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A mutant of arginine decarboxylase and its application in the production of agmatine

A technology of arginine decarboxylase and agmatine, which is applied in the biological field and can solve problems such as the limitation of agmatine production

Active Publication Date: 2022-04-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the chemical synthesis method, the production of agmatine by the enzymatic conversion method has the advantages of simple steps, less by-products, and environmental protection. However, due to the product inhibition of arginine decarboxylase used in the existing enzymatic conversion method, in In the presence of the product agmatine, the enzymatic activity of arginine decarboxylase is inhibited, therefore, the existing arginine decarboxylase is often severely limited in yield when applied to the production of agmatine

Method used

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  • A mutant of arginine decarboxylase and its application in the production of agmatine
  • A mutant of arginine decarboxylase and its application in the production of agmatine
  • A mutant of arginine decarboxylase and its application in the production of agmatine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Preparation of Arginine Decarboxylase Mutants

[0046] Specific steps are as follows:

[0047] Synthetic nucleotide sequence is as the gene speA of coding arginine decarboxylase shown in SEQ ID NO.2; The gene speA of the coding arginine decarboxylase that will obtain and pET-28a (+) plasmid pass through EcoR I / BamH I After double enzyme digestion and ligation, the ligation product was obtained; the ligation product was transformed into Escherichia coli BL21 to obtain the transformation product; the transformation product was spread on LB solid medium (containing 50 μg·mL -1 Kanamycin), cultured upside down in a constant temperature incubator at 37°C for 8-12 hours to obtain transformants; pick transformants and inoculate them into LB liquid medium, and shake the flask for 8-12 hours at 37°C and 180rpm Afterwards, the plasmid was extracted for enzyme digestion verification (for the verification results, see figure 1 ) and sequencing verification, if the veri...

Embodiment 2

[0063] Example 2: Product inhibition of arginine decarboxylase mutants to agmatine

[0064] Specific steps are as follows:

[0065] Add concentrations of 0, 5, 10, 20, 50, 100 g / L (corresponding to 0.0000, 0.0385, 0.0769, 0.1538, 0.3846, 0.7692 mol / L) agmatine, to obtain reaction systems containing different concentrations of agmatine; use the reaction systems containing different concentrations of agmatine to detect the wild-type arginine decarboxylase and arginine decarboxylation obtained in Example 1, respectively. The specific enzyme activity of the pure enzyme of enzyme mutant I534D and arginine decarboxylase mutant D535W (detection result is shown in Table 1); Obtain wild-type arginine decarboxylase, arginine decarboxylase mutation according to the detection result of specific enzyme activity Half-inhibition constant of body I534D and arginine decarboxylase mutant D535W to agmatine; wherein, half-inhibition constant=the corresponding concentration of agmatine when the ...

Embodiment 3

[0069] Embodiment 3: the production of agmatine (whole cell transformation method+recombinant Escherichia coli)

[0070] Specific steps are as follows:

[0071]The transformation system was added with 20g / L L-arginine, 4mmol / L MgSO 4 , the Tris-HCL buffer (pH 8.5, 50mmol / L) of 7mmol / L pyridoxal phosphate (PLP); the recombinant Escherichia coli BL21 / pET-28a-speA, BL21 / pET-28a-speA that embodiment 1 obtains -1 and BL21 / pET-28a-speA-2 cells were added to the transformation system in an amount of 30g / L, and transformed at 37°C and 220r / min for 12h to obtain transformation liquid.

[0072] Detect the content of agmatine and the conversion rate of L-arginine in the conversion liquid, and the detection results are shown in Table 2;

[0073] Wherein, the calculation formula of L-arginine conversion rate is as follows:

[0074] The conversion rate of L-arginine=the amount of agmatine produced (mol) / the initial amount of L-arginine added (mol)×100%.

[0075] It can be seen from Tabl...

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Abstract

The invention discloses an arginine decarboxylase mutant and its application in the production of agmatine, belonging to the field of biotechnology. The present invention provides arginine decarboxylase mutants I534D and D535W that are less affected by product inhibition, wherein the arginine decarboxylase mutant I534D is obtained by adding the amino acid sequence of wild-type arginine as shown in SEQ ID NO.1 The 534th isoleucine of decarboxylase is mutated to aspartic acid, and its half-inhibition constant can reach 0.66mol / L, which is 5.5 times higher than that of wild-type arginine decarboxylase; arginine decarboxylase mutant D535W is obtained by mutating the 535th aspartic acid of the wild-type arginine decarboxylase shown in SEQ ID NO.1 to tryptophan, and its half-inhibition constant can reach 0.50 mol / L, compared with Wild-type arginine decarboxylase was increased 4.2-fold.

Description

technical field [0001] The invention relates to an arginine decarboxylase mutant and its application in the production of agmatine, which belongs to the field of biotechnology. Background technique [0002] Agmatine is a derivative of arginine. Studies have shown that agmatine has biological activities such as lowering blood sugar, lowering blood pressure, diuresis, anti-inflammation, anti-depression, and inhibiting cell proliferation. The antagonism of (NMDA) receptor is strong and long-lasting, and it has the withdrawal effect on animal morphine dependence, and it is a kind of detoxification drug with great development value. Therefore, agmatine has a very broad market in the field of medicine. [0003] At present, the industry mainly uses two methods of chemical synthesis and enzymatic conversion to produce agmatine. Among them, the chemical synthesis method generally uses compounds such as 1,4-butanediamine, diethyl adipate, 1,4-dibromobutane and potassium phthalimide...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/70C12N15/77C12N1/21C12P13/00C12R1/19C12R1/15
CPCC12N9/88C12N15/70C12N15/77C12P13/001C12Y401/01019
Inventor 徐美娟饶志明许家钰王怡杨凤玉杨套伟张显
Owner JIANGNAN UNIV
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