CN107586761B - Post-extraction process for producing liver detoxification enzyme by pichia pastoris - Google Patents
Post-extraction process for producing liver detoxification enzyme by pichia pastoris Download PDFInfo
- Publication number
- CN107586761B CN107586761B CN201710955530.5A CN201710955530A CN107586761B CN 107586761 B CN107586761 B CN 107586761B CN 201710955530 A CN201710955530 A CN 201710955530A CN 107586761 B CN107586761 B CN 107586761B
- Authority
- CN
- China
- Prior art keywords
- liver detoxification
- detoxification enzyme
- pichia pastoris
- post
- extraction process
- 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.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides a post-extraction process for producing liver detoxification enzyme by pichia pastoris, which comprises the following steps: the method comprises the steps of sequentially carrying out centrifugation, ultrafiltration, cation exchange chromatography column chromatography, elution, concentration and spray drying on pichia pastoris fermentation liquor to obtain the liver detoxification enzyme. The method has the advantages of simple operation, environmental protection and low energy consumption, and does not need to use a large amount of inorganic salt compared with a salting-out method. The extracted liver detoxification enzyme has high purity and good stability.
Description
Technical Field
The invention relates to a post-extraction process of enzyme, in particular to a post-extraction process for producing liver detoxification enzyme by pichia pastoris, and belongs to the technical field of enzyme engineering.
Background
The extraction of the liver detoxification enzyme refers to the process of extracting the liver detoxification enzyme from the liver detoxification enzyme fermentation liquor. Because a large amount of culture medium components are required to be added in the fermentation process of the pichia pastoris, and other byproducts are also generated in the fermentation process of the pichia pastoris, a series of processes such as salting out, organic solution precipitation and the like are required in the process of extracting and purifying the liver detoxification enzyme.
The traditional method needs to consume a large amount of inorganic salt, has high energy consumption and is not environment-friendly; the prepared liver detoxification enzyme has high impurity protein content, and the structure of the liver detoxification enzyme is extremely easy to damage in the extraction process, so that the loss rate is high, and the extraction efficiency is low.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a post-extraction process for producing liver detoxification enzyme by pichia pastoris, so as to realize the following purposes:
(1) the energy consumption is reduced, and the problem that a large amount of inorganic salt is required to be used for salting out in the prior art is solved;
(2) improving the purity of the prepared liver detoxification enzyme;
(3) reducing the loss rate of the liver detoxification enzyme in the extraction process.
In order to solve the technical problems, the following technical scheme is adopted:
a post-extraction process for producing liver detoxification enzyme by pichia pastoris, which comprises the following steps: the pichia pastoris fermentation liquor is sequentially subjected to centrifugation, ultrafiltration, cation exchange chromatography column passing, elution, concentration and spray drying to obtain the liver detoxification enzyme dry powder.
The centrifugation: centrifuging the pichia pastoris fermentation liquor by adopting a centrifuge, and collecting supernatant; the centrifugation: the rotating speed is 15000 and 20000 rpm/min, and the centrifugal efficiency is 2.5-3.5T/h.
The ultrafiltration: and (3) carrying out ultrafiltration on the centrifugal supernatant through a 100kda hollow fiber column, and then carrying out ultrafiltration through a 10kda hollow fiber column to obtain the liver detoxification enzyme solution.
The cation exchange chromatography column: equilibrating the cation exchange chromatography column with phosphate buffer to ph 6.0; adjusting pH of the liver detoxification enzyme solution to 6.0, and passing through a cation exchange chromatography column with flow rate controlled at 1.5-2.5 BV/h.
The elution is as follows: eluting the ion exchange column with 7.0 pH potassium dihydrogen phosphate-sodium hydroxide buffer solution, and collecting eluate when OD280 of the eluate reaches 0.08; eluting with glycine-sodium hydroxide buffer solution with pH of 9.0 to obtain ion exchange column, and collecting eluate.
The concentration: concentrating with reverse osmosis membrane with pore diameter of 0.0001um under 1.1 MPa.
The spray drying: and sequentially adding skim milk, trehalose and sorbitol into the obtained liver detoxification enzyme concentrated solution, wherein the mass ratio of the skim milk to the trehalose to the sorbitol is 1:1: 1.
Ultrafiltration is carried out on the 100kda hollow fiber column: the height of the hollow fiber column is 2360mm, the diameter is 225mm, and the ultrafiltration speed is 2T/h.
And (3) ultrafiltration by using a 10kda hollow fiber column: the height of the hollow fiber column is 2280mm, the diameter is 160mm, and the ultrafiltration speed is 1.2T/h.
The prepared liver detoxification enzyme dry powder comprises the following components: the titer is above 840000U/g.
Compared with the prior art, the invention has the beneficial effects that:
(1) the method has the advantages of simple operation, environmental protection and low energy consumption, and does not need to use a large amount of inorganic salt compared with a salting-out method.
(2) The purity of the extracted liver detoxification enzyme is high, and the content of the hybrid protein in the liver detoxification enzyme is lower than 2.5 percent; compared with the traditional organic solvent precipitation method, the content of impurity protein in the obtained liver detoxification enzyme is reduced by about 10 percent.
(3) The extraction process can well protect the stability of the liver detoxification enzyme, avoid the damage of high temperature to the liver detoxification enzyme, and the loss rate of the liver detoxification enzyme in the extraction process is lower than 1 percent; while the conventional process results in a loss of 5%.
(4) The titer of the liver detoxification enzyme dry powder extracted by the extraction process is above 840000U/g.
Detailed Description
Example 1 post-extraction process for producing liver detoxification enzyme by pichia pastoris
The production process of the liver detoxification enzyme comprises the preparation of liver detoxification enzyme fermentation liquor and the post-extraction process of producing the liver detoxification enzyme by pichia pastoris;
the preparation of the liver detoxification enzyme fermentation liquor comprises the following steps:
firstly, culturing an activated and preserved recombinant pichia pastoris strain to prepare a fermentation seed solution, and then transferring the fermentation seed solution into a 15T fermentation culture medium according to 15% of inoculation amount, wherein the content of effective components of the fermentation culture medium is as follows: yeast extract 20g/L, peptone 13.3g/L, biotin 6.7ug/L, glycerol 46.7mL/L, K2HPO 410 g/L; performing high-density fermentation for 144h to obtain the pichia pastoris fermentation liquor.
The adopted strain is recombinant pichia pastoris containing liver detoxification enzyme gene, belongs to transgenic engineering bacteria, and the preservation number of the recombinant pichia pastoris is CCTCC M2017278.
Through detection, the OD600=2.1 of the obtained pichia pastoris fermentation liquor, the concentration is 368g/L, and the titer of the liver detoxification enzyme is 40000U/g.
The post-extraction process for producing the liver detoxification enzyme by pichia pastoris, namely the process for extracting the liver detoxification enzyme from the pichia pastoris fermentation liquor, comprises the following steps:
step 1 centrifugation
Centrifuging 15T of pichia pastoris fermentation liquor at high speed by adopting a drum centrifuge, wherein the centrifugal rotating speed is 18000rpm/min, the fermentation liquor centrifugal efficiency in the whole centrifugal process is 3T/h, removing thalli and fermentation liquor residues, and collecting supernatant, wherein the supernatant is about 12T, namely four fifths of the volume of the fermentation liquor; the detection shows that the liver detoxification enzyme titer of the supernatant is 50000U/g.
Step 2 Ultrafiltration
Taking the supernatant collected in the step 1, and firstly carrying out ultrafiltration by using a 100kda hollow fiber column: the height is 2360mm, the diameter is 225mm, the flow rate of the supernatant fluid passing through the hollow fiber column is 2T/h, and macromolecular heteroprotein in the liver detoxification enzyme solution is removed;
ultrafiltering the obtained filtrate with 10kda hollow fiber column to remove small molecular foreign protein in the liver detoxification enzyme solution, wherein the height is 2280mm, the diameter is 160mm, the flow rate of the supernatant passing through the hollow fiber column is 1.2T/h, and collecting the filtrate to obtain about 11.3T liver detoxification enzyme solution; the detection proves that the liver detoxification enzyme titer of the liver detoxification enzyme solution is 53100U/g.
Step 3 passing through cation exchange chromatographic column
Preparing a CM Sepharose Fast Flow cation exchange chromatographic column;
the CM Sepharose Fast Flow cation exchange column was equilibrated with 0.2mol/L phosphate buffer until the effluent had a ph of 6.0.
Taking the liver detoxification enzyme solution prepared in the step 2, and adjusting the ph of the liver detoxification enzyme solution to 6.0 by using a phosphate buffer solution with the concentration of 0.2 mol/L; then, pumping the liver detoxification enzyme solution with ph =6.0 into a balanced CM Sepharose Fast Flow cation exchange chromatographic column, and controlling the Flow rate at 2 BV/h; the liver detoxification enzymes may be bound to an ion exchange column, through which most of the rest of the molecules may pass directly.
(in ion exchange, BV/h is usually a representation of the space flow rate, i.e.the average amount of liquid flowing through a unit volume of resin per unit time (h) in the column, sometimes referred to as specific volume (sv) or resin Bed Volume (BV) in short times.)
Step 4 elution
Firstly, eluting the ion exchange column by using a potassium dihydrogen phosphate-sodium hydroxide buffer solution with the ph of 7.0, wherein the elution speed of the buffer solution is 50 ml/min; when OD280 of the eluent reaches 0.08, the elution is finished, and the impurity protein is washed away, and the eluent is not collected in the process. Then eluting the ion exchange column by using a glycine-sodium hydroxide buffer solution with ph of 9.0, and collecting eluent; when the OD280 of the eluate was below 0.08, the eluate collection was complete. The collected eluent is high-purity liver detoxification enzyme solution, and is about 3.4T. The liver detoxification enzyme titer of the eluent is 154000U/g through detection.
Step 5 concentration
And (4) concentrating the high-purity liver detoxification enzyme solution collected in the step (4) by using a reverse osmosis membrane to remove partial water molecules and inorganic salts in the liquid, wherein the aperture of the reverse osmosis membrane is 0.0001um, the pressure is controlled to be 1.1Mpa, and the concentration is carried out for 3 hours to obtain 760kg of liver detoxification enzyme concentrated solution. The detection shows that the liver detoxification enzyme titer of the liver detoxification enzyme concentrated solution is 650000U/g.
Step 6 spray drying
Sequentially adding skim milk, trehalose and sorbitol into the liver detoxification enzyme concentrated solution obtained in the step 5, wherein the adding amount of the skim milk, the trehalose and the sorbitol is 7% of the mass of the liver detoxification enzyme concentrated solution; starting stirring, stirring for 0.5h, and stirring speed is 150 r/min.
Pumping the uniformly stirred materials into a spray drying tower at the speed of 0.12T/h through a screw pump, and carrying out spray drying; temperature of the spray drying: the temperature of the air inlet is 150 ℃, the temperature of the air outlet is 70 ℃, and the evaporated water amount per hour is 100 Kg;
550kg of liver detoxification enzyme dry powder is obtained. The detection shows that the liver detoxification enzyme titer of the liver detoxification enzyme dry powder is 850000U/g.
The liver detoxification enzyme can also be called as aflatoxin detoxification enzyme.
Except for special description, the proportions are mass ratios, and the percentages are mass percentages.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. The foregoing is illustrative of the best mode of the invention, and details not described herein are within the common general knowledge of a person of ordinary skill in the art; the scope of the present invention is defined by the appended claims, and any equivalent modifications based on the technical teaching of the present invention are also within the scope of the present invention.
Claims (3)
1. A post-extraction process for producing liver detoxification enzyme by pichia pastoris is characterized in that: the post-extraction process comprises the following steps: sequentially carrying out centrifugation, ultrafiltration, cation exchange chromatography column chromatography, elution, concentration and spray drying on the pichia pastoris fermentation liquor to obtain liver detoxification enzyme;
the OD600 of the pichia pastoris fermentation liquor =2.1, the concentration is 368g/L, and the titer of the liver detoxification enzyme is 40000U/g;
and (3) ultrafiltration: the centrifugal supernatant is firstly ultrafiltered by a 100kda hollow fiber column which is: the height is 2360mm, the diameter is 225mm, the flow rate of the supernatant fluid passing through the hollow fiber column is 2T/h, and macromolecular heteroprotein in the liver detoxification enzyme solution is removed;
performing ultrafiltration on the obtained filtrate by using a 10kda hollow fiber column, removing micromolecular heteroprotein in the liver detoxification enzyme solution, wherein the height is 2280mm, the diameter is 160mm, the flow rate of the supernatant passing through the hollow fiber column is 1.2T/h, and collecting the filtrate, wherein the liver detoxification enzyme titer of the liver detoxification enzyme solution is 53100U/g;
the cation exchange chromatography column: using 0.2mol/L phosphate buffer solution to balance the CM Sepharose Fast Flow cation exchange chromatographic column until the pH value of the effluent is 6.0; adjusting the pH value of the liver detoxification enzyme solution to 6.0 by using a phosphate buffer solution with the concentration of 0.2 mol/L; then passing through a cation exchange chromatographic column, and controlling the flow rate at 2 BV/h;
and (3) centrifuging: centrifuging the pichia pastoris fermentation liquor at high speed by adopting a drum centrifuge, wherein the centrifugal rotating speed is 18000rpm, the centrifugal efficiency of the fermentation liquor in the whole centrifugation process is 3T/h, removing thalli and fermentation liquor residues, and collecting supernatant, wherein the liver detoxification enzyme titer of the supernatant is 50000U/g;
and (3) eluting: eluting the ion exchange column with potassium dihydrogen phosphate-sodium hydroxide buffer solution with pH of 7.0, wherein the elution speed of the buffer solution is 50ml/min, and the elution is not collected when OD280 of the eluate reaches 0.08; eluting the ion exchange column by using a glycine-sodium hydroxide buffer solution with the pH value of 9.0, collecting the eluent, and finishing the collection of the eluent when the OD280 of the eluent is lower than 0.08; the liver detoxification enzyme titer of the eluent is 154000U/g;
and (3) concentrating: concentrating with reverse osmosis membrane with pore diameter of 0.0001um under 1.1Mpa for 3 hr, wherein the liver detoxification enzyme titer of the concentrated solution is 650000U/g.
2. The post-extraction process for producing liver detoxification enzyme by pichia pastoris, according to claim 1, wherein the post-extraction process comprises the following steps: the spray drying: and sequentially adding skim milk, trehalose and sorbitol into the obtained liver detoxification enzyme concentrated solution, wherein the mass ratio of the skim milk to the trehalose to the sorbitol is 1:1: 1.
3. The post-extraction process for producing liver detoxification enzyme by pichia pastoris, according to claim 1, wherein the post-extraction process comprises the following steps: the prepared liver detoxification enzyme dry powder comprises the following components: the titer is above 840000U/g.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710955530.5A CN107586761B (en) | 2017-10-14 | 2017-10-14 | Post-extraction process for producing liver detoxification enzyme by pichia pastoris |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710955530.5A CN107586761B (en) | 2017-10-14 | 2017-10-14 | Post-extraction process for producing liver detoxification enzyme by pichia pastoris |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107586761A CN107586761A (en) | 2018-01-16 |
| CN107586761B true CN107586761B (en) | 2021-03-30 |
Family
ID=61053469
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710955530.5A Active CN107586761B (en) | 2017-10-14 | 2017-10-14 | Post-extraction process for producing liver detoxification enzyme by pichia pastoris |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107586761B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050260704A1 (en) * | 2004-05-20 | 2005-11-24 | National Research Development Corporation | Novel thrombolytic enzyme and a process for its preparation |
-
2017
- 2017-10-14 CN CN201710955530.5A patent/CN107586761B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050260704A1 (en) * | 2004-05-20 | 2005-11-24 | National Research Development Corporation | Novel thrombolytic enzyme and a process for its preparation |
Non-Patent Citations (3)
| Title |
|---|
| 乳糖酶基因在毕赤酵母中的表达鉴定发酵工艺优化及发酵液后处理的研究;苏全;《中国优秀硕士学位论文全文数据库 基础科学辑》;20080315(第03期);A006-64页 * |
| 基因工程α-半乳糖苷酶的制备及其性质研究;高新等;《生物工程学报》;20030331;第19卷(第2期);223-226页 * |
| 黄曲霉毒素解毒酶基因的克隆与表达研究;丁炜;《中国优秀硕士学位论文全文数据库 基础科学辑》;20101215(第12期);A006-39页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107586761A (en) | 2018-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9139856B2 (en) | Process for production of galactooligosaccharides (GOS) | |
| CN113150120B (en) | Separation and purification method of porcine myoglobin in fermentation liquor | |
| CN109486876A (en) | A method of threonine is extracted and is purified in fermentation | |
| CN102276849A (en) | Method for extracting gutta-percha from folium cortex eucommiae | |
| CN103614398B (en) | The encoding gene of cis-Epoxysuccinic acid hydratase, the polypeptide of its coding and related application thereof | |
| CN108070032A (en) | A kind of purification process of recombination human source collagen | |
| CN104073537A (en) | Preparation method for active collagen from compound protease | |
| CN104726478A (en) | Recombinant Escherichia coli for expressing arginine deiminase gene and application of recombinant Escherichia coli | |
| CN104974032A (en) | Method of separation and extraction of D-lactic acid from sodium D-lactate fermentation liquid | |
| CN101665448B (en) | Method for extracting coronatine from fermentation liquor by using membrane separation technique | |
| CN107586761B (en) | Post-extraction process for producing liver detoxification enzyme by pichia pastoris | |
| CN110628744A (en) | Method for separating and purifying esterifying enzyme from strong aromatic yeast | |
| CN110607329A (en) | Method for producing propionic acid by fermentation | |
| CN108774273B (en) | Trehalose crystallization process | |
| CN103305575A (en) | Method for preparing active collagen from compound protease | |
| CN103992992A (en) | Coding gene of (+) gamma-lactamase in Sulfolobus solfataricus P2, and application thereof | |
| CN102127128A (en) | New process for extracting stevioside by utilizing enzyme coupling membrane separation technology | |
| CN103539688B (en) | A kind of method of separation and Extraction Serine from Corynebacterium glutamicum fermented liquid | |
| US7572612B2 (en) | Method of production of para-hydroxycinnamic acid using a thermostable TAL enzyme | |
| CN108570098B (en) | Separation and purification method of multiple antigen components of pertussis | |
| CN110527690A (en) | A kind of heat resistant type tannase and its application | |
| CN108315315B (en) | Method for preparing ferulic acid decarboxylase | |
| CN111139275B (en) | Improved method for preparing mussel oligosaccharide by using yeast | |
| CN107827976B (en) | A kind of ulinastatin purification process based on drainage column | |
| CN115927517A (en) | Preparation process of humanized collagen |
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 |