Lysine decarboxylase cad a mutant and its application in pentanediamine production

Abstract

The application discloses a lysine decarboxylase CadA mutant and application thereof in production of pentamethylene diamine, and belongs to the field of bioengineering. D542R / H600E The optimal mutant CadA of the lysine decarboxylase exhibits significantly enhanced enzyme activity under neutral and alkaline conditions: the enzyme activity is 7.5 times that of the wild type under pH 7.0, is 5.0 times, 5.6 times and 0.2 times that of the wild type under pH 8.0, pH 9.0 and pH 10.0 respectively. The optimal mutant of the lysine decarboxylase is applied to production of pentamethylene diamine of Escherichia coli, and the yield of the pentamethylene diamine reaches 318.5 g / L. The application improves the conversion efficiency of the lysine decarboxylase in neutral and alkaline environments, improves the synthesis speed of the pentamethylene diamine in neutral and alkaline environments, and lays a foundation for efficient synthesis of the pentamethylene diamine.

Description

Technical Field

[0001] This invention relates to a lysine decarboxylase CadA mutant and its application in the production of pentanediamine, 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 undergo condensation polymerization with diacids such as succinic acid and biosuccinic acid to form polyamide PA5X series polymer materials. These materials are renewable, low-carbon, and possess excellent mechanical properties and chemical stability, thus having 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... D542R / H600E The enzyme activity at pH 7.0 was 7.5 times that of the wild type, and at pH 8.0, pH 9.0, and pH 10.0, it was increased by 5.0-fold, 5.6-fold, and 0.2-fold respectively compared to the wild type, effectively increasing the stability of CadA enzyme in neutral and alkaline pH environments. The optimal enzyme activity of the CadA mutant was determined. D542R / H600E 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 D542R CadA H600Eand CadA D542R / H600E .

[0006] Said CadA D542R / H600E The amino acid sequence of the enzyme (SEQ ID NO.3): MNVIAILNHMGVYFKEEPIRELHRALERLNFQIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEEISKMNENLPLYAFANTYSTLDVSLNDLRLQISFFEYALGAAEDIANKIKQTTDEYINTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSLFYDFFGPNTMKSDISISVSELGSLLDHSGPHKEAEQYIARVFNADRSYMVTNGTSTANKIVGMYSAPAGSTILIDRNCHKSLTHLMMMSDVTPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDFIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGIVASTETAAAMMKGNAGKRLINGSIERAIKFRKEIKRLRTESDGWFFDVWQPDHIDTTECWPLRSDSTWHGFKNIDNEHMYLDPIKVTLLTPGMEKDGTMSDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIRKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRIQELAQNIHKLIVHENLPDLMYRAFEVLPTMVMTPYAAFQKELHGMTEEVYLDEMVGRINANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLKEESKK encoding the CadA D542R / H600E The nucleotide sequence of the enzyme (SEQ ID NO.4): This invention also provides the optimal lysine decarboxylase mutant CadA. D542R / H600E Application in catalyzing the removal of the α-carboxyl group from lysine to generate pentamethylenediamine.

[0007] The present invention also provides a CadA-containing D542R / H600E The method for constructing engineered strains containing mutant genes is as follows: Gene amplification from the genome of Escherichia coli BL21(DE3) using primers CadA-F and CadA-R cadA .use Nco I and EcoR I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, the product was ligated using T4 DNA ligase. cadA The ligation product was ligated into pETDuet-1. The ligation product was introduced into *E. coli* JM109, and screening was performed by colony PCR and Sanger sequencing to finally construct the plasmid pETDuet-CadA. The mutant gene CadA was amplified from pETDuet-CadA using primers D542R-F and D542R-R. D542R ,use Nde I and Xho I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, CadA was ligated using T4 DNA ligase. D542R The plasmid pETDuet-1 was ligated into pETDuet-1, 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-CadA. D542R Using primers H600E-F and H600E-R, from pETDuet-CadA D542R amplified mutant gene CadA D542R / H600E ,use Nde I and Xho I performed double digestion of the PCR product and pETDuet-1 plasmid, and after purification, CadA was ligated using T4 DNA ligase. D542R / H600E The plasmid pETDuet-1 was ligated into pETDuet-1, 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-CadA. D542R / H600E The recombinant plasmid pETDuet-CadA D542R / H600E Transform to E. coli BL2(DE3) was used to obtain a recombinant strain for pentanediamine synthesis. pETduet-CadA was then transformed into [a specific strain] in the same manner. E. coli BL2(DE3) was used to obtain a recombinant strain for pentanediamine synthesis as a control strain.

[0008] The present invention also provides a gene encoding the lysine decarboxylase mutant described above or a recombinant vector carrying the gene.

[0009] The present invention also provides recombinant cells expressing the above-mentioned mutants or carrying the above-mentioned genes or the recombinant vectors.

[0010] In one embodiment of the present invention, the recombinant cells are bacteria or fungi as host cells.

[0011] 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.

[0012] 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 aspartic acid at position 542 of lysine decarboxylase as shown in SEQ ID NO.1 to arginine; Alternatively, the aspartic acid at position 542 of the lysine decarboxylase, as shown in SEQ ID NO.1, may be mutated to arginine, and the histidine at position 600 may be mutated to glutamic acid.

[0013] The present invention also provides a genetically engineered bacterium that expresses the above-mentioned lysine decarboxylase mutant.

[0014] 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.

[0015] 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.

[0016] 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 500~600 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 °C, 200~300 rpm, 10~12 h, and the initial pH is 6.0.

[0017] 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.

[0018] Beneficial effects (1) In this invention, the lysine decarboxylase gene is modified by semi-rational site-directed mutagenesis. The aspartic acid D at position 542 and the histidine H at position 600 of the lysine decarboxylase are mutated to arginine R and glutamic acid E, respectively, resulting in the lysine decarboxylase mutant CadA. D542R / H600E At pH 7.0, the enzyme activity was 7.5 times that of the wild type. At pH 8.0, pH 9.0 and pH 10.0, the activity was increased by 5.0 times, 5.6 times and 0.2 times, respectively, compared with the wild type, 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 pentamethylenediamine, 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 pentamethylenediamine. Attached Figure Description

[0019] Figure 1 Lysine is converted to pentamethylenediamine by lysine decarboxylase. Detailed Implementation

[0020] 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.

[0021] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

[0022] 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 to determine the lysine 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. After centrifugation at 12000 r / min for 10 min, the supernatant was collected, derivatized, and used for HPLC analysis to determine the pentanediamine yield. The unit enzyme activity was defined as U / g = 1 mole pentanediamine / min / g protein.

[0023] Table 1: Reaction system for detecting lysine decarboxylase activity

[0024] 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.

[0025] 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 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 Lysine Mutation Site The specific steps are as follows: Lysine decarboxylase CadA is a key enzyme catalyzing the conversion of L-lysine to pentanediamine, and its catalytic process depends on the cofactor pyridoxal phosphate (PLP). CadA's active form is the decameric form, but this structure is only stable at pH < 6; under neutral or alkaline conditions, it readily depolymerizes into oligomers, leading to a significant decrease in enzyme activity. To improve the stability of CadA under neutral and alkaline conditions, its structural characteristics were analyzed.

[0028] The results showed that there were four negatively charged acidic residues (D561, D542, D309) at the decadal pentamer ring interface.

[0029] These residues cannot neutralize their charge under neutral / alkaline conditions, resulting in significant electrostatic repulsion and affecting decamer formation. Therefore, the acidic residues were replaced with basic amino acids (lysine, arginine, or histidine) to reduce interfacial electrostatic repulsion and promote decamer assembly. Furthermore, the C-terminal domain (CTD, residues 564-715) of CadA is known to constitute a channel for substrate entry into the active site. This region contains multiple positively charged residues (H600, H685), which may repel positively charged L-lysine residues, thereby reducing substrate entry efficiency.

[0030] Therefore, the above residues were replaced with acidic amino acids (glutamic acid or aspartic acid) to enhance substrate affinity.

[0031] Example 2: 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 cadAThe 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. D542R CadA H600E and CadA D542R / H600E Mutant 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 ligated into pETDuet-1 and introduced into E. coli JM109. The recombinant plasmid pETDuet-CadA was finally obtained by screening by colony PCR and Sanger sequencing.

[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 D542R-F and D542R-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. D542R Mutant recombinant plasmid pETDuet-CadA D542R .

[0034] pETDuet-CadA was subjected to full-plasmid PCR using primers H600E-F and H600E-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. H600E Mutant recombinant plasmid pETDuet-CadA H600E .

[0035] pETDuet-CadA was stimulated using primers H600E-F and H600E-R. D542R 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. D542R / H600E Mutant recombinant plasmid pETDuet-CadA D542R / H600E .

[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 3: 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 D542R , E. coli BL21 / pETDuet- CadA H600E , E. coliBL21 / pETDuet- CadA D542R / H600E 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 freezing and 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 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., the reaction system containing dithiothreitol, pyridoxal phosphate, lysine, and potassium sodium phosphate buffer, the lysine decarboxylase CadA or mutant pure 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 D542R / H600E At pH 7.0, the enzyme activity was 7.5 times that of the wild type. At pH 8.0, pH 9.0, and pH 10.0, the activity was increased by 5.0 times, 5.6 times, and 0.2 times, respectively, compared with the wild type, effectively increasing the stability of CadA enzyme in neutral and alkaline pH environments.

[0043] Example 5: 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. coliBL21 / pETDuet- CadA D542R , E. coli BL21 / pETDuet- CadA H600E , E. coli BL21 / pETDuet-CadA D542R / H600E 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 incubated at 37°C and 250 r / min for 12 h to activate the bacterial strain and prepare the seed culture.

[0044] (2) The activated strain was transferred to 100 mL LB liquid medium at an inoculation rate of 2% (v / v) of the prepared seed culture 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°C and 5000 rpm for 15 min, and take the precipitate as wet cells.

[0046] 2. Whole-cell preparation of pentamethylenediamine 2 L reaction system: 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.

[0047] 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.

[0048] 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).

[0049] The results are shown in Table 4 below: Table 4: Pentanediamine yield of different strains

[0050] The results showed that using the combined mutant CadA D542R / H600E The recombinant strain was reacted in a 5-liter bioreactor for 10 h, achieving a yield of 318.5 g / L of pentanediamine.

[0051] 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 lysine decarboxylase mutant, characterized in that, The lysine decarboxylase mutant is, The lysine decarboxylase with the amino acid sequence shown in SEQ ID NO.1 was obtained by mutating aspartic acid at position 542 to arginine. Alternatively, it can be obtained by mutating aspartic acid at position 542 of the lysine decarboxylase, as shown in SEQ ID NO.1, to arginine, and simultaneously mutating histidine at position 600 to glutamic acid.

2. The gene encoding the lysine decarboxylase mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. A recombinant enzyme catalyst containing the lysine decarboxylase mutant of claim 1, characterized in that, It is any 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.

5. 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 aspartic acid at position 542 of the lysine decarboxylase, as shown in SEQ ID NO.1, to arginine. Alternatively, the aspartic acid at position 542 of the lysine decarboxylase, as shown in SEQ ID NO.1, may be mutated to arginine, and the histidine at position 600 may be mutated to glutamic acid.

6. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria expressed the lysine decarboxylase mutant of claim 1, and the genetically engineered bacteria was Escherichia coli.

7. A method for synthesizing pentamethylenediamine, characterized in that, The method involves using lysine as a substrate and employing the mutant described in claim 1 or the recombinase catalyst described in claim 4, or the genetically engineered bacteria described in claim 6 or its lysate or fermentation broth as a catalyst, to catalytically convert and synthesize pentamethylenediamine.

8. The method according to claim 7, characterized in that, 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 500~600 g / L and coenzyme PLP at a concentration of 0.08~0.12 mmol / L.

9. The method according to claim 8, characterized in that, The reaction conditions were: 35~40 °C, 200~300 rpm, 10~12 h.

10. The mutant of claim 1, the recombinase catalyst of claim 4, the genetically engineered bacteria of claim 6, or the method of any one of claims 7-9, is used in the preparation of pentanediamine.

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