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CN-122012637-A - Biosynthesis method of nylon salt

CN122012637ACN 122012637 ACN122012637 ACN 122012637ACN-122012637-A

Abstract

The invention discloses a biosynthesis method of nylon salt, and belongs to the field of bioengineering. The present invention provides an arginine decarboxylase mutant AdiA T204E/H736E with an optimal pH up-regulation. The mutant AdiA T204E/H736E maintains higher activity in the whole alkaline range of pH 7.0-9.0. The invention further expresses the mutant AdiA T204E/H736E in escherichia coli to construct a genetically engineered bacterium with the yield of butanediamine reaching 138 g/L, further takes arginine regulated by adipic acid as a substrate, generates butanediamine under the action of the genetically engineered bacterium, further heats the butanediamine to react with adipate ions, and directly extracts and purifies nylon salt from the reaction liquid.

Inventors

  • DENG YU
  • LI GUOHUI
  • Fu Zimeng
  • Li Chouqiang
  • MAO YIN
  • ZHOU SHENGHU
  • ZHAO YUNYING

Assignees

  • 江南大学

Dates

Publication Date
20260512
Application Date
20260120

Claims (10)

  1. 1. A method for biosynthesis of nylon salt, comprising the steps of: S1, taking arginine regulated by adipic acid, terephthalic acid or sebacic acid as a substrate of genetic engineering bacteria for producing butanediamine, catalyzing the arginine to generate butanediamine at the temperature of 30-40 ℃, and removing thalli to obtain supernatant containing butanediamine; S2, heating the supernatant, decoloring, continuously reacting at 50-70 ℃ to generate nylon salt, and extracting and separating from the reaction solution to obtain the nylon salt.
  2. 2. The synthetic method according to claim 1, wherein the genetically engineered bacteria producing butanediamine express arginine decarboxylase mutant adiA T204E/H736E , arginine decarboxylase SpeA and agmatinase SpeB in a host, wherein the arginine decarboxylase mutant adiA T204E/H736E is obtained by mutating threonine at position 204 of arginine decarboxylase with the amino acid sequence shown in SEQ ID NO.1 to glutamic acid and mutating histidine at position 736 to glutamic acid.
  3. 3. The method according to claim 1, wherein arginine is added in the step S1 at a concentration of 100 to 300 g/L.
  4. 4. The method according to claim 1 or 3, wherein in step S1, the genetically engineered bacterium is subjected to cell culture to obtain wet cells, and the wet cells are added to the catalytic reaction system at a concentration of 60 to 100 g/L.
  5. 5. The method according to claim 4, wherein in step S1, the catalytic reaction system further comprises 0.5-1.5 mM PLP,3-5 mM MgSO 4 ·7H 2 O,10-14 mM MnSO 4 ·H 2 O,0.3-0.5 mM DTT,0.5-1.5 mM IPTG,0.5-1.5 mM ampicillin.
  6. 6. The method according to claim 1, wherein the catalytic reaction time is 15 to 30 hours.
  7. 7. The method according to claim 1, wherein in step S2, adipic acid, terephthalic acid or sebacic acid is gradually and slowly added to adjust pH to 7.5, and adipic acid, terephthalic acid or sebacic acid is not added.
  8. 8. The synthesis method according to claim 2, wherein the genetically engineered bacteria include but are not limited to escherichia coli genetically engineered bacteria, bacillus subtilis genetically engineered bacteria, corynebacterium glutamicum genetically engineered bacteria, and yeast genetically engineered bacteria, the amino acid sequence of arginine decarboxylase SpeA is SEQ ID NO.4, and the amino acid sequence of agmatinase SpeB is SEQ ID NO. 5.
  9. 9. The method of claim 1, wherein the nylon salt is a nylon 46 salt, a nylon 4T salt, or a nylon 410 salt.
  10. 10. The use of the synthetic method according to any one of claims 1 to 9 in the preparation of nylon products, wherein the use is to polymerize the prepared nylon salt in an autoclave to obtain the nylon product, and the nylon product is nylon 46, nylon 4T or nylon 410.

Description

Biosynthesis method of nylon salt Technical Field The invention relates to a biosynthesis method of nylon salt, and belongs to the field of bioengineering. Background The polyamide (Polyamide, PA) is a high-molecular compound prepared by polycondensation of monomers with amino and carboxyl, and a large amount of natural polyamide such as polypeptide exists in nature, and the artificial polyamide is various nylon products such as nylon fabrics, engineering parts and the like. The very large part of the polyamide product is determined by the monomer unit structure. Polyamide products having a butanediamine unit structure include polyamide46 (Polyamide, PA 46), polyamide4T (Polyamide T, PA 4T), and polyamide410 (Polyamide, PA 410). They are obtained by polymerizing the monomers adipic acid, p-benzoic acid, sebacic acid and butanediamine, respectively. Nylon 4T belongs to semi-aromatic polyamide (high-temperature nylon), is an important engineering plastic, and has the characteristics of high melting point, good heat resistance, excellent dimensional stability, excellent mechanical strength and the like. Nylon 46 and nylon 410 are aliphatic polyamides, have highly symmetrical molecular structures, and have the characteristics of high crystallinity, high rigidity, high strength, high temperature resistance and the like. The method for synthesizing polyamide includes high temperature solution polycondensation, low temperature solution polycondensation, direct melt polycondensation, amine transesterification, interfacial polycondensation, etc. The high-temperature solution method adopts water as a reaction solvent, and has lower production cost. The process comprises three steps of salification, prepolymerization and solid phase polycondensation. Nylon 46 salt, nylon 4T salt, nylon 410 salt are precursor materials of PA46, PA4T, PA, 410, respectively. The raw material butanediamine of the nylon salt is not limited to chemical synthesis at present, and the butanediamine is prepared by modifying the metabolic pathway of engineering strains and utilizing amino acids with wide sources, so that the high-efficiency biosynthesis of butanediamine is realized. The direct production of nylon salts using biosynthesized butanediamine is currently less studied, most of which use butanediamine obtained from the reaction of acrylonitrile and hydrocyanic acid. The biosynthesis of butanediamine requires a method to be found for extraction, and the extraction of pure butanediamine from fermentation broth is difficult. If the nylon salt can be directly generated by reaction in the fermentation broth, the problem of difficult extraction of the butanediamine is solved. The yield of butanediamine in the fermentation liquor is improved, and the method becomes a key step of nylon salt. Arginine decarboxylase AdiA is an important member of the E.coli acid stress response system, a typical decameric higher order homooligomeric protein, forming a bilayer pentacyclic structure from five dimers. The enzyme catalyzes the decarboxylation of L-arginine to form agmatine, and is a key enzyme for synthesizing butanediamine (putrescine, an important nylon monomer) by a biological method. However, the assembly of the AdiA decamer structure is highly pH dependent, being stable only under acidic conditions (pH < 6.0) since its surface is rich in acidic amino acids, protonates the charge in an acidic environment, promotes oligomerization, and under neutral and basic conditions acidic amino acid deprotonation creates electrostatic repulsion, leading to deagglomeration into dimers or monomers, with significant loss of enzyme activity. This pH dependence severely limits the whole cell catalytic application of AdiA in neutral or alkaline environments, whereas in industrial fermentations the intracellular environment is usually neutral and the butanediamine product is alkaline again, resulting in the inability of traditional AdiA to be efficiently used for large scale biosynthesis of butanediamine. Currently, various engineering strategies have been used to promote protein oligomerization, such as modulating interfacial residue complementarity, introducing disulfide bonds or electrostatic attraction, increasing interfacial hydrophobicity, etc., which are readily implemented in dimeric and tetrameric proteins. However, for AdiA such higher order decameric proteins, the existing strategies have difficulty in effectively achieving stable oligomeric structures under medium alkaline conditions due to complex interfacial interactions (involving both meridional and latitudinal dimensional interfaces), and great difficulty in molecular dynamics simulation. In addition, similar pH control mechanisms exist for other acid-induced decarboxylases such as lysine decarboxylase CadA, limiting efficient biosynthesis of polyamines. Therefore, development of a novel rational engineering strategy is needed, and accurate modification is performed on the interface of the high-ord