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Carbamyl phosphate synthetase mutant with effect of uridylic acid feedback inhibition resistance and application thereof

A carbamoyl phosphate and mutant technology, applied in the field of enzyme engineering, can solve problems such as reduced sensitivity

Active Publication Date: 2019-10-18
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Sylviane Delannay et al. (Journal of Molecular Biology, 1999,286(4):1217-1228) pointed out that the 937-1073 region is the allosteric binding site of regulatory factors including IMP, UMP and ornithine, and was discovered by site-directed mutagenesis , the mutation of S948 to F reduced the sensitivity of CPase to IMP and UMP

Method used

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  • Carbamyl phosphate synthetase mutant with effect of uridylic acid feedback inhibition resistance and application thereof
  • Carbamyl phosphate synthetase mutant with effect of uridylic acid feedback inhibition resistance and application thereof
  • Carbamyl phosphate synthetase mutant with effect of uridylic acid feedback inhibition resistance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Mutation of the conserved site at the C-terminus of carbamyl phosphate synthase, recombinant expression plasmid

[0024] Four carbamoyl phosphate synthetases from different sources, including Escherichia coli, Corynebacterium glutamicum, Bacillus amyloliquefaciens and Bacillus subtilis, were selected for homologous sequence alignment of their allosteric regulatory domains ( figure 2 ), from which several conserved sites were selected, in order to reduce the binding of UMP, the hydrophilic amino acid residues were mutated into hydrophobic residues, or the size of the residues was enlarged to increase its steric hindrance, and the mutation sites The design was included in the primer sequence, and the sequence was sent to Shanghai Bioengineering Co., Ltd. for synthesis. The specific primer sequence is shown in Table 1.

[0025] Table 1 Sequence list of primers for site-directed mutagenesis

[0026]

[0027]

[0028] Preparation of the vector: PCR amplifi...

Embodiment 2

[0032] Example 2: cepPCR directed evolution of carbamoyl phosphate synthase

[0033] The carbamoyl phosphate synthase large subunit CarB from E.coli MG1655 has a total of 1073 amino acids and is divided into 4 functional domains, namely f1:1-403aa is the carboxylic acid phosphate synthesis domain, f2:404-553aa is the oligomerization domain F3:554-936aa is the carbamoyl phosphate synthesis domain and F4:937-1073aa is the allosteric regulatory domain. By changing the PCR reaction system and simulating natural evolution, site mutations are introduced into the large subunit CarB. The specific reaction system is as follows: wild-type carAB is used as a template, and cepPCR-F and cepPCR-R of each domain are used as upstream and downstream. Primers (see Table 2 for primer sequences), 1 μl of Taq DNA Polymerase, dATP, dGTP concentrations of 0.2 mM, dTTP, dCTP concentrations of 1 mM, Mg 2+ Concentration is 3mM, Mn 2+ The concentration is 0.2mM, add sterile water to a total volume of ...

Embodiment 3

[0050] Embodiment 3: expression and protein purification of mutant enzyme

[0051] Inoculate the 9 strains of recombinant bacteria obtained in Example 1 and Example 2 and Rosetta / pET28a-CarAB expressing wild type into 50ml LB containing 50μg / ml, shake at 37°C and 220rpm for 6h, and then transfer the bacteria Into 100ml of LB, 37°C, 220rpm culture to OD 600 =0.6-0.8, add IPTG with a final concentration of 0.2mM and induce at 18°C ​​and 220rpm for 16h. Centrifuge at 6000rpm for 10min to collect the bacteria.

[0052] The protein was purified by Ni-NTA nickel column affinity chromatography. Resuspend the bacteria with 50ml of buffer solution (20mM Tris-HCl; 500mMNaCl; 10mM imidazole), then crush it by a pressure breaker, centrifuge at 6000rpm at 4°C for 20min, take the supernatant and load it onto a nickel column that has been equilibrated with buffer. The adsorbed protein was then gradiently eluted with loading buffers containing different concentrations of imidazole (0-150mM...

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Abstract

The invention provides a carbamyl phosphate synthetase mutant with an effect of uridylic acid feedback inhibition resistance and application thereof. The mutant is a mutant K1006A obtained by mutatingthe 1006 lysine into alanine or a mutant N1015F obtained by mutating the 1015 asparagine into phenylalanine, based on the carbamyl phosphate synthetase having an amino acid sequence as shown in the SEQ ID NO.1. Compared with a wild type Ec-CarAB, the mutant enzyme N1015F and 1006A have the advantages of respectively showing relief of feedback inhibition of the inhibitor UMP and improvement of catalytic activity. The initial enzyme activity of the N1015F is not variable in comparison with that of WT, and the activity of the N1015F is invariant along with increase of the concentration of the inhibitor UMP. The initial enzyme activity of the K1006A is increased by 12 percent than that of the wild type enzyme, the enzyme activity of the K1006A is gradually reduced along with increase of the concentration of the UMP, the K1006A is saturated under 20mM UMP concentration, and the relative enzyme activity of the K1006A still can be kept at 65 percent or more.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to a uridine monophosphate (UMP)-resistant carbamoyl phosphate synthetase mutant and application thereof. Background technique [0002] Carbamyl phosphate synthase (CPS) is divided into two types, CPS Ⅰ and CPS Ⅱ, both of which are isoenzymes. CPS Ⅰ is a key enzyme involved in the urea cycle in the energy metabolism of liver cells, which can remove excess ammonia in cells. Deletion will lead to hyperammonemia. CPSⅡ (EC 6.3.5.5) is the first key enzyme in the pyrimidine de novo synthesis pathway. It uses glutamine, bicarbonate, and 2Mg-ATP as substrates to catalyze the formation of glutamate, Phosphate and 2Mg-ADP, the carbamoyl phosphate produced by the reaction mainly has two metabolic pathways. One is coupled with aspartate carbamoylase to participate in the de novo synthesis of pyrimidine nucleosides; the other is coupled with ornithine carbamoylase to synthesize orni...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N1/21C12P19/30C12R1/19
CPCC12N9/93C12P19/305C12Y603/05005
Inventor 李志敏沈苏
Owner EAST CHINA UNIV OF SCI & TECH
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