CN104726504B - The Laccase Catalyzed synthetic method of 5,5 '-dehydrogenation, two Acetovanillone - Google Patents
The Laccase Catalyzed synthetic method of 5,5 '-dehydrogenation, two Acetovanillone Download PDFInfo
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- CN104726504B CN104726504B CN201510142163.8A CN201510142163A CN104726504B CN 104726504 B CN104726504 B CN 104726504B CN 201510142163 A CN201510142163 A CN 201510142163A CN 104726504 B CN104726504 B CN 104726504B
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- acetovanillone
- dehydrogenation
- laccase
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- sodium
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- DFYRUELUNQRZTB-UHFFFAOYSA-N apocynin Chemical compound COC1=CC(C(C)=O)=CC=C1O DFYRUELUNQRZTB-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 108010029541 Laccase Proteins 0.000 title claims abstract description 27
- 238000006356 dehydrogenation reaction Methods 0.000 title claims abstract description 20
- 238000010189 synthetic method Methods 0.000 title claims abstract description 9
- 239000000047 product Substances 0.000 claims abstract description 21
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 238000005215 recombination Methods 0.000 claims abstract description 11
- 230000006798 recombination Effects 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 8
- 239000007853 buffer solution Substances 0.000 claims abstract description 7
- 238000005406 washing Methods 0.000 claims abstract description 7
- 239000002244 precipitate Substances 0.000 claims abstract description 4
- FDPIMTJIUBPUKL-UHFFFAOYSA-N pentan-3-one Chemical compound CCC(=O)CC FDPIMTJIUBPUKL-UHFFFAOYSA-N 0.000 claims description 4
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- IEHIPPDDMCEYPI-UHFFFAOYSA-L disodium propanedioate propanedioic acid Chemical compound [Na+].[Na+].OC(=O)CC(O)=O.[O-]C(=O)CC([O-])=O IEHIPPDDMCEYPI-UHFFFAOYSA-L 0.000 claims description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 claims description 2
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- GZLGNNHEHXBCBI-UHFFFAOYSA-L [Na+].[Na+].OC(=O)C(O)C(O)C(O)=O.[O-]C(=O)C(O)C(O)C([O-])=O Chemical compound [Na+].[Na+].OC(=O)C(O)C(O)C(O)=O.[O-]C(=O)C(O)C(O)C([O-])=O GZLGNNHEHXBCBI-UHFFFAOYSA-L 0.000 claims 1
- 230000003139 buffering effect Effects 0.000 claims 1
- OVYQSRKFHNKIBM-UHFFFAOYSA-N butanedioic acid Chemical compound OC(=O)CCC(O)=O.OC(=O)CCC(O)=O OVYQSRKFHNKIBM-UHFFFAOYSA-N 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 5
- 239000007800 oxidant agent Substances 0.000 abstract description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 abstract description 3
- 239000002253 acid Substances 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 2
- 239000013043 chemical agent Substances 0.000 abstract description 2
- 229930014626 natural product Natural products 0.000 abstract description 2
- 231100000252 nontoxic Toxicity 0.000 abstract description 2
- 230000003000 nontoxic effect Effects 0.000 abstract description 2
- 239000001301 oxygen Substances 0.000 abstract description 2
- 229910052760 oxygen Inorganic materials 0.000 abstract description 2
- 238000000034 method Methods 0.000 description 7
- 238000001556 precipitation Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 206010013786 Dry skin Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000003973 paint Substances 0.000 description 3
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- HLNDPICGHQGWSU-UHFFFAOYSA-N 1-[3-(5-acetyl-2-hydroxy-3-methoxyphenyl)-4-hydroxy-5-methoxyphenyl]ethanone Chemical compound COC1=CC(C(C)=O)=CC(C=2C(=C(OC)C=C(C=2)C(C)=O)O)=C1O HLNDPICGHQGWSU-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- LHGVFZTZFXWLCP-UHFFFAOYSA-N guaiacol Chemical compound COC1=CC=CC=C1O LHGVFZTZFXWLCP-UHFFFAOYSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000033116 oxidation-reduction process Effects 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical class NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- -1 Aromatic amine compound Chemical class 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 229930185605 Bisphenol Natural products 0.000 description 1
- 108010031396 Catechol oxidase Proteins 0.000 description 1
- 102000030523 Catechol oxidase Human genes 0.000 description 1
- 239000005944 Chlorpyrifos Substances 0.000 description 1
- 241001269238 Data Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 102000004722 NADPH Oxidases Human genes 0.000 description 1
- 108010002998 NADPH Oxidases Proteins 0.000 description 1
- 208000027089 Parkinsonian disease Diseases 0.000 description 1
- 206010034010 Parkinsonism Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 235000009499 Vanilla fragrans Nutrition 0.000 description 1
- 244000263375 Vanilla tahitensis Species 0.000 description 1
- 235000012036 Vanilla tahitensis Nutrition 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- IISBACLAFKSPIT-UHFFFAOYSA-N bisphenol A Chemical compound C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 IISBACLAFKSPIT-UHFFFAOYSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- SBPBAQFWLVIOKP-UHFFFAOYSA-N chlorpyrifos Chemical compound CCOP(=S)(OCC)OC1=NC(Cl)=C(Cl)C=C1Cl SBPBAQFWLVIOKP-UHFFFAOYSA-N 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- SILCDLWESNHZKB-UHFFFAOYSA-L disodium 4-hydroxy-4-oxobutanoate Chemical compound [Na+].[Na+].OC(=O)CCC([O-])=O.OC(=O)CCC([O-])=O SILCDLWESNHZKB-UHFFFAOYSA-L 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229960001867 guaiacol Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000001226 reprecipitation Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000001433 sodium tartrate Substances 0.000 description 1
- 229960002167 sodium tartrate Drugs 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 description 1
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 description 1
- 235000012141 vanillin Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of Laccase Catalyzed synthetic methods of two Acetovanillone of 5,5 ' dehydrogenation.Include the following steps：Acetovanillone is dissolved in the buffer solution that water or pH are 3.5 ~ 6.0, is configured to solution, control temperature is added recombination laccase, is reacted in shaking table at 20 ~ 60 °C；It staticly settles after reaction, washing precipitate, it is dry at not higher than 50 DEG C, obtain 5,5 ' dehydrogenation of product, two Acetovanillone.The reaction raw materials and recombination laccase itself of the present invention are natural products, nontoxic, using the oxygen in air in reaction process, need not add other oxidants；Reaction condition of the present invention is mild, can carry out at room temperature, and product is single, is easy to purify, and is not related to the chemical agents such as soda acid in purification step, and product purity is high.
Description
Technical field
The present invention relates to living things catalysis and field of bioconversion, and in particular to a kind of paint of two Acetovanillone of 5,5 '-dehydrogenation
Enzymatic synthesis method.
Background technology
Acetovanillone is a kind of active material separated out of plant, can specifically inhibit nadph oxidase
Activity has anti-oxidant and anti-inflammatory activity, and be used to treat parkinsonism.In nature, 5,5 '-dehydrogenation, two Acetovanillone
（Dehydrodiacetovanillone, CAS：29799-22-2）It is the metabolite of Acetovanillone, is catching for superoxide anion
Agent is obtained, is considered that there is higher activity, but the product only micro presence in vivo than Acetovanillone.
At present using Acetovanillone as there are mainly two types of the methods of two Acetovanillone of Material synthesis 5,5 '-dehydrogenation.One is changes
Method uses persulfate for oxidant, using ferrous sulfate as initiator, reacts 5min at 100 °C.At the end of reaction, production
Object precipitates because not soluble in water, but simultaneously some molysite, persulfate and sulfate also can coprecipitation get off.Subsequently need
Use NH4OH or NaOH solution re-dissolve sediment, then carry out reprecipitation with HCl solution, to remove impurity, finally with boiling
Water repeatedly washs precipitation, and efficiency of pcr product is about 60%.In the technique, since persulfate oxidation is stronger, side chain is often caused
Fracture, to form low molecular weight substance；It might have tripolymer generation simultaneously.Although as it can be seen that the chemical synthesis reaction time
It is short, but subsequent purification is complex, need to consume the chemicals such as more soda acid, while products obtained therefrom purity and yield compared with
It is low.Another method for synthesizing a small amount of 5,5 '-dehydrogenation, two Acetovanillone is to utilize peroxidase, is most typically utilized peppery
Root peroxidase, with H2O2For oxidant, is reacted in pH=4, by filtration washing, obtain product.
Laccase is a kind of polyphenol oxidase of cupric, and oxidation-reduction potential is relatively low, oxidable single phenol, bis-phenol, amino phenols and
Aromatic amine compound, can be directly with O2Redox is realized for electron transit mediator, and unique by-product is exactly after reaction
Water.Recombination laccase is to utilize molecular biotechnology, the product being prepared by DNA recombinant expression, has purity high, active
High, the advantages that oxidation-reduction potential is high.
Invention content
The present invention provides a kind of Laccase Catalyzed preparation 5 environmental-friendly, simple for process, efficient, production cost is low,
The method of 5 '-dehydrogenation, two Acetovanillone lays the foundation for its further production and application.
A kind of Laccase Catalyzed synthetic method of two Acetovanillone of 5,5 '-dehydrogenation, includes the following steps：
（1）Acetovanillone is dissolved in the buffer solution that water or pH are 3.5 ~ 6.0, is configured to solution, control temperature exists
20 ~ 60 °C, recombination laccase is added, is reacted in shaking table；
（2）It staticly settles after reaction, washing precipitate, it is dry at not higher than 50 DEG C, obtain 5,5 '-dehydrogenation of product
Two Acetovanillones.
Step（1）In, the molar concentration of the Acetovanillone solution is 33 mmol/L；Shaking speed is 120~150
Rpm, reaction time are more than 4 h；The recombination laccase dosage is 3.0~6.0 U/mol Acetovanillones.
Step（1）In, the buffer solution is selected from Acetic acid-sodium acetate, phosphoric acid-sodium phosphate, citric acid-sodium citrate, winestone
One kind in acid-sodium tartrate, succinic acid-sodium succinate, malonic acid-sodium malonate aqueous solution.
Compared with prior art, it is an advantage of the invention that：
1. reaction raw materials and recombination laccase itself are natural products, nontoxic, using the oxygen in air in reaction process, no
It needs to add other oxidants；
2. reaction condition is mild, can carry out at room temperature；
3. product is single, it is easy to purify, is not related to the chemical agents such as soda acid in purification step, product purity is high.
Description of the drawings
Fig. 1 is raw material Acetovanillone（a）With embodiment 1（b）, embodiment 2（c）The HPLC of product schemes.
Specific implementation mode
（3）The recombination laccase of the present invention presses bibliography（Xie, H. F., Li, Q., Wang, M. M., and
Zhao, L. G. (2013) Production of a Recombinant Laccase from Pichia pastoris
and Biodegradation of Chlorpyrifos in a Laccase/Vanillin System, J Microbiol Biotechn23, 864-871.）Middle method is prepared and is purified.
The assay method of laccase activity of the present invention is：Using 1cm light path cuvettes, uv-spectrophotometric is utilized
Meter measures, Detection wavelength 465nm.Buffer solution is the citric acid-sodium citrate buffer solution of the pH 5.0 of 50mM, and 2mL is anti-
It answers and contains 1.8 mL buffer solutions, 0.9 mL, 2.5 mM guaiacol solution and 0.1 mL in system by certain diluted paint
Enzyme.It measures and is carried out at 30 °C, record the variation of 3 min absorbance As, the changing value of average minute clock absorbance is calculated
Laccase activity is calculated as follows in A, and wherein absorptivity is 1.2 ' 104 M-1×cm-1：
Embodiment 1
6.6 mmol Acetovanillones are dissolved in 200mL water, recombination paint is added by the dosage of 6.0 U/mol Acetovanillones
Enzyme reacts 4 h in 30 °C of 150rpm shaking tables, and after reaction, precipitation is filtered, and 100 °C of hot water washings is used in combination to precipitate 3 times,
In 50 °C of dryings, two Acetovanillone product of white powder dehydrogenation, purity 96.9%, yield 94% are obtained.
Embodiment 2
6.6 mmol Acetovanillones are dissolved in 5.0 citric acid-sodium citrate buffers of pH of a concentration of 50 mM of 200mL
In, recombination laccase is added by the dosage of 3.0 U/mol Acetovanillones, 24 h are reacted in 30 °C of 150rpm shaking tables, reaction terminates
Afterwards, precipitation is filtered, is used in combination 100 °C of hot water washings to precipitate 3 times, in 50 °C of dryings, obtains two vanilla of pale yellow powder shape dehydrogenation
Ethyl ketone product, purity 97.0%, yield 88%.
Embodiment 3
6.6 mmol Acetovanillones are dissolved in 200mL water, color suede leather lid is added by the dosage of 6.0 U/mol Acetovanillones
Bacterium source laccase, reacts 4 h in 30 °C of 150rpm shaking tables, and after reaction, precipitation is filtered, and 100 °C of hot water washings are used in combination
Precipitation 3 times, in 50 °C of dryings, obtains pale yellow powder shape product, purity 83.3%, yield 81%.
Fig. 1 is the HPLC spectrograms of raw material and product in embodiment 1 and embodiment 2.Wherein：When the reservation of raw material Acetovanillone
Between be 10.7~10.9min.1 product of embodiment, 2 product of embodiment retention time be respectively 14.6 and 14.5 min.
1 Acetovanillone of table and embodiment 1 and 2 product of embodiment1H-NMR modal datas
（Solvent：DMSO-d6）
* with solvent overlap of peaks.
It can be seen from the data in Table 1 that the hydrogen for 2 product of embodiment 1 and embodiment polymerizeing by Laccase Catalyzed is composed
Data are consistent with document, meet 5,5 '-dehydrogenation, two Acetovanillone chemical constitution.
Claims (5)
1. the Laccase Catalyzed synthetic method of 5,5 '-dehydrogenation of one kind, two Acetovanillone, which is characterized in that include the following steps：
（1）Acetovanillone is dissolved in the buffer solution that water or pH are 3.5 ~ 6.0, is configured to solution, control temperature 20 ~
60 °C, recombination laccase is added, is reacted in shaking table；
（2）It staticly settles after reaction, washing precipitate, it is dry at not higher than 50 DEG C, it is fragrant to obtain 5,5 '-dehydrogenation of product two
Careless ethyl ketone.
2. as described in claim 15, the Laccase Catalyzed synthetic method of 5 '-dehydrogenation, two Acetovanillone, which is characterized in that described
Acetovanillone solution molar concentration be 33 mmol/L.
3. as described in claim 15, the Laccase Catalyzed synthetic method of 5 '-dehydrogenation, two Acetovanillone, which is characterized in that shaking table
Rotating speed is 120~150 rpm, and the reaction time is more than 4 h.
4. as described in claim 15, the Laccase Catalyzed synthetic method of 5 '-dehydrogenation, two Acetovanillone, which is characterized in that recombination
Laccase dosage is 3.0~6.0 U/mol Acetovanillones.
5. as described in claim 15, the Laccase Catalyzed synthetic method of 5 '-dehydrogenation, two Acetovanillone, which is characterized in that buffering
Liquid is selected from Acetic acid-sodium acetate, phosphoric acid-sodium phosphate, citric acid-sodium citrate, tartaric acid-sodium tartrate, succinic acid-succinic acid
One kind in sodium, malonic acid-sodium malonate aqueous solution.
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| CN101967497A (en) * | 2010-07-15 | 2011-02-09 | 池州方达科技有限公司 | Preparation of 2-(6'-methoxy-2'-naphthyl) allyl alcohol by laccase method |
| CN102643886A (en) * | 2012-04-13 | 2012-08-22 | 东华大学 | Method for preparing polymerized rutin by laccase mediator system catalysis |
| CN103088076A (en) * | 2013-01-29 | 2013-05-08 | 南京理工大学 | Method for preparing benzoyl formic acid and R-mandelic acid by coupling reaction of S- mandelic acid dehydrogenase and laccase |
| CN103710363A (en) * | 2013-12-16 | 2014-04-09 | 南京林业大学 | Laccase gene Lac6 and expression protein and application thereof |
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| US9475916B2 (en) * | 2010-12-20 | 2016-10-25 | Allnex Austria Gmbh | Process for curing surface-coating compositions |
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| CN103710363A (en) * | 2013-12-16 | 2014-04-09 | 南京林业大学 | Laccase gene Lac6 and expression protein and application thereof |
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