Lysine decarboxylase mutant and application thereof in synthesis of pentamethylene diamine

Abstract

The invention discloses a high-efficiency lysine decarboxylase mutant and an application of the high-efficiency lysine decarboxylase mutant in synthesis of pentamethylene diamine. A series of mutants including a single mutant (such as N218G, T222V, C244P and the like), a double mutant and a three mutant are obtained by carrying out multi-site mutation on wild type lysine decarboxylase (SEQ ID NO.2) from Klebsiella grimontii. The catalytic efficiency of the mutant is remarkably improved, and the activity of the mutant is improved by 5.0-10.2 times compared with that of a wild type enzyme. According to the method disclosed by the invention, the substrate conversion rate of 99% or above can be realized within 2 hours through an optimized process of constructing an expression vector and a genetically engineered bacterium, inducing expression of lysine decarboxylase and synthesizing 1, 5-pentanediamine by utilizing a whole-cell catalysis technology. Compared with a traditional chemical method and a natural enzyme method, the method disclosed by the invention has the advantages of simplified process, high catalytic efficiency, low production cost and the like, and is suitable for producing bio-based 1, 5-pentamethylene diamine and nylon 56 salt.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to lysine decarboxylase mutants and their application in the synthesis of pentanediamine. Background Technology

[0002] Lysine decarboxylase (LDC) is a key enzyme catalyzing the decarboxylation of L-lysine to produce 1,5-pentanediamine (cadaverine). The reaction uses pyridoxal phosphate (PLP) as a coenzyme, and the product, 1,5-pentanediamine, serves as the core monomer of bio-based polyamides, holding significant application value in the synthesis of high-performance materials such as Nylon 56 and Nylon 510. Compared to traditional petroleum-based nylons, bio-based nylon 5X not only possesses lightweight, high hygroscopicity, and excellent mechanical properties, but also reduces dependence on non-renewable resources, promoting green chemical development. However, the industrial production of pentanediamine still faces many challenges: chemical methods rely on toxic raw materials (such as hydrogen cyanide) and expensive catalysts, causing severe environmental pollution; fermentation methods suffer from low substrate conversion rates, numerous byproducts, and complex separation and purification processes; while enzymatic methods can directly catalyze the decarboxylation of lysine, the poor stability of natural LDC in industrial environments, low immobilization efficiency, and high coenzyme costs limit its large-scale application.

[0003] In recent years, genetically engineered lysine decarboxylases have become a research hotspot. For example, using *E. coli* CadA as a commonly used starting sequence, mutants with enhanced thermostability or activity (such as mutations at Val3, Ala590, and Glu690 sites) have been obtained through directed evolution or rational design. However, the activity increase is mostly less than 20%, and defects such as enzyme activity decay and insufficient reusability still exist in high pH or long-term reactions. Although the mutant disclosed in patent CN 112899261 A has made breakthroughs in activity and stability, it is still difficult to meet the industrial catalysis demand for efficient and low-cost enzyme preparations. In addition, most existing mutants are derived from *E. coli*, and their decameric structure is prone to depolymerization and inactivation under alkaline conditions, further limiting catalytic efficiency.

[0004] In conclusion, developing enzyme preparations with higher enzyme activity and the ability to meet the requirements of efficient industrial catalysis is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a performance-enhanced lysine decarboxylase mutant for the synthesis of pentanediamine, thereby solving the problem that existing lysine decarboxylases have low enzyme activity and cannot meet the requirements of efficient industrial catalysis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A lysine decarboxylase mutant, which is any of the following mutants: Mutant 1, wherein mutant 1 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the 218 Asn position is mutated to Gly; Mutant 2, wherein mutant 2 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein Thr at position 222 is mutated to Val; Mutant 3, wherein mutant 3 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the 224th amino acid codon is mutated from AAC to AAT; Mutant 4, wherein mutant 4 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein Cys at position 244 is mutated to Pro; Mutant 5, wherein mutant 5 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the 247 Ser position is mutated to Cys; Mutant 6, wherein mutant 6 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Leu at position 248 is mutated to Arg; Mutant 7 is a mutant formed by synonymously mutating Thr(ACC) at position 304 to Thr(ACA) based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2. Mutant 8, wherein mutant 8 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Asp at position 330 is mutated to Lys; Mutant 9, wherein mutant 9 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Ser at position 364 is mutated to Cys; Mutant 10 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, in which the Ser at position 376 is mutated to Asn.

[0007] The nucleotide sequence of the gene encoding the wild-type lysine decarboxylase is shown in SEQ ID NO. 1.

[0008] The present invention also provides a recombinant plasmid comprising a gene encoding any of the above-described lysine decarboxylase mutants.

[0009] The original expression vector of the recombinant plasmid can be any of the commonly used expression vectors in the field for expressing the target gene in Escherichia coli. Preferably, the original expression vector of the recombinant plasmid is a pET series vector, more preferably a pET 29a vector.

[0010] The present invention also provides a recombinant strain comprising the above-described recombinant plasmid.

[0011] Preferably, the recombinant strain originates from *Escherichia coli*. Escherichia coli BL21(DE3) can also be other bacteria, such as Bacillus subtilis, Corynebacterium glutamicum, yeast, Streptomyces, Penicillium, or filamentous fungi such as Cephalosporium.

[0012] The present invention also provides a method for constructing the recombinant plasmid as described above, the method comprising the following steps: The recombinant plasmid is obtained by inserting a nucleotide sequence encoding the amino acid sequence described above between the restriction enzyme sites of the original expression vector.

[0013] The construction method specifically includes the following steps: Klebsiella pneumoniae ( Klebsiella grimontii A recombinant plasmid was obtained by inserting a gene fragment of wild-type lysine decarboxylase from the species into the NdeI and EcoRI restriction sites of the original expression vector pET-29a.

[0014] The above recombinant plasmid was transformed to [a specific target] using a thermal shock method. Escherichia coli Recombinant bacteria can be obtained from BL21(DE3).

[0015] A method for expressing the above-mentioned lysine decarboxylase mutant includes the following steps: transforming the above-mentioned recombinant plasmid into an *E. coli* strain to obtain a recombinant strain; activating the recombinant strain and inoculating it into LB medium or fermentation medium, waiting for the bacterial cell OD... 600 When the value reaches 0.6-1.2, IPTG is added to induce the expression of the lysine decarboxylase mutant; the final concentration of IPTG in the culture medium is 0.1-1 mM, the induction conditions are 16-37℃, and the induction time is 4-48 h.

[0016] The present invention also provides a method for whole-cell catalytic synthesis of 1,5-pentanediamine, wherein the above-mentioned recombinant bacteria are fermented and cultured, and then added to a reaction system containing L-lysine to convert L-lysine into 1,5-pentanediamine.

[0017] Preferably, the reaction system comprises the following components at the following concentrations: recombinant bacteria at 14,000-45,000 U / g (based on enzyme activity), L-lysine at 0.75M-1.4M, PLP at 0.024mM-0.048mM, pH 6.3-6.8, reaction temperature at 37℃, and reaction time at 2h.

[0018] More preferably, the pH is 6.5; the reaction temperature is 37°C; the cofactor PLP is 0.024 mM; and the catalytic reaction can be completed in 2 hours.

[0019] The beneficial effects of this invention are as follows: Based on traditional chemical synthesis and enzymatic methods, this invention identifies potential target residues by analyzing the active site of lysine decarboxylase. Combined with saturation mutagenesis technology, the enzyme molecule is modified, resulting in a lysine decarboxylase mutant that exhibits higher catalytic efficiency compared to wild-type lysine decarboxylase. Using L-lysine as a substrate, and through a specific catalytic process, it can be used to synthesize 1,5-pentanediamine with a conversion rate exceeding 99% within 2 hours. Compared to previous chemical synthesis and enzymatic methods, this invention offers shorter reaction time, lower cost, and promising application prospects.

[0020] This invention proposes a method based on Klebsiella pneumoniae (Klebsiella pneumoniae) Klebsiella grimontii A novel lysine decarboxylase mutant derived from [source missing]. Through key site modification, this mutant significantly enhanced enzyme activity, reaching 10.2 times higher than the wild type, achieving the highest enzyme activity reported to date. Simultaneously, it reduced catalyst usage or shortened catalytic reaction time, enabling the development of a new process for preparing 1,5-pentanediamine and nylon 56 salt. Attached Figure Description

[0021] Figure 1 This is the pET29a-CadA-WT recombinant plasmid map. Detailed Implementation

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of pentanediamine per minute under specific conditions, defined as 1 U. The instrument used was a Shimadzu LC2030 high-performance liquid chromatograph.

[0024] Enzyme activity assay conditions: A 1 mL enzyme activity reaction system consisted of 900 μL of L-lysine hydrochloride (final concentration 1.6 M) and PLP (final concentration 0.36 mM), and 100 μL of enzyme solution. The reaction conditions were: 37℃, 220 rpm shaker for 10 min. To terminate the reaction, 100 μL of reaction solution was added to 900 μL of pure water, mixed thoroughly, placed in a 100℃ metal bath for 2 min, cooled to room temperature, and centrifuged for 2 min. The solution was then diluted 10-fold with pure water, filtered through a 0.45 μm membrane, and subjected to HPLC analysis.

[0025] Chromatographic methods: Shimadzu LC-1260 high-performance liquid chromatograph; column: C18-AQ column, 5μm, 4.6×250mm; detector: LC-ELSD evaporative light detector, temperature 85℃, flow rate 2.1 mL / min; mobile phase A: 500 mL water + 500 μL trifluoroacetic acid + 100 μL heptafluorobutyric acid (pH 2.0), mobile phase B: acetonitrile, mobile phase A: mobile phase B = 85:15; flow rate: 0.6 mL / min, column temperature: 30℃, injection volume: 10 μL.

[0026] Example 1: Construction of wild-type strain of lysine decarboxylase GenScript Biotech optimized the CadA sequence of Klebsiella grimontii using codons, synthesized the entire gene sequence (nucleotide sequence shown in SEQ ID NO. 1), and cloned it into the vector pET29a to obtain the recombinant plasmid pET29a-CadA-WT, the map of which is shown below. Figure 1 As shown.

[0027] Example 2: Obtaining a lysine decarboxylase mutant strain Twenty-two amino acid residues were randomly selected: N218, G219, T220, S221, T222, N224, N243, C244, H245, S247, L248, T304, Y308, D330, A332, W333, S364, H366, K367, Q374, A375, and S376. Single-site saturation mutagenesis was performed on these 22 sites. Four mutation primers were designed to uniformly and without redundancy introduce the twenty natural amino acids into the mutation library. The primers for the mutation library are detailed in Table 1.

[0028] Table 1: Primers for Mutant Library

[0029] 1. Design degenerate primers and construct a recombinant mutant plasmid library. Primer preparation: 2.5 nmol of primer powder was centrifuged to collect the contents at the bottom of the tube. Then, 25 μL of sterile ddH2O was added to the tube to prepare a 100 μM primer stock solution. Next, the primer stock solution was diluted to obtain a 10 μM primer dilution, which will be used in the subsequent PCR system. In the PCR system, primers F1, F2, F3, and F4 were mixed in a ratio of 12:6:1:1 (i.e., 12 μL, 6 μL, 1 μL, and 1 μL of premixed F1-F4 were added respectively, resulting in a final primer concentration of 5 μM).

[0030] To improve the synthesis capacity of wild-type lysine decarboxylase, the recombinant vector pET29a-CadA-WT constructed in Example 1 was used as a template, and two rounds of PCR reactions were performed using primers corresponding to each mutant. The PCR system and reaction procedure for the first round are shown in Tables 2 and 3. The PCR system and reaction procedure for the second round are shown in Tables 4 and 5.

[0031] Table 2: PCR System

[0032] Table 3: PCR reaction procedure

[0033] Table 4: PCR System

[0034] Table 5: PCR reaction procedure

[0035] 2. PCR product transformation Take 10 μL of the second-round PCR amplification product and add 0.5 μL of... Dpn I enzyme, digested at 37°C for 3 hours, and 1 μL of the digested product was electroporated into E. coli BL21(DE3) competent cells were cultured in a shaker at 37°C and 220 rpm for 1 h. 200 μL, 300 μL, and 400 μL of culture medium were then spread onto kanamycin-resistant (50 μg / mL) LB agar plates and incubated at 37°C. Single colonies grew after 12-16 h, forming the mutant library.

[0036] Example 3: High-throughput screening of lysine decarboxylase mutant library and acquisition of single-point mutant strains 3.1 Strain preparation All single colonies were scraped from a plate used in Example 2 and sent for sequencing to verify the diversity of the mutant library. Afterwards, negative control bacteria were picked up separately using sterile toothpicks. E. coliBL21(DE3) / pET29a, positive control bacteria E. coli Single colonies of BL21(DE3) / pET29a-CadA-WT and mutant libraries grown on LB agar plates were inoculated into 96-well plates for culture (300 μL of LB medium and kanamycin were added to each well). The plates were incubated at 37°C and 800 rpm with shaking for 12 h.

[0037] Next, 120 μL of overnight culture was transferred to a new 96-well plate, 80 μL of 50% glycerol was added, and the plate was capped and stored at -80℃. Simultaneously, 800 μL of TB medium was added to the 96-well deep-well plate, along with isopropyl-β-D-thiogalactoside (IPTG) and kanamycin (final concentrations of 0.1 mM and 50 μg / mL, respectively). The plate was then cultured at 25℃ and 800 rpm for 12 h with shaking for protein expression.

[0038] 3.2 High-throughput screening method Transfer 300 µL of the cultured bacterial solution to each well of a 96-well plate. Add 100 µL of 10% lysine solution (pH 5.5, concentration in 0.4 M acetate-sodium acetate buffer) to each well. Then add PLP (8 mM stock solution) to each well to achieve a final concentration of 0.1 mM and mix thoroughly. Incubate the 96-well plate at 37°C and 800 rpm for 30 min on a shaker. Afterward, terminate the reaction and centrifuge for 10 min.

[0039] After the reaction was complete, 100 µL of the reaction solution was transferred to each well of a 96-well microplate, and 5 µL of mixed indicator (bromocresol purple and bromothymol blue mixed in a 1:1 ratio) was added to each well for color development. The clones with the most obvious color changes were identified by reading the values ​​at 595 nm using a microplate reader or by directly observing the color differences.

[0040] Based on the results of the ELISA reader test, strains with values ​​higher than the positive control were selected and sent to Suzhou Genewiz Co., Ltd. for sequencing analysis.

[0041] Example 4: Expression and enzyme activity evaluation of wild-type lysine decarboxylase and mutant strains The constructed recombinant E. coli E. coliBL21 / pET29a-CadA-WT and its mutant strains were activated by streaking on solid LB agar plates containing 50 μg / mL kanamycin. After incubation at 37°C for 12-24 h, single colonies were picked and transferred to 10 mL of LB liquid medium supplemented with 50 μg / mL kanamycin. The culture was then incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the culture was inoculated into 100 mL of LB medium (kanamycin concentration 50 μg / mL) and incubated at 37°C with shaking at 220 rpm for approximately 4-6 h. The culture was then analyzed when the OD value of the bacterial culture reached a certain level. 600 When the value reaches 0.6, add an appropriate concentration (0.1mM) of IPTG to induce the expression of lysine decarboxylase and its mutant, and continue to culture at 25℃ and 220rpm for no more than 20h.

[0042] After cultivation, the bacterial cells were collected by centrifugation at 9000 rpm and 4℃ for 10 min. The cells were then disrupted by sonication, and the supernatant was collected by centrifugation again to obtain the crude enzyme solution of the strain. Finally, the content of pentanediamine in the crude enzyme solution was determined by HPLC to evaluate the enzyme activity. The mutation sites and enzyme activity results of each mutant strain are shown in Table 6.

[0043] Table 6: Mutation sites of strains and enzyme activity assay results

[0044] Based on the enzyme activity comparison of the above strains, the preferred strains are selected. E. coli BL21(DE3) / pET29a-CadA-N218G-5 (mutant 1) E. coli BL21(DE3) / pET29a-CadA-T222V-24 (mutant 2) E. coli BL21(DE3) / pET29a-CadA-N224N-37 (mutant 3) E. coli BL21(DE3) / pET29a-CadA-C244P-6 (mutant 4) E. coli BL21(DE3) / pET29a-CadA-S247C-17 (mutant 5) E. coli BL21(DE3) / pET29a-CadA-L248R-53 (mutant 6) E. coli BL21(DE3) / pET29a-CadA-T304T-9 (mutant 7) E. coli BL21(DE3) / pET29a-CadA-D330K-5 (mutant 8) E. coliBL21(DE3) / pET29a-CadA-S364C-10 (mutant 9) E. coli The BL21(DE3) / pET29a-CadA-S376N-22 (mutant 10) strain was used for combined mutation.

[0045] The main reason for the increased enzyme activity after mutation in the above-mentioned strains is that the changes at key sites optimized the enzyme's structure, stability, substrate binding capacity, and catalytic efficiency. These mutations may function through the following mechanisms: eliminating steric hindrance and increasing the flexibility of the active site; introducing new interactions (such as disulfide bonds and charge interactions); optimizing the stability of local or global structures; and improving the enzyme's affinity for substrates or coenzymes.

[0046] Example 5: Two-site mutation of lysine decarboxylase and its expression and enzyme activity evaluation Using the same method as in Example 2, double mutants were constructed one by one based on the single mutant. The primers for the double-site mutation are shown in Table 7, the PCR system is shown in Table 8, and the PCR reaction procedure is the same as the second round of PCR reaction procedure in Example 2 (see Table 5).

[0047] For example, double mutant expression plasmids N218G / N224N and N218G / L248R were constructed based on the N218G mutant. The template was pET29a-CadA-N218G plasmid, and the primers were N224N-fw and N224N-rev, and L248R-fw and L248R-rev. The recombinant plasmids were named pET29a-CadA-N218G / N224N and pET29a-CadA-N218G / L248R.

[0048] The above plasmids were sequenced by Genewiz, and plasmids with correct sequences were transformed into... E. coli BL21(DE3) was used to obtain a recombinant strain with the CadA mutant.

[0049] All recombinant bacteria and wild-type controls were evaluated using the same shake-flask expression and catalytic system as in Example 4. The enzyme activity results of the dual-site combined mutant strains are shown in Table 9.

[0050] Table 7: Primers for Two-Site Combination Mutation

[0051] Table 8: PCR System

[0052] Table 9: Enzyme activity of dual-site combined mutant strains

[0053] Example 6: Three-point mutations of lysine decarboxylase and their expression and enzyme activity evaluation Using the same method as in Example 5, three-site mutants were constructed one by one based on the two-site mutants. The primers for the three-site mutations are shown in Table 7, and the PCR system is shown in Table 8. The PCR reaction procedure was the same as the second round of PCR reaction procedure in Example 2 (see Table 5).

[0054] For example, a three-point mutant N218G / N224N / L248R was constructed based on the N218G / N224N mutant, using the template pET29a-CadA-N218G / N224N plasmid and primers L248R-fw and L248R-rev. The recombinant plasmid was named pET29a-CadA-N218G / N224N / L248R expression plasmid.

[0055] The above plasmids were sequenced by Genewiz, and plasmids with correct sequences were transformed into... E. coli BL21(DE3) was used to obtain a recombinant strain with the CadA mutant.

[0056] All recombinant bacteria and wild-type controls were evaluated using the same shake-flask expression and catalytic assay as in Example 4. The enzyme activity results of the three-site combination mutant strains are shown in Table 10.

[0057] Table 10: Enzyme activity of three-site combination mutant strains

[0058] Example 7: Whole-cell catalytic biosynthesis of 1,5-pentanediamine Whole-cell catalytic reaction (1L): The mutant 4 cells collected by centrifugation were resuspended in 500mL of water (OD). 600 Add 220 g / L L-lysine and 0.024 mM PLP to a solution at 37°C, 220 rpm, and pH 6.3-6.8 for 2 h to obtain a reaction solution. Detect pentanediamine and its content using the above HPLC method.

[0059] To determine the optimal reaction conditions, experiments were conducted using different reaction systems. Taking a 1L reaction system as an example, when studying the effect of different temperatures on the reaction, the following parameters were kept constant: L-lysine 220g / L, PLP 0.024mM, crude enzyme solution 14000U, and pH 6.5. The reaction temperature was set at 30-40℃, 220rpm, and the reaction time was 2h. The results are shown in Table 11. It can be seen that when the reaction temperature is between 30-40℃, the formation rate of pentanediamine is better at the end of the reaction, with the optimal value at 37℃.

[0060] Table 11: Effect of different temperatures on pentamethylenediamine

[0061] When studying the effect of different substrate concentrations on the reaction, the following parameters were kept constant: PLP 0.024 mM, crude enzyme solution 14000 U, pH 6.5, reaction temperature 37℃, and 220 rpm. The concentration of L-lysine was kept between 220 g / L and 400 g / L. After 2 hours of reaction, the results are shown in Table 12. The results indicate that the formation rate of pentanediamine was better at the end of the reaction when the L-lysine concentration was between 220 g / L and 400 g / L, with the optimal concentration at 300 g / L.

[0062] Table 12: Effect of different substrate concentrations on pentanediamine

[0063] Based on the above experimental results, the optimal reaction conditions were determined as follows: taking a 1L reaction system as an example, L-lysine 300g / L, PLP 0.024mM, crude enzyme solution 14000U, pH 6.5, reaction temperature 37℃, 220rpm, reaction time 2h. Under these conditions, mutant strains 1-10 could all complete the conversion of L-lysine to pentanediamine after 2h of reaction. The L-lysine conversion rate could reach over 99%, and at most 220.3g / L of pentanediamine could be generated.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lysine decarboxylase mutant, characterized in that, It is any of the following mutants: Mutant 1, wherein mutant 1 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Asn at position 218 is mutated to Gly; Mutant 2, wherein mutant 2 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein Thr at position 222 is mutated to Val; Mutant 3, wherein mutant 3 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Asn(AAC) at position 224 is synonymously mutated to Asn(AAT). Mutant 4, wherein mutant 4 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein Cys at position 244 is mutated to Pro; Mutant 5, wherein the mutant 5 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Ser at position 247 is mutated to Cys; Mutant 6, wherein the mutant 6 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Leu at position 248 is mutated to Arg; Mutant 7, wherein mutant 7 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein Thr(ACC) at position 304 is synonymously mutated to Thr(ACA). Mutant 8, wherein the mutant 8 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Asp at position 330 is mutated to Lys; Mutant 9, wherein mutant 9 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Ser at position 364 is mutated to Cys; Mutant 10, wherein the mutant 10 is based on the wild-type lysine decarboxylase shown in SEQ ID NO. 2, wherein the Ser at position 376 is mutated to Asn.

2. A recombinant plasmid, characterized in that, It includes a gene encoding any of the lysine decarboxylase mutants of claim 1.

3. The recombinant plasmid according to claim 2, characterized in that, The original expression vector for the recombinant plasmid was the pET29a vector.

4. A recombinant bacterial strain, characterized in that, It contains the recombinant plasmid as described in claim 2 or claim 3.

5. The recombinant strain according to claim 4, characterized in that, The recombinant strain originates from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, yeast, Streptomyces, Penicillium, or Cephalosporium.

6. A method for expressing a lysine decarboxylase mutant, characterized in that, The process includes the following steps: transforming the recombinant plasmid as described in claim 2 or 3 into the starting strain to obtain a recombinant strain; activating the recombinant strain and inoculating it into LB medium or fermentation medium, waiting for the bacterial cells to OD... 600 When the value reaches 0.6-1.2, IPTG is added to induce the expression of the lysine decarboxylase mutant; the final concentration of IPTG in the culture medium is 0.1-1 mM, the induction conditions are 16-37℃, and the induction time is 4-48 h.

7. The use of the lysine decarboxylase mutant according to claim 1 in the synthesis of 1,5-pentanediamine and nylon 56 salt.

8. The application according to claim 7, characterized in that, The mutant was prepared into an immobilized mutant enzyme using whole-cell catalysis technology and applied to the synthesis of 1,5-pentanediamine and nylon 56 salt.

9. The application according to claim 7, characterized in that, After fermentation, the recombinant bacteria were added to a reaction system containing L-lysine.

10. The application according to claim 9, characterized in that, The reaction system includes the following components at the following concentrations: recombinant strain with 14,000-45,000 U / g of enzyme activity, L-lysine 0.75M-1.4M, PLP 0.024mM-0.048mM, pH 6.3-6.8, reaction temperature 37℃, and reaction time 2h.

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