WO2018014453A1 - 一种提高重组人源胶原蛋白生产水平的发酵工艺 - Google Patents

一种提高重组人源胶原蛋白生产水平的发酵工艺 Download PDF

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WO2018014453A1
WO2018014453A1 PCT/CN2016/102435 CN2016102435W WO2018014453A1 WO 2018014453 A1 WO2018014453 A1 WO 2018014453A1 CN 2016102435 W CN2016102435 W CN 2016102435W WO 2018014453 A1 WO2018014453 A1 WO 2018014453A1
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fermentation
added
recombinant human
hours
sodium pyruvate
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French (fr)
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杨树林
杜尔凤
黄建民
高力虎
赵健烽
陶海
冯丽萍
周爱梅
季乐
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江苏江山聚源生物技术有限公司
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Priority to EP16909380.4A priority Critical patent/EP3473726B8/en
Priority to KR1020197001826A priority patent/KR102132132B1/ko
Priority to US16/318,556 priority patent/US11136373B2/en
Publication of WO2018014453A1 publication Critical patent/WO2018014453A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • 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
    • C12N1/165—Yeast isolates
    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the invention belongs to the technical field of biological fermentation and relates to a fermentation process for improving the production level of recombinant human collagen.
  • Collagen is an important protein in animals. It is widely distributed in tissues such as skin, cartilage and blood vessels. It is involved in cell migration, differentiation and reproduction, and plays an important role in maintaining the normal physiological functions of cells, tissues and organs. It is widely used in the fields of feed, beauty, cosmetics and medicine.
  • collagen raw materials are mainly obtained by physicochemical methods such as acid, alkali, heating, and the like, such as pigs, cattle and other animal skin, bones and other tissues, and then isolated and purified.
  • the collagen component obtained by the above method is complicated, has poor water solubility, and has a virus hazard due to the animal tissue, which limits the application of collagen in medicine.
  • Chinese patent 201110327865.5 constructed a genetically engineered strain of Pichia pastoris recombinant human collagen, which was fermented by genetic engineering bacteria.
  • the human-derived collagen has a fermentation cycle of 136 hours, a protein expression of 16 g/L, and a fermentation level of 0.118 g/L ⁇ h. Although the protein expression is increased, the fermentation cycle is too long and the fermentation level is low. Therefore, further shortening the fermentation time, increasing the protein expression level, and further improving the fermentation level are beneficial to the industrialized large-scale production of collagen, and can bring practical application value to industrial production.
  • the object of the present invention is to provide a fermentation process for improving the production level of recombinant human collagen.
  • the fermentation cycle increased the expression of recombinant human collagen and further increased the production level of recombinant human collagen.
  • a fermentation process for improving the production level of recombinant human collagen comprises the following steps: inoculating the Pichia pastoris seed into a sterilized fermentation medium, and after 14 to 18 hours of fermentation, adding methanol to induce expression. At the same time, sodium pyruvate is added to the fermentation medium, wherein the sodium pyruvate is added in an amount of 0.01 to 10 g/L.
  • Pichia pastoris described in the present invention is Pichia pastoris, which was deposited on June 29, 2011 at the General Microbiology Center of the China Microbial Culture Collection Management Committee, with the preservation number CGMCC No. 5021, and in China. It is fully disclosed in patent 201110327865.5.
  • the sodium pyruvate is added in an amount of from 0.1 g/L to 1 g/L.
  • the sodium pyruvate is added in a manner of addition.
  • the Pichia pastoris fermentation medium of the present invention adopts the existing formula and can be: 85% H 3 PO 4 6.6 to 26.7 mL/L, CaSO 4 ⁇ 2H 2 O 0.3 to 1.175 g/L, K 2 SO 4 4.5 ⁇ 18.2 g/L, MgSO 4 ⁇ 7H 2 O 3.7 to 14.9 g/L, KOH 1.0 to 4.13 g/L, glycerin 10.0 to 40.0 g/L, and PTM1 solution 0.435 to 4.35 mL/L.
  • the inoculation amount of the Pichia pastoris seed solution is 8-12%
  • the fermentation temperature is 28-30 ° C
  • the pH of the ammonia water is 5.0-5.2
  • the dissolved oxygen is not less than 20%
  • the methanol induction time is 90 to 120 hours.
  • the remarkable effect of the present invention is as follows: in the fermentation process, the sodium pyruvate is added in the methanol-induced expression stage, the biosynthesis rate of the recombinant human collagen is increased, and the continuous flow is added.
  • the method can further increase the biosynthesis rate of recombinant human collagen, shorten the fermentation time, increase the expression of recombinant human collagen, increase the fermentation level by more than 20%, and save the production cost, especially suitable for recombinant human collagen.
  • the industrialized large-scale production of protein can bring huge practical application value to industrial production.
  • the strain used is Pichia pastoris expressing recombinant human collagen, Pichia pastoris, and the preservation number is CGMCC NO. 5021, and the storage date is June 29, 2011. On the day, the depositary is the General Microbiology Center of the China Microbial Culture Collection Management Committee.
  • the PTM1 solution of the present invention is referred to the operation manual of Invitrogen, and the specific formula is: CuSO 4 ⁇ 5H 2 O 6.0g / L; NaI 0.08g / L; MnSO 4 ⁇ H 2 O 3.0g / L; NaMoO 4 ⁇ 2H 2 O 0.2g/L; H 3 BO 3 0.02g/L; CoCl 2 0.5g/L; ZnCl 2 20.0g/L; FeSO 4 ⁇ 7H 2 O 65.0g/L; biotin 0.2g/L; H 2 SO 4 5.0 mL/L, and sterilized by filtration through a 0.22 m filter, and stored at room temperature.
  • Fermentation medium 85% H 3 PO 4 26.7 mL / L; CaSO 4 ⁇ 2H 2 O 1.175 g / L; K 2 SO 4 18.2 g / L; MgSO 4 ⁇ 7H 2 O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 4.35 mL / L.
  • the seed solution was added to a 1000 L fermentor containing 500 L of fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the tank pressure was 0.05 MPa, and the air flow rate and the number of revolutions were adjusted so that the dissolved oxygen (DO) was >30%.
  • DO dissolved oxygen
  • methanol was added to the methanol-inducing culture stage, and sodium pyruvate was added at a time, and the amount of the fermentation medium was 0.01 g/L. Adjust the rotation speed, air flow rate and methanol speed to make the dissolved oxygen (DO)>20%.
  • DO dissolved oxygen
  • the fermentation is finished, the fermentation liquid is collected, the supernatant of the fermentation liquid is obtained by centrifugation, and the recombinant human collagen concentration is finally determined by HPLC.
  • the fermentation cycle was 112 hours, and the fermentation production level was 0.143 g/L ⁇ h, which was 23.3% higher than that of the control group.
  • Fermentation medium 85% H 3 PO 4 26.7 mL / L; CaSO 4 ⁇ 2H 2 O 1.175 g / L; K 2 SO 4 18.2 g / L; MgSO 4 ⁇ 7H 2 O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 4.35 mL / L.
  • the seed liquid was added to a 1000 L fermentor containing 500 L of fermentation medium according to a 10% inoculation amount, and the initial stirring speed was 200 rpm, the tank pressure was 0.05 MPa, and the air flow rate was adjusted.
  • the rotational speed gives a dissolved oxygen (DO) > 30%.
  • DO dissolved oxygen
  • methanol is added to the methanol-inducing culture stage, and sodium pyruvate is added at a time, and the amount is 10 g/L. Adjust the rotation speed, air flow rate and methanol speed to make the dissolved oxygen (DO)>20%.
  • DO dissolved oxygen
  • the fermentation is finished, the fermentation liquid is collected, the supernatant of the fermentation liquid is obtained by centrifugation, and the recombinant human collagen concentration is finally determined by HPLC.
  • the fermentation cycle was 112 hours, and the fermentation production level was 0.150 g/L ⁇ h, which was 29.3% higher than that of the control group.
  • Fermentation medium 85% H 3 PO 4 26.7 mL / L; CaSO 4 ⁇ 2H 2 O 1.175 g / L; K 2 SO 4 18.2 g / L; MgSO 4 ⁇ 7H 2 O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 4.35 mL / L.
  • the seed solution was added to a 1000 L fermentor containing 500 L of fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the tank pressure was 0.05 MPa, and the air flow rate and the number of revolutions were adjusted so that the dissolved oxygen (DO) was >30%.
  • DO dissolved oxygen
  • the carbon source is exhausted, the dissolved oxygen rises abruptly, 50% glycerol is added, and the carbon source is added.
  • the wet weight of the bacteria is 216 g/L, and the glycerin is stopped, and the fermentation time is 17 hours.
  • methanol is added to the methanol-inducing culture stage, and sodium pyruvate is added at a time, and the amount is 0.1 g/L.
  • Fermentation medium 85% H 3 PO 4 26.7 mL / L; CaSO 4 ⁇ 2H 2 O 1.175 g / L; K 2 SO 4 18.2 g / L; MgSO 4 ⁇ 7H 2 O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 4.35 mL / L.
  • the seed solution was added to a 1000 L fermentor containing 500 L of fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the tank pressure was 0.05 MPa, and the air flow rate and the number of revolutions were adjusted so that the dissolved oxygen (DO) was >30%.
  • DO dissolved oxygen
  • the carbon source is exhausted, the dissolved oxygen rises abruptly, 50% glycerol is added, and the carbon source is added.
  • the wet weight of the bacteria is 213 g/L, and the glycerin is stopped, and the fermentation time is 18 hours.
  • methanol is added to the methanol-inducing culture stage, and the sodium pyruvate solution is continuously added to the end of the fermentation, and the total addition amount is 0.1 g/L. Adjust the rotation speed, air flow rate and methanol speed to make dissolved oxygen (DO)>20%.
  • DO dissolved oxygen
  • the fermentation is finished, the fermentation liquid is collected, the supernatant of the fermentation liquid is obtained by centrifugation, and the recombinant human collagen concentration is finally determined by HPLC.
  • the fermentation cycle was 108 hours, and the fermentation production level was 0.169 g/L ⁇ h, which was 45.3% higher than that of the control group.
  • Fermentation medium 85% H 3 PO 4 26.7 mL / L; CaSO 4 ⁇ 2H 2 O 1.175 g / L; K 2 SO 4 18.2 g / L; MgSO 4 ⁇ 7H 2 O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 4.35 mL / L.
  • the seed solution was added to a 1000 L fermentor containing 500 L of fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the tank pressure was 0.05 MPa, and the air flow rate and the number of revolutions were adjusted so that the dissolved oxygen (DO) was >30%.
  • DO dissolved oxygen
  • the carbon source is exhausted, the dissolved oxygen rises abruptly, and 50% glycerol is added to the surface to supplement the carbon source.
  • the wet weight of the bacteria is 212g/L, and the glycerin is stopped.
  • the fermentation time was 16 hours.
  • methanol is added to the methanol-inducing culture stage, and the sodium pyruvate solution is continuously added to the end of the fermentation, and the total amount is 1 g/L. Adjust the rotation speed, air flow rate and methanol speed to make the dissolved oxygen (DO)>20%.
  • DO dissolved oxygen
  • the fermentation is finished, the fermentation liquid is collected, the supernatant of the fermentation liquid is obtained by centrifugation, and the recombinant human collagen concentration is finally determined by HPLC.
  • the fermentation cycle was 112 hours, and the fermentation production level was 0.161 g/L ⁇ h, which was 38.8% higher than that of the control group.
  • Fermentation medium 85% H 3 PO 4 26.7 mL / L; CaSO 4 ⁇ 2H 2 O 1.175 g / L; K 2 SO 4 18.2 g / L; MgSO 4 ⁇ 7H 2 O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM1 4.35 mL / L.
  • the seed solution was added to a 1000 L fermentor containing 500 L of fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the tank pressure was 0.05 MPa, and the air flow rate and the number of revolutions were adjusted so that the dissolved oxygen (DO) was >30%.
  • DO dissolved oxygen
  • methanol is added to the methanol-inducing culture stage, and sodium pyruvate is added at a time, and the amount is 1 g/L. Adjust the rotation speed, air flow rate and methanol speed to make the dissolved oxygen (DO)>20%.
  • DO dissolved oxygen
  • the fermentation is finished, the fermentation liquid is collected, the supernatant of the fermentation liquid is obtained by centrifugation, and the recombinant human collagen concentration is finally determined by HPLC.
  • the fermentation cycle was 113 hours, and the fermentation production level was 0.155 g/L ⁇ h, which was 33.6% higher than that of the control group.
  • Fermentation medium 85% H 3 PO 4 6.6 mL / L; CaSO 4 ⁇ 2H 2 O 0.3 g / L; K 2 SO 4 4.5 g / L; MgSO 4 ⁇ 7H 2 O 3.7 g / L; KOH 1g / L Glycerin 10.0 g / L; PTM1 0.435 mL / L.
  • the seed solution was added to a 1000 L fermentor containing 500 L of fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the tank pressure was 0.05 MPa, and the air flow rate and the number of revolutions were adjusted so that the dissolved oxygen (DO) was >30%.
  • DO dissolved oxygen
  • methanol is added to the methanol-inducing culture stage, and sodium pyruvate is added at a time, and the amount is 1 g/L. Adjust the rotation speed, air flow rate and methanol speed to make the dissolved oxygen (DO)>20%.
  • DO dissolved oxygen
  • the fermentation is finished, the fermentation liquid is collected, the supernatant of the fermentation liquid is obtained by centrifugation, and the recombinant human collagen concentration is finally determined by HPLC.
  • the ratio was 17.1 g/L, the fermentation cycle was 113 hours, and the fermentation production level was 0.151 g/L ⁇ h, which was 30.2% higher than that of the control group.
  • the fermentation medium was a basic salt medium (BSM, reference CN102443057B): 85% H3PO4 26.7 mL/L; CaSO4 ⁇ 2H2O 1.175 g/L; K2SO4 18.2 g/L; MgSO4 ⁇ 7H2O 14.9 g/L; KOH 4.13 g/L Glycerol 40.0 g / L; PTM1 4.35 mL / L.
  • BSM basic salt medium
  • the seed solution was added to a 1000 L fermentor containing 500 L of fermentation medium at an inoculation amount of 10%, the initial stirring speed was 200 rpm, the tank pressure was 0.05 MPa, and the air flow rate and the number of revolutions were adjusted so that the dissolved oxygen (DO) was >30%.
  • DO dissolved oxygen
  • the methanol is added to the methanol-inducing culture stage, and the dissolved oxygen (DO) is adjusted by adjusting the rotation speed, the tank pressure, the air flow rate, and the flow rate of the methanol. >20%, after induction of fermentation for 120h, the fermentation was terminated, the fermentation broth was collected, and the supernatant of the fermentation broth was obtained by centrifugation.
  • the final recombinant human collagen concentration was 15.8 g/L
  • the fermentation cycle was 136 hours
  • the fermentation production level was 0.116. g/L ⁇ h.

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Abstract

本发明提供了一种提高重组人源胶原蛋白生产水平的发酵工艺，首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加入丙酮酸钠，其中丙酮酸钠的加入量为0.01-10g/L。

Description

一种提高重组人源胶原蛋白生产水平的发酵工艺 技术领域
本发明属于生物发酵技术领域，涉及一种提高重组人源胶原蛋白生产水平的发酵工艺。
背景技术
胶原蛋白是动物体内一类重要的蛋白质，广泛分布于皮肤、软骨、血管等组织，参与细胞的迁移、分化和繁殖，对维护细胞、组织、器官的正常生理功能起着重要的作用，在食品、饲料、美容、化妆品、医药等领域应用普遍。目前，胶原蛋白原料主要通过酸、碱、加热等物理化学方法处理如猪、牛等动物皮肤、骨骼等组织后分离纯化获得。但是，上述方法得到的胶原蛋白成分复杂，水溶性差，并且由于来自动物组织，存在病毒隐患，限制了胶原蛋白在医药方面的应用。
随着DNA重组技术的迅速发展，各种宿主细胞被选为基因工程菌用于表达胶原蛋白。与传统工艺相比，胶原蛋白的基因工程菌发酵工艺具有生产原料易得、环保、产品质量稳定等优点，但是也存在着发酵周期较长，生产效率较低等问题。中国专利201010602214.8公开了一种毕赤酵母表达重组类人胶原蛋白的生产方法，采用巴斯德毕赤酵母Pichia pastoris C13为菌种，重组胶原蛋白表达量为5g/L，发酵周期99小时，发酵水平为0.051g/L·h，发酵周期虽短，但蛋白表达量和发酵水平过低。中国专利201110327865.5构建了一种重组人源胶原蛋白的巴氏毕赤酵母基因工程菌，基因工程菌经发酵培养得到重 组人源胶原蛋白，发酵周期为136小时，蛋白表达量为16g/L，发酵水平为0.118g/L·h，蛋白表达量虽有提高，但是发酵周期过长，发酵水平较低。因此，进一步地缩短发酵时间，提高蛋白表达量，进而提高发酵水平，有利于胶原蛋白的工业化大规模生产，能够为产业化生产带来实际应用价值。
发明内容
本发明的目的在于提供一种提高重组人源胶原蛋白生产水平的发酵工艺，通过在发酵过程中加入0.01g/L-10g/L的丙酮酸钠，调节毕赤酵母菌的生长代谢速度，缩短了发酵周期，提高了重组人源胶原蛋白的表达量，进一步提升了重组人源胶原蛋白的生产水平。
本发明的技术方案如下：
一种提高重组人源胶原蛋白生产水平的发酵工艺，包括以下步骤：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加入丙酮酸钠，其中丙酮酸钠的加入量为0.01～10g/L。
本发明所述的毕赤酵母为巴斯德毕赤酵母Pichia pastoris，已于2011年6月29日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.5021，并在中国专利201110327865.5中充分公开。
优选地，所述的丙酮酸钠的加入量为0.1g/L～1g/L。
优选的，所述的丙酮酸钠的加入方式为流加。
本发明所述的毕赤酵母发酵培养基采用现有配方，可以是：85％ H3PO4 6.6～26.7mL/L，CaSO4·2H2O 0.3～1.175g/L，K2SO4 4.5～18.2g/L，MgSO4·7H2O 3.7～14.9g/L，KOH1.0～4.13g/L，甘油10.0～40.0g/L，PTM1溶液0.435～4.35mL/L。
本发明的发酵工艺中，毕赤酵母菌种液的接种量为8～12％，发酵温度为28～30℃，氨水调节pH为5.0～5.2，溶氧不低于20％，甲醇诱导时间为90～120小时。
与现有技术相比，本发明的显著效果如下：本发明在发酵过程中，选取在甲醇诱导表达阶段加入丙酮酸钠，提高了重组人源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人源胶原蛋白的生物合成速率，发酵时间缩短，同时重组人源胶原蛋白的表达量增加，发酵水平提高了20％以上，节约了生产成本，特别适用于重组人源胶原蛋白的工业化大规模生产，能够为产业化生产带来巨大的实际应用价值。
具体实施方式
在本发明的具体实施例中，采用的菌株为表达重组人源胶原蛋白的毕赤酵母，为巴斯德毕赤酵母Pichia pastoris，保藏编号为CGMCC NO.5021，保藏日期为2011年6月29日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心。
本发明所述的PTM1溶液参照Invitrogen公司的操作手册，具体配方为：CuSO4·5H2O 6.0g/L；NaI0.08g/L；MnSO4·H2O 3.0g/L；NaMoO4·2H2O 0.2g/L；H3BO3 0.02g/L；CoCl2 0.5g/L；ZnCl2 20.0g/L；FeSO4·7H2O 65.0g/L；生物素0.2g/L；H2SO4 5.0mL/L，用0.22m的 滤膜过滤除菌，室温保存。
下面结合实施例和附图对本发明作进一步详述。
实施例1
发酵培养基：85％H3PO4 26.7mL/L；CaSO4·2H2O 1.175g/L；K2SO4 18.2g/L；MgSO4·7H2O 14.9g/L；KOH 4.13g/L；甘油40.0g/L；PTM1 4.35mL/L。
将种子液按照10％的接种量加到含有500L发酵培养基的1000L发酵罐中，初始搅拌转速200rpm、罐压0.05MPa，调节空气流量和转速使溶氧(DO)＞30％。当碳源耗尽后，溶氧陡然上升，开始流加50％甘油，补充碳源，至菌体湿重为212g/L，停止补加甘油，此时发酵培养时间为16小时。甘油耗尽后开始流加甲醇，进入甲醇诱导培养阶段，同时一次性加入丙酮酸钠，加入量为0.01g/L发酵培养基。调节转速、空气流量和流加甲醇速度使溶氧(DO)＞20％，诱导发酵96h后，结束发酵，收集发酵液，离心获得发酵液上清，经HPLC检测，最终重组人源胶原蛋白浓度为16.0g/L，发酵周期112小时，发酵生产水平为0.143g/L·h，与对照组相比提高23.3％。
实施例2
发酵培养基：85％H3PO4 26.7mL/L；CaSO4·2H2O 1.175g/L；K2SO4 18.2g/L；MgSO4·7H2O 14.9g/L；KOH 4.13g/L；甘油40.0g/L；PTM1 4.35mL/L。
将种子液按照10％的接种量加到含有500L发酵培养基的1000L发酵罐中，初始搅拌转速200rpm、罐压0.05MPa，调节空气流量和 转速使溶氧(DO)＞30％。当碳源耗尽后，溶氧陡然上升，开始流加50％甘油，补充碳源，至菌体湿重为210g/L，停止补加甘油，此时发酵培养时间为16小时。甘油耗尽后开始流加甲醇，进入甲醇诱导培养阶段，同时一次性加入丙酮酸钠，加入量为10g/L。调节转速、空气流量和流加甲醇速度使溶氧(DO)＞20％，诱导发酵96h后，结束发酵，收集发酵液，离心获得发酵液上清，经HPLC检测，最终重组人源胶原蛋白浓度为16.8g/L，发酵周期112小时，发酵生产水平为0.150g/L·h，与对照组相比提高29.3％。
实施例3
发酵培养基：85％H3PO4 26.7mL/L；CaSO4·2H2O 1.175g/L；K2SO4 18.2g/L；MgSO4·7H2O 14.9g/L；KOH 4.13g/L；甘油40.0g/L；PTM1 4.35mL/L。
将种子液按照10％的接种量加到含有500L发酵培养基的1000L发酵罐中，初始搅拌转速200rpm、罐压0.05MPa，调节空气流量和转速使溶氧(DO)＞30％。当碳源耗尽后，溶氧陡然上升，开始流加50％甘油，补充碳源，至菌体湿重为216g/L，停止补加甘油，此时发酵培养时间为17小时。甘油耗尽后开始流加甲醇，进入甲醇诱导培养阶段，同时一次性加入丙酮酸钠，加入量为0.1g/L。调节转速、空气流量和流加甲醇速度使溶氧(DO)＞20％，诱导发酵96h后，结束发酵，收集发酵液，离心获得发酵液上清，经HPLC检测，最终重组人源胶原蛋白浓度为17.8g/L，发酵周期113小时，发酵生产水平为0.158g/L·h。与对照组相比提高36.2％。
实施例4
发酵培养基：85％H3PO4 26.7mL/L；CaSO4·2H2O 1.175g/L；K2SO4 18.2g/L；MgSO4·7H2O 14.9g/L；KOH 4.13g/L；甘油40.0g/L；PTM1 4.35mL/L。
将种子液按照10％的接种量加到含有500L发酵培养基的1000L发酵罐中，初始搅拌转速200rpm、罐压0.05MPa，调节空气流量和转速使溶氧(DO)＞30％。当碳源耗尽后，溶氧陡然上升，开始流加50％甘油，补充碳源，至菌体湿重为213g/L，停止补加甘油，此时发酵培养时间为18小时。甘油耗尽后开始流加甲醇，进入甲醇诱导培养阶段，同时连续流加丙酮酸钠溶液至发酵结束，总加入量为0.1g/L。调节转速、空气流量和流加甲醇速度使溶氧(DO)＞20％，诱导发酵90h后，结束发酵，收集发酵液，离心获得发酵液上清，经HPLC检测，最终重组人源胶原蛋白浓度为18.2g/L，发酵周期108小时，发酵生产水平为0.169g/L·h，与对照组相比提高45.3％。
实施例5
发酵培养基：85％H3PO4 26.7mL/L；CaSO4·2H2O 1.175g/L；K2SO4 18.2g/L；MgSO4·7H2O 14.9g/L；KOH 4.13g/L；甘油40.0g/L；PTM1 4.35mL/L。
将种子液按照10％的接种量加到含有500L发酵培养基的1000L发酵罐中，初始搅拌转速200rpm、罐压0.05MPa，调节空气流量和转速使溶氧(DO)＞30％。当碳源耗尽后，溶氧陡然上升，开始流加50％甘油，补充碳源，至菌体湿重为212g/L，停止补加甘油，此时发 酵培养时间为16小时。甘油耗尽后开始流加甲醇，进入甲醇诱导培养阶段，同时连续流加丙酮酸钠溶液至发酵结束，总加入量为1g/L。调节转速、空气流量和流加甲醇速度使溶氧(DO)＞20％，诱导发酵96h后，结束发酵，收集发酵液，离心获得发酵液上清，经HPLC检测，最终重组人源胶原蛋白浓度为18.0g/L，发酵周期112小时，发酵生产水平为0.161g/L·h，与对照组相比提高38.8％。
实施例6
发酵培养基：85％H3PO4 26.7mL/L；CaSO4·2H2O 1.175g/L；K2SO4 18.2g/L；MgSO4·7H2O 14.9g/L；KOH 4.13g/L；甘油40.0g/L；PTM1 4.35mL/L。
将种子液按照10％的接种量加到含有500L发酵培养基的1000L发酵罐中，初始搅拌转速200rpm、罐压0.05MPa，调节空气流量和转速使溶氧(DO)＞30％。当碳源耗尽后，溶氧陡然上升，开始流加50％甘油，补充碳源，至菌体湿重为210g/L，停止补加甘油，此时发酵培养时间为17小时。甘油耗尽后开始流加甲醇，进入甲醇诱导培养阶段，同时一次性加入丙酮酸钠，加入量为1g/L。调节转速、空气流量和流加甲醇速度使溶氧(DO)＞20％，诱导发酵96h后，结束发酵，收集发酵液，离心获得发酵液上清，经HPLC检测，最终重组人源胶原蛋白浓度为17.5g/L，发酵周期113小时，发酵生产水平为0.155g/L·h，与对照组相比提高33.6％。
实施例7
发酵培养基：85％H3PO4 6.6mL/L；CaSO4·2H2O 0.3g/L；K2SO4  4.5g/L；MgSO4·7H2O 3.7g/L；KOH 1g/L；甘油10.0g/L；PTM1 0.435mL/L。
将种子液按照10％的接种量加到含有500L发酵培养基的1000L发酵罐中，初始搅拌转速200rpm、罐压0.05MPa，调节空气流量和转速使溶氧(DO)＞30％。当碳源耗尽后，溶氧陡然上升，开始流加50％甘油，补充碳源，至菌体湿重为210g/L，停止补加甘油，此时发酵培养时间为17小时。甘油耗尽后开始流加甲醇，进入甲醇诱导培养阶段，同时一次性加入丙酮酸钠，加入量为1g/L。调节转速、空气流量和流加甲醇速度使溶氧(DO)＞20％，诱导发酵96h后，结束发酵，收集发酵液，离心获得发酵液上清，经HPLC检测，最终重组人源胶原蛋白浓度为17.1g/L，发酵周期113小时，发酵生产水平为0.151g/L·h，与对照组相比提高30.2％。
对比例
发酵培养基为基础盐培养基(BSM，参考CN102443057B)：85％H3PO4 26.7mL/L；CaSO4·2H2O 1.175g/L；K2SO4 18.2g/L；MgSO4·7H2O 14.9g/L；KOH 4.13g/L；甘油40.0g/L；PTM1 4.35mL/L。
将种子液按照10％的接种量加到含有500L发酵培养基的1000L发酵罐中，初始搅拌转速200rpm、罐压0.05MPa，调节空气流量和转速使溶氧(DO)＞30％。当碳源耗尽后，溶氧陡然上升，开始流加50％甘油，补充碳源，至菌体湿重为218g/L，停止补加甘油，此时发酵培养时间为16小时。甘油耗尽后开始流加甲醇，进入甲醇诱导培养阶段，通过调节转速、罐压、空气流量和流加甲醇速度使溶氧(DO) ＞20％，诱导发酵120h后，结束发酵，收集发酵液，离心获得发酵液上清，经HPLC检测，最终重组人源胶原蛋白浓度为15.8g/L，发酵周期136小时，发酵生产水平为0.116g/L·h。

Claims (6)

  1. 一种提高重组人源胶原蛋白生产水平的发酵工艺，其特征在于，包括以下步骤：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加入丙酮酸钠，其中丙酮酸钠的加入量为0.01～10g/L。
  2. 根据权利要求1所述的发酵工艺，其特征在于，所述的丙酮酸钠的加入量为0.1g/L～1g/L。
  3. 根据权利要求1所述的发酵工艺，其特征在于，所述的毕赤酵母为巴斯德毕赤酵母Pichia pastoris，保藏编号为CGMCC No.5021。
  4. 根据权利要求1或2所述的发酵工艺，其特征在于，所述的丙酮酸钠的加入方式为流加。
  5. 根据权利要求1所述的发酵工艺，其特征在于，所述的毕赤酵母发酵培养基的配方为85％H3PO46.6～26.7mL/L，CaSO4·2H2O0.3～1.175g/L，K2SO44.5～18.2g/L，MgSO4·7H2O3.7～14.9g/L，KOH1.0～4.13g/L，甘油10.0～40.0g/L，PTM1溶液0.435～4.35mL/L。
  6. 根据权利要求1所述的发酵工艺，其特征在于，发酵工艺中，毕赤酵母菌种液的接种量为8～12％，发酵温度为28～30℃，氨水调节pH为5.0～5.2，溶氧不低于20％，甲醇诱导时间为90～120小时。
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US11136373B2 (en) 2021-10-05
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CN106119322A (zh) 2016-11-16
CN106119322B (zh) 2019-10-15
KR20190018725A (ko) 2019-02-25
EP3473726A4 (en) 2019-04-24
EP3473726B8 (en) 2019-10-16
US20190241645A1 (en) 2019-08-08

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