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Glutamate oxidase mutant, nucleic acid molecule, applications of glutamate oxidase mutant, and method for preparing ketoglutaric acid

A glutamate oxidase and nucleic acid molecule technology, which is applied in the field of ketoglutarate preparation, can solve the problems of insufficient L-glutamate oxidase catalytic properties, long production cycle, low conversion rate and the like

Active Publication Date: 2019-02-01
NANJING REDWOOD FINE CHEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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
In 2014, Chen Jian and others from Jiangnan University (China Invention Patent Application No. 201410132063.2) adopted the method of whole cell transformation to produce a-ketoglutarate, but the catalytic properties of the L-glutamic acid oxidase used were not good enough. And the conversion rate is low, within 24 hours, the conversion rate is only 59.6%, and the output is 7.7g / L

Method used

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  • Glutamate oxidase mutant, nucleic acid molecule, applications of glutamate oxidase mutant, and method for preparing ketoglutaric acid
  • Glutamate oxidase mutant, nucleic acid molecule, applications of glutamate oxidase mutant, and method for preparing ketoglutaric acid
  • Glutamate oxidase mutant, nucleic acid molecule, applications of glutamate oxidase mutant, and method for preparing ketoglutaric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Construction of Genetically Engineered Bacteria Expressing Glutamate Oxidase Mutants

[0035] (1) On the basis of the wild sequence of Streptomyces glutamate oxidase, artificially design, the designed gene sequence is shown in SEQ ID NO.2, and the amino acid sequence of the glutamate oxidase mutant encoded by it is as follows Shown in SEQ ID NO.1. The above-mentioned gene sequence is synthesized by the method of whole gene synthesis.

[0036] (2) The synthetic gene sequence was cloned into the pDK6 plasmid through EcoR1 and Pst1 2 restriction sites, and the above-constructed plasmid was transferred into Escherichia coli DH5α competent cells, and after positive transformants were selected and identified by sequencing, the recombinant expression vector was obtained , named pDK6-12 plasmid (see figure 1 ).

[0037] (3) The recombinant expression vector pDK6-12 plasmid was transformed into Escherichia coli BL21(DE3) strain to obtain genetically engineered bacteria capabl...

Embodiment 2

[0039] Preparation of glutamate oxidase mutants

[0040] (1) Streak the genetically engineered bacteria used to express the Streptomyces glutamic acid oxidase mutant prepared in Example 1 on the kanamycin-resistant LB solid medium, and cultivate it in a biochemical incubator at 37°C Overnight, select a larger colony and inoculate it into LB liquid medium containing kanamycin resistance, and culture it on a shaker at 37°C 220rpm for 6-8h, or at 30°C 200rpm overnight to obtain a primary seed culture solution.

[0041] (2) Inoculate the primary seed culture liquid into the TB liquid medium containing kanamycin resistance according to the inoculation amount of 1%, and cultivate it in a shaker at 37° C. 220 rpm for 4 to 6 hours until the bacterial liquid is turbid by visual inspection, and obtain Secondary seed culture fluid.

[0042] (3) According to the inoculum amount of 1%, the secondary seeds are inoculated into the fermenter for cultivation. Initial control: 37°C aeration a...

experiment example

[0047] Experiments using the above bacteria to catalyze the formation of ketoglutaric acid from glutamic acid

[0048] Enzyme reaction: the reaction system is 8 liters

[0049] 1. Weigh 200 grams of frozen bacteria (approximately 2.5% of the transformation system), 80 mL of 400,000 units of catalase, dilute to 5 liters of tap water (pH is approximately 6.4), keep warm at 35 ° C, 400 rpm, 1 vvm, Tank pressure 0.05Mpa.

[0050] 2. Take by weighing 800 grams of sodium glutamate monohydrate (about 10% of the conversion system), dissolve it with 3 liters of tap water, add the conversion system with a peristaltic pump flow, and the flow rate is 1.2 liters / hour. During the period, the pH was controlled at about 6.5 with 1% hydrochloric acid. Samples were taken every hour to detect ketoglutaric acid, and the conversion was completed in about 3 hours.

[0051] Ketoglutarate detection

[0052] 1. Standard configuration: Accurately weigh 0.008 g of ketoglutaric acid standard (above 9...

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Abstract

The present invention discloses a glutamate oxidase mutant, a nucleic acid molecule, applications of the glutamate oxidase mutant, and a method for preparing ketoglutaric acid, wherein the amino acidsequence of the glutamate oxidase mutant is represented by SEQ ID NO.1. According to the present invention, the glutamate oxidase mutant can catalyze the formation of ketoglutaric acid from glutamic acid, and has high catalytic ability compared with the wild type glutamate oxidase.

Description

technical field [0001] The invention relates to the technical field of ketoglutarate preparation, in particular to a glutamic acid oxidase mutant, nucleic acid molecule and application thereof and a method for preparing 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 g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12P7/50
CPCC12N9/0022C12P7/50
Inventor 吴法浩李钢高仰哲
Owner NANJING REDWOOD FINE CHEM CO LTD
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