Process for the preparation of a nylon monomer and its use in the synthesis of nylon fibers

Abstract

This invention discloses a method for preparing nylon monomers and their application in the synthesis of nylon fibers, belonging to the field of bioengineering. The optimal mutant CadA for lysine decarboxylase provided by this invention... S446R / H685E The enzyme activity at pH 6.0 was 3.3 times that of the wild type, and at pH 7.0, pH 8.0, and pH 9.0, it was 8.3 times, 7.0 times, and 8.6 times that of the wild type, respectively, effectively increasing the stability of CadA enzyme in neutral and alkaline pH environments. Applying the optimal lysine decarboxylase mutant to the production of pentanediamine from *E. coli*, the yield of pentanediamine reached 352.7 g / L. This invention improves the conversion efficiency of lysine decarboxylase in neutral and alkaline environments, and increases the synthesis rate of pentanediamine in neutral and alkaline environments, laying the foundation for the efficient synthesis of pentanediamine.

Description

Technical Field

[0001] This invention relates to a method for preparing nylon monomers and their application in the synthesis of nylon fibers, belonging to the field of bioengineering. Background Technology

[0002] Cadaverine, also known as cadaverine, is a key platform compound in the synthesis of bio-based polymer materials. As one of the core monomers of polyamide (nylon) materials, cadaverine can be used to generate polyamide PA5X series polymer materials, which are renewable, low-carbon, and have excellent mechanical properties and chemical stability. Therefore, they have broad commercial demand in fields such as biodegradable materials, textiles, engineering plastics, and automotive lightweighting.

[0003] Currently, pentanediamine is mainly produced through whole-cell catalysis of lysine. Lysine decarboxylase (CadA) plays a crucial role in the decarboxylation of lysine to pentanediamine, being a key enzyme determining the yield and conversion rate. However, the optimal pH for naturally derived CadA is around 6, and its activity is significantly inhibited within the pH range of 7-10. This mismatch with the neutral to alkaline environment commonly encountered in practical catalytic processes limits the efficient synthesis of bio-based pentanediamine. Furthermore, insufficient enzyme activity at high pH conditions can lead to reduced substrate utilization and prolonged reaction cycles, impacting the stability and economics of industrial production. Therefore, developing CadA mutants that maintain high activity and stability in neutral to alkaline environments is a key technological direction for improving the yield and efficiency of bio-based pentanediamine production. Summary of the Invention

[0004] The purpose of this invention is to solve the problem of inhibited enzyme activity of lysine decarboxylase (CadA) under near-neutral or alkaline conditions, and further to solve the problem of low synthesis rate of pentanediamine in neutral or alkaline environments. This invention provides a lysine decarboxylase mutant with optimal pH upregulation, wherein the obtained lysine decarboxylase mutant CadA... S446R / H685E The enzyme activity was 3.3 times that of the wild type at pH 6.0, and 8.3 times, 7.0 times, and 8.6 times that of the wild type at pH 7.0, pH 8.0, and pH 9.0, respectively, effectively increasing the stability of CadA enzyme in neutral and alkaline pH environments. The optimal enzyme activity was achieved with the CadA mutant. S446R / H685E It was applied to the biosynthesis of pentanediamine, increasing the yield of pentanediamine.

[0005] This invention provides a lysine decarboxylase CadA mutant, obtained by mutating wild-type lysine decarboxylase CadA, specifically: CadA S446R CadA S446R / H685E .

[0006] The CadA S446R Amino acid sequence of the enzyme (SEQ ID NO.3): MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTERDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHHNLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKEESKK The nucleotide sequence encoding the CadA S446R enzyme (SEQ ID NO.4): The CadA S446R / H685E enzyme's amino acid sequence (SEQ ID NO.5): MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTERDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHHNLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAEYPGFETDIHGAYRQADGRYTVKVLKEESKK The nucleotide sequence encoding the CadA S446R / H685E enzyme (SEQ ID NO.6): This invention also provides the optimal lysine decarboxylase mutant CadA. S446R CadA S446R / H685E Application in catalyzing the removal of the α-carboxyl group from lysine to generate pentamethylenediamine.

[0007] The present invention also provides a gene encoding the lysine decarboxylase mutant described above or a recombinant vector carrying the gene.

[0008] The present invention also provides recombinant cells expressing the above-mentioned mutants or carrying the above-mentioned genes or the recombinant vectors.

[0009] In one embodiment of the present invention, the recombinant cells are bacteria or fungi as host cells.

[0010] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned lysine decarboxylase mutant, which is any one of the following forms: (1) Cultivate recombinant expression transformants containing the lysine decarboxylase mutant, and isolate transformant cells containing the recombinant lysine decarboxylase mutant enzyme; (2) Culture the recombinant expression transformant containing the lysine decarboxylase mutant, isolate the transformant cells containing the recombinant lysine decarboxylase mutant enzyme, and break the transformant cells containing the recombinant lysine decarboxylase mutant enzyme to obtain the cell lysate. (3) Cultivate recombinant expression transformants containing the lysine decarboxylase mutant, isolate transformant cells containing the recombinant lysine decarboxylase mutant enzyme, break the transformant cells containing the recombinant lysine decarboxylase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant lysine decarboxylase mutant enzyme to obtain lyophilized enzyme powder.

[0011] The present invention also provides a method for improving the enzyme activity of lysine decarboxylase under neutral and alkaline conditions or for increasing the yield of lysine decarboxylase in the preparation of pentanediamine, wherein the method comprises mutating serine at position 446 of lysine decarboxylase as shown in SEQ ID NO.1 to arginine; Alternatively, the serine at position 446 of the lysine decarboxylase, as shown in SEQ ID NO.1, may be mutated to arginine, and the histidine at position 685 may be mutated to glutamic acid.

[0012] The present invention also provides a genetically engineered bacterium that expresses the above-mentioned lysine decarboxylase mutant.

[0013] 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.

[0014] The present invention also provides a method for synthesizing pentanediamine, wherein the method comprises using lysine as a substrate and employing the mutant, the recombinant cell, or the recombinase catalyst, or the genetically engineered bacteria, its lysate, or its fermentation broth as a catalyst, to catalytically convert and synthesize pentanediamine.

[0015] In one embodiment of the present invention, the amount of the genetically engineered bacteria added is 10~20 g / L, and the reaction system also contains L-lysine hydrochloride at a concentration of 500~1000 g / L and coenzyme PLP at a concentration of 0.08~0.12 mmol / L. In one embodiment of the present invention, the reaction conditions are: 35~40 ℃, 200~300 rpm, 10~12 h, and the initial pH is 6.0.

[0016] The present invention also provides a method for synthesizing nylon fibers, the method comprising the following steps: (1) Using lysine as a substrate, the above mutant or the above recombinant cell or the above recombinase catalyst, or the above genetically engineered bacteria or its lysate or its fermentation broth as a catalyst, the reaction is carried out to obtain a reaction solution; (2) Preparation of Nylon 56 Salt Adipic acid was slowly added to the reaction solution to adjust the pH to 6-8. After stirring the reaction, the solution was concentrated and crystallized. The solution was then filtered and dried to obtain the nylon salt. (3) Preparation of nylon fibers PA56 fiber is obtained by spinning nylon 56 salt as raw material.

[0017] In one embodiment of the present invention, the amount of the genetically engineered bacteria added is 10~20 g / L, and the reaction system also contains L-lysine hydrochloride at a concentration of 500~1000 g / L and coenzyme PLP at a concentration of 0.08~0.12 mmol / L. The reaction conditions were: 35-40 °C, 200-300 rpm, 10-12 h, and the initial pH was 6.0.

[0018] In one embodiment of the present invention, the temperature of the stirring reaction is 45-55°C and the reaction time is 1-5 h.

[0019] In one embodiment of the present invention, concentration is carried out by rotary evaporation at 55-65°C until saturation, and crystallization is carried out by cooling at 0-10°C for 10-20 hours.

[0020] The present invention also provides the use of the above-mentioned mutant, recombinant cell, recombinase catalyst, genetically engineered bacteria, or method in the preparation of pentamethylenediamine or products containing pentamethylenediamine.

[0021] Beneficial effects (1) This invention modifies the lysine decarboxylase gene through semi-rational site-directed mutagenesis, wherein the resulting lysine decarboxylase mutant CadA S446R / H685E At pH 6.0, the enzyme activity was 3.3 times that of the wild type, and at pH 7.0, pH 8.0 and pH 9.0, it was 8.3 times, 7.0 times and 8.6 times that of the wild type, respectively, effectively increasing the stability of CadA enzyme in neutral and alkaline pH environments; (2) The optimal pH of the lysine decarboxylase mutant obtained in this invention is upregulated (in the process of preparing pentanediamine, the pH increases rapidly from the initial 6.0. The mutant of this invention can quickly adapt to the increase of pH, so no additional pH adjustment is required during the reaction process), and the enzyme activity stability in neutral and alkaline pH ranges is improved, which is more suitable for the conditions required for industrial microbial catalysis, laying the foundation for efficient synthesis of pentanediamine. Attached Figure Description

[0022] Picture 1 Morphology diagram of PA56 fiber.

[0023] Picture 2 Tensile properties of PA56 fiber. Detailed Implementation

[0024] 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.

[0025] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0026] The detection methods involved in the following embodiments are as follows: The method for detecting lysine decarboxylase activity is as follows: Prepare a 200 µL enzyme activity reaction system according to Table 1, and determine the lysine decarboxylase activity. The reaction system was precisely reacted at 37℃ for 10 min, and the reaction was terminated by adding 20 µL of 40% trichloroacetic acid. The reaction solution was cooled in an ice-water bath. Centrifuged at 12000 r / min for 10 min, and the supernatant was used for derivatization and HPLC analysis to determine the pentanediamine yield. Unit enzyme activity was defined as U / g = 1 mole pentanediamine / min / g protein.

[0027] Table 1: Reaction system for detecting lysine decarboxylase activity

[0028] 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.

[0029] HPLC detection of pentamethylenediamine 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 heptanediamine), mix well, and adjust the pH to 10 with saturated NaOH solution. Then add 1 mL of the derivatization reagent dansyl chloride (5 g / L, dissolved in acetone). Incubate the mixture in a light-protected water bath at 60℃ 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 it for HPLC detection.

[0030] 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.

[0031] Example 1: Preparation of the CadA mutant of lysine decarboxylase Through primers CadA -F and CadA -R from E. coli Lysine decarboxylase gene amplified in the BL21(DE3) genome cadA The enzyme was double-digested and ligated into the pETDuet-1 expression plasmid to construct the recombinant plasmid pETDuet-CadA. CadA was then constructed by performing full-plasmid PCR on the pETDuet-CadA plasmid. H685E CadA S446R CadA S446R / H685EMutant recombinant expression plasmid.

[0032] The specific steps are as follows: Through primers CadA -F and CadA -R from E. coli Lysine decarboxylase gene amplified in the BL21(DE3) genome cadA ,use Nde I and Xho I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. cadA The ligation product was introduced into E. coli JM109 and screened by colony PCR and Sanger sequencing to finally obtain the recombinant plasmid pETDuet-CadA.

[0033] Wild-type lysine decarboxylase CadA amino acid sequence (SEQ ID NO.1): MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHHNLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKEESKK The nucleotide sequence encoding wild-type lysine decarboxylase CadA (SEQ ID NO.2): pETDuet-CadA was subjected to full-plasmid PCR using primers H685E-F and H685E-R. The PCR product was purified and then used. 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 CadA. H685E Mutant recombinant plasmid pETDuet-CadA H685E .

[0034] pETDuet-CadA was subjected to full-plasmid PCR using primers S446R-F and S446R-R. The PCR product was purified and then used. 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 CadA. S446R Mutant recombinant plasmid pETDuet-CadA S446R .

[0035] pETDuet-CadA was stimulated using primers S446R-F and S446R-R. H685E Perform whole-plasmid PCR, and use the PCR products after purification. 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 CadA. S446R / H685E Mutant recombinant plasmid pETDuet-CadA S446R / H685E .

[0036] Table 2: Primer Sequence Listing

[0037] The recombinant plasmids containing the mutants were introduced into Escherichia coli BL21 to prepare recombinant strains expressing the mutants.

[0038] Example 2: Expression of mutant lysine decarboxylase and purification of CadA The specific steps are as follows: The recombinant plasmids expressing the wild-type CadA gene and its mutant gene, prepared in Example 2, were introduced into Escherichia coli BL21 to prepare recombinant strains ( E.Coli BL21 / pETDuet-CadA, E.Coli BL21 / pETDuet-CadA H685E , E.Coli BL21 / pETDuet- CadA S446R , E.ColiBL21 / pETDuet- CadA S446R / H685E This allows CadA and its mutant genes to be expressed in E. coli BL21 with an N-terminal 6×His tag.

[0039] All expression strains were cultured in 500 mL LB medium at 37°C for 3 hours, followed by induction of protein expression by culturing with 0.1 mmol / L IPTG at 30°C for 12 hours. The cultured bacterial 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.

[0040] Example 3: 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., the reaction system containing dithiothreitol, pyridoxal phosphate, lysine, and potassium sodium phosphate buffer, the lysine decarboxylase CadA or mutant purified enzyme solution prepared in Example 3 was added to the reaction system. The final concentration is shown in Table 1. The reaction system was reacted at 37°C for 10 min, and the reaction was terminated by adding 20 µL of 40% trichloroacetic acid. 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 pentanediamine yield. The unit enzyme activity was defined as U / g = 1 mole pentanediamine / min / g protein.

[0041] The results are shown in Table 3 below: Table 3: Enzyme activity under different pH conditions

[0042] The results show: HPLC analysis revealed that the mutant CadA S446R / H685E The enzyme activity was 3.3 times that of the wild type at pH 6.0, and 8.3 times, 7.0 times, and 8.6 times that of the wild type at pH 7.0, pH 8.0, and pH 9.0, respectively, effectively increasing the stability of CadA enzyme in neutral and alkaline pH environments.

[0043] Example 4: Recombinant bacteria containing mutant lysine decarboxylase produce pentamethylenediamine The specific steps are as follows: 1. Preparation of wet mycelium (1) The recombinant pentamethylenediamine strain preserved in glycerol tubes ( E.Coli BL21 / pETDuet-CadA, E.Coli BL21 / pETDuet- CadAH685E , E.Coli BL21 / pETDuet- CadA S446R , E.Coli BL21 / pETDuet-CadA S446R / H685E After streaking and separating the bacteria on LB plates, a single colony was picked and inoculated into 20 mL of LB medium. The culture was then carried out at 37°C and 250 r / min for 12 h to activate the bacterial strain and prepare the seed culture.

[0044] (2) The prepared seed culture was inoculated into 100 mL LB liquid medium at a 2% (v / v) inoculation rate and cultured at 37°C for 2.5 h. Then, 0.1 mmol / L IPTG was added for induction. cadA Gene expression was performed, and the mixture was cultured at 30°C for 16 h to prepare the fermentation broth.

[0045] (3) Centrifuge the prepared fermentation broth at 4℃ and 5000 rpm for 15 min, and take the precipitate as wet cells.

[0046] 2. Whole-cell preparation of pentamethylenediamine Whole-cell catalysis was conducted in a 5 L bioreactor (working volume 2 L). The reaction system consisted of the following: L-lysine hydrochloride was dissolved in 100 mM PBS buffer (pH 6.0), and the substrate was added to the reactor in two equal batches at 0 h and 2 h, respectively. The final total substrate concentration in the reaction system was 639 g / L; the wet cell concentration was 13 g / L; and the coenzyme PLP concentration was 0.1 mmol / L. The entire catalytic system was carried out in a 5 L bioreactor at 37 °C, 200 rpm, and an initial pH of 6.0 (no pH adjustment was required during the reaction). The reaction was conducted in a closed system for 10 h.

[0047] Samples were taken every hour (from 0 to 10 hours) and centrifuged at 10,000 g for 1 minute to remove cells. Finally, the amount of pentanediamine produced was determined by high-performance liquid chromatography (HPLC).

[0048] The results are shown in Table 4 below: Table 4: Pentanediamine yield of different strains

[0049] The results showed that using the combined mutant CadA S446R / H685E The recombinant strain was reacted in a 5-liter bioreactor for 10 h, achieving a yield of 352.7 g / L of pentanediamine.

[0050] Example 5: A method for synthesizing nylon fibers 1. Synthesis of Nylon Fibers The method includes the following steps: (1) The concentration of L-lysine hydrochloride was 585 g / L (dissolved in 100 mM PBS buffer, pH 6.0), the final wet bacterial concentration was 13 g / L, and the coenzyme PLP concentration was 0.1 mmol / L.

[0051] The entire catalytic system was carried out in a 5 L bioreactor at 37 °C, 200 rpm, and an initial pH of 6.0 (no pH adjustment was required during the reaction). The reaction was carried out in a closed system for 10 h to obtain the reaction solution. (2) Preparation of Nylon 56 Salt The reaction solution was centrifuged, and the supernatant was collected. The supernatant was heated to 50°C, and activated carbon was added at 15% of the mass of pentanediamine in the reaction solution (liquid phase detection). Adipic acid was gradually and slowly added to adjust the pH until it reached 7, at which point no more adipic acid was added. The mixture was stirred at 50°C and 200 rpm for 2 hours, then concentrated to saturation by rotary evaporation at 60°C. The solution was then cooled and crystallized at 4°C for 12 hours. After the crystals were fully separated, they were filtered and dried in a vacuum drying oven at 65°C to obtain nylon 56 salt.

[0052] (3) Preparation of nylon fibers PA56 was successfully prepared by solid-state polycondensation to achieve molecular chain growth using nylon 56 salt as raw material, and PA56 fibers were obtained by melt spinning. The specific steps are as follows: Solid-phase polycondensation process: 3 kg of dried nylon 56 salt and 30 g of sodium hypophosphite were added to the polycondensation reactor. After three cycles of evacuation and nitrogen purging, the reactor was sealed at normal pressure, and stirring was started to raise the temperature. After condensation water was generated, the pressure increased. When the pressure reached 2 MPa, the temperature was held for 30 minutes, then slowly evacuated for 10 minutes to reduce the pressure to normal. Immediately afterwards, a vacuum was drawn until the pressure was below 10 kPa. The temperature was held for another 3 hours, then nitrogen purging was applied, and the mixture was cooled to below 70°C before being discharged and finely ground. The resulting product is PA56. Picture 1 ).

[0053] Melt spinning process: PA56 was dried in a forced-air drying oven at 80℃ for 6 hours. The dried material was then fed into a single-screw extruder with extruder heating temperatures of 150℃, 200℃, 260℃, and 220℃, a screw speed of 440 r / min, a screw diameter of 65 mm, and a length-to-diameter ratio of 36:1. After melting, the material was fed into the spinning section and extruded through a spinneret. It was then rapidly drawn into nylon 56 fibers by a winding device. The spinneret had 24 concentrically arranged, equally spaced holes with an outer diameter of 50 mm and a winding speed of 2000 m / min.

[0054] Nylon fibers were prepared.

[0055] 2. The tensile properties of PA56 fiber were tested according to the national standard (GB / T 1040.1-2025). The results are as follows Picture 2 As shown.

[0056] 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, characterized in that, The genetically engineered bacteria expressed a lysine decarboxylase mutant; the lysine decarboxylase mutant was obtained by mutating serine at position 446 of the lysine decarboxylase as shown in SEQ ID NO.1 to arginine; or by mutating serine at position 446 of the lysine decarboxylase as shown in SEQ ID NO.1 to arginine, and simultaneously mutating histidine at position 685 to glutamic acid; Preferably, the genetically engineered bacteria include, but are not limited to, genetically engineered Escherichia coli, genetically engineered Bacillus subtilis, genetically engineered Corynebacterium glutamicum, and genetically engineered yeast.

2. A recombinant enzyme catalyst containing a lysine decarboxylase mutant, characterized in that, The lysine decarboxylase mutant is obtained by mutating serine at position 446 of the lysine decarboxylase shown in SEQ ID NO.1 to arginine; or by mutating serine at position 446 of the lysine decarboxylase shown in SEQ ID NO.1 to arginine, and simultaneously mutating histidine at position 685 to glutamic acid; the recombinase catalyst is any one of the following forms: (1) Cultivate recombinant expression transformants containing the lysine decarboxylase mutant, and isolate transformant cells containing the recombinant lysine decarboxylase mutant enzyme; (2) Culture the recombinant expression transformant containing the lysine decarboxylase mutant, isolate the transformant cells containing the recombinant lysine decarboxylase mutant enzyme, and break the transformant cells containing the recombinant lysine decarboxylase mutant enzyme to obtain the cell lysate. (3) Cultivate recombinant expression transformants containing the lysine decarboxylase mutant, isolate transformant cells containing the recombinant lysine decarboxylase mutant enzyme, break the transformant cells containing the recombinant lysine decarboxylase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant lysine decarboxylase mutant enzyme to obtain lyophilized enzyme powder.

3. A lysine decarboxylase mutant, characterized in that, The lysine decarboxylase mutant is obtained by mutating serine at position 446 of the lysine decarboxylase shown in SEQ ID NO.1 to arginine; or by mutating serine at position 446 of the lysine decarboxylase shown in SEQ ID NO.1 to arginine, and simultaneously mutating histidine at position 685 to glutamic acid.

4. A gene encoding the lysine decarboxylase mutant of claim 3.

5. A recombinant vector carrying the gene of claim 4.

6. A recombinant cell expressing the lysine decarboxylase mutant of claim 3 or carrying the gene of claim 4 or the recombinant vector of claim 5, preferably, the recombinant cell is a bacterial or fungal host cell.

7. A method for improving the enzyme activity of lysine decarboxylase under neutral and alkaline conditions or for increasing the yield of lysine decarboxylase in the preparation of pentanediamine, characterized in that, The method involves mutating serine at position 446 of the lysine decarboxylase shown in SEQ ID NO.1 to arginine; or mutating serine at position 446 of the lysine decarboxylase shown in SEQ ID NO.1 to arginine, while simultaneously mutating histidine at position 685 to glutamic acid.

8. A method for synthesizing pentamethylenediamine, characterized in that, The method is to use lysine as a substrate, and employ the lysine decarboxylase mutant of claim 3, the recombinant cell of claim 6, or the recombinant enzyme catalyst of claim 2, or the genetically engineered bacteria of claim 1, or its lysate or fermentation broth, as a catalyst to catalytically convert and synthesize pentanediamine. Preferably, the amount of the genetically engineered bacteria added is 10~20 g / L, and the reaction system also contains L-lysine hydrochloride at a concentration of 500~1000 g / L and coenzyme PLP at a concentration of 0.08~0.12 mmol / L. Preferably, the reaction conditions are: 35~40 ℃, 200~300 rpm, 10~12 h, and the initial pH is 6.

0.

9. A method for synthesizing nylon fibers, characterized in that, The method includes the following steps: (1) Using lysine as a substrate, the reaction is carried out using the lysine decarboxylase mutant of claim 3, the recombinant cell of claim 6, or the recombinase catalyst of claim 2, or the genetically engineered bacteria of claim 1, its lysate, or its fermentation broth as a catalyst, to obtain a reaction solution; preferably, the amount of the genetically engineered bacteria added is 10~20 g / L, and the reaction system also contains L-lysine hydrochloride at a concentration of 500~1000 g / L and coenzyme PLP at 0.08~0.12 mmol / L; The reaction conditions were: 35-40 °C, 200-300 rpm, 10-12 h, and the initial pH was 6.

0. (2) Preparation of Nylon 56 salt Adipic acid is slowly added to the reaction solution to adjust the pH to 6-8. After stirring the reaction, the solution is concentrated and crystallized. The solution is then filtered and dried to obtain the nylon salt. Preferably, the temperature of the stirring reaction is 45-55°C and the reaction time is 1-5 h. Preferably, the concentration is carried out by rotary evaporation at 55-65°C until saturation, and the crystallization is carried out by cooling the solution at 0-10°C for 10-20 h. (3) Preparation of nylon fibers PA56 fiber is obtained by spinning nylon 56 salt as raw material.

10. The lysine decarboxylase mutant of claim 3, the recombinant cell of claim 6, or the recombinase catalyst of claim 2, or the genetically engineered bacteria of claim 1, used in the preparation of pentanediamine or products containing pentanediamine.

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