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Method for alpha-ketoglutarate production by catalysis of L-glutamate dehydrogenase

A technology of glutamic acid dehydrogenase and ketoglutarate, which is applied in the field of biochemistry, can solve the problems of high cost, heavy pollution, complicated steps and the like, and achieves the effects of low production cost, good reaction selectivity and low raw materials

Active Publication Date: 2017-06-13
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention discloses a method for producing α-ketoglutaric acid catalyzed by L-glutamic acid dehydrogenase to solve the problems of heavy pollution, high cost and cumbersome steps in the prior art

Method used

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  • Method for alpha-ketoglutarate production by catalysis of L-glutamate dehydrogenase
  • Method for alpha-ketoglutarate production by catalysis of L-glutamate dehydrogenase
  • Method for alpha-ketoglutarate production by catalysis of L-glutamate dehydrogenase

Examples

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Effect test

Embodiment 1

[0035] Example 1: L-glutamate dehydrogenase catalyzes the production of α-ketoglutarate at different NAD+ concentrations

[0036] At 30°C, in 10ml pH8 Tris-HCl reaction system, L-glutamic acid 15mM, NAD+, 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride 1mM, catalase 500U / mL, L-glutamate dehydrogenase 5U / mL, the reaction solution was connected to the outside air. Take 200 μL of the reaction solution after reacting for 48 hours, add 800 μL of water to dilute, and detect the reaction progress by liquid phase. The results are shown in Table 1.

[0037] Table 1

[0038] NAD+concentration(mM) Yield(%) 0.5 52.1 1 90.7 1.5 75.2 2 50.5 2.5 46.8 3 43.7

Embodiment 2

[0039] Example 2: L-glutamate dehydrogenase catalyzes the production of α-ketoglutarate at different concentrations of 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride

[0040] At 30°C, in 10ml pH8 Tris-HCl reaction system, L-glutamic acid 15mM, NAD+1mM, 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride, catalase 500U / mL, L-glutamate dehydrogenase 5U / mL, the reaction solution is connected with the outside air. Take 200 μL of the 48-h reaction solution, add 800 μL of water to dilute, and detect the reaction progress by liquid phase. The results are shown in Table 2.

[0041] Table 2

[0042] 7-Trifluoromethyl-1,10-ethyleneisoalloxazine chloride (mM) Yield(%) 0.5 47.6 1 90.7 1.5 72.7 2 63.7 2.5 58.2 3 54.1

Embodiment 3

[0043] Example 3: L-glutamate dehydrogenase catalyzes the production of α-ketoglutarate under different pH types

[0044]At 30°C, in 10ml of pH8 different buffer systems, L-glutamic acid 15mM, NAD+1mM, 7-trifluoromethyl-1,10-ethyleneisoalloxazine chloride 1mM, catalase 500U / mL, L-glutamate dehydrogenase 5U / mL, the reaction solution is connected with the outside air. Take 200 μL of the reaction solution after reacting for 48 hours, add 800 μL of water to dilute, and detect the reaction progress by liquid phase. The results are shown in Table 3.

[0045] table 3

[0046] pH type Yield(%) Tris-HCl 90.7 KPi 50.4 CBS 60.2 CPBS 71.8 barbiturate - hydrochloric acid 53.6

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Abstract

The invention discloses a method for alpha-ketoglutarate production by catalysis of L-glutamate dehydrogenase. Under conditions of existence of a hydrogen peroxide scavenging agent and in buffer solution, alpha-ketoglutarate is generated by oxidation reaction of L-glutamic acid or salts thereof under catalysis of L-glutamate dehydrogenase and cofactors. The L-glutamate dehydrogenase depending on the cofactors obtained by organic small-molecular catalytic regeneration is adopted for catalysis of L-glutamic acid or salts thereof to generate alpha-ketoglutarate through oxidation reaction, low cost of raw material and low production cost are realized, a production process is simple and easy to operate and free of environment pollution, resource recycling is realized, and a theoretical foundation is laid for industrial efficient production of alpha-ketoglutarate. The L-glutamic acid or salts thereof are directly used as raw materials to generate alpha-ketoglutarate through one-step reaction, and high reaction selectivity and high yield are realized.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to a method for catalyzing and producing alpha-ketoglutarate by using L-glutamic acid dehydrogenase. Background technique [0002] α-Ketoglutaric acid is an important dicarboxylic acid in the tricarboxylic acid cycle and amino acid metabolism. It participates in the formation of amino acids and the transport of nitrogen. Wide range of applications. At present, a new application field is the thermal polycondensation of α-ketoglutaric acid and triols (glycerol, 1,2,4-butanetriol, 1,2,6-hexanetriol), and the resulting polymer triol -α-ketoglutarate has potential applications in biomedicine. At present, there are three production methods of α-ketoglutaric acid: (1) chemical synthesis method, multi-step synthesis using diethyl succinate and diethyl oxalate as raw materials; (2) microbial fermentation method, bacteria and yeast; (3) Biocatalysis. Yarrowia lipolytica WSH-Z06 is ...

Claims

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Application Information

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IPC IPC(8): C12P7/50
CPCC12P7/50Y02P20/584
Inventor 应汉杰濮玲玲朱晨杰谭卓涛张晓
Owner NANJING UNIV OF TECH
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