Meso-diaminopimelic acid dehydrogenase mutants with improved catalytic efficiency

A technology of diaminopimelate dehydrogenase and mesization, applied in the field of genetic engineering

Active Publication Date: 2019-11-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no research report on the modification of the substrate channel of meso-diaminopimelate dehydrogenase to improve the enzyme activity.

Method used

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  • Meso-diaminopimelic acid dehydrogenase mutants with improved catalytic efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of meso-diaminopimelate dehydrogenase mutant

[0028] With the pET-28a recombinant plasmid containing the meso-diaminopimelate dehydrogenase gene (shown in SEQ ID NO.1) derived from Ureibacillus thermosphaericus (Ureibacillus thermosphaericus) method build) as a template.

[0029] Use the oligonucleotide fragment containing the mutation point as the homology arm to design upstream and downstream primers. The specific primers are as follows (the bold and underlined are the mutation sites):

[0030]

[0031] The mutant plasmids were constructed by inverse PCR of the whole plasmid.

[0032] PCR amplification system: template 1 μL, upstream and downstream primers 0.5 μL, 2×Phanta Max Master Mix polymerase 10 μL, sterilized ddH 2 O 8 μL, total reaction system 20 μL. PCR reaction conditions: 95°C pre-denaturation, 5min; 95°C denaturation, 30s, 58°C annealing, 30s, 72°C extension, 2min, 30 cycles; 72°C full extension, 10min.

[0033] The PCR prod...

Embodiment 2

[0035] Example 2: Induced expression of meso-diaminopimelate dehydrogenase mutants

[0036] Inoculate the engineered meso-diaminopimelate dehydrogenase mutant engineered bacteria constructed in Example 1 into LB liquid medium containing 50 μg / mL kanamycin, cultivate overnight at 37°C and 180 r / min, and then transfer In 50mL of LB medium. The inoculum size is 1%, the culture temperature is 37°C, and the rotation speed is 180r / min. Grow to OD 600After reaching 0.6-0.9, add IPTG with a final concentration of 0.5mM for induction. The induction temperature is lowered to 16°C. After 14 hours of induction, the bacteria are collected by centrifugation at 8000rpm at 4°C for 10 minutes, and stored in a -70°C refrigerator for later use.

Embodiment 3

[0037] Example 3: Separation and purification of meso-diaminopimelate dehydrogenase mutant

[0038] Take the wet bacterial cells collected in Example 2, wash twice with 10mL of 50mM PBS buffer solution of pH 7.5, resuspend in 10mL of 50mM PBS buffer solution of pH 7.5, oscillate and shake well, and break under ultrasonic waves for 1s , stop for 3s, the total duration is 15min. The cell lysate was centrifuged at 12,000 rpm for 20 min to remove cell debris, and the supernatant, namely the crude enzyme solution, was collected and filtered with a 0.22 μm filter membrane for subsequent separation and purification of the enzyme.

[0039] The purification column is a Ni-NTA column with a packing volume of 5mL, and the loading equilibration buffer M 0 (20mM Tris, 500mMNaCl, pH 7.4) equilibrate the Ni-NTA column, load the crude enzyme solution at a rate of 0.5mL / min, and use the loading equilibration buffer M 0 Elution to remove unadsorbed protein, and finally with elution buffer M ...

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Abstract

The invention discloses meso-diaminopimelic acid dehydrogenase mutants with improved catalytic efficiency, and belongs to the technical field of gene engineering. According to the invention, single-site/double-site mutation is carried out on meso-diaminopimelic acid dehydrogenase shown as SEQ ID NO.2, and mutant enzymes D94A, W123K, D94A/W123K and W148K are successfully expressed and purified in escherichia coli. Enzyme activity measurement finds that the three meso-diaminopimelic acid dehydrogenase mutants have higher catalytic activity than the wild type, for example, the efficiencies of catalyzing phenylpyruvic acid by the mutant enzymes DAPDH-D94A, DAPDH-W123K and DAPDH-D94A/W123K are respectively improved by 3.5 times, 1.5 times and 1.3 times, and the enzyme activity of key enzymes issuccessfully improved.

Description

technical field [0001] The invention relates to a meso-diaminopimelate dehydrogenase mutant with improved catalytic efficiency, which belongs to the technical field of genetic engineering. Background technique [0002] D-phenylalanine has great application value in food, industry, agriculture, biomedicine, etc., such as strontium ranelate dry suspension for the treatment of diabetes, nateglinide tablets for the treatment of diabetes, etc. . Meso-diaminopimelate dehydrogenase (DAPDH, EC 1.4.1.16) has been widely used in the preparation of D-type aromatic amino acids such as D-phenylalanine (Akita H, et al. Highly stable meso- diaminopimelate dehydrogenase from an Ureibacillus thermosphaericus strain A1 isolated from a Japanese compost: purification, characterization and sequencing. AMB Express, 2011, 1:43). And some studies have carried out site-directed mutagenesis on meso-diaminopimelate dehydrogenase to modify its properties, all of which focus on modifying the vicinity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21C12P13/22C12R1/19
CPCC12N9/0016C12N15/70C12P13/222C12Y104/01016
Inventor 饶志明陈佳杰徐美娟杨套伟张显
Owner JIANGNAN UNIV
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