Process for the preparation and purification of butylenediamine

Abstract

This invention discloses a method for preparing and purifying butanediamine, belonging to the field of bioengineering. This invention provides a novel arginine decarboxylase mutant with a specific enzyme activity of 47.1 U / g under alkaline conditions, significantly higher than the original wild-type enzyme (3 U / g). This invention systematically optimizes the purification process, successfully developing a novel purification flow consisting of three key steps: desalting, extraction, and rotary evaporation. Verification showed that the purified butanediamine product, as detected by high-performance liquid chromatography, achieved a purity of 99.5% and a yield of 89.3%. This invention achieves efficient separation and purification of high-purity butanediamine, laying a solid material foundation and providing technical support for its subsequent use in the synthesis of high-performance nylon products such as PA46, PA410, and PA4T.

Description

Technical Field

[0001] This invention relates to a method for preparing and purifying butanediamine, belonging to the field of bioengineering. Background Technology

[0002] Butylene diamine (Putrescine), chemically known as 1,4-butanediamine, also widely called putrescine, belongs to the amino acid derivative category. As a molecule playing a crucial role in life processes, 1,4-butanediamine participates in and regulates cellular metabolic processes in various organisms, playing a vital role in maintaining normal physiological functions. Simultaneously, 1,4-butanediamine exhibits extremely high application value in the industrial field, particularly in engineering plastics, pharmaceutical synthesis, agrochemical manufacturing, and surfactant production. One of its primary uses is as an important precursor in the synthesis of polyamide materials, enabling the production of various high-performance polyamide products, including PA46, PA410, and PA4T. These materials, due to their excellent mechanical properties and heat resistance, have been widely used in many key areas such as the textile industry, machinery and chemical industry, electronics and electrical appliance manufacturing, and the automotive industry.

[0003] Currently, the production of butanediamine still mainly relies on traditional chemical synthesis methods. These methods typically use toxic chemicals as raw materials, and the entire production process faces a series of technical challenges, such as harsh reaction conditions, extremely high requirements for equipment corrosion resistance and high pressure resistance, the need for expensive catalysts, high hydrogen operating pressure, and high overall energy consumption. These factors collectively constitute high technical barriers in the butanediamine synthesis process, limiting its large-scale production and application expansion. Therefore, in recent years, new methods utilizing synthetic biology techniques to directly convert renewable biomass raw materials into butanediamine by modifying microbial cells or constructing efficient enzyme catalytic systems have attracted increasing attention. This bio-manufacturing route not only effectively avoids many technical bottlenecks in traditional chemical processes but also exhibits significant environmentally friendly characteristics, such as reducing toxic waste emissions and energy consumption, and possesses potential economic advantages, thus providing an important and promising solution for the green and sustainable production of butanediamine. Summary of the Invention

[0004] [Technical Solution]

[0005] The first objective of this invention is to provide an arginine decarboxylase mutant, which is obtained by simultaneously mutating glutamic acid at position 482 to arginine, histidine at position 749 to aspartic acid, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid, based on the amino acid sequence shown in SEQ ID NO.1.

[0006] A second object of the present invention is to provide a vector encoding the gene of the arginine decarboxylase mutant.

[0007] A third object of the present invention is to provide recombinant cells expressing the arginine decarboxylase mutant or the vector.

[0008] Preferably, the recombinant cells use bacteria or fungi as host cells.

[0009] A fourth objective of this invention is to provide a recombinant strain expressing the arginine decarboxylase mutant, arginine decarboxylase SpeA, and guanidine aminoase SpeB.

[0010] In one embodiment of the present invention, the recombinant strain uses Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or yeast as the host.

[0011] In one embodiment of the present invention, the amino acid sequence of the guanidine amino acid enzyme SpeB is shown in SEQ ID NO.3, and the amino acid sequence of the arginine decarboxylase SpeA is shown in SEQ ID NO.4.

[0012] A fifth object of the present invention is to provide a whole-cell catalyst containing the recombinant strain.

[0013] The sixth objective of this invention is to provide a method for the continuous catalytic synthesis of butanediamine, wherein butanediamine is prepared by using arginine as a substrate and catalyzing a reaction with the arginine decarboxylase mutant, the recombinant strain, or the whole-cell catalyst.

[0014] In one embodiment of the present invention, the method is as follows:

[0015] (1) The recombinant strain or the whole-cell catalyst is fermented to obtain a fermentation broth;

[0016] (2) Let the fermentation broth stand for 20-30 minutes. After the cells settle naturally, remove the supernatant and add the substrate arginine directly to catalyze the reaction to synthesize butanediamine.

[0017] (3) After the catalytic reaction is complete, repeat step (2) at least once and collect and combine all the fermentation broth.

[0018] In one embodiment of the present invention, a CO2 atmosphere is maintained throughout the reaction.

[0019] In one embodiment of the present invention, the CO2 atmosphere is continuously introduced at a flow rate of 0.5~2 vvm.

[0020] In one embodiment of the present invention, the amount of arginine added is 200 g / L to 400 g / L.

[0021] In one embodiment of the present invention, the reaction conditions are a temperature of 35~45℃, a rotation speed of 200~400 r / min, and an initial pH of 7.0.

[0022] In one embodiment of the present invention, the fermentation broth is purified.

[0023] In one embodiment of the present invention, the purification step is as follows:

[0024] (1) Adjust the pH of the fermentation broth to 9.5~13.5, and react at 40~100℃ and 100~300 rpm for 1~7 h;

[0025] (2) Filtration, and extract butanediamine from the filtrate using n-butanol.

[0026] In one embodiment of the present invention, the extraction conditions are 25~75℃, 100~300 rpm for 1~5 hours.

[0027] In one embodiment of the present invention, the volume ratio of n-butanol to filtrate is (2~7):5.

[0028] Preferably, the pH of the fermentation broth is adjusted to 13.5, and the reaction is carried out at 80°C and 200 rpm for 5 h.

[0029] Preferably, the extraction conditions are 65°C, 100~300 rpm for 1 h.

[0030] Preferably, the volume ratio of n-butanol to filtrate is 5:5.

[0031] The seventh objective of this invention is to provide a method for improving the activity of arginine decarboxylase, wherein the method involves mutating glutamic acid at position 482 of the arginine decarboxylase as shown in SEQ ID NO.1 to arginine, histidine at position 749 to aspartic acid, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid.

[0032] An eighth object of the present invention is to provide the use of the arginine decarboxylase mutant, or the vector, or the recombinant cell, or the recombinant strain, or the whole-cell catalyst in the preparation of PA46, PA410, and PA4T.

[0033] [Beneficial Effects]

[0034] (1) This invention provides a novel arginine decarboxylase mutant, which exhibits the highest specific enzyme activity (47.1 U / g) under alkaline conditions (pH 7.0), significantly improving catalytic performance compared to the original wild-type enzyme (specific enzyme activity of only 3 U / g). This mutant effectively improves the enzyme's catalytic efficiency and has significant potential for industrial applications.

[0035] (2) Regarding the post-processing of the fermentation broth, this invention systematically optimized the purification process and successfully developed a novel purification flow consisting of three key steps: desalting, extraction, and rotary evaporation. Verification showed that the purified butanediamine product, as detected by high-performance liquid chromatography (HPLC), achieved a purity of 99.5% and a yield of 89.3%. This invention achieves efficient separation and purification of high-purity butanediamine, laying a solid material foundation and providing technical support for its subsequent use in the synthesis of high-performance nylon products such as PA46, PA410, and PA4T. Detailed Implementation

[0036] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0037] 1. The culture medium involved in the following examples

[0038] LB solid culture: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 2 g / L agar powder.

[0039] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0040] SOB medium: 5 g / L yeast extract, 20 g / L peptone, 0.5 g / L sodium chloride, 0.95 g / L MgCl2 and 0.186 g / L KCl.

[0041] 2. The biomaterials involved in the following examples

[0042] The plasmids pETDuet-adiA, pETDuet-SpeB, and pETDuet-speA-speB are disclosed in the application document with publication number CN117535274A.

[0043] 3. The detection methods involved in the following embodiments

[0044] (1) Method for detecting arginine decarboxylase activity:

[0045] Prepare a 200 µL enzyme activity reaction system according to Table 1, and determine the arginine decarboxylase activity. The reaction system was incubated at 37℃ 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. After centrifugation at 12000 r / min for 10 min, the supernatant was collected, derivatized, and used for HPLC determination of butanediamine yield. The enzyme activity unit is defined as U·g. -1 = 1 mole of butanediamine / min·g -1 protein.

[0046] The 15 mmol / L potassium sodium phosphate buffer solution was prepared as follows: the pH of 15 mmol / L KH2PO4 was adjusted to 7.0 using 15 mmol / L Na2HPO4.

[0047] Table 1. Reaction system for detecting arginine decarboxylase activity

[0048]

[0049] (2) HPLC detection of butanediamine

[0050] 1) Sample derivatization and extraction

[0051] The pre-column derivatization procedure for dansyl chloride was as follows: After centrifuging the fermentation broth at 12000 r / min for 10 min, 500 μL of the supernatant was taken as the sample. 500 μL of saturated NaHCO3 solution and an internal standard (5 μL of 10 g / L heptamethylenediamine) were added and mixed thoroughly. The pH was adjusted to 10 with saturated NaOH solution, followed by the addition of 1 mL of the derivatization reagent dansyl chloride (5 g / L, soluble in acetone). The mixture was incubated in a light-protected water bath at 60℃ for 30 min. Extraction was performed for 10 min with 2 mL of anhydrous diethyl ether, and the upper organic phase was collected. This extraction process was repeated twice. The two organic phases were mixed and dried using a nitrogen evaporator to remove the diethyl ether. The derivatized compound was dissolved in 500 μL of acetonitrile solution, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC.

[0052] 2) HPLC chromatographic determination

[0053] Chromatographic conditions:

[0054] 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.

[0055] Example 1 Preparation of Arginine Decarboxylase AdiA Mutant

[0056] The amino acid sequence of the wild-type arginine decarboxylase AdiA is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. Using the recombinant plasmid pETDuet-adiA as a template, primers containing the mutation site were designed (Table 2), and full-plasmid PCR was performed on the pETDuet-adiA plasmid using the corresponding primers in Table 2 to construct the mutant recombinant expression plasmid.

[0057] pETDuet-adiA E482R Using recombinant plasmid pETDuet-adiA as a template, primers were used... adiA Full plasmid PCR was performed using E482-F / R, and the PCR product was transformed into E. coli JM109. After overnight culture, colonies were picked for PCR and sequencing verification, finally yielding the mutant recombinant plasmid pETDuet-adiA. E482R .

[0058] pETDuet-adiA H749D Using recombinant plasmid pETDuet-adiA as a template, primers were used... adiA Full plasmid PCR was performed using the -H749D-F / R strain. The PCR product was transformed into E. coli JM109, cultured overnight, and colonies were picked for PCR and sequencing verification. The mutant recombinant plasmid pETDuet-adiA was finally obtained. H749D .

[0059] pETDuet-adiA E482R-H749D Using the single mutant plasmid pETDuet-adiA E482R Using a template, through primers adiA Full plasmid PCR was performed using the -H749D-F / R strain. The PCR product was transformed into E. coli JM109, cultured overnight, and colonies were picked for PCR and sequencing verification. The mutant recombinant plasmid pETDuet-adiA was finally obtained.E482R-H749D .

[0060] pETDuet-adiA E482R-H749D-E467K-H736E : Using recombinant plasmid pETDuet-adiA E482R-H749D Using a template, through primers adiA -H736E-F / R adiA The -E467K-F / R plasmid was subjected to full plasmid PCR sequentially. The PCR product was transformed into E. coli JM109, cultured overnight, and colonies were picked for PCR and sequencing verification. Finally, the mutant recombinant plasmid pETDuet-adiA was obtained. E482R-H749D-E467K-H736E .

[0061] Table 2 Primer sequences involved in the examples

[0062]

[0063] Example 2 Expression of mutant arginine decarboxylase

[0064] pETDuet-adiA, pETDuet-SpeB, pETDuet-speA-speB, and the mutant plasmid constructed in Example 1 were transformed into Escherichia coli BL21(DE3) to obtain recombinant bacteria carrying guanidine amino acid enzyme SpeB, arginine decarboxylase AdiA, or arginine decarboxylase AdiA mutants, respectively.

[0065] All recombinant bacteria were first inoculated into 20 mL of LB liquid medium at 50 μL and cultured overnight at 37°C and 250 r / min as seed culture. Then, the seed culture was inoculated into 500 mL of SOB medium at a 1% (v / v) inoculation rate and cultured at 37°C for 3 h. IPTG was then added to a final concentration of 0.1 mmol / L, and the culture was incubated overnight 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 of Tris-HCl buffer, and then sonicated. The supernatant was purified by affinity chromatography using a Ni-NTA Superflow resin column.

[0066] Pure enzyme solutions of wild-type AdiA, guanidine amino acid enzyme SpeB, and various mutants were prepared.

[0067] According to the reaction system in Table 1, that is, to the reaction system containing dithiothreitol, EDTA, pyridoxal phosphate (PLP), MgSO4, arginine, and guanidine amino acid enzyme SpeB pure enzyme solution in potassium sodium phosphate buffer, add arginine decarboxylase AdiA or mutant pure enzyme solution, and the final concentration is shown in Table 1.

[0068] The results showed that the enzyme activity detection results of each mutant were different for WT and AdiA. E482R AdiA H749D AdiA E482R-H749D AdiA E482R-H749D-E467K-H736E The enzyme activities were 3 U / g, 11.5 U / g, 12.8 U / g, 31.5 U / g, and 47.1 U / g, respectively.

[0069] Example 3: Production of Butanediamine by Recombinant Bacteria Containing Mutant Arginine Decarboxylase

[0070] (1) Construction of recombinant bacteria

[0071] Through primers adiA -F and adiA -R (Table 2) Amplified the mutant gene from the mutant constructed in Example 1, and ligated the mutant gene into the recombinant plasmid pETDuet-speA-speB expressing guanidine amino acid enzyme SpeB and arginine decarboxylase SpeA, to obtain the mutant-containing recombinant plasmid for the preparation of butanediamine:

[0072] Through primers adiA -F and adiA -R is the mutant pETDuet-adiA constructed from Example 1. E482R The mutant gene E482R was amplified in the middle, using Nde I and Xho The PCR product and the pETDuet-speA-speB plasmid were double-digested. After purification, the mutant gene was ligated into pETDuet-speA-speB using T4 DNA ligase. The ligation product was introduced into E. coli JM109, and screening was performed by colony PCR and Sanger sequencing to obtain the recombinant plasmid pETDuet-speA-adiA. E482R -speB. The same method was used to prepare the recombinant plasmid pETDuet-speA-adiA-speB expressing the wild type and the recombinant plasmid pETDuet-speA-adiA expressing the mutant. H749D -speB、pETDuet-speA-adiA E482R-H749D -speB、pETDuet-speA-adiA E482R-H749D-E467K-H736E -speB.

[0073] The recombinant plasmid pETDuet-speA-adiA E482R -speB conversion to E. coli BL21(DE3) was used to obtain a recombinant strain for butanediamine synthesis. E. coli BL21(DE3) speA-E482R-speB .

[0074] Recombinant strains containing wild-type adiA were obtained using the same method. E. coli BL21(DE3) speA-adiA-speB and recombinant strains containing mutants E. coli BL21(DE3) speA-H749D-speB , E. coli BL21(DE3) speA-E482R-H749D-speB , E. coli BL21(DE3) speA-E482R-H749D-E467K-H736E-speB .

[0075] (2) Recombinant bacterial culture

[0076] The butanediamine recombinant strain preserved in the glycerol tube in step (1) E. coli BL21(DE3) speA-E482R-speB , E. coli BL21(DE3) speA-H749D-speB , E. coli BL21(DE3) speA-E482R-H749D-speB , E. coli BL21(DE3) speA-E482R-H749D-E467K-H736E-speB After streaking and isolating the bacteria on LB plates, a single colony was picked and inoculated into 50 mL of LB liquid medium and cultured overnight (12-16 h) at 37°C and 200 r / min to obtain the primary seed culture.

[0077] The primary seed culture was inoculated into 1 L LB liquid medium at an inoculation rate of 2% (v / v), and ampicillin was added to a final concentration of 100 mg / L. The medium was then placed in a constant temperature shaker at 37℃ and cultured at 250 r / min for 2.5 h. After cooling to 30℃, 0.1 mmol / L IPTG was added to induce expression for 16 h to prepare the fermentation broth.

[0078] 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.

[0079] (3) Whole-cell catalytic synthesis of butanediamine

[0080] 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.

[0081] Reaction system: In a 5 L fermenter, the working volume was 3 L, including: arginine total concentration 348.4 g / L (added in batches), 0.1 mmol / L PLP, and finally, the volume was increased to 3 L with PBS buffer (pH 7.0) to achieve cell OD. 600 Approximately 80.

[0082] Reaction conditions: catalytic temperature 42℃, stirring speed 300 r / min, continuous CO2 gas flow (flow rate 1 vvm), reaction time 12 h.

[0083] The prepared fermentation broth was allowed to stand for 30 minutes until it naturally settled to the bottom of the fermenter. Then, the supernatant was extracted and 2.7 L of fresh substrate was added directly to carry out the reaction.

[0084] The catalysis was completed when the arginine in the catalytic system was exhausted. Stirring was stopped, and the cells in the fermenter settled naturally within 30 minutes during the settling process. Subsequently, 2.7 L of supernatant was slowly released through the drain valve on the side wall of the tank, leaving only 300 mL of concentrated cell solution. The remaining cells in the fermenter participated in the second round of catalytic reaction.

[0085] Adding 2.7 L of fresh substrate to a 5L fermenter still maintains the cell OD. 600 The concentration was set to 80, and the cells underwent a second catalytic reaction until the arginine in the catalytic system was depleted. Catalysis was then complete, stirring was stopped, and the cells in the fermenter settled naturally within 30 minutes. Subsequently, the upper layer of reaction liquid was drained, and the remaining cells in the tank participated in the third round of catalytic reaction.

[0086] Table 3. Yield of Butanediamine

[0087]

[0088] Example 5: Separation and purification process of butanediamine

[0089] Collect and combine the recombinant bacteria from Example 4 E. coli BL21(DE3) speA-E482R-H749D-E467K-H736E-speB The reaction solution obtained after three rounds of catalytic reaction was used for the separation and purification of butanediamine. The specific experimental steps are as follows:

[0090] Take the recombinant bacteria from Example 4 E. coli BL21(DE3) speA-E482R-H749D-E467K-H736E-speBThe fermentation broth was adjusted to pH > 9.0 with NaOH solution and reacted at 80℃ and 200 rpm for 5 h. After the reaction, the solid precipitate was removed by filtration and the filtrate was collected. The obtained filtrate was extracted with n-butanol at a volume ratio of 2:5 (n-butanol: filtrate) at 40℃ and 200 rpm for 1 h. After standing and separation, the concentrations of butanediamine in the organic phase and aqueous phase were measured, and the extraction rate was calculated according to formula (1):

[0091] Extraction rate =

[0092] Where m1 refers to the mass (g) of butanediamine in the organic phase; and m2 refers to the mass (g) of butanediamine in the aqueous phase.

[0093] (1) Optimization of desalination pH:

[0094] Three 100 mL aliquots of fermentation broth were taken, and the pH was adjusted to 9.5, 11.5, and 13.5 respectively using NaOH solution. The mixtures were reacted at 80℃ and 200 rpm for 5 h. After the reaction, the mixture was filtered to remove the solid precipitate, and the filtrate was collected. The resulting filtrate was extracted with n-butanol at a volume ratio of 2:5 (n-butanol:filtrate) at 40℃ and 200 rpm for 1 h. After standing and separating the layers, the concentrations of butanediamine in the organic and aqueous phases were measured, and the extraction rate was calculated.

[0095] (2) Optimization of desalination temperature:

[0096] Four 100 mL aliquots of fermentation broth were taken, and the pH was adjusted to 13.5. Each aliquot was then subjected to a water bath at different temperatures (40℃, 60℃, 80℃, and 100℃) at 200 rpm for 5 h. After the reaction, the aliquots were filtered to remove the solid precipitate, and the filtrate was collected. The filtrate was then extracted with n-butanol at a volume ratio of 2:5 (n-butanol:filtrate) at 55℃ and 200 rpm for 1 h. After standing and separation, the concentrations of butanediamine in the organic and aqueous phases were measured, and the extraction rate was calculated.

[0097] (3) Optimization of desalination time:

[0098] Four 100 mL aliquots of fermentation broth were taken, the pH was adjusted to 13.5, and the mixtures were reacted at 80 °C and 200 rpm for 1 h, 3 h, 5 h, and 7 h, respectively. After the reaction was completed, the mixture was filtered to remove the solid precipitate, and the filtrate was collected. The resulting filtrate was extracted with n-butanol at a volume ratio of 2:5 (n-butanol:filtrate) at 55 °C and 200 rpm for 1 h. After standing and separating the layers, the concentrations of butanediamine in the organic and aqueous phases were measured, and the extraction rate was calculated.

[0099] (4) Optimization of extraction temperature:

[0100] The pH of the fermentation broth was adjusted to 13.5, and the reaction was carried out at 80℃ and 200 rpm for 5 h, respectively. After the reaction, the mixture was filtered to remove the solid precipitate, and the filtrate was collected. Subsequently, four 100 mL aliquots of the filtrate were taken and extracted at different temperatures (25℃, 45℃, 65℃, and 75℃) using n-butanol at a volume ratio of 2:5 (n-butanol:filtrate) at 200 rpm for 1 h. After standing and separation, the concentration of butanediamine in the organic and aqueous phases was measured, and the extraction rate was calculated.

[0101] (5) Optimization of extraction time:

[0102] Take 100 mL of each of the six fermentation broths, adjust the pH to 13.5, and react at 80℃ and 200 rpm for 5 h. After the reaction, filter to remove the solid precipitate and collect the filtrate. Extract the filtrate with n-butanol at a volume ratio of 2:5 (n-butanol:filtrate) at 65℃ and 200 rpm for 0.5 h, 1 h, 2 h, 3 h, 4 h, and 5 h, respectively. After standing and separating the layers, determine the concentration of butanediamine in the organic and aqueous phases and calculate the extraction rate.

[0103] (6) Optimization of extraction ratio:

[0104] Take 100 mL of each of the six fermentation broths prepared in Example 4, adjust the pH to 13.5, and react at 80℃ and 200 rpm for 5 h. After the reaction, filter to remove solid precipitate and collect the filtrate. Extract the filtrate with n-butanol at 65℃ and 200 rpm for 1 h at volume ratios of 2:5, 3:5, 4:5, 5:5, 6:5, and 7:5 (n-butanol:filtrate), respectively. After standing and separating the layers, determine the concentration of butanediamine in the organic and aqueous phases and calculate the extraction rate.

[0105] A "desalting-extraction-rotary evaporation" process was developed, and the optimal purification conditions (pH 13.5, 80℃, 5 h) and extraction conditions (65℃, 1 h, n-butanol extraction ratio 5:5) were determined. The butanediamine sample obtained after rotary evaporation had a purity of 99.5% and a yield of 89.3% as determined by liquid chromatography. The separation and purification of high-purity butanediamine was successfully achieved, laying the foundation for the subsequent production of nylon 46.

[0106] 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. An arginine decarboxylase mutant, characterized in that, The mutant was obtained by simultaneously mutating glutamic acid at position 482 to arginine, histidine at position 749 to aspartic acid, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid, based on the amino acid sequence shown in SEQ ID NO.

1.

2. A vector carrying the gene encoding the arginine decarboxylase mutant of claim 1.

3. Recombinant cells expressing the arginine decarboxylase mutant of claim 1 or the vector of claim 2.

4. A recombinant bacterial strain, characterized in that, The arginine decarboxylase mutant, arginine decarboxylase SpeA, and guanidine amino acid enzyme SpeB as described in claim 1 are expressed; the amino acid sequence of guanidine amino acid enzyme SpeB is shown in SEQ ID NO.3, and the amino acid sequence of arginine decarboxylase SpeA is shown in SEQ ID NO.

4.

5. A whole-cell catalyst, characterized in that, It contains the recombinant strain described in claim 4.

6. A method for producing butanediamine, characterized in that, The method involves preparing butanediamine using arginine as a substrate and catalyzing a reaction with the recombinant strain described in claim 4 or the whole-cell catalyst described in claim 5. The method is as follows: (1) The recombinant strain of claim 4 or the whole-cell catalyst of claim 5 is fermented to obtain a fermentation broth; (2) Let the fermentation broth stand for 20-30 min. After the cells settle naturally, remove the supernatant, add the substrate arginine, and carry out the catalytic reaction to synthesize butanediamine. (3) After the catalytic reaction is complete, repeat step (2) at least once and collect and combine all the fermentation broth.

7. The method according to claim 6, characterized in that, The fermentation broth was purified.

8. The method according to claim 7, characterized in that, The purification steps are as follows: (1) Adjust the pH of the fermentation broth to 9.5~13.5, and react at 40~100℃ and 100~300 rpm for 1~7 h; (2) Filtration, and extraction of butanediamine from the filtrate using n-butanol; The extraction conditions are 25~75℃, 100~300 rpm for 1~5 h; The volume ratio of n-butanol to filtrate is (2~7):

5.

9. A method for increasing the activity of arginine decarboxylase, characterized in that, The method involves mutating glutamic acid at position 482 of the arginine decarboxylase shown in SEQ ID NO.1 to arginine, histidine at position 749 to aspartic acid, glutamic acid at position 467 to lysine, and histidine at position 736 to glutamic acid.

10. The arginine decarboxylase mutant of claim 1, or the vector of claim 2, or the recombinant cell of claim 3, or the recombinant strain of claim 4, or the whole-cell catalyst of claim 5, or the method of any one of claims 6 to 8, used in the preparation of butanediamine or products containing butanediamine or in the preparation of nylon products with butanediamine as a precursor; wherein the nylon products include PA46, PA410, and PA4T.

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