阅读说明：本技术 一种补加竞争性蛋白抑制重组人源胶原蛋白降解的生产工艺 (Production process for inhibiting degradation of recombinant human collagen by supplementing competitive protein ) 是由 殷世清 朱永真 张甲庆 徐靖洋 张凤龙 李艳琪 于 2021-10-13 设计创作，主要内容包括：本发明公开了一种补加竞争性蛋白抑制重组人源胶原蛋白降解的生产工艺,属于生物工程发酵技术领域,具体是在毕赤酵母发酵的诱导阶段,向发酵液中一次性补加竞争性蛋白来抑制人源胶原蛋白的降解,同时,发酵结束后,要进行升温处理,来灭活发酵液的酶活。本发明的生产工艺可保证胶原蛋白在纯化过程中的稳定性,有效地提高了产量和纯度,本工艺稳定易实现,适合大规模发酵生产。(The invention discloses a production process for inhibiting degradation of recombinant human collagen by supplementing competitive protein, belonging to the technical field of bioengineering fermentation. The production process can ensure the stability of the collagen in the purification process, effectively improve the yield and the purity, is stable and easy to realize, and is suitable for large-scale fermentation production.)

1. A production process for inhibiting degradation of recombinant human collagen by supplementing competitive protein is characterized in that competitive protein is supplemented into fermentation liquor at one time to inhibit degradation of human collagen in an induction stage of fermentation of pichia pastoris, and meanwhile, after the fermentation is finished, temperature rise treatment is carried out to inactivate enzyme activity of the fermentation liquor.

2. The process according to claim 1, wherein the competitive protein is one or more of yeast peptone, tryptone peptone, bovine bone peptone, fish peptone, soybean peptone, acid hydrolyzed casein, etc.

3. The production process according to claim 2, wherein the preferred competitive protein added is acid hydrolyzed casein.

4. The process according to claim 3, wherein the acid hydrolyzed casein is added in an amount of 15 g/L.

5. The process according to claim 1, wherein the enzyme deactivation is carried out at a temperature of 60-75 ℃ for 5-30 min.

6. The production process according to claim 5, wherein the temperature is increased to inactivate the enzyme preferably at 65 ℃ for 30 min.

The technical field is as follows:

the invention belongs to the technical field of bioengineering fermentation, and particularly relates to a production process for inhibiting degradation of recombinant human collagen by a method of supplementing competitive protein.

Background art:

the collagen is a fibrin in human body, accounts for 25% -33% of the protein content of human body, is mainly present in skin, muscle, skeleton and other parts, and has the effects of improving the elasticity of skin and muscle, enhancing the combination of calcium and bone cells, protecting the health of skeleton and the like.

The collagen has unique biological characteristics, can be widely applied to the fields of beauty, clinical medicine, food, chemical industry and the like, and with the expansion of Chinese skin care, beauty, nutrition and health care requirements, the market demand of the collagen can meet the continuous expansion stage, and meanwhile, the industrial standard degree needs to be further improved.

The human collagen gene is integrated into pichia pastoris after being processed by adopting a genetic engineering method, and a collagen product with the sequence consistent with that of natural collagen is produced by a fermentation method. Different from natural collagen, the recombinant collagen is in a single-chain structure, the natural collagen is in a triple-helix structure, and the stability of the recombinant collagen is inferior to that of the natural collagen, so the expression technology of the recombinant collagen is difficult, and the product is easy to degrade. The recombinant collagen has the advantages of large-scale production, stable product and no risk of virus pollution of animal sources. Therefore, it is very interesting to develop a method for inhibiting the degradation of recombinant human collagen during fermentation.

The invention content is as follows:

the invention aims to provide a production process for inhibiting the degradation of recombinant human collagen by supplementing competitive protein. Competitive protein is supplemented into fermentation liquor in the induction stage of pichia pastoris fermentation to competitively combine protease, reduce the degradation of the protease on the human collagen and improve the purity and yield of the collagen.

The process for inhibiting the degradation of the recombinant human collagen comprises the following steps:

1) screening for different competitive proteins

In the invention, the inventor screens different sources and different kinds of proteins, and the proteins from different sources comprise one or a compound of more of yeast peptone, tryptone, bovine bone peptone, fish peptone, soybean peptone, acid hydrolyzed casein and the like.

2) Addition ratio of competitive protein

In the invention, the inventor optimizes the adding proportion of the competitive protein, and finds that 0.5-2.5% of the competitive protein is added into the fermentation liquor in the induction stage of pichia pastoris fermentation, so that the degradation of the recombinant human collagen can be effectively inhibited, the stability of the recombinant human collagen is improved to 84% from 35% without adding the competitive protein, and the purity and the yield of the collagen are improved.

3) After the fermentation is finished, heating to inactivate enzyme

After fermentation is finished, the fermentation liquor is heated, so that the stability of the recombinant human collagen can be improved, the collagen is kept stable all the time in the purification process and is not degraded, the temperature for heating and enzyme deactivation is 60-75 ℃, and the time is 5-30 min. After the temperature rise is finished, the temperature is quickly reduced to 25 ℃. After the step, the recombinant human collagen can keep the stability for 24 hours at room temperature (25-30 ℃) and keep the stability for 48 hours at 4 ℃.

Compared with the prior art, the invention has the following advantages:

the innovation points of the invention are as follows: in the induction stage of fermentation, competitive protein is added into the fermentation liquor and is competitively combined with the protein in the fermentation liquor, so that the degradation of the collagen by protease is reduced, the stability of the collagen in the fermentation liquor is improved, and the yield and the purity are effectively improved. The process is stable and easy to realize, and is suitable for large-scale fermentation production.

Drawings

FIG. 1 is a graph showing the results of examples 1 to 6.

FIG. 2 is a graph showing the results of experimental groups of different parameters of example 6.

FIG. 3 is an electrophoretogram of conventional fermented human collagen.

FIG. 4 is an electrophoretogram of human collagen fermented by adding competitive protein in example 6.

Detailed Description

The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the embodiments are described only for illustrating the present invention and should not limit the present invention described in detail in the claims.

Examples 1 to 6

After the induction of the recombinant pichia pastoris is finished, DO is more than 20%, the methanol feeding is stable, sterilized competitive protein is added at one time according to the addition amount of 15g/L, and then the normal methanol feeding induction is carried out. After fermentation, the temperature is raised to 65 ℃ for 30min, and after the temperature is raised, the temperature is rapidly reduced to 25 ℃. Detecting the yield of the recombinant human collagen (including degraded protein), and detecting the yield of the full-sequence target collagen in the recombinant human collagen. Thus determining the stability of the recombinant human collagen.

In examples 1-6, several competing proteins such as yeast peptone, tryptone, bovine bone peptone, fish peptone, soy peptone, acid hydrolyzed casein were selected, and the results are shown in FIG. 1.

Example 7

This example performs a comparative study of different parameters on acid hydrolyzed casein as competitive protein.

Example 6 was selected for further studies with the addition of acid hydrolyzed casein, protein yield and protein stability of interest at different temperature increases and incubation times. And performing grouping experiments according to the combination of different temperature rise temperatures and heat preservation time.

After the induction of the recombinant pichia pastoris is finished, DO is more than 20%, the methanol feeding is stable, the sterilized acid hydrolyzed casein is added at one time according to the addition amount of 15g/L, and then the normal methanol feeding is used for induction. After fermentation is finished, heating, wherein the heating temperature is 60-75 ℃, the heat preservation time is 10-30min, and after heating is finished, rapidly cooling to 25 ℃. And (3) detecting the yield of the recombinant human collagen (including degraded protein) and detecting the yield of the full-sequence target collagen in the recombinant human collagen, thereby determining the stability of the recombinant human collagen, and the specific result is shown in figure 2.

Example 8 comparative example

This example provides a set of comparative experiments, in particular comparing the conventional fermentation process of the prior art with the method of example 6, and subjecting the proteins obtained by both methods to electrophoresis.

The conventional fermentation of the production process in the prior art is to perform normal methanol feeding induction with DO being more than 20% after the induction of the recombinant pichia pastoris is finished. After fermentation, the extraction process is normally carried out, the yield of recombinant human collagen (including degraded protein) reaches 17.2g/L, wherein the amount of the full-sequence target collagen is 2.8 g/L. The stability of the recombinant human collagen is 16.2%, and the electrophoresis chart is shown in figures 3 and 4.