CN104130990A - Beta-galactosidase and application thereof - Google Patents
Beta-galactosidase and application thereof Download PDFInfo
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- CN104130990A CN104130990A CN201410390773.5A CN201410390773A CN104130990A CN 104130990 A CN104130990 A CN 104130990A CN 201410390773 A CN201410390773 A CN 201410390773A CN 104130990 A CN104130990 A CN 104130990A
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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Abstract
The invention discloses beta-galactosidase and application thereof. The application method comprises the following steps: cloning beta-galactosidase genes bgl from bacillus coagulans into an expression vector pETDuet-1, transforming the beta-galactosidase genes into Escherichia coli BL21 (DE3), thus obtaining the beta-galactosidase with the expression quantity of 2031U/mg. The expressed beta-galactosidase has the optimal pH value of 6.0 and the optimal temperature of 60 DEG C. The recombinant beta-galactosidase can be used for lactose hydrolysis.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of beta-galactosidase enzymes and application thereof.
Background technology
Beta-galactosidase enzymes (EC3.2.1.238) formal name used at school is β-D-galactoside galactohydrolase, conventional Sumylact L by name, and this enzyme energy catalysis semi-lactosi glycosidic bond hydrolysis, hydrolyzes lactose generates glucose and semi-lactosi.Lactose is distinctive carbohydrate in Mammals milk, and in milk, most carbohydrates are lactose, but lactose only has after being hydrolyzed into glucose and semi-lactosi by beta-galactosidase enzymes and could be absorbed by body small intestinal cell.After the just birth of most of Mammalss, in body, betagalactosidase activity is higher, but afterwards with age activity decline gradually, final lactase deficiencies also causes lactose intolerance.Aisa people and African are the crowds of lactose intolerance that the most easily occurs in the world, and this class crowd takes in after lactose because lactose can not directly be entered large intestine by intestinal absorption, stimulates intestinal peristalsis to accelerate, and causes the symptoms such as borborygmus, diarrhoea.
Beta-galactosidase enzymes is widely used in Dairy Industry.In milk and diary product process, add safe beta-galactosidase enzymes, make its digestion for glucose and semi-lactosi, such milk is known as low lactose milk.Low lactose milk not only can meet the needs of normal consumer, the person that more can meet lactose intolerance, the edible milk-product of congenital beta-Galactosidase deficiency person, fully absorbs the needs of milk-product nutrition.At present, abroad realize already commercially producing of low lactose milk, be domesticly still in the junior stage." easyproization milk " that Inner Mongolia Yili Industry Group Co., Ltd releases is the domestic a low lactose milk that can effectively solve lactose intolerance or lactase defect problem, its core technology is lactose hydrolysis technology, be exactly by adding beta-galactosidase enzymes, more than 90% lactose in milk being hydrolyzed.
Beta-galactosidase enzymes wide material sources, commercial beta-galactosidase enzymes is mainly derived from microorganism at present, comprises intestinal bacteria (Escherichia coli), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), kluyveromyces (Kluyveromyces), saccharomyces fragilis (Saccharomyces fragilis) etc.Bacillus coagulans (Bacillus coagulans) is that its optimum growth temperature, 55 ℃ of left and right, has fast hydrolyzing lactose fermentation and produces sour characteristic through the milk-acid bacteria of a kind of " generally believing safety " of U.S. FDA approval.Also from the beta-galactosidase gene of Bacillus coagulans, be not cloned at present the report of expression, and the protein sequence difference of the protein sequence of this beta-galactosidase enzymes and other beta-galactosidase enzymess of having reported is very large.
Summary of the invention
Technical problem to be solved by this invention, is to provide a kind of new beta-galactosidase enzymes, for the acquisition of beta-galactosidase enzymes provides new enzyme source.
The technical problem that the present invention also will solve is to provide the application of above-mentioned beta-galactosidase enzymes.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A beta-galactosidase enzymes, its aminoacid sequence is as shown in SEQ ID No:2.
The encode gene order of above-mentioned beta-galactosidase enzymes, its nucleotide sequence is as shown in SEQ ID No:1.This gene source is in Bacillus coagulans NL01.
Beta-galactosidase gene of the present invention also comprises encoding to have the variant form with the SEQ ID No.1 sequence of beta-galactosidase enzymes identical function albumen from Bacillus coagulans NL01.These variant forms comprise: the disappearance of several Nucleotide, insertion/or replace, and at the several Nucleotide of 5 ' and 3 ' end interpolation.
A recombinant vectors, it is the carrier that has comprised nucleotide sequence shown in SEQ ID No:1.
Wherein, described carrier is pETDuet-1.
A recombinant bacterium, it is the bacterial strain that has comprised nucleotide sequence shown in SEQ ID No:1.
Wherein, described bacterial strain is intestinal bacteria.
The production method of above-mentioned beta-galactosidase enzymes recombinase is:
(1) beta-galactosidase gene from Bacillus coagulans NL01 is cloned into coli expression carrier pETDuet-1, transform e. coli bl21 (DE3), amicillin resistance screening obtains the recombinant bacterial strain that contains beta-galactosidase gene recombinant plasmid;
(2) above-mentioned recombinant bacterial strain is inoculated in to liquid nutrient medium, works as OD
600nmreach that after 0.4～0.8, to add final concentration be the IPTG abduction delivering of 0.1～1mmol/L, induction time is 3～12 hours, and inducing temperature is 16～37 ℃;
(3) utilize Ni affinity column to carry out the purifying of beta-galactosidase enzymes recombinase, obtain the pure enzyme of electrophoresis of beta-galactosidase enzymes recombinase.
The application of above-mentioned beta-galactosidase enzymes in lactose hydrolysis.Beta-galactosidase enzymes recombinase optimal pH of the present invention is 6.0, still has vigor over half in the scope of pH5.0～7.0, and in pH6.0～8.0, scope internal stability is better, and this pH scope is applicable to the soda acid scope of most of milk-product; Optimum temperuture is 60 ℃, and 50 ℃ better with stability inferior, has higher industrial value.
Beneficial effect: the present invention has cloned and recombinant expressed Bacillus coagulans beta-galactosidase enzymes first, and the enzyme work of recombinase, up to 2031U/mg, has higher pH stability and thermostability.The acquisition that the present invention is beta-galactosidase enzymes provides new source, and by high efficiency recombinant expressed for industrial production provides a large amount of beta-galactosidase enzymess, can be widely used in Dairy Industry.
Accompanying drawing explanation
Fig. 1 is optimal pH and the pH Detection of Stability result of beta-galactosidase enzymes.
Fig. 2 is optimum temperuture and the temperature stability detected result of beta-galactosidase enzymes.
Fig. 3 is the impact of metal ion on betagalactosidase activity.
Fig. 4 is the TLC detected result of beta-galactosidase enzymes hydrolyzes lactose.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1: the pcr amplification of Bacillus coagulans NL01 beta-galactosidase gene.
Adopt the genome of Promega company to extract test kit, extract the genomic dna of Bacillus coagulans NL01.
Design primer, introducing can be inserted EcoR I and the Sal I restriction enzyme site of pETDuet-1 plasmid (Novagen), and sequence is as follows:
gal.f：5’-GAATTCGATGTTAAAAAAACAAGAAAAATTTTATTATG-3’
gal.r：5’-GTCGACCTATTTTTCAATTACCTGCAAAATTTTCA-3’
The Bacillus coagulans NL01 genomic dna extracting of take is template, adopts above-mentioned primer to carry out pcr amplification.Pcr amplification is totally 100 μ L, containing ultrapure water 64 μ L, 5 * FastPfu PCR Buffer20 μ L, and dNTPs0.25mmol/L, each 0.2～0.4 μ mol/L of primer, template 50～200ng, the FastPfu DNA Polymerase5U of TransGen company.Pcr amplification condition is: 95 ℃ of denaturations 2 minutes, by 30 circulations of following parameter (72 ℃ are extended 1 minute for 95 ℃ of sex change 20 seconds, 60 ℃ of annealing 20 seconds), after loop ends, 72 ℃ are extended 5 minutes.1% agarose gel electrophoresis analytical results for PCR reaction product.PCR obtains after a large amount of goal gene, and the electrophoretic separation of condensing PCR product utilizes the DNA recovery test kit of Promega company from gel, to reclaim beta-galactosidase gene.
Clone and the sequencing of embodiment 2:PCR product.
The PCR product of embodiment 1 is reclaimed and is connected to the pEasy-Blunt cloning vector of TransGen company, transform intestinal bacteria Mach1-T1 competent cell, coating penbritin is dull and stereotyped, screens positive recombinant bacterial strain order-checking.Order-checking is completed by Beijing six directions Hua Da genome company, and sequencing result is carried out to sequential analysis, shows that PCR product is 1998bp, and 665 amino acid of encoding, as shown in SEQ ID No:1.
Embodiment 3: Bacillus coagulans NL01 beta-galactosidase gene is at colibacillary expression and purification
The correct recombinant bacterial strain that embodiment 2 is screened is inoculated in 5mL containing the LB liquid nutrient medium of 100 μ g/mL penbritins, and 37 ℃ of shaking tables are cultivated 8～12 hours, extract recombinant plasmid, with EcoR I and Sal I enzyme, cut 1～3 hour.Adopt same enzyme blanking method to process pETDuet-1 plasmid expression vector, enzyme is cut product and is carried out agarose gel electrophoresis separation and purification.Adopt 22 ℃ of connections of T4DNA ligase enzyme of NEB company to use CaCl after 1 hour
2method transforms e. coli bl21 (DE3) competent cell, and coating penbritin is dull and stereotyped, screens positive recombinant bacterial strain and extracts plasmid, the recombinant plasmid that EcoR I and Sal I double digestion evaluation and screening arrive.The bacterial strain called after E.coli BL21 (pETDuet-gal) that contains correct recombinant plasmid.Correct recombinant plasmid is checked order, the gene order recording is as shown in SEQ ID No.1 simultaneously.
E.coli BL21 (pETDuet-gal) recombinant bacterial strain is inoculated in to 50mL containing the LB liquid nutrient medium of 100 μ g/mL penbritins, 37 ℃ of shaking tables are cultivated 8～12 hours, transfer and in 1L, contain the LB liquid nutrient medium of 100 μ g/mL penbritins, 37 ℃ of shaking tables are cultivated, as nutrient solution OD
600nmreach that after 0.4～0.8, to add final concentration be the IPTG abduction delivering of 0.1～1mmol/L, induction time is 3～12 hours, and inducing temperature is 16～37 ℃.After inducing culture finishes, in 12,000 revs/min centrifugal 5 minutes, obtain cell precipitation, after physiological saline washed twice, be resuspended in the phosphate buffered saline buffer of 100mL pH7.4,50mmol/L.In mixture of ice and water, utilize ultrasonic disruption cell walls, the broken time is 20～40 minutes, the solution after broken wall with 16,000 revs/min centrifugal 30～60 minutes, gained supernatant liquor is crude enzyme liquid.
Because pETDuet-1 plasmid N end has 6His label, with HisTrap HP5mL Ni affinity column (GE Healthcare), can remove fast in crude enzyme liquid not with the foreign protein of 6His label, obtain the pure beta-galactosidase enzymes of electrophoresis.
Above-mentioned beta-galactosidase enzymes enzyme activity determination method:
Be totally 1mL, containing 700 μ L deionized waters, the potassium phosphate buffer of 100 μ L500mmol/L pH6.0, the oNPG of 100 μ L20mmol/L, blending incubation, after 5 minutes, adds the enzyme liquid of 100 μ L through suitably diluting, mix, 55 ℃ of temperature are bathed 10 minutes, add immediately the Na of 2mL0.5mmol/L
2cO
3termination reaction is the growing amount of oNP with the light absorption value at spectrophotometer measurement 410nm place.The unit of activity of enzyme is defined as: it is 1 unit of activity U that hydrolysis in 1 minute generates the enzyme amount of 1 μ mol oNP.
Embodiment 4: the basic enzymatic property of Bacillus coagulans NL01 restructuring beta-galactosidase enzymes
(1) pH and the temperature impact on enzymic activity
By the damping fluid configuration enzyme reaction system of different pH, the relative enzyme of measuring under different pH according to enzyme activity determination method described in embodiment 3 is lived.Result shows, the optimal pH of enzyme is 6.0 (Figure 1A).Enzyme is preserved in the damping fluid of above-mentioned different pH after 1 hour, measured relative enzyme and live in the damping fluid reaction system of pH6.0, result shows, enzyme is the most stable (Figure 1B) in the scope of pH6.5～8.0.By enzyme 4 ℃ of preservations in the potassium phosphate buffer of pH7.0, after 2 months, to measure enzyme and live, result shows enzyme basic not decline alive.
Above-mentioned different pH damping fluid is respectively: 50mmol/L acetic acid-sodium acetate buffer pH4.0～5.5,50mmol/L dipotassium hydrogen phosphate-potassium phosphate buffer pH6.0～8.0.
Dipotassium hydrogen phosphate-potassium phosphate buffer configuration enzyme reaction system with 50mmol/L, pH6.0, according to enzyme activity determination method described in embodiment 3, measuring relative enzyme under differing temps lives, result shows, enzyme optimal reactive temperature is 55～60 ℃ (Fig. 2 A).Enzyme is preserved under above-mentioned differing temps after 1 hour, measured relative enzyme and live under 60 ℃ of conditions, with the enzyme activity of preserving under 4 ℃ of conditions, be defined as 100%, result shows, enzyme is more stable below 50 ℃, and relative activity remains on more than 85% (Fig. 2 B).
Above-mentioned differing temps is respectively: 40 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃.
(2) impact of part metals ions enzyme activity
Take oNPG as substrate, and in enzyme is lived reaction system, adding respectively final concentration is each metal ion species of 2mmol/L, measures relative enzyme and lives.Result shows, Mn
2+to enzyme, work has promoter action, Mg
2+, Na
+, Co
2+, Ni
2+, K
+to enzyme, work has slight restraining effect, Ca
2+, Zn
2+, Cu
2+to enzyme, work has strongly inhibited effect.
Embodiment 5: Bacillus coagulans NL01 restructuring beta-galactosidase enzymes be take the hydrolysis reaction that lactose is substrate
With dipotassium hydrogen phosphate-potassium phosphate buffer of 50mmol/L, pH6.0, configure the lactose solution of 20g/L, add the pure enzyme of 5 μ L in 55 ℃ of reactions 1 hour, 100 ℃ are boiled 5 minutes inactivator liquid, then carry out thin-layer chromatography (TLC) analysis.TLC thin plate (Silica gel60, Merk) after point sample, at developing agent (propyl carbinol: ethanol: water=5:3:2, v/v/v) exhibition layer in, after taking-up thin plate dries, spray painting developer (in 20% sulphuric acid soln containing 0.5% 3,5-orcin), 120 ℃ of bakings make the colour developing of sugared spot for 10 minutes.As shown in Figure 4, lactose decomposition product has corresponding glucose spot and semi-lactosi spot, is that beta-galactosidase enzymes hydrolyzes lactose generates.
Claims (8)
1. a beta-galactosidase enzymes, is characterized in that, its aminoacid sequence is as shown in SEQ ID No:2.
2. a gene order for coding beta-galactosidase enzymes claimed in claim 1, is characterized in that, its nucleotide sequence is as shown in SEQ ID No:1.
3. a recombinant vectors, is characterized in that, it is the carrier that has comprised nucleotide sequence shown in SEQ ID No:1.
4. recombinant vectors according to claim 3, is characterized in that, described carrier is pETDuet-1.
5. a recombinant bacterium, is characterized in that, it is the bacterial strain that has comprised nucleotide sequence shown in SEQ ID No:1.
6. recombinant bacterium according to claim 5, is characterized in that, described bacterial strain is intestinal bacteria.
7. the application of beta-galactosidase enzymes claimed in claim 1 in lactose hydrolysis.
8. application according to claim 7, is characterized in that, beta-galactosidase enzymes optimal pH is 6.0, still has vigor over half in the scope of pH5.0～7.0, and in pH6.0～8.0, scope internal stability is better; Optimum temperuture is 60 ℃, and 50 ℃ better with stability inferior.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182307A (en) * | 2018-10-11 | 2019-01-11 | 山东隆科特酶制剂有限公司 | A kind of method that bacillus coagulans liquid fermentation produces beta galactosidase |
| CN109207402A (en) * | 2018-10-11 | 2019-01-15 | 山东隆科特酶制剂有限公司 | One bacillus coagulans and its liquid fermentation enzyme producing method |
| CN109295038A (en) * | 2018-10-25 | 2019-02-01 | 安徽大学 | A kind of beta galactosidase, its encoding gene and its application |
| CN113493777A (en) * | 2021-06-25 | 2021-10-12 | 青岛大学 | Beta-galactosidase and application thereof in lactose degradation |
| WO2021212723A1 (en) * | 2020-04-22 | 2021-10-28 | 青岛大学 | Beta-galactosidase gala and use thereof |
-
2014
- 2014-08-08 CN CN201410390773.5A patent/CN104130990A/en active Pending
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109182307A (en) * | 2018-10-11 | 2019-01-11 | 山东隆科特酶制剂有限公司 | A kind of method that bacillus coagulans liquid fermentation produces beta galactosidase |
| CN109207402A (en) * | 2018-10-11 | 2019-01-15 | 山东隆科特酶制剂有限公司 | One bacillus coagulans and its liquid fermentation enzyme producing method |
| CN109182307B (en) * | 2018-10-11 | 2021-07-23 | 山东隆科特酶制剂有限公司 | Method for producing beta-galactosidase by liquid fermentation of bacillus coagulans |
| CN109207402B (en) * | 2018-10-11 | 2021-07-23 | 山东隆科特酶制剂有限公司 | Bacillus coagulans and liquid fermentation enzyme production method thereof |
| CN109295038A (en) * | 2018-10-25 | 2019-02-01 | 安徽大学 | A kind of beta galactosidase, its encoding gene and its application |
| CN109295038B (en) * | 2018-10-25 | 2021-09-17 | 安徽大学 | Beta-galactosidase, coding gene and application thereof |
| WO2021212723A1 (en) * | 2020-04-22 | 2021-10-28 | 青岛大学 | Beta-galactosidase gala and use thereof |
| CN113493777A (en) * | 2021-06-25 | 2021-10-12 | 青岛大学 | Beta-galactosidase and application thereof in lactose degradation |
| CN113493777B (en) * | 2021-06-25 | 2023-11-07 | 青岛大学 | Beta-galactosidase and application thereof in lactose degradation |
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Application publication date: 20141105 |