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Enzymic method for producing [alpha]-ketoglutaric acid

A ketoglutaric acid and enzymatic technology, which is applied in the field of enzymatic production of alpha-ketoglutaric acid, can solve the problems of complex extraction and refining processes, high total cost, long production cycle and the like

Inactive Publication Date: 2014-10-22
SHANGHAI RES & DEV CENT OF INDAL BIOTECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, biological fermentation also has its disadvantages: long production cycle, low yield, mixing of various components in the product and fermentation liquid, resulting in complicated extraction and refining process, and the total cost is still high
Long Liu et al. (Long Liu, G S Hossain, et al. Journal of Biotechnology, 2013, 164:97-104) used L-glutamic acid as a substrate to deaminate α-ketone using L-amino acid deaminase Glutaric acid, but its output is low, only less than 12.6g / L, and there is product inhibition in the reaction, it is still difficult to industrialize

Method used

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  • Enzymic method for producing [alpha]-ketoglutaric acid
  • Enzymic method for producing [alpha]-ketoglutaric acid
  • Enzymic method for producing [alpha]-ketoglutaric acid

Examples

Experimental program
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Effect test

Embodiment 1

[0129] Example 1. Construction of highly expressed L-glutamic acid oxidase, cloning and expression of L-glutamic acid oxidase derived from Streptomyces sp X-119-6

[0130] The L-glutamic acid oxidase gene LGOX (NCBI accession number: AB085623; SEQ ID NO.: 1) derived from Streptomyces sp X-119-6 was synthesized to obtain the plasmid pUC57-Lgox.

[0131] Digest pUC57-Lgox with NdeI and HindIII at 37°C for 3-6 hours. The enzyme digestion system is: pUC57-Logx 43μL, Buffer R 5μL, NdeI 1μL, HindIII 1μL, nucleic acid electrophoresis and gel recovery kit to recover 2.1kb Lgox fragment.

[0132] The recovered L-gox fragment was ligated with the expression vector pET28b treated by the same enzyme digestion with T4 DNA ligase at 16°C overnight, transformed E. coli DH5α competent cells with the calcium chloride method, and spread on the LB plate containing Kan, 37 Cultivate overnight. Pick a single clone and inoculate it in LB test tubes for culture, extract the plasmid with a plasmid ...

Embodiment 2

[0134] Embodiment 2, the purification of enzyme

[0135] After fermentation, it was centrifuged at 8000 rpm to obtain wet bacteria. The enzyme activity of the wet bacteria obtained by fermentation was 610 U / g. The enzyme activity of L-glutamic acid oxidase was determined and purified according to the literature Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp. X-119-6.Arima, J.;Tamura,T.;Kusakabe,H.;Ashiuchi,M.;Yagi,T.;Tanaka,H.;Inagaki,K.; J.Biochem.134, 805-812 (2003), the purified enzyme has a specific activity of 52 U / mg.

Embodiment 3

[0136] Example 3, Conversion and preparation of α-ketoglutarate with LGOX-containing cell lysate

[0137] Use a fermenter or other containers with good oxygen supply conditions for the reaction, the reaction system contains 100g / L sodium glutamate, add L-glutamic acid oxidase cell lysate according to the final concentration of 4000U / L, according to the final concentration of 1500000U / L Add catalase (Novozymes Terminox Ultra 2000L), use 5M sodium hydroxide or hydrochloric acid to control the pH between 7.0-8.0, the temperature is 35°C, the ventilation volume is 1vvm, and the tank pressure is 0.01 mpa. Control the dissolved oxygen at 25% with the adjustment of the ventilation rate. When the dissolved oxygen rises, add 50g / L of sodium glutamate, the dissolved oxygen drops, and continue to transform until the dissolved oxygen rises and the transformation ends. The content of α-ketoglutaric acid determined by HPLC was 111.8g / L, and the molar conversion rate based on the volume chan...

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Abstract

The invention discloses an enzymic method for producing [alpha]-ketoglutaric acid. The method includes a step of carrying out a catalytic oxidation reaction, catalyzed by L-glutamic oxidase, to L-glutamic or a salt thereof in the presence of a hydrogen peroxide scavenger to obtain [alpha]-ketoglutaric acid. The method is short in production period, is high in product concentration and yield, is low in environment-protective pressure and is suitable for large-scale industrial production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for enzymatically producing alpha-ketoglutarate. Background technique [0002] α-Ketoglutaric acid (α-KG) is an important biological compound. It is a keto acid product of glutamic acid deamination, and is an intermediate product of the tricarboxylic acid cycle, and participates in the synthesis of amino acids, proteins, vitamins and energy metabolism in organisms. α-Ketoglutarate is widely used in food, medicine, fine chemical and feed industries. In particular, L-arginine α-ketoglutarate with a molar ratio of 1:1 and L-arginine α-ketoglutarate with a molar ratio of 2:1 are currently increasingly sold in the international market An amino acid salt health product, as a functional nutrition enhancer and liver-protecting drug, is mainly used to enhance physical fitness, promote rapid muscle growth and recovery, promote the absorption of nutrients and energy by li...

Claims

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Application Information

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IPC IPC(8): C12P7/50C12N9/08C12N9/06
Inventor 陶荣盛朱傅赟孙梁栋沈正权沈青郑云陈成蒋宇杨晟
Owner SHANGHAI RES & DEV CENT OF INDAL BIOTECH
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