CN103275953B - Beta-galactosidase with broadened nucleoside substrate specificity - Google Patents
Beta-galactosidase with broadened nucleoside substrate specificity Download PDFInfo
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- CN103275953B CN103275953B CN201310257432.6A CN201310257432A CN103275953B CN 103275953 B CN103275953 B CN 103275953B CN 201310257432 A CN201310257432 A CN 201310257432A CN 103275953 B CN103275953 B CN 103275953B
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Abstract
The invention relates to beta-galactosidase with broadened nucleoside substrate specificity. An amino acid sequence of the beta-galactosidase with the broadened nucleoside substrate specificity is shown as SEQ ID No. 1 (sequence identifier number 1). The invention further provides a gene for encoding the beta-galactosidase with the broadened nucleoside substrate specificity. The beta-galactosidase with the broadened nucleoside substrate specificity has new receptor selectivity, can conduct transglycosylation on a phenolic compound which is difficult to glycosylate or cannot be glycosylated by the natural beta-galactosidase, can serve as a powerful tool to modify the phenolic compound and relevant drug molecules, and has a wide application prospect.
Description
Technical field
The present invention relates to a kind of beta-galactosidase enzymes that glycosides substrate specificity is widened that turns, relate in particular to and a kind of phenolic compound is turned to the beta-galactosidase enzymes that glycosyl efficiency is high, belong to zymoprotein field of engineering technology.
Technical background
Beta-galactosidase enzymes (β-galactosidase, EC3.2.1.23) is the important glycoside hydrolase of a class, and catalysis beta galactose glycosidic bond is hydrolyzed.This fermentoid is extensively present in animal, plant and microorganism, can generate semi-lactosi and glucose by hydrolyzes lactose (Gal β 1 → 4Glc), in foodstuffs industry, have a wide range of applications, within 1998, classified as foodstuff additive new variety by the Food Additives Used in China stdn council, for being hydrolyzed the lactose of cow's milk and other milk-product, solve lactose intolerant patient's Dairy Consumption problem etc.The beta-galactosidase enzymes of some kind, in hydrolyzes lactose molecule, also has the galactosyl of turning effect,, take lactose or its hydrolysate as acceptor, turns glycosyl complex functionality oligomeric galactose.
Be accompanied by recent years the rise of glycosylation engineering, the glycosyl activity that turns of beta-galactosidase enzymes is extended gradually, be used to the synthetic multiple galactoside compound that has significant application value in fields such as grocery trade, industry, pharmacy industries, comprising oligosaccharides, alkyl glycoside, aromatic base glucosides and glycosyl conjugate etc.At present the beta-galactosidase enzymes of report take monose, oligosaccharides, simple glucosides, alcohols and other complex compound when acceptor carries out Transglycosylation, glycosylation mostly occurs on the hydroxyl of acceptor molecule sugar ring or alkyl, and the galactosylation of aromatic nucleus hydroxyl only has an example report, the beta-galactosidase enzymes that adopts Kluyveromyces lactis (Kluyveromyces lactis) to originate carries out glycosylation modified to Resorcinol, the Resorcinol galactoside of generation has improved 1.19 times than the anti-oxidant activity of Resorcinol heteroside (arbutin).
Phenolic compound is the typical compound with aromatic nucleus hydroxyl, there is very important pharmacological activity, it is glycosylation modified significant, but because phenolic compound phenyl ring hydroxyl nucleophilicity is lower, and natural Glycosylase carries out glycosylation modified difficulty take this compounds as acceptor larger.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of beta-galactosidase enzymes that glycosides substrate specificity is widened that turns is provided.
Turn the beta-galactosidase enzymes that glycosides substrate specificity is widened, aminoacid sequence is as shown in SEQ ID No.1.This turns the beta-galactosidase enzymes that glycosides substrate specificity widens and derives from the beta-galactosidase enzymes that GenBank accession number is ACE06986.1, is operated its 980th tryptophane phenylalanine replaced by molecular biology.
The above-mentioned gene that turns the beta-galactosidase enzymes that glycosides substrate specificity widens of encoding, nucleotide sequence is as shown in SEQ ID No.2.
A kind of expression vector that has inserted the above-mentioned gene that turns the beta-galactosidase enzymes that glycosides substrate specificity widens of encoding.
A kind of reconstitution cell, contains above-mentioned expression vector.
Preferably, described reconstitution cell is by obtaining after above-mentioned expression vector transformed competence colibacillus e. coli bl21 (DE3).
The above-mentioned preparation method who turns the beta-galactosidase enzymes that glycosides substrate specificity widens:
The beta-galactosidase gene sequence (proteins encoded GenBank accession number is ACE06986.1) that synthetic GenBank accession number is EU734748.1, be connected on pET-21b (+) plasmid, transform e. coli bl21 (DE3), then extract recombinant plasmid, take plasmid as template, add mutant primer, adopt sudden change test kit (full formula gold Easy Mutagenesis System, Beijing), in this enzyme gene order, introduce sudden change by pcr amplification reaction, PCR product is after Dpn I enzyme (TaKaRa) is processed, transform e. coli bl21 (DE3).Then according to delivering document [Synthesis of galactosyl sucralose by β-galactosidase from Lactobacillus bulgaricus L3, Food Chem, 2012,134:269-275.] described method prepares beta-galactosidase enzymes mutant enzyme.
The application of above-mentioned beta-galactosidase enzymes in glycosylation modified phenolic compound, step is as follows:
(1) adopting phosphoric acid buffer preparation lactose concn is 0.1M～0.3M, and phenols acceptor density 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) step (1) is made to reaction system and reacts 20～60 minutes in 37～45 ℃ of water-baths, boil termination reaction, 10000～12000 revs/min centrifugal 20 minutes, get supernatant liquor;
(3) supernatant liquor step (2) being made, after separating, being dried, makes phenols glycoside compounds.
Preferred according to the present invention, the phenols acceptor in described step (1) is selected from coffic acid, Phloroglucinol or trans-resveratrol.
Preferred according to the present invention, the phosphoric acid buffer in 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 described step (1) is 50mM, the potassium phosphate buffer of pH7.0.
Preferred according to the present invention, the reaction conditions in described step (2) is 45 ℃ of water-baths 45 minutes.
Preferred according to the present invention, the termination reaction condition of boiling in described step (2) is 100 ℃ and boils 5 minutes.
Preferred according to the present invention, separation in described step (3) adopts preparative thin layer chromatography board to separate, the Bio-gel P2 chromatographic column of model Silica gel60F254 (Merck, Germany) or employing specification 15mm × 100cm is carried out chromatographic separation.
Preferred according to the present invention, being dried as lyophilize in described step (3).
Further preferred according to the present invention, in described step (3), also comprise that the thin-layer chromatography while separation detects, merge the step of the product that migration distance is identical.
The step that above-mentioned thin-layer chromatography detects is as follows:
After thin layer chromatography board point sample, in developing agent, launch, after spray painting developer, within 5 minutes, make sugared spot colour developing 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 0.5wt% 3, the solution of 5-orcin.
The relevant developing agent of trans-resveratrol is made by acetonitrile and water 85:15 mixing by volume.
Beneficial effect
The invention provides a kind of new beta-galactosidase enzymes that turns glycosides substrate specificity that possesses, this enzyme is the mutant enzyme obtaining by the natural beta-galactosidase enzymes of molecular modification, show the new glycosyl acceptor substrate specificity that turns, can be difficult to glycosylated polyhydroxy phenol receptoroid as coffic acid, Phloroglucinol and cannot glycosylated phenols carry out galactosylation modification as trans-resveratrol natural beta-galactosidase enzymes enzyme, can be used as glycosylation modified toolenzyme, aspect phenolic compound processing and drug molecule modification, having application prospect.
Accompanying drawing explanation
Fig. 1 is the mass spectrum of coffic acid galactoside;
Fig. 2 is the mass spectrum of trans-resveratrol galactoside.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described further, but institute of the present invention protection domain is not limited to this.
PET-21b (+) plasmid is purchased from Invitrogen company;
E. coli bl21 (DE3) is purchased from Invitrogen company.
Embodiment 1: turn acquisition and the zymoprotein preparation of the gene of the beta-galactosidase enzymes that glycosides substrate specificity widens
The beta-galactosidase gene sequence (proteins encoded GenBank accession number is ACE06986.1) that synthetic GenBank accession number is EU734748.1, be connected on pET-21b (+) plasmid, transform e. coli bl21 (DE3), then extract recombinant plasmid, take plasmid as template, 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
In primer, TTT coding phenylalanine, replaces with phenylalanine by PCR by 980 tryptophanes;
Pcr amplification condition is: 95 ℃ of denaturations 5 minutes; (72 ℃ are extended 7 minutes for 95 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds to react 20 circulations; After 20 loop ends, 72 ℃ are extended 10 minutes.
PCR product is after Dpn I (TaKaRa) enzyme is processed, transform e. coli bl21 (DE3), the beta-galactosidase enzymes mutant enzyme gene obtaining 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.
Prepare 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 by Xylene Brilliant Cyanine G method.
Embodiment 2: utilize the synthetic coffic acid galactoside of sudden change beta-galactosidase enzymes
With pH7.0,50mM potassium phosphate buffer preparation reaction system 50mL, lactose final concentration is 0.2M, and coffic acid final concentration is 0.1M, and the addition of enzyme is 4 μ g/mL.45 ℃ of reactions, after 45 minutes, 100 ℃ are boiled 5 minutes, termination reaction.
By centrifugal 20 minutes 12000 revs/min of the reaction solutions after boiling, draw supernatant liquor 1mL, with the Bio-gel P2 chromatographic column separation of specification 15mm × 100cm, take water as moving phase, flow velocity 0.2mL/ minute, collects elution samples, thin-layer chromatography detects, merge the identical product of migration distance, after lyophilize, make powder, be glucosides product;
Getting above-mentioned glucosides product dilute with water is that mass percent is 1% solution, carries out mass spectroscopy, the feature molecular ion peak [M-H] of target product
-for m/z341.09(as shown in Figure 1), judge that molecular weight of product is 342, turns out to be coffic acid galactoside.
The step that above-mentioned thin-layer chromatography detects is as follows:
After thin layer chromatography board point sample, in developing agent, launch, after spray painting developer, within 5 minutes, make sugared spot colour developing 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 0.5wt% 3, the solution of 5-orcin.
Embodiment 3: utilize sudden change beta-galactosidase enzymes synthesizing resveratrol galactoside
It is 0.2M that reaction system adopts the lactose solution final concentration of phosphoric acid buffer preparation, the trans-resveratrol final concentration of acetone preparation is 0.05M, the beta-galactosidase enzymes addition of aminoacid sequence as shown in SEQ ID NO.1 is 10 μ g/mL, in 40 ℃ of water-baths, reacts 20 minutes, boils termination reaction.
By centrifugal 20 minutes of 12000 revs/min of reaction solutions after boiling, draw supernatant liquor, at Preparative TLC chromatoplate (PLC Silica gel60F254, Merck) point sample exhibition layer.After exhibition layer finishes, on chromatoplate, get the wide bar shaped platelet colour developing of 1cm every 10cm, judge the position of target carbohydrate in thin layer chromatography board.Then scraping thin layer chromatography board not color development area containing the silica gel powder of target glucosides, it is dissolved in the water again, centrifuging and taking supernatant, makes powder after lyophilize, be glucosides product.
Thin-layer chromatography detects as embodiment 2, and difference is that developing agent is by acetonitrile and water 85:15 mixed preparing by volume.
Getting above-mentioned trans-resveratrol galactoside dilute with water is that mass percent is 1% solution, carries out mass spectroscopy, and instrument is Shimadzu LCMS-IT-TOF mass spectrograph (Japan), the feature molecular ion peak [M-H] of target product
-for m/z389.11(as shown in Figure 2), judge that molecular weight of product is 390, turns out to be trans-resveratrol galactoside.
Claims (5)
1. turn the beta-galactosidase enzymes that glycosides substrate specificity is widened, aminoacid sequence is as shown in SEQ ID No.1.
2. the above-mentioned gene that turns the beta-galactosidase enzymes that glycosides substrate specificity widens of coding, nucleotide sequence is as shown in SEQ ID No.2.
3. one kind has been inserted the expression vector of gene described in claim 2.
4. a reconstitution cell, contains expression vector described in claim 3.
5. reconstitution cell as claimed in claim 4, is characterized in that, described reconstitution cell is by obtaining after above-mentioned expression vector transformed competence colibacillus e. coli bl21 (DE3).
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6875601B1 (en) * | 1998-05-22 | 2005-04-05 | Compagnie Gervais Danone | Mutant Lactobacillus bulgaricus strains free from β-galactoside activity |
| CN101899463A (en) * | 2010-07-05 | 2010-12-01 | 山东大学 | Beta-galactosidase with cellulose adsorption zone and application thereof |
| CN102337254A (en) * | 2011-08-26 | 2012-02-01 | 江南大学 | Mutant of beta-galactosidase and preparation method and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6875601B1 (en) * | 1998-05-22 | 2005-04-05 | Compagnie Gervais Danone | Mutant Lactobacillus bulgaricus strains free from β-galactoside activity |
| CN101899463A (en) * | 2010-07-05 | 2010-12-01 | 山东大学 | Beta-galactosidase with cellulose adsorption zone and application thereof |
| CN102337254A (en) * | 2011-08-26 | 2012-02-01 | 江南大学 | Mutant of beta-galactosidase and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Lili Lu, et al..Synthesis of galactosyl sucralose by b-galactosidase from Lactobacillus bulgaricus L3.《Food Chemistry》.2012,第134卷269-275. |
| Synthesis of galactosyl sucralose by b-galactosidase from Lactobacillus bulgaricus L3;Lili Lu, et al.;《Food Chemistry》;20120301;第134卷;269-275 * |
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