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Method for producing S-adenosylmethionine through fermentation of recombinant pichia pastoris

A technology of adenosylmethionine and genetically engineered bacteria, applied in microorganism-based methods, biochemical equipment and methods, fermentation, etc., to achieve the effects of high SAM yield, short fermentation cycle, and shortened fermentation time

Inactive Publication Date: 2009-11-04
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, fermentation amplification is also a difficulty in the large-scale and high-yield preparation of SAM

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] (1) Seed cultivation

[0019] Use an inoculation needle to scrape a few colonies on the well-grown slope and connect them to the liquid seed medium. The volume of the bottle is 25mL / 250mL, and cultured on a shaker at 30°C and 200rpm for 16 hours. The composition of the seed medium is (prepared according to 1L): 10g of yeast powder, 20g of peptone, 20g of glucose, and the rest is water; pH5.0.

[0020] (2) Fermentation culture

[0021] 100ml OD 600 Seed medium containing seeds of value 5 was inoculated into a 3.7 L fermenter (KLF2000 3.7L, Bioengineering AG, Switzerland) containing fermentation medium at an inoculum equivalent to 5% (v / v). Fermentation medium (prepared according to 1L) is: H 3 PO 4 26.7mL, CaSO 4 0.93g, K 2 SO 4 18.2g, MgSO 4 ·7H 2 O 14.9g, KOH 4.13g, glycerin 40g, PTM 14.35mL, the rest is water, adjust the pH to 5.0 with 28-30% (w / v) ammonia water. Among them, PTM1 (prepared by 1L): CuSO 4 ·5H 2 O 6.0g, KI0.08g, MnSO 4 ·H 2 O 3.0g, Na 2 M...

Embodiment 2

[0024] (1) Seed cultivation

[0025] Use an inoculation needle to scrape a few bacterial colonies on the long inclined surface and connect them to the liquid seed medium. The amount of bottling is 25ml / 250ml, and cultured on a shaker at 32°C and 250rpm for 20 hours. The composition of the seed medium is (prepared according to 1L): 10g of yeast powder, 20g of peptone, 20g of glycerin, and the rest is water; pH8.0.

[0026] (2) Fermentation culture

[0027] 200ml OD 600 Seed medium containing seeds with a value of 10 was inoculated into a 3.7 L fermenter containing fermentation medium at an inoculum equivalent to 10% (v / v). Fermentation medium (prepared according to 1L) is: H 3 PO 4 26.7mL, CaSO 4 0.93g, K 2 SO 4 18.2g, MgSO 4 ·7H 2 O 14.9g, KOH 4.13g, glycerin 40g, PTM 14.35mL, the rest is water, adjust the pH to 5.0 with 28-30% (w / v) ammonia water. Among them, PTM1 (prepared by 1L): CuSO 4 ·5H 2 O 6.0g, KI0.08g, MnSO 4 ·H 2 O 3.0g, Na2 MoO 4 2H 2 O0.2g, H 3 BO...

Embodiment 3

[0030] (1) Seed cultivation

[0031] Use an inoculation needle to scrape a few bacterial colonies on a well-grown slope and connect them to the liquid seed culture medium. The amount of bottling is 20ml / 250ml, and cultured on a shaker at 28°C and 100rpm for 18 hours. The composition of the seed medium is (prepared according to 1L): 10g of yeast powder, 20g of peptone, 20g of glycerin, and the rest is water; pH8.0.

[0032] (2) Fermentation culture

[0033] 120ml OD 600 Seed medium containing seeds with a value of 5 was inoculated into a 3.7 L fermenter containing fermentation medium at an inoculum equivalent to 6% (v / v). Fermentation medium (prepared according to 1L) is: H 3 PO 4 26.7mL, CaSO 4 0.93g, K 2 SO 4 18.2g, MgSO 4 ·7H 2 O 14.9g, KOH 4.13g, glycerin 40g, PTM 14.35mL, the rest is water, adjust the pH to 5.0 with 28-30% (w / v) ammonia water. Among them, PTM1 (prepared by 1L): CuSO 4 ·5H 2 O 6.0g, KI0.08g, MnSO 4 ·H 2 O 3.0g, Na 2 MoO 4 2H 2 O0.2g, H 3 B...

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Abstract

The invention discloses a method for producing S-adenosylmethionine through fermentation of recombinant pichia pastoris, which comprises that strain seed liquid of the recombinant pichia pastoris of a S-adenosylmethionine synthetase gene integrated with a heterologous expression gene is inoculated into a fermentation medium for fermentation culture, and is characterized in that: the inoculum size of the strain seed liquid of the recombinant pichia pastoris into the fermentation medium is between 5 and 10(v / v)percent; the culture condition in the fermentation culture is that the temperature is between 28 and 32 DEG C, the pH is between 3.0 and 8.0, and the dissolved oxygen level is kept between 30 and 100 percent; and when the strain seed liquid is cultured until the microbial density is between 80 and 120 g / L, the fermentation medium is added with methanol to begin methanol induction, the concentration of the methanol is controlled to be between 0.3 and 1.0(w / v)percent, and the fermentation medium is simultaneously added with L-methionine of which the concentration is controlled to be between 12.5 and 100mmol / L and ammonium sulphate in which the ammonium ion concentration is controlled to be between 300 and 600mmol / L. The method for producing the S-adenosylmethionine through the fermentation can accumulate SAM yield over 11.6g / L in 100h of fermentation time, so the method has short fermentation period, high SAM yield and simple and convenient operation, and can be used for preparing the SAM in mass production.

Description

technical field [0001] The invention belongs to the field of biological fermentation, and in particular relates to a method for producing S-adenosylmethionine by fermenting Pichia pastoris genetically engineered bacteria. Background technique [0002] Adenosylmethionine (S-adenosylmethionine, SAM) widely exists in animals and plants, and is an important biological intermediate. The molecular formula of SAM is C 15 h 22 N 6 o 5 S, with a molecular weight of 398.44, crystallizes as a solid and is easily soluble in water. SAM has two configurations, R-type and S-type, and only S-type SAM has biological activity in organisms. Under neutral and alkaline conditions, it is easy to absorb moisture, resulting in a conformational change to R-SAM, which is biologically inactive. S-SAM can be stored for a long time under low temperature, acidic and dry conditions. The formation of SAM in organisms is obtained by the reaction of adenosine triphosphate and methionine catalyzed by a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/40C12R1/84
Inventor 张鲁嘉张建国王学东魏东芝
Owner EAST CHINA UNIV OF SCI & TECH
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