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Method for efficiently producing L-glutamate oxidase

A glutamate oxidase, high-efficiency expression technology, applied in the fields of fermentation engineering and enzyme engineering, can solve the problems of α-KG difficulty, safety problems, raw material sources, etc., and achieve the effect of high-efficiency expression

Active Publication Date: 2015-07-01
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the industrial production of α-KG mainly adopts organic synthesis, which involves a series of complex chemical reaction processes, which causes a series of problems in raw material sources, environmental pollution, etc.
At the same time, due to serious safety problems in the process of chemically synthesizing α-KG, it is difficult to directly use α-KG in the fields of food, medicine and cosmetics.

Method used

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  • Method for efficiently producing L-glutamate oxidase
  • Method for efficiently producing L-glutamate oxidase
  • Method for efficiently producing L-glutamate oxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Optimization of Lactose Inducing Conditions

[0030] Using the recombinant strain FMME089 (Panqing Niu, Enzymatic production of α-ketoglutaric acid from L-glutamic acid via L-glutamateoxidase, Journal of Biotechnology, 2014, 56-62) constructed in our laboratory as the starting strain, the conditions for lactose induction were optimized.

[0031] 1) Inoculate the recombinant strain in LB medium, OD 600 When it is between 0.5-0.6, add 1, 3, 5, 7 g / L lactose respectively to induce 4 hours at 37°C, and compare the expression of LGOX. It was found that the lactose induction effect of 5g / L was the best and the enzyme activity reached 4.53U / mL ( figure 1 A).

[0032] 2) On the basis of the optimal lactose induction concentration, the expression levels of LGOX were measured at 3, 4, 5, and 6 hours after induction, respectively. The results showed that the effect of induction for 5h was the best, and the enzyme activity reached 4.73U / mL ( figure 1 B).

Embodiment 2

[0033] Embodiment 2 batch fermentation

[0034] The recombinant bacteria were first inoculated on the LB seed medium for 10-12 hours, and then transferred to a 5L fermenter containing 2LLB medium with a 4% inoculation amount. The initial fermentation conditions are: rotation speed 300rpm, temperature 37°C, ventilation rate 1vvm. When OD600=0.5-0.7, the final concentration of 5g / L lactose was used for induction, and the bacterial concentration and enzyme activity expression at different time points were measured. The results showed that the enzyme activity reached the maximum value of 9.43U / mL 4 hours after induction.

Embodiment 3

[0035] Embodiment 3 fed-batch fermentation based on DO-stat

[0036] The starting condition of the fermenter is the same as that of batch fermentation. Before feeding, control the DO to be above 30%. When the DO suddenly rises, start feeding, and control the DO-related feeding. Whenever the DO is higher than 30%, it will be fed for a while. material. The composition of feed solution was 500g / L glycerol, and 5g / L lactose was used for induction when the cell concentration no longer increased. After 16 hours of fermentation, the cell concentration OD=47.7, and the enzyme activity was 35.55U / mL ( image 3 ).

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Abstract

The invention discloses a method for efficiently producing L-glutamate oxidase and belongs to the technical fields of fermentation engineering and enzyme engineering. In the method disclosed by the invention, the optimal induction condition for L-glutamate oxidase recombinant bacteria is inducted for 5h in the presence of 5g / L lactose at 25-30 DEG C; through researches on different feeding modes of batch feeding fermentation, a manner of combining index feeding with Do-stat is beneficial to increase for a bacteria density; through optimization for induction condition of batch fermentation, inducing lasts for 12h in the presence of 10g / L lactose under the condition of a logarithmic phase OD600 of 40, and the highest enzyme activity reaches 156.1U / mL; high-expression recombinant wet bacteria are added in buffer solution containing 110g / L of glutamic acid with a pH value of 8.0, and converted for 24h at 37 DEG C to produce 107.9g / L of alpha-ketoglutaric acid, the conversion rate is 90% or above, and the needed bacterium solution amount is only 1 / 50 of that of shake-flask fermentation.

Description

technical field [0001] The invention relates to a method for efficiently producing L-glutamic acid oxidase, which belongs to the technical fields of fermentation engineering and enzyme engineering. Background technique [0002] α-ketoglutarate (α-KG for short), as an important high-value fine chemical (200,000-250,000 yuan / ton), is widely used in industrial fields such as food, medicine, chemical industry and cosmetics. . In the field of medicine, α-KG can reduce the burden on the kidneys of patients with kidney disease, reduce complications, and promote rapid recovery after surgery; compounded with amino acids such as arginine, it can quickly help athletes replenish energy, and is widely used in functional nutritional enhancement In addition, due to the special chemical properties of α-KG, it is widely used in the chemical synthesis industry. With the continuous expansion of the application field of α-KG, the demand for α-KG in the domestic and international markets conti...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N1/21C12P7/50C12R1/19
CPCC12N9/0022C12P7/50C12Y104/03011
Inventor 刘立明樊祥臣陈乐乐
Owner JIANGNAN UNIV
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