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CN-122012358-A - Method for biologically synthesizing butanediamine and application of butanediamine in preparation of nylon 46 salt

CN122012358ACN 122012358 ACN122012358 ACN 122012358ACN-122012358-A

Abstract

The invention discloses a method for biologically synthesizing butanediamine and application thereof in preparing nylon 46 salt, and belongs to the field of bioengineering. An arginine decarboxylase mutant AdiA T195E/E467K with an optimal pH up-regulation was obtained. The mutant AdiA T195E/E467K maintains higher activity in the whole alkaline range of pH 7.0-9.0. The invention further expresses the mutant AdiA T195E/E467K in escherichia coli, constructs a genetically engineered bacterium with the yield of the butanediamine reaching 135 g/L, decolorizes and reacts the generated fermentation supernatant to form salt, and separates the salt to obtain nylon 46 salt.

Inventors

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

Assignees

  • 江南大学

Dates

Publication Date
20260512
Application Date
20260120

Claims (10)

  1. 1. A genetic engineering bacterium for producing butanediamine is characterized in that the genetic engineering bacterium expresses an arginine decarboxylase mutant adiA T195E/E467K , an arginine decarboxylase SpeA and an agmatinase SpeB in a host, wherein the arginine decarboxylase mutant adiA T195E/E467K is obtained by mutating threonine at 195 th site of the arginine decarboxylase with an amino acid sequence shown as SEQ ID NO.1 into glutamic acid and mutating glutamic acid at 467 th site into lysine.
  2. 2. The genetically engineered bacterium of claim 1, wherein the expression vector is a pET series expression vector, preferably the expression vector is a petguet-1 expression vector; Preferably, 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 saccharomycete genetically engineered bacteria.
  3. 3. The genetically engineered bacterium of claim 2, wherein the arginine decarboxylase SpeA has the amino acid sequence of SEQ ID NO.4 and the agmatinase SpeB has the amino acid sequence of SEQ ID NO. 3.
  4. 4. The genetically engineered bacterium of any one of claims 1-3, wherein the genetically engineered bacterium is used for preparing butanediamine or products containing butanediamine or nylon products.
  5. 5. The preparation method of the nylon 46 salt is characterized by comprising the following steps: S1, culturing the genetically engineered bacterium of any one of claims 1-3 to obtain wet thalli; S2, using arginine regulated by adipic acid as a substrate of the genetic engineering bacteria, catalyzing arginine to generate butanediamine at the temperature of 30-40 ℃, and removing thalli to obtain supernatant containing butanediamine; S3, heating the supernatant, decoloring, continuously reacting at 50-70 ℃ to generate nylon salt, and extracting and separating from the reaction solution to obtain nylon 46 salt.
  6. 6. The method according to claim 5, wherein arginine is added at a concentration of 100 to 300 g/L in the step S2.
  7. 7. The method according to claim 5 or 6, wherein in the step S2, the wet cells are added to the catalytic reaction system at a concentration of 60 to 100 g/L.
  8. 8. The process of claim 7, 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.
  9. 9. The method according to claim 5, wherein the catalytic reaction time is 15 to 30 hours.
  10. 10. The process according to claim 5, wherein in step S3, adipic acid is gradually and slowly added to adjust pH to 7.5, and adipic acid is not added any more.

Description

Method for biologically synthesizing butanediamine and application of butanediamine in preparation of nylon 46 salt Technical Field The invention relates to a method for biologically synthesizing butanediamine and application thereof in preparing nylon 46 salt, belonging to the field of bioengineering. Background At present, the synthesis of butanediamine by utilizing a microbial engineering strain to construct a butanediamine anabolism pathway has been applied to factories and is produced in a large scale, butanediamine is a raw material of nylon polymers such as polyamide 46, polyamide 4T and the like, and the cost of industrially producing the nylon polymers is reduced by adopting the biosynthesized butanediamine. The biosynthesis butanediamine is directly combined with the synthesis of the nylon polymer, so that the difficulty and loss of independent extraction of butanediamine are reduced. Butanediamine reacts with different acids to form different nylon product precursors, wherein butanediamine reacts with adipic acid to form nylon 46 salts. Nylon 46 salt is a precursor of polyamide46 (Polyamide, PA 46). 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 polyamide46 (Polyamide, PA 46) has high symmetry in molecular structure, high crystallinity, high rigidity, high strength, high temperature resistance and other characteristics. The advent of PA46 filled the blank of common engineering plastics (PA 6, PA66, PBT) and specialty materials (LCP, PPS, PEEK), called "super nylon", which was used mainly in the automotive and electrical electronics fields. Since the polymerization temperature of PA46 is higher than the melting point of PA46 and butanediamine is easily volatilized and cyclized, the polymerization of PA46 generally proceeds through three steps of salt formation-prepolymerization-solid phase polymerization. How to increase the yield of the butanediamine becomes the key of the nylon 46 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-order oligodecarboxylase, so that the pH adaptability of the enzyme is shifted to the neutral-alkaline state, the catalytic efficiency of the enzyme is improved, and the green large-scale production of nylon 46 is promoted. Disclosure of Invention The invention aims to solve the problem that the enzyme activity of arginine decarboxylase (AdiA) is inhibited under the condition of approaching to neutrality, obtain an arginine decarboxylase mutant with the optimal pH being up-regulated, construct genetic engineering bacteria capable of efficiently producing butanediamine based on the arginine decarboxyl