CN1651574A - Enzymatic synthesis of plant type melanine - Google Patents

Enzymatic synthesis of plant type melanine Download PDF

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Publication number
CN1651574A
CN1651574A CNA2004100041084A CN200410004108A CN1651574A CN 1651574 A CN1651574 A CN 1651574A CN A2004100041084 A CNA2004100041084 A CN A2004100041084A CN 200410004108 A CN200410004108 A CN 200410004108A CN 1651574 A CN1651574 A CN 1651574A
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enzyme
melanochrome
derivative
plant type
zymin
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王敬文
张金萍
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Research Institute of Subtropical Forestry of Chinese Academy of Forestry
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Research Institute of Subtropical Forestry of Chinese Academy of Forestry
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

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Abstract

A process for synthesizing the vegetative melanin by enzyme method includes such steps as preparing polyphenol oxidase and phenol from plant, creating enzymatic reaction system in buffering phosphate solution, enzymatic reaction to synthesize malanin, separating and purifying.

Description

Plant type melanochrome enzyme process is synthetic
Technical field the invention belongs to biological chemistry goods field
Technical background melanochrome is biological protectiveness pigment, extensively is present in animal, plant and the microorganism, and black have and absorbs ultraviolet, anti-oxidant, as to eliminate free radical effect, and biological organism is played provide protection.Form by melanic structure, can be divided into three kinds of patterns, i.e. eumelanin (eumelanin), pheomelanins (phaeomelanin) and different melanochrome (allpmelanin).The eumelanin color is dark-brown or black, is transformed and generates via DOPA (Dopa) approach by tyrosine, and this melanochrome mainly is distributed in animal cuticle and the hair cell, and in internal organ film or the tissue, can be described as animal-type melanochrome.Pheomelanins is the cystine derivatives of eumelanin, and sulfur atom-containing, color are reddish brown or yellow, are common in the hair of animal.Different melanochrome mainly is distributed in the plant, platymiscium type melanochrome, and being similar to eumelanin is brown or black, it is mainly generated via poly hydroxyl naphthalene approach by dihydric phenols such as catechol, trihydric phenol.
Nicolaus et al proposes, and animal source melanochrome belongs to the indoles type more, and plant-sourced melanochrome belongs to the pyrocatechol type more.Porta is to be skeleton with indoles or benzothiazine according to its structural unit after further research, and animal source melanochrome is divided into eumelanin (eumelanin) and pheomelanins (pheomelanin).
Wang Yan etc. have studied the infrared spectrum characterization of several natural black pigment molecular structures, propose hair melanochrome and belong to eumelanin and pheomelanins mixed type melanochrome, Pericarpium Musae melanochrome, sunflower seeds melanochrome all belong to pyrocatechol type melanochrome, be that polyphenol substance is converted into hydroxyquinone under the effect of polyphenoloxidase and phenolic hydroxyl group enzyme, and then polymerization formation.They think that also Semen Sesami Nigrum melanochrome is the mixed type melanochrome of indoles type and pyrocatechol type simultaneously.Affirmations such as Yin Peiyu contain the phenols conjugated structure in the Semen Sesami Nigrum melanochrome, prove that tentatively Semen Sesami Nigrum melanochrome has the melanic constitutional features of catechol type, are made of catechol, Resorcinol and micro-catechuic acid, do not contain indoles type component.According to these research, show plant type melanochrome mainly be by monohydric phenol,
Phenols components such as dihydric phenol are synthetic under the effect of polyphenoloxidase and phenolic hydroxyl group enzyme.
Summary of the invention the present invention forms according to the plant type melanin structure, and with the synthetic plant type melanochrome of zymochemistry method, bare bones of the present invention is as follows:
1. the enzyme preparation of polyphenol oxidase enzyme system
From multiple polyphenol oxidase enzyme active high vegetable material and mushroom material, separate, produce the polyphenol oxidase enzyme comprising vegetable matters such as pears, apple, potato, sweet potato, bamboo shoots, alder, China fir, pine, mandarin jacket wood, mushroom, Lasiosphaera fenzliis, comprise the phenolic hydroxyl group enzyme.Vegetable matter is pulverized homogenate in phosphoric acid buffer PH7.0, centrifugal 15～20 minutes of 5000rpm removes slag and obtains supernatant liquor, adds to make the ammonium sulfate that reaches 40～50% saturation ratios, saltouts in 4 ℃ of refrigerators, centrifugal collection zymoprotein precipitation.Zymoprotein is dissolved in small volume phosphoric acid buffer (PH7.0), the desalination of last Sephadex G-25 post, and the phosphoric acid buffer wash-out is collected enzyme liquid.Add freezing acetone or alcohol in the enzyme liquid and be settled out zymoprotein, centrifugal collection zymoprotein, lyophilize gets the powder zymin, enzyme activity 11000～15000 units/g.
2. enzymatic is synthetic
Dissolve 0.1～2.0g Resorcinol, pyrocatechol, pyrogallol, gallic acid, tyrosine in the 1000ml phosphoric acid buffer (PH8.0) separately, or its different combination, transfer PH to 8.0 with 2MNaOH, 35～42 ℃ of insulations, add above-mentioned zymin 0.5～1.0g, continue insulation 3～5 hours, stirring that does not stop or ventilation.The blackening gradually of reaction solution color is up to thick black.
3. separation and purification
After the enzymatic building-up reactions finishes, add 6N HCL and transfer PH≤4, melanochrome is flocks separates out, left standstill 3～4 hours, and the supernatant liquor that inclines, centrifugal or filtration collecting precipitation, the deionized water thorough washing, lyophilize obtains the melanochrome crude product.
The melanochrome product often with protein or other compositions take place in conjunction with and separate out with precipitation, need carry out purification process.The melanochrome dried powder was joined among the 6N HCL backwash 16～24 hours, and backwash finishes the back standing over night, the supernatant liquor that inclines, and centrifugal collecting precipitation, the deionized water thorough washing, lyophilize obtains the pure product of melanochrome.Perhaps, with melanochrome powder suspension (0.5g/ liter) in trypsin solution, transfer PH7～8,40 ℃ insulation 2～3 hours, after the enzymic digestion reaction finishes, transfer PH≤4, leave standstill the back melanin deposition and get off the supernatant liquor that inclines, centrifugal collecting precipitation, the deionized water thorough washing obtains the pure product of melanochrome through lyophilize.
4. physico-chemical property detects
Though different (animal, plant, microorganism), the structure differences (indoles type, phenol quinoid) in melanochrome source, but the physico-chemical property that is shown is the same (Nicolaus R.A.et al, 1964), the common characteristic ultraviolet absorption is promptly arranged, water insoluble, be insoluble to acid, be insoluble to common organic solvents, dissolve in alkaline water, can be by KMnO 4Or H 2O 2Oxidation and fading etc. detects according to these physico-chemical properties, and detected result shows that obtained product all has these melanic physico-chemical properties, and successfully enzymatic has synthesized plant type melanochrome.
Embodiment
Example 1.
With cross-fertilize seed potato that 100g gathered in the crops 4 ℃ of precoolings 2 hours, remove the peel subsequently, shred, in ice bath, add an amount of 0.1mol/L phosphoric acid buffer (PH7.0) homogenate, centrifugal 15～20 minutes of 5000rpm, add ammonium sulfate in the supernatant liquor and reach 50% saturation ratio, in 4 ℃ of refrigerators, saltout centrifugal collection zymoprotein precipitation.Zymoprotein is dissolved in small volume 0.1mol/L phosphoric acid buffer (PH7.0), the desalination of last Sephadex G-25 post, the phosphoric acid buffer wash-out is collected enzyme liquid.Add 2.5 times of (V/V)-18 ℃ freezing acetone in the enzyme liquid, the centrifugal collection zymoprotein of 8000rpm obtains zymin 1.8g through lyophilize.Measure the required enzyme amount of L-Dopa generation 1mmol dopachrome with the MansonH.S. method and be defined as an enzyme activity unit, Dopachrome is 3600mol/L at the optical extinction coefficient of 475nm -1.cm -1, the zymin vitality test result that the present invention makes is 6744 units/g.
Example 2.
The new fresh mushroom of 100g 4 ℃ of precoolings 2 hours, is added an amount of 0.1mol/L phosphoric acid buffer (PH7.0) homogenate in ice bath, centrifugal 15～20 minutes of 5000rpm, ammonium sulfate reaches 50% saturation ratio in the supernatant liquor, saltouts centrifugal collecting precipitation in 4 ℃ of refrigerators.Obtain precipitation is dissolved in small volume phosphoric acid buffer (PH7.0), and elutriant is collected in the desalination of last Sephadex G-25 post, add 2.5 times of (V/V)-18 ℃ freezing acetone, separate out the zymoprotein precipitation, the centrifugal collection zymoprotein of 8000rpm obtains zymin 4.26g through lyophilize.The enzymic activity that records by example 1 method is 7546 units/g.
Example 3.
With pyrocatechol 1g and yellow soda ash 0.2g with a little hot water dissolving after, add in the 1000ml0.2mol/L phosphoric acid buffer (PH8.0), heat to 40～42 ℃, add example 1 zymin 1.0～1.5g, add CuSO 4.7H 2O 50mg, 40～42 ℃ are incubated 3～5 hours in water-bath, do not stop to stir.After reaction finishes, add 6N HCL and transfer PH≤4, the melanochrome flocks is separated out, left standstill 5 hours, and the supernatant liquor that inclines, centrifugal collection flocks, lyophilize obtains melanochrome phase product 1.24g.
Example 4.
With pyrocatechol 1g and yellow soda ash 0.2g with a little hot water dissolving after, add in the 1000ml 0.2mol/L phosphoric acid buffer (PH8.0), heat to 40～42 ℃, add example 2 zymins 1.0～1.2g, add CuSO 4.7H 2O 50mg, 40～42 ℃ are incubated 3～5 hours in water-bath, do not stop to stir.After reaction finishes, add 6N HCL and transfer PH≤4, the melanochrome flocks is separated out, left standstill 5 hours, and the supernatant liquor that inclines, centrifugal collecting precipitation, lyophilize obtains melanochrome crude product 0.98g.
Example 5.
With Resorcinol 1g and yellow soda ash 0.2g with a little hot water dissolving after, join in the 1000ml 0.2mol/L phosphoric acid buffer (PH8.0), heat to 40～42 ℃, add example 1 zymin 1.0～1.5g, add CuSO 4.7H 2O 50mg is incubated 3～5 hours in water-bath, do not stop to stir.After reaction finishes, add 6N HCL and transfer PH≤4, the melanochrome flocks is separated out, left standstill 5 hours, and the supernatant liquor that inclines, centrifugal collecting precipitation, lyophilize obtains melanochrome crude product 1.2g.
Example 6.
With pyrogallol 1g and 0.2g yellow soda ash with a little hot water dissolving after, join in the 1000ml 0.2mol/L phosphoric acid buffer (PH8.0), heat to 40～42 ℃, add example 1 zymin 1.0～1.5g, add CuSO 4.7H 2O 50mg, 40～42 ℃ are incubated 3～5 hours in water-bath, do not stop to stir.After reaction finishes, add 6N HCL and transfer PH≤4, the melanochrome flocks is separated out, left standstill 5 hours, and the supernatant liquor that inclines, centrifugal collecting precipitation, lyophilize obtains melanochrome crude product 1.05g.
Example 7.
With gallic acid 1g and 0.2g yellow soda ash with a little hot water dissolving after, join in the 1000ml 0.2mol/L phosphoric acid buffer (PH8.0), heat to 40～42 ℃, add example 1 zymin 1.0～1.5g, add CuSO 4.7H 2O 50mg, 40～42 ℃ are incubated 3～5 hours in water-bath, do not stop to stir.After reaction finishes, add 6N HCL and transfer PH≤4, the melanochrome flocks is separated out, left standstill 5 hours, and the supernatant liquor that inclines, centrifugal collecting precipitation, lyophilize obtains melanochrome crude product 0.92g.
Example 8.
With example 3,4,5,6, the 7 melanochrome crude products of being produced respectively take by weighing 1g, heat backwash 18～24 hours respectively in 50ml 6N HCL, cooled and filtered, the deionized water thorough washing, 105 ℃ of dryings, the purifying melanin that obtains is respectively 0.53g, 0.62g, 0.46g, 0.48g, 0.37g.
Example 9.
With example 3,4,5,6, the 7 melanochrome crude products of being produced respectively take by weighing 1g, join respectively in the 100ml phosphoric acid buffer (PH6.8), be heated to 40～42 ℃, add trypsinase 500mg more respectively, 40～42 ℃ are incubated 2～3 hours, add 6N HCL and transfer PH≤4, centrifugal collecting precipitation, and use the deionized water thorough washing, and 105 ℃ of dryings, the purifying melanin that obtains is respectively 0.43g, 0.63g, 0.41g, 0.46g, 0.40g.
Example 10.
Will be by example 3,4,5,6, the synthetic melanic physico-chemical property of producing of plant type of 7 enzyme process is measured, and the Dopa-melanine (Dopa melanochrome) that produces with Sigma company and the melanochrome of producing from vegetable material horse-chestnut (Aesculushippocastanicum), Sunflower Receptacle (Heliantus annus) by Kereatea et al method compare:
(1) ultraviolet and visible absorption are the strongest in the short-wave band absorption, increase to absorb with wavelength and weaken;
(2) the same with Sunflower Receptacle melanochrome, water insoluble with Dopa melanochrome, horse-chestnut, be insoluble to general common organic solvent;
(3) dissolving fully in 0.5mol/LNaOH or KOH solution;
(4) be insoluble to acid, some precipitation in PH＜5;
(5) FeCl 3Precipitate when existing;
(6) KMnO 4And H 2O 2Can oxidation and make and fade
(7) can make AgNO 3Regeneration NH 4Solution
These character all show example 3,4, and 5,6,7 enzymatic synthetic products are plant type melanochrome.
The present invention makes a living to produce and gets plant type melanochrome new way is provided, and technology is simple, produces conveniently, and is with low cost.
Nicolaus?R.A.，Piattellim.，Fattorusso?E.Tetrahedron，1964，20：1163?Porta?G.J.Invest.Dermatol，1980，75：122
Wang Yan etc., assay office, 1996, the 15 volumes, the 6th phase, 63-35.
Yin Peiyu etc., the gas chromatography/mass spectrometry method is identified melanic structure type in the Semen Sesami Nigrum, chromatogram, calendar year 2001 the 19th is rolled up the 3rd phase, 208-209.
Manson?H.S.Oxidase.Aun.Rev.Biochem.1965，34，595-634.
Kerestes?J.J.et?al.Biological?active?fraction?of?vegetable?melanin，process?for?itsproduction?and?its?use，United?States?Patent?Application，20020041905.April?11，2002.

Claims (8)

1. the present invention proposes with enzymic synthesis production and produce the melanic new way novel method of plant type.
2. by claim 1, the melanic new way of plant type, novel method are produced in enzymic synthesis production, comprise the separating and purifying of separation preparation, enzyme reaction substrate, enzymatic reaction system composition, enzymatic reaction condition and enzymatic reaction product of zymin.
3. by claim 2, related enzyme comprises all enzyme classes that participates in synthesis of melanin and corresponding coenzyme, prothetic group and the activation factor etc. in the polyphenol oxidase enzyme system, this zymin that comprises the polyphenol oxidase enzyme system, both comprised the enzyme crude extract, also comprised through partially purified zymin powder and solution.
4. by claim 3, all enzyme classes of related polyphenol oxidase enzyme system are to produce by the method that is widely known by the people from each plant material and microbial material (comprising fungi).
5. by claim 2, enzyme reaction substrate comprises monohydric phenol and derivative (tyrosine, tyrasamine, DOPA, Dopamine HCL etc. and derivative thereof), dihydric phenol and derivative (Resorcinol, pyrocatechol, this diphenol, catechuic acid, isocatechin etc. and derivative thereof) thereof, trihydric phenol and derivative (pyrogallol, gallic acid etc. and derivative thereof) thereof.
6. by claim 2, the synthetic plant type melanin reactions system of enzymatic forms and comprises the substrate 0.1-3.0g/ liter in phosphoric acid buffer (PH6-9), the claim 5, the enzyme system 5000-100000 units per liter in the claim 3.
7. by claim 2, transfer PH≤4 precipitations to separate out melanochrome, melanochrome is separated from enzyme reaction solution with acid.
8. by claim 2, handle the synthetic melanochrome crude product of producing of enzyme process, make the melanochrome product be able to purifying with 3-9N HCL backwash 10-30 hour or with the pancreatin hydrolysis digestion method.
CNA2004100041084A 2004-02-06 2004-02-06 Enzymatic synthesis of plant type melanine Pending CN1651574A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102090421A (en) * 2010-11-30 2011-06-15 中国农业科学院生物技术研究所 Biologically active substance and application thereof to prevention and control of soybean cyst nematode No.4 microspecies
CN102399824A (en) * 2010-09-13 2012-04-04 浙江医药股份有限公司新昌制药厂 Method for preparing plant carbon black substitute by using leaves of Vaccinium plants as raw material
CN106140107A (en) * 2015-03-27 2016-11-23 西南林业大学 A kind of method that natural black pigment base resin is prepared in ammonia catalytic thermal solidification
CN106146773A (en) * 2015-03-29 2016-11-23 西南林业大学 A kind of method of the natural melanin synthesized base resin of direct acid catalysis
CN111778291A (en) * 2020-06-23 2020-10-16 南京师范大学 Method for increasing melanin production by fermenting or converting sunflower meal and sunflower shells by using microorganisms or enzymes
CN111826406A (en) * 2020-07-20 2020-10-27 西安幸福未来化妆品有限公司 Preparation method and application of melanin precursor dopaquinone
CN113875990A (en) * 2021-10-11 2022-01-04 中北大学 Ferrous iron with antioxidant activity and preparation method thereof

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102399824A (en) * 2010-09-13 2012-04-04 浙江医药股份有限公司新昌制药厂 Method for preparing plant carbon black substitute by using leaves of Vaccinium plants as raw material
CN102399824B (en) * 2010-09-13 2013-08-07 浙江医药股份有限公司新昌制药厂 Method for preparing plant carbon black substitute by using leaves of Vaccinium plants as raw material
CN102090421A (en) * 2010-11-30 2011-06-15 中国农业科学院生物技术研究所 Biologically active substance and application thereof to prevention and control of soybean cyst nematode No.4 microspecies
CN102090421B (en) * 2010-11-30 2013-06-26 中国农业科学院生物技术研究所 Biologically active substance and application thereof to prevention and control of soybean cyst nematode No.4 microspecies
CN106140107A (en) * 2015-03-27 2016-11-23 西南林业大学 A kind of method that natural black pigment base resin is prepared in ammonia catalytic thermal solidification
CN106140107B (en) * 2015-03-27 2019-06-21 西南林业大学 A kind of method that ammonia catalysis-heat cure prepares natural black pigment base resin
CN106146773A (en) * 2015-03-29 2016-11-23 西南林业大学 A kind of method of the natural melanin synthesized base resin of direct acid catalysis
CN106146773B (en) * 2015-03-29 2018-05-04 西南林业大学 A kind of method of the directly natural melanin synthesized base resin of acid catalysis
CN111778291A (en) * 2020-06-23 2020-10-16 南京师范大学 Method for increasing melanin production by fermenting or converting sunflower meal and sunflower shells by using microorganisms or enzymes
CN111826406A (en) * 2020-07-20 2020-10-27 西安幸福未来化妆品有限公司 Preparation method and application of melanin precursor dopaquinone
CN113875990A (en) * 2021-10-11 2022-01-04 中北大学 Ferrous iron with antioxidant activity and preparation method thereof

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