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N-acetylgutamate kinase mutant with heat stability improved remarkably

A technology of glutamic acid kinase and thermal stability, which is applied in the field of bioengineering and can solve the problems of poor thermal stability of NAGK

Active Publication Date: 2016-08-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, although the overexpression of NAGK was obtained by cloning the N-acetylglutamate kinase gene (argB) and introducing it into other strains, the thermal stability of NAGK is not good, that is, the half-life of NAGK is 36h at 37°C, while L-arginine Sour fermentation cycle is 96h

Method used

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  • N-acetylgutamate kinase mutant with heat stability improved remarkably
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Embodiment 1

[0019] Construction of embodiment 1 mutant expression plasmid and acquisition of recombinant E.coli BL21 strain

[0020] According to the argB gene sequence of Corynebacterium crenatum SYPA5-5, design the two primers of the N-acetylglutamate kinase coding gene, and then design the PCR point mutation intermediate primer according to the amino acid site to be mutated:

[0021] The mutant gene was amplified in vitro by overlap extension PCR.

[0022] The primers used for site-directed mutagenesis were:

[0023] PargB F: 5'-CGCGAATTCATGAATGACTTGATCAAAG-3' (EcoRI)

[0024] PargB R: 5'-CGCGTCGACTTACAGTTCCCCATCCTTG-3'(SalI)

[0025] Parg B K234T Fm: 5'-GTG TCCAAGATCA CTGCCACCGA GCTG-3'

[0026] Parg B K234T Rm: 5'-CAGCTCGGTG GCAGTGATCT TGGACAC-3'

[0027] Extracting the genome of Corynebacterium bacilli SYPA5-5 as a template;

[0028] PCR reaction conditions: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 72°C for 60s...

Embodiment 2

[0029] Retransform the recombinant plasmid with correct sequencing into E.coli BL21(DE3) competent cells to obtain recombinant E.coli BL21 strain Example 2 Expression of mutant N-acetylglutamate kinase and Ni-NTA purification

[0030] Inoculate the recombinants stored in cryopreserved tubes into LB medium containing kanamycin (final concentration: 50 μg / mL), culture overnight at 37°C with shaking, transfer at 1% inoculum size, and culture at 37°C until the OD is about 0.6- 0.8, add human IPTG to a final concentration of 1 mmol / L, and induce expression overnight at 16°C. Centrifuge the overnight induced expression bacterial solution at 10000r / min, 4°C for 15min, collect the bacterial cells, suspend the bacterial cells with Tris-HCI (pH8.0) buffer solution, break the cells by ultrasonic, and then filter through a 0.45μm filter membrane, select the expression The vector pET-28a contains 6His-Tag, NAGK is purified by Ni-NTA, and the pure NAGK enzyme protein is analyzed by SDS-PAGE...

Embodiment 3

[0031] The enzyme activity assay of embodiment 3 mutants

[0032] Carry out the enzymatic reaction of the pure enzyme solution obtained in the previous step: the total volume of the enzymatic reaction is 3mL, containing 595mmol / L Tris-HCl (pH8.0), 20mmol / L N-acetylglutamic acid, 20mmol / L MgCl 2 , 20mmol / L ATP disodium salt, 149mmol / LNH 2 0H . HCI and an appropriate amount of crude enzyme solution; after reacting at 37°C for 1 hour, add 1 mL of reaction termination solution (1.0mol / L HCI containing 5% FeCl 3 . 6H 2 0,4% trichloroacetic acid) to terminate the reaction; centrifuge, take the supernatant to measure the absorbance value A of N-acetylglutamic acid hydroxamic acid540 . The values ​​of Km, Vm and Kat were measured by changing the concentration of the substrate-N-acetylglutamic acid (NAG) and using the double reciprocal method. The results obtained are shown in Table 1: N-acetylglutamate kinase mutant K234T NAGK The kinetic parameters of the wild type (CgNAGK) are...

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Abstract

The invention discloses an N-acetylgutamate kinase mutant with heat stability improved remarkably and belongs to the field of bioengineering .The gene of N-acetylgutamate kinase from Corynebacterium crenatum SYPA5-5 is subjected to site-specific mutagenesis, so that lysine K at the site 234 of the encoded amino acid sequence is mutated into threonine T .the half-life period of the N-acetylgutamate kinase mutant at 37 DEG C is prolonged to be 112 h from 36 h of a wild type N-acetylgutamate kinase mutant, which is 3.11 times of the length before mutation .The N-acetylgutamate kinase mutant can better meet the production requirement of arginine, and lays a foundation for efficient synthesis of L-arginine.

Description

technical field [0001] The invention relates to an N-acetylglutamic acid kinase mutant with significantly improved thermal stability, which belongs to the technical field of bioengineering. Background technique [0002] L-arginine is a basic semi-essential amino acid, which has many physiological functions. It is an essential amino acid for the synthesis of cytoplasmic protein and nuclear protein; as the only source of ammonia, it participates in the synthesis of creatine; as an important intermediate in the urea cycle, it plays a role in eliminating excess ammonia in the liver, preventing excessive ammonia accumulation and causing poisoning ; It also has the function of regulating human immunity, can inhibit tumor growth, and promote wounded tissue healing. Moreover, arginine is the direct precursor of nitric oxide, urea, ornithine and sarcosine, an important element for the synthesis of myostatin, and is used for the synthesis of polyamine, citrulline and glutamine. Ther...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/77
CPCC12N9/1217C12Y207/02008
Inventor 饶志明徐美娟张景景杨套伟张显陈肖峰
Owner JIANGNAN UNIV
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