Biosynthesis method of nylon salt

Abstract

The invention discloses a biosynthesis method of nylon salt, and belongs to the field of bioengineering. According to the arginine decarboxylase mutant AdiAT204E / H736E, the arginine decarboxylase mutant AdiAT204E / H736E which is most suitable for up-regulation of pH is obtained. The mutant AdiAT204E / H736E keeps relatively high activity in the whole neutral and alkaline range of pH (Potential of Hydrogen) of 7.0 to 9.0. Further, the mutant AdiAT204E / H736E is expressed in escherichia coli, a genetically engineered bacterium with butanediamine yield reaching 138 g / L is constructed, arginine regulated by adipic acid is further used as a substrate, butanediamine is generated under the action of the genetically engineered bacterium, butanediamine and adipate ions are further heated to react, and the butanediamine with adipate ions is obtained. And nylon salt is directly extracted and purified from the reaction liquid.

Description

Technical Field

[0001] This invention relates to a method for the biosynthesis of nylon salts, belonging to the field of bioengineering. Background Technology

[0002] Polyamide (PA) is a polymer compound produced by the polycondensation reaction of monomers with amino and carboxyl groups. Natural polyamides, such as polypeptides, exist in large quantities in nature; artificial polyamides are what people refer to as various nylon products, such as nylon fabrics and engineering parts. The characteristics of polyamide products are mostly determined by the monomer unit structure. Polyamide products with butanediamine as the unit structure include polyamide 46 (PA46), polyamide 4T (PA4T), and polyamide 410 (PA410). They are obtained by polymerizing adipic acid, terebenzoic acid, sebacic acid, and butanediamine, respectively. Nylon 4T belongs to semi-aromatic polyamides (high-temperature nylon) and is an important engineering plastic with high melting point, good heat resistance, excellent dimensional stability, and superior mechanical strength. Nylon 46 and Nylon 410 belong to aliphatic polyamides with highly symmetrical molecular structures, exhibiting high crystallinity, high rigidity, high strength, and high-temperature resistance.

[0003] Polyamides can be synthesized through various methods, including high-temperature solution polycondensation, low-temperature solution polycondensation, direct melt polycondensation, amine-ester exchange polymerization, and interfacial polycondensation. Among these, the high-temperature solution method uses water as the reaction solvent, resulting in lower production costs. This process involves three steps: salt formation, prepolymerization, and solid-state polycondensation. Nylon 46 salt, Nylon 4T salt, and Nylon 410 salt are the precursors for PA46, PA4T, and PA410, respectively.

[0004] The raw material for nylon salts, butanediamine, is no longer limited to chemical synthesis. By modifying the metabolic pathways of engineered strains, butanediamine can be prepared using widely available amino acids, achieving efficient biosynthesis of butanediamine. Compared to industrial production, this method has the advantages of being green, sustainable, and less polluting. Currently, there is limited research on the direct production of nylon salts from biosynthesized butanediamine; most studies still use butanediamine obtained from the reaction of acrylonitrile and hydrogen cyanide.

[0005] The biosynthetic butanediamine requires extraction, but extracting pure butanediamine from fermentation broth is difficult. If nylon salts could be directly generated in the fermentation broth, the extraction difficulty of butanediamine could be solved. Increasing the yield of butanediamine in the fermentation broth has become a key step in the production of nylon salts. Arginine decarboxylase AdiA is an important member of the E. coli acid stress response system, a typical decadal high-order homologous oligomeric protein, consisting of five dimers forming a bilayer pentacyclic structure. This enzyme catalyzes the decarboxylation of L-arginine to generate guanidine butanediamine, a key enzyme in the biosynthesis of butanediamine (putrescine, an important nylon monomer). However, the assembly of the AdiA decadal structure is highly pH-dependent, stable only under acidic conditions (pH < 6.0). This is because its surface is rich in acidic amino acids, which protonate and neutralize the charge in an acidic environment, promoting oligomerization. Under neutral and alkaline conditions, the acidic amino acids deprotonate, generating electrostatic repulsion, causing the decadal to depolymerize into dimers or monomers, resulting in a significant loss of enzyme activity. This pH dependence severely limits the whole-cell catalytic application of AdiA in neutral or alkaline environments. In industrial fermentation, the intracellular environment is usually neutral, while the butanediamine product is alkaline, which makes it impossible for traditional AdiA to be used efficiently for the large-scale biosynthesis of butanediamine.

[0006] Currently, various engineering strategies have been developed to promote protein oligomerization, such as regulating interfacial residue complementarity, introducing disulfide bonds or electrostatic attraction, and improving interfacial hydrophobicity. These strategies are easily implemented in dimer and tetramer proteins. However, for higher-order decadal proteins like AdiA, due to the complex interfacial interactions (involving both meridional and latitudinal interfaces) and the difficulty in molecular dynamics simulations, existing strategies struggle to effectively achieve oligomeric structural stability under moderately alkaline conditions. Furthermore, other acid-inducible decarboxylases, such as lysine decarboxylase CadA, also exhibit similar pH-regulating mechanisms, limiting the efficient biosynthesis of polyamine compounds.

[0007] Therefore, there is an urgent need to develop a new rational engineering strategy to precisely modify the interface of higher-order oligomeric decarboxylases, so as to shift the pH of the enzymes towards neutral and alkaline conditions, thereby improving the catalytic efficiency of the enzymes and promoting the green and large-scale production of nylon. Summary of the Invention

[0008] The purpose of this invention is to first solve the problem of arginine decarboxylase (AdiA) activity being inhibited under near-neutral conditions, and to obtain an arginine decarboxylase mutant with optimal pH upregulation. Furthermore, based on the arginine decarboxylase mutant with optimal pH upregulation, a genetically engineered bacterium capable of efficiently producing butanediamine was constructed. This invention further uses adipic acid-regulated arginine as a substrate. Arginine is converted into butanediamine under the action of the genetically engineered strain. Further heating causes the butanediamine to react with adipic acid ions, and the reaction solution is directly used for the extraction and purification of nylon salt.

[0009] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: The first objective of this invention is to provide a method for the biosynthesis of nylon salts, comprising the following steps: S1. Arginine regulated by adipic acid, terebenzoic acid, and sebacic acid is used as a substrate for the genetically engineered bacteria that produce butanediamine. Arginine is catalyzed to produce butanediamine at 30-40℃. The bacterial cells are removed to obtain a supernatant containing butanediamine. S2. The supernatant is heated and decolorized. After decolorization, the reaction continues at 50-70℃ to generate nylon salt. The nylon salt is then extracted and separated from the reaction solution.

[0010] In one embodiment of the present invention, the genetically engineered bacteria that produce butanediamine are those that express an arginine decarboxylase mutant in a host. adiA T204E / H736E Arginine decarboxylase SpeA and guanidine amino acid enzyme SpeB, wherein the arginine decarboxylase mutant adiA T204E / H736E It is obtained by mutating threonine at position 204 of the arginine decarboxylase, as shown in SEQ ID NO.1, to glutamic acid, and mutating histidine at position 736 to glutamic acid.

[0011] In one embodiment of the present invention, in step S1, arginine is added at a concentration of 100~300 g / L.

[0012] In one embodiment of the present invention, in step S1, the genetically engineered bacteria are obtained by cell culture to obtain wet bacterial cells, and added to the catalytic reaction system at a wet bacterial cell concentration of 60-100 g / L.

[0013] In one embodiment of the present invention, in step S1, the system of the catalytic reaction further includes 0.5-1.5 mM MPLP, 3-5 mM MgSO4·7H2O, 10-14 mM MnSO4·H2O, 0.3-0.5 mM DTT, 0.5-1.5 mM IPTG, and 0.5-1.5 mM ampicillin.

[0014] In one embodiment of the present invention, the catalytic reaction time is 15-30 hours.

[0015] In one embodiment of the present invention, in step S2, decolorization is performed using activated carbon.

[0016] In one embodiment of the present invention, the amount of activated carbon added is 10%-20% of the mass of butanediamine.

[0017] In one embodiment of the present invention, heating before decolorization involves heating the supernatant to 60-80°C.

[0018] In one embodiment of the present invention, in step S2, when the nylon salt is generated, adipic acid, terephthalic acid or sebacic acid is gradually and slowly added to adjust the pH. After the pH is adjusted to 7.5, no more adipic acid, terephthalic acid or sebacic acid is added.

[0019] In one embodiment of the present invention, the genetically engineered bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.

[0020] In one embodiment of the present invention, the amino acid sequence of the arginine decarboxylase SpeA is SEQ ID NO.4, and the amino acid sequence of the guanidine amino acid enzyme SpeB is shown in SEQ ID NO.5.

[0021] In one embodiment of the present invention, the nylon salt is nylon 46 salt, nylon 4T salt, or nylon 410 salt.

[0022] In one embodiment of the present invention, when the nylon salt is nylon 46 salt, adipic acid is used to adjust the pH of the arginine solution.

[0023] In one embodiment of the present invention, when the nylon salt is nylon 4T salt, terebenzoic acid is used to adjust the pH of the arginine solution.

[0024] In one embodiment of the present invention, when the nylon salt is nylon 410 salt, sebacic acid is used to adjust the pH of the arginine solution.

[0025] A second objective of this invention is to provide the application of the method in the preparation of nylon products, wherein the prepared nylon salt is polymerized in an autoclave to obtain the nylon product, wherein the nylon product is nylon 46, nylon 4T, or nylon 410.

[0026] Beneficial effects Following the rational copolymerization modification strategy described in this invention, the meridional interface and zonal channel interface of arginine decarboxylase were rationally modified, either separately or simultaneously, to obtain an arginine decarboxylase mutant, AdiA, with optimal pH upregulation. T204E / H736E The mutant AdiA T204E / H736E It maintains high activity throughout the entire moderately alkaline range of pH 7.0-9.0. Furthermore, this invention utilizes the mutant AdiA... T204E / H736E A genetically engineered bacterium producing 138 g / L of butanediamine was constructed by expressing it in Escherichia coli. Arginine regulated by adipic acid was then used as a substrate, and arginine was converted into butanediamine under the action of the genetically engineered strain. The butanediamine was further heated to react with adipic acid ions, and the reaction solution was directly used to extract and purify nylon salt. Detailed Implementation

[0027] The culture media involved in the following examples: LB solid culture: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 2 g / L agar powder.

[0028] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0029] The detection methods involved in the following embodiments are as follows: The method for detecting arginine decarboxylase activity is as follows: Prepare a 200 µL enzyme activity reaction system according to Table 1, and determine the arginine decarboxylase activity. The reaction system was precisely reacted at 37°C for 10 min, and the reaction was terminated by adding 20 µL of 40% trichloroacetic acid. The reaction solution was then cooled in an ice-water bath. The reaction was then accelerated at 12000 r·min. -1 Centrifuge for 10 min, collect the supernatant, derivatize it, and use it for HPLC detection of butanediamine yield. Unit enzyme activity is defined as U / g = 1 mol butanediamine / min / g protein.

[0030] Table 1: Reaction system for arginine decarboxylase activity detection

[0031] The 15 mmol / L potassium sodium phosphate buffer solution is prepared as follows: the pH value of 15 mmol / L KH2PO4 is adjusted to the corresponding pH value using 15 mmol / L Na2HPO4.

[0032] HPLC detection of butanediamine 1) Sample derivatization and extraction The pre-column derivatization procedure for dansyl chloride is as follows: After centrifuging the fermentation broth or reaction solution at 12000 r / min for 10 min, take 500 μL of the supernatant as the sample. Add 500 μL of saturated NaHCO3 solution and internal standard (5 μL of 10 g / L heptamethylenediamine), mix thoroughly, and adjust the pH to 10 with saturated NaOH solution. Then add 1 mL of the derivatization reagent dansyl chloride (5 g / L, soluble in acetone). Incubate the mixture in a light-protected water bath at 60°C for 30 min, add 2 mL of anhydrous diethyl ether, and extract for 10 min. Collect the upper organic phase. Repeat the extraction operation twice, mix the two organic phases, and dry them with nitrogen to remove the diethyl ether. Dissolve the derivatized compound in 500 μL of acetonitrile solution, filter through a 0.22 μm filter membrane, and use for HPLC detection.

[0033] 2) HPLC chromatographic determination Chromatographic conditions: High-performance liquid chromatography (HPLC) separation of diamine dansyl chloride derivatives was performed on a C18 column at a separation temperature of 30°C and a UV detection wavelength of 254 nm. The injection volume was 10 μL. Mobile phase A was ultrapure water, and mobile phase B was HPLC-grade acetonitrile. Both mobile phases were filtered through a 0.22 μm filter before use. The gradient elution program was set as follows: 0–4 min, 55%–70% B; 4–6 min, 70% B; 6–11 min, 70% B; 11–12 min, 95% B; 12–13 min, 95% B; 13–16 min, 55% B. The total flow rate was set to 0.7 mL / min.

[0034] Example 1: Determination of the AdiA mutation site in arginine decarboxylase The specific steps are as follows: Arginine decarboxylase AdiA catalyzes the synthesis of guanidinediamine from arginine and is a key enzyme in the butanediamine (ADC) synthesis pathway. Its catalytic reaction requires pyridoxal phosphate (PLP) to assist in decarboxylation and proton transfer, but does not consume PLP. The effective quaternary structure of AdiA is a decameric, but the decameric can only form in environments with pH < 6, resulting in an optimal reaction pH of slightly acidic (pH 5.4). Based on the crystal structure of the AdiA decameric (PDB ID: 2VYC), interface analysis was performed using PyMOL 2.5 software. The AdiA decameric consists of a bilayer pentacyclic structure composed of five dimers, exhibiting centrosymmetry. The meridional interface, located between the pentacyclic monomers, mainly involves acidic amino acid enrichment regions and exhibits significant electrostatic repulsion under moderately alkaline conditions. The latitudinal interface, located between the dimers, simultaneously forms substrate channels. The CAVER tool was used to predict these substrate channels, which directly affect the binding and transport of the substrate arginine. The surface of AdiA is rich in acidic amino acids, which results in the decadomer being stable only at pH < 6.0.

[0035] Based on the above research, T204 and H736 were identified as mutation sites for arginine decarboxylase AdiA.

[0036] Example 2: Preparation of Arginine Decarboxylase AdiA Mutant The specific steps are as follows: Through primers adiA -F and adiA -R from E. coli Amplification of arginine decarboxylase gene in the BL21(DE3) genome adiA ,use Nde I and XhoI performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. adiA The ligation product was introduced into E. coli JM109 and screened by colony PCR and Sanger sequencing to finally obtain the recombinant plasmid pETDuet-adiA.

[0037] The amino acid sequence (SEQ ID NO.1) of the wild-type arginine decarboxylase is as follows: MKVLIVESEFLHQDTWVGNAVERLADALSQQNVTVIKSTSFDDGFAILSSNEAIDCLMFSYQMEHPDEHQNVRQLIGKLHERQQNVPVFLLGDREKALAAMDRDLLELVDEFAWILEDTADFIAGRAVAAMTRYRQQLLPPLFSALMKYSDIHEYSWAAPGHQGGVGFTKTPAGRFYHDYYGENLFRT DMGIERTSLGSLLDHTGAFGESEKYAARVFGADRSWSVVVGTSGSNRTIMQACMTDNDVVVVDRNCHKSIEQGLMLTGAKPVYMVPSRNRYGIIGPIYPQEMQPETLQKKISESPLTKDKAGQKPSYCVVTNCTYDGVCYNAKEAQDLLEKTSDRLHFDEAWYGYARFNPIYADHYAMRGEPGDHNGPT VFATHSTHKLLNALSQASYIHVREGRGAINFSRFNQAYMMHATTSPLYAICASNDVAVSMMDGNSGLSLTQEVIDEAVDFRQAMARLYKEFTADGSWFFKPWNKEVVTDPQTGKTYDFADAPTKLLTTVQDCWVMHPGESWHGFKDIPDNWSMLDPIKVSILAPGMGEDGELEETGVPAALVTAWLGRH GIVPTRTTDFQIMFLFSMGVTRGKWGTLVNTLCSFKRHYDANTPLAQVMPELVEQYPDTYANMGIHDLGDTMFAWLKENNPGARLNAAYSGLPVAEVTPREAYNAIVDNNVELVSIENLPGRIAANSVIPYPPGIPMLLSGENFGDKNSPQVSYLRSLQSWDHHFPGFEHETEGTEIIDGIYHVMCVKA The nucleotide sequence encoding the arginine decarboxylase is as follows: Through primers adiAT204E -F and adiAT204E -R was used to perform full-plasmid PCR on pETDuet-adiA, and the PCR product was purified before use. Dpn The bacteria were digested with enzyme I, and the digestion product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain AdiA. T204E Mutant recombinant plasmid pETDuet-AdiA T204E .

[0038] Through primers adiAT204D -F and adiAT204D -R was used to perform full-plasmid PCR on pETDuet-adiA, and the PCR product was purified before use. Dpn The bacteria were digested with enzyme I, and the digestion product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain AdiA. T204D The mutant recombinant plasmid pETDuet-AdiA T204D .

[0039] Through primers adiAH736E -F4 and adiAH736E -R4 for pETDuet-adiA T204E Perform whole-plasmid PCR, and use the PCR products after purification. Dpn The bacteria were digested with enzyme I, and the digestion product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain AdiA. T204E / H736E Mutant recombinant plasmid pETDuet-AdiA T204E / H736E .

[0040] Table 2: Primer Sequence Listing

[0041] Example 3: Expression of mutant arginine decarboxylase and purification of AdiA The specific steps are as follows: The recombinant plasmids expressing the AdiA gene and its mutants, and the recombinant plasmid expressing the guanidine aminotransferase SpeB gene, prepared in Example 2, were expressed in *E. coli* BL21 with an N-terminal 6-His tag. All expression strains were cultured in 500 mL SOB medium at 37°C for 3 hours, followed by overnight culture with 0.1 mmol / L IPTG at 30°C to induce protein expression. The overnight cultured cells were collected by refrigerated centrifugation at 8000 rpm for 5 min, resuspended in 50 mL Tris-HCl buffer, and then sonicated. The supernatant was purified by affinity chromatography using a Ni-NTA Superflow resin column.

[0042] The method for preparing the recombinant vector expressing the guanidine aminotransferase SpeB gene is as follows: First, it is digested with double enzymes (the enzyme sites are: Nde I, Xho I) The ligation method was used to construct the guanidine amino acid enzyme SpeB overexpression plasmid pETDuet-SpeB; the amino acid sequence of SpeB (SEQ ID NO.3) is as follows: MSTLGHQYDNSLVSNAFGFLRLPMNFQPYDSDSDWVITGVPFDMATSGRAGGRHGPAAIRQVSTNLAWEHNRFPWNFDMRERLNVVDCGDLVYAFGDAREMSEKLQAHAEKLLAAGKRMLSFGGDHFVTLPLLRAHAKHFGKMALVHFDAHTD TYANGCEFDHGTMFYTAPKEGLIDPNHSVQIGIRTEFDKDNGFTVLDACQVNDRSVDDVIAQVKQIVGDMPVYLTFDIDCLDPAFAPGTGTPVIGGLTSDRAIKLVRGLKDLNIVGMDVVEVAPAYDQSEITALAAATLALEMLYIQAAKKGE* Pure enzyme solutions of wild-type AdiA, AdiA mutant, and guanidine amino acid enzyme SpeB were prepared.

[0043] Example 4: Enzyme activity assay of wild-type and mutant enzymes at different pH values The specific steps are as follows: Following the reaction system in Table 1, i.e., adding arginine decarboxylase AdiA or mutant pure enzyme solution to a potassium sodium phosphate buffer containing dithiothreitol, EDTA, pyridoxal phosphate (PLP), MgSO4, arginine, and guanidine amino acid enzyme pure solution, to achieve the final concentration shown in Table 1, the reaction system was incubated at 37°C for 10 min. The reaction was terminated by adding 20 µL of 40% trichloroacetic acid, and the reaction solution was cooled in an ice-water bath. After centrifugation at 12000 r / min for 10 min, the supernatant was derivatized and used for HPLC detection of butanediamine yield. The unit enzyme activity was defined as U / g = 1 mole of butanediamine / min / g protein.

[0044] The enzyme activity detection results of each mutant by HPLC are shown in Table 3 below. The results show that the mutant AdiA T204E / H736E It maintains high activity within the pH range of 7.0–9.0.

[0045] Table 3

[0046] Example 5: Construction of genetically engineered bacteria containing mutant arginine decarboxylase The specific steps are as follows: 1. Construction of genetically engineered bacteria Through primers speA -F and speA -R amplifies the gene from the genome of Escherichia coli BL21(DE3). speA .use Nco I and EcoR I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. speA The ligation product was ligated into pETDuet-1. The ligation product was introduced into E. coli JM109, and screened by colony PCR and Sanger sequencing to finally construct the plasmid pETDuet-speA.

[0047] Through primers speB -F and speB -R amplifies the gene from the genome of Escherichia coli BL21(DE3). speB .

[0048] use EcoR I and Hind III. The PCR product and pETDuet-speA plasmid were double-digested, and the purified products were then ligated using T4 DNA ligase. speBLigation was performed on pETDuet-speA. The ligation product was introduced into *E. coli* JM109, and screened by colony PCR and Sanger sequencing to finally construct the plasmid pETDuet-speA-speB. Primers were then used to... adiA -F and adiA -R from pETduet- adiA T204E / H736E amplified mutant gene adiA T204E / H736E ,use Nde I and Xho I performed double digestion of the PCR product and the pETDuet-speA-speB plasmid, and after purification, the product was ligated using T4 DNA ligase. adiA T204E / H736E The plasmid pETDuet-speA-speB was ligated into pETDuet-speA-speB, and the ligation product was introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to obtain the recombinant plasmid pETDuet-speA-. adiA T204E / H736E -speB. The recombinant plasmid pETDuet-speA- adi T204E / H736E -speB conversion to E. coli BL2(DE3) was used to obtain the butanediamine-synthesizing genetically engineered bacterium PUT1.

[0049] In the same way, using wild type adiA Gene replacement adiA T204E / H736E Gene, construct pETDuet-speA-adiA-speB recombinant plasmid, and transform it into E. coli BL2(DE3) was used to obtain the butanediamine-synthesizing genetically engineered bacterium PUT0 as a control strain.

[0050] Table 4: Primer Sequence Listing

[0051] The amino acid sequence (SEQ ID NO.4) of the arginine decarboxylase SpeA is as follows: * 2. Preparation of wet mycelium (1) After the recombinant butanediamine strains PUT0 and PUT1 stored in glycerol tubes were streaked on LB solid plates containing 50 μg / mL kanamycin, single colonies were picked and inoculated into 50 mL LB liquid medium and cultured in a shaker at 37 ℃ and 250 r / min for 12 h to prepare seed culture.

[0052] (2) The prepared seed culture was transferred to 1 L LB medium containing the same antibiotic at an inoculation rate of 2% (v / v) and cultured at 37 °C until OD. 600 The temperature was lowered to 30 °C at ≈15-20 °C, and 0.1 mmol / L IPTG was added to induce expression for 16 h to prepare the fermentation broth.

[0053] (3) After induction, the prepared fermentation broth was centrifuged at 4 ℃ and 8000 r· / min for 20 min to collect the cells. The cells were washed twice with 50 mmol / L phosphate buffer at pH 7.0 and then resuspended to obtain wet cells.

[0054] 3. Whole-cell catalytic preparation of butanediamine Whole-cell catalysis was conducted in a 5 L bioreactor with a working volume of 3 L. The reaction system composition was as follows: total L-arginine concentration of 348.4 g / L (added in batches), and wet cell concentration equivalent to OD0.05. 600 ≈80 (approximately 80 g wet weight / L), PLP 0.1 mmol / L, temperature 42 ℃, rotation speed 300 r / min, initial pH 7.0, closed reaction, reaction time 12 h.

[0055] Samples were taken every 2 hours. After centrifugation at 12,000 r / min, the supernatant was derivatized with dansyl chloride, and the putrescine concentration was detected by HPLC.

[0056] The results are shown in Table 5: Table 5:

[0057] Finally, the optimal double mutant AdiA of this invention was utilized. T204E / H736E The recombinant whole-cell catalyst achieved a putrescine yield of 138 g / L within 12 h.

[0058] Example 6: Whole-cell transformation of nylon 46 salt by genetically engineered bacteria containing mutant arginine decarboxylase The specific steps are as follows: The wet bacterial cells prepared in Example 5 were used for the catalytic reaction. The reaction system consisted of 80 g / L wet bacterial cells, 200 g / L arginine (pH adjusted to 7.0 with adipic acid), 1 mM PLP, 4 mM MgSO4·7H2O, 12 mM MnSO4·H2O, 0.4 mM MTT, 1 mM IPTG, and 1 mM ampicillin. The reaction was carried out at 37°C for 24 h to obtain a reaction solution containing butanediamine.

[0059] The reaction solution was centrifuged, and the supernatant was collected. The mixture was heated to 70°C, and activated carbon was added at 17% of the mass of butanediamine. The mixture was stirred at 70°C for 0.5 hours, filtered, and then the reaction was continued in a 60°C water bath. After the reaction started, adipic acid was gradually and slowly added to adjust the pH until it reached 7.5. No more adipic acid was added after the pH was adjusted. After the reaction was complete, the mixture was distilled under reduced pressure and freeze-dried under vacuum to obtain nylon 46 salt, with a yield of 81.34%.

[0060] Furthermore, nylon 46 salt was polymerized in a high-pressure reactor to obtain high molecular weight nylon 46, and the melting point of the prepared nylon 46 was 290℃.

[0061] Example 7: Optimization of Arginine Concentration in the Reaction System The specific steps are as follows: Arginine was added to water at different final concentrations (200 g / L, 300 g / L, 400 g / L), and the pH was adjusted to 7.0 with adipic acid. The solubility of arginine in water was observed. Arginine itself has low solubility in water, limiting the substrate concentration. Adjusting arginine to neutral pH with an acidic reagent can improve its solubility in water. However, the solubility of arginine-adipate formed by adjusting arginine concentration with adipic acid is limited; compared to adjusting the concentration of arginine in water to 400 g / L with hydrochloric acid, the upper limit for adjusting arginine concentration with adipic acid is 300 g / L. When the added arginine concentration reached 400 g / L, the solution became gel-like with the addition of adipic acid, making it unsuitable as a substrate for addition to the bacterial catalytic system.

[0062] Example 8: Whole-cell transformation of nylon 4T salt by genetically engineered bacteria containing mutant arginine decarboxylase The specific steps are as follows: The wet bacterial cells prepared in Example 5 were used for the catalytic reaction. The reaction system consisted of 80 g / L wet bacterial cells, 200 g / L arginine (pH adjusted to 7.0 with terebenzoic acid), 1 mM PLP, 4 mM MgSO4·7H2O, 12 mM MnSO4·H2O, 0.4 mM DTT, 1 mM IPTG, and 1 mM ampicillin. The reaction was carried out at 37°C for 24 h to obtain a reaction solution containing butanediamine.

[0063] The reaction solution was centrifuged, and the supernatant was collected. The mixture was heated to 70°C, and activated carbon was added at 17% of the mass of butanediamine. The mixture was stirred at 70°C for 0.5 hours, filtered, and then the reaction was continued in a 60°C water bath. After the reaction began, terebenzoic acid was gradually and slowly added to adjust the pH until it reached 7.5. No more terebenzoic acid was added after this adjustment. After the reaction was complete, the mixture was distilled under reduced pressure and freeze-dried under vacuum to obtain nylon 4T salt.

[0064] The nylon 4T salt was further polymerized in a high-pressure reactor to obtain the nylon 4T product. The melting point of the prepared nylon 4T is 325℃.

[0065] Example 9: Whole-cell transformation of nylon 410 salt by genetically engineered bacteria containing mutant arginine decarboxylase The specific steps are as follows: The wet bacterial cells prepared in Example 5 were used for the catalytic reaction. The reaction system consisted of 80 g / L wet bacterial cells, 200 g / L arginine (pH adjusted to 7.0 with sebacic acid), 1 mM PLP, 4 mM MgSO4·7H2O, 12 mM MnSO4·H2O, 0.4 mM MTT, 1 mM IPTG, and 1 mM ampicillin. The reaction was carried out at 37°C for 24 h to obtain a reaction solution containing butanediamine.

[0066] The reaction solution was centrifuged, and the supernatant was collected. The mixture was heated to 70°C, and activated carbon was added at 17% of the mass of butanediamine. The mixture was stirred at 70°C for 0.5 hours, filtered, and then the reaction was continued in a 60°C water bath. After the reaction began, sebacic acid was gradually and slowly added to adjust the pH until it reached 7.5. No more sebacic acid was added after the pH was adjusted. After the reaction was complete, the mixture was distilled under reduced pressure and freeze-dried under vacuum to obtain nylon 410 salt.

[0067] The nylon 410 salt was further polymerized in a high-pressure reactor to obtain the nylon 410 product. The melting point of the prepared nylon 410 was 250℃.

[0068] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for the biosynthesis of nylon salts, characterized in that, Includes the following steps: S1. Arginine modified with adipic acid, terephthalic acid or sebacic acid is used as a substrate for the genetically engineered bacteria that produce butanediamine. Arginine is catalyzed to produce butanediamine at 30~40℃. The bacterial cells are removed to obtain a supernatant containing butanediamine. S2. The supernatant is heated and decolorized. After decolorization, the reaction continues at 50-70℃ to generate nylon salt. The nylon salt is then extracted and separated from the reaction solution.

2. The synthesis method according to claim 1, characterized in that, The genetically engineered bacteria that produce butanediamine express an arginine decarboxylase mutant in the host. adiA T204E / H736E Arginine decarboxylase SpeA and guanidine amino acid enzyme SpeB, wherein the arginine decarboxylase mutant adiA T204E / H736E It is obtained by mutating threonine at position 204 of the arginine decarboxylase, as shown in SEQ ID NO.1, to glutamic acid, and mutating histidine at position 736 to glutamic acid.

3. The synthesis method according to claim 1, characterized in that, In step S1, arginine is added at a concentration of 100~300 g / L.

4. The synthesis method according to claim 1 or 3, characterized in that, In step S1, the genetically engineered bacteria are obtained by cell culture and wet cells are added to the catalytic reaction system at a wet cell concentration of 60-100 g / L.

5. The synthesis method according to claim 4, characterized in that, In step S1, the catalytic reaction system also includes 0.5-1.5 mM PLP, 3-5 mM MgSO4·7H2O, 10-14 mM MnSO4·H2O, 0.3-0.5 mM DTT, 0.5-1.5 mM IPTG, and 0.5-1.5 mM ampicillin.

6. The synthesis method according to claim 1, characterized in that, The catalytic reaction time is 15~30h.

7. The synthesis method according to claim 1, characterized in that, In step S2, when the nylon salt is generated, adipic acid, terephthalic acid, or sebacic acid are gradually and slowly added to adjust the pH. Once the pH reaches 7.5, no more adipic acid, terephthalic acid, or sebacic acid are added.

8. The synthesis method according to claim 2, characterized in that, The genetically engineered bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast; the amino acid sequence of the arginine decarboxylase SpeA is SEQ ID NO.4, and the amino acid sequence of the guanidine aminoase SpeB is shown in SEQ ID NO.

5.

9. The synthesis method according to claim 1, characterized in that, The nylon salt is nylon 46 salt, nylon 4T salt, or nylon 410 salt.

10. The application of the synthesis method according to any one of claims 1 to 9 in the preparation of nylon products, characterized in that, The application involves polymerizing the prepared nylon salt in a high-pressure autoclave to obtain the nylon product, wherein the nylon product is nylon 46, nylon 4T, or nylon 410.

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