CN103275953A - Beta-galactosidase with broadened nucleoside substrate specificity - Google Patents
Beta-galactosidase with broadened nucleoside substrate specificity Download PDFInfo
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- CN103275953A CN103275953A CN2013102574326A CN201310257432A CN103275953A CN 103275953 A CN103275953 A CN 103275953A CN 2013102574326 A CN2013102574326 A CN 2013102574326A CN 201310257432 A CN201310257432 A CN 201310257432A CN 103275953 A CN103275953 A CN 103275953A
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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 the glycosides substrate specificity is widened that changes, relating in particular to a kind ofly changes the high beta-galactosidase enzymes of glycosyl efficient to phenolic compound, belongs to the zymoprotein field of engineering technology.
Technical background
(β-galactosidase EC3.2.1.23) is the important glycoside hydrolase of a class to beta-galactosidase enzymes, catalysis beta galactose glycosidic bond generation hydrolysis.This fermentoid extensively is present in animal, plant and the microorganism, (Gal β 1 → 4Glc) generates semi-lactosi and glucose to the energy hydrolyzes lactose, in foodstuffs industry, have a wide range of applications, classified as the foodstuff additive new variety by the Food Additives Used in China stdn council in 1998, the lactose that is used for hydrolysis cow's milk and other milk-product solves lactose intolerant patient's dairy products consumption problem etc.The beta-galactosidase enzymes of some kind also has the effect of the galactosyl of commentaries on classics in the hydrolyzes lactose molecule, be acceptor with lactose or its hydrolysate namely, changes glycosyl complex functionality oligomeric galactose.
Be accompanied by the rise of glycosylation engineering in recent years, the commentaries on classics glycosyl activity of beta-galactosidase enzymes is extended gradually, be used to the synthetic multiple galactoside compound that significant application value is arranged 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 is being that acceptor is when changeing the glycosyl reaction with monose, oligosaccharides, simple glucosides, alcohols and other complex compound, 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 namely 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 for typically having the compound of aromatic nucleus hydroxyl, has very important pharmacological activity, it is glycosylation modified significant, but because phenolic compound phenyl ring hydroxyl nucleophilicity is lower, and it is bigger that natural Glycosylase is with this compounds that acceptor carries out glycosylation modified difficulty.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of beta-galactosidase enzymes that the glycosides substrate specificity is widened that changes is provided.
A kind ofly change the beta-galactosidase enzymes that the glycosides substrate specificity is widened, aminoacid sequence is shown in SEQ ID No.1.The beta-galactosidase enzymes that this commentaries on classics glycosides substrate specificity is widened derives from the beta-galactosidase enzymes that the GenBank accession number is ACE06986.1, by the molecular biology operation its 980th tryptophane is replaced with phenylalanine.
The gene of the beta-galactosidase enzymes that a kind of above-mentioned commentaries on classics glycosides substrate specificity of encoding is widened, nucleotide sequence is shown in SEQ ID No.2.
A kind of expression carrier of having inserted the beta-galactosidase enzymes that the above-mentioned commentaries on classics glycosides substrate specificity of encoding widens.
A kind of reconstitution cell contains above-mentioned expression vector.
Preferably, described reconstitution cell is by obtaining above-mentioned expression vector transformed competence colibacillus e. coli bl21 (DE3) back.
The preparation method of the beta-galactosidase enzymes that above-mentioned commentaries on classics glycosides substrate specificity is widened:
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, 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, the PCR product after Dpn I enzyme (TaKaRa) is handled, transformed into escherichia 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 the 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 the phenols acceptor density 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 phenols glycoside compounds.
Preferred according to the present invention, the phenols acceptor in the described step (1) is selected from coffic acid, Phloroglucinol or trans-resveratrol.
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, separation in the described step (3) adopts preparation type thin layer chromatography board to separate, and model Silica gel60F254 (Merck, Germany) or adopt the Bio-gel P2 chromatographic column of specification 15mm * 100cm to carry out chromatographic separation.
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.
85:15 is mixed by volume by acetonitrile and water for the relevant developing agent of trans-resveratrol.
Beneficial effect
The invention provides a kind of beta-galactosidase enzymes that possesses new commentaries on classics glycosides substrate specificity, this enzyme is the mutant enzyme that obtains by the natural beta-galactosidase enzymes of molecular modification, shown new commentaries on classics glycosyl acceptor substrate specificity, can to natural beta-galactosidase enzymes enzyme be difficult to glycosylated polyhydroxy phenol receptoroid such as coffic acid, Phloroglucinol and can't glycosylated phenols such as trans-resveratrol carry out galactosylation and modify, can be used as glycosylation modified toolenzyme, aspect phenolic compound processing and the drug molecule modification application prospect is being arranged.
Description of drawings
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 thereto.
PET-21b (+) plasmid is available from Invitrogen company;
E. coli bl21 (DE3) is available from Invitrogen company.
Embodiment 1: the acquisition of the gene of the beta-galactosidase enzymes that commentaries on classics glycosides substrate specificity is widened and zymoprotein preparation
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 (TaKaRa) enzyme 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.
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, the lactose final concentration is 0.2M, and the coffic acid 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.
12000 rev/mins of reaction solutions after will boiling are centrifugal 20 minutes, draw supernatant liquor 1mL, Bio-gel P2 chromatographic column with specification 15mm * 100cm is separated, be moving phase with water, flow velocity 0.2mL/ minute, collect elution samples, thin-layer chromatography detects, merge the identical product of migration distance, make powder after the lyophilize, be the 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 the coffic acid galactoside.
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.
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 shown in SEQ ID NO.1 is 10 μ g/mL, and reaction is 20 minutes in 40 ℃ of water-baths, boils termination reaction.
Centrifugal 20 minutes of 12000 rev/mins of reaction solutions after will boiling are drawn supernatant liquor, at preparation thin layer chromatography board (PLC Silica 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 thin layer chromatography board.Scrape then get thin layer chromatography board not color development area contain the silica gel powder of target glucosides, it is dissolved in the water again, the centrifuging and taking supernatant is made powder after the lyophilize, be the glucosides product.
Thin-layer chromatography detects as embodiment 2, and difference is developing agent 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 used instrument is Tianjin, island 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 the trans-resveratrol galactoside.
Claims (5)
1. one kind is changeed the beta-galactosidase enzymes that the glycosides substrate specificity is widened, and aminoacid sequence is shown in SEQ ID No.1.
2. the gene of the beta-galactosidase enzymes widened of the above-mentioned commentaries on classics glycosides substrate specificity of coding, nucleotide sequence is shown in SEQ ID No.2.
3. expression carrier of having inserted the beta-galactosidase enzymes that the described commentaries on classics glycosides of coding claim 2 substrate specificity widens.
4. a reconstitution cell contains the described expression vector of claim 3.
5. reconstitution cell as claimed in claim 4 is characterized in that, described reconstitution cell is by obtaining above-mentioned expression vector transformed competence colibacillus e. coli bl21 (DE3) back.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110678479A (en) * | 2017-04-11 | 2020-01-10 | 科·汉森有限公司 | Lactase with improved performance |
| CN111518790A (en) * | 2018-11-27 | 2020-08-11 | 江南大学 | Sucrose hydrolase mutant and preparation method and application thereof |
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 (1)
| Title |
|---|
| LILI LU, ET AL.: "Synthesis of galactosyl sucralose by b-galactosidase from Lactobacillus bulgaricus L3", 《FOOD CHEMISTRY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110678479A (en) * | 2017-04-11 | 2020-01-10 | 科·汉森有限公司 | Lactase with improved performance |
| CN111518790A (en) * | 2018-11-27 | 2020-08-11 | 江南大学 | Sucrose hydrolase mutant and preparation method and application thereof |
| CN111518790B (en) * | 2018-11-27 | 2021-11-23 | 江南大学 | Sucrose hydrolase mutant and preparation method and application thereof |
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