CN103276029B - A kind of preparation method of pyrocatechol galactoside - Google Patents
A kind of preparation method of pyrocatechol galactoside Download PDFInfo
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- CN103276029B CN103276029B CN201310255797.5A CN201310255797A CN103276029B CN 103276029 B CN103276029 B CN 103276029B CN 201310255797 A CN201310255797 A CN 201310255797A CN 103276029 B CN103276029 B CN 103276029B
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Abstract
The present invention relates to a kind of preparation method of pyrocatechol galactoside, comprise the steps: that (1) adopts phosphoric acid buffer preparation lactose concn to be 0.1M ~ 0.3M, pyrocatechol concentration is 0.05M ~ 0.2M, and the beta-galactosidase enzymes addition of aminoacid sequence as shown in SEQ ID NO.1 is the reaction system of 2 μ g ~ 10 μ g/mL; (2), after reaction system being reacted, termination reaction is boiled, centrifugal, get supernatant liquor; (3) by supernatant liquor, after separation, drying, obtained pyrocatechol galactoside.Adopt the beta-galactosidase enzymes synthesizing o-dihydroxybenzene galactoside through sudden change, product is compared with its raw material pyrocatechol, containing galactosyl, add water-soluble and stability, this compound self can be used as chemosynthesis intermediate in order to the new high added value compound of derivative synthesis.
Description
Technical field
The present invention relates to the preparation method of pyrocatechol glycosides derivatives, particularly a kind of method utilizing beta-galactosidase enzymes mutant enzyme synthesizing o-dihydroxybenzene galactoside, belongs to sugar engineering technical field.
Technical background
Phenolic compound is the compound of a class with aromatic ring structure, has multiple physiology and pharmacological effects, as catechol there is antioxygenation, Avarol can suppress copying of HIV.The glycosylation of phenolic compound not only can increase water-soluble, can also improve pharmacological property and curative effect.Such as: Resorcinol has toxicity, Resorcinol-alpha-glucosaccharase (arbutin) then has antibacterial and skin lightening effects; Sub-Eugenol has the function of maintenance hair owing to suppressing 5α-reductase, but it easily distils, have special odor, sub-Eugenol-beta-glucoside that chemical method glycosylation is formed can the slow metabolism by the microorganism that scalp exists, degraded release phenol type structure, is conducive to the performance of drug effect gradually.Can say, new phenolic compound glucosides has new pharmacological property, the phenolic compound different in kind that different glycosylation is modified, and such as Resorcinol galactoside anti-oxidant activity increases 1.19 times than arbutin.
Pyrocatechol is a kind of basic raw material of fine chemicals, and be widely used in the industries such as agricultural chemicals, spices, medicine, dyestuff, polymkeric substance, market potential is huge.The exploitation of current catechol derivatives adopts chemical method more, enzyme process derivatize only has a routine relevant report, adopt alpha-glucosidase to carry out glucosyl group modification to the catechin compounds with pyrocatechol structure, the enzyme process galactosyl that there is no pyrocatechol modifies report.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of preparation method of pyrocatechol galactoside is provided.
A preparation method for pyrocatechol galactoside, comprises the steps:
(1) adopt phosphoric acid buffer preparation lactose concn to be 0.1M ~ 0.3M, pyrocatechol concentration is 0.05M ~ 0.2M, and the beta-galactosidase enzymes addition of aminoacid sequence as shown in SEQ ID NO.1 is the reaction system of 2 μ g ~ 10 μ g/mL;
(2) obtained for step (1) reaction system reacted 20 ~ 60 minutes in 37 ~ 45 DEG C of water-baths, boil termination reaction, 10000 ~ 12000 revs/min centrifugal 20 minutes, gets supernatant liquor;
(3) by the supernatant liquor that step (2) is obtained, after separation, drying, obtained pyrocatechol galactoside.
Preferred according to the present invention, in described step (1), the beta-galactosidase enzymes of aminoacid sequence as shown in SEQ ID NO.1 selects GenBank accession number to be ACE06986.1, is replaced by the 980th tryptophane phenylalanine, obtained.Beta-galactosidase enzymes after replacement can according to delivering document [Synthesis of galactosyl sucralose by β-galactosidase fromLactobacillus bulgaricus L3, Food Chem, 2012,134:269-275] described method prepares zymoprotein.Above-mentioned beta-galactosidase enzymes has pyrocatechol and significantly turns glycosyl activity.
Preferred according to the present invention, the phosphoric acid buffer in described step (1) is the buffer solution of potassium phosphate of concentration 10 ~ 100mM, pH6 ~ 8; Preferred further according to the present invention, the damping fluid in described step (1) is the potassium phosphate buffer of 50mM, pH7.0.
Preferred according to the present invention, the reaction conditions in described step (2) is 45 DEG C of water-baths 45 minutes.
Preferred according to the present invention, the termination reaction condition of boiling in described step (2) is 100 DEG C and boils 5 minutes.
Preferred according to the present invention, the separation in described step (3) adopts preparative thin layer chromatography board to be separated, model Silica gel60F254 (Merck, Germany).
Preferred according to the present invention, the drying in described step (3) is lyophilize.
Preferred further according to the present invention, thin-layer chromatography when also comprising separation in described step (3) detects, and merges the step of the identical product of migration distance.
The step that above-mentioned thin-layer chromatography detects is as follows:
Launch in developing agent after thin layer chromatography board point sample, after spray painting developer, within 5 minutes, make sugared spot development in 120 DEG C of bakings.
Above-mentioned developing agent is by propyl carbinol, dehydrated alcohol and water 5:3:2 mixed preparing by volume; Developer to be volume percent be 20% sulfuric acid and concentration be the solution of 3, the 5-orcins of 0.5wt%.
Beneficial effect
The present invention take pyrocatechol as raw material, with cheap lactose substrate for galactosyl donor, adopt the beta-galactosidase enzymes synthesizing o-dihydroxybenzene galactoside through sudden change, product is compared with its raw material pyrocatechol, containing galactosyl, add water-soluble and stability.This compound self can be used as a kind of chemosynthesis intermediate, in order to the high added value compound that derivative synthesis is new, has potential using value in each side such as chemical industry, medicine.
Accompanying drawing explanation
Fig. 1 is the mass spectrum of pyrocatechol galactoside;
Fig. 2 is the hydrogen spectrum of pyrocatechol galactoside;
Fig. 3 is that the hydrogen hydrogen of pyrocatechol galactoside is correlated with nuclear magnetic spectrum;
Fig. 4 is the hydrocarbon directly related nuclear magnetic spectrum of pyrocatechol galactoside;
Fig. 5 is the hydrocarbon long-range relevant nuclear magnetic spectrum of pyrocatechol galactoside.
Embodiment
Below in conjunction with embodiment, the present invention will be further described, but institute of the present invention protection domain is not limited thereto.
Biological material source
PET-21b (+) plasmid is purchased from Invitrogen company;
E. coli bl21 (DE3) is purchased from Invitrogen company.
Embodiment 1
A preparation method for pyrocatechol galactoside, step is as follows:
1. the preparation of beta-galactosidase enzymes
Synthetic GenBank accession number is the beta-galactosidase gene sequence (proteins encoded GenBank accession number is ACE06986.1) of EU734748.1, be connected on pET-21b (+) plasmid, transformation of E. coli BL21 (DE3), then recombinant plasmid is extracted, take plasmid as template, mutagenesis kit (full formula gold Easy Mutagenesis System, Beijing) is adopted in this enzyme gene order, to introduce sudden change by PCR;
Upstream primer is: 5 '-CGGGGATGACTCC
tTTgGGCAGAAGGTCCA-3 '; SEQ ID NO.3
Downstream primer is: 5 '-
aAAgGAGTCATCCCCGCCGACCCCCATCTG-3 '; SEQ ID NO.4
980 tryptophanes are replaced with phenylalanine by PCR by TTT encoding phenylalanine in primer;
Pcr amplification condition is: 95 DEG C of denaturations 5 minutes; (72 DEG C extend 7 minutes for 95 DEG C of sex change 30 seconds, 55 DEG C of annealing 30 seconds to react 20 circulations; After 20 loop ends, 72 DEG C extend 10 minutes.
PCR primer is after Dpn I enzyme (TaKaRa) process, transformation of E. coli BL21 (DE3), the beta-galactosidase enzymes mutant enzyme gene obtained is through order-checking, and nucleotide sequence is as shown in SEQ ID NO.2, and the aminoacid sequence of its coding is as shown in SEQ ID NO.1.
According to delivering document [Synthesis of galactosyl sucralose by β-galactosidase from Lactobacillusbulgaricus L3, Food Chem, 2012,134:269-275] described method prepares beta-galactosidase enzymes mutant enzyme.The enzyme amount of beta-galactosidase enzymes enzyme liquid is measured with Coomassie Brilliant Blue.
2. beta-galactosidase enzymes catalyzes and synthesizes pyrocatechol galactoside
With pH7.0,50mM potassium phosphate buffer preparation reaction system 50mL, lactose final concentration is 0.2M, and pyrocatechol final concentration is 0.1M, and the addition of enzyme is 4 μ g/mL.45 DEG C of reactions after 45 minutes, 100 DEG C are boiled 5 minutes, termination reaction.
3. the purifying of pyrocatechol galactoside
By centrifugal 20 minutes of the reaction solution 12000 revs/min after boiling, Aspirate supernatant, at Preparative TLC chromatoplate (PLCSilica gel60F254, Merck) point sample exhibition layer.After exhibition layer terminates, chromatoplate gets the wide bar shaped platelet colour developing of 1cm every 10cm, judges the position of target carbohydrate on chromatoplate.Then the non-color development area of scraping chromatoplate is containing the silica gel powder of target glucosides, and it be again dissolved in the water, centrifuging and taking supernatant, after lyophilize, gained powder is pyrocatechol galactoside.
The step that above-mentioned thin-layer chromatography detects is as follows:
Thin layer chromatography board point sample, launches in developing agent, after spray painting developer, within 5 minutes, makes sugared spot development in 120 DEG C of bakings.
Above-mentioned developing agent is by propyl carbinol, dehydrated alcohol and water 5:3:2 mixed preparing by volume; Developer to be volume percent be 20% sulfuric acid and concentration be the solution of 3, the 5-orcins of 0.5wt%.
4. the Structural Identification of pyrocatechol galactoside
To get above-mentioned pyrocatechol galactoside dilute with water be mass percent be 1% solution, carry out mass spectroscopy, the characteristic molecular quasi-molecular ions [M+Na] of target product
+for m/z295.07(as shown in Figure 1), judge that molecular weight of product is 272, with expection molecular weight of product consistent.
5mg powder is dissolved in deuterated water, carry out nuclear-magnetism parsing, comprehensive hydrogen spectrum (as shown in Figure 2), hydrogen hydrogen Correlated Spectroscopy (COSY) (as shown in Figure 3), hydrocarbon directly related spectrum (HSQC) (as shown in Figure 4), hydrocarbon long-range Correlated Spectroscopy (HMBC) (as shown in Figure 5), determine the chemical shiftsum coupling constant that each position is hydrocarbon, identify that the product structure of new synthesis is pyrocatechol-β-D-galactopyranoside.
1h NMR shows galactose proton signal and is positioned at δ 4.95 ~ 3.69ppm, and on phenyl ring, proton signal is δ 7.12 ~ 6.86ppm.Chemical shift 4.95ppm can observe the characteristic bimodal signal of semi-lactosi anomeric proton, coupling constant is 7.8Hz, infer that galactosyl is connected with pyrocatechol molecule by β key, can observe that in HMBC figure the C-1 ' (δ 144.8) of pyrocatechol phenyl ring and semi-lactosi anomeric proton H-1 (δ 4.95) exist coupled signal, confirm the existence of beta galactose glycosidic bond.
Pyrocatechol-β-D-galactopyranoside the chemical structural formula obtained is as follows:
Above-mentioned mass spectroscopy instrument is Shimadzu LCMS-IT-TOF mass spectrograph (Japan); Nmr analysis instrument is AVANCE600 type superconduction super shielding fourier transform NMR spectrometer (Bruker company of Switzerland).
Embodiment 2
A preparation method for pyrocatechol-β-D-galactopyranoside, step is as follows:
(1) adopt phosphoric acid buffer preparation lactose concn to be 0.2M, pyrocatechol concentration is 0.05M, and the beta-galactosidase enzymes addition of aminoacid sequence as shown in SEQID NO.1 is the reaction system of 10 μ g/mL;
(2) obtained for step (1) reaction system reacted 20 minutes in 37 DEG C of water-baths, boil termination reaction, 12000 revs/min centrifugal 20 minutes, gets supernatant liquor;
(3) pyrocatechol galactoside purifying with detect with embodiment 1.
Claims (9)
1. a preparation method for pyrocatechol galactoside, comprises the steps:
(1) adopt phosphoric acid buffer preparation lactose concn to be 0.1M ~ 0.3M, pyrocatechol concentration is 0.05M ~ 0.2M, and the beta-galactosidase enzymes addition of aminoacid sequence as shown in SEQ ID NO.1 is the reaction system of 2 μ g ~ 10 μ g/mL;
(2) obtained for step (1) reaction system reacted 20 ~ 60 minutes in 37 ~ 45 DEG C of water-baths, boil termination reaction, 10000 ~ 12000 revs/min centrifugal 20 minutes, gets supernatant liquor;
(3) by the supernatant liquor that step (2) is obtained, after separation, drying, obtained pyrocatechol galactoside.
2. preparation method as claimed in claim 1, it is characterized in that, the phosphoric acid buffer in described step (1) is the buffer solution of potassium phosphate of concentration 10 ~ 100mM, pH6 ~ 8.
3. preparation method as claimed in claim 2, it is characterized in that, the damping fluid in described step (1) is the potassium phosphate buffer of 50mM, pH7.0.
4. preparation method as claimed in claim 1, it is characterized in that, the reaction conditions in described step (2) is 45 DEG C of water-baths 45 minutes.
5. preparation method as claimed in claim 1, it is characterized in that, the termination reaction condition of boiling in described step (2) is 100 DEG C and boils 5 minutes.
6. preparation method as claimed in claim 1, is characterized in that, the separation in described step (3) adopts preparative thin layer chromatography board to be separated.
7. preparation method as claimed in claim 1, it is characterized in that, the drying in described step (3) is lyophilize.
8. preparation method as claimed in claim 1, is characterized in that, thin-layer chromatography when also comprising separation in described step (3) detects, merges the step of the identical product of migration distance.
9. preparation method as claimed in claim 8, is characterized in that, the step that above-mentioned thin-layer chromatography detects is as follows:
Launch in developing agent after thin layer chromatography board point sample, after spray painting developer, within 5 minutes, make sugared spot development in 120 DEG C of bakings;
Above-mentioned developing agent is by propyl carbinol, dehydrated alcohol and water 5:3:2 mixed preparing by volume; Developer to be volume percent be 20% sulfuric acid and concentration be the solution of 3, the 5-orcins of 0.5wt%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5710133A (en) * | 1993-05-07 | 1998-01-20 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | α-glycosyl derivative of catecholamine or its salt, and its preparation and uses |
| CN102241708A (en) * | 2011-05-10 | 2011-11-16 | 山东大学 | Galactoside compound and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5710133A (en) * | 1993-05-07 | 1998-01-20 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | α-glycosyl derivative of catecholamine or its salt, and its preparation and uses |
| CN102241708A (en) * | 2011-05-10 | 2011-11-16 | 山东大学 | Galactoside compound and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| accession EU734748.1;Lu,L. et al.;《Genbank》;20120608;全文 * |
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