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A kind of glutamate dehydrogenase mutant and its application

A technology for glutamate dehydrogenase and mutants, which is applied to glutamate dehydrogenase mutants and their application fields, can solve the problems of low optical purity of products, cumbersome processes, etc., and achieves improved catalytic activity and wide industrial application prospects Effect

Active Publication Date: 2022-04-15
宿迁市江南大学产业技术研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In chemical synthesis, there are often problems of cumbersome process and low optical purity of the product

Method used

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  • A kind of glutamate dehydrogenase mutant and its application
  • A kind of glutamate dehydrogenase mutant and its application
  • A kind of glutamate dehydrogenase mutant and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Embodiment 1: Construction of glutamate dehydrogenase mutant library

[0097] figure 1 is the crystal structure of glutamate dehydrogenase derived from bovine liver (PDB: 6dhd), which shows the interaction mode of the substrate glutamate and the enzyme; the amino acid residues 115K and 350N shown in the stick structure are related to the substrate The two oxygens of the main chain carboxyl group of glutamic acid interact to anchor the substrate main chain; it is found by sequence comparison (such as figure 2 Shown), these two amino acid residues are conserved sites in glutamate dehydrogenase, so the two sites are used as transformation objects.

[0098] The recombinant plasmid pET-28a-GluDH and the recombinant strain E.coli BL21(DE3) / pET-28a- GluDH, the recombinant strain E.coli BL21(DE3) / pET-28a-GluDH was named E.coli BL21(DE3) / pET-28a-WT1, and inoculated into LB solid medium for culture.

[0099] According to the target gene sequence upstream and downstream of the...

Embodiment 2

[0105] Embodiment 2: Primary screening of glutamate dehydrogenase mutant library

[0106] Specific steps are as follows:

[0107] (1) Pick the single colony of the recombinant bacterium BL21(DE3) / pET-28a-fusion protein obtained in Example 1, and transfer it to a 96-well deep-well plate (each well contains 300 μL LB liquid medium and 50 μg / mL kanamycin), and incubated at 37°C and 200rpm for 10-12h.

[0108] (2) Transfer 50 μL culture from each well obtained after incubation in step (1) to the second 96-deep well plate (each well contains 400 μL LB liquid medium and 50 μg / mL kanamycin in the corresponding wells in the prime), and a second 96-deep well plate was incubated at 37°C and 250rpm for 3-4h. Afterwards, 50 μL of LB liquid medium containing 2 mM IPTG was added to each well of the second 96 deep-well plate to induce protein expression, and then the second 96 deep-well plate was incubated at 20 °C and 250 rpm for 16- 18h.

[0109] (3) The product obtained in step (2) i...

Embodiment 3

[0112] Embodiment 3: the preparation of crude enzyme liquid of glutamic acid dehydrogenase

[0113] Specific steps are as follows:

[0114] (1) Strain activation: the E.coli BL21(DE3) / pET-28a-WT1 obtained in Example 1 and the genetically engineered bacteria containing the glutamate dehydrogenase mutant gene obtained in Example 2 were respectively: BL21(DE3) / pET-28a-K116S / N348L, BL21(DE3) / pET-28a-K116E / N348M, BL21(DE3) / pET-28a-K116Q / N348M were inoculated into test tubes containing 5 mL of LB liquid medium, Cultivate at 37° C. and 200 rpm for 6-8 hours to obtain seed liquid.

[0115] (2) Preparation of crude enzyme solution: respectively inoculate the activated seed solution into a conical flask containing 100mL LB liquid medium at an inoculum size of 2% (v / v), and cultivate for 2-3h at 37°C and 200rpm , to OD 600 After adding IPTG to a final concentration of 0.1mmol / L, continue to cultivate at 17°C and 200rpm for 12-17h to obtain a fermentation broth; respectively centrifuge...

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Abstract

The invention discloses a glutamic acid dehydrogenase mutant and application thereof, and belongs to the technical fields of enzyme engineering and microorganism engineering. Adopt the method provided by the present invention to provide a kind of method of brand-new biosynthetic (R)-4-aminovaleric acid; Wild-type glutamate dehydrogenase has no measurable catalytic activity, and the mutant of the present invention all has Catalytic activity, and mutant K116S / N348L, K116E / N348M, the catalytic activity of K116Q / N348M can respectively reach 1.87U / mg, 3.16U / mg, 4.55U / mg, prove that the technical scheme of the present invention has realized 4-oxo The catalytic activity of valeric acid is created from scratch and improved, and the biosynthesis of (R)-4-aminovaleric acid provided by the present invention is green, environmentally friendly and efficient, and has a wide range of industrial application prospects.

Description

technical field [0001] The invention relates to a glutamic acid dehydrogenase mutant and application thereof, belonging to the technical fields of enzyme engineering and microbial engineering. Background technique [0002] SacubitriI is an active ingredient drug of the anti-heart failure drug entrento, a neprilysin inhibitor, and it is combined with valsartan, an angiotensin II receptor blocker, to reduce the risk of heart failure in patients with heart failure. Vascular death and hospitalization for chronic heart failure (NYHA class II-IV) and reduced ejection fraction. Optically pure (R)-4-aminovaleric acid is the chiral building block of the compound Sacubitril; in addition (R)-4-aminovaleric acid itself is a γ-amino acid, which can be used to synthesize hybrid peptides with physiological activity or special structure research; which makes (R)-4-aminovaleric acid have important application value. Asymmetric reductive amination of 4-oxopentanoic acid using engineered glu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P41/00C12P13/00C12R1/19
CPCC12N9/0016C12N15/70C12P41/006C12P13/005C12Y104/01002
Inventor 穆晓清徐岩周峰
Owner 宿迁市江南大学产业技术研究院
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