L-lysine decarboxylase derived from klebsiella grimontii and use thereof

Abstract

The application discloses L-lysine decarboxylase derived from Klebsiella grimontii and application thereof. The L-lysine decarboxylase disclosed by the application has an amino acid sequence shown in SEQ ID NO:1, and a mutant thereof has an amino acid sequence shown in SEQ ID NO:3. The L-lysine decarboxylase and the mutant thereof provided by the application can efficiently catalyze synthesis of 1,5-pentanediamine.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to an L-lysine decarboxylase derived from Klebsiella grimontii and its application. Background Technology

[0002] Pentylene diamine, also known as cadaverine, can be polymerized with diacids to synthesize high-molecular-weight polyamide materials (i.e., nylon). Globally, approximately 7 million tons of polyamide materials are produced annually, consuming a large amount of petrochemical resources. Petrochemical resources are non-renewable; therefore, the biosynthesis of pentylene diamine, a crucial monomer for polyamides, represents a future technological trend with significant economic and ecological implications. The enzymatic synthesis of 1,5-pentylene diamine is also of great importance for the synthesis of polyamides, polyurethanes, and other high-molecular-weight polymers. L-lysine decarboxylase (LDC, enzyme classification number EC4.1.1.18) exists in various microorganisms, and its main biological function is to catalyze the decarboxylation of L-lysine to produce 1,5-pentylene diamine.

[0003] Since the current technology for stable and efficient catalytic production of 1,5-pentanediamine is mostly achieved through lysine decarboxylases derived from Escherichia coli, if other sources of lysine decarboxylases can be discovered to achieve efficient and stable production of 1,5-pentanediamine, it will further promote the industrial production of 1,5-pentanediamine. Summary of the Invention

[0004] The purpose of this invention is to provide an L-lysine decarboxylase derived from Klebsiella grimontii, in order to solve the problem that existing L-lysine decarboxylases have unsatisfactory performance in terms of activity and stability.

[0005] Specifically, the present invention obtains L-lysine decarboxylase from Klebsiella grimontii, the amino acid sequence of which is shown in SEQ ID NO:1 and the nucleotide sequence of which is shown in SEQ ID NO:2. Furthermore, it is mutated to obtain an L-lysine decarboxylase mutant with significantly improved activity and stability, the amino acid sequence of which is shown in SEQ ID NO:3 and the nucleotide sequence of which is shown in SEQ ID NO:4.

[0006] In a first aspect, the present invention provides an L-lysine decarboxylase having the amino acid sequence shown in SEQ ID NO:1.

[0007] The L-lysine decarboxylase provided by this invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically, for example, by using recombinant technology to express it from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants).

[0008] In some embodiments, the L-lysine decarboxylase is obtained by introducing a recombinant vector containing its encoding gene into Escherichia coli (e.g., E.coli BL21(DE3)) to obtain a recombinant genetically engineered bacterium, and then inducing the expression of the recombinant genetically engineered bacterium to obtain the lysine decarboxylase.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned L-lysine decarboxylase, having the nucleotide sequence shown in SEQ ID NO:2.

[0010] The nucleic acid molecules provided by this invention can usually be obtained by PCR amplification or artificial synthesis.

[0011] In a third aspect, the present invention provides a mutant of the above-mentioned L-lysine decarboxylase, having the amino acid sequence shown in SEQ ID NO:3, and whose catalytic activity and stability are further improved compared with the above-mentioned L-lysine decarboxylase. Compared with the amino acid sequence of the L-lysine decarboxylase shown in SEQ ID NO:1, this L-lysine decarboxylase mutant has the following mutations: Arg at position 24 is mutated to Leu, Cys at position 67 is mutated to Gly, Ala at position 135 is mutated to Gly, Tyr at position 145 is mutated to Cys, Thr at position 237 is mutated to Ser, Lys at position 290 is mutated to Glu, Trp at position 454 is mutated to Arg, and Glu at position 611 is mutated to Val.

[0012] The mutant of L-lysine decarboxylase provided by the present invention can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically, for example, by using recombinant technology to express it from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants).

[0013] In some embodiments, the L-lysine decarboxylase is obtained by introducing a recombinant vector containing its encoding gene into Escherichia coli (e.g., E.coli BL21(DE3)) to obtain a recombinant genetically engineered bacterium, and then inducing expression of the recombinant genetically engineered bacterium to obtain a mutant of L-lysine decarboxylase.

[0014] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the mutant of the above-mentioned L-lysine decarboxylase, having the nucleotide sequence shown in SEQ ID NO:4.

[0015] The nucleic acid molecules provided by this invention can usually be obtained by PCR amplification or artificial synthesis.

[0016] In a fifth aspect, the present invention provides a recombinant vector comprising any of the nucleic acid molecules described above.

[0017] The recombinant vector includes a cloning vector and an expression vector, wherein the cloning vector is used to replicate the relevant sequence and the expression vector is used to express the relevant gene.

[0018] In some embodiments, the recombinant vector is pET-KgLDC or pET-ΔKgLDC68, which is obtained by replacing the sequence between the XbaI and BamHI restriction sites of pET-28a(+) with the nucleic acid molecule encoding the L-lysine decarboxylase or the nucleic acid molecule of the mutant L-lysine decarboxylase, respectively, while keeping the rest of the sequence unchanged.

[0019] In a sixth aspect, the present invention provides a recombinant cell comprising any of the recombinant vectors described above.

[0020] In some embodiments, the recombinant cells are induced to produce the above-mentioned L-lysine decarboxylase or a mutant of the above-mentioned L-lysine decarboxylase.

[0021] In some implementations, the method for constructing the recombinant cells includes the following:

[0022] The recombinant vector was transformed into host cells and induced to obtain mutants expressing the above-mentioned L-lysine decarboxylase or the above-mentioned L-lysine decarboxylase.

[0023] Furthermore, the recombinant vector is any of the recombinant vectors described above, and the host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., preferably Escherichia coli BL21(DE3).

[0024] In some embodiments, the recombinant cells are recombinant bacteria W and recombinant bacteria 68, and the recombinant cells can be recombinant genetically engineered bacteria. The culture medium used when the recombinant genetically engineered bacteria express L-lysine decarboxylase or its mutants can be any culture medium in the art that allows the recombinant genetically engineered bacteria to grow and produce the L-lysine decarboxylase or its mutants of the present invention, preferably LB medium.

[0025] There are no special requirements for the culture method and culture conditions, as long as the genetically engineered bacteria can grow normally and express L-lysine decarboxylase or its mutant.

[0026] More specifically, the above-mentioned method for constructing recombinant cells includes the following steps:

[0027] (1) Amplification of the L-lysine decarboxylase gene KgLDC;

[0028] (2) Obtaining the mutant ΔKgLDC of the L-lysine decarboxylase gene;

[0029] (3) Construction of recombinant expression plasmids pET-KgLDC and pET-ΔKgLDC;

[0030] (4) Transform the recombinant expression plasmids pET-KgLDC and pET-ΔKgLDC into host cells;

[0031] (5) Positive clones were obtained by screening with resistant culture medium.

[0032] In a seventh aspect, the present invention provides a method for preparing an L-lysine decarboxylase or a mutant thereof, comprising:

[0033] The recombinant cells described above were induced to undergo culture to obtain a culture.

[0034] Isolate the above-mentioned L-lysine decarboxylase or a mutant of the above-mentioned L-lysine decarboxylase from the culture;

[0035] The methods for inducing recombinant cell culture and for isolating L-lysine decarboxylase or its mutants from the culture are conventional methods in this field. For example, after activating the recombinant strain, it is inoculated into LB medium or fermentation medium, and the cell OD is... 600 When the value reaches 0.6-30, IPTG is added to induce the expression of L-lysine decarboxylase or its mutant; the final concentration of IPTG in the culture medium is 0.1-1 mM, the induction conditions are 30℃, and the induction time is 12 h.

[0036] In an eighth aspect, the present invention provides the use of the above-described L-lysine decarboxylase, any of the above-described nucleic acid molecules, mutants of the above-described L-lysine decarboxylase, the above-described recombinant vector, the above-described recombinant cells, and / or the L-lysine decarboxylase or mutants thereof prepared by the above-described methods in the preparation of 1,5-pentanediamine.

[0037] In a ninth aspect, the present invention provides a method for preparing 1,5-pentanediamine, comprising the following steps: using any of the above-described L-lysine decarboxylases, any of the above-described L-lysine decarboxylase mutants, any of the above-described recombinant cells and / or the L-lysine decarboxylase or its mutants prepared by the above method as catalysts to catalyze the reaction of L-lysine to obtain 1,5-pentanediamine.

[0038] In some embodiments, the temperature in the above catalytic reaction is 20-30°C, for example 24°C, 25°C, 26°C, or any value or range between these values, preferably 25°C;

[0039] The catalytic reaction includes L-lysine and pyridoxal phosphate as a coenzyme;

[0040] The final concentration of L-lysine is 200-300 g / L, such as 200 g / L, 210 g / L, 220 g / L, 230 g / L, 240 g / L, 250 g / L, 260 g / L, or any value or range between these values, preferably 250 g / L.

[0041] The final concentration of pyridoxal phosphate is 0.005-0.015M, for example 0.008M, 0.01M, 0.012M, or any value or range between these values, preferably 0.01M.

[0042] In some embodiments, the above-described catalytic reaction includes catalyzing the reaction of L-lysine with any of the recombinant cells described above to obtain 1,5-pentanediamine;

[0043] Specifically, the recombinant cells or their lyophilized powder can be used as catalysts for whole-cell catalytic production of 1,5-pentanediamine. The amount of catalyst used is 250-750 mg / g L-lysine, preferably 500 mg / g L-lysine.

[0044] It should be understood that the L-lysine decarboxylase or its mutants described in this invention can be used in whole-cell engineered bacteria, in unpurified crude enzyme form, or in partially or completely purified enzyme form. Furthermore, the L-lysine decarboxylase or its mutants of this invention can be prepared into immobilized enzymes or catalysts in immobilized cell form using immobilization techniques known in the art.

[0045] The L-lysine decarboxylase and its mutant of the present invention can efficiently catalyze the synthesis of 1,5-pentanediamine. In particular, the mutant, under suitable conditions, with L-lysine as substrate and pyridoxal phosphate as coenzyme, has a conversion rate of 93.6% and an enzyme activity of 1520.6 U / L, which is 2.91 times that of the original enzyme, and its stability is also better than that of the original enzyme.

[0046] Specifically, this invention uses an L-lysine decarboxylase derived from *Klebsiella grimontii* as the original sequence and obtains an L-lysine decarboxylase mutant with improved enzyme activity and stability via error-prone PCR. This allows for a reduction in catalyst usage or a shorter reaction time, while also exhibiting good stability, thereby further reducing production costs. This invention advances the industrialization process of biological production of 1,5-pentanediamine and nylon 56 salt. Attached Figure Description

[0047] Figure 1The HPLC chromatogram is for 1,5-pentanediamine standard.

[0048] Figure 2 This is the HPLC chromatogram of L-lysine standard.

[0049] Figure 3 This is the HPLC chromatogram of the mutant enzyme reaction solution.

[0050] Figures 1-3 In this context, CAD represents 1,5-pentanediamine, and Lys represents L-lysine. Detailed Implementation

[0051] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0052] Klebsiella grimontii was disclosed in the literature "Hubbard A, Newire E, Botelho J, et al. Isolation of an antimicrobial-resistant, biofilm-forming, Klebsiella grimontii isolate from a reusable water bottle[J]. Microbiology Open, 2020, 9." and is available to the public from Wanhua Chemical Group Co., Ltd.

[0053] pET-28a(+) is a product of Sangon Biotech (Shanghai) Co., Ltd., with product catalog number B540183.

[0054] Example 1: Amplification of the L-lysine decarboxylase gene KgLDC from Klebsiella grimontii

[0055] 1. Extract genomic DNA from Klebsiella grimontii.

[0056] 2. Using the genomic DNA obtained in step 1 as a template, PCR was performed using primer 1 (5'-cggataacaattcccctctagaatgaacgttatcgcaatcat-3', SEQ ID NO:5) and primer 2 (5'-cggagctcgaattcggatccttatttgttgttttcttctct-3', SEQ ID NO:6) as primer pairs to obtain a PCR amplification fragment containing the L-lysine decarboxylase gene KgLDC. The nucleotide sequence of the L-lysine decarboxylase gene is shown in SEQ ID NO:2, and the amino acid sequence of the L-lysine decarboxylase it encodes is shown in SEQ ID NO:1.

[0057] Example 2: Obtaining the gene sequence of the L-lysine decarboxylase mutant using error-prone PCR technology

[0058] Error-prone PCR methods modify and increase the natural error rate of polymerases based on standard PCR. Taq polymerase is the most commonly used error-prone PCR polymerase. Compared to the basic PCR reaction (1.5 mM), error-prone PCR reactions typically contain a higher concentration of magnesium chloride (7 mM) to stabilize non-complementary pairs. Additionally, manganese chloride can be added to further increase the error rate. This example utilizes error-prone PCR technology to obtain a mutant of the L-lysine decarboxylase gene.

[0059] Using the PCR amplification fragment from Example 1 as a template, and primers 1 and 2 as primer pairs, the following error-prone PCR was performed, resulting in 125 mutants of the L-lysine decarboxylase gene, namely ΔkgLDC1-ΔkgLDC125.

[0060] The 50 μL error-prone PCR system is as follows: 5 μL 10× error-prone PCR buffer (500 mmol / L KCl, 70 mmol / L MgCl, 100 mmol / L Tris-HCl pH 8.3, 0.1% (w / v, gelatin)); 0.5 mmol / L dATP and dGTP, 2.5 mmol / L dCTP and dTTP; half primers, 40 pmol each; template DNA 2.0 μL; MgCl2 7 mmol / L; MnCl2 0.3 mmol / L; Taq DNA polymerase 2.5 U; ddH2O added to 50 μL. PCR amplification conditions: 94℃ for 3 min; 94℃ for 1 min, 59℃ for 1 min, 72℃ for 2 min, 30 cycles; 72℃ for 10 min.

[0061] Example 3: Cloning of the L-lysine decarboxylase gene and construction of an expression strain

[0062] 1. The PCR amplification fragment containing the L-lysine decarboxylase gene obtained in Example 1 was digested with XbaI and BamHI to obtain the gene fragment; pET-28a(+) was digested with XbaI and BamHI to obtain the vector fragment; the gene fragment and the vector fragment were ligated to obtain the recombinant expression plasmid, which was named pET-KgLDC. The plasmid was sent for sequencing, and the results were consistent with expectations.

[0063] 2. The recombinant expression plasmid pET-KgLDC obtained in step 1 was transformed into E. coli DH5α competent cells by heat shock. The cells were then plated on LB solid medium containing 25 μg / mL kanamycin to obtain the corresponding single clone strain, which was named H. After amplification and plasmid extraction, the pET-KgLDC plasmid was obtained. The obtained plasmid was transformed into E. coli BL21(DE3) by chemical transformation. The cells were then plated on LB solid medium containing 25 μg / mL kanamycin and screened to obtain the recombinant strain W expressing L-lysine decarboxylase.

[0064] Example 4: Cloning of L-lysine decarboxylase gene mutant and construction of expression strain

[0065] Following the method in Example 3, recombinant plasmids pET-ΔKgLDC1-pET-ΔKgLDC125 were constructed using 125 mutants of the L-lysine decarboxylase gene obtained in Example 2, and recombinant bacteria 1-recombinant bacteria 125 expressing L-lysine decarboxylase mutants were obtained.

[0066] Example 5: Obtaining an L-lysine decarboxylase mutant with high catalytic efficiency using high-throughput screening.

[0067] The single-clone colonies verified by PCR in Examples 3 and 4 were cultured in 5 mL of LB medium, and IPTG was added to induce protein expression. The OD of the bacterial culture was measured after 12 h. 600 The value was adjusted, and the bacterial solution was diluted with water to achieve the desired OD value. 600 The value was approximately 2. 50 μL of the diluted bacterial culture was added to each well of a 96-well plate, along with 100 μL of a 500 g / L L-lysine aqueous solution and 10 μL of a 0.2 M pyridoxal phosphate aqueous solution. Water was then added to a final volume of 200 μL. The mixture was reacted at 25 °C and 300 rpm for 36 h to obtain the reaction solution. After the reaction, the protein was inactivated by heating at 95 °C for 5 min.

[0068] Dissolve 162 mg of bromocresol purple (BCP) in 1 ml of 100% ethanol, and dilute with water to 2 ml to prepare the BCP stock solution. Add 180 μl of 500 mM sodium acetate buffer (pH 6.0), 10 μl of reaction solution, and 10 μl of the BCP stock solution to a 96-well plate. Measure the absorbance at 595 nm using a UV-Vis spectrophotometer. Higher L-lysine decarboxylase activity results in higher 1,5-pentanediamine production and a higher pH of the reaction solution.

[0069] The strain corresponding to the reaction solution with the highest absorbance (recombinant strain 68) underwent plasmid extraction. The extracted plasmid was sequenced, yielding the gene sequence encoding the L-lysine decarboxylase mutant, as shown in SEQ ID NO:4. The amino acid sequence of the L-lysine decarboxylase mutant encoded by this sequence is shown in SEQ ID NO:3. Compared to the amino acid sequence of the L-lysine decarboxylase shown in SEQ ID NO:1, this L-lysine decarboxylase mutant exhibits the following mutations: Arg at position 24 is mutated to Leu, Cys at position 67 is mutated to Gly, Ala at position 135 is mutated to Gly, Tyr at position 145 is mutated to Cys, Thr at position 237 is mutated to Ser, Lys at position 290 is mutated to Glu, Trp at position 454 is mutated to Arg, and Glu at position 611 is mutated to Val.

[0070] Example 6: Preparation of enzymes and determination of enzyme activity

[0071] The recombinant strain W obtained in Example 3 and the recombinant strain 68 screened in Example 5 were respectively subjected to scale-up culture. After scale-up culture, the fermentation broth was subjected to conventional treatments such as centrifugation (8000 rpm, 10 min), cell disruption, and freeze drying to prepare freeze-dried powders of L-lysine decarboxylase (original enzyme) and its mutant (mutant enzyme) and stored at -80℃.

[0072] The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of 1,5-pentanediamine per minute or the amount of enzyme required to consume 1 μmol of substrate L-lysine per minute under the reaction conditions of Example 5 (final concentration of lyophilized powder is 500 mg / g L-lysine).

[0073] The enzyme activities of the original enzyme and the mutant enzyme were 521.9 U / L and 1520.6 U / L, respectively. Compared with the original enzyme, the activity of the mutant enzyme was 2.91 times. HPLC analysis of the residual L-lysine yielded conversion rates of 60.8% and 93.6% for the two enzymes, respectively. The specific HPLC method is as follows:

[0074] Take 1 mL of the reaction solution (or its dilution), add 300 μL of NaHCO3-NaOH buffer solution (1 mol / L NaOH solution added to saturated NaHCO3 solution, adjust the pH to 9.5), then add 1 mL of 5 mg / mL dansyl chloride aqueous solution, sonicate for 10 min under light-protected conditions, let stand at room temperature for 10 min, finally adjust to 5 mL with acetonitrile, filter through a 0.45 μm filter membrane, and then test on the instrument.

[0075] Chromatographic column: Diamonsil C18 (4.6mm×250mm, 5μm) column; column temperature: 30℃; mobile phase: water:acetonitrile (volume ratio) = 25:75; flow rate: 1mL / min; sample loading volume: 10μl; detection wavelength: 254nm.

[0076] The HPLC chromatograms of 1,5-pentanediamine standard and L-lysine standard are shown below. Figure 1 and 2 As shown, the retention time of 1,5-pentanediamine was 9.756 min, and the retention time of L-lysine was 3.804 min.

[0077] The HPLC chromatogram of the mutant enzyme reaction solution is shown below. Figure 3 As shown, the retention time of 1,5-pentanediamine was 9.756 min, and the retention time of L-lysine was 3.804 min.

[0078] Example 7: Stability test of L-lysine decarboxylase mutant

[0079] Take a small amount of the lyophilized powder of the original enzyme and mutant enzyme obtained in Example 6, and react it according to the reaction conditions in Example 5. After the reaction is completed, centrifuge to recover the enzyme and react it again. The conversion rates of the two enzymes in each batch are shown in Table 1.

[0080] Table 1

[0081]

[0082] The results showed that the mutant enzyme had good reusability, and the conversion rate could still reach more than 85% after 4 batches of reaction. Compared with the original enzyme, it had better stability. After 4 batches of reaction, the activity of the original enzyme decreased by 25.7%, while the activity of the mutant enzyme only decreased by 5.1%.

[0083] sequence

[0084] SEQ ID NO:1

[0085] MNVIAIMNHMGVYFKEEPIRELHRALERLDFRIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELCEDISKMNEYMPLYAFANTYSTLDVSLNDLRMQVRFFEYALGAAEDIANKIKQNTDEYIDTILPPLTKALFKYVREGKYTFCTPGHMGGTAFQKSPVGSIFYDFFGSNTMKSDISISVSELGSLLDHSGPHKEAEEYIARVFNAERSYMVTNGTSTANKIVGMYSAPAGSTVLIDRNCHKSLTHLMMMSDITPIYFRPTRNAYGILGGIPQSEFQHATIAKRVKETPNATWPVHAVITNSTYDGLLYNTDYIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGVVASTETAAAMMKGNAGKRLIDGSIERSIKFRKEIKRLKGESDGWFFDVWQPEHIDGAECWPLRSDSAWHGFKNIDNEHMYLDPIKVTLLTPGMKKDGTMDDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRVQELAQNIHKLIEHHNLPDLMFRAFEVLPTMMITPYAAFQKELHGQTEEVYLEEMVGRVNANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLREENNK

[0086] SEQ ID NO:2

[0087]

[0088] SEQ ID NO:3

[0089] MNVIAIMNHMGVYFKEEPIRELHLALERLDFRIVYPNDRDDLLKLIENNARLCGVIFDWDKYNLELGEDISKMNEYMPLYAFANTYSTLDVSLNDLRMQVRFFEYALGAAEDIANKIKQNTDEYIDTILPPLTKGLFKYVREGKCTFCTPGHMGGTAFQKSPVGSIFYDFFGSNTMKSDISISVSELGSLLDHSGPHKEAEEYIARVFNAERSYMVTNGTSTANKIVGMYSAPAGSSVLIDRNCHKSLTHLMMMSDITPIYFRPTRNAYGILGGIPQSEFQHATIAKRVEETPNATWPVHAVITNSTYDGLLYNTDYIKKTLDVKSIHFDSAWVPYTNFSPIYEGKCGMSGGRVEGKVIYETQSTHKLLAAFSQASMIHVKGDVNEETFNEAYMMHTTTSPHYGVVASTETAAAMMKGNAGKRLIDGSIERSIKFRKEIKRLKGESDGWFFDVRQPEHIDGAECWPLRSDSAWHGFKNIDNEHMYLDPIKVTLLTPGMKKDGTMDDFGIPASIVAKYLDEHGIVVEKTGPYNLLFLFSIGIDKTKALSLLRALTDFKRAFDLNLRVKNMLPSLYREDPEFYENMRVQELAQNIHKLIEHHNLPDLMFRAFVVLPTMMITPYAAFQKELHGQTEEVYLEEMVGRVNANMILPYPPGVPLVMPGEMITEESRPVLEFLQMLCEIGAHYPGFETDIHGAYRQADGRYTVKVLREENNK

[0090] SEQ ID NO:4

[0091]

Claims

1. An L-lysine decarboxylase mutant, the amino acid sequence of which is shown in SEQ ID NO:

3.

2. A nucleic acid molecule encoding the L-lysine decarboxylase mutant of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

4.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. A recombinant cell comprising the recombinant vector of claim 3.

5. A method for preparing an L-lysine decarboxylase mutant, comprising the following steps: The recombinant cells described in claim 4 were induced and cultured to obtain a culture. The L-lysine decarboxylase mutant of claim 1 was isolated from the culture.

6. The use of the L-lysine decarboxylase mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, the recombinant cell of claim 4, or the L-lysine decarboxylase mutant prepared by the preparation method of claim 5 in the preparation of 1,5-pentanediamine.

7. A method for preparing 1,5-pentanediamine, comprising the following steps: using the L-lysine decarboxylase mutant of claim 1, the recombinant cell of claim 4, or the L-lysine decarboxylase mutant prepared by the method of claim 5 as a catalyst to catalyze the reaction of L-lysine to obtain 1,5-pentanediamine.

8. The preparation method according to claim 7, characterized in that: In the catalytic reaction, the temperature is 20-30°C, and the catalytic reaction includes L-lysine and pyridoxal phosphate, wherein L-lysine is used as the substrate and pyridoxal phosphate is used as the coenzyme.

9. The preparation method according to claim 8, characterized in that: The final concentration of L-lysine is 200-300 g / L, and the final concentration of pyridoxal phosphate is 0.005-0.015 M.

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