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An N-acetylglutamate kinase mutant with improved catalytic efficiency

A glutamate kinase and catalytic efficiency technology, applied in the field of N-acetylglutamate kinase mutants, can solve the problems of weak catalytic activity, poor thermal stability, and low specific enzyme activity

Active Publication Date: 2019-05-21
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 has been obtained by cloning the N-acetylglutamate kinase gene (argB) and introducing it into other bacterial strains, the specific enzyme activity of NAGK is not high, that is, the catalytic activity of NAGK is weak, and the thermostability is not good That is, at 37°C, the half-life of NAGK is 36h, while the fermentation cycle of L-arginine is 96h

Method used

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  • An N-acetylglutamate kinase mutant with improved catalytic efficiency
  • An N-acetylglutamate kinase mutant with improved catalytic efficiency

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Experimental program
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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 I74V Fm: 5'-CGGTGGTGGACCTCAGGTTTCTGAGATGC-3'

[0026] Parg B I74V Rm: 5'-GCATCTCAGAAACCTGAGGTCCACCACCG-3'

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

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

Embodiment 2

[0030] Embodiment 2 Expression of mutant N-acetylglutamate kinase and Ni-NTA purification

[0031] 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, 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, and a specific band with a molecular weight of about 36kDa is detected. The pure NAGK enzyme is used for protein The concentration and enzyme ...

Embodiment 3

[0032] The catalytic constant comparison of embodiment 3 wild enzyme and mutant enzyme

[0033] The pure enzyme solution obtained in the previous step was subjected to an enzymatic reaction: the total volume of the enzymatic reaction was 3mL, containing 595mmol / LTris-HCl (pH8.0), 20mmol / L N-acetylglutamic acid, 20mmol / L MgCl 2 , 20mmol / L ATP disodium salt, 149mmol / L NH 2 0H·HCl and an appropriate amount of crude enzyme solution; after reacting at 37°C for 1 h, add 1 mL of reaction termination solution (1.0mol / L HCI containing 5% FeCl3 ·6H 2 0,4% trichloroacetic acid) to stop the reaction; centrifuge, take the supernatant to measure the absorbance value A of N-acetylglutamic acid hydroxamic acid 540 . The values ​​of Km, Vm and Kcat 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 I74V NAGK Catalytic efficiency (Kcat / Km) ...

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Abstract

The invention discloses an N-acetylgutamate kinase mutant with improved catalysis efficiency and belongs to the field of biological engineering .A gene of N-acetylgutamate kinase coming from Corynebacteriaum crenatum SYPA5-5 is subjected to site-directed mutation, so that the amino acid sequence encoded by the gene is mutated from isoleucine I to valine V at the 74th site .Catalysis efficiency (Kcat / Km) of the N-acetylgutamate kinase mutant is improved to 843 mM<-1>min<-1> from 513 mM<-1>min<-1> of a wild type, and is 1.64 times of the catalysis efficiency before mutation .Heat stability of the mutant is also improved .The N-acetylgutamate kinase mutant better meets production requirements of arginine, and a foundation is laid for efficient synthesis of L-arginine.

Description

technical field [0001] The invention relates to an N-acetylglutamate kinase mutant with improved catalytic efficiency, 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. Therefore, L-argini...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/12C12N15/54
CPCC12N9/1217C12Y207/02008
Inventor 饶志明徐美娟张景景杨套伟张显葛小勋
Owner JIANGNAN UNIV
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