CN114606214A - Method for synchronously purifying lipase, acid protease and alkaline protease - Google Patents
Method for synchronously purifying lipase, acid protease and alkaline protease Download PDFInfo
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- 102000004882 Lipase Human genes 0.000 title claims abstract description 28
- 108090001060 Lipase Proteins 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 25
- 108091005658 Basic proteases Proteins 0.000 title claims abstract description 23
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 22
- 239000004365 Protease Substances 0.000 title claims abstract description 21
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 20
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 42
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 42
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 42
- 102000004190 Enzymes Human genes 0.000 claims abstract description 39
- 108090000790 Enzymes Proteins 0.000 claims abstract description 39
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 39
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 39
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 28
- 238000000746 purification Methods 0.000 claims abstract description 24
- 230000000694 effects Effects 0.000 claims abstract description 22
- 238000005185 salting out Methods 0.000 claims abstract description 17
- 239000011780 sodium chloride Substances 0.000 claims abstract description 14
- 239000011324 bead Substances 0.000 claims abstract description 10
- 238000001742 protein purification Methods 0.000 claims abstract description 9
- 239000012535 impurity Substances 0.000 claims abstract description 6
- 238000005571 anion exchange chromatography Methods 0.000 claims abstract description 5
- 238000005349 anion exchange Methods 0.000 claims abstract description 4
- 238000005516 engineering process Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 27
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 13
- 239000007853 buffer solution Substances 0.000 claims description 12
- 238000001962 electrophoresis Methods 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 230000001502 supplementing effect Effects 0.000 claims description 4
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000004367 Lipase Substances 0.000 abstract description 14
- 235000019421 lipase Nutrition 0.000 abstract description 14
- 108091005508 Acid proteases Proteins 0.000 abstract description 6
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 abstract 2
- 229910001870 ammonium persulfate Inorganic materials 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 35
- 235000019419 proteases Nutrition 0.000 description 14
- 238000000502 dialysis Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 239000000843 powder Substances 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 230000002378 acidificating effect Effects 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 235000013405 beer Nutrition 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 229940079919 digestives enzyme preparation Drugs 0.000 description 2
- 150000002148 esters Chemical group 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
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- 206010011224 Cough Diseases 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
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- 238000005119 centrifugation Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 238000005457 optimization Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- 238000012545 processing Methods 0.000 description 1
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- 231100000331 toxic Toxicity 0.000 description 1
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- 238000005303 weighing Methods 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The invention relates to the technical field of protein purification, and discloses a method for synchronously purifying lipase, acid protease and alkaline protease, which adopts ammonium sulfate salting-out and Q Beads 6FF anion chromatography to purify three proteins, and the protein purification technology is carried out through the following steps: determining the saturation of ammonium sulfate corresponding to three kinds of protein when the protein is separated out by an ammonium persulfate salting-out method; removing impurities from 50% ammonium sulfate salt-separated protein and 50% ammonium sulfate, adding 80% ammonium sulfate salt-separated protein into QBeads 6FF anion exchange column, eluting with 50-400mM NaCl solution, collecting eluate of each component, and measuring protein concentration and enzyme activity; the method for synchronously purifying the lipase, the acid protease and the alkaline protease improves the purification multiple of the lipase by 63.24 times, the purification multiple of the acid protease by 10.33 times and the purification multiple of the alkaline protease by 31.69 times.
Description
Technical Field
The invention relates to the technical field of protein purification, in particular to a method for synchronously purifying lipase, acid protease and alkaline protease.
Background
Lipase is one of important industrial enzyme preparations, can catalyze reactions such as lipolysis, ester exchange, ester synthesis and the like, and is widely applied to the industrial fields such as energy, oil processing, food, medicine and the like. Lipases are ubiquitous in nature in plant seeds, animal tissues and microorganisms, with industrial lipases being sourced from microorganisms. The catalyst has the advantages of mild reaction conditions, high catalytic rate, good stereoselectivity, wide substrate specificity, convenient extraction and potential unlimited supply, and is widely used in the industrial fields.
In the modern society, due to the rapid development of food industry, consumer awareness of food safety and health is increasing, and the demand for flour and bean products is increasing. If good flour is produced, high-quality raw materials are needed, so that catalytic enzyme needs to be added in food treatment, the catalytic efficiency of the enzyme is particularly high and is more than 100 times higher than that of a common catalyst, the baking quality of bread can be improved, and the quality guarantee period of products can be prolonged.
The protease is an enzyme which can hydrolyze peptide bonds in protein and generate amino acids or small peptides, is one of three enzyme preparations in the world, accounts for 60 percent of the global enzyme preparation sales amount, is mainly derived from animals, plants and microorganisms, generally does not contain toxic and harmful substances, can be inactivated after the hydrolysis reaction is completed, and therefore, has wide application in the food industry. The acidic proteinase is an enzyme suitable for hydrolyzing protein in an acidic environment, is mainly used for fur softening and unhairing treatment, can also be used for low-temperature dyeing of wool and beer clarification process, and has the functions of diminishing inflammation, relieving cough, reducing phlegm and promoting digestion. In the process of beer clarification, protease is required to be added to degrade suspended matters in beer so as to improve the clarity of the beer, and in addition, the utilization rate of a substrate can be improved, and the cost is reduced.
The invention researches the separation and purification of lipase, acid protease and alkaline protease, provides a method for synchronously purifying three proteins of lipase, acid protease and alkaline protease, and has the advantages of simple operation, less purification steps and relatively good purification effect. The invention is rarely reported in each document and patent, has certain innovativeness and has good industrial application prospect.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for synchronously purifying lipase, acid protease and alkaline protease, which adopts ammonium sulfate salting-out and Q Beads 6FF anion chromatography to purify three proteins, and the protein purification technology is carried out by the following steps:
step one, determining the saturation of ammonium sulfate corresponding to three kinds of protein when the three kinds of protein are separated out by an ammonium sulfate salt separation method;
removing impurities from the protein precipitated by 50% ammonium sulfate and 50% ammonium sulfate, then supplementing the protein precipitated by 80% ammonium sulfate to a Q Beads 6FF anion exchange column, eluting by using 50-400mM NaCl solution, collecting eluates of each component, and measuring protein concentration and enzyme activity;
and step three, performing SDS-PAGE electrophoresis on the collected enzyme solution to determine the purification effects of the three proteins.
Preferably, in the step one, ammonium sulfate precipitation proteins with different saturation degrees of 40-80% are prepared, and the enzyme solution after salting out needs dialysis to remove salt.
Preferably, the NaCl solution in step two is prepared by using a buffer solution corresponding to the dissolution of different proteins, and filtered by a 0.45 μm filter membrane before being loaded on the column.
Preferably, the flow rate in the elution process in the second step is controlled to be 1mL/min, and the collected eluate is subjected to SDS-PAGE electrophoresis detection after concentration.
Compared with the prior art, the invention has the following beneficial effects: the method for synchronously purifying the lipase, the acid protease and the alkaline protease is simple and easy to operate, has relatively few purification steps, and finally improves the purification multiple of the lipase by 63.24 times, the purification multiple of the acid protease by 10.33 times and the purification multiple of the alkaline protease by 31.69 times.
Drawings
Other features, objects and advantages of the present application will become more apparent upon reading of the following detailed description of non-limiting embodiments thereof, made with reference to the accompanying drawings in which:
FIG. 1 is a graph showing fractional salting-out of ammonium sulfate;
FIG. 2 is a crude enzyme solution electrophoretogram;
FIG. 3 is a salting-out electrophoretogram of ammonium sulfate with different saturation degrees;
FIG. 4 is an electrophoretogram of purified lipase, acid protease and alkaline protease.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The drawings are only for purposes of illustration and are not intended to be limiting, certain elements of the drawings may be omitted, enlarged or reduced to better illustrate the embodiments of the present invention, and do not represent the size of the actual product, and it is understood that some well-known structures, elements and descriptions thereof in the drawings may be omitted for persons skilled in the art.
In the description of the present invention, it should be further noted that, unless otherwise explicitly stated or limited, the terms "disposed" and "connected" should be interpreted broadly, and may be, for example, a fixed connection, a movable connection, a detachable connection, an integrated connection, a mechanical connection, an electrical connection, a direct connection, an indirect connection via an intermediate medium, and a communication between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations. The present application will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
Referring to FIGS. 1-4, a method for simultaneous purification of lipase, acid protease and alkaline protease using ammonium sulfate salting-out and Q Beads 6FF anion chromatography for three protein purification techniques by the following steps:
step one, determining the saturation of ammonium sulfate corresponding to three kinds of protein when the three kinds of protein are separated out by an ammonium sulfate salt separation method;
removing impurities from the protein precipitated by 50% ammonium sulfate and 50% ammonium sulfate, then supplementing the protein precipitated by 80% ammonium sulfate to a Q Beads 6FF anion exchange column, eluting by using 50-400mM NaCl solution, collecting eluates of each component, and measuring protein concentration and enzyme activity;
and step three, performing SDS-PAGE electrophoresis on the collected enzyme solution to determine the purification effects of the three proteins.
Wherein, in the step one, 40-80% of ammonium sulfate precipitation protein with different saturation degrees needs to be prepared, and the enzyme solution after salting out needs to be dialyzed for desalting.
Wherein, the NaCl solution in the second step needs to be prepared by buffer solution corresponding to different proteins, and the NaCl solution needs to be filtered by a filter membrane of 0.45 μm before being loaded on the column.
And controlling the flow rate to be 1mL/min in the elution process in the step two, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection on the collected eluent after concentration.
The invention adopts ammonium sulfate salting-out and Q Beads 6FF anion chromatography to purify three proteins. The protein purification technology is carried out by the following steps: (1) mixing enzyme powder: diluting buffer solution at a ratio of 1:10, mixing, centrifuging at 8,000rpm for 5min, adding ground ammonium sulfate powder into the supernatant, standing for 3h, centrifuging at 8,000rpm for 5min, performing gradient salting-out, and collecting the supernatant and precipitate of each gradient, and desalting in dialysis bag.
The enzyme solution after salting out with 5 times of column volume is loaded on a Q column at the flow rate of 1mL/min (the enzyme solution is filtered and purified by a 0.22 mu m filter head before loading).
Preparing 50-400mM NaCl solution, eluting the Q column according to 5 times of column volume gradient, collecting the eluent of each gradient, and performing enzyme activity and protein determination after concentration.
The concentrated solution was subjected to SDS-PAGE, and the effect of the purified protein was observed.
Wherein the enzyme powder in the step (1) is provided by Jiangsu Youyou Biotech limited, and the buffer solution is selected from the following components: after optimization, the buffer for purifying lipase was phosphate buffer (pH 7.5), and the buffer for purifying acidic protease and alkaline protease was Tris-HCl (pH 7.6). The saturation degree of ammonium sulfate selected by the purified lipase is 50%, and the saturation degree of acidic protease and alkaline protease is 80% (50% ammonium sulfate is removed and then is supplemented to 80%).
Wherein the NaCl solution in the step (3) is prepared by using a corresponding buffer solution. The collected eluate was concentrated by centrifugation for 20min using a 3kDa molecular weight cut-off concentration tube.
It should be noted that the enzyme powder of the present invention is provided by yoyo biotechnology limited of Jiangsu, and the ion chromatography packing (Q Beads 6FF) in the protein purification process is purchased from Changzhou Tiandi people and company. The enzyme activity of the three enzymes is determined by adopting a test method published by the national standard of the people's republic of China, and is described in GB/T23535-.
Example 1
Weighing 1g of enzyme powder and a corresponding buffer solution (the buffer solution adopted by the purified lipase is phosphate buffer solution with pH 7.5, and the buffer solution for purifying the acidic protease and the alkaline protease is Tris-HCl with pH 7.6), uniformly mixing according to a ratio of 1:10, centrifuging at 8,000rpm for 5min, adding pre-ground ammonium sulfate powder into supernatant according to 40-80% of saturation (slowly stirring while adding), standing at 4 ℃ for 4h, centrifuging, collecting precipitate, dissolving the precipitate in the corresponding buffer solution again, putting a dialysis bag with molecular weight cutoff of 3kDa, clamping two ends, putting the precipitate into the buffer solution, dialyzing at 4 ℃ for 12h, collecting enzyme solutions of each component after dialysis, determining enzyme activity of the precipitate and the supernatant, and drawing a ammonium sulfate fractional salting-out graph. And finally collecting enzyme liquid precipitated by the ammonium sulfate with each saturation degree, and carrying out SDS-PAGE electrophoresis to determine the corresponding ammonium sulfate saturation degrees when three target proteins are separated out, wherein the results are shown in figures 1 and 3.
This example illustrates that when the ammonium sulfate saturation reaches 50%, there is precipitation and enzyme activity is detected in the supernatant, the enzyme activity of the precipitated fraction increases with increasing ammonium sulfate saturation, and the enzyme activity of the supernatant decreases with increasing ammonium sulfate saturation. When the saturation degree of ammonium sulfate reaches 80%, the enzyme activity in the precipitate reaches the maximum value, and the enzyme activity in the supernatant is hardly detected. Therefore, it was confirmed that all proteins in the enzyme solution could be extracted by 80% saturation with ammonium sulfate.
Example 2
The dialyzed crude enzyme solution (0.45 μm filter) was applied to a Q Beads 6FF ion chromatography column equilibrated with buffer, and the flow rate was controlled at 1 mL/min. Then gradient elution is carried out by 5 times of column volume of 50-400mM NaCl solution (corresponding buffer solution configuration), the flow rate is controlled at 1mL/min, and the eluates of each gradient are collected and put into a dialysis bag to be kept stand for 10h at 4 ℃ for dialysis and desalination. After dialysis, loading into a concentration tube with molecular weight cutoff of 3kDa, centrifuging at 8,000rpm for 20min, collecting concentrated solution, and performing protein concentration and enzyme activity determination and SDS-PAGE electrophoresis to observe protein purification effect.
This example illustrates that lipase was salted out by 50% ammonium sulfate, eluted with 50mM NaCl, and the most purified after concentration, with a purification factor of 63.24; salting out the protease by 80% ammonium sulfate, eluting by 300mM NaCl, and concentrating to obtain the best purification effect, wherein the purification multiple reaches 10.33 times; the amylase is eluted by 350mM NaCl, and the purification effect is best after concentration, and the purification multiple reaches 31.69 times. The detailed data are shown in the following table:
TABLE 1 purification of Lipase
TABLE 2 purification of proteases
TABLE 3 purification of Amylase
Example 3
The purified three enzyme solutions were subjected to SDS-PAGE electrophoresis, and the kit was purchased from Shanghai, and the results are shown in FIG. 4, wherein: 1, hole: marker, 2 holes: crude enzyme solution, 3 wells: salting out with 50% ammonium sulfate, 4-hole: removing impurities with 50% ammonium sulfate, supplementing to 80% for salting out, and performing 5-hole: lipase, 6 wells: acid protease, 7-well: an alkaline protease;
this example shows that, through SDS-PAGE electrophorograms, we can find that three enzymes, lipase, acid protease and alkaline protease, have better purification effect, single and obvious band, and no serious impurity protein pollution, and by calculation, we can obtain: the relative molecular mass of the lipase is about 36.2kDa, the relative molecular mass of the acid protease is about 61.7kDa, and the relative molecular mass of the alkaline protease is about 49.8 kDa.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. A method for synchronously purifying lipase, acid protease and alkaline protease, which adopts ammonium sulfate salting-out and Q Beads 6FF anion chromatography to purify three proteins, and is characterized in that the protein purification technology is carried out by the following steps:
step one, determining the saturation of ammonium sulfate corresponding to three kinds of protein when the three kinds of protein are separated out by an ammonium sulfate salt separation method;
removing impurities from the protein precipitated by 50% ammonium sulfate and 50% ammonium sulfate, then supplementing the protein precipitated by 80% ammonium sulfate to a Q Beads 6FF anion exchange column, eluting by using 50-400mM NaCl solution, collecting eluates of each component, and measuring protein concentration and enzyme activity;
and step three, performing SDS-PAGE electrophoresis on the collected enzyme solution to determine the purification effects of the three proteins.
2. The method for simultaneously purifying lipase, acid protease and alkaline protease according to claim 1, wherein: in the first step, 40-80% ammonium sulfate precipitation protein with different saturation degrees needs to be prepared, and the enzyme solution after salting out needs to be dialyzed to remove salt.
3. The method for simultaneously purifying lipase, acid protease and alkaline protease according to claim 1, wherein: in the second step, the NaCl solution is prepared by using a buffer solution corresponding to the dissolution of different proteins, and is filtered by a 0.45-micron filter membrane before being loaded on the column.
4. The method for simultaneous purification of lipase, acid protease and alkaline protease according to claim 1, characterized in that: and controlling the flow rate to be 1mL/min in the elution process in the step two, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection on the collected eluent after concentration.
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