CN113150120A - Method for separating and purifying porcine myoglobin in fermentation liquor - Google Patents
Method for separating and purifying porcine myoglobin in fermentation liquor Download PDFInfo
- Publication number
- CN113150120A CN113150120A CN202110558529.5A CN202110558529A CN113150120A CN 113150120 A CN113150120 A CN 113150120A CN 202110558529 A CN202110558529 A CN 202110558529A CN 113150120 A CN113150120 A CN 113150120A
- Authority
- CN
- China
- Prior art keywords
- myoglobin
- fermentation liquor
- fermentation
- porcine
- tris
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
Abstract
The invention discloses a method for separating and purifying porcine myoglobin in fermentation liquor, belonging to the technical field of biology. The method comprises the steps of creatively adding ammonium sulfate with different concentrations step by step into fermentation liquor, concentrating the fermentation liquor of recombinant pichia pastoris for expressing the porcine myoglobin, further performing ultrafiltration concentration on the fermentation liquor, and purifying the concentrated solution to obtain the myoglobin with the purity of 88.04 percent and the purification rate of 66.05 percent, thereby realizing the purification of the porcine myoglobin from the fermentation liquor, solving the problem that the prior porcine myoglobin fermentation liquor is difficult to purify, and realizing the high-efficiency purification of the porcine myoglobin fermentation liquor. The separation and purification method has the advantages of high specificity, mild operation conditions, simple steps, good purification effect, high recovery rate, low cost and high speed, and is beneficial to expanded production.
Description
Technical Field
The invention relates to a method for separating and purifying porcine myoglobin in fermentation liquor, belonging to the technical field of biology.
Background
Myoglobin is a single subunit heme protein found in muscle in some invertebrates, and in cardiac and skeletal muscle in most vertebrates. Myoglobin can be reversibly combined with oxygen in muscle tissue sarcoplasm, has the functions of transporting and storing oxygen, and endows the muscle tissue with bright red color. In recent years, with the development of meat analogue technology, the addition of myoglobin can make the meat analogue more realistically simulate the colour of real meat.
At present, affinity chromatography, ion exchange chromatography and gel filtration chromatography are commonly used for separating and purifying proteins from fermentation liquor, and most of the methods rely on AKTA protein purifiers. In most reports, purification using a His tag in affinity chromatography is most commonly performed by binding a target protein to nickel ions and then eluting the resulting product with imidazole. Any purification tag commonly used in enzyme preparations cannot be used in food processing, so purification with His-tag cannot be performed. Thus, conventional protein purification methods cannot be directly applied to the separation of food-grade proteins.
The purification of protein firstly solves the problem of the concentration of the fermentation liquor. When the pichia pastoris recombinant bacteria are used for fermentation production of the porcine myoglobin, the fermentation liquor contains the defoaming agent, and the defoaming agent can cause irreversible dissolution reaction on most ultrafiltration membranes, so that the target protein cannot be concentrated. Secondly, the problems of protein degradation, easy degradation of the porcine myoglobin and industrial large-scale purification production are solved.
Disclosure of Invention
In order to solve the problems of the separation and purification of fermentation liquor obtained by fermenting recombinant pichia pastoris at present, the invention creatively improves the separation and purification process. Aiming at the problem of fermentation liquor concentration, the fermentation liquor contains a defoaming agent, and the defoaming agent can cause irreversible dissolution reaction on most ultrafiltration membranes, so that the target protein cannot be concentrated. The ultrafiltration cups are therefore not suitable for the concentration of porcine myoglobin. Salting-out can achieve the purpose of concentrating the fermentation broth, but is not suitable for treating large amounts of fermentation broth because of the loss caused during salting-out. In addition, concentration of porcine myoglobin using Vivaflow 200 ultrafiltration membrane membranes was attempted, which allows for rapid concentration of the broth and effectively avoids loss of protein. In order to purify porcine myoglobin with high efficiency, three food-grade purification methods without affinity tags are tried in the patent. The following three types can be mainly classified: salting out-desalting-gel filtration chromatography (AKTA), salting out-desalting-DEAE anion exchange chromatography (AKTA), and ultrafiltration concentration-Q anion exchange chromatography (gravity column). However, the AKTA protein purification apparatus has a low throughput and a low purification efficiency, and is therefore unsuitable for large-scale industrial production. The use of the gravity column can improve the purification treatment capacity, reduce the purification time and avoid the degradation of the porcine myoglobin. The ultrafiltration concentration-Q anion exchange chromatography (gravity column) has the advantages of simple operation, low cost, high purification rate and the like, and can be used for large-scale industrial production.
The first object of the present invention is to provide a method for separating and purifying porcine myoglobin from a microbial fermentation broth, wherein the method comprises the steps of (a) to (c) separating and purifying porcine myoglobin from the fermentation broth;
(a) salting out-desalting-anion exchange method;
(b) salting out, desalting, and gel filtration chromatography;
(c) concentration-anion exchange;
the fermentation liquor is obtained by fermenting recombinant pichia pastoris for expressing the porcine myoglobin, and the NCBI Reference Sequence of the porcine myoglobin is NP-999401.1.
In one embodiment, the recombinant pichia pastoris expresses porcine myoglobin by taking pichia pastoris X33 as a host;
the preparation of the fermentation liquor comprises the following steps:
(1) first-order seed culture: activating the preserved recombinant pichia pastoris, and culturing for 16-18h at the temperature of 15-35 ℃ and the rpm of 100-600;
(2) secondary seed culture: inoculating the primary seed solution into a seed culture medium in an amount of 1-5% by volume, and culturing at 15-35 ℃ and 100-600 rpm to OD600＝6～8；
(3) Fermentation culture: inoculating the seed solution into a fermentation culture medium, culturing in a fermentation tank, controlling DO to be 20-40%, preferably controlling DO to be 30%, stirring at a rotation speed of 200-800rpm, and ventilating at a flow rate of 0.5-2.5 VVM;
(4) preparation of fermentation supernatant: after fermenting for at least 84h, taking the fermentation liquor, centrifuging to remove thalli, carrying out vacuum filtration on the supernatant, and collecting to obtain the fermentation liquor.
In one embodiment, the porcine myoglobin is concentrated by addition of ammonium sulfate.
In one embodiment, ammonium sulfate powder is slowly added into the fermentation supernatant, stirred until the concentration of ammonium sulfate reaches 50-60% saturation, and kept standing for 2 hours at 1-4 ℃; centrifuging at 4 ℃ for 25-35 min at 5000-10000 g, and collecting the supernatant; adding ammonium sulfate powder into the supernatant until the concentration of ammonium sulfate reaches 60-70%, and standing overnight at 1-4 ℃; centrifuging at 5000-10000 g for 25-35 min, and collecting precipitate; the pellet was reconstituted with 10mM Tris-HCl buffer, pH 9.20 to give a concentrate.
In one embodiment, when the salting-out-desalting-anion exchange method is adopted, the obtained concentrated solution is loaded into a desalting column, is equilibrated and eluted by a Tris-HCl buffer solution with the concentration of 10-15 mM and the pH value of 9.0-10.0, and an elution peak before the change of the conductivity is collected by detecting the conductivity, and a desalted sample is collected; loading the obtained desalted sample into an anion exchange column, balancing with 10-15 mM Tris-HCl buffer solution with the pH value of 9.0-10.0, performing gradient elution with 1-2M NaCl buffer solution, and collecting a second elution peak by detecting UV280 nm to obtain the purified porcine myoglobin.
In one embodiment, the anion exchange chromatography packing material is DEAE-Sepharose, Q Beads 6FF, respectively.
In one embodiment, when salting-out-desalting-gel filtration chromatography is adopted, the obtained concentrated solution is loaded into a desalting column, is equilibrated and eluted by a Tris-HCl buffer solution with the concentration of 10-15 mM and the pH value of 9.0-10.0, and an elution peak before the change of the conductivity is collected by detecting the conductivity, and a desalted sample is collected; and loading the obtained desalted sample into a gel filtration chromatographic column, balancing with 10-15 mM Tris-HCl buffer solution with pH of 9.0-10.0, eluting with 10-15 mM Tris-HCl buffer solution with pH of 9.0-10.0, and collecting an elution peak by detecting UV280 nm to obtain the purified porcine myoglobin.
In one embodiment, the packing for gel filtration chromatography is Superdex.
In one embodiment, when the concentration-anion exchange method is adopted, the obtained concentrated solution is further concentrated by using a membrane package, the obtained concentrated supernatant is loaded into an anion exchange column, is balanced by using 10-15 mM Tris-HCl buffer solution with the pH value of 9.0-10.0, is subjected to gradient elution by using 1-2M NaCl buffer solution, and the eluent with the elution concentration of 20% NaCl is collected, namely the myoglobin pure product.
In one embodiment, the membrane package is: vivaflow 200 tangential flow filtration membrane package.
In one embodiment, elution is performed with buffer B solution, and the salinity of buffer B is 10% -90%.
In one embodiment, the myoglobin having a molecular weight greater than 10kDa is intercepted by a membrane.
In one embodiment, the packing material of the desalting column is Sephadex G-25.
In one embodiment, the membrane material of the concentrating membrane is polypropylene.
In one embodiment, the purified porcine myoglobin can be subjected to extraction of hemoglobin and optical detection.
Has the advantages that: the method comprises the steps of creatively adding ammonium sulfate with different concentrations step by step into fermentation liquor, concentrating the fermentation liquor of recombinant pichia pastoris for expressing the porcine myoglobin, further performing ultrafiltration concentration on the fermentation liquor, and purifying the concentrated solution to obtain the myoglobin with the purity of 88.04 percent and the purification rate of 66.05 percent, thereby realizing the purification of the porcine myoglobin from the fermentation liquor, solving the problem that the prior porcine myoglobin fermentation liquor is difficult to purify, and realizing the high-efficiency purification of the porcine myoglobin fermentation liquor.
Drawings
FIG. 1 is the electrophoresis chart of the different salting-out concentrated proteins by ammonium sulfate in example 3.
FIG. 2 is an electrophoresis diagram of a protein purified by desalting-gel filtration chromatography and desalting-DEAE anion exchange chromatography.
FIG. 3 is an electrophoretogram of protein purified by ultrafiltration concentration-Q anion exchange chromatography.
FIG. 4 is a graph showing the comparison of purity and purification rate of porcine myoglobin purified by different methods.
FIG. 5 is a drawing showing the extraction and analysis of hemoglobin from porcine myoglobin.
Detailed Description
Determination of protein content: the detection is carried out by using a Bradford protein concentration determination kit developed by Biyunnan biotechnological research institute, and the specific operation steps refer to the kit use instructions.
BMGY medium composition: 20g peptone, 10g glycerol, 10g yeast extract, 4X 10-4g biotin.
Example 1 construction of recombinant bacteria expressing Pichia pastoris of porcine myoglobin
Construction of the Pastoris X33- α GAP-Mb Strain:
the porcine myoglobin gene (nucleotide sequence shown in SEQ ID NO: 1) is connected to the multiple cloning site of the integrative expression vector pGAPZ alpha A to construct the recombinant plasmid pGAPZ alpha-A-Mb.
The constructed recombinant plasmid pGAPZ alpha-A-Mb is transformed into escherichia coli DH5 alpha, a transformation liquid is coated on an LB plate containing 20 mu g/mLzeocin, a monoclonal is grown at 37 ℃, after colony PCR and sequencing verification is carried out on the monoclonal, a plasmid is extracted from a correct positive clone, the extracted plasmid is respectively transformed into pichia pastoris X33 through an electric transformation method, and the recombinant bacterium P.
EXAMPLE 2 preparation of fermentation broth
The preparation of the fermentation liquor comprises the following steps:
1. first-order seed culture: carrying out streak culture on recombinant Pichia pastoris X33-alpha GAP-Mb stored at the temperature of-80 ℃, selecting a single colony to be inoculated into a YPD seed culture medium, and culturing for 16-18h at the temperature of 30 ℃ and the speed of 220 rpm;
2. secondary seed culture: inoculating the primary seed solution into YPD secondary seed culture medium at 1%, and culturing at 30 deg.C and 220rpm to OD600＝8～10；
3. Fermentation culture: the seed solution was inoculated at 10% inoculum size into a 5L fermentor containing 1.8L of fermentation medium containing 10g/L glycerol and 20mg/L final hemoglobin. Fermenting according to a 30% DO-Stat fermentation strategy, wherein the ventilation rate is 1.5VVM, and the stirring speed is 200-800 rpm. After fermentation for about 12h, glycerol is exhausted, and feeding is started at the moment. The speed of feeding is mainly automatically controlled by DO and stirring, when the DO is greater than 30%, glycerol is fed, the addition of glycerol flow just meets the requirement of thallus growth, and when the DO is less than 30%, the stirring speed is increased (the initial rotating speed is 200rpm, and the highest rotating speed is 800 rpm). Adding heme with final concentration of 50mg/L, 100mg/L, 150mg/L and 200mg/L respectively during feeding glycerol, and fermenting;
4. preparation of fermentation supernatant: after fermenting for at least 84h, taking the fermentation liquor, centrifuging to remove thalli, carrying out vacuum filtration on the supernatant, and collecting.
Example 3 separation and purification of porcine myoglobin in fermentation broth by salting-out-desalting-anion exchange chromatography
Salting out of fermentation liquor: precipitating by one-step method, slowly adding ammonium sulfate powder into the fermentation supernatant, stirring until the ammonium sulfate concentration reaches 30%, 40%, 50%, 60%, 70%, 80% of ammonium sulfate saturation, and standing at 4 deg.C for 2 hr; the pellet was collected by centrifugation at 10000g for 30min at 4 ℃ and reconstituted with 10mM Tris-HCl buffer, pH 9.20, and analyzed by SDS-PAGE validation. FIG. 1 shows the results of salting-out, in which lanes 1-6 correspond to 30% ammonium sulfate, 40% ammonium sulfate, 50% ammonium sulfate, 60% ammonium sulfate, 70% ammonium sulfate, and 80% ammonium sulfate, respectively. As can be seen from the figure, when the concentration of ammonium sulfate is 60%, the porcine myoglobin is salted out in a large amount, and when the concentration of ammonium sulfate is 80%, the porcine myoglobin is not concentrated any more. Therefore, ammonium sulfate of 50% and 70% concentration was selected for two-stage salting-out.
1. Two-stage ammonium sulfate precipitation (salting out): slowly adding ammonium sulfate powder into the fermentation supernatant, stirring until the concentration of ammonium sulfate reaches 50% saturation, and standing at 4 deg.C for 2 hr; centrifuging at 4 deg.C at 10000g for 30min to collect supernatant; adding ammonium sulfate powder into the supernatant until the concentration of ammonium sulfate reaches 70%, and standing overnight at 4 deg.C; centrifuging at 10000g for 30min, and collecting precipitate; the pellet was reconstituted with 10mM Tris-HCl buffer, pH 9.20;
2. desalting: loading the solution obtained in the step 1 into a desalting column Sephadex G-25, balancing and eluting the solution by using 10mM Tris-HCl buffer solution with the pH value of 9.20, and collecting an elution peak before the change of the conductivity by detecting the conductivity;
3. anion exchange chromatography: loading the desalted sample obtained in the step 2 into an anion exchange column DEAE-Sepharose, carrying out equilibration by using 10mM Tris-HCl buffer solution with pH 9.20, carrying out gradient elution by using 1M NaCl buffer solution, and collecting a second elution peak by detecting UV280 nm.
The samples obtained in step 2 and step 3 were analyzed by SDS-PAGE, and the results are shown in FIG. 2, wherein lane 1 shows the desalting results, lane 3 shows the purification results, the purity is 81.60%, and the purification recovery is 48.25%.
Example 4 separation and purification of porcine myoglobin in fermentation broth by salting-out, desalting and gel filtration chromatography
1. Two-stage ammonium sulfate precipitation: the same procedure as in example 3 for ammonium sulfate precipitation;
2. desalting: loading the solution obtained in the step 1 into a desalting column, balancing and eluting with 10mM Tris-HCl buffer solution with pH of 9.20, and collecting an elution peak before conductivity change by detecting conductivity;
3. gel filtration chromatography: the desalted sample obtained in step 2 was loaded on a Superdex gel filtration chromatography column, equilibrated with 10mM Tris-HCl buffer pH 9.20, and eluted with 10mM Tris-HCl buffer pH 9.20, and the peak was collected by detecting UV280 nm.
The samples obtained in step 3 were analyzed by SDS-PAGE, and the results are shown in lane 2 of FIG. 2, with 100% purity and 14.84% recovery.
Example 5 concentration-anion exchange chromatography for isolation and purification of porcine myoglobin from fermentation broth
1. Sample preparation: the pH of the concentrated (two stage ammonium sulphate precipitation step as described in example 3) broth was adjusted to 9.20 with Tris base and the broth was filtered through a 0.45 μm filter. Then the fermentation liquor is inoculated into a Vivaflow 200 membrane-covered reflux system, and myoglobin with the molecular weight of more than 10kDa is intercepted and concentrated. The specific operation steps of membrane package reflux are as follows: the Vivaflow 200 membranes were attached as described to form a reflux system. Then carrying out ultrafiltration concentration under the action of a peristaltic pump to obtain an ultrafiltration concentrated solution.
2. Q anion exchange column packing balance: the Q Beads 6FF anionic packing was column equilibrated with 50 column volumes of buffer A solution.
3. Loading: a sample of 1 column volume was added and the sample flowed down spontaneously due to gravity. And after the liquid is drained, adding buffer A solution to balance the sample.
4. And (3) elution: and (3) carrying out gradient elution on the sample by using buffer B solutions with different concentrations (10% of buffer B and 30% -90% of buffer B respectively), and collecting the eluent.
The eluate was analyzed by SDS-PAGE, and the result is shown in FIG. 4, wherein lane 1 of FIG. A corresponds to the purification result, the purity is 88.04%, and the highest purification rate can reach 66.05%; panel B corresponds to gradient elution, 20% buffer B eluate in lane 2, 10% buffer B (10% salt concentration) and 30% -90% buffer B in lanes 1 and 3-9, respectively.
Example 6 hemoglobin extraction and optical detection
1. The acid acetone method. The purified porcine myoglobin was treated with acidic acetone solution (V)Acetone (II)：VHydrochloric acid3: 100) extraction was carried out for 40 minutes, and then the pH of the solution was adjusted to neutral using 1M NaOH. 3000g, centrifuged for 5 minutes to give a supernatant and acetone was removed using a rotary evaporator. The pH of the resulting solution was adjusted to 5-7 with 1M HCl and precipitation of heme occurred. 3000g, centrifuged for 15 minutes and washed twice with distilled water.
2. Detecting the heme extracted by the above method by optical detection method. And (3) full-wavelength scanning (respectively adding 200 mu L of purified liquid into a 96-well plate, and then performing wavelength scanning at 200-800 nm), and determining a characteristic peak. Then, the corresponding response value was measured under the characteristic peak, and the detection result is shown in fig. 5.
The results show that when the exogenous substrate heme concentration is 150mg/L, the amount of heme on myoglobin binding is at most, and is 0.22mol of heme per mol of porcine myoglobin.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
Jiangsu Donghui Biotechnology Co.,Ltd.
<120> method for separating and purifying porcine myoglobin in fermentation liquor
<130> BAA210623A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 465
<212> DNA
<213> Artificial sequence
<400> 1
atgggtttgt ctgatggtga atggcaattg gttttaaatg tttggggtaa agttgaagct 60
gatgttgcag gtcatggtca agaagttttg atcagattgt ttaaaggtca tccagaaact 120
ttggaaaagt tcgataagtt taaacatttg aagtctgaag atgaaatgaa ggcttcagaa 180
gatttgaaga aacatggtaa cactgttttg acagctttgg gtggtatttt gaaaaagaaa 240
ggtcatcatg aagcagaatt gactccatta gctcaatctc atgcaacaaa gcataagatc 300
cctgttaagt atttggaatt catttctgaa gcaatcatcc aagttttaca atcaaaacat 360
cctggtgact ttggtgctga tgcacaaggt gctatgtcaa aggcattgga attgtttaga 420
aacgatatgg ctgcaaagta caaggaatta ggttttcaag gttaa 465
Claims (10)
1. A method for separating and purifying porcine myoglobin from a microbial fermentation broth, which is characterized in that the porcine myoglobin is separated and purified from the fermentation broth by the method of any one of (a) to (c);
(a) salting out-desalting-anion exchange method;
(b) salting out, desalting, and gel filtration chromatography;
(c) concentration-anion exchange;
the fermentation liquor is obtained by fermenting recombinant pichia pastoris for expressing the porcine myoglobin, and the NCBI Reference Sequence of the porcine myoglobin is NP-999401.1.
2. The method of claim 1, wherein the recombinant pichia pastoris is pichia pastoris X33 used as a host for expressing porcine myoglobin;
the preparation of the fermentation liquor comprises the following steps:
(1) first-order seed culture: activating the preserved recombinant pichia pastoris, and culturing for 16-18h at the temperature of 15-35 ℃ and the rpm of 100-600;
(2) secondary seed culture: inoculating the primary seed solution into a seed culture medium in an amount of 1-5% by volume, and culturing at 15-35 ℃ and 100-600 rpm to OD600＝6～8；
(3) Fermentation culture: inoculating the seed solution into a fermentation culture medium, culturing in a fermentation tank, controlling DO to be 20-40%, stirring at a rotation speed of 200-800rpm, and ventilating at a flow rate of 0.5-2.5 VVM;
(4) preparation of fermentation supernatant: after fermenting for at least 84h, taking the fermentation liquor, centrifuging to remove thalli, carrying out vacuum filtration on the supernatant, and collecting to obtain the fermentation liquor.
3. The method according to claim 1 or 2, wherein the porcine myoglobin is concentrated by adding ammonium sulfate.
4. The method according to claim 3, characterized in that ammonium sulfate powder is slowly added to the fermentation supernatant and stirred until the ammonium sulfate concentration reaches 50-60% saturation, and the mixture is left standing at 1-4 ℃ for 2 hours; centrifuging at 4 ℃ for 25-35 min at 5000-10000 g, and collecting the supernatant; adding ammonium sulfate powder into the supernatant until the concentration of ammonium sulfate reaches 60-70%, and standing overnight at 1-4 ℃; centrifuging at 5000-10000 g for 25-35 min, and collecting precipitate; the pellet was reconstituted with 10mM Tris-HCl buffer, pH 9.20 to give a concentrate.
5. The method of claim 4, wherein when the salting-out-desalting-anion exchange method is used, the obtained concentrated solution is loaded into a desalting column, equilibrated and eluted with 10 to 15mM Tris-HCl buffer solution having pH of 9.0 to 10.0, and subjected to conductivity detection, and an elution peak before a change in conductivity is collected to obtain a desalted sample; loading the obtained desalted sample into an anion exchange column, balancing with 10-15 mM Tris-HCl buffer solution with the pH value of 9.0-10.0, performing gradient elution with 1-2M NaCl buffer solution, and collecting a second elution peak by detecting UV280 nm to obtain the purified porcine myoglobin.
6. The method of claim 4, wherein when salting-out-desalting-gel filtration chromatography is used, the obtained concentrated solution is loaded into a desalting column, equilibrated and eluted with 10 to 15mM Tris-HCl buffer solution having pH of 9.0 to 10.0, and subjected to conductivity detection, and an elution peak before a change in conductivity is collected and collected to obtain a desalted sample; and loading the obtained desalted sample into a gel filtration chromatographic column, balancing with 10-15 mM Tris-HCl buffer solution with pH of 9.0-10.0, eluting with 10-15 mM Tris-HCl buffer solution with pH of 9.0-10.0, and collecting an elution peak by detecting UV280 nm to obtain the purified porcine myoglobin.
7. The method of claim 4, wherein when the concentration-anion exchange method is adopted, the obtained concentrated solution is further concentrated by using a membrane pack, the obtained concentrated supernatant is loaded into an anion exchange column, the anion exchange column is equilibrated by using 10-15 mM Tris-HCl buffer solution with the pH value of 9.0-10.0, then gradient elution is carried out by using 1-2M NaCl buffer solution, and the eluent with the elution concentration of 20% NaCl is collected, namely the myoglobin pure product.
8. The method of claim 7, wherein the membrane package is: vivaflow 200 tangential flow filtration membrane package.
9. The method according to claim 8, wherein elution is performed with buffer B solution, and the salinity of buffer B is 10-90%.
10. The method of claim 8, wherein the myoglobin having a molecular weight greater than 10kDa is intercepted using a membrane module.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110558529.5A CN113150120B (en) | 2021-05-21 | 2021-05-21 | Separation and purification method of porcine myoglobin in fermentation liquor |
| PCT/CN2021/126637 WO2022242033A1 (en) | 2021-05-21 | 2021-10-27 | Genetically engineered bacterium for producing porcine myoglobin, and fermentation and purification of same |
| US17/743,716 US11639515B2 (en) | 2021-05-21 | 2022-05-13 | Genetically engineered strain for producing porcine myoglobin and food-grade fermentation and purification thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110558529.5A CN113150120B (en) | 2021-05-21 | 2021-05-21 | Separation and purification method of porcine myoglobin in fermentation liquor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113150120A true CN113150120A (en) | 2021-07-23 |
| CN113150120B CN113150120B (en) | 2022-09-30 |
Family
ID=76877154
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110558529.5A Active CN113150120B (en) | 2021-05-21 | 2021-05-21 | Separation and purification method of porcine myoglobin in fermentation liquor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113150120B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114409763A (en) * | 2022-03-15 | 2022-04-29 | 湖南华腾制药有限公司 | Purification method of recombinant fibronectin peptide |
| WO2022242033A1 (en) * | 2021-05-21 | 2022-11-24 | 江苏东汇生物科技有限公司 | Genetically engineered bacterium for producing porcine myoglobin, and fermentation and purification of same |
| US11639515B2 (en) | 2021-05-21 | 2023-05-02 | Taixing Dongsheng Bio-Tech Co., Ltd | Genetically engineered strain for producing porcine myoglobin and food-grade fermentation and purification thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63146899A (en) * | 1987-11-17 | 1988-06-18 | Daiichi Rajio Isotope Kenkyusho:Kk | Production of purified myoglobin |
| US5118796A (en) * | 1987-12-09 | 1992-06-02 | Centocor, Incorporated | Efficient large-scale purification of immunoglobulins and derivatives |
| CN1800202A (en) * | 2006-01-20 | 2006-07-12 | 南京师范大学 | Method for extracting purified high ferro myoglobins from cardiac muscle |
| CN101575373A (en) * | 2009-06-12 | 2009-11-11 | 中国人民解放军第三军医大学野战外科研究所 | Preparation method of hemoglobin extract |
| US20100261842A1 (en) * | 2005-09-20 | 2010-10-14 | Nandu Deorkar | Purified vegetarian protein A and process for production thereof |
| CN105745332A (en) * | 2013-09-11 | 2016-07-06 | 非凡食品有限公司 | Secretion of heme-containing polypeptides |
| CN110105445A (en) * | 2019-04-28 | 2019-08-09 | 甘肃省农业科学院农产品贮藏加工研究所 | The extracting method of myoglobins in a kind of beef |
| CN112321720A (en) * | 2020-11-05 | 2021-02-05 | 无锡和邦生物科技有限公司 | Method for purifying exenatide human serum albumin fusion protein |
| CN112592890A (en) * | 2020-12-17 | 2021-04-02 | 江南大学 | Method for promoting proliferation of muscle stem cells |
| WO2021140489A1 (en) * | 2020-01-10 | 2021-07-15 | Intron Biotechnology, Inc. | A method for preparing porcine myoglobin using escherichia coli |
-
2021
- 2021-05-21 CN CN202110558529.5A patent/CN113150120B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63146899A (en) * | 1987-11-17 | 1988-06-18 | Daiichi Rajio Isotope Kenkyusho:Kk | Production of purified myoglobin |
| US5118796A (en) * | 1987-12-09 | 1992-06-02 | Centocor, Incorporated | Efficient large-scale purification of immunoglobulins and derivatives |
| US20100261842A1 (en) * | 2005-09-20 | 2010-10-14 | Nandu Deorkar | Purified vegetarian protein A and process for production thereof |
| CN1800202A (en) * | 2006-01-20 | 2006-07-12 | 南京师范大学 | Method for extracting purified high ferro myoglobins from cardiac muscle |
| CN101575373A (en) * | 2009-06-12 | 2009-11-11 | 中国人民解放军第三军医大学野战外科研究所 | Preparation method of hemoglobin extract |
| CN105745332A (en) * | 2013-09-11 | 2016-07-06 | 非凡食品有限公司 | Secretion of heme-containing polypeptides |
| CN110105445A (en) * | 2019-04-28 | 2019-08-09 | 甘肃省农业科学院农产品贮藏加工研究所 | The extracting method of myoglobins in a kind of beef |
| WO2021140489A1 (en) * | 2020-01-10 | 2021-07-15 | Intron Biotechnology, Inc. | A method for preparing porcine myoglobin using escherichia coli |
| CN112321720A (en) * | 2020-11-05 | 2021-02-05 | 无锡和邦生物科技有限公司 | Method for purifying exenatide human serum albumin fusion protein |
| CN112592890A (en) * | 2020-12-17 | 2021-04-02 | 江南大学 | Method for promoting proliferation of muscle stem cells |
Non-Patent Citations (10)
| Title |
|---|
| GENBANK: "Myoglobin[Sus scrofa]", 《GENBANK》 * |
| 刘立华主编: "《制药工程专业实验》", 31 July 2018 * |
| 周勃等: "从猪血中分离纯化高纯度的猪血红蛋白", 《色谱》 * |
| 唐朝晖主编: "《生命科学综合设计实验指南》", 30 September 2018 * |
| 崔大伟等: "人肌红蛋白基因的克隆、表达及其抗体制备", 《中国生物制品学杂志》 * |
| 张敏等: "高铁含量猪血红蛋白的提取纯化及特性研究", 《食品工业科技》 * |
| 汤祥明等: "猪心肌中高铁肌红蛋白的提取和纯化", 《畜牧与兽医》 * |
| 郝月绰等: "皱褶假丝酵母脂肪酶CRL1在毕赤酵母中的高效表达及其催化合成维生素E醋酸酯", 《现代食品科技》 * |
| 郝木强等: "重组低出血抗凝蛋白在毕赤酵母中的中试发酵工艺研究及其纯化与鉴定", 《生物技术通讯》 * |
| 陶伟娟等: "胞红蛋白酵母表达载体构建与表达纯化", 《药物生物技术》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022242033A1 (en) * | 2021-05-21 | 2022-11-24 | 江苏东汇生物科技有限公司 | Genetically engineered bacterium for producing porcine myoglobin, and fermentation and purification of same |
| US11639515B2 (en) | 2021-05-21 | 2023-05-02 | Taixing Dongsheng Bio-Tech Co., Ltd | Genetically engineered strain for producing porcine myoglobin and food-grade fermentation and purification thereof |
| CN114409763A (en) * | 2022-03-15 | 2022-04-29 | 湖南华腾制药有限公司 | Purification method of recombinant fibronectin peptide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113150120B (en) | 2022-09-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113150120B (en) | Separation and purification method of porcine myoglobin in fermentation liquor | |
| CN102839165B (en) | Gene mutation type recombined protease K and industrialized production method thereof | |
| CN110117601B (en) | Grifola frondosa glucan synthase, encoding gene and application thereof | |
| CN114410635B (en) | Venetian fusarium endogenous U6 promoter and gene editing method based on CRISPR/Cas9 | |
| US11325953B2 (en) | Protein of ‘dangshan suli’ having function of promoting growth of pollen tube, encoding gene PBRTTS1 and use thereof | |
| CN113667682B (en) | YH66-RS11190 gene mutant and application thereof in preparation of L-valine | |
| CN110964096A (en) | Preparation method of recombinant human C-reactive protein | |
| US11639515B2 (en) | Genetically engineered strain for producing porcine myoglobin and food-grade fermentation and purification thereof | |
| CN110862978B (en) | Preparation method of recombinant halophilic archaea protease | |
| CN114181288B (en) | Process for producing L-valine, gene used therefor and protein encoded by the gene | |
| CN114958813B (en) | Alkaline protease SP4, mutant, coding gene and application thereof | |
| CN108034642B (en) | Glucose oxidase CnGOD19 and improved enzyme, gene and application thereof | |
| CN111484557A (en) | Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds | |
| CN111139257B (en) | Pear PMEI protein in-vitro expression method and application thereof | |
| CN114349831A (en) | aspA gene mutant, recombinant bacterium and method for preparing L-valine | |
| WO2022242033A1 (en) | Genetically engineered bacterium for producing porcine myoglobin, and fermentation and purification of same | |
| CN110951716B (en) | Circumscribed alginate lyase VsAly7D, recombinant strain thereof and application thereof | |
| CN113480622A (en) | Method for preparing and purifying recombinant pneumolysin | |
| CN108191971B (en) | Preparation method of recombinant human metallothionein III alpha fragment pure product | |
| CN110468115B (en) | Aspergillus niger cis-epoxy succinate hydrolase gene and application thereof | |
| CN112522234B (en) | Preparation method of restriction endonuclease FseI | |
| CN110452891B (en) | Penicillium expansum cis-epoxy succinate hydrolase gene and application thereof | |
| CN111004318B (en) | Purification method of rhPTH (1-34) protein stock solution | |
| CN115747187B (en) | Recombinase UvsX and expression gene and application thereof | |
| CN114134125B (en) | Preparation method and application of halophilic archaea nitrite reductase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230914 Address after: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province Patentee after: Jiangnan University Patentee after: TAIXING DONGSHENG BIO-TECH CO.,LTD. Address before: 214000 1800 Lihu Avenue, Binhu District, Wuxi, Jiangsu Patentee before: Jiangnan University Patentee before: Jiangsu Donghui Biotechnology Co.,Ltd. |