Glutamate dehydrogenase mutant and application thereof

A technique for glutamate dehydrogenase and mutants, which is applied in the field of glutamate dehydrogenase mutants and its applications, and can solve the problems of cumbersome process and low optical purity of products

Active Publication Date: 2021-01-29
宿迁市江南大学产业技术研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In chemical synthesis, there are often problems of cu

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  • Glutamate dehydrogenase mutant and application thereof
  • Glutamate dehydrogenase mutant and application thereof
  • Glutamate dehydrogenase mutant and application thereof

Examples

Experimental program
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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 glutamate dehydrogenase mutant and application thereof, and belongs to the technical field of enzyme engineering and microbial engineering. By adopting the method provided bythe invention, a brand-new method for synthesizing (R) -4-aminovaleric acid by a biological method is provided; wild glutamate dehydrogenase has no measurable catalytic activity, but the glutamate dehydrogenase mutant has catalytic activity, and the catalytic activity of the mutants K116S/N348L, K116E/N348M and K116Q/N348M can reach 1.87 U/mg, 3.16 U/mg and 4.55 U/mg respectively, so that the catalytic activity of the 4-oxovaleric acid is improved from zero. The biological method for synthesizing (R) -4-aminovaleric acid provided by the invention is green, environment-friendly and efficient,and has a wide industrial application prospect.

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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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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