CN111777680B - Separation and purification process for improving stability of recombinant collagen solution - Google Patents

Separation and purification process for improving stability of recombinant collagen solution Download PDF

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Publication number
CN111777680B
CN111777680B CN202010609943.XA CN202010609943A CN111777680B CN 111777680 B CN111777680 B CN 111777680B CN 202010609943 A CN202010609943 A CN 202010609943A CN 111777680 B CN111777680 B CN 111777680B
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separation
recombinant collagen
purification process
membrane
pichia pastoris
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CN111777680A (en
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杜尔凤
许乔林
闻帅
冯志平
刘宇乾
赵健烽
黄建民
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Zhejiang Zhuji Juyuan Biotechnology Co ltd
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Zhejiang Zhuji Juyuan Biotechnology Co ltd
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]

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  • Health & Medical Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention discloses a separation and purification process for improving the stability of a recombinant collagen solution. The process comprises the steps of centrifugally separating pichia pastoris fermentation liquid, collecting supernatant, adding acid to adjust the pH to 3-4, filtering by a ceramic membrane to remove impurities, concentrating by a nanofiltration membrane to deacidify until the pH is 5.5+/-0.5, and obtaining the high-purity recombinant collagen solution. The method is simple to operate, the purity of the recombinant collagen obtained by separation and purification can reach 97.16%, the stability of the recombinant collagen in aqueous solution and the purity of the product are obviously improved, and the method is suitable for large-scale production by using membrane filtering equipment in the separation and purification process of the recombinant collagen.

Description

Separation and purification process for improving stability of recombinant collagen solution
Technical Field
The invention belongs to the technical field of separation and purification of biological products, and relates to a separation and purification process for improving stability of a recombinant collagen solution.
Background
Collagen is a family of proteins with the most abundant content in animals, and is widely used as a natural biological material in the fields of foods, cosmetics and medical treatment. However, most of the collagen is derived from animal skin, bones and other tissues of animals such as pigs, cattle, fish and the like, and is mainly obtained by physical and chemical treatment such as acid, alkali, heating and the like, the raw material components are complex, the batch diversity is large, the hidden danger of viruses exists, a large amount of acid-base wastewater is generated in the production process, the environmental pollution is serious, the immunogenicity needs to be increased and removed due to the heterology of the animals and the human body, and the further application of the collagen in medicine is limited.
Along with the development of modern DNA recombination technology, various host cells are selected as genetic engineering bacteria for constructing and expressing collagen, so that the defects of animal-derived collagen are overcome. Chinese patent 201110327865.5 constructs a Pichia pastoris genetically engineered strain of recombinant collagen, the strain is fermented and cultured to obtain the recombinant collagen, the fermentation period is 136 hours, and the protein expression quantity is 16g/L. The fermentation liquor is collected after centrifugation, and the supernatant is filtered by a filter membrane and separated and purified by Sephadex G100 gel column chromatography, so that the method has higher cost in large-scale production. Patent application CN104561201a utilizes ultrafiltration and nanofiltration membrane equipment to produce human-like collagen. The membrane filtration process is simple and is suitable for industrial mass production. However, in the process of membrane filtration, the recombinant collagen is separated from the environment of the fermentation broth, circulates at a high speed in the membrane equipment, is difficult to completely maintain the hydrolytic stability, and partial protein is degraded, so that the activity of the product is reduced.
Disclosure of Invention
The invention aims to provide a separation and purification process for improving the stability of a recombinant collagen solution, which is beneficial to mass production by using membrane filtration equipment by reducing the pH value of the recombinant collagen solution and changing the pH environment of the recombinant collagen solution to cause the change of charges and covalent bonds of protein molecules.
The technical scheme for realizing the purpose of the invention is as follows:
a separation and purification process for improving stability of recombinant collagen solution comprises the following specific steps: and (3) centrifugally separating the pichia pastoris fermentation broth, collecting supernatant, adding acid to adjust the pH to 3-4, filtering by a ceramic membrane to remove impurities, concentrating by a nanofiltration membrane, and deacidifying until the pH is 5.5+/-0.5 to obtain the recombinant collagen solution.
The Pichia pastoris is Pichia pastoris, and is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No.5021 in the year 2011, 6 and 29, and fully disclosed in China patent 201110327865.5.
Preferably, the pH is adjusted to 3.2 to 3.7, more preferably 3.5, by adding an acid.
Preferably, the acid is phosphoric acid or acetic acid, preferably phosphoric acid.
The ceramic membrane disclosed by the invention adopts a ceramic membrane conventionally used in the field, and the pore diameter of the membrane core of the ceramic membrane is 20-200 nm, preferably 100nm.
Preferably, the nanofiltration membrane adopts a nanofiltration membrane which is conventionally used in the field, and the pore diameter of the membrane core of the nanofiltration membrane is 3 KD-7 KD, preferably 5KD.
Compared with the prior art, the invention has the following remarkable effects: the invention discovers that the stability of the recombinant collagen is inversely related to the pH for the first time. In the separation and purification process of the recombinant collagen, the pH of the centrifugal supernatant is regulated to 3-4 by adding acid, the pH environment of the recombinant collagen solution is changed, and the change of the electric charge and covalent bonds of protein molecules is caused, so that the recombinant collagen is more stable, the purity of the recombinant collagen is improved, and the large-scale production by using membrane filtering equipment is facilitated.
Detailed Description
In the specific embodiment of the invention, the adopted strain is Pichia pastoris for expressing recombinant collagen, the preservation number is CGMCC NO.5021, the preservation date is 2011, the month is 29, and the preservation unit is China general microbiological culture Collection center of China Committee for culture Collection of microorganisms.
The invention will be further described in detail with reference to specific examples.
Example 1
(1) Centrifuging Pichia pastoris fermentation broth, and collecting supernatant;
(2) Adding phosphoric acid into the supernatant to adjust the pH to 4.0;
(3) Filtering 200L supernatant by washing with 100nm ceramic membrane, and collecting dialysate 300L;
(4) Concentrating and deacidifying the dialysate by adopting a nanofiltration membrane core of 5KD, adding purified water for concentration each time, sampling and measuring pH until the pH is 5.5 to obtain high-concentration concentrated solution 21L, and detecting the purity of the recombinant collagen to be 94.52%.
Example 2
(1) Centrifuging Pichia pastoris fermentation broth, and collecting supernatant;
(2) Adding phosphoric acid into the supernatant to adjust the pH to 3.7;
(3) Filtering 200L supernatant by washing with 100nm ceramic membrane, and collecting dialysate 300L;
(4) Concentrating and deacidifying the dialysate by adopting a nanofiltration membrane core of 5KD, adding purified water for concentration each time, sampling and measuring pH until the pH is 5.5, obtaining high-concentration concentrated solution 20L, and detecting the purity of the recombinant collagen to be 95.83%.
Example 3
(1) Centrifuging Pichia pastoris fermentation broth, and collecting supernatant;
(2) Adding phosphoric acid into the supernatant to adjust the pH to 3.5;
(3) Filtering 200L supernatant by washing with 100nm ceramic membrane, and collecting dialysate 300L;
(4) Concentrating and deacidifying the dialysate by adopting a nanofiltration membrane core of 5KD, adding purified water for concentration each time, sampling and measuring pH until the pH is 5.5, obtaining high-concentration concentrated solution of 20.5L, and detecting the purity of the recombinant collagen to be 97.16%.
Example 4
(1) Centrifuging Pichia pastoris fermentation broth, and collecting supernatant;
(2) Adding phosphoric acid into the supernatant to adjust the pH to 3.2;
(3) Filtering 200L supernatant by washing with 100nm ceramic membrane, and collecting dialysate 300L;
(4) Concentrating and deacidifying the dialysate by adopting a nanofiltration membrane core of 5KD, adding purified water for concentration each time, sampling and measuring pH until the pH is 5.5 to obtain a high-concentration concentrated solution 22L, and detecting the purity of the recombinant collagen to be 95.20%.
Example 5
(1) Centrifuging Pichia pastoris fermentation broth, and collecting supernatant;
(2) Adding phosphoric acid into the supernatant to adjust the pH to 3.0;
(3) Filtering 200L supernatant by washing with 100nm ceramic membrane, and collecting dialysate 300L;
(4) Concentrating and deacidifying the dialysate by adopting a nanofiltration membrane core of 5KD, adding purified water for concentration each time, sampling and measuring pH until the pH is 5.5, obtaining high-concentration concentrated solution 20L, and detecting the purity of the recombinant collagen to be 94.81%.
Comparative example
(1) The Pichia pastoris fermentation broth is centrifuged and the supernatant is collected.
(2) Filtering 200L supernatant by washing with 100nm ceramic membrane, and collecting dialysate 300L;
(3) Concentrating and deacidifying the dialysate by adopting a nanofiltration membrane core of 5KD, adding purified water for concentration each time, sampling and measuring pH until the pH is 5.5 to obtain high-concentration concentrated solution 21L, and detecting the purity of the recombinant collagen to be 92.33%.

Claims (7)

1. A separation and purification process for improving stability of recombinant collagen solution is characterized by comprising the following specific steps: centrifugally separating a pichia pastoris fermentation broth, collecting a supernatant, adding acid to adjust the pH to 3-4, filtering by a ceramic membrane to remove impurities, concentrating and deacidifying by a nanofiltration membrane until the pH is 5.5+/-0.5 to obtain a recombinant collagen solution, wherein pichia pastoris is pichia pastorisPichia pastorisThe preservation number is CGMCC No.5021, and the acid is phosphoric acid or acetic acid.
2. The separation and purification process according to claim 1, wherein the pH is adjusted to 3.2 to 3.7 by adding an acid.
3. The separation and purification process according to claim 1, wherein the pH is adjusted to 3.5 by adding an acid.
4. The separation and purification process according to claim 1, wherein the ceramic membrane has a membrane core pore size of 20-200 nm.
5. The separation and purification process according to claim 1, wherein the ceramic membrane has a membrane core pore size of 100nm.
6. The separation and purification process according to claim 1, wherein the nanofiltration membrane has a membrane core pore size of 3kd to 7kd.
7. The separation and purification process according to claim 1, wherein the nanofiltration membrane has a membrane core pore size of 5KD.
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CN112625120B (en) * 2020-12-25 2023-07-04 浙江诸暨聚源生物技术有限公司 Process for continuous large-scale production of recombinant collagen
CN114470333B (en) * 2022-03-09 2023-05-23 哈尔滨敷尔佳科技股份有限公司 Preparation method of crosslinked recombinant collagen gel

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CN104561201A (en) * 2014-12-29 2015-04-29 山东鲁抗医药股份有限公司 Method for extracting recombinant human-like collagen with membrane filter method

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CN106191181B (en) * 2016-07-22 2019-12-13 江苏江山聚源生物技术有限公司 Fermentation process for expressing recombinant protein by pichia pastoris

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CN104561201A (en) * 2014-12-29 2015-04-29 山东鲁抗医药股份有限公司 Method for extracting recombinant human-like collagen with membrane filter method

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