Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase

A technology of glutamic acid oxidase and ketoglutarate, which is applied in the biological field, can solve the problems of small processing capacity, high cost, and large time consumption, and achieve the effect of meeting large-scale production and reducing production cost and use cost

Inactive Publication Date: 2016-02-17
ZHEJIANG DEQING HUINING BIOTECH
View PDF10 Cites 13 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although affinity chromatography has the characteristics of high selectivity, high activity recovery and high purity, and has become one of the most effective techniques for purifying biological macromolecules such as pro

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase
  • Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase
  • Method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0031] The specific implementation manner of the present invention will be described in detail below in conjunction with the accompanying drawings.

[0032] Such as Figure 1 to Figure 8 A shown method utilizing L-glutamic acid oxidase to catalyze the production of α-ketoglutarate, the following steps further describe the present invention:

[0033] Synthesis of step 1, L-glutamic acid oxidase gene:

[0034] Whole Gene Synthesis Based on E. coli Codon Bias Streptomycessp The L-glutamic acid oxidase gene LGOX (SEQ ID NO.1) derived from X-119-6, and entrusted Shanghai Jierui Biotechnology Co., Ltd. to carry out the total synthesis of the L-glutamic acid oxidase gene. The ends were cut with NcoI and XhoI respectively and connected to pET28a(+) to obtain the fusion expression vector pET28a-LGOX.

[0035] Step 2, construction of fusion expression vector pET-LGOX-ELP:

[0036] According to the L-glutamate oxidase gene sequence, design a pair of primers, upstream primer: 5'-CATG...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a method for producing alpha-oxoglutarate under catalysis of L-glutamate oxidase. The method comprises the following steps: performing optimal design and synthesis on codon of an L-glutamate oxidase (L-GOX) gene; constructing a fusion expression vector pET-28a-LGOX-ELP of the L-GOX and Elastin-Like Proteins (ELP), transferring the vector into escherichia coli Rosetta (DE3), selecting a converter for cultivation, adding an inducer for expression, and obtaining recombined L-glutamate oxidase fusion protein crude enzyme fluid; performing purification by an ITC method, thus obtaining recombined L-glutamate oxidase fusion proteins, performing enzyme digestion on the recombined L-glutamate oxidase fusion proteins by protease, and obtaining mature L-glutamate oxidase; producing oxoglutarate under the catalysis of the mature L-glutamate oxidase. The method is simple, quick and efficient, can be used for large-scale separation and purification of L-glutamate oxidase, and is higher in actual application value in the enzyme production of oxoglutarate.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for catalyzing and producing alpha-ketoglutarate by utilizing L-glutamic acid oxidase. Background technique [0002] α-ketoglutarate (α-KG) is an important biological compound. As one of the important intermediate metabolites of the tricarboxylic acid cycle, α-ketoglutarate participates in important physiological processes such as amino acid, sugar, protein and fat metabolism, and plays an important role in the regulation of microbial carbon and nitrogen metabolism. Due to the importance of α-ketoglutaric acid in cell metabolism, it is widely used in food, medicine, fine chemical and cosmetic industries, and the market demand is huge. In particular, L-arginine-α-ketoglutarate (1:1) and L-arginine-α-ketoglutarate (2:1) is an amino acid with increasing sales in the international market Salt health products, as functional nutrition enhancers and liver-protecting d...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12P7/50C12N15/53C12N15/70C12N9/06
Inventor 徐志南唐云平沈颂娣朱晓仙黄保苗王鹏
Owner ZHEJIANG DEQING HUINING BIOTECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap