Fermentation method and application of XVII type recombinant collagen

Abstract

The invention particularly discloses a fermentation method and application of XVII type recombinant collagen, and relates to the technical field of biological fermentation. The fermentation method provided by the invention comprises a fermentation methanol induced expression stage, continuous fed-batch of amino acids of different types and different proportions, regulation of growth metabolism of pichia pastoris and improvement of the synthesis rate of recombinant collagen, the fermentation yield can be improved by 69.8%, and the fermentation time can be shortened by 12-24 h.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, and in particular to a fermentation method for type XVII recombinant collagen and its application. Background Technology

[0002] Pichia pastoris ( Pichia pastoris Pichia pastoris is a widely used eukaryotic expression system for recombinant protein production. Its fermentation process is significantly influenced by culture medium composition, culture conditions, and metabolic regulation. Amino acids, as key substrates for cell growth and protein synthesis, play a crucial role in the fermentation performance of Pichia pastoris (such as cell growth, protein expression, metabolic flux distribution, and byproduct accumulation). Amino acids are not only monomers for protein synthesis but also participate in energy metabolism, signal transduction, and stress response. Therefore, studying the effects of amino acids on Pichia pastoris fermentation is of great significance for improving the yield and quality of recombinant proteins.

[0003] The effects of amino acids on the growth of Pichia pastoris: 1. Promoting Cell Proliferation: In basal media (such as BSM), certain amino acids (e.g., lysine, histidine, leucine) may be limiting factors in Pichia pastoris. Exogenous supplementation of these amino acids can significantly increase biomass and shorten the lag phase. For example, glutamine (Gln) serves as the main nitrogen source, supporting the TCA cycle and nucleotide synthesis. Branched-chain amino acids (BCAAs, such as leucine, isoleucine, valine) affect energy metabolism and cell membrane synthesis.

[0004] 2. Nitrogen source utilization efficiency: Compared to inorganic nitrogen (such as (NH4)2SO4), amino acids are more easily absorbed as organic nitrogen sources, reducing energy consumption and improving carbon utilization efficiency. Amino acids can reduce the accumulation of byproducts. When carbon sources are limited, Pichia pastoris may shift to fermentation metabolism, accumulating ethanol or acetic acid and inhibiting growth. Supplementing with amino acids can maintain oxidative phosphorylation and reduce harmful metabolites.

[0005] Serine (Ser) and glycine (Gly) are involved in one-carbon metabolism, affecting NADPH supply, and thus affecting the balance of reducing power. At the same time, amino acids are direct precursors of collagen synthesis, especially glycine, proline. Glycine is the most abundant amino acid in collagen, accounting for about one-third of the total molecular weight. The shortage of glycine will directly lead to the interruption of peptide chain synthesis, and it is the most primary limiting amino acid. Proline is the second most abundant amino acid in collagen, and a deficiency of proline will significantly reduce the synthesis rate and yield of collagen. Lysine is involved in collagen structure and is also a substrate for post-translational modification, affecting the structural stability and function of collagen. Methionine and cysteine improve the tolerance and utilization efficiency of the cell to methanol, significantly increase the yield and quality (correct folding rate) of the exogenous protein, and reduce the accumulation of toxic by-products (such as formaldehyde and hydrogen peroxide). In summary, amino acids affect the fermentation performance of Pichia pastoris through various mechanisms, and reasonable regulation of their supply can significantly improve cell growth and protein yield. SUMMARY

[0006] (I) Technical problems solved In view of this, one of the main purposes of the present application is to provide a fermentation method of type XVII recombinant collagen, which comprises adding amino acids to the fermentation broth, and the fermentation yield can be increased by 69.8%, and the fermentation time can be shortened by 12-24h.

[0007] (II) Technical solutions In order to achieve the above-mentioned purpose, the present application provides a fermentation method of type XVII recombinant collagen, which comprises adding amino acids to the fermentation broth, and the amino acids include glycine, proline, lysine, glutamine, methionine, cysteine and / or L-hydroxyproline.

[0008] In one embodiment, the amino acids include glycine and cysteine.

[0009] In one embodiment, the concentration ratio of cysteine and glycine is 1: (1-5).

[0010] In one embodiment, the concentration ratio of cysteine and glycine is 1:2.

[0011] In one embodiment, the fermentation method comprises adding amino acids during the induction period after starvation.

[0012] In one embodiment, the fermentation method comprises: S1: adding glycine after the glycine in the fermentation medium is consumed; S2: stopping adding glycine and entering the starvation period after the glycine is consumed; S3: adding methanol to enter the induction period, and adding amino acids at the same time.

[0013] In one embodiment, the criterion for adding glycerol is that the dissolved oxygen of the fermentation broth rises to 50-75%.

[0014] In one embodiment, the criterion for stopping the addition of glycerol is that the wet weight of the cells reaches 200-230 g / L.

[0015] In one embodiment, the starvation period is maintained for 20-40 min.

[0016] In one embodiment, the dissolved oxygen of the fermentation broth during the induction period is 10-25%.

[0017] In one embodiment, the rate of adding the amino acid is 0.5-1 mM / L / h.

[0018] In one embodiment, the fermentation medium comprises: yeast peptone 1-4 g / L, yeast extract 1-4 g / L, 85% phosphoric acid 8-12 mL / L, calcium sulfate 0.23-0.69 g / L, potassium sulfate 6.37-11.83 g / L, magnesium sulfate heptahydrate 5.25-9.75 g / L, potassium hydroxide 1-4 g / L, glycerol 20-60 g / L, antifoam agent 0.35-0.65 g / L, PTM1 3.0-5.65 mL / L, and biotin 3-8 mg / L.

[0019] In one embodiment, the cells for fermentation are Pichia pastoris cells.

[0020] In one embodiment, the fermentation method specifically comprises: S1: adding glycerol when the dissolved oxygen of the fermentation broth rises to 50-75% after the glycerol in the fermentation medium is consumed; S2: stopping the addition of glycerol when the wet weight of the cells reaches 200-230 g / L, and entering a starvation period of 20-40 min after the glycerol is consumed; S3: adding methanol to enter an induction period, and adding amino acids.

[0021] In one embodiment, the fermentation method specifically comprises: S1: adding glycerol when the dissolved oxygen of the fermentation broth rises to 50-75% after the glycerol in the fermentation medium is consumed; S2: stopping the addition of glycerol when the wet weight of the cells reaches 200-230 g / L, and entering a starvation period of 20-40 min after the glycerol is consumed; S3: adding methanol to enter an induction period, controlling the dissolved oxygen of the fermentation broth to be 10-25%, and adding cysteine and glycine at a rate of 0.5-1 mM / L / h.

[0022] In one embodiment, the fermentation method specifically comprises: S1: after the glycerol in the fermentation medium is consumed, the dissolved oxygen in the fermentation broth is added to 50-75% when the glycerol is added; S2: when the wet weight of the bacteria reaches 200-230 g / L, the glycerol is stopped and the fermentation enters the starvation phase after the glycerol is consumed, and is maintained for 20-40 min; S3: methanol is added to enter the induction phase, the dissolved oxygen in the fermentation broth is controlled at 10-25%, and cysteine and glycine with a concentration ratio of 1:2 are added at a rate of 0.5-1 mM / L / h.

[0023] In another aspect, the application provides the use of the fermentation method described above in the preparation of type XVII recombinant collagen.

[0024] In another aspect, the application also provides a type XVII recombinant collagen obtained by the fermentation method described above.

[0025] (Three) beneficial effects The application provides a fermentation method for type XVII recombinant collagen and its application. Compared with the prior art, the application has the following beneficial effects: 1. By increasing key amino acids during the methanol induction expression stage of fermentation, the growth metabolism of Pichia pastoris is adjusted to increase protein synthesis. The fermentation yield can be increased by 69.8%, and the fermentation time can be shortened by 12-24 h. Pichia pastoris JY-17 3 3 growth metabolism, to increase protein synthesis. The fermentation yield can be increased by 69.8%, and the fermentation time can be shortened by 12-24 h. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the embodiments of the application clearer, the technical scheme in the embodiments of the application will be described clearly and completely below in conjunction with the embodiments of the application. Obviously, the described embodiments are part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0027] Terms and definitions As used herein, "containing", "having" or "including" includes "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting essentially of", "consisting essentially of", and "consisting of" are sub-concepts of "containing", "having" or "including".

[0028] The experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the technical field, unless otherwise specified.

[0029] Example 1 Fermentation of type XVII recombinant collagen #1: Fermentation medium: 85% H3PO4 8.9ml / L, CaSO4 2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4 7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L. (PTM1 solution refers to the manual of Pichia pastoris of Invitrogen Company, and the specific formula includes: sodium iodide 0.08g / L, copper sulfate pentahydrate 6.0g / L, manganese sulfate monohydrate 3.0g / L, sodium molybdate 0.2g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc sulfate 20.0g / L, ferrous sulfate heptahydrate 65.0g / L, biotin 0.2g / L, sulfuric acid 5.0mL / L, and 0.22um filter membrane is sterilized by filtration.) Yeast extract 1-4g / L, yeast extract 1-4g / L, 85% phosphoric acid 8-12mL / L, calcium sulfate 0.23-0.69g / L, potassium sulfate 6.37-11.83g / L, magnesium sulfate heptahydrate 5.25-9.75g / L, potassium hydroxide 1-4g / L, glycerol 40g / L, antifoam 0.35-0.65g / L, PTM1 3.0-5.65mL / L, biotin 3-8mg / L.

[0030] The fermentation process of type XVII recombinant collagen includes the following steps: The Pichia pastoris Pichia pastoris JY-17 3 3According to the inoculation amount of 5%, inoculate in a 5L fermenter with a medium volume of 2L. In the basic culture stage, the culture temperature is 30℃, the pH is adjusted to 5.2 with ammonia water, the dissolved oxygen is not less than 25%, the tank pressure is maintained at 0.03MPa, and the initial stirring speed is 250rpm. After 15-18h of culture, the glycerol in the basic medium is consumed, the dissolved oxygen quickly rises to 50-75%, 50% glycerol is added to make the wet weight of the bacterial body reach 200-230g / L, and glycerol addition is stopped until it is consumed. After stopping the addition of glycerol, starve for 30min, and then enter the methanol addition stage for expressing recombinant protein, the tank pressure is 0.03MPa, the pH is adjusted to 5.8, the dissolved oxygen is controlled at 10~25%, and at the same time, glycine is added, the addition speed is 1mM / L / h, and the fermentation is stopped after 84h of methanol induction. In the methanol induction stage, the fermentation broth is collected every 6h, centrifuged at 15000rpm, the supernatant is collected, and the content of recombinant collagen is detected by SDS-PAGE.

[0031] Example 2 Fermentation of type XVII recombinant collagen #2: Fermentation medium: 85% H3PO4 8.9ml / L, CaSO4 2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4 7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L. (PTM1 solution refers to the manual of Pichia pastoris of Invitrogen Company, and the specific formula includes: sodium iodide 0.08g / L, copper sulfate pentahydrate 6.0g / L, manganese sulfate monohydrate 3.0g / L, sodium molybdate 0.2g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc sulfate 20.0g / L, ferrous sulfate heptahydrate 65.0g / L, biotin 0.2g / L, sulfuric acid 5.0mL / L, and 0.22um filter membrane is sterilized by filtration.) Yeast extract 1-4g / L, yeast extract 1-4g / L, 85% phosphoric acid 8-12mL / L, calcium sulfate 0.23-0.69g / L, potassium sulfate 6.37-11.83g / L, magnesium sulfate heptahydrate 5.25-9.75g / L, potassium hydroxide 1-4g / L, glycerol 40g / L, antifoam 0.35-0.65g / L, PTM1 3.0-5.65mL / L, biotin 3-8mg / L.

[0032] The fermentation process of the recombinant collagen of type XVII includes the following steps: Pichia pastoris JY-173 3According to the inoculation amount of 5%, inoculate in a 5L fermenter, and the medium volume is 2L. In the basic culture stage, the culture temperature is 30℃, the pH is adjusted to 5.2 by ammonia water, the dissolved oxygen is not less than 25%, the tank pressure is maintained at 0.03MPa, and the initial stirring speed is 250rpm. After 15-18h of culture, the glycerol in the basic material is consumed, the dissolved oxygen quickly rises to 50-75%, 50% glycerol is added to make the wet weight of the bacteria reach 200-230g / L, and glycerol is stopped until it is consumed. After stopping the glycerol addition, starve for 30min, and then enter the methanol addition stage for expressing the recombinant protein. The tank pressure is 0.03MPa, the pH is adjusted to 5.8, the dissolved oxygen is controlled at 10-25%, and at the same time, proline is added at a flow rate of 1mM / L / h. After 84h of methanol induction, the fermentation is stopped. In the methanol induction stage, the fermentation broth is collected every 6h, centrifuged at 15000rpm, and the supernatant is collected. The content of recombinant collagen protein is detected by SDS-PAGE.

[0033] Example 3 Fermentation of recombinant collagen of type XVII #3: Fermentation medium: 85% H3PO4 8.9ml / L, CaSO4 2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4 7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L. (PTM1 solution refers to the manual of Pichia pastoris of Invitrogen Company, and the specific formula includes: sodium iodide 0.08g / L, copper sulfate pentahydrate 6.0g / L, manganese sulfate monohydrate 3.0g / L, sodium molybdate 0.2g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc sulfate 20.0g / L, ferrous sulfate heptahydrate 65.0g / L, biotin 0.2g / L, sulfuric acid 5.0mL / L, and 0.22um filter membrane is sterilized by filtration.) Yeast extract 1-4g / L, yeast extract 1-4g / L, 85% phosphoric acid 8-12mL / L, calcium sulfate 0.23-0.69g / L, potassium sulfate 6.37-11.83g / L, magnesium sulfate heptahydrate 5.25-9.75g / L, potassium hydroxide 1-4g / L, glycerol 40g / L, antifoam 0.35-0.65g / L, PTM1 3.0-5.65mL / L, biotin 3-8mg / L.

[0034] The fermentation process of the recombinant collagen of type XVII includes the following steps: The Pichia pastoris Pichia pastoris JY-17 3 3According to the inoculation amount of 5%, inoculate in a 5L fermenter with a medium volume of 2L. In the basic culture stage, the culture temperature is 30℃, the pH is adjusted to 5.2 with ammonia water, the dissolved oxygen is not less than 25%, the tank pressure is maintained at 0.03MPa, and the initial stirring speed is 250rpm. After 15-18h of culture, the glycerol in the basic medium is consumed, the dissolved oxygen quickly rises to 50-75%, 50% glycerol is added to make the wet weight of the bacterial body reach 200-230g / L, and glycerol addition is stopped until it is consumed. After stopping the addition of glycerol, starve for 30min, and then enter the methanol addition stage for expressing recombinant protein, the tank pressure is 0.03MPa, the pH is adjusted to 5.8, the dissolved oxygen is controlled at 10-25%, and at the same time, lysine is added at a flow rate of 0.5mM / L / h. After 84h of methanol induction, the fermentation is terminated. During the methanol induction stage, the fermentation broth is collected every 6h, centrifuged at 15000rpm, and the supernatant is collected. The content of recombinant collagen protein is detected by SDS-PAGE.

[0035] Example 4 Fermentation of recombinant collagen of type XVII Fermentation medium: 85% H3PO4 8.9ml / L, CaSO4 2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4 7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L. (PTM1 solution refers to the manual of Pichia pastoris of Invitrogen Company, and the specific formula includes: sodium iodide 0.08g / L, copper sulfate pentahydrate 6.0g / L, manganese sulfate monohydrate 3.0g / L, sodium molybdate 0.2g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc sulfate 20.0g / L, ferrous sulfate heptahydrate 65.0g / L, biotin 0.2g / L, sulfuric acid 5.0mL / L, and 0.22um filter membrane is sterilized by filtration.) Yeast extract 1-4g / L, yeast extract 1-4g / L, 85% phosphoric acid 8-12mL / L, calcium sulfate 0.23-0.69g / L, potassium sulfate 6.37-11.83g / L, magnesium sulfate heptahydrate 5.25-9.75g / L, potassium hydroxide 1-4g / L, glycerol 40g / L, antifoam 0.35-0.65g / L, PTM1 3.0-5.65mL / L, biotin 3-8mg / L.

[0036] The fermentation process of the type XVII recombinant collagen protein includes the following steps: Pichia pastoris JY-173 3According to the inoculation amount of 5%, inoculate in a 5L fermenter, and the medium volume is 2L. In the basic culture stage, the culture temperature is 30℃, the pH is adjusted to 5.2 by ammonia water, the dissolved oxygen is not less than 25%, the tank pressure is maintained at 0.03MPa, and the initial stirring speed is 250rpm. After 15-18h of culture, the glycerol in the basic material is consumed, the dissolved oxygen quickly rises to 50-75%, 50% glycerol is added to make the wet weight of the bacteria reach 200-230g / L, and glycerol is stopped until it is consumed. After stopping the glycerol addition, starve for 30min, and then enter the methanol addition stage for expressing the recombinant protein, the tank pressure is 0.03MPa, the pH is adjusted to 5.8, the dissolved oxygen is controlled at 10-25%, and glutamine is added at the same time, the addition speed is 0.5mM / L / h, and the fermentation is stopped after 84h of methanol induction. In the methanol induction stage, the fermentation broth is collected every 6h, centrifuged at 15000rpm, the supernatant is collected, and the content of recombinant collagen protein is detected by SDS-PAGE.

[0037] Example 5 Fermentation of type XVII recombinant collagen protein #5: Fermentation medium: 85% H3PO4 8.9ml / L, CaSO4 2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4 7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L. (PTM1 solution refers to the manual of Pichia pastoris of Invitrogen Company, and the specific formula includes: sodium iodide 0.08g / L, copper sulfate pentahydrate 6.0g / L, manganese sulfate monohydrate 3.0g / L, sodium molybdate 0.2g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc sulfate 20.0g / L, ferrous sulfate heptahydrate 65.0g / L, biotin 0.2g / L, sulfuric acid 5.0mL / L, and 0.22um filter membrane is sterilized by filtration.) Yeast extract 1-4g / L, yeast extract 1-4g / L, 85% phosphoric acid 8-12mL / L, calcium sulfate 0.23-0.69g / L, potassium sulfate 6.37-11.83g / L, magnesium sulfate heptahydrate 5.25-9.75g / L, potassium hydroxide 1-4g / L, glycerol 40g / L, antifoam 0.35-0.65g / L, PTM1 3.0-5.65mL / L, biotin 3-8mg / L.

[0038] The fermentation process of type XVII recombinant collagen includes the following steps: The Pichia pastoris Pichia pastoris JY-17 3 3According to the inoculation amount of 5%, inoculate in a 5L fermenter, and the medium volume is 2L. In the basic culture stage, the culture temperature is 30℃, the pH is adjusted to 5.2 by ammonia water, the dissolved oxygen is not less than 25%, the tank pressure is maintained at 0.03MPa, and the initial stirring speed is 250rpm. After 15-18h of culture, the glycerol in the basic medium is consumed, the dissolved oxygen quickly rises to 50-75%, 50% glycerol is added to make the wet weight of the bacterial body reach 200-230g / L, and glycerol is stopped until it is consumed. After stopping the addition of glycerol, starve for 30min, and then enter the methanol addition stage for expressing recombinant protein, the tank pressure is 0.03MPa, the pH is adjusted to 5.8, the dissolved oxygen is controlled at 10~25%, and at the same time, methionine and proline are added at a concentration ratio of 1:2, and the flow rate is 1mM / L / h. After 84h of methanol induction, the fermentation is stopped. In the methanol induction stage, the fermentation broth is collected every 6h, centrifuged at 15000rpm, the supernatant is collected, and the content of recombinant collagen is detected by SDS-PAGE.

[0039] Example 6 Fermentation of type XVII recombinant collagen #6: Fermentation medium: 85% H3PO4 8.9ml / L, CaSO4·2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4·7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L. (PTM1 solution refers to the manual of Pichia pastoris of Invitrogen Company, and the specific formula includes: sodium iodide 0.08g / L, copper sulfate pentahydrate 6.0g / L, manganese sulfate monohydrate 3.0g / L, sodium molybdate 0.2g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc sulfate 20.0g / L, ferrous sulfate heptahydrate 65.0g / L, biotin 0.2g / L, sulfuric acid 5.0mL / L, and 0.22um filter membrane is sterilized by filtration.) Yeast extract 1-4g / L, yeast extract 1-4g / L, 85% phosphoric acid 8-12mL / L, calcium sulfate 0.23-0.69g / L, potassium sulfate 6.37-11.83g / L, magnesium sulfate heptahydrate 5.25-9.75g / L, potassium hydroxide 1-4g / L, glycerol 40g / L, antifoam 0.35-0.65g / L, PTM1 3.0-5.65mL / L, biotin 3-8mg / L.

[0040] The fermentation process of type XVII recombinant collagen includes the following steps: The Pichia pastoris Pichia pastoris JY-17 3 3According to the inoculation amount of 5%, inoculate in a 5L fermenter with a medium volume of 2L. In the basic culture stage, the culture temperature is 30℃, the pH is adjusted to 5.2 with ammonia water, the dissolved oxygen is not less than 25%, the tank pressure is maintained at 0.03MPa, and the initial stirring speed is 250rpm. After 15-18h of culture, the glycerol in the basic medium is consumed, the dissolved oxygen quickly rises to 50-75%, 50% glycerol is added to make the wet weight of the bacterial body reach 200-230g / L, and glycerol addition is stopped until it is consumed. After stopping the addition of glycerol, starve for 30min, and then enter the methanol addition stage for expressing recombinant protein, the tank pressure is 0.03MPa, the pH is adjusted to 5.8, the dissolved oxygen is controlled at 10~25%, and at the same time, cysteine and glycine are added at a concentration ratio of 1:2, and the addition speed is 1mM / L / h. After 84h of methanol induction, the fermentation is stopped. In the methanol induction stage, the fermentation broth is collected every 6h, centrifuged at 15000rpm, the supernatant is collected, and the content of recombinant collagen is detected by SDS-PAGE.

[0041] Comparative Example 1 Fermentation of Type XVII Recombinant Collagen: Fermentation medium: 85% H3PO4 8.9ml / L, CaSO4 2H2O 1.03g / L, K2SO4 15.9g / L, MgSO4 7H2O 15.5g / L, KOH 3.82g / L, glycerol 40.0g / L, PTM1 solution 3.91ml / L. (PTM1 solution refers to the manual of Pichia pastoris of Invitrogen Company, and the specific formula includes: sodium iodide 0.08g / L, copper sulfate pentahydrate 6.0g / L, manganese sulfate monohydrate 3.0g / L, sodium molybdate 0.2g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc sulfate 20.0g / L, ferrous sulfate heptahydrate 65.0g / L, biotin 0.2g / L, sulfuric acid 5.0mL / L, and 0.22um filter membrane is sterilized by filtration.) Yeast extract 1-4g / L, yeast extract 1-4g / L, 85% phosphoric acid 8-12mL / L, calcium sulfate 0.23-0.69g / L, potassium sulfate 6.37-11.83g / L, magnesium sulfate heptahydrate 5.25-9.75g / L, potassium hydroxide 1-4g / L, glycerol 40g / L, antifoam 0.35-0.65g / L, PTM1 3.0-5.65mL / L, biotin 3-8mg / L.

[0042] The fermentation process of the recombinant collagen of type XVII includes the following steps: Pichia pastoris JY-173 3According to the inoculation amount of 5%, inoculate in a 5L fermenter with a medium volume of 2L. In the basic culture stage, the culture temperature is 30℃, the pH is adjusted to 5.2 with ammonia water, the dissolved oxygen is not less than 25%, the tank pressure is maintained at 0.03MPa, and the initial stirring speed is 250rpm. After 15-18h of culture, the glycerol in the basic medium is consumed, the dissolved oxygen quickly rises to 50-75%, 50% glycerol is added to make the wet weight of the bacteria reach 200-230g / L, and glycerol addition is stopped until it is consumed. After stopping the addition of glycerol, starve for 30min, and then enter the methanol addition stage for expressing recombinant protein, with tank pressure of 0.03MPa, pH adjusted to 5.8, dissolved oxygen controlled at 10-25%, and fermentation terminated after 84h of methanol induction. In the methanol induction stage, the fermentation broth is collected every 6h, centrifuged at 15000rpm, and the supernatant is collected for detection of the content of recombinant collagen by SDS-PAGE.

[0043] Comparative Example 2 Fermentation of recombinant collagen of type XVII: Fermentation medium: 85% H3PO4 8.9 ml / L, CaSO4 2H2O 1.03 g / L, K2SO4 15.9 g / L, MgSO4 7H2O 15.5 g / L, KOH 3.82 g / L, glycerol 40.0 g / L, PTM1 solution 3.91 ml / L. (PTM1 solution refers to the manual of Pichia pastoris of Invitrogen Company, and the specific formula includes: sodium iodide 0.08 g / L, copper sulfate pentahydrate 6.0 g / L, manganese sulfate monohydrate 3.0 g / L, sodium molybdate 0.2 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc sulfate 20.0 g / L, ferrous sulfate heptahydrate 65.0 g / L, biotin 0.2 g / L, sulfuric acid 5.0 mL / L, and 0.22 um filter membrane is used for sterilization.) Yeast extract 1-4 g / L, yeast extract 1-4 g / L, 85% phosphoric acid 8-12 mL / L, calcium sulfate 0.23-0.69 g / L, potassium sulfate 6.37-11.83 g / L, magnesium sulfate heptahydrate 5.25-9.75 g / L, potassium hydroxide 1-4 g / L, glycerol 40 g / L, antifoam 0.35-0.65 g / L, PTM1 3.0-5.65 mL / L, biotin 3-8 mg / L.

[0044] The fermentation process of type XVII recombinant collagen includes the following steps: Pichia pastoris Pichia pastoris JY-17 3 3According to the inoculation amount of 5%, inoculate in a 5L fermenter with a medium volume of 2L. In the basic culture stage, the culture temperature is 30°C, the pH is adjusted to 5.2 with ammonia water, the dissolved oxygen is not less than 25%, the tank pressure is maintained at 0.03MPa, and the initial stirring speed is 250rpm. After 15-18h of culture, the glycerol in the basic medium is consumed, the dissolved oxygen quickly rises to 50-75%, 50% glycerol is added to make the wet weight of the bacterial body reach 200-230g / L, and glycerol addition is stopped until it is consumed. After stopping the addition of glycerol, starve for 30min, and then enter the methanol addition stage for expressing recombinant protein, the tank pressure is 0.03MPa, the pH is adjusted to 5.8, the dissolved oxygen is controlled at 10-25%, and L-hydroxyproline is added at the same time, the addition speed is 0.5mM / L / h, and the fermentation is stopped after 84h of methanol induction. The fermentation is stopped after 84h of methanol induction. In the methanol induction stage, the fermentation broth is collected every 6h, centrifuged at 15000rpm, the supernatant is collected, and the content of recombinant collagen is detected by SDS-PAGE.

[0045] The fermentation results are shown in Table 1: Table 1 Fermentation results Comparing example 1-6 and comparative example 1, methanol induction stage, while adding different kinds of amino acids and different proportions of amino acids, the fermentation level of type XV recombinant collagen is obviously improved, wherein the fermentation level of type XV recombinant collagen of example 6 is higher than that of comparative example 1 by about 69.8% in 84h of methanol induction, and the fermentation time can be shortened by 12-24h accordingly. Comparing example 1-6 and comparative example 2, it can be seen that during the methanol induction stage, the addition of L-hydroxyproline does not obviously improve the yield of recombinant collagen. It is considered that Pichia pastoris lacks a metabolic pathway for efficiently degrading and utilizing L-hydroxyproline as a carbon source, and L-hydroxyproline cannot be rapidly consumed. L-hydroxyproline is highly similar in structure to L-proline, and it can competitively inhibit the absorption and transport system of Pichia pastoris cells to proline, and does not obviously improve the expression of collagen.

[0046] It should be noted that the relational terms herein such as first and second, and the like, are used solely to distinguish one from another entity or action, without necessarily requiring or implying any such actual relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element preceded by "comprises... a" does not, without more constraints, foreclose the existence of additional identical elements in the process, method, article, or apparatus that comprises the recited element.

[0047] The above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A fermentation method of a type XVII recombinant collagen, characterized by, The fermentation method comprises adding amino acids into the fermentation liquor, and the amino acids include glycine, proline, lysine, glutamine, methionine, cysteine and / or L-hydroxyproline.

2. The fermentation process according to claim 1, characterized in that, The amino acids include glycine and cysteine.

3. The fermentation process of claim 2, wherein, The concentration ratio of the cysteine and glycine is 1: (1-5).

4. The fermentation process of claim 3, wherein, The concentration ratio of the cysteine and glycine is 1:

2.

5. The fermentation process according to any one of claims 1 to 4, characterized in that, The fermentation method comprises adding amino acids in the induction period after the starvation.

6. The fermentation process of claim 5, wherein, The fermentation method comprises: S1: adding glycine after the glycine in the fermentation medium is consumed; S2: stopping the addition of glycine and entering the starvation period after the glycine is consumed; S3: adding methanol and amino acids to enter the induction period.

7. The fermentation process of claim 6, wherein, The judgment standard for adding glycine is that the dissolved oxygen in the fermentation liquor rises to 50-75%.

8. The fermentation process of claim 6, wherein, The judgment standard for stopping the addition of glycine is that the wet weight of the bacteria reaches 200-230 g / L.

9. The fermentation process of claim 6, wherein, The starvation period is kept for 20-40 min.

10. The fermentation process of claim 6, wherein, The dissolved oxygen in the fermentation liquor in the induction period is 10-25%.

11. The fermentation process of claim 6, wherein, The addition rate of the amino acids is 0.5-1 mM / L / h.

12. The fermentation process of claim 6, wherein, The fermentation medium comprises: yeast protein peptone 1-4 g / L, yeast extract 1-4 g / L, 85% phosphoric acid 8-12 mL / L, calcium sulfate 0.23-0.69 g / L, potassium sulfate 6.37-11.83 g / L, magnesium sulfate heptahydrate 5.25-9.75 g / L, potassium hydroxide 1-4 g / L, glycine 20-60 g / L, antifoaming agent 0.35-0.65 g / L, PTM1 3.0-5.65 mL / L, and biotin 3-8 mg / L.

13. The fermentation process according to any one of claims 6 to 12, characterized in that, The fermented cell is a Pichia pastoris cell.

14. The use of the fermentation method in any one of claims 1-13 in the preparation of type XVII recombinant collagen.

15. A recombinant collagen of type XI V, characterized in that, The fermentation method in any one of claims 1-13.

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