Recombinant Pichia pastoris culture medium, and culture method and application thereof

A technology of Pichia pastoris and a culturing method, which is applied in the field of medium for recombinant Pichia pastoris, can solve the problems of easy to produce precipitation substances, complex components, high price and the like, and achieves the effects of convenient configuration, simple components and strong applicability

Inactive Publication Date: 2015-11-11
GUANGDONG HINAPHARM PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a medium for recombinant Pichia pastoris and its cultivation method an

Method used

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  • Recombinant Pichia pastoris culture medium, and culture method and application thereof
  • Recombinant Pichia pastoris culture medium, and culture method and application thereof
  • Recombinant Pichia pastoris culture medium, and culture method and application thereof

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preparation example Construction

[0027] The preparation method of the medium of the above-mentioned recombinant Pichia pastoris of the present invention comprises the steps of: replacing the BMGY medium with cheap glucose, corn steep liquor, ammonium sulfate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate according to the BMGY medium components Components in, and then design an orthogonal table to test, and screen to obtain the culture medium of the recombinant Pichia pastoris. The main improvement of the present invention is that, based on the physiological characteristics and metabolic laws of Pichia pastoris, by adjusting the carbon-to-nitrogen ratio (C:N) balance of the medium formula, the culture medium of recombinant Pichia pastoris with the optimal formula is screened and obtained The culture medium of the recombinant Pichia pastoris is conducive to the high-density reproduction and growth of bacteria.

[0028] Specifically, the configuration steps of BMGY medium are as follows: 10g...

Embodiment 1

[0042] Example 1: High-density cultivation of recombinant Pichia pastoris GS115-ple

[0043] (1) Rejuvenation of strains:

[0044] In order to ensure that the obtained strains are pure recombinant strains, it is necessary to screen the strains again under stress-resistant conditions. Streak the bacteria stored in the glycerol tube with an inoculation loop on the YPD agar plate containing 200ug / mL bleomycin, culture statically at 28°C for 48 hours, and wait for obvious single colonies to grow, then set aside.

[0045] (2) Shake flask seed culture:

[0046] Pick 2 single colonies with large colonies from the YPD agar plate in (1) and inoculate them into the shake flask medium, and inoculate 100mL of liquid in a 500mL triangular flask, and culture at 180rpm for 24h. The culture medium of the shake flask recombinant Pichia pastoris comprises: 20 g / L of glucose, 20 g / L of peptone, and 10 g / L of yeast extract powder.

[0047] (3) High-density fermentation culture in fermenter:

...

Embodiment 2

[0050] Embodiment 2: the high-density cultivation of recombinant Pichia pastoris X33-ple

[0051] (1) Rejuvenation of strains:

[0052] In order to ensure that the obtained strains are pure recombinant strains, it is necessary to screen the strains again under stress-resistant conditions. Streak the bacteria stored in the glycerol tube with an inoculation loop on the YPD agar plate containing 300ug / mL bleomycin, and culture it statically at 30°C for 72 hours. When a single colony grows obviously, it is ready for use.

[0053] (2) Shake flask seed culture:

[0054] Pick 3 single colonies with large colonies from the YPD agar plate in (1) and inoculate them into the shake flask medium, and inoculate 100mL of liquid in a 500mL triangular flask, and culture at 200rpm for 20h. The ingredients of the shake flask culture medium include glucose 20g / L, peptone 20g / L, and yeast extract powder 10g / L.

[0055] (3) High-density fermentation culture in fermenter:

[0056] The bacterial ...

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Abstract

The invention discloses a recombinant Pichia pastoris culture medium, and a culture method and application thereof. The recombinant Pichia pastoris culture medium is prepared from cheap glucose, corn steep liquor, partial inorganic salts and a small amount of yeast extract. The recombinant Pichia pastoris culture medium is convenient to prepare and suitable for industrial production. The glycerol strain is subjected to bleomycin-containing YPD agar plate rejuvenation and sequentially subjected to seed culture, fermentation tank early-stage culture and fermentation tank later-stage fed-batch nutrient solution culture; and after the recombinant Pichia pastoris is cultured for 18-24 hours, the diluted coating culture counting detection indicates that the yeast living cell number in the fermentation liquid can reach 4 billion/mL or above, the wet bacterium weight can reach 180-220 g/L, and the glucose-to-yeast yield is 0.43-0.46 which is approximate to the theoretical value 0.5. The concentration of the metabolism byproduct ethanol in the whole culture process is almost 0, and the bacterium concentration and wet bacterium weight can quickly increase to the target values, thereby effectively shortening the culture period, enhancing the equipment utilization rate and lowering the production cost.

Description

technical field [0001] The invention relates to the technical field of biological culture, in particular to a culture medium of recombinant Pichia pastoris and its culture method and application. Background technique [0002] Pichia pastoris (Pichia pastoris) is a single-celled lower eukaryote. It is a eukaryotic expression system that has been studied in recent years. Post-translational processing and modification, non-production of toxic substances and other characteristics. The currently reported Pichia pastoris culture medium includes BMGY, BMMY and BSM. Among them, BMGY and BMMY medium are mostly used in laboratory research, and the components are complex and the configuration is cumbersome; on the other hand, organic nitrogen sources such as YNB, yeast extract powder, and peptone are needed, which are expensive and not suitable for industrial production. BSM medium is an inorganic salt medium, its components are simple, easy to configure, but it is easy to produce se...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12R1/84
Inventor 闫凯周玉岩逯佩凤叶滔冯蓓
Owner GUANGDONG HINAPHARM PHARMA CO LTD
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