Biosynthesis method of butanediamine and application of butanediamine in preparation of nylon 46 salt
By modifying the T195E/E467K site of the arginine decarboxylase AdiA, a genetically engineered strain was constructed, solving the problem of reduced enzyme activity of AdiA under neutral or alkaline conditions, and realizing efficient production of butanediamine and preparation of nylon 46 salt.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2026-01-20
- Publication Date
- 2026-05-12
AI Technical Summary
In existing technologies, the activity of arginine decarboxylase AdiA is significantly reduced under neutral or alkaline conditions, which limits the large-scale biosynthesis of butanediamine and makes it difficult to apply efficiently in industrial fermentation.
By mutating the arginine decarboxylase AdiA, especially by modifying the T195E/E467K site, a mutant AdiAT195E/E467K with optimal pH upregulation was formed. A genetically engineered strain was then constructed, and combined with a suitable expression vector and catalytic conditions, the enzyme achieved highly efficient catalysis.
It maintains high activity under neutral to alkaline conditions, achieving efficient production of butanediamine with a yield of 135 g/L. Furthermore, it improves the preparation efficiency of nylon 46 salt by reacting with adipic acid to generate nylon 46 salt.
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Abstract
Description
Technical Field
[0001] This invention relates to a method for biosynthesizing butanediamine and its application in the preparation of nylon 46 salt, belonging to the field of bioengineering. Background Technology
[0002] Currently, the use of microbial engineered strains to construct a butylene diamine (BED) biosynthetic pathway for BED synthesis has been applied in factories and is being used for large-scale production. BED is a raw material for nylon polymers such as polyamide 46 and polyamide 4T. Using biosynthesized BED reduces the cost of industrial production of nylon polymers. Directly combining biosynthesized BED with nylon polymer synthesis reduces the difficulty and loss associated with separate BED extraction. BED reacts with different acids to generate different nylon product precursors; for example, BED reacts with adipic acid to generate nylon 46 salt.
[0003] Nylon 46 salt is a precursor to polyamide 46 (PA46). Polyamide (PA) is a polymer compound produced by the condensation polymerization 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. Polyamide 46 (PA46) has a highly symmetrical molecular structure and possesses properties such as high crystallinity, high rigidity, high strength, and high temperature resistance. The emergence of PA46 filled the gap between ordinary engineering plastics (PA6, PA66, PBT) and special materials (LCP, PPS, PEEK), and it is known as "super nylon," primarily used in the automotive and electronics industries. Because the polymerization temperature of PA46 is higher than its melting point, and butanediamine is easily volatile and prone to cyclization, the polymerization of PA46 typically involves three steps: salt formation, prepolymerization, and solid-state polymerization.
[0004] Increasing the yield of butanediamine has become crucial for Nylon 46 salt production. Arginine decarboxylase AdiA, an important member of the E. coli acid stress response system, is 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 produce guanidinediamine, a key enzyme in the biosynthesis of butanediamine (an important nylon monomer). However, the assembly of the AdiA decadal structure is highly pH-dependent, remaining stable only under acidic conditions (pH < 6.0). This is because its surface is rich in acidic amino acids, which protonate in an acidic environment to neutralize the charge, 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.
[0005] 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.
[0006] 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 adaptation of the enzyme towards neutral and alkaline conditions, thereby improving the catalytic efficiency of the enzyme and promoting the green and large-scale production of nylon 46. Summary of the Invention
[0007] 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.
[0008] 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 genetically engineered bacterium for producing butanediamine, wherein the bacterium expresses an arginine decarboxylase mutant in a host. to A T195E / E467K Arginine decarboxylase SpeA and guanidine amino acid enzyme SpeB, wherein the arginine decarboxylase mutant to A T195E / E467K It is obtained by mutating threonine at position 195 of the arginine decarboxylase, as shown in SEQ ID NO.1, to glutamic acid, and mutating glutamic acid at position 467 to lysine.
[0009] In one embodiment of the present invention, the expression vector is a pET series expression vector, preferably a pETDuet-1 expression vector.
[0010] 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.
[0011] In one embodiment of the present invention, the amino acid sequence of arginine decarboxylase SpeA is SEQ ID NO.4, and the amino acid sequence of guanidine amino acid enzyme SpeB is shown in SEQ ID NO.3. A second object of the present invention is to provide the application of the genetically engineered bacteria or the method described herein in the preparation of butanediamine or products containing butanediamine or in the preparation of nylon products.
[0012] A third objective of this invention is to provide a method for preparing nylon 46 salt, comprising the following steps: S1. The genetically engineered bacteria are cultured to obtain wet bacterial cells; S2. Using adipic acid-regulated arginine as the substrate of the genetically engineered bacteria, the arginine is catalyzed to generate butanediamine at 30-40℃, and the bacterial cells are removed to obtain a supernatant containing butanediamine. S3. After heating the supernatant, decolorize it. After decolorization, continue the reaction at 50-70℃ to generate nylon salt. Extract and separate nylon 46 salt from the reaction solution.
[0013] In one embodiment of the present invention, in step S2, arginine is added at a concentration of 100~300 g / L.
[0014] In one embodiment of the present invention, in step S2, wet bacterial cells are added to the catalytic reaction system at a concentration of 60-100 g / L.
[0015] 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.
[0016] In one embodiment of the present invention, the catalytic reaction time is 15-30 h.
[0017] In one embodiment of the present invention, in step S3, when the nylon salt is generated by the reaction, adipic acid is gradually and slowly added to adjust the pH, and no more adipic acid is added after the pH is adjusted to 7.5.
[0018] 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. T195E / E467K The mutant AdiA T195E / E467K It maintains high activity throughout the entire moderately alkaline range of pH 7.0-9.0. Furthermore, this invention utilizes the mutant AdiA... T195E / E467K The strain was expressed in Escherichia coli, and a genetically engineered bacterium with a yield of 135 g / L was constructed. The fermentation supernatant was decolorized and reacted to form salt, and nylon 46 salt was separated. Detailed Implementation
[0019] 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.
[0020] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.
[0021] 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.
[0022] Table 1: Reaction system for arginine decarboxylase activity detection
[0023] 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.
[0024] The method for detecting butanediamine residue in nylon 46 salt is as follows: Take 0.1 g of the obtained nylon 46 salt sample into 1 mL of methanol solution, sonicate for 30 min, centrifuge at 12000 rpm for 5 min, take 30 μl of supernatant as the sample, add 60 μl of methanol, 3 μl of DDEMM (200 mM), 47 μl of water, and 180 μl of borate buffer (0.05 M, pH=9), incubate at 70℃ for 2 h, filter through a 0.22 μm filter membrane and use for HPLC detection.
[0025] 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.
[0026] 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.
[0027] 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 makes the decadomer stable only at pH < 6.0.
[0028] Based on the above research, T195 and E467 were identified as mutation sites for arginine decarboxylase AdiA.
[0029] Example 2: Preparation of Arginine Decarboxylase AdiA Mutant The specific steps are as follows: Through primers to A -F and to A -R from E. coli Amplification of arginine decarboxylase gene in the BL21(DE3) genome to A ,use Yes 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. to A The ligation product was ligated into pETDuet-1 and introduced into E. coli JM109. The recombinant plasmid pETDuet-adiA was finally obtained by screening by colony PCR and Sanger sequencing.
[0030] 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 adiAT195E -F and adiAT195E -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 digested product was then introduced into E. coli JM109. Screening was performed by colony PCR and Sanger sequencing to finally obtain adiA. T195E Mutant recombinant plasmid pETDuet-adiA T195E .
[0031] Through primers adiAE467K -F and adiAE467K -R3 for pETDuet-adiA T195E 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. T195E / E467K The mutant recombinant plasmid pETDuet-AdiA T195E / E467K .
[0032] Table 2: Primer Sequence Listing
[0033] 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.
[0034] 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: Yes 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.
[0035] 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.
[0036] The enzyme activity detection results of each mutant by HPLC are shown in Table 3 below. The results show that the mutant AdiA T195E / E467K It maintains high activity within the pH range of 7.0–9.0.
[0037] Table 3
[0038] 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 hope -F and hope-R amplifies the gene from the genome of Escherichia coli BL21(DE3). hope .use No 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. hope 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.
[0039] Through primers hope -F and hope -R amplifies the gene from the genome of Escherichia coli BL21(DE3). hope .
[0040] 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. hope Ligation 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... to A -F and to A -R from pETduet- to A T195E / E467K amplified mutant gene to A T195E / E467K ,use Yes 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. to A T195E / E467K 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-. to A T195E / E467K -speB. The recombinant plasmid pETDuet-speA- to A T195E / E467K -speB conversion to E. coli BL2(DE3) was used to obtain the butanediamine-synthesizing genetically engineered bacterium PUT0.
[0041] In the same way, using wild type to A Gene replacement to A T195E / E467KGene, 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.
[0042] Table 4: Primer Sequence Listing
[0043] 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.
[0044] (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.
[0045] (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.
[0046] 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.
[0047] 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.
[0048] The results are shown in Table 5: Table 5:
[0049] Finally, the optimal double mutant AdiA of this invention was utilized. T195E / E467K The recombinant whole-cell catalyst achieved a butanediamine yield of 135 g / L within 12 h.
[0050] 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.
[0051] 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, 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 80.67%.
[0052] 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 genetically engineered bacterium for producing butanediamine, characterized in that, The genetically engineered bacteria expressed an arginine decarboxylase mutant in the host. adiA T195E / E467K Arginine decarboxylase SpeA and guanidine amino acid enzyme SpeB, wherein the arginine decarboxylase mutant adiA T195E / E467K It is obtained by mutating threonine at position 195 of the arginine decarboxylase, as shown in SEQ ID NO.1, to glutamic acid, and mutating glutamic acid at position 467 to lysine.
2. The genetically engineered bacterium according to claim 1, characterized in that, The expression vector is a pET series expression vector, preferably a pETDuet-1 expression vector; Preferably, the genetically engineered bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.
3. The genetically engineered bacterium according to claim 2, characterized in that, The amino acid sequence of arginine decarboxylase SpeA is shown in SEQ ID NO.4, and the amino acid sequence of guanidine amino acid enzyme SpeB is shown in SEQ ID NO.
3.
4. The genetically engineered bacteria according to any one of claims 1 to 3 are used in the preparation of butanediamine or products containing butanediamine or in the preparation of nylon products.
5. A method for preparing nylon 46 salt, characterized in that, Includes the following steps: S1. The genetically engineered bacteria according to any one of claims 1 to 3 are cultured to obtain wet bacterial cells; S2. Using adipic acid-regulated arginine as the substrate of the genetically engineered bacteria, the arginine is catalyzed to generate butanediamine at 30-40℃, and the bacterial cells are removed to obtain a supernatant containing butanediamine. S3. After heating the supernatant, decolorize it. After decolorization, continue the reaction at 50-70℃ to generate nylon salt. Extract and separate nylon 46 salt from the reaction solution.
6. The preparation method according to claim 5, characterized in that, In step S2, arginine is added at a concentration of 100~300 g / L.
7. The preparation method according to claim 5 or 6, characterized in that, In step S2, wet bacterial cells are added to the catalytic reaction system at a concentration of 60-100 g / L.
8. The preparation method according to claim 7, 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.
9. The preparation method according to claim 5, characterized in that, The catalytic reaction time is 15~30h.
10. The preparation method according to claim 5, characterized in that, In step S3, when the nylon salt is produced, adipic acid is gradually and slowly added to adjust the pH. Once the pH reaches 7.5, no more adipic acid is added.