Diaminopimelate dehydrogenase mutant and application thereof

A technology of diaminopimelate dehydrogenase and mutants, which is applied in the field of genetic engineering, can solve problems such as the transformation of coenzyme preference in the absence of diaminopimelate dehydrogenase, and achieve low price and good stability Effect

Pending Publication Date: 2021-10-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the modification of coenzyme prefere

Method used

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  • Diaminopimelate dehydrogenase mutant and application thereof
  • Diaminopimelate dehydrogenase mutant and application thereof
  • Diaminopimelate dehydrogenase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Construction of expression plasmid pET-28a-ddh

[0027] It is derived from C. glutamicum XQ-5 diaminopimelic acid dehydrogenated DAPDH, which is connected to the pET-28a plasmid through restriction enzyme sites EcoRI and XholI.

Embodiment 2

[0028] Example 2: Transforming the plasmid into E.coli BL21(DE3) for expression

[0029] The recombinant expression plasmid pET-28a-ddh was transformed into E.coli BL21(DE3), and the recombinant expression strain was screened by culturing on LB+Kan solid medium at 37°C. The starting strain and the recombinant strain were first inoculated into LB 10ml liquid vials for primary seed liquid culture for 10-12 hours, then transferred to liquid TB medium with 2% inoculation amount, and cultivated to OD in a shaker at 37°C 600 Between 0.5 and 0.6, a final concentration of 0.5 mM IPTG was added, and the expression was induced at 16° C. for 20 h. Collect the cells after expression, wash twice with PBS buffer (pH 7.4), suspend the cells and control them at the same OD 600 , and then break the bacteria with an ultrasonic breaker, and centrifuge to get the supernatant to obtain the crude enzyme solution. Such as figure 1 , SDS-PAGE was carried out on the crude enzyme solution, and it wa...

Embodiment 3

[0030] Embodiment 3: Transform the mutant plasmid into E.coli BL21 (DE3) and measure the kinetic parameters of enzyme activity and enzyme after mutation

[0031] Primers were designed to mutate the 36-arginine of the enzyme DAPDH. Mutations (Arg36Ala, Arg36Glu, Arg36Leu, Arg36Phe, Arg36Cys, Arg36Tyr) were performed using pET-28a-ddh as a template through a point mutation kit. The obtained plasmid containing the mutated gene was transformed into E. coli BL21(DE3). The recombinant strain E.coli BL21(DE3)pET-28a-ddh Arg36Ala Inoculate them in LB 10ml liquid vials for primary seed liquid culture for 10-12 hours, then transfer to liquid TB medium with 2% inoculum amount, and culture in a shaker at 37°C until OD 600 Between 0.5 and 0.6, a final concentration of 0.5 mM IPTG was added, and the expression was induced at 16° C. for 20 h. Collect the cells after expression, wash twice with PBS buffer (pH 7.4), suspend the cells and control them at the same OD 600 , and then break the...

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PUM

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Abstract

The invention discloses a diaminopimelate dehydrogenase mutant and an application thereof. According to the invention, diaminopimelate dehydrogenase from C.glutamicum XQ-5 is mutated, Arg at the 36 site is mutated into Glu, the activity of an enzyme taking NADH as a coenzyme is greater than that of an enzyme taking NADPH as a coenzyme, and the preference transformation of the coenzyme is implemented to a certain extent. Compared with the coenzyme NADP (H), the coenzyme NAD (H) has the advantages of good stability, low price, wider coenzyme cyclic regeneration method and the like, and can promote the industrial application of the enzyme.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a mutant of diaminopimelic acid dehydrogenase and its application. Background technique [0002] Diaminopimelate dehydrogenase (diaminopimelate dehydrogenase) is encoded by the gene ddh. Diaminopimelate dehydrogenase catalyzes the reversible reaction of ammonia, NADPH, and tetrahydrodipicolinate to form mesodiaminopimelate, the immediate precursor of L-lysine in the bacterial lysine biosynthetic pathway body. Since mammals lack this metabolic pathway, inhibitors of enzymes in this pathway are useful as antibiotics or herbicides. This enzyme was first found in Bacillus, Sphaeroides, Corynebacterium glutamicum and Brevibacterium. [0003] Diaminopimelate dehydrogenase is a dimer that displays the specificity of the stereotropic meso-DAP isoform It is a homodimer of structurally distinct subunits. Each subunit consists of three domains. The N-terminal domain contai...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12N15/56C12N15/70C12N1/21C12P13/08C12R1/19
CPCC12N9/0016C12N15/70C12P13/08C12Y104/01016Y02A50/30
Inventor 徐建中刘宁吴慧雯
Owner JIANGNAN UNIV
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