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DNA molecules encoding arginine deiminase and uses thereof

An arginine deiminase and DNA molecule technology, applied in the field of L-citrulline production, can solve the problems of excess substrate, unfavorable long-term preservation and use of the enzyme, low catalytic ability and temperature stability, etc. The cost of production and use, the effect of high salt concentration resistance, good thermal stability

Pending Publication Date: 2021-11-02
河北远大九孚生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The liquid enzyme activity reached 200000-270000U / L, and the arginine dosage was 258g / L, and the pH was adjusted to 6-7 with concentrated hydrochloric acid. After 5.5 hours of conversion, the conversion rate reached 96.5%. Although the activity after mutation was improved increase, but when the substrate concentration is greater than 200g / L, there will still be substrate remaining after the reaction
[0008] The Chinese patent application with publication number CN106591271A discloses a mutant of arginine deiminase, which is a mutant in which glycine near the active center of the enzyme is mutated into proline, and the mutant has improved enzyme activity compared with the wild enzyme 1.5 times, the half-life at 40 ℃ has been improved by 5.43 times, which solves the problem of low catalytic ability and temperature stability when arginine deiminase catalyzes citrulline synthesis, but the substrate concentration of this reaction is 100g / L, the pH is 6.0-6.5, the conversion rate is 95%, and the half-life at 40°C is about 100min, which is not conducive to the long-term preservation and use of the enzyme

Method used

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  • DNA molecules encoding arginine deiminase and uses thereof
  • DNA molecules encoding arginine deiminase and uses thereof
  • DNA molecules encoding arginine deiminase and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Codon Optimization of Nucleotide Sequence Encoding Arginine Deiminase The protein sequence of arginine deiminase was obtained from NCBI database. According to the protein sequence information (SEQ ID NO:2) of arginine deiminase (ADI) of Pseudomonas aeruginosa strain3 Re2-7 and the codon preference rule of Escherichia coli, the gene sequence is coded Sub-optimization (SEQ ID N NO: 1), after designing and adding EcoRI and HindIII restriction sites at both ends of the gene, it was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for artificial synthesis.

[0053] SEQ ID NO:1

[0054] ATGATGTCAACCGAAAAAACGAAACTGGGTGTTCATAGCGAAGCTGGCAAACTGCGTAAGGTGATGGTCTGTTCTCCGGGTCTGGCTCATCAGCGTCTGACCCCGAGCAACTGCGACGAACTGCTGTTTGATGACGTTATTTGGGTCAATCAAGCCAAACGCGACCATTTTGATTTCGTGACCAAGATGCGTGAACGCGGCATTGATGTTCTGGAAATGCACAACCTGCTGACCGAAACGATCCAGAATCCGGAAGCTCTGAAATGGATTCTGGACCGTAAGATCACGGCAGATTCGGTTGGCCTGGGTCTGACCTCAGAACTGCGTTCGTGGCTGGAAAGCCTGGAACCGCGTAAACTGGCGGAATATCTGATTGGCGGTGT...

Embodiment 2

[0058] Example 2. Construction of strains expressing arginine deiminase

[0059] After designing and adding EcoRI and HindIII restriction sites at both ends of SEQ ID NO:1, it was sent to Shanghai Sangon Co., Ltd. for artificial synthesis and constructed on the pET21a plasmid. The plasmid was transformed into Escherichia coli BL21(DE3) (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) by the heat shock method. After culturing overnight at 37°C, positive transformants were selected and sequenced again. The sequenced correct transformants were genetically engineered. bacteria.

Embodiment 3

[0060] Example 3. Bacterial strain culture expressing arginine deiminase

[0061] 3.1 Colony morphology: pale yellow, rounded edges, smooth surface, and small bumps.

[0062] 3.2 Medium:

[0063] Seed medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, adjust the pH to 7.0-7.2 with 30% (w / v) sodium hydroxide aqueous solution, sterilize at 121°C, sterilize Bacteria time was 20 minutes; sterile filtered ampicillin sodium aqueous solution (final concentration 75 mg / L) was added before inoculation.

[0064] Seed bottle liquid volume 20-40ml / 250ml seed bottle, triangular flask or fermentation bottle liquid volume 50-100ml / 500ml triangular flask or fermentation bottle.

[0065]Fermentation medium: yeast extract 15g / L, sodium chloride 3g / L, ammonium chloride 2g / L, dipotassium hydrogen phosphate 0.75g / L, potassium dihydrogen phosphate 0.75g / L, magnesium sulfate heptahydrate 0.75g / L, pH7.0-7.2, sterilization temperature 121°C, sterilization time 20 minutes; liquid vol...

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Abstract

The invention provides a DNA (deoxyribonucleic acid) molecule for coding arginine deiminase. The DNA molecule comprises a nucleotide sequence as shown in SEQ ID NO: 1. The invention also provides an expression vector containing the DNA molecule disclosed by the invention and a genetically engineered bacterium for expressing the arginine deiminase. The invention also provides a method for preparing L-citrulline by using the engineering bacterium provided by the invention. The genetically engineered bacterium provided by the invention has very high capability of catalyzing arginine to generate L-citrulline, and the expression efficiency is greatly improved, so the production and use cost is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a DNA molecule encoding arginine deiminase, and also relates to a vector containing the DNA molecule and its use for producing L-citrulline. Background technique [0002] L-citrulline, also known as carbamoylornithine, was first isolated from watermelon juice by Ugataro et al. in 1914, and it was later confirmed by Wada Mitsutoku as an amino acid. Citrulline exists in the seeds of Cucurbitaceae plants in a free state. So far, people have successfully isolated citrulline from watermelon juice, wild watermelon leaves, walnut kernels, walnut seedlings and seeds. [0003] Citrulline is a non-protein amino acid. In recent years, many foreign studies have shown that citrulline has many important physiological functions, such as scavenging free radicals, serving as an indicator of allogeneic rejection, vasodilation, stabilizing blood pressure, and diagnosing rheumatoid Arthritis, anti-oxidat...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/78C12N15/70C12N1/21C12P13/10C12R1/19
CPCC12N9/78C12N15/70C12P13/10C12Y305/03006
Inventor 李哲余伟李佳韩烁张松赵桥
Owner 河北远大九孚生物科技有限公司