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Method for synthesizing alpha-ketoglutaric acid by biological conversion method

A technology of ketoglutaric acid and biotransformation, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of high production costs

Active Publication Date: 2015-12-23
ZHEJIANG SHUREN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In high-sugar and low-nitrogen medium, add dehydrogenase inhibitors to prevent α-ketoglutarate from being further metabolized, thereby promoting the accumulation of α-ketoglutarate, so that cheap L-glutamic acid can be converted For α-ketoglutarate with a higher price, it solves the problem of high production cost of the current fermentation method, enzyme method and whole cell conversion method, and has high industrial application value

Method used

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  • Method for synthesizing alpha-ketoglutaric acid by biological conversion method

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1: Screening of transformed yeast strains

[0027] Seven yeast strains were purchased from the market, including 2 strains of Saccharomyces cerevisiae, 1 strain of Pichia pastoris, 1 strain of Candida guilliermond, 1 strain of Sporidiobolus johnsonii, One strain of Kluyveromyces marxianus and one strain of Candida tropicalis. Among them, Kluyveromyces marxianus (Kluyveromyces marxianus) was purchased from the American Type Culture Collection, with the preservation number ATCC36534.

[0028] Firstly, pick a ring full of bacteria from the above-mentioned yeast slant strains stored in the refrigerator, inoculate it in a fresh slant medium, and culture the slant in a biochemical incubator at 30°C for 36 hours to obtain an activated yeast strain slant. Pick 4 full rings of bacteria from the activated strain slant, inoculate into a 250mL Erlenmeyer flask containing 50mL of transformation medium, and shake the flask at 30°C and 200r / min for 24h after transformation (c...

Embodiment 2

[0034] Example 2: H 2 o 2 Choice of Concentration

[0035] After confirming that Kluyveromyces marxense ATCC36534 was used as the strain for transforming L-glutamic acid into α-ketoglutarate, H 2 o 2 As an inhibitor of α-ketoglutarate dehydrogenase, H 2 o 2 the optimal concentration of addition.

[0036] First, pick 1 full ring of bacterial cells from the Kluyveromyces marxense ATCC36534 slant strain stored in the refrigerator, inoculate it on a fresh slant medium, and culture the slant surface in a biochemical incubator at 30°C for 24 hours to obtain an activated Kluyveromyces marxii strain ATCC36534 inclined plane. Pick 2 rings of bacteria from the activated strain slant and put them into 50mL seed medium (in a 250mL Erlenmeyer flask). Seed culture is based on a constant temperature shaking shaker at 30°C, 200r / min shaking culture for 24h, and dry The seed solution with a bacterium concentration of 5.35g / L. Use a sterile pipette to draw 5 mL of seed solution of ATCC3...

Embodiment 3

[0040] Embodiment 3: the selection of methotrexate concentration

[0041] After confirming that Kluyveromyces marxense ATCC36534 was used as the strain for converting glutamic acid into α-ketoglutarate, methotrexate was added to the medium as an inhibitor of α-ketoglutarate dehydrogenase. This implementation class The optimal concentration of methotrexate was selected for optimization.

[0042] First, pick 1 full ring of bacterial cells from the Kluyveromyces marxense ATCC36534 slant strain stored in the refrigerator, inoculate it on a fresh slant medium, and culture the slant surface in a biochemical incubator at 30°C for 24 hours to obtain an activated Kluyveromyces marxii strain ATCC36534 inclined plane. Pick 2 rings of bacteria from the activated strain slant and put them in a Erlenmeyer flask containing 50mL of seed medium (in a 250mL Erlenmeyer flask). Seed culture is based on a 30°C constant temperature shaking shaker, 200r / min shaking culture for 24h , to obtain a dr...

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Abstract

The invention aims at providing a method for synthesizing alpha-ketoglutaric acid by a biological conversion method. According to the method, L-glutamic acid is used as a substrate; in a conversion culture solution obtained from kluyveromyces marxianus ATCC36534 through being cultured via a conversion culture substrate, alpha-ketoglutaricdehydrogenase inhibitors are added; the synthesis conversion culture is performed under the oscillation conditions of 25 to 30 DEG C and 200 to 250 r / min; after the synthesis conversion culture is completed, the alpha-ketoglutaric acid is obtained through separating and purifying the synthesis conversion solution; the L-glutamic acid is converted into alpha-ketoglutaric acid by yeast cells in the growth state; the alpha-ketoglutaricdehydrogenase inhibitors are added; the further metabolism consumption is blocked, so that a great amount of alpha-ketoglutaric acid is accumulated in the culture medium; when the feeing concentration of the substrate L-glutamic acid is 50g / L, the mol conversion rate can reach 83.2 percent. The low-value L-glutamic acid is converted into high-value alpha-ketoglutaric acid; the large-scale industrial application can be realized; a production process has the advantages of short period, high conversion rate, small environment pollution and the like.

Description

(1) Technical field [0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a method for synthesizing α-ketoglutaric acid by a biotransformation method. (2) Background technology [0002] α-ketoglutaric acid (α-ketoglutaric acid), also known as α-carbonyl glutaric acid, 2-oxoglutaric acid or α-glutaric acid, CAS number is 328-50-7. α-Ketoglutarate is one of the important intermediates in the tricarboxylic acid (TCA) cycle, which plays an important role in cellular substance metabolism and energy metabolism. As an important intermediate, it is an important precursor for the synthesis of various amino acids and vitamins. α-Ketoglutaric acid has important application prospects in the fields of medicine, organic synthesis, and nutritional enhancers. At present, its main application areas include: as a component of sports functional drinks, as an intermediate in organic synthesis, and as a supporting tool for liver function testi...

Claims

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Application Information

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IPC IPC(8): C12P7/50C12R1/645
Inventor 陈虹陈蔚青张建芬柯薇陆胤梅建凤
Owner ZHEJIANG SHUREN UNIV
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