CN104059895A - Separating and purifying method of yarrowia lipolytica W29 lipase - Google Patents
Separating and purifying method of yarrowia lipolytica W29 lipase Download PDFInfo
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- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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Abstract
The invention discloses a separating and purifying method of yarrowia lipolytica W29 lipase. The method comprises the following specific steps of (1), adding saturated ammonium sulfate liquor containing EDTA (ethylene diamine tetraacetic acid) into fermentation liquid supernatant containing yarrowia lipolytica W29 lipase, so that final saturation level of the liquor is 30-55%; then, regulating pH to 4.5-6.5, centrifuging to obtain precipitates after placing for 10 hours on ice; (2), carrying out dialysis and desalting onto precipitates obtained in the step (1) to obtain crude enzyme liquor; and (3), centrifuging the crude enzyme liquor obtained in the step (2) at a low speed, carrying out DEAE-Sepharose FF gel filtration chromatography, collecting eluant, mixing with glycerol after concentrating eluant by PEG20000, and placing at (-)20 DEG C for storage. The separating and purifying method of the yarrowia lipolytica W29 lipase has the advantages of high purifying multiple and high recovery rate, and is simple to operate.
Description
Technical field
The present invention relates to separating and purifying technology field, relate in particular to a kind of Yarrowia lipolytica W
29the separation purification method of lipase.
Background technology
Yarrowia lipolytica (Y.lipolitica) is found in nineteen forty-two first, is a kind of unconventional property, non-pathogenic, strict good foster yeast, belongs to ascus yeast.Often live in the place of being rich in the material such as lipid, protein containing meat salad, soy sauce etc., its growth temperature is 25～30 DEG C, existence pH3.0～8.0, can utilize hydrophobic substance as ethanol, oils, lipid acid, alkanes is as chloralkane, polymethylation, and carbohydrate is grown as carbon source as glucose sugar, sucrose, maltose etc.Y.lipolitica can produce the product such as a large amount of organic acid (as a-ketoglutaric acid, citric acid, isocitric acid etc.), extracellular enzyme (the outer aspartic protease of born of the same parents, the outer Sumizyme MP of born of the same parents, extracellular lipase and esterase ester, the outer Phosphoric acid esterase of born of the same parents, the outer RNase of born of the same parents, a-mannosidase etc.), its protein excretion pattern has typicalness, the model animals that is often used to Study on Protein secretion, makes it all be widely used at scientific research, industrial circle based on above characteristic.Particularly its utilization to oil wastewater processing aspect, as Wu Lan etc. finds Y.lipolitica W
29salad oil and food and drink oil waste water are had to higher degradation capability, be that 25～30 DEG C, pH6.0～7.0, incubation time are under the condition of 50h, shaking speed 200r/min in temperature, salad oil to oil-contg≤2000mg/L and the degradation rate of food and drink waste water have reached 93.3% and 85.08%, and find Y.lipolitica W
29by producing lipase, grease is degraded.
1948, Nelson and Peters reported first Yarrowia lipolytica can produce significant quantities of fat enzyme, it can the reaction such as catalytic hydrolysis, esterification, transesterification, ammonia solution in out-phase or organic phase, and substrate is extensive, has very high substrate specificity, regioselectivity and enantioselectivity.These advantages make it have industrial use widely, as environmental protection, washing, organic synthesis, grease processing, food-processing, medicine are synthetic, bioenergy etc.The discoveries such as Gerardo are done carbon source and inductor, add tween-80 in substratum, reduce air flow with sweet oil or Semen Maydis oil can make Y.lipolitica improve the output of lipase; Destain etc. utilize the method for sudden change to improve the enzymatic productivity of Y.lipolitica, make its enzymatic productivity improve nearly 30 times; The method of the utilization mutagenesis such as Ficker filters out removes the Y.lipolitica bacterial classification that glucose suppresses, and it produces the work of enzyme enzyme and has improved 10 times; The utilization molecular biology methods such as Pignede have obtained Lip2 gene from Y.lipolitica, find to express Lip2 and have lipase characteristic, but when knocking out after Lip2 gene, Y.lipolitica can also normal growth on the flat board taking tributyrin as carbon source only, and the approach that also has other degraded tributyrin in Y.lipolitica is described.
The lipase in different microorganisms source has different catalysis characteristicses and catalysis activity, and its moiety is also had nothing in common with each other, and therefore the method for separating and purifying lipase and step are also different.Microbial lipase separation and purification is mainly by two large technology: 1) precipitation technology, mainly comprises ammonium sulfate, ethanol and acetone precipitation; 2) chromatographic technique, mainly comprises that ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel-filtration etc. complete.In order to obtain maximum the enzyme rate of recovery and purification, conventionally extract in purge process and adopt multistep method of purification at lipase.Generally first use operating unit concentrating sample non-specific, low resolution, with this protein concentrate sample and improve target protein concentration, remove topmost impurity; Then adopt high-resolution operating unit as ion exchange chromatography, gel permeation chromatography, hydrophobic chromatography etc.The latter generally has highly selective and separates before sweeping ion exchange chromatography and affinity chromatography be placed on, and this class separation small scale of gel permeation chromatography, the slow operating unit of segregation rate are placed on finally, can effectively improve like this separation efficiency, reduce loss of proteins.
Therefore, find simple lipase separation purification method for Yarrowia lipolytica W
29utilize important in inhibiting.
Summary of the invention
The object of this invention is to provide a kind of Yarrowia lipolytica W
29the separation purification method of lipase.
The present invention adopts following technical scheme:
Yarrowia lipolytica W of the present invention
29the concrete steps of the separation purification method of lipase are as follows:
(1) toward containing Yarrowia lipolytica W
29in the fermented supernatant fluid of lipase, add the saturated ammonium sulphate solution that contains EDTA, making its whole saturation ratio is 30-55%, then regulates pH to 4.5-6.5, places after 10h on ice, carries out centrifugal being precipitated;
(2) precipitation step (1) being obtained, the desalination of dialysing, obtains crude enzyme liquid;
(3) crude enzyme liquid step (2) being obtained carries out low-speed centrifugal, then, by DEAE-SepharoseFF gel permeation chromatography, then collects elutriant, and elutriant, after PEG20000 is concentrated, is mixed with glycerine, puts at-20 DEG C and preserves.
In step (1), the ultimate density of EDTA in solution is 1mM.
In step (1), the whole saturation ratio of ammoniumsulphate soln is preferably 45%.
In step (1), use sulfuric acid or ammoniacal liquor to regulate pH value.
In step (1), preferably regulate pH to 6.0.
In step (1), centrifugal is after centrifugal 15min, to get precipitation under 4 DEG C, 14000r/min.
In step (2), the concrete steps of dialysis desalination are, the precipitation that step (1) is obtained adds in dialysis tubing, the total amount adding is the 1/2-2/3 of dialysis tubing volume, clamps and is inverted with dialysis clamp, and be 1:30 by precipitation with damping fluid volume ratio, carefully the dialysis tubing of above-mentioned application of sample sealing is put into the 25mMpH7.5Tris-HCL damping fluid of 4 DEG C of precoolings, 4 DEG C of low rate mixing dialysis systems, 4h changes dialysis buffer liquid one time, changes altogether 3 times.
In step (3), elutriant after PEG20000 is concentrated, with glycerine by volume 1:1 mix.
Positively effect of the present invention is as follows:
Yarrowia lipolytica W of the present invention
29the separation purification method of lipase has simple to operate, purification height and the high advantage of the rate of recovery.
Brief description of the drawings
Fig. 1 is the schematic diagram of the impact of pH on ammonium sulfate precipitation lipase.
Fig. 2 is the schematic diagram of the impact of saturation ratio on ammonium sulfate precipitation lipase.
Fig. 3 is the Yarrowia lipolytica W of the embodiment of the present invention 3
29the DEAE Sepharose fast flow column chromatography figure of lipase.
Embodiment
The following examples are to describe in further detail of the present invention.
Embodiment 1
(1) toward containing Yarrowia lipolytica W
29in the fermented supernatant fluid of lipase, add the saturated ammonium sulphate solution that contains EDTA, making its whole saturation ratio is 30%, then regulates pH to 4.5, places after 10h on ice, carries out centrifugal being precipitated;
(2) precipitation step (1) being obtained, the desalination of dialysing, obtains crude enzyme liquid;
(3) crude enzyme liquid step (2) being obtained carries out low-speed centrifugal, then, by DEAE-SepharoseFF gel permeation chromatography, then collects elutriant, and elutriant, after PEG20000 is concentrated, is mixed with glycerine, puts at-20 DEG C and preserves.
In step (1), the ultimate density of EDTA in solution is 1mM.
In step (1), use sulfuric acid or ammoniacal liquor to regulate pH value.
In step (1), centrifugal is after centrifugal 15min, to get precipitation under 4 DEG C, 14000r/min.
In step (2), the concrete steps of dialysis desalination are first dialysis tubing to be put into 2% (w/v) NaCO
3boiling water bath 10min in (containing 1mM EDTA) solution, distilled water cleans, then boils dialysis tubing 10min with 1mM EDTA, cooling stand-by with distilled water cleaning.Then be that the precipitation that step (1) is obtained adds in dialysis tubing, the total amount adding is about the 1/2-2/3 of dialysis tubing volume, clamp and be inverted with dialysis clamp, be 1:30 by precipitation with damping fluid volume ratio, carefully the dialysis tubing of above-mentioned application of sample sealing is put into the 25mM pH7.5Tris-HCL damping fluid of 4 DEG C of precoolings, 4 DEG C of low rate mixing dialysis systems, 4h changes dialysis buffer liquid one time, changes altogether 3 times.
In step (3), elutriant after PEG20000 is concentrated, with glycerine by volume 1:1 mix.
Embodiment 2
(1) toward containing Yarrowia lipolytica W
29in the fermented supernatant fluid of lipase, add the saturated ammonium sulphate solution that contains EDTA, making its whole saturation ratio is 55%, then regulates pH to 6.5, places after 10h on ice, carries out centrifugal being precipitated;
(2) precipitation step (1) being obtained, the desalination of dialysing, obtains crude enzyme liquid;
(3) crude enzyme liquid step (2) being obtained carries out low-speed centrifugal, then, by DEAE-Sepharose FF gel permeation chromatography, then collects elutriant, and elutriant, after PEG20000 is concentrated, is mixed with glycerine, puts at-20 DEG C and preserves.
In step (1), the ultimate density of EDTA in solution is 1mM.
In step (1), use sulfuric acid or ammoniacal liquor to regulate pH value.
In step (1), centrifugal is after centrifugal 15min, to get precipitation under 4 DEG C, 14000r/min.
In step (2), the concrete steps of dialysis desalination are first dialysis tubing to be put into 2% (w/v) NaCO
3boiling water bath 10min in (containing 1mMEDTA) solution, distilled water cleans, then boils dialysis tubing 10min with 1mMEDTA, coolingly cleans stand-by with distilled water.Then be that the precipitation that step (1) is obtained adds in dialysis tubing, the total amount adding is about the 1/2-2/3 of dialysis tubing volume, clamp and be inverted with dialysis clamp, be 1:30 by precipitation with damping fluid volume ratio, carefully the dialysis tubing of above-mentioned application of sample sealing is put into the 25mMpH7.5Tris-HCL damping fluid of 4 DEG C of precoolings, 4 DEG C of low rate mixing dialysis systems, 4h changes dialysis buffer liquid one time, changes altogether 3 times.
In step (3), elutriant after PEG20000 is concentrated, with glycerine by volume 1:1 mix.
Embodiment 3
(1) toward containing Yarrowia lipolytica W
29in the fermented supernatant fluid of lipase, add the saturated ammonium sulphate solution that contains EDTA, making its whole saturation ratio is 45%, then regulates pH to 6.0, places after 10h on ice, carries out centrifugal being precipitated;
(2) precipitation step (1) being obtained, the desalination of dialysing, obtains crude enzyme liquid;
(3) crude enzyme liquid step (2) being obtained carries out low-speed centrifugal, then, by DEAE-SepharoseFF gel permeation chromatography, then collects elutriant, and elutriant, after PEG20000 is concentrated, is mixed with glycerine, puts at-20 DEG C and preserves.
In step (1), the ultimate density of EDTA in solution is 1mM.
In step (1), use sulfuric acid or ammoniacal liquor to regulate pH value.
In step (1), centrifugal is after centrifugal 15min, to get precipitation under 4 DEG C, 14000r/min.
In step (2), the concrete steps of dialysis desalination are first dialysis tubing to be put into 2% (w/v) NaCO
3boiling water bath 10min in (containing 1mMEDTA) solution, distilled water cleans, then boils dialysis tubing 10min with 1mMEDTA, coolingly cleans stand-by with distilled water.Then be that the precipitation that step (1) is obtained adds in dialysis tubing, the total amount adding is about the 1/2-2/3 of dialysis tubing volume, clamp and be inverted with dialysis clamp, be 1:30 by precipitation with damping fluid volume ratio, carefully the dialysis tubing of above-mentioned application of sample sealing is put into the 25mMpH7.5Tris-HCL damping fluid of 4 DEG C of precoolings, 4 DEG C of low rate mixing dialysis systems, 4h changes dialysis buffer liquid one time, changes altogether 3 times.
In step (3), elutriant after PEG20000 is concentrated, with glycerine by volume 1:1 mix.
Detection method:
1, enzyme is lived:
Enzyme assay adopts alkali formula volumetry.Taking 5mL sweet oil emulsion as substrate, add 4mL phosphoric acid buffer (25mmol/L, pH7.0), again with enzyme liquid 1mL composition 10mL reaction system, after 40 DEG C of water-bath 10min, add 15mL95% ethanol termination reaction, the lipid acid amount of enzymatic hydrolysis gained is made indicator with 1% phenolphthalein, records through 0.05mol/LNaOH titration.The present invention is 40 DEG C in temperature, under the condition of pH=7.0, produces the required enzyme amount of the free fat enzyme acid of 1U/moL be defined as a U of Ge Meihuo unit, international unit (U/mL) with lipase hydrolysis triolein per minute.
2, protein content:
The mensuration of protein content adopts Xylene Brilliant Cyanine G method.
3, than vigor (U/mg):
The enzyme work of every mL enzyme liquid is multiplied by the enzyme liquid cumulative volume of acquisition, then divided by total protein concentration.
4, purification:
Ratio vigor by specific activity of enzyme after every single step purification divided by enzyme in initial fermented supernatant fluid.
5, the rate of recovery:
By the total enzyme amount obtaining after every single step purification divided by the initial total enzyme amount of fermented supernatant fluid.
The saturated ammonium sulphate solution that contains EDTA of the embodiment of the present invention 3 steps (1) and the sedimentation effect that does not contain the ammoniumsulphate soln of EDTA are contrasted, and experimental result is as shown in table 1:
Table 1
As can be seen from Table 1, ammonium sulfate saturated solution adds EDTA can significantly improve the effect of ammonium sulfate precipitation lipase, its reason may be exactly in ammonium sulfate saturated solution, to contain micro-heavy metal, thereby it can make lipase sex change lose activity, and EDETATE SODIUM can form complex compound and form precipitation with heavy metal, reduce the effect of heavy metal to lipase.
Fig. 1 is the schematic diagram of the impact of pH on ammonium sulfate precipitation lipase, and as can be seen from Figure 1, pH is large and present certain rule on ammonium sulfate precipitation lipase impact, first along with the rising of pH, the effect of ammonium sulfate precipitation lipase constantly strengthens, and is 6.0 o'clock to pH, and it is best that its effect reaches, along with the further rising of pH, the effect of ammonium sulfate precipitation lipase but declines rapidly afterwards.
Fig. 2 is the schematic diagram of the impact of saturation ratio on ammonium sulfate precipitation lipase.As can be seen from Figure 2, the amount of ammonium sulfate precipitation lipase is along with the rule that ammonium sulfate saturation ratio changes is: first increase along with the increase of the full degree of ammonium sulfate always, in the time that saturation ratio is 45%, lipase precipitation capacity reaches balance substantially, afterwards along with the increase lipase precipitation capacity of ammonium sulfate concentrations does not significantly increase.All in all saturation ratio is higher than 45% time, and its lipase sedimentation effect is better.
Fig. 3 is the Yarrowia lipolytica W of the embodiment of the present invention 3
29the DEAE Sepharose fast flow column chromatography figure of lipase, as can be seen from Figure 3, chromatography curve presents 2 protein elution peaks, between protein peak, also have and intersect, and in two protein peaks, there is lipase activity to have (as Fig. 3-6 pipe and Fig. 8-11 pipe), collect respectively 3-6 pipe and 8-11 pipe elutriant, measure the volume of protein concn, enzymic activity and gained sample.Take a morsel and carry out PAGE electrophoresis and carry out Purity, the sample after all the other purifying is concentrated through PEG20000 respectively, and gained sample mixes in 1:1 ratio with glycerine, in-20 DEG C of Refrigerator stores.Electrophoresis result shows: after purifying, obtained 2 kinds of lipase, purifying enzyme 1 molecular weight is 37-38kD, and purifying enzyme 2 molecular weight are 41-42kD.
Measure respectively Yarrowia lipolytica W in embodiment 3
29the crude enzyme liquid of lipase, the liquid of saltouing, ion exchange chromatography liquid, the cumulative volume of concentrated solution, Tot Prot, total activity, calculate than vigor, purification multiple, the rate of recovery it, and statistics is in table 2 and table 3.
Table 2 Yarrowia lipolytica W
29the purification yield of purifying enzyme 1
Table 3 Yarrowia lipolytica W
29the purification yield of purifying enzyme 2
Can find out that from table 2 and table 3 fermented supernatant fluid is after the purification steps such as ammonium sulfate precipitation and DEAE-SepharoseFF column chromatography, PEG20000 be concentrated, purifying enzyme 1 purifying 27 times, the rate of recovery is 4.99%, specific activity is 167.73U/mg, purifying enzyme 2 purifying 38 times, the rate of recovery is 4.20%, and specific activity is 233.89U/mg.From table, also can find out that purifying enzyme 1 and purifying enzyme 2 have improved respectively 8.7 times and 7.8 times than vigor in anion-exchange chromatography process, its protein content has reduced respectively 30 times and 16.6 times simultaneously.
Result shows, Y.lipolitica W
29extracellular lipase is obtaining concentrated and purification effect preferably after the purification step such as ammonium sulfate precipitation, anionresin.
Although illustrated and described embodiments of the invention, for the ordinary skill in the art, be appreciated that without departing from the principles and spirit of the present invention and can carry out multiple variation, amendment, replacement and modification to these embodiment, scope of the present invention is limited by claims and equivalent thereof.
Claims (8)
1. a Yarrowia lipolytica W
29the separation purification method of lipase, is characterized in that: the concrete steps of described method are as follows:
(1) toward containing Yarrowia lipolytica W
29in the fermented supernatant fluid of lipase, add the saturated ammonium sulphate solution that contains EDTA, making its whole saturation ratio is 30-55%, then regulates pH to 4.5-6.5, places after 10h on ice, carries out centrifugal being precipitated;
(2) precipitation step (1) being obtained, the desalination of dialysing, obtains crude enzyme liquid;
(3) crude enzyme liquid step (2) being obtained carries out low-speed centrifugal, then, by DEAE-SepharoseFF gel permeation chromatography, then collects elutriant, and elutriant, after PEG20000 is concentrated, is mixed with glycerine, puts at-20 DEG C and preserves.
2. Yarrowia lipolytica W as claimed in claim 1
29the separation purification method of lipase, is characterized in that:
In step (1), the ultimate density of EDTA in solution is 1mM.
3. Yarrowia lipolytica W as claimed in claim 1
29the separation purification method of lipase, is characterized in that:
In step (1), the whole saturation ratio of ammoniumsulphate soln is 45%.
4. Yarrowia lipolytica W as claimed in claim 1
29the separation purification method of lipase, is characterized in that:
In step (1), use sulfuric acid or ammoniacal liquor to regulate pH value.
5. Yarrowia lipolytica W as claimed in claim 1
29the separation purification method of lipase, is characterized in that:
In step (1), regulate pH to 6.0.
6. Yarrowia lipolytica W as claimed in claim 1
29the separation purification method of lipase, is characterized in that:
In step (1), centrifugal is after centrifugal 15min, to get precipitation under 4 DEG C, 14000r/min.
7. Yarrowia lipolytica W as claimed in claim 1
29the separation purification method of lipase, is characterized in that:
In step (2), the step of dialysis desalination is, the precipitation that step (1) is obtained adds in dialysis tubing, the total amount adding is the 1/2-2/3 of dialysis tubing volume, clamps and is inverted with dialysis clamp, and be 1:30 by precipitation with damping fluid volume ratio, carefully the dialysis tubing of above-mentioned application of sample sealing is put into the 25mMpH7.5Tris-HCL damping fluid of 4 DEG C of precoolings, 4 DEG C of low rate mixing dialysis systems, 4h changes dialysis buffer liquid one time, changes altogether 3 times.
8. Yarrowia lipolytica W as claimed in claim 1
29the separation purification method of lipase, is characterized in that: in step (3), elutriant after PEG20000 is concentrated, with glycerine by volume 1:1 mix.
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Cited By (3)
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| CN105543194A (en) * | 2016-01-22 | 2016-05-04 | 领先生物农业股份有限公司 | Method for extracting lipase from vegetable oil containing microorganism fermentation liquid |
| CN108094714A (en) * | 2017-12-01 | 2018-06-01 | 杭州柏普泰生物科技有限公司 | A kind of feed addictive for promoting Acipenser baeri growth and preparation method and application |
| CN110628744A (en) * | 2019-10-24 | 2019-12-31 | 宜宾五粮液股份有限公司 | Method for separating and purifying esterifying enzyme from strong aromatic yeast |
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| CN101735967A (en) * | 2009-11-11 | 2010-06-16 | 南京工业大学 | Organic solvent resisting lipase, application thereof and bacteria for producing same |
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| CN101735967A (en) * | 2009-11-11 | 2010-06-16 | 南京工业大学 | Organic solvent resisting lipase, application thereof and bacteria for producing same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543194A (en) * | 2016-01-22 | 2016-05-04 | 领先生物农业股份有限公司 | Method for extracting lipase from vegetable oil containing microorganism fermentation liquid |
| CN108094714A (en) * | 2017-12-01 | 2018-06-01 | 杭州柏普泰生物科技有限公司 | A kind of feed addictive for promoting Acipenser baeri growth and preparation method and application |
| CN110628744A (en) * | 2019-10-24 | 2019-12-31 | 宜宾五粮液股份有限公司 | Method for separating and purifying esterifying enzyme from strong aromatic yeast |
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