CN103275941A - Method for preparing pyruvic oxidase - Google Patents
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- CN103275941A CN103275941A CN201310236929XA CN201310236929A CN103275941A CN 103275941 A CN103275941 A CN 103275941A CN 201310236929X A CN201310236929X A CN 201310236929XA CN 201310236929 A CN201310236929 A CN 201310236929A CN 103275941 A CN103275941 A CN 103275941A
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Abstract
The invention relates to a method for preparing pyruvic oxidase. The method comprises the steps as follows: (1), cultivation of microbial cells containing the pyruvic oxidase, centrifugal collection of bacterial cells, and cell disruption; (2), preparation of a polyethylene glycol/inorganic salt two-aqueous phase extraction system solution; (3), three times of extraction separation by the aid of a two-aqueous phase extraction system, and obtaining of an inorganic salt solution of the pyruvic oxidase; and (4), flirtation and concentration of an ultrafiltration membrane, freezing and drying of a trapped fluid, and preparation of a pyruvate oxidase product. The method can prepare the pyruvic oxidase continuously in a large scale, the preparation technology of the oxidase is simple and convenient, the production period is short, the operation condition is mild, the recovery rate of the oxidase activity is high, and the production cost of the pyruvic oxidase is lower.
Description
Technical field
The present invention relates to a kind of method for preparing pyruvic oxidase, particularly utilize aqueous two phase extraction technique to prepare the method for pyruvic oxidase, belong to technical field of bioengineering.
Background technology
Pyruvic oxidase (Pyruvate oxidase, EC1.2.3.3) be a kind of broad-spectrum clinical diagnosis zymin, be mainly used in the vitality test of alanine aminotransferase in the blood (ALT) and aspartic transaminase (AST), in addition, the mensuration that also is used for compounds such as pyruvate kinase determination of activity and pyruvic acid, inorganic phosphate, urea.
Microbe fermentation method is adopted in the preparation of pyruvic oxidase, namely have the microorganism cells of high reactivity pyruvic oxidase such as plant lactobacillus (Lactobacillus plantarum), aerococcus viridans acquisitions such as (Aerococcus viridans) by cultivation, its preparation process comprises the cultivation of microorganism cells and the separation and purification of enzyme.In the production cost of pyruvic oxidase constituted, the cost of lower procedures such as separation and purification occupied sizable ratio, and therefore, how adopting efficiently, isolation technique is the technical issues that need to address during pyruvic oxidase is produced.
At present, the separation purification method of pyruvic oxidase mainly adopts salting-out process, gel chromatography, ion exchange method etc., as Tao Jiaquan (East China University of Science's Master's thesis, 2007) when carrying out pyruvic oxidase research, it is that 1.21U/mg, the enzyme rate of recovery are 23.1%, the purifying multiple is 7.1 that pyruvic oxidase in the recombination bacillus coli is adopted the purification process of Ni-IDA affinity chromatography, the specific activity of enzyme of the pyruvic oxidase that obtains; The method of three step of ammonium sulfate precipitation, DEAE-Sepharose ion-exchange, UltrogelAcA34 gel-filtration purifying is also adopted in this research, and the specific activity of enzyme of the pyruvic oxidase that obtains is that 1.1U/mg, the enzyme rate of recovery are 29.8%, the purifying multiple is 10.Chinese patent literature CN101659943A(application number 200910092897.4) adopt nickel post affinity chromatography, ammonium sulfate precipitation, sephadex G-25 chromatography column desalting method to obtain pyruvic oxidase.Problems such as aforesaid method exists complex process, production cycle to grow, enzymatic activity recovery is low, can not produce continuously, thus caused the production cost of pyruvic oxidase higher.Therefore, improve existing production technology and to develop new production technology imperative with the production cost that reduces pyruvic oxidase.
Aqueous two phase extraction technique is the bioseparation technology that development in recent years is got up, and is the effective ways of separation and purification biologically active substance.After aqueous two-phase system referred to two kinds of different types of aqueous solutions of polymers mixing or a kind of aqueous solutions of polymers and a kind of salt solution mix, when both concentration reached certain value, mixed solution left standstill two liquid phase systems that the back layering produces.The principle of aqueous two-phase system extraction is based on the selectivity of biological substance in system and distributes.Aqueous two-phase extraction has the operational condition gentleness, treatment capacity is big, separating step is few, energy consumption is low, and organic solvent-free is residual, is easy to advantages such as industrialization operation.Aqueous two-phase extraction has been widely used in the aspects such as separation and purification that technical field of bioengineering carries out biomacromolecules such as bio-transformation, protein (enzyme) and nucleic acid at present.
Double-aqueous phase system commonly used in biochemical separation engineering is polymer/polymer system and polymer/inorganic salt system, and the polymer/polymer system is as polyoxyethylene glycol (PEG)/dextran (Dex) system.Because the viscosity of the following middle dextran mutually that PEG/ dextran system forms is very high, produces spawn through regular meeting, make the process control of solution produce difficulty, limited the application of PEG/ dextran system.
Except polymer/polymer system and polymer/inorganic salt system, also have some organic solvents (as alcohols) and inorganic salt also can form double-aqueous phase system, this system also can be used for the separation of protein (enzyme), as Chinese patent literature CN101693735A(application number 200910309154.8), the method of a kind of aqueous two-phase extraction isolated protein and enzyme is disclosed, its characteristics are directly to join polyvalent alcohol and inorganic salt in protein or the enzyme solution in proportion, stir, leave standstill under the room temperature, form two-phase, protein or enzyme mainly are assigned to phase, and keep higher activity, thereby reach effective separation and Extraction purpose.But because the molecular weight of polyvalent alcohol is little a lot of than the molecular weight of polymkeric substance (as PEG), polyvalent alcohol and inorganic salt are when forming double-aqueous phase system, both need to have at least a kind of higher concentration (with respect to polyoxyethylene glycol/inorganic salt system) that has in system, polyvalent alcohol is as a kind of organic solvent, when its concentration is higher, enzyme vigor in its solution can descend, and along with the increasing of its concentration, enzymic activity descends also to be increased; When inorganic salt concentration was higher, zymoprotein can produce precipitation because of salting out, thereby caused the decline of enzymatic activity recovery.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide that a kind of technology is simple, enzymatic activity recovery is high, production cost is low, be convenient to the preparation method of the pyruvic oxidase of large-scale production.
Method of the present invention is to utilize polyoxyethylene glycol/inorganic salt aqueous two phase extraction technique to prepare pyruvic oxidase, reduces production costs thereby reach, and obtains the purpose of better economical effectiveness.
For achieving the above object, the concrete technical scheme taked of the present invention is as follows:
A kind of method of utilizing two phase aqueous extraction system to prepare pyruvic oxidase may further comprise the steps:
(1) microorganism cells that will contain pyruvic oxidase is cultivated, and gets cell culture fluid, and is centrifugal, makes the wet thallus cell, and somatic cells is resuspended in the deionized water, makes cell suspending liquid, and smudge cells obtains cytoclasis liquid;
(2) preparation inorganic salt solution, in this inorganic salt solution, add polyoxyethylene glycol, the mass percent concentration of inorganic salt is that the mass percent concentration of 10～20wt%, polyoxyethylene glycol is 20～30wt% in total system, mixes, and makes polyoxyethylene glycol/inorganic salt double-aqueous phase system solution;
(3) add polyoxyethylene glycol/inorganic salt double-aqueous phase system solution that step (2) makes in the cytoclasis liquid that makes to step (1), the volume ratio of double-aqueous phase system solution and cytoclasis liquid is 1:(1～2), mix, leave standstill phase-splitting, make under the inorganic salt on the phase solution and the polyoxyethylene glycol that contains pyruvic oxidase solution mutually;
(4) add inorganic salt in the phase solution on the polyoxyethylene glycol that makes to step (3), making the mass concentration of inorganic salt in total system is 6～10wt%, mix, leave standstill phase-splitting, make on the polyoxyethylene glycol of phase solution and the secondary separation that contains pyruvic oxidase under the inorganic salt of secondary separation solution mutually;
(5) add inorganic salt in the phase solution on the polyoxyethylene glycol that makes to step (4), making the mass concentration of inorganic salt in total system is 5～6wt%, the pH value of regulator solution is 5.5～6.0, mix, leave standstill phase-splitting, make under the inorganic salt that contain pyruvic oxidase on the phase solution and polyoxyethylene glycol solution mutually;
(6) phase solution under the inorganic salt that contain pyruvic oxidase that step (5) is made adopts ultrafiltration membrance filter to concentrate, and collects trapped fluid, and lyophilize makes pyruvic oxidase.
Preferred according to the present invention, the centrifugal condition in the described step (1) is: the centrifugal 10～20min of 3000～5000r/min.
Preferred according to the present invention, the cell mass percent concentration in the cell suspending liquid in the described step (1) is 25～35%.
Preferred according to the present invention, the smudge cells in the described step (1) adopts the high pressure cell crusher, and working pressure is 50～60MPa, and service temperature is 5～15 ℃.
Preferred according to the present invention, the inorganic salt in described step (2), (3), (4), (5), (6) are selected from one of ammonium sulfate, sodium sulfate, Sodium phosphate dibasic, potassiumphosphate or Seignette salt;
Preferred according to the present invention, the molecular weight polyethylene glycol in described step (2), (3), (4), (5) is one of 1000,1500 or 2000;
Preferred according to the present invention, the phase-splitting time of leaving standstill in described step (3), (4), (5) is 10～60min.
Preferred according to the present invention, the ultra-filtration membrane operating pressure in the described step (6) is 0.1～0.3MPa, and the molecular weight cut-off of film is 40000～50000Dal, and service temperature is 25 ℃.
Preferred according to the present invention, described step (5) also comprises phase solution on the polyoxyethylene glycol after the ultrafiltration membrance filter of recovery usefulness concentrates, participates in the step of polyoxyethylene glycol in the preparation steps (2)/inorganic salt double-aqueous phase system solution.
Further preferred according to the present invention, the molecular weight cut-off of the ultra-filtration membrane of described recovery usefulness is 2500Dal.
Beneficial effect
1, preparation method of the present invention can realize serialization, the mass-producing of pyruvic oxidase preparation, is convenient to its suitability for industrialized production.
2, the present invention is according to the characteristics of pyruvic oxidase, adopt polyoxyethylene glycol/inorganic salt aqueous two phase extraction technique to prepare pyruvic oxidase, density difference is little between the two-phase that this extraction system forms, viscosity is low, more be applicable to the gravity settling phase-splitting, and operational condition gentleness, enzymatic activity recovery height, energy consumption low, be easy to automatic control.
3, preparation method's technology of the present invention is easy, with short production cycle, is easy to engineering and amplifies, and polyoxyethylene glycol can be recycled, and has greatly reduced the production cost of pyruvic oxidase.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is specifically described or is described further, purpose is that methods of this invention will be better understood, but protection scope of the present invention is not limited to following embodiment.
The vitality test step of pyruvic oxidase is as follows among the present invention:
(1) measuring principle
Pyruvic oxidase makes the generation hydrogen peroxide that reacts of the endogenous pyruvic acid in the sample, and hydrogen peroxide generates water and oxygen under catalatic effect, to consume the endogenous pyruvic acid in the sample, the following reaction of its catalysis:
By above reaction formula as can be known, can determine that the enzyme of pyruvic oxidase is alive by the absorbance of measuring quinonimine dye at 550nm wavelength place.
(2) measure reagent
(A) potassium primary phosphate-citrate buffer solution of reaction mixture 1:1mol/L (pH5.8) 2.0mL, the flavin adenine dinucleotide 0.1mL of 2mmol/L, the thiaminpyrophosphate ester 0.2mL of 3mmol/L, the 4-aminoantipyrene solution 1.0mL of 15mmol/L, the superoxide enzyme solution 1.0mL of 50U/mL, distilled water 1.7mL.
(B) the 2,4 dichloro phenol 2.0mL of reaction mixture 2:0.2%, the Adlerika 2.0mL of 0.15mol/L.
(C) the Potassium pyruvate solution of substrate solution: 1.0mol/L.
(D) reaction terminating liquid: the EDTA disodium salt of 3.72mg is dissolved in the water of 100mL.
(E) potassium primary phosphate-citrate buffer solution of enzyme diluent: 50mmol/L (pH5.8).
(3) measurement operation process
(A) get the reaction mixture 1 of 0.6mL, the reaction mixture 2 of 0.3mL and the substrate solution of 0.1mL, fully mix, be heated to 37 ℃ then.
(B) be 0.1～0.2U/mL with the enzyme diluent with pyruvic oxidase diluted sample to enzyme activity unit, the sample diluting liquid of getting 0.02mL then joins in the mixed solution of step (A) formation, and mixing is at 37 ℃ of following constant temperature 10min.
(C) add reaction terminating liquid 2.0mL, at 37 ℃ of following balance 5min.
(D) get the above-mentioned enzyme solution of 0.1mL, in the spectrophotometer (Tianjin, island UV-2450 spectrophotometer) of 37 ℃ of constant temperature, under 550nm, measure absorbance (A
1).
(E) replace the pyruvic oxidase sample by the above-mentioned steps operation with the enzyme diluent, measure absorbance (A
2).
(F) calculate Δ A value: Δ A=A
1-A
2
(4) enzyme activity unit definition: under these conditions, the enzyme amount of 1 minute internal consumption 1 μ mol pyruvic acid is 1U.
(5) formula is calculated in enzyme work:
Enzyme activity (U/mg)=(0.82 * Δ A * D
f) ÷ C
Wherein: 10 is the reaction times; Vt is cumulative volume 3.02mL; Vs is sample volume 0.02mL; D
fBe pyruvic oxidase dilution of sample multiple; 1/2 is 2mol H in the reaction
2O
2Produce the quinonimine dye material of 1mol; 36.88 be the molar absorptivity (cm of quinonimine dye under condition determination
2/ μ mol); C is the concentration (mg/mL) of enzyme in the solution.
Can calculate the rate of recovery of enzyme activity according to the enzyme activity determination method of above-mentioned pyruvic oxidase; Under cytoclasis liquid and the inorganic salt mutually the Protein content in the solution adopt Xylene Brilliant Cyanine G G-250 staining to measure; The purifying multiple equals the ratio of the ratio enzyme activity in the phase solution and the ratio enzyme activity of cytoclasis liquid under the inorganic salt.
Raw material sources:
Polyoxyethylene glycol is available from Dow Chemical company, and the pyruvic oxidase sample is available from the graceful bio tech ltd of last Hypon, and pyruvic acid is composed the bio tech ltd that shakes available from Shanghai, and peroxidase is good and bio tech ltd available from Shanghai.
Embodiment 1
The used bacterial classification of present embodiment is plant lactobacillus (Lactobacillus plantarum) ATCC8014, and this bacterial classification is available from U.S. representative microbial DSMZ (ATCC).
Get bacterial classification plant lactobacillus (Lactobacillus plantarum) ATCC8014 that produces pyruvic oxidase and be inoculated in the triangular flask that the 20mL seed culture medium is housed, 30 ℃ leave standstill cultivate 24h after, make liquid seeds; Then by inoculum size 10%(V/V) be inoculated in the fermention medium of 200mL, 37 ℃, 250r/min shaking table cultivation 18h make the somatic cells nutrient solution that contains pyruvic oxidase.Produce enzyme activity after testing and can reach 1562U/L.
Seed culture medium (g/L): peptone 10, extractum carnis 10, yeast extract paste 5, glucose 20, sodium acetate 5, dibasic ammonium citrate 2, tween-80 1, K
2HPO
42, MgSO
41, MnSO
40.5, pH value 6.5.
Fermention medium (g/L): peptone 10, extractum carnis 10, yeast extract paste 5, glucose 20, sodium acetate 3, dibasic ammonium citrate 2, tween-80 1, K
2HPO
42, MgSO
40.5, MnSO
40.2, pH value 6.5.
A kind of method of utilizing two phase aqueous extraction system to prepare pyruvic oxidase may further comprise the steps:
(1) will contain plant lactobacillus cell culture fluid 1000mL centrifugal 20min under the 3000r/min condition of pyruvic oxidase, make the wet thallus cell, somatic cells is resuspended in the deionized water, make the suspension that the cell mass concentration is 35wt%, be cooled to about 10 ℃, the APV-2000-1 type high pressure cell crusher that adopts German APV company to produce carries out three cytoclasis, and the crusher working pressure is 60MPa, makes the cytoclasis solution 198mL that contains pyruvic oxidase.
(2) preparation ammonium sulfate solution 200mL, add molecular weight in this solution and be 1000 polyoxyethylene glycol (PEG1000), making the mass percent concentration of ammonium sulfate in total system is 15wt%, the mass percent concentration of polyoxyethylene glycol in total system is 25wt%, mix, make polyoxyethylene glycol/ammonium sulfate double-aqueous phase system solution.
(3) add polyoxyethylene glycol/ammonium sulfate double-aqueous phase system solution that step (2) makes by equal-volume in the cytoclasis liquid that makes to step (1), after stirring the 10min mixing, leave standstill the 50min phase-splitting, make under the ammonium sulfate on the phase solution 118mL and polyoxyethylene glycol solution 278mL mutually.
The materials such as foreign protein, polysaccharide and nucleic acid that contain pyruvic oxidase and small part on the polyoxyethylene glycol in the phase solution contain whole cell debriss and materials such as most foreign protein, polysaccharide and nucleic acid in the phase solution under the ammonium sulfate.Through aqueous two-phase extraction separation for the first time, the enzymatic activity recovery of pyruvic oxidase reaches 98.3% after testing.
(4) add ammonium sulfate in the phase solution on the polyoxyethylene glycol that makes to step (3), making the mass concentration of ammonium sulfate in solution is 10wt%, after stirring the 10min mixing, leave standstill the 40min phase-splitting, make on the polyoxyethylene glycol of phase solution 122mL and the secondary separation that contains pyruvic oxidase under the ammonium sulfate of secondary separation solution 156mL mutually.
Contain the foreign protein that contains pyruvic oxidase and trace on the polyoxyethylene glycol of secondary separation of pyruvic oxidase in the phase solution, contain materials such as foreign protein, polysaccharide and nucleic acid under the ammonium sulfate of secondary separation in the phase solution.Through aqueous two-phase extraction separation for the second time, the enzymatic activity recovery of pyruvic oxidase reaches 96.7% after testing.
(5) add ammonium sulfate in the phase solution on the polyoxyethylene glycol of the secondary separation that contains pyruvic oxidase that makes to step (4), making the mass concentration of ammonium sulfate in solution is 6wt%, transferring the pH value of solution with hydrochloric acid is 5.7, after stirring the 10min mixing, leave standstill the 30min phase-splitting, make under the ammonium sulfate that contains pyruvic oxidase on the phase solution 55mL and polyoxyethylene glycol solution 101mL mutually.
Contain polyoxyethylene glycol and foreign protein on the polyoxyethylene glycol in the phase solution, contain under the ammonium sulfate of pyruvic oxidase and contain pyruvic oxidase and ammonium sulfate in the phase solution.It is the ultrafiltration membrance filter of 2500Dal that last phase solution is adopted molecular weight cut-off, except foreigh protein removing, collects the polyoxyethylene glycol filtered liquid, and polyoxyethylene glycol can be recycled.Through aqueous two-phase extraction separation for the third time, the enzymatic activity recovery of pyruvic oxidase reaches 94.4% after testing.
(6) phase solution under the ammonium sulfate that step (5) is made, adopting molecular weight cut-off under 0.15MPa pressure is that the ultrafiltration membrance filter of 40000Dal concentrates, and removes ammonium sulfate and residual polyoxyethylene glycol, collects trapped fluid, the trapped fluid lyophilize makes pyruvic oxidase 428mg.
The enzymatic activity recovery of pyruvic oxidase reaches 93.2% after testing, and the purifying multiple reaches about 7.5.
Embodiment 2
As the embodiment 1 described method of utilizing two phase aqueous extraction system to prepare pyruvic oxidase, difference is:
In the step (2), preparation aqueous sodium persulfate solution 200mL, add molecular weight in this solution and be 1500 polyoxyethylene glycol (PEG1500), making the mass percent concentration of sodium sulfate in total system is 16wt%, the mass percent concentration of polyoxyethylene glycol in total system is 26wt%, mix, make polyoxyethylene glycol/sodium sulfate double-aqueous phase system solution.
The enzymatic activity recovery of pyruvic oxidase reaches 92.1% after testing, and the purifying multiple reaches about 7.3.
Embodiment 3
As the embodiment 1 described method of utilizing two phase aqueous extraction system to prepare pyruvic oxidase, difference is:
In the step (2), preparation soluble tartrate sodium water solution 200mL, add molecular weight in this solution and be 1000 polyoxyethylene glycol (PEG1000), making the mass percent concentration of Seignette salt in total system is 20wt%, the mass percent concentration of polyoxyethylene glycol in total system is 20wt%, mix, make polyoxyethylene glycol/Seignette salt double-aqueous phase system solution.
The enzymatic activity recovery of pyruvic oxidase reaches 90.6% after testing, and the purifying multiple reaches about 7.1.
Embodiment 4
As the embodiment 1 described method of utilizing two phase aqueous extraction system to extract pyruvic oxidase, difference is:
In the step (2), preparation potassiumphosphate aqueous solution 200mL, add molecular weight in this solution and be 1500 polyoxyethylene glycol (PEG1500), making the mass percent concentration of potassiumphosphate in total system is 10wt%, the mass percent concentration of polyoxyethylene glycol in total system is 30wt%, mix, make polyoxyethylene glycol/potassiumphosphate double-aqueous phase system solution.
The enzymatic activity recovery of pyruvic oxidase reaches 91.2% after testing, and the purifying multiple reaches about 7.2.
Embodiment 5
As the embodiment 1 described method of utilizing two phase aqueous extraction system to extract pyruvic oxidase, difference is:
In the step (2), preparation Sodium phosphate dibasic aqueous solution 200mL, add molecular weight in this solution and be 1500 polyoxyethylene glycol (PEG1500), making the mass percent concentration of Sodium phosphate dibasic in total system is 20wt%, the mass percent concentration of polyoxyethylene glycol in total system is 20wt%, mix, make polyoxyethylene glycol/Sodium phosphate dibasic double-aqueous phase system solution.
The enzymatic activity recovery of pyruvic oxidase reaches 90.5% after testing, and the purifying multiple reaches about 7.1.
Embodiment 6
The used bacterial classification of present embodiment is aerococcus viridans (Aerococcus viridans) ATCC11563, and this bacterial classification is available from U.S. representative microbial DSMZ (ATCC).
Get bacterial classification aerococcus viridans (Aerococcus viridans) ATCC11563 that produces pyruvic oxidase and be inoculated in the triangular flask that the 20mL seed culture medium is housed, after 30 ℃, 150r/min shaking table are cultivated 24h, make liquid seeds; Be inoculated in the fermention medium of 200mL by inoculum size 10% then, 30 ℃, 250r/min shaking table cultivation 18h make the somatic cells nutrient solution that contains pyruvic oxidase.Produce enzyme activity after testing and can reach 1253U/L.
Seed culture medium (g/L): glucose 10, yeast extract paste 5, peptone 5, NaCl5, KH
2PO
42, MgSO
41, MnSO
41, pH value 7.0.
Fermention medium (g/L): glucose 20, yeast extract paste 10, peptone 10, extractum carnis 5, NaCl3, KH
2PO
41, MgSO
40.5, pH value 6.5.
A kind of method of utilizing two phase aqueous extraction system to prepare pyruvic oxidase may further comprise the steps:
(1) will contain aerococcus viridans cell culture fluid 1000mL centrifugal 10min under the 5000r/min condition of pyruvic oxidase, make the wet thallus cell, somatic cells is resuspended in the deionized water, make the suspension that the cell mass concentration is 30wt%, be cooled to about 10 ℃, the APV-2000-1 type high pressure cell crusher that adopts German APV company to produce carries out four cytoclasis, and the crusher working pressure is 50MPa, makes the cytoclasis solution 186mL that contains pyruvic oxidase.
(2) preparation ammonium sulfate solution 200mL, add molecular weight in this solution and be 2000 polyoxyethylene glycol (PEG2000), making the mass percent concentration of ammonium sulfate in total system is 10wt%, the mass percent concentration of polyoxyethylene glycol in total system is 30wt%, mix, make polyoxyethylene glycol/ammonium sulfate double-aqueous phase system solution.
(3) add polyoxyethylene glycol/ammonium sulfate double-aqueous phase system solution that step (2) makes by equal-volume in the cytoclasis liquid that makes to step (1), after stirring the 10min mixing, leave standstill the 45min phase-splitting, make under the ammonium sulfate on the phase solution 112mL and polyoxyethylene glycol solution 260mL mutually.
The materials such as foreign protein, polysaccharide and nucleic acid that contain pyruvic oxidase and small part on the polyoxyethylene glycol in the phase solution contain whole cell debriss and materials such as most foreign protein, polysaccharide and nucleic acid in the phase solution under the ammonium sulfate.Through aqueous two-phase extraction separation for the first time, the enzymatic activity recovery of pyruvic oxidase reaches 98.1% after testing.
(4) add ammonium sulfate in the phase solution on the polyoxyethylene glycol that makes to step (3), making the mass concentration of ammonium sulfate in total system is 6wt%, after stirring the 10min mixing, leave standstill the 40min phase-splitting, make on the polyoxyethylene glycol of phase solution 114mL and the secondary separation that contains pyruvic oxidase under the ammonium sulfate of secondary separation solution 146mL mutually.
Contain the foreign protein that contains pyruvic oxidase and trace on the polyoxyethylene glycol of secondary separation of pyruvic oxidase in the phase solution, contain materials such as foreign protein, polysaccharide and nucleic acid under the secondary separation ammonium sulfate in the phase solution.Through aqueous two-phase extraction separation for the second time, the enzymatic activity recovery of pyruvic oxidase reaches 96.5% after testing.
(5) add ammonium sulfate in the phase solution on the polyoxyethylene glycol of the secondary separation that contains pyruvic oxidase that makes to step (4), making the mass concentration of ammonium sulfate in total system is 5wt%, transferring the pH value of solution with hydrochloric acid is 6.0, after stirring the 10min mixing, leave standstill the 30min phase-splitting, make under the ammonium sulfate that contains pyruvic oxidase on the phase solution 51mL and polyoxyethylene glycol solution 95mL mutually.
Contain polyoxyethylene glycol and foreign protein on the polyoxyethylene glycol in the phase solution, contain pyruvic oxidase and ammonium sulfate under the ammonium sulfate in the phase solution.It is the ultrafiltration membrance filter of 2500Dal that last phase solution is adopted molecular weight cut-off, except foreigh protein removing, collects the polyoxyethylene glycol filtered liquid, and polyoxyethylene glycol can be recycled.Through aqueous two-phase extraction separation for the third time, the enzymatic activity recovery of pyruvic oxidase reaches 94.2% after testing.
(6) phase solution under the ammonium sulfate that step (5) is made, adopting molecular weight cut-off under 0.2MPa pressure is that the ultrafiltration membrance filter of 50000Dal concentrates, and removes ammonium sulfate and residual polyoxyethylene glycol, collects trapped fluid, the trapped fluid lyophilize makes pyruvic oxidase 356mg.
The enzymatic activity recovery of pyruvic oxidase reaches 92.8% after testing, and the purifying multiple reaches about 7.4.
Embodiment 7
As the embodiment 6 described methods of utilizing two phase aqueous extraction system to prepare pyruvic oxidase, difference is:
In the step (3), add polyoxyethylene glycol/ammonium sulfate double-aqueous phase system solution that step (2) makes in the cytoclasis liquid that makes to step (1), the volume ratio of double-aqueous phase system solution and cytoclasis liquid is 1:2, after stirring the 15min mixing, leave standstill the 25min phase-splitting, make under the ammonium sulfate on the phase solution 84mL and polyoxyethylene glycol solution 195mL mutually.
The enzymatic activity recovery of pyruvic oxidase reaches 90.1% after testing, and the purifying multiple reaches about 7.0.
Comparative Examples 1
A kind of method of utilizing two phase aqueous extraction system to prepare pyruvic oxidase may further comprise the steps:
(1) with the step (1) of embodiment 1.
(2) preparation ammonium sulfate solution 200mL, in this solution, add 1, the 4-butyleneglycol, making the mass percent concentration of ammonium sulfate in total system is 40wt%, the mass percent concentration of 1,4-butyleneglycol in total system is 56wt%, mixes, make 1,4-butyleneglycol/ammonium sulfate double-aqueous phase system solution.
(3) in the cytoclasis liquid that makes to step (1) by equal-volume add that step (2) makes 1,4-butyleneglycol/ammonium sulfate double-aqueous phase system solution, after stirring the 10min mixing, leave standstill the 50min phase-splitting, make phase solution 107mL and 1 under the ammonium sulfate, phase solution 289mL contains pyruvic oxidase in the phase solution on 1, the 4-butyleneglycol on the 4-butyleneglycol.Through aqueous two-phase extraction separation for the first time, the enzymatic activity recovery of pyruvic oxidase reaches 92.5% after testing.
(4) to step (3) make 1, add ammonium sulfate in the phase solution on the 4-butyleneglycol, making the mass concentration of ammonium sulfate in solution is 20wt%, after stirring the 10min mixing, leave standstill the 40min phase-splitting, make under the ammonium sulfate phase solution 166mL on the phase solution 123mL and 1,4-butyleneglycol, contain pyruvic oxidase in the phase solution on 1, the 4-butyleneglycol.Through aqueous two-phase extraction separation for the second time, the enzymatic activity recovery of pyruvic oxidase reaches 85.6% after testing.
(5) to step (4) make 1, add ammonium sulfate in the phase solution on the 4-butyleneglycol, making the mass concentration of ammonium sulfate in solution is 15wt%, transferring the pH value of solution with hydrochloric acid is 5.7, after stirring the 10min mixing, leave standstill the 30min phase-splitting, make under the ammonium sulfate phase solution 109mL on the phase solution 57mL and 1,4-butyleneglycol.Contain 1,4-butyleneglycol and foreign protein on 1, the 4-butyleneglycol in the phase solution, contain pyruvic oxidase and ammonium sulfate under the ammonium sulfate in the phase solution.Through aqueous two-phase extraction separation for the third time, the enzymatic activity recovery of pyruvic oxidase reaches 81.3% after testing.
(6) phase solution under the ammonium sulfate that step (5) is made, adopting molecular weight cut-off under 0.15MPa pressure is that the ultrafiltration membrance filter of 40000Dal concentrates, and removes ammonium sulfate and residual 1, the 4-butyleneglycol, collect trapped fluid, the trapped fluid lyophilize makes pyruvic oxidase 372mg.
The enzymatic activity recovery of pyruvic oxidase reaches 80.1% after testing, and the purifying multiple reaches about 5.7.
In Comparative Examples 1, the gap of enzymatic activity recovery and embodiment 1 is all bigger in each extraction process, and enzymatic activity recovery has descended 13.1% during to step (5).In embodiment 1 and Comparative Examples 1, three extracting operations of enzyme are identical, and difference is that the material of formation double water-phase in Comparative Examples 1 is 1,4-butyleneglycol and ammonium sulfate.Because 1, the molecular weight of 4-butyleneglycol (90.12) be the PEG of embodiment 1 molecular weight (1000) 9%, its molecular ratio PEG is little many, so 1,4-butyleneglycol and ammonium sulfate are when forming double-aqueous phase system, PEG and the ammonium sulfate concentrations of the concentration ratio PEG/ ammonium sulfate system of 1,4-butyleneglycol and ammonium sulfate are high more than 1 times.The ammonium sulfate of high density makes that the ionic concn of solution is strong excessively, and zymoprotein can produce precipitation because of salting out, and high density 1, the 4-butyleneglycol also makes enzymic activity descend; And pyruvic oxidase is to be made of four subunits, and when it is in 1 of higher concentration, in the time of in 4-butyleneglycol and the ammoniumsulphate soln, the combination between the subunit can weaken, and the active centre of enzyme also can influencedly change, thereby also causes the decline of enzyme activity.
In addition, in embodiment 1, polyoxyethylene glycol can adopt ultrafiltration membrance filter, filtered liquid concentrates the back and collects PEG, and PEG can be recycled, and 1, the 4-butyleneglycol is difficult to adopt lower-cost ultra-filtration membrane concentrating and separating because molecular size and water molecules approach, and its use cost is higher relatively.
Therefore, adopt polyoxyethylene glycol/inorganic salt double-aqueous phase system to prepare the operational path that pyruvic oxidase is a kind of enzymatic activity recovery height, production cost is lower.
Claims (10)
1. method of utilizing two phase aqueous extraction system to prepare pyruvic oxidase may further comprise the steps:
(1) microorganism cells that will contain pyruvic oxidase is cultivated, and gets cell culture fluid, and is centrifugal, makes the wet thallus cell, and somatic cells is resuspended in the deionized water, makes cell suspending liquid, and smudge cells obtains cytoclasis liquid;
(2) preparation inorganic salt solution, in this inorganic salt solution, add polyoxyethylene glycol, the mass percent concentration of inorganic salt is that the mass percent concentration of 10～20wt%, polyoxyethylene glycol is 20～30wt% in total system, mixes, and makes polyoxyethylene glycol/inorganic salt double-aqueous phase system solution;
(3) add polyoxyethylene glycol/inorganic salt double-aqueous phase system solution that step (2) makes in the cytoclasis liquid that makes to step (1), the volume ratio of double-aqueous phase system solution and cytoclasis liquid is 1:(1～2), mix, leave standstill phase-splitting, make under the inorganic salt on the phase solution and the polyoxyethylene glycol that contains pyruvic oxidase solution mutually;
(4) add inorganic salt in the phase solution on the polyoxyethylene glycol that makes to step (3), making the mass concentration of inorganic salt in total system is 6～10wt%, mix, leave standstill phase-splitting, make on the polyoxyethylene glycol of phase solution and the secondary separation that contains pyruvic oxidase under the inorganic salt of secondary separation solution mutually;
(5) add inorganic salt in the phase solution on the polyoxyethylene glycol that makes to step (4), making the mass concentration of inorganic salt in total system is 5～6wt%, the pH value of regulator solution is 5.5～6.0, mix, leave standstill phase-splitting, make under the inorganic salt that contain pyruvic oxidase on the phase solution and polyoxyethylene glycol solution mutually;
(6) phase solution under the inorganic salt that contain pyruvic oxidase that step (5) is made adopts ultrafiltration membrance filter to concentrate, and collects trapped fluid, and lyophilize makes pyruvic oxidase.
2. the method for claim 1 is characterized in that, the centrifugal condition in the described step (1) is: the centrifugal 10～20min of 3000～5000r/min.
3. the method for claim 1 is characterized in that, the cell mass percent concentration in the cell suspending liquid in the described step (1) is 25～35%.
4. the method for claim 1 is characterized in that, the smudge cells in the described step (1) adopts the high pressure cell crusher, and working pressure is 50～60MPa, and service temperature is 5～15 ℃.
5. the method for claim 1 is characterized in that, the inorganic salt in described step (2), (3), (4), (5), (6) are selected from one of ammonium sulfate, sodium sulfate, Sodium phosphate dibasic, potassiumphosphate or Seignette salt.
6. the method for claim 1 is characterized in that, the molecular weight polyethylene glycol in described step (2), (3), (4), (5) is one of 1000,1500 or 2000.
7. the method for claim 1 is characterized in that, the phase-splitting time of leaving standstill in described step (3), (4), (5) is 10～60min.
8. the method for claim 1 is characterized in that, the ultra-filtration membrane operating pressure in the described step (6) is 0.1～0.3MPa, and the molecular weight cut-off of film is 40000～50000Dal, and service temperature is 25 ℃.
9. the method for claim 1 is characterized in that, described step (5) also comprises phase solution on the polyoxyethylene glycol after the ultrafiltration membrance filter of recovery usefulness concentrates, participates in the step of polyoxyethylene glycol in the preparation steps (2)/inorganic salt double-aqueous phase system solution.
10. method as claimed in claim 9 is characterized in that, the molecular weight cut-off of the ultra-filtration membrane of described recovery usefulness is 2500Dal.
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