阅读说明：本技术 调节ptm1添加方式提高重组胶原蛋白生产水平的发酵工艺 (Fermentation process for improving production level of recombinant collagen by adjusting addition mode of PTM1 ) 是由 许乔林 杜尔凤 闻帅 冯志平 刘宇乾 赵健烽 黄建民 于 2020-06-30 设计创作，主要内容包括：本发明公开了一种调节PTM1添加方式提高重组胶原蛋白生产水平的发酵工艺。所述工艺先将毕赤酵母菌种液接种到灭菌的发酵培养基中,发酵培养14～18小时后,开始补加甲醇进行诱导表达,同时向发酵培养基中流加PTM1,PTM1流加速度控制在30-300ml/h。本发明在发酵过程中,选取在甲醇诱导表达阶段将PTM1与甲醇分开添加至发酵培养基中,提高重组胶原蛋白的生物合成速率,并且采用连续流加的方式,能够进一步提高重组胶原蛋白的生物合成速率,同时重组胶原蛋白的表达量增加,发酵水平提高20％以上,节约生产成本,特别适用于重组胶原蛋白的工业化大规模生产。(The invention discloses a fermentation process for improving the production level of recombinant collagen by adjusting the addition mode of PTM 1. The process comprises the steps of firstly inoculating a pichia pastoris strain liquid into a sterilized fermentation culture medium, after fermentation culture is carried out for 14-18 hours, beginning to supplement methanol for induction expression, and simultaneously adding PTM1 into the fermentation culture medium in a flowing mode, wherein the adding speed of PTM1 is controlled to be 30-300 ml/h. In the fermentation process, PTM1 and methanol are separately added into a fermentation culture medium at a methanol induction expression stage, so that the biosynthesis rate of the recombinant collagen is improved, the biosynthesis rate of the recombinant collagen can be further improved by adopting a continuous fed-batch mode, the expression amount of the recombinant collagen is increased, the fermentation level is improved by more than 20%, the production cost is saved, and the method is particularly suitable for industrial large-scale production of the recombinant collagen.)

1. The fermentation process for improving the production level of the recombinant collagen by adjusting the addition mode of the PTM1 is characterized by comprising the following steps: inoculating the pichia pastoris strain liquid into a sterilized fermentation culture medium, after fermentation culture is carried out for 14-18 hours, beginning to feed methanol for induction expression, and simultaneously feeding a PTM1 solution into the fermentation culture medium, wherein the feeding speed of the PTM1 is 30-300 ml/h.

2. The fermentation process according to claim 1, wherein the feeding speed of the PTM1 is 100-200 ml/h.

3. The fermentation process of claim 1, wherein the Pichia pastoris is Pichia pastoris, with the accession number CGMCC No. 5021.

4. The fermentation process of claim 1, wherein the fermentation medium is formulated as: 85% H₃PO₄6.6～26.7mL/L，CaSO₄·2H₂O 0.3～1.175g/L，K₂SO₄4.5～18.2g/L，MgSO₄·7H₂O3.7-14.9 g/L, KOH 1.0-4.13 g/L, glycerol 10.0-40.0 g/L, and PTM1 solution 0.435-4.35 mL/L.

5. The fermentation process of claim 1, wherein the formula of the PTM1 solution is as follows: CuSO₄·5H₂O 6.0g/L；NaI 0.08g/L；MnSO₄·H₂O 3.0g/L；NaMoO₄·2H2O 0.2g/L；H₃BO₃0.02g/L；CoCl₂0.5g/L；ZnCl₂20.0g/L；FeSO₄·7H₂O65.0 g/L; biotin 0.2 g/L; h₂SO₄5.0mL/L。

6. The fermentation process of claim 1, wherein the inoculation amount of the pichia pastoris strain liquid is 8-12%, the fermentation temperature is 28-30 ℃, the pH value is adjusted to 5.0-5.2 by ammonia water, the dissolved oxygen is not less than 20%, and the methanol induction time is 90-120 hours.

Technical Field

The invention belongs to the technical field of biological fermentation, and relates to a fermentation process for improving the production level of recombinant collagen by adjusting the addition mode of PTM 1.

Background

Collagen is an important protein in animal bodies, is widely distributed in tissues such as skin, cartilage, blood vessels and the like, participates in migration, differentiation and propagation of cells, plays an important role in maintaining normal physiological functions of cells, tissues and organs, and is widely applied to the fields of food, feed, beauty treatment, cosmetics, medicines and the like. At present, collagen raw materials are mainly obtained by separating and purifying animal skin, bones and other tissues of pigs, cows and the like after being treated by physical and chemical methods such as acid, alkali, heating and the like. However, the collagen obtained by the above method has a complex composition and poor water solubility, and is derived from animal tissues, which has a virus hazard and limits the application of collagen in medicine.

With the rapid development of DNA recombination technology, various host cells are selected as genetically engineered bacteria for expressing collagen. Compared with the traditional process, the fermentation process of the genetically engineered bacteria of the collagen has the advantages of easily obtained production raw materials, environmental protection, stable product quality and the like, but also has the problems of longer fermentation period, lower production efficiency and the like. Chinese patent 20181004890.8 discloses a methanol fed-batch fermentation process for expressing recombinant collagen by pichia pastoris, the yield of the collagen exceeds 20g/L, the yield is improved by more than 5%, and the recovery rate of the recombinant collagen reaches more than 75%. Although the expression level of the recombinant collagen is improved, the improvement range is not large. Therefore, it is necessary to further increase the protein expression level and further increase the fermentation level, which is beneficial to the industrial mass production of collagen and brings practical application value to the industrial production.

Disclosure of Invention

The invention aims to provide a fermentation process for improving the production level of recombinant collagen by adjusting the addition mode of PTM1, which adjusts the growth and metabolism speed of pichia pastoris by separately adding methanol and PTM1 in the fermentation process, improves the expression quantity of the recombinant collagen and further improves the production level of the recombinant collagen.

The technical scheme for realizing the purpose of the invention is as follows:

the fermentation process for improving the production level of the recombinant collagen by adjusting the addition mode of the PTM1 comprises the following steps: inoculating the pichia pastoris strain liquid into a sterilized fermentation culture medium, after fermentation culture is carried out for 14-18 hours, beginning to feed methanol for induction expression, and simultaneously feeding a PTM1 solution into the fermentation culture medium, wherein the feeding speed of the PTM1 is 30-300 ml/h.

Preferably, the adding speed of the PTM1 flow is 100-200 ml/h.

The Pichia pastoris is Pichia pastoris, has been preserved in the common microorganism center of China general microbiological culture Collection management Committee in 2011, 6 and 29, has the preservation number of CGMCC No.5021, and is fully disclosed in Chinese patent 201110327865.5.

The pichia pastoris fermentation medium adopts the existing formula and can be: 85% H₃PO₄6.6～26.7mL/L，CaSO₄·2H₂O 0.3～1.175g/L，K₂SO₄4.5～18.2g/L，MgSO₄·7H₂O3.7-14.9 g/L, KOH 1.0-4.13 g/L, glycerol 10.0-40.0 g/L, and PTM1 solution 0.435-4.35 mL/L.

The PTM1 solution of the invention adopts the existing formula, and can be: CuSO₄·5H₂O 6.0g/L；NaI 0.08g/L；MnSO₄·H₂O 3.0g/L；NaMoO₄·2H2O 0.2g/L；H₃BO₃0.02g/L；CoCl₂0.5g/L；ZnCl₂20.0g/L；FeSO₄·7H₂O65.0 g/L; biotin 0.2 g/L; h₂SO₄5.0mL/L。

In the fermentation process, the inoculation amount of the pichia pastoris strain liquid is 8-12%, the fermentation temperature is 28-30 ℃, the pH value is adjusted to 5.0-5.2 by ammonia water, the dissolved oxygen is not lower than 20%, and the methanol induction time is 90-120 hours.

Compared with the prior art, the invention has the following remarkable effects: in the fermentation process, the PTM1 solution is added in the stage of methanol induction expression, so that the biosynthesis rate of the recombinant collagen is improved, the expression quantity is improved by adopting a continuous feeding mode, the fermentation level is improved by 20 percent to the maximum extent, the production cost is greatly saved, and the method is particularly suitable for industrial large-scale production of the recombinant collagen.

Detailed Description

In the specific embodiment of the invention, the adopted bacterial strain is Pichia pastoris for expressing recombinant collagen, namely Pichia pastoris, the preservation number is CGMCC NO.5021, the preservation date is 2011, 6 and 29 days, and the preservation unit is the China general microbiological culture Collection center.

The PTM1 solution refers to an operation manual of Invitrogen company, and the specific formula is as follows: CuSO₄·5H₂O 6.0g/L；NaI 0.08g/L；MnSO₄·H₂O 3.0g/L；NaMoO₄·2H2O 0.2g/L；H₃BO₃0.02g/L；CoCl₂0.5g/L；ZnCl₂20.0g/L；FeSO₄·7H₂O65.0 g/L; biotin 0.2 g/L; h₂SO₄5.0mL/L, filter-sterilized with a 0.22 μm filter and stored at room temperature.

The present invention will be described in more detail with reference to examples.