CN101709293A - Method for separating and purifying dextran dextrinase from gluconobater oxydans - Google Patents
Method for separating and purifying dextran dextrinase from gluconobater oxydans Download PDFInfo
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- CN101709293A CN101709293A CN200910242774A CN200910242774A CN101709293A CN 101709293 A CN101709293 A CN 101709293A CN 200910242774 A CN200910242774 A CN 200910242774A CN 200910242774 A CN200910242774 A CN 200910242774A CN 101709293 A CN101709293 A CN 101709293A
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- dextrinase
- dextran
- ammonium sulfate
- sulfate precipitation
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Abstract
The invention relates to a method for separating and purifying dextran dextrinase from gluconobater oxydans, comprising the following steps of: firstly, collecting thalli by centrifugation, suspending by using phosphate buffer, crushing cells by using ultrasonic waves, , collecting a supernatant by centrifugation; and finally carrying out protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography and gel chromatography to obtain the high-purity dextran dextrinase by separation. The method has the advantages of simplicity, good repeatability and high protein yield.
Description
Technical field
The invention belongs to the protein purification field, particularly, the present invention relates to a kind of from bacillus of oxidizing glucose the method for separation and purification dextran dextrinase.
Background technology
Dextran (dextran), have another name called dextran, it is a kind of high molecular polymer that forms by the glucose unit dehydration, as a kind of extracellular products that generates by the enzyme catalysis of secretion property, it be find the earliest in the world and early as the microbial polysaccharide of main plasma substitute, its chemical structure mainly is by the α of glucose-1, the linear long molecular chain that 6-key head and the tail dehydrating condensation forms, the α-1 that in molecular structure, contains different ratios, 2, α-1,3 and α-1, the side chain of 4-glycosidic link, molecular formula is (C
6H
10O
5) n.
Dextran has been widely used in a plurality of fields such as medicine, food, stratographic analysis because of multiple advantages such as it is safe, nontoxic, good biocompatibilities.The dextran colloidal solution has expanding blood volume, keeps the effect of blood pressure, and first aid usefulness can be used as plasma substitute when confessing blood and traumatic shock.China's pharmacopeia 1990 editions is listed dextran and compound preparation thereof in.That uses clinically often has three kinds of specifications: macrodex, Dextran 40, Dextran 20.In foodstuffs industry, dextran as low calorie foodstuff additive, has been used for the production of multiple drink and food because of its retentiveness, toughness.Also but instead of part maltose is made the weighting material of soft heart chocolate or is added in the brewer's malt and go, and improves the foaminess of goods.In petroleum industry, dextran can be used as oil well sludge additive.It also can be applicable to fine chemistry industry, makes sephadex, has been widely used in the molecular sieve chromatography technology at present.In addition, in food, animal-feed and cosmetics production, received suitable concern as potential benefit source of students material.
Discover that certain endonuclease capable that derives from the bacillus of oxidizing glucose (Gluconobacter oxydans) utilizes maltodextrin and the synthetic dextran of starch partial hydrolysate, this kind of enzyme be named as dextran dextrinase (dextran dextrinase, DDase).But the α-1 on this enzyme catalysis donor non reducing end, the 4-glucopyranosyl is transferred on the non reducing end of acceptor, forms α-1, the 6-glycosidic link.Wherein main reaction pattern is α on the donor-1, the fracture of 4-glycosidic link, and glucose unit is transferred to and forms α-1 on the acceptor, the 6-glycosidic link.The bacillus of oxidizing glucose dextran dextrinase can utilize the starch derivative dextrin to synthesize dextran, this dextran can also be as beneficial source of students materials such as food fibre, low fat food, low calorific food weighting agent and swelling agents because of its particular structure characteristic (side chain is many).
In recent years, the research of relevant bacillus of oxidizing glucose dextran and dextran dextrinase (DDase) rarely has report.In addition, up to the present, the domestic report of not relevant bacillus of oxidizing glucose dextran dextrinase separating and purifying technology method as yet.
Summary of the invention
The object of the invention provide a kind of from bacillus of oxidizing glucose the method for separation and purification dextran dextrinase.
For achieving the above object, provided by the invention from bacillus of oxidizing glucose the method for separation and purification dextran dextrinase, key step is as follows:
1), and, adopts the supersonic method smudge cells, clear enzyme solution in centrifugal collection under the 0-4 ℃ of condition with the phosphoric acid buffer suspension with the fermented liquid centrifugal collecting cell;
2) go up the clear enzyme solution part obtains containing dextran dextrinase through protamine sulfate precipitation and ammonium sulfate precipitation crude enzyme liquid;
3) crude enzyme liquid obtains the thick component of dextran dextrinase through the hydrophobic chromatography purifying;
4) the thick component of dextran dextrinase obtains refined dextrose acid anhydride dextrinase stoste through the gel permeation chromatography purifying.
In the described method, the protamine sulfate in the protamine sulfate precipitation process and the mass ratio of total protein are 0.1～10%.
In the described method, in the ammonium sulfate precipitation process final concentration of ammonium sulfate by weight volume ratio be 20～80%.
In the described method, in the step 3 in the initial damping fluid of hydrophobic chromatography purge process the concentration of ammonium sulfate be 0.5～3M, elution process adopts the stepwise elution method.
In the described method, be to adopt Phenyl hydrophobic chromatography column separating purification.
In the described method, the elution flow rate of gel permeation chromatography purifying is 0.3mL/min～1mL/min in the step 4.
In the described method, be to adopt Superdex 200PG gel chromatography column chromatography purification.
The remarkable advantage that the present invention compares with prior art is: this method is easy, good reproducibility and proteic yield height.
Embodiment
Process of the present invention is substantially:
1) fermented liquid centrifugal collecting cell, the usefulness phosphoric acid buffer is resuspended, adopts the supersonic method smudge cells, clear enzyme solution in the 0-4 ℃ of following centrifugal collection.
2) upwards clear enzyme solution partly adds protamine sulfate, and the mass ratio of this protamine sulfate and total protein is 0.1～10%, centrifugally removes the nucleic acid material that smudge cells produces, and reduces the viscosity of solution simultaneously; Upwards add ammonium sulfate in the clear enzyme solution again, the final concentration of this ammonium sulfate is 20～80% (w/v), and centrifugation foreign protein and acquisition contain the crude enzyme liquid of dextran dextrinase.
3) crude enzyme liquid is splined on Phenyl hydrophobic chromatography post, contains the ammonium sulfate of 0.5～3M in the initial damping fluid, and the wash-out stage is adopted the stepwise elution method, and purifying obtains the thick component of dextran dextrinase.
4) the thick component of dextran dextrinase is splined on Superdex 200PG gel chromatography chromatography column, and the setting elution flow rate is 0.3mL/min-1mL/min, and purifying obtains refined dextrose acid anhydride dextrinase.
Others are because the disclosure of this paper is conspicuous to those skilled in the art.
Below be further described by a specific embodiment.
Embodiment 1
The acquisition of crude enzyme liquid in the step 1) born of the same parents
Get the nutrient solution of 100mL, 4 ℃ of frozen centrifugation collecting cells are resuspended in the phosphoric acid buffer (pH 6.47) of 50mM again.Adopt the supersonic method smudge cells, and under 4 ℃ of conditions the centrifugal 20min of 10000r/min, clear enzyme solution in the collection the results are shown in Table 1.
Step 2) protamine sulfate precipitation
Add a certain amount of protamine sulfate in the born of the same parents in step 1 in the crude enzyme liquid, make its addition reach 1% of total protein concentration, low temperature slowly stirs 1 hour (h), and the high speed frozen centrifugation is removed nucleic acid material, the results are shown in Table 1.
The step 3) ammonium sulfate precipitation
Add the ammonium sulfate of 40% (mass volume ratio) in the crude enzyme liquid in step 2, low temperature slowly stirs 1 hour (h), and the high speed frozen centrifugation is removed foreign protein, and supernatant is the crude enzyme liquid that contains dextran dextrinase, the results are shown in Table 1.
Step 4) hydrophobic chromatography separation purifying technique
The crude enzyme liquid of getting in the 5mL step 3 is splined on Phenyl hydrophobic chromatography post, carries out purifying with the quick protein purification workstation of AKTA (known technology).Initial damping fluid is 50mM phosphoric acid buffer (pH 6.47, include the ammonium sulfate of 2M), adopts the straight line gradient elution sample of ammonium sulfate 2M～0, receives required component according to the light absorption value of 280nm.Each active ingredient merged and concentrate obtain the thick component of dextran dextrinase, the results are shown in Table 1.
Step 5) gel chromatography technology
The thick component of getting in the 2mL step 4 is splined on Superdex 200PG gel chromatographic columns, carries out purifying with the quick protein purification workstation of AKTA.Elution buffer is 50mM phosphoric acid buffer (pH 6.47, include the NaCl of 0.1M), and flow velocity is set at 0.5mL/min, receives required component according to the light absorption value of 280nm, the results are shown in Table 1.
Table 1: the purifying of bacillus of oxidizing glucose dextran dextrinase
Claims (7)
1. the method for a separation and purification dextran dextrinase from bacillus of oxidizing glucose, key step is as follows:
1), and, adopts the supersonic method smudge cells, clear enzyme solution in centrifugal collection under the 0-4 ℃ of condition with the phosphoric acid buffer suspension with the fermented liquid centrifugal collecting cell;
2) go up the clear enzyme solution part obtains containing dextran dextrinase through protamine sulfate precipitation and ammonium sulfate precipitation crude enzyme liquid;
3) crude enzyme liquid obtains the thick component of dextran dextrinase through the hydrophobic chromatography purifying;
4) the thick component of dextran dextrinase obtains refined dextrose acid anhydride dextrinase stoste through the gel permeation chromatography purifying.
2. the method for claim 1, wherein the protamine sulfate in the protamine sulfate precipitation process and the mass ratio of total protein are 0.1～10%.
The method of claim 1, wherein in the ammonium sulfate precipitation process final concentration of ammonium sulfate by weight volume ratio count 20～80%.
The method of claim 1, wherein in the step 3 in the initial damping fluid of hydrophobic chromatography purge process the concentration of ammonium sulfate be 0.5～3M, elution process adopts the stepwise elution method.
5. as claim 1 or 4 described methods, wherein, be to adopt Phenyl hydrophobic chromatography column separating purification.
The method of claim 1, wherein in the step 4 elution flow rate of gel permeation chromatography purifying be 0.3mL/min～1mL/min.
7. as claim 1 or 6 described methods, wherein, be to adopt Superdex 200PG gel chromatography column chromatography purification.
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| CN200910242774A CN101709293A (en) | 2009-12-16 | 2009-12-16 | Method for separating and purifying dextran dextrinase from gluconobater oxydans |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321597A (en) * | 2011-10-09 | 2012-01-18 | 中国海洋大学 | Dextran dextrinase for preparing dextran |
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2009
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321597A (en) * | 2011-10-09 | 2012-01-18 | 中国海洋大学 | Dextran dextrinase for preparing dextran |
| CN102321597B (en) * | 2011-10-09 | 2013-10-02 | 中国海洋大学 | Dextran dextrinase for preparing dextran |
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Open date: 20100519 |