CN103276029A - Preparation method of pyrocatechol galactoside - Google Patents

Abstract

The invention relates to a preparation method of pyrocatechol galactoside, which comprises the following steps of: (1) preparing a reaction system with a phosphate buffer, (2) boiling to terminate reaction after the reaction of the reaction system, centrifuging, and taking a supernate, and (3) separating and drying the supernate, and obtaining pyrocatechol galactoside, wherein for the reaction system, the lactose concentration is 0.1-0.3M; the pyrocatechol concentration is 0.05-0.2M; the addition level of beta-galactosidase with an amino acid sequence shown as SEQ ID NO. 1 (sequence identifier number 1) is 2-10 micrograms/mL. Since mutational beta-galactosidase is used for synthesizing pyrocatechol galactoside, a product contains galactosyl compared with the raw material, pyrocatechol; the water solubility and stability are improved; and the compound can serve as a chemical synthesis intermediate for deriving and synthesizing a new compound with a high added value.

Description

A kind of preparation method of pyrocatechol galactoside

Technical field

The present invention relates to the preparation method of pyrocatechol glycosides derivatives, particularly a kind of method of utilizing beta-galactosidase enzymes mutant enzyme synthesizing o-dihydroxybenzene galactoside belongs to sugared field of engineering technology.

Technical background

Phenolic compound is the compound that a class has aromatic ring structure, has multiple physiology and pharmacology function, as catechol have antioxygenation, Avarol can suppress copying of HIV etc.The glycosylation of phenolic compound not only can increase water-soluble, can also improve pharmacological property and curative effect.For example: Resorcinol has toxicity, and Resorcinol-alpha-glucosaccharase (arbutin) then has antibiotic and the skin whitening effect; Sub-Eugenol has the function of maintenance hair owing to suppressing 5, but its easy distillation has special odor, the slow metabolism of microorganism that sub-Eugenol-beta-glucoside that the chemical method glycosylation forms can be existed on the scalp, degraded discharges the phenols structure gradually, is conducive to the performance of drug effect.We can say that new phenolic compound glucosides has new pharmacological property, the phenolic compound different in kind that different glycosylation is modified, for example Resorcinol galactoside anti-oxidant activity has increased 1.19 times than arbutin.

Pyrocatechol is a kind of basic raw material of fine chemicals, is widely used in industries such as agricultural chemicals, spices, medicine, dyestuff, polymkeric substance, and market potential is huge.Chemical method is adopted in the exploitation of catechol derivatives at present more, the enzyme process derivatize only has a routine relevant report, adopt alpha-glucosidase that the catechin compounds that has the pyrocatechol structure is carried out glucosyl group and modify, still do not have the enzyme process galactosyl of pyrocatechol and modify 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 kind of preparation method of pyrocatechol galactoside comprises the steps:

(1) adopting phosphoric acid buffer preparation lactose concn is 0.1M～0.3M, and pyrocatechol concentration is 0.05M～0.2M, and the beta-galactosidase enzymes addition of aminoacid sequence shown in SEQ ID NO.1 is the reaction system of 2 μ g～10 μ g/mL;

(2) step (1) is made reaction system and in 37～45 ℃ of water-baths, reacted 20～60 minutes, boil termination reaction, 10000～12000 rev/mins centrifugal 20 minutes, get supernatant liquor;

(3) supernatant liquor that step (2) is made after separation, drying, makes the pyrocatechol galactoside.

Preferred according to the present invention, it is ACE06986.1 that the middle beta-galactosidase enzymes of aminoacid sequence shown in SEQ ID NO.1 of described step (1) selected the GenBank accession number for use, and the 980th tryptophane replaced with phenylalanine, makes.Beta-galactosidase enzymes after the replacement can be 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 remarkable commentaries on classics glycosyl activity to pyrocatechol.

Preferred according to the present invention, the phosphoric acid buffer in the described step (1) is concentration 10～100mM, the buffer solution of potassium phosphate of pH6～8; Further preferred according to the present invention, the damping fluid in the described step (1) is 50mM, the potassium phosphate buffer of pH7.0.

Preferred according to the present invention, the reaction conditions in the described step (2) is 45 ℃ of water-baths 45 minutes.

Preferred according to the present invention, the termination reaction condition of boiling in the described step (2) is 100 ℃ and boiled 5 minutes.

Preferred according to the present invention, the separation in the described step (3) adopts preparation type thin layer chromatography board to separate, and model Silica gel60F254 (Merck, Germany).

Preferred according to the present invention, the drying in the described step (3) is lyophilize.

Further preferred according to the present invention, comprise also in the described step (3) that the thin-layer chromatography when separating detects, merge the step of the identical product of migration distance.

The step that above-mentioned thin-layer chromatography detects is as follows:

In developing agent, launch behind the thin layer chromatography board point sample, behind the spray painting developer, made sugared spot colour developing in 5 minutes in 120 ℃ of bakings.

Above-mentioned developing agent is by propyl carbinol, dehydrated alcohol and water 5:3:2 mixed preparing by volume; Developer is that volume percent is that 20% sulfuric acid and concentration are 3 of 0.5wt%, the solution of 5-orcin.

Beneficial effect

The present invention is raw material with the pyrocatechol, be the galactosyl donor with cheap lactose substrate, adopt the beta-galactosidase enzymes synthesizing o-dihydroxybenzene galactoside through sudden change, product is compared with its raw material pyrocatechol, contain galactosyl, increased water-soluble and stable.This compound self can be used as a kind of chemosynthesis intermediate, in order to the synthetic new high added value compound of deriving, has potential using value in each side such as chemical industry, medicine.

Description of drawings

Fig. 1 is the mass spectrum of pyrocatechol galactoside;

Fig. 2 is the hydrogen spectrum of pyrocatechol galactoside;

Fig. 3 is the relevant nuclear magnetic spectrum of the hydrogen hydrogen of pyrocatechol galactoside;

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

The present invention will be further described below in conjunction with embodiment, but institute of the present invention protection domain is not limited thereto.

Biological material source

PET-21b (+) plasmid is available from Invitrogen company;

E. coli bl21 (DE3) is available from Invitrogen company.

Embodiment 1

A kind of preparation method of 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, transformed into escherichia coli BL21 (DE3), extract recombinant plasmid then, be template with the plasmid, adopt sudden change test kit (full formula gold Easy Mutagenesis System, Beijing) 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

TTT coding phenylalanine replaces with phenylalanine by PCR with 980 tryptophanes in the primer;

The pcr amplification condition is: 95 ℃ of pre-sex change 5 minutes; (72 ℃ were extended 7 minutes for 95 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds to react 20 circulations; 72 ℃ were extended 10 minutes after 20 loop ends.

The PCR product after Dpn I enzyme (TaKaRa) is handled, transformed into escherichia coli BL21 (DE3), the beta-galactosidase enzymes mutant enzyme gene of acquisition is through order-checking, nucleotide sequence is shown in SEQ ID NO.2, its amino acid sequence coded is shown in SEQ ID NO.1.

Prepare the beta-galactosidase enzymes mutant enzyme according to delivering the described method of document [Synthesis of galactosyl sucralose by β-galactosidase from Lactobacillus bulgaricus L3, Food Chem, 2012,134:269-275].Measure the enzyme amount of beta-galactosidase enzymes enzyme liquid with the Xylene Brilliant Cyanine G method.

2. beta-galactosidase enzymes catalyzes and synthesizes the pyrocatechol galactoside

With pH7.0,50mM potassium phosphate buffer preparation reaction system 50mL, the lactose final concentration is 0.2M, and the pyrocatechol final concentration is 0.1M, and the addition of enzyme is 4 μ g/mL.After 45 minutes, 100 ℃ were boiled termination reaction 5 minutes 45 ℃ of reactions.

3. the purifying of pyrocatechol galactoside

Centrifugal 20 minutes of 12000 rev/mins of reaction solutions after will boiling are drawn supernatant liquor, at preparation thin layer chromatography board (PLCSilica gel60F254, Merck) point sample exhibition layer.After the exhibition layer finishes, on chromatoplate, get the wide bar shaped platelet colour developing of 1cm every 10cm, judge the position of target carbohydrate on chromatoplate.Scrape then get chromatoplate not color development area contain the silica gel powder of target glucosides, it is dissolved in the water again, the centrifuging and taking supernatant, the gained powder is the pyrocatechol galactoside after the lyophilize.

The step that above-mentioned thin-layer chromatography detects is as follows:

The thin layer chromatography board point sample launches in developing agent, behind the spray painting developer, makes sugared spot colour developing in 5 minutes in 120 ℃ of bakings.

Above-mentioned developing agent is by propyl carbinol, dehydrated alcohol and water 5:3:2 mixed preparing by volume; Developer is that volume percent is that 20% sulfuric acid and concentration are 3 of 0.5wt%, the solution of 5-orcin.

4. the structure of pyrocatechol galactoside is identified

Getting above-mentioned pyrocatechol galactoside dilute with water is that mass percent is 1% solution, carries out mass spectroscopy, the feature molecular ion peak [M+Na] of target product ⁺For m/z295.07(as shown in Figure 1), judge that molecular weight of product is 272, consistent with the expection molecular weight of product.

The 5mg powder is dissolved in deuterium in the water, carrying out nuclear-magnetism resolves, comprehensive hydrogen spectrum (as shown in Figure 2), relevant (COSY) (as shown in Figure 3), hydrocarbon directly related spectrum (HSQC) (as shown in Figure 4), the hydrocarbon long-range relevant spectrum (HMBC) (as shown in Figure 5) of composing of hydrogen hydrogen, determine chemical shift and coupling constant that each position is hydrocarbon, identify that new synthetic product structure is pyrocatechol-β-D-galactopyranoside. ¹H NMR shows that the galactose proton signal is positioned at δ 4.95～3.69ppm, and proton signal is δ 7.12～6.86ppm on the phenyl ring.Chemical shift 4.95ppm can observe the bimodal signal of feature of semi-lactosi anomeric proton, coupling constant is 7.8Hz, infer that galactosyl links to each other with the pyrocatechol molecule by the β key, there is coupled signal in the C-1 ' (δ 144.8) that can observe the pyrocatechol phenyl ring in HMBC figure with semi-lactosi anomeric proton H-1 (δ 4.95), has confirmed the existence of beta galactose glycosidic bond.

Pyrocatechol-the β that obtains-D-galactopyranoside chemical structural formula is as follows:

The used instrument of above-mentioned mass spectroscopy is Tianjin, island LCMS-IT-TOF mass spectrograph (Japan); The used instrument of nmr analysis is the super shielding of AVANCE600 type superconduction fourier transform NMR spectrometer (Switzerland Bruker company).

Embodiment 2

The preparation method of a kind of pyrocatechol-β-D-galactopyranoside, step is as follows:

(1) adopting phosphoric acid buffer preparation lactose concn is 0.2M, and pyrocatechol concentration is 0.05M, and the beta-galactosidase enzymes addition of aminoacid sequence shown in SEQID NO.1 is the reaction system of 10 μ g/mL;

(2) step (1) is made reaction system and in 37 ℃ of water-baths, reacted 20 minutes, boil termination reaction, 12000 rev/mins centrifugal 20 minutes, get supernatant liquor;

(3) purifying of pyrocatechol galactoside and detection are with embodiment 1.

Claims (9)

1. the preparation method of a pyrocatechol galactoside comprises the steps:

(1) adopting phosphoric acid buffer preparation lactose concn is 0.1M～0.3M, and pyrocatechol concentration is 0.05M～0.2M, and the beta-galactosidase enzymes addition of aminoacid sequence shown in SEQ ID NO.1 is the reaction system of 2 μ g～10 μ g/mL;

(2) step (1) is made reaction system and in 37～45 ℃ of water-baths, reacted 20～60 minutes, boil termination reaction, 10000～12000 rev/mins centrifugal 20 minutes, get supernatant liquor;

(3) supernatant liquor that step (2) is made after separation, drying, makes the pyrocatechol galactoside.

2. preparation method as claimed in claim 1 is characterized in that, the phosphoric acid buffer in the described step (1) is concentration 10～100mM, the buffer solution of potassium phosphate of pH6～8.

3. preparation method as claimed in claim 2 is characterized in that, the damping fluid in the described step (1) is 50mM, the potassium phosphate buffer of pH7.0.

4. preparation method as claimed in claim 1 is characterized in that, the reaction conditions in the described step (2) is 45 ℃ of water-baths 45 minutes.

5. preparation method as claimed in claim 1 is characterized in that, the termination reaction condition of boiling in the described step (2) is 100 ℃ and boiled 5 minutes.

6. preparation method as claimed in claim 1 is characterized in that, the separation in the described step (3) adopts preparation type thin layer chromatography board to separate.

7. preparation method as claimed in claim 1 is characterized in that, the drying in the described step (3) is lyophilize.

8. preparation method as claimed in claim 1 is characterized in that, also comprises the step of the product that thin-layer chromatography detects, the merging migration distance is identical when separating in the described step (3).

9. preparation method as claimed in claim 8 is characterized in that, the step that above-mentioned thin-layer chromatography detects is as follows:

In developing agent, launch behind the thin layer chromatography board point sample, behind the spray painting developer, made sugared spot colour developing in 5 minutes in 120 ℃ of bakings;

Above-mentioned developing agent is by propyl carbinol, dehydrated alcohol and water 5:3:2 mixed preparing by volume; Developer is that volume percent is that 20% sulfuric acid and concentration are 3 of 0.5wt%, the solution of 5-orcin.

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