dissolve in organic solvent and obtain organic layer, and make organic layer evaporation obtain catechol-type structural material.
6. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, single phenolic structural material is the tyrosine residues on polymer.
7. a high molecular crosslink reaction method, is characterized in that, utilizes by the catechol-type structural material utilizing tyrosine oxidase to prepare from the method that single phenolic structural material prepares catechol-type structural material according to claim 6.

Claims (7)

1. utilize tyrosine oxidase to prepare a method for catechol-type structural material from single phenolic structural material, it is characterized in that, comprise the following steps (a) to the step of in step (c),
Step (a), is imported reducing power and makes the quinonoid structure material generated by the peroxidation of tyrosine oxidase be reduced to catechol-type structural material;
Step (b), utilizes transition metal ion or boron ion to make the catechol-type structural material of generation form coordinate bond;
Step (c), imports reducing power and methionine(Met)-tyrosine oxidase is activated.
2. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, the reducing power used in step (a) or step (c) provides from the material be selected from the group that is made up of electrochemistry, nicotinamide adenine dinucleotide reduced, NADPH, xitix, halfcystine, P-coumaric acid, azanol, catechol, pyrogallol and coffic acid.
3. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, single phenolic structural material is selected from the group be made up of Tyrosine Amino acid, tyrosol, trans-resveratrol, daidzein, genistein, apigenin, Phloretin, phenol, P-coumaric acid, p-NP, p-cresol, 4-Vinyl phenol and Ortho-Aminophenol.
4. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, catechol-type structural material be selected from by luteolin, 3-hydroxyl Phloretin, 3 '-adjacent hydroxyl daidzein, orobol, piceatannol, Hydroxytyrosol, DOPA, coffic acid, 3,4-dihydroxyl oil of mirbane, 3, in the group of 4-orcin and 3,4-dihydroxy-benzene ethene composition.
5. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, also comprise the step (d) of separation and purification catechol-type structural material, in above-mentioned steps (d), dissolving comprises the resultant of catechol-type structural material to obtain water layer in organic solvent, after the pH of the water layer that reduction obtains, also dissolve in organic solvent and obtain organic layer, and make organic layer evaporation obtain catechol-type structural material.
6. the tyrosine oxidase that utilizes according to claim 1 prepares the method for catechol-type structural material from single phenolic structural material, it is characterized in that, single phenolic structural material is the tyrosine residues on polymer.
7. a high molecular crosslink reaction method, is characterized in that, utilizes by the catechol-type structural material utilizing tyrosine oxidase to prepare from the method that single phenolic structural material prepares catechol-type structural material according to claim 6.
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R.STEPHEN BROWN，等: "A Substrate Recycling Assay for Phenolic Compounds Using Tyrosinase and NADH", 《ANALYTICAL BIOCHEMISTRY》 *
芶琳: "组氨酸残基的化学修饰对酪氨酸酶活性和构象的影响", 《四川农业大学硕士学位论文》 *

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* Cited by examiner, † Cited by third party
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CN109825518A (en) * 2019-02-25 2019-05-31 佐登妮丝（广州）美容化妆品有限公司 The preparation method and its usage of 3 '-hydroxy dye geniteins
CN110283797A (en) * 2019-06-20 2019-09-27 天津科技大学 A kind of tyrosinase and its gene, engineering bacteria and preparation method

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