An L-lysine decarboxylase derived from Pseudomonas mendocina and its application

Abstract

The application discloses L-lysine decarboxylase derived from Pseudomonas mendocina 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 Pseudomonas mendocina and its application. Background Technology

[0002] 1,5-Pentanediamine, also known as cadaverine, has the molecular formula C5H. 14 N2, with a molecular weight of 102.18, is flammable, toxic, and highly irritating. 1,5-Pentanediamine has a wide range of applications. Industrially, as an important industrial chemical raw material, its derived bio-polyamides can be widely used in various polyamide products. It can replace hexamethylenediamine, a bio-amine produced by traditional chemical methods, and can be polymerized with diacids to synthesize high-quality polymer materials—novel nylon 56. In agriculture, it can be used to regulate plant senescence, promote the development of stamens and pistils, improve fruit development, and increase fruit yield. In medicine, it is an effective treatment for dysentery and also an important pharmaceutical intermediate.

[0003] The chemical synthesis of 1,5-pentanediamine mostly uses non-renewable petroleum as a raw material, which not only severely pollutes the environment and hinders sustainable development, but also faces problems such as petroleum resource depletion and price increases. Therefore, the bio-enzymatic synthesis of 1,5-pentanediamine is of great significance for the synthesis of high molecular polymers such as polyamides and polyurethanes. 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-pentanediamine. In recent years, biocatalysis has mainly used L-lysine degrading enzymes derived from Escherichia coli, but the activity of L-lysine degrading enzymes decreases sharply when the pH increases. Since both L-lysine and 1,5-pentanediamine are alkaline substances, L-lysine decarboxylase needs to tolerate a high pH. Summary of the Invention

[0004] The purpose of this invention is to provide an L-lysine decarboxylase derived from Pseudomonas mendocina, in order to solve the problems of unsatisfactory performance of existing L-lysine decarboxylases in terms of activity and alkali resistance.

[0005] Specifically, the present invention obtains L-lysine decarboxylase from Pseudomonas mendocina, 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 alkali resistance, 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 alkali resistance 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: Met at position 79 is mutated to Arg, Ala at position 140 is mutated to Val, Ala at position 229 is mutated to Val, Ala at position 369 is mutated to Val, Val at position 406 is mutated to Ala, Arg at position 467 is mutated to Pro, Ala at position 508 is mutated to Val, and Ala at position 611 is mutated to Gly.

[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-pmLDC or pET-ΔpmLDC108, 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 108, 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 enables 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 pmLDC;

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

[0029] (3) Construction of recombinant expression plasmids pET-pmLDC and pET-ΔpmLDC;

[0030] (4) Transform the recombinant expression plasmids pET-pmLDC and pET-ΔpmLDC 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 the above-mentioned L-lysine decarboxylase, a mutant of the above-mentioned L-lysine decarboxylase, the above-mentioned recombinant cells and / or the L-lysine decarboxylase or a mutant thereof 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°C-30°C, for example 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°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 g / L to 300 g / L, such as 200 g / L, 210 g / L, 220 g / L, 225 g / L, 230 g / L, 240 g / L, 250 g / L, 260 g / L, 270 g / L, 280 g / L, 290 g / L, 300 g / L, or any value or range between these values, preferably 250 g / L;

[0041] The final concentration of pyridoxal phosphate is 0.005M-0.015M, for example 0.005M, 0.006M, 0.007M, 0.008M, 0.009M, 0.01M, 0.015M, 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 described above 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, for example, 250 mg / g L-lysine, 300 mg / g L-lysine, 350 mg / g L-lysine, 400 mg / g L-lysine, 450 mg / g L-lysine, 500 mg / g L-lysine, 550 mg / g L-lysine, 600 mg / g L-lysine, 650 mg / g L-lysine, 700 mg / g L-lysine, 750 mg / g L-lysine, or any value or range between these values, 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 89.4% and an enzyme activity of 1042.6 U / L, which is 2.15 times that of the original enzyme. It also has better alkali resistance than the original enzyme.

[0046] In particular, this invention uses an L-lysine decarboxylase derived from Pseudomonas mendocina as the original sequence to obtain a mutant enzyme that enhances the activity and alkali resistance of L-lysine decarboxylase. This can reduce the amount of catalyst used or shorten the reaction time, and it can tolerate higher pH values, thereby further reducing production costs and promoting the industrialization of biological production of 1,5-pentanediamine and nylon 56 salt. Attached Figure Description

[0047] Figure 1 The 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] Pseudomonas mendocina was disclosed in the literature “Yen KM, Karl MR, Blatt LM, et al. Cloning and characterization of a Pseudomonas mendocina KR1 genecluster encoding toluene-4-monooxygenase.[J]. Journal of Bacteriology, 1991, 173(17):5315-5327.” 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 from Pseudomonas mendocina

[0055] 1. Extract genomic DNA from Pseudomonas mendocina.

[0056] 2. Using the genomic DNA obtained in step 1 as a template, PCR was performed using primer 1 (5'-gcggataacaattcccctctagaatgtataaagatctcaagtttc-3', SEQ ID NO:5) and primer 2 (5'-gacggagctcgaattcggatccttattcgaccaggcagtcca-3', SEQ ID NO:6) to obtain a PCR amplification fragment containing the L-lysine decarboxylase gene pmLDC. 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 ΔpmLDC1-ΔpmLDC125.

[0060] The error-prone PCR reaction system consisted of: 5 μl of 10× amplification buffer, 4 μl each of the four dNTP mixtures (2.5 mmol / L), 50 pmol of each primer, 1.5 μg of template DNA, 0.5 μL of Taq DNA polymerase, and Mg... 2+ 7 mmol / L, add double-distilled water to 50 μl.

[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-pmLDC. The plasmid was sent for sequencing, and the results were consistent with expectations.

[0063] 2. The recombinant expression plasmid pET-pmLDC obtained in step 1 was heat-shocked into E. coli DH5α competent cells and cultured 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-pmLDC plasmid was obtained. The obtained plasmid was chemically transformed into E. coli BL21(DE3) and screened on LB solid medium containing 25 μg / mL kanamycin 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-ΔpmLDC1-pET-ΔpmLDC125 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 bacterial culture was diluted with water to an OD value of 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, and 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] Take 5 μL of the obtained reaction solution and add water to make up to 50 μL. Add the mixture to a 96-well plate with 50 μL of 1 mol / L Na2CO3 aqueous solution and 50 μL of 10 mol / L TNBs (2,4,6-trinitrobenzenesulfonic acid) aqueous solution and react at 42 °C for 6 min. Then, take 100 μL of the cooled reaction mixture and add it to a 96-well deep plate. Add 500 μL of toluene and shake to extract for 2 min. Allow the mixture to stand at room temperature for 3 min to separate the phases. Pipette 100 μL of the upper extract and 100 μL of anhydrous ethanol into the 96-well plate and mix them. Measure the absorbance at 340 nm using a microplate reader.

[0069] The strain corresponding to the reaction solution with the highest absorbance (recombinant strain 108) 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: Met at position 79 is mutated to Arg, Ala at position 140 is mutated to Val, Ala at position 229 is mutated to Val, Ala at position 369 is mutated to Val, Val at position 406 is mutated to Ala, Arg at position 467 is mutated to Pro, Ala at position 508 is mutated to Val, and Ala at position 611 is mutated to Gly.

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

[0071] The recombinant strain W obtained in Example 3 and the recombinant strain 108 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 485.6 U / L and 1042.6 U / L, respectively. Compared with the original enzyme, the activity of the mutant enzyme was 2.15 times. HPLC analysis of the residual L-lysine yielded conversion rates of 50.8% and 89.4% 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 3.768 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: Alkali tolerance 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 them according to the reaction conditions in Example 5. Adjust the initial pH value to 7, 8, 9, 10 and 11 respectively. The conversion rates of the two enzymes in each reaction are shown in Table 1.

[0080] Table 1

[0081]

[0082] The results showed that the mutant enzyme had better alkali resistance. Even at an initial pH of 11, the conversion rate could still reach over 80%, which was better than that of the original enzyme. After the reaction was carried out at pH 11, the activity of the original enzyme decreased by 49.21%, while the activity of the mutant enzyme only decreased by 9.5%.

[0083] sequence

[0084] SEQ ID NO:1

[0085] MYKDLKFPVLIVHRDIKADTVAGDRVRAIAQELTQDGFSILPTANAAEGRIVASTHHGLACILVAAEGAGENSRLLQDMVELIRVARVRAPQLPIFALGEQVTIENAPAEAMADLNHLRGILYLYEDTVSFLARQVARAAHNYLDGLLPPFFKALVQHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEARAARNFGADHTFFVINGTSTANKIVWHSMVGRDDLVLVDRNCHKSILHSIIMTGAIPLYLCPERNELGIIGPIPLSEFSRESIQAKIEASPLARGRAPKVKLAVVTNSTYDGLCYNAEMVKQALGDSVEVLHFDEAWYAYAAFHEFYAGRYGMGTQCDEQSPLVFTTHSTHKLLAAFSQASMIHVQDGGQRQLDRDRFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRRALANVRRNLSAEDWWFSIWQPGAADGADSLSTADWVLQPDADWHGFGEVASDYVLLDPIKVTLVMPGLNAAGKLEQQGIPAAVVSKFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLLTELLEFKRSYDANLPLIDVLPSIAHAGGGRYQGMGLRDLCDALHGCYRENATAKALKSMYTALPELAIKPADAYDRLVRGEVEAVPIDQLQGRIAAVMLVPYPPGIPLIMPGERFTAATRSILHYLSFARTFDQAFPGFDIDVHGLQTEAGEYCVDCLVE

[0086] SEQ ID NO:2

[0087]

[0088] SEQ ID NO:3

[0089] MYKDLKFPVLIVHRDIKADTVAGDRVRAIAQELTQDGFSILPTANAAEGRIVASTHHGLACILVAAEGAGENSRLLQDRVELIRVARVRAPQLPIFALGEQVTIENAPAEAMADLNHLRGILYLYEDTVSFLARQVARAVHNYLDGLLPPFFKALVQHTAQSNYSWHTPGHGGGVAYRKSPVGQAFHQFFGENTLRSDLSVSVPELGSLLDHTGPLAEAEARAARNFGVDHTFFVINGTSTANKIVWHSMVGRDDLVLVDRNCHKSILHSIIMTGAIPLYLCPERNELGIIGPIPLSEFSRESIQAKIEASPLARGRAPKVKLAVVTNSTYDGLCYNAEMVKQALGDSVEVLHFDEAWYAYAAFHEFYVGRYGMGTQCDEQSPLVFTTHSTHKLLAAFSQASMIHAQDGGQRQLDRDRFNEAFMMHISTSPQYGIIASLDVASAMMEGPAGRSLIQETFDEALSFRPALANVRRNLSAEDWWFSIWQPGAADGADSLSTADWVLQPDVDWHGFGEVASDYVLLDPIKVTLVMPGLNAAGKLEQQGIPAAVVSKFLWERGLVVEKTGLYSFLVLFSMGITKGKWSTLLTELLEFKRSYDANLPLIDVLPSIGHAGGGRYQGMGLRDLCDALHGCYRENATAKALKSMYTALPELAIKPADAYDRLVRGEVEAVPIDQLQGRIAAVMLVPYPPGIPLIMPGERFTAATRSILHYLSFARTFDQAFPGFDIDVHGLQTEAGEYCVDCLVE

[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: The temperature in the catalytic reaction is 20-30°C.

9. The preparation method according to claim 7, characterized in that: The catalytic reaction comprises L-lysine and pyridoxal phosphate.

10. The preparation method according to claim 9, 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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