CN103571806A - Method for rapidly separating and purifying dextran sucrase through two aqueous phase extraction - Google Patents

Method for rapidly separating and purifying dextran sucrase through two aqueous phase extraction Download PDF

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CN103571806A
CN103571806A CN201310560298.7A CN201310560298A CN103571806A CN 103571806 A CN103571806 A CN 103571806A CN 201310560298 A CN201310560298 A CN 201310560298A CN 103571806 A CN103571806 A CN 103571806A
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dextran
dextransucrase
aqueous
purifying
peg
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陈山
刘玫
梁赢
张义平
姚晓麦
浦媛媛
邹青松
黄磊
谢政
相萍萍
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Guangxi University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01005—Dextransucrase (2.4.1.5)

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Abstract

The invention discloses a method for rapidly separating and purifying dextran sucrase through two aqueous phase extraction. The inventor creates the method by adding polyethylene glycol (PEG) and exogenous glucan so as to construct a PEG/Dextran two aqueous phase system. According to the method, the problems of low enzyme activity recovery, difficulty in operation and low separation purity due to single method in the existing methods for separating the dextran sucrase through ammonium sulfate precipitation and membrane separation are solved. The method is environment-friendly and safe and efficient, is simple to operate, takes short time, and can be used for rapidly separating and purifying the dextran sucrase in short time.

Description

The method of aqueous two-phase extraction fast separating and purifying dextransucrase
Technical field
The invention belongs to bioseparation field of engineering technology, relate in particular to a kind of method of aqueous two-phase extraction fast separating and purifying dextransucrase.
Background technology
Dextransucrase (Dextransucrase, phylogenetic systematics EC2.4.1.5) belong to sugared acid anhydride lytic enzyme 70 families (Family70), by Leuconostoc mesenteroides fermentation, obtained, it is the glycosyltransferase of the synthetic dextran of a kind of catalysis sucrose, the dextran catalyzing and synthesizing can be used as plasma substitute, in industries such as food, medicine, chemical industry, all has a wide range of applications.But the dextransucrase that Leuconostoc mesenteroides fermentation obtains contains plurality of impurities, mix with product dextran, so that the unusual thickness of crude enzyme liquid, caused great difficulty to the separation and purification of enzyme.
At present, there is several different methods to can be used for the separation and purification of dextransucrase, as ammonium sulfate precipitation method, ultrafiltration, organic solvent precipitation method, chromatographic separation etc.Ammonium sulfate precipitation method has damaged the activity of dextransucrase to a great extent, and when surpassing 80% ammonium sulfate processing by mass concentration, the enzyme rate of recovery alive is no more than 10%.Though ultrafiltration process has the higher enzyme rate of recovery alive, crude enzyme liquid viscosity is high, easily causes film to stop up, and membrane flux declines very fast, is not suitable for industrial applications.The precipitation specificity of organic solvent precipitation method is higher than ammonium sulfate precipitation method, and precipitation is without desalination, and it is comparatively easy to filter, still easily causes the deactivation of enzyme.Chromatographic separation is general to be combined with other purification process (as ultrafiltration, saltout, dextranase degrades) etc., although it can obtain the enzyme that purity is higher, but easily cause, enzyme pollutes, enzyme is lived, and loss is serious, treatment capacity is few, can only, for laboratory scale, be difficult to carry out suitability for industrialized production.
Aqueous two-phase extraction refers to that two kinds of hydrophilic polymer aqueous solution can form double water-phase under certain condition, utilize separated object partition ratio in two-phase different and realize separated technology, this method has easy phase-splitting, cheap, extracting efficient is gentle, nontoxic, extract loss of activity is little, removal of impurities ability is strong, percentage extraction is high, can operate continuously, the advantage such as safe ready, can be widely used in product separation and the purifying such as protein, enzyme, nucleic acid, amino acid, polypeptide, organoid.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of simple to operate, aqueous two-phase extraction fast separating and purifying dextransucrase rapidly and efficiently, with improve the enzyme of dextransucrase live the rate of recovery, save the purifying time.
For solving the problems of the technologies described above, the present invention by the following technical solutions: the method for aqueous two-phase extraction fast separating and purifying dextransucrase, comprises the following steps:
(1) with Leuconostoc mesenteroides fermentation, produce dextransucrase and prepare dextransucrase crude enzyme liquid;
(2) in the crude enzyme liquid of step (1), add polyoxyethylene glycol (PEG) and exogenous dextran (Dextran) structure PEG/Dextran double-aqueous phase system;
(3) the PEG/Dextran double-aqueous phase system stand at low temperature layering of step (2) is separated with high speed centrifugation;
(4) the separated lower dialysis mutually of step (3) is concentrated and obtains dextransucrase (containing dextran).
Step (1) is undertaken by following operation: after Leuconostoc mesenteroides CICC21724 is brought back to life in No. 43 culture medium slant test tubes, be seeded to solid medium No. 43; After growing healthy and strong single bacterium colony, the single colony inoculation of picking 2 ring is to seed culture medium; Until culture propagation during to logarithmic phase, with the inoculum size of 2% (v/v), be inoculated in culture medium, cultivate 20-26h; Then by nutrient solution centrifugal removal thalline under 12000r/min, 4 ℃, the condition of 20min, collect supernatant liquor and obtain dextransucrase crude enzyme liquid.
No. 43 solid culture based formulas are sucrose 130g/L, peptone 2.0g/L, KH 2pO 40.3g/L, Na 2hPO 41.4g/L, agar 20.0g/L, pH7.0-7.2; Seed culture based formulas is sucrose 50g/L, yeast extract paste 10g/L, peptone 10g/L, KH 2pO 40.3g/L, Na 2hPO 41.4g/L, pH6.8; Culture medium formula is sucrose 50g/L, yeast extract paste 12.5g/L, extractum carnis 20g/L, KH 2pO 41.0g/L, Na 2hPO 43.6g/L, pH7.0-7.2.
The condition that solid medium is cultivated bacterial classification is 25 ± 3 ℃; The condition that seed culture medium and culture medium are cultivated bacterial classification is 25 ± 3 ℃, 150 ± 50r/min, and liquid amount is 20% of contained container standard capacity.
In step (2), PEG/Dextran double-aqueous phase system is configured to: crude enzyme liquid 20mL, add certain density PEG solution and exogenous Dextran solution, and with sterilized water, complementing to system total mass is 40g; The molecular weight of PEG is that the quality final concentration (w/w) of 6000, PEG6000 is 15%; The molecular weight of exogenous Dextran is 10,000,40,000 or 70,000, the quality final concentration (w/w) of Dextran-T10 is 0.8%～2.4%, the quality final concentration (w/w) that the quality final concentration (w/w) of Dextran-T40 is 0.8%～2.4%, Dextran-T70 is 0.4%～2.0%.
In step (2), hold the Beckman centrifuge tube that PEG/Dextran double-aqueous phase system adopts 50mL.
Step (3) is undertaken by following operation: build after PEG/Dextran double-aqueous phase system, evenly mix, be placed in the standing 1h of refrigerator of 4 ℃, then utilize high speed freezing centrifuge under 12000r/min, 4 ℃ of conditions after centrifugal 20min, outwell phase, by lower, use mutually sodium-acetate buffer (pH5.4) dilution of 0.02mol/L to be settled to 15mL.
In step (4), dialysing and selecting molecular weight is the dialysis tubing of 7000Da.
For the problem existing in current dextransucrase preparation process, contriver builds PEG/Dextran double-aqueous phase system by adding polyoxyethylene glycol and exogenous dextran, thereby has set up the method for aqueous two-phase extraction fast separating and purifying dextransucrase of the present invention.This method extracting efficient is gentle, and under optimum extraction condition, the enzyme of the dextransucrase rate of recovery alive can reach 95.99%, than vigor, is 29.90U/mg, and purification is 1.86 times; In gained dextransucrase, only contain dextran (work has certain protection and stabilization to enzyme), impurity is few, can be used for research and the production of the synthetic dextran of enzyme process.Meanwhile, the present invention extracts consuming time short, simple to operate, applicable to sharp separation dextransucrase.
Embodiment
Following embodiment used medium and culture condition are as follows:
Solid culture based formulas is sucrose 130g/L, peptone 2.0g/L, KH 2pO 40.3g/L, Na 2hPO 41.4g/L, agar 20.0g/L, pH7.0-7.2; Seed culture based formulas is sucrose 50g/L, yeast extract paste 10g/L, peptone 10g/L, KH 2pO 40.3g/L, Na 2hPO 41.4g/L, pH6.8; Culture medium formula is sucrose 50g/L, yeast extract paste 12.5g/L, extractum carnis 20g/L, KH 2pO 41.0g/L, Na 2hPO 43.6g/L, pH7.0-7.2.
The condition that solid medium is cultivated bacterial classification is 25 ± 3 ℃; The condition that seed culture medium and culture medium are cultivated bacterial classification is 25 ± 3 ℃, 150 ± 50r/min, and liquid amount is 20% of contained container standard capacity.
Embodiment 1
After Leuconostoc mesenteroides CICC21724 is brought back to life in No. 43 culture medium slant test tubes, be seeded to solid medium No. 43; After growing healthy and strong single bacterium colony, the single colony inoculation of picking 2 ring is to seed culture medium; Until culture propagation during to logarithmic phase, with the inoculum size of 2% (v/v), be inoculated in culture medium, cultivate 20-26h; Then by nutrient solution centrifugal removal thalline under 12000r/min, 4 ℃, the condition of 20min, collect supernatant liquor and obtain dextransucrase crude enzyme liquid.
Get the Beckman centrifuge tube that crude enzyme liquid 20mL is placed in 50mL, add respectively the PEG6000 solution of 12mL mass concentration 50% and the Dextran-T10 solution of 1.60mL mass concentration 20%, make its quality final concentration (w/w) be respectively 15% and 0.8%, with sterilized water, complementing to system total mass is 40g, is built into PEG/Dextran double-aqueous phase system.Build after PEG/Dextran double-aqueous phase system, fully shake up even mixing, the standing 1h of refrigerator that is placed in 4 ℃ makes phase-splitting abundant, then utilize high speed freezing centrifuge under 12000r/min, 4 ℃ of conditions after centrifugal 20min, outwell phase, by lower, use mutually sodium-acetate buffer (pH5.4) dilution of 0.02mol/L to be settled to 15mL, the enzyme rate of recovery alive that records dextransucrase is 79.61%, than vigor, be 25.90U/mg, purification is 1.61 times.Then, selecting molecular weight is the dialysis tubing of 7000Da, and the lower dialysis mutually of separation is concentrated and obtain dextransucrase (containing dextran).
Embodiment 2
Prepare dextransucrase crude enzyme liquid with embodiment 1.
Get the Beckman centrifuge tube that crude enzyme liquid 20mL is placed in 50mL, add respectively the PEG6000 solution of 12mL mass concentration 50% and the Dextran-T10 solution of 4.80mL mass concentration 20%, make its quality final concentration (w/w) be respectively 15% and 2.4%, with sterilized water, complementing to system total mass is 40g, is built into PEG/Dextran double-aqueous phase system.Build after PEG/Dextran double-aqueous phase system, fully shake up even mixing, the standing 1h of refrigerator that is placed in 4 ℃ makes phase-splitting abundant, then utilize high speed freezing centrifuge under 12000r/min, 4 ℃ of conditions after centrifugal 20min, outwell phase, by lower, use mutually sodium-acetate buffer (pH5.4) dilution of 0.02mol/L to be settled to 15mL, the enzyme rate of recovery alive that records dextransucrase is 66.81%, than vigor, be 20.23U/mg, purification is 1.26 times.Then, selecting molecular weight is the dialysis tubing of 7000Da, and the lower dialysis mutually of separation is concentrated and obtain dextransucrase (containing dextran).
Embodiment 3
Prepare dextransucrase crude enzyme liquid with embodiment 1.
Get the Beckman centrifuge tube that crude enzyme liquid 20mL is placed in 50mL, add respectively the PEG6000 solution of 12mL mass concentration 50% and the Dextran-T40 solution of 1.28mL mass concentration 25%, make its quality final concentration (w/w) be respectively 15% and 0.8%, with sterilized water, complementing to system total mass is 40g, is built into PEG/Dextran double-aqueous phase system.Build after PEG/Dextran double-aqueous phase system, fully shake up even mixing, the standing 1h of refrigerator that is placed in 4 ℃ makes phase-splitting abundant, then utilize high speed freezing centrifuge under 12000r/min, 4 ℃ of conditions after centrifugal 20min, outwell phase, by lower, use mutually sodium-acetate buffer (pH5.4) dilution of 0.02mol/L to be settled to 15mL, the enzyme rate of recovery alive that records dextransucrase is 89.90%, than vigor, be 32.58U/mg, purification is 1.48 times.Then, selecting molecular weight is the dialysis tubing of 7000Da, and the lower dialysis mutually of separation is concentrated and obtain dextransucrase (containing dextran).
Embodiment 4
Prepare dextransucrase crude enzyme liquid with embodiment 1.
Get the Beckman centrifuge tube that crude enzyme liquid 20mL is placed in 50mL, add respectively the PEG6000 solution of 12mL mass concentration 50% and the Dextran-T40 solution of 3.20mL mass concentration 20%, make its quality final concentration (w/w) be respectively 15% and 2.0%, with sterilized water, complementing to system total mass is 40g, is built into PEG/Dextran double-aqueous phase system.Build after PEG/Dextran double-aqueous phase system, fully shake up even mixing, the standing 1h of refrigerator that is placed in 4 ℃ makes phase-splitting abundant, then utilize high speed freezing centrifuge under 12000r/min, 4 ℃ of conditions after centrifugal 20min, outwell phase, by lower, use mutually sodium-acetate buffer (pH5.4) dilution of 0.02mol/L to be settled to 15mL, the enzyme rate of recovery alive that records dextransucrase is 72.39%, than vigor, be 23.04U/mg, purification is 1.05 times.Then, selecting molecular weight is the dialysis tubing of 7000Da, and the lower dialysis mutually of separation is concentrated and obtain dextransucrase (containing dextran).
Embodiment 5
Prepare dextransucrase crude enzyme liquid with embodiment 1.
Get the Beckman centrifuge tube that crude enzyme liquid 20mL is placed in 50mL, add respectively the PEG6000 solution of 12mL mass concentration 50% and the Dextran-T70 solution of 0.80mL mass concentration 20%, make its quality final concentration (w/w) be respectively 15% and 0.4%, with sterilized water, complementing to system total mass is 40g, is built into PEG/Dextran double-aqueous phase system.Build after PEG/Dextran double-aqueous phase system, fully shake up even mixing, the standing 1h of refrigerator that is placed in 4 ℃ makes phase-splitting abundant, then utilize high speed freezing centrifuge under 12000r/min, 4 ℃ of conditions after centrifugal 20min, outwell phase, by lower, use mutually sodium-acetate buffer (pH5.4) dilution of 0.02mol/L to be settled to 15mL, the enzyme rate of recovery alive that records dextransucrase is 67.84%, than vigor, be 15.25U/mg, purification is 1.14 times.Then, selecting molecular weight is the dialysis tubing of 7000Da, and the lower dialysis mutually of separation is concentrated and obtain dextransucrase (containing dextran).
Embodiment 6
Prepare dextransucrase crude enzyme liquid with embodiment 1.
Get the Beckman centrifuge tube that crude enzyme liquid 20mL is placed in 50mL, add respectively the PEG6000 solution of 12mL mass concentration 50% and the Dextran-T70 solution of 4.00mL mass concentration 20%, make its quality final concentration (w/w) be respectively 15% and 2.0%, with sterilized water, complementing to system total mass is 40g, is built into PEG/Dextran double-aqueous phase system.Build after PEG/Dextran double-aqueous phase system, fully shake up even mixing, the standing 1h of refrigerator that is placed in 4 ℃ makes phase-splitting abundant, then utilize high speed freezing centrifuge under 12000r/min, 4 ℃ of conditions after centrifugal 20min, outwell phase, by lower, use mutually sodium-acetate buffer (pH5.4) dilution of 0.02mol/L to be settled to 15mL, the enzyme rate of recovery alive that records dextransucrase is 58.42%, than vigor, be 13.62U/mg, purification is 1.02 times.Then, selecting molecular weight is the dialysis tubing of 7000Da, and the lower dialysis mutually of separation is concentrated and obtain dextransucrase (containing dextran).

Claims (8)

1. a method for aqueous two-phase extraction fast separating and purifying dextransucrase, is characterized in that comprising the following steps:
(1) with Leuconostoc mesenteroides fermentation, produce dextransucrase and prepare dextransucrase crude enzyme liquid;
(2) in the crude enzyme liquid of step (1), add polyoxyethylene glycol and exogenous dextran structure PEG/Dextran double-aqueous phase system;
(3) the PEG/Dextran double-aqueous phase system stand at low temperature layering of step (2) is separated with high speed centrifugation;
(4) the separated lower dialysis mutually of step (3) is concentrated and obtains dextransucrase.
2. the method for aqueous two-phase extraction fast separating and purifying dextransucrase according to claim 1, it is characterized in that step (1) undertaken by following operation: after Leuconostoc mesenteroides CICC21724 is brought back to life in No. 43 culture medium slant test tubes, be seeded to solid medium No. 43; After growing healthy and strong single bacterium colony, the single colony inoculation of picking 2 ring is to seed culture medium; Until culture propagation, during to logarithmic phase, the inoculum size with 2% is inoculated in culture medium, cultivates 20-26h; Then by nutrient solution centrifugal removal thalline under 12000r/min, 4 ℃, the condition of 20min, collect supernatant liquor and obtain dextransucrase crude enzyme liquid.
3. the method for aqueous two-phase extraction fast separating and purifying dextransucrase according to claim 2, is characterized in that: described No. 43 solid culture based formulas are sucrose 130g/L, peptone 2.0g/L, KH 2pO 40.3g/L, Na 2hPO 41.4g/L, agar 20.0g/L, pH7.0-7.2; Described seed culture based formulas is sucrose 50g/L, yeast extract paste 10g/L, peptone 10g/L, KH 2pO 40.3g/L, Na 2hPO 41.4g/L, pH6.8; Described culture medium formula is sucrose 50g/L, yeast extract paste 12.5g/L, extractum carnis 20g/L, KH 2pO 41.0g/L, Na 2hPO 43.6g/L, pH7.0-7.2.
4. the method for aqueous two-phase extraction fast separating and purifying dextransucrase according to claim 2, is characterized in that: the condition that described solid medium is cultivated bacterial classification is 25 ± 3 ℃; The condition that described seed culture medium and culture medium are cultivated bacterial classification is 25 ± 3 ℃, 150 ± 50r/min, and liquid amount is 20% of contained container standard capacity.
5. the method for aqueous two-phase extraction fast separating and purifying dextransucrase according to claim 1, it is characterized in that in step (2), PEG/Dextran double-aqueous phase system is configured to: crude enzyme liquid 20mL, add certain density PEG solution and exogenous Dextran solution, with sterilized water, complementing to system total mass is 40g; The molecular weight of described PEG is that the quality final concentration of 6000, PEG6000 is 15%; The molecular weight of described exogenous Dextran be 10,000,40,000 or the quality final concentration of 70,000, the Dextran-T10 quality final concentration that is 0.8%～2.4%, Dextran-T40 be 0.8%～2.4%, Dextran-T70 quality final concentration is 0.4%～2.0%.
6. the method for aqueous two-phase extraction fast separating and purifying dextransucrase according to claim 5, is characterized in that holding in step (2) the Beckman centrifuge tube that PEG/Dextran double-aqueous phase system adopts 50mL.
7. the method for aqueous two-phase extraction fast separating and purifying dextransucrase according to claim 6, it is characterized in that step (3) undertaken by following operation: build after PEG/Dextran double-aqueous phase system, evenly mix, be placed in the standing 1h of refrigerator of 4 ℃, then utilize high speed freezing centrifuge under 12000r/min, 4 ℃ of conditions after centrifugal 20min, outwell phase, by lower, with the sodium-acetate buffer dilution of 0.02mol/L pH5.4, be settled to 15mL mutually.
8. the method for aqueous two-phase extraction fast separating and purifying dextransucrase according to claim 1, is characterized in that it is the dialysis tubing of 7000Da that molecular weight is selected in the middle dialysis of step (4).
CN201310560298.7A 2013-11-12 2013-11-12 Method for rapidly separating and purifying dextran sucrase through two aqueous phase extraction Pending CN103571806A (en)

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CN104761424A (en) * 2015-03-31 2015-07-08 川渝中烟工业有限责任公司 Method for extracting free amino acids from tobacco branches
CN109852648A (en) * 2019-04-11 2019-06-07 广西大学 The method that enzyme process prepares dextran selenium polymers
CN114686544A (en) * 2020-12-30 2022-07-01 广东省科学院生物工程研究所 Method for spontaneously regulating hydrolysis to generate alpha-glucan with specific molecular weight by utilizing three-water-phase system and application of method

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104761424A (en) * 2015-03-31 2015-07-08 川渝中烟工业有限责任公司 Method for extracting free amino acids from tobacco branches
CN109852648A (en) * 2019-04-11 2019-06-07 广西大学 The method that enzyme process prepares dextran selenium polymers
CN109852648B (en) * 2019-04-11 2022-08-09 广西大学 Method for preparing dextran selenium polymer by enzyme method
CN114686544A (en) * 2020-12-30 2022-07-01 广东省科学院生物工程研究所 Method for spontaneously regulating hydrolysis to generate alpha-glucan with specific molecular weight by utilizing three-water-phase system and application of method

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Application publication date: 20140212