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Strain MQO-160 for generating L-glutamate oxidase and application of strain

A technology of glutamic acid oxidase and bacterial strains, applied in the field of microorganisms, can solve the problems of high price, restrictions on the application and development of L-glutamic acid oxidase, single source, etc. Effect

Inactive Publication Date: 2017-01-11
SHANDONG MINQIANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high price and single source seriously restrict the application and development of L-glutamic acid oxidase

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034]Example 1 Biological characteristics of bacterial strain MQO-160

[0035] The basal and aerial hyphae of the strain MQO-160 are both milky white, the mycelium has more septa, and the aerial mycelium has more dendritic branches. Some mycelia are in the vigorous growth stage and have a bud-like structure. The strain produces a golden yellow or purple red soluble pigment. The spores of the strain are oval, spherical or cylindrical, with a smooth surface and spiral-shaped spore filaments.

Embodiment 2

[0036] Example 2 Preparation of L-glutamic acid oxidase by strain MQO-160

[0037] Proceed as follows:

[0038] (1) Slant culture: inoculate the strain MQO-160 on the slant medium under aseptic conditions for inverted culture, the culture temperature is 28°C, and the culture time is 48h;

[0039] Incline medium: soluble starch 20g, KNO 3 1g, K 2 HPO 4 0.5g, MgSO 4 ·7H 2 O 0.5g, NaCl 0.5g, FeSO 4 ·7H 2 O 0.01g, agar 20g, add pure water to 1L, adjust pH to 7.4-7.6 with NaOH, sterilize at 115°C for 15min.

[0040] (2) Expanded cultivation of seeds: Pick colonies from the slant medium in step (1), dilute with water to obtain a bacterial solution, inoculate the bacterial solution onto the seed medium for expanded cultivation, and the inoculation amount is 10% (v / v), the temperature of the expanded culture is 28°C, the rotation speed of the shaker is 120r / min, and the culture time is 12h.

[0041] Seed medium: 30g sucrose, 6g yeast extract, (NH 4 ) 2 SO 4 6g, CaCO 3 ...

Embodiment 3

[0046] Example 3 Production of α-ketoglutarate by microbial enzymatic method (specifically, L-glutamic acid is transformed into α-ketoglutarate by L-glutamic acid oxidase)

[0047] Specific steps are as follows:

[0048] (1) Preparation of crude enzyme liquid: the fermentation liquid of L-glutamic acid oxidase is first filtered through a ceramic membrane to remove bacteria, and the supernatant is concentrated 50 times through a reverse osmosis membrane to obtain L-glutamate for enzyme conversion. Crude enzyme solution of amino acid oxidase.

[0049] (2) Transformation culture: Add crude enzyme solution of L-glutamic acid oxidase, H 2 o 2 Enzymes and MnCl 2 And the substrate L-sodium glutamate, 37 ℃, 200r / min transformation 24h, obtain the conversion solution containing a-ketoglutaric acid; The final concentration of phosphate buffer is 50mmol, the concentration of L-glutamic acid oxidase The final concentration is 15U / ml, H 2 o 2 The final concentration of enzyme is 20U / ...

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Abstract

The invention relates to a strain MQO-160 for generating L-glutamate oxidase and application of the strain. The strain MQO-160 has the preservation number of CGMCC No.12892, the preservation organization of the China General Microbiological Culture Collection Center, the preservation date of August 22nd, 2016 and the preservation address of the apartment No.3 of the yard No.1 in the Beichen West Road in Chaoyang District of Beijing. The invention further comprises the application of the strain MQO-160 to preparation of L-glutamate oxidase. The bottleneck defect of generating alpha-oxoglutarate through an L-glutamate oxidase conversion method is overcome, and the strain MQO-160 is low in price, easy to obtain, simple in process and high in product purity.

Description

technical field [0001] The invention relates to a bacterial strain MQO-160 producing L-glutamic acid oxidase and its application, belonging to the technical field of microorganisms. Background technique [0002] L-glutamate oxidase (EC 1.4.3.11, L-glutamate oxidase, GLOD) is an L-amino acid oxidase with flavin adenine dinucleotide (FAD) as a coenzyme, which was discovered in the early 1980s A new enzyme, which was first isolated and purified from snake venom, mouse kidney, invertebrates and microorganisms. L-glutamate oxidase can oxidize the deamination of L-glutamate to produce α-ketoglutarate and hydrogen peroxide without adding exogenous cofactors. GLOD has a high degree of stereoselectivity for catalytic reaction substrates, high catalytic efficiency and mild reaction conditions, and has been widely used in various fields such as food, light industry, chemical industry, medicine, environmental protection, energy and scientific research. Since the enzyme was discovered ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/06C12P7/50C07C51/42C07C59/347C12R1/465
CPCC12N9/0022C12P7/50C12Y104/03011C07C51/42C12R2001/465C12N1/205C07C59/347
Inventor 胡安红闫洪波蔡晓洲徐凯黄巧娣张斌
Owner SHANDONG MINQIANG BIOTECH
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