Recombinant escherichia coli for producing L-lysine decarboxylase LDC1 and application of recombinant escherichia coli in synthesis of pentamethylene diamine
By screening and optimizing the L-lysine decarboxylase LDC1 of Panasonic 1.19 and constructing recombinant E. coli, the problem of the decline in activity of existing enzymes under alkaline conditions was solved, and the production of 1,5-pentanediamine was achieved efficiently, which was suitable for industrial applications.
Patent Information
- Application Number
- CN202510274586.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-10
- Publication Date
- 2025-04-25
AI Technical Summary
The existing L-lysine decarboxylase has decreased activity under alkaline conditions and insufficient stability, resulting in limited use in industrial applications.
L-lysine decarboxylase LDC1 derived from Panasonic 1.19 was obtained through screening, and its nucleotide sequence was optimized to construct recombinant E. coli that can express LDC1, improving its activity and stability under alkaline conditions.
The production of 1,5-pentanediamine was achieved with high efficiency catalyzing of L-lysine decarboxylase LDC1 at high concentrations of lysine, with the output increased from 12.3g/L to 62.2g/L, and maintained 60% activity at pH 8, which is suitable for industrial production.
Smart Images
Figure CN119876202A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of genetic engineering bacteria and relates to a recombinant Escherichia coli producing L-lysine decarboxylase LDC1 and application of the recombinant Escherichia coli in the synthesis of pentamethylenediamine. Background Art
[0002] 1,5-Pentanediamine is an important industrial bio-based platform chemical. Due to the increasing concern about resource consumption and environmental issues, it has become inevitable to produce bio-based industrial products such as biofuels and biopolymers through sustainable "green" routes to replace petrochemical products. 1,5-Pentanediamine is a five-carbon diamine, known as one of the most promising bio-based platform chemicals, with great industrial application value. Its applications mainly include as a basic raw material for polyamide (commonly known as nylon), promoting plant growth, and as a promoter, chelating agent, polyurethane, additive, and antidiarrheal drug in the medical field.
[0003] Traditional production of pentamethylenediamine relies on chemical synthesis methods, which not only consume a large amount of fossil resources, but also cause environmental pollution, are not in line with the principle of sustainable development, and face a series of problems such as depletion of oil resources and rising prices.
[0004] 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. With the development of biotechnology, the use of LDC to catalyze the production of 1,5-pentanediamine through biological pathways has become an emerging environmentally friendly way to produce pentamethylenediamine. At present, LDCs from microorganisms such as Hafnia alvei, Klebsiella quasipneumoniae and Escherichia coli have been widely studied. However, the activity of these L-lysine decarboxylases is generally susceptible to pH and is easily inactivated under alkaline conditions. In addition, the stability of wild-type L-lysine decarboxylase under alkaline conditions still has a lot of room for improvement. Therefore, there are still few L-lysine decarboxylases that can be used for industrial applications. For example, OSIRE Tolbert et al. reported an L-lysine decarboxylase SmcadA, which has the highest activity at pH 6.0, and when the pH increases, the activity decreases (OSIRE Tolbert, YANG Taowei, QIAO Zhina, et al. Molecular modification of lysine decarboxylase for catalytic synthesis of pentanediamine [J]. Food and Fermentation Industries, 2022, 48 (1): 8-14). The optimal pH of the L-lysine decarboxylase HaLDC from Hafnia alvei is 6.5, and the alkaline condition is not reached (LIU SM, QI HS. Molecular engineering and immobilization of lysine decarboxylase for synthesis of 1,5-diaminopentane: areview. Chin J Biotech, 2022, 38 (12): 4403-4419.). Therefore, the development of a new LDC with high catalytic activity and high stability is of great significance for the industrial production of pentamethylenediamine. So far, there has been no relevant research report on L-lysine decarboxylase from Pantoea sp.1.19. Summary of the invention
[0005] The present invention obtains an L-lysine decarboxylase LDC1 from Pantoea 1.19 through screening, optimizes its nucleotide sequence, and constructs a recombinant Escherichia coli that can express LDC1, so that it can efficiently catalyze L-lysine to produce 1,5-pentanediamine.
[0006] The technical solution of the present invention is as follows:
[0007] The nucleotide sequence of the LDC1 gene encoding L-lysine decarboxylase derived from Pantoea 1.19 is shown in SEQ ID No.2.
[0008] A recombinant expression vector is a recombinant expression vector pET28a-LDC1 containing a nucleotide sequence as shown in SEQ ID No. 2 and encoding an L-lysine decarboxylase LDC1 gene derived from Pantoea 1.19.
[0009] The method for constructing the above-mentioned recombinant expression vector specifically comprises the following steps: performing high-fidelity PCR amplification on the L-lysine decarboxylase LDC1 gene sequence derived from Pantoea 1.19, inserting the target fragment after gel recovery and purification into the EcoRI and XhoI restriction sites of the pET28a vector, and obtaining the recombinant expression vector pET28a-LDC1 after verification by colony PCR and sequencing.
[0010] The recombinant Escherichia coli producing the above-mentioned L-lysine decarboxylase LDC1 contains the above-mentioned recombinant expression vector.
[0011] The specific steps of the construction method of the above recombinant Escherichia coli are as follows:
[0012] The above recombinant expression vector pET28a-LDC1 was linearized by restriction enzyme digestion and then transformed into competent Escherichia coli BL21 (DE3). After resistance screening, the correct transformants were selected by colony PCR to obtain recombinant Escherichia coli BL21 (DE3)-LDC1.
[0013] The application of the above recombinant Escherichia coli in the production of L-lysine decarboxylase LDC1 comprises the following specific steps:
[0014] The above recombinant E. coli was inoculated into the culture medium and cultured until the bacterial OD 600 After the pH value is 0.6-0.8, IPTG is added to a final concentration of 0.5-0.7 mM for induction. The induction temperature is 25-30°C and the induction time is 16-18 hours. After the induction, the wet cells are collected by centrifugation, the cell pellet is resuspended and ultrasonically broken, the supernatant is collected by centrifugation and purified to obtain L-lysine decarboxylase LDC1.
[0015] The application of the above recombinant Escherichia coli in the synthesis of pentamethylenediamine comprises the following specific steps:
[0016] The above recombinant E. coli was inoculated into the culture medium and cultured until the bacterial OD 600After the pH value reaches 0.5-0.8, IPTG is added to a final concentration of 0.5-0.7 mM for induction, and L-lysine and coenzyme pyridoxal phosphate (PLP) are added to continue the culture to synthesize pentamethylenediamine.
[0017] Furthermore, the induction temperature was 37°C and the induction time was 16 h.
[0018] Furthermore, the final concentration of L-lysine was 100 g / L, and the final concentration of the coenzyme pyridoxal phosphate was 0.1 mM.
[0019] The L-lysine decarboxylase LDC1 catalytic reaction system of the present invention has a fast reaction speed and a high conversion rate. Through the optimization of the whole cell induction conditions, under the condition of L-lysine concentration of 100 g / L, after 24 hours of reaction, the 1,5-pentanediamine production is increased from 12.3 g / L to 62.2 g / L, showing a significant increase in production. The L-lysine decarboxylase of the present invention has high activity and alkaline resistance, with 60% activity remaining after incubation for 12 hours at pH 8, and is not easily inactivated under alkaline conditions, and has a very broad application prospect. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 Schematic diagram of the construction of recombinant plasmid pET28a(+)-LDC1.
[0021] Figure 2 It is the SDS-PAGE analysis profile of the crude enzyme solution, wherein 1: LDC1 expression at 20°C (fermentation solution after centrifugation), 2, 4: LDC1 expression at 20°C (cell lysate supernatant), 3, 5: LDC1 expression at 20°C (cell lysate precipitate), M: marker.
[0022] Figure 3 The optimal pH (a) and pH stability test diagram (b) of L-lysine decarboxylase LDC1.
[0023] Figure 4 The optimum temperature (a) and temperature stability test diagram (b) of L-lysine decarboxylase LDC1.
[0024] Figure 5 The graph shows the optimization results of L-lysine decarboxylase LDC1 induction time (a), induction temperature (b) and IPTG concentration (c).
[0025] Figure 6Figure 1 shows the optimization of the whole-cell catalytic conditions of L-lysine decarboxylase LDC1. The yield of 1,5-pentanediamine after 24 h of reaction at a concentration of 100 g / L L-lysine. (a) shows the L-lysine consumption at different induction times, (b) shows the 1,5-pentanediamine yield at different induction times, (c) shows the L-lysine consumption and 1,5-pentanediamine yield at different induction temperatures, and (d) shows the yield at different inoculation OD values. 600 L-lysine consumption and 1,5-pentanediamine production. DETAILED DESCRIPTION
[0026] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.
[0027] Example 1
[0028] 1. Construction of recombinant expression vector pET28a-LDC1:
[0029] The amino acid sequence of L-lysine decarboxylase LDC1 from Pantoea 1.19 is shown in SEQ ID No.1. The coding gene sequence of L-lysine decarboxylase LDC1 was obtained by codon optimization of its coding gene, and the nucleotide sequence of L-lysine decarboxylase LDC1 coding gene was synthesized by Nine Days Gene Company (SEQ ID No.2), which was inserted between the EcoRI and XhoI restriction sites of the pET28a vector to obtain the recombinant expression vector pET28a-LDC1, which was verified to be correct by sequencing. The schematic diagram of the construction of the recombinant expression vector pET28a-LDC1 is shown in Figure 1 .
[0030] 2. Preparation of recombinant Escherichia coli:
[0031] Take out the competent E. coli BL21 (DE3) cells from the -80℃ refrigerator and place them on ice for 5 minutes. Then take 1-2μL of the recombinant expression vector pET28a-LDC1 and add it to the competent cells. After mixing, place it in an ice bath for 30 minutes to allow the plasmid to attach to the surface of the competent cells. Then heat shock it in a 42℃ water bath for 60-90s, and quickly place it in an ice bath for 2 minutes. Then add 800μL of sterile LB culture medium and culture it in a shaker at 37℃ and 220rpm for 1 hour. Finally, centrifuge at 4000rpm for 5 minutes, discard the supernatant, apply the bacterial solution to the LB solid plate containing the corresponding antibiotic resistance, and culture it in an incubator at 37℃ overnight, inverted, for no more than 16 hours. Finally, the correct transformant was obtained by colony PCR to obtain the recombinant E. coli BL21 (DE3) -LDC1.
[0032] 3. Shake flask fermentation:
[0033] A single colony of recombinant E. coli BL21(DE3)-LDC1 was picked and inoculated into 10 mL of autoclaved medium. The medium formula was: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, ampicillin 100 mg / L. After culturing at 37°C and 200 rpm for 20 h, it was inoculated into 50 mL of autoclaved LB medium at a volume ratio of 1:100. The culture was continued at 37°C until the bacterial OD reached 0. 600 The pH value was 0.6-0.8, IPTG was added to a final concentration of 0.5mM, and after continuing to culture at 20°C and 200rpm for 20h, the culture solution was centrifuged at 4°C and 6000rpm for 5min to collect wet bacteria. Then, a PBS solution with a pH of 6.5 was added to resuspend the bacterial precipitate, and then ultrasonically disrupted. After the solution became clear and transparent, it was centrifuged at 4°C and 8000rpm for 6min to separate the supernatant and bacterial precipitate. The supernatant was used as the extracted crude enzyme solution. The protein concentration of the crude enzyme solution was measured by Bradford method to be 2.05mg / mL. The crude enzyme solution was detected by SDS-PAGE and stored in a refrigerator at 4°C.
[0034] The specific steps of SDS-PAGE detection are as follows: take 200 μL of cell disruption solution and centrifuge at 12,000 rpm for 5 minutes, aspirate the supernatant and place it in another clean centrifuge tube, use 200 μL of distilled water to wash the precipitate once and resuspend it. Take 20 μL of the supernatant and precipitate suspension in a PCR tube, add 5 μL of 5×Protein SDS-PAGE Loading Buffer, and use a PCR instrument to denature at 99°C for 10 minutes. The sample is concentrated and separated in a concentrated gel (5%) and a separation gel (8%) using an electrophoresis instrument. The obtained separation gel is stained for 10 minutes and decolorized until the protein bands are clear, and the molecular weight and expression of LDC in the gel are analyzed. After the purification, the LDCs are analyzed using the same method to observe the LDC collection and whether a single protein is obtained.
[0035] The results of SDS-PAGE are shown in Figure 2 .from Figure 2 It can be seen that the target protein is overexpressed in the recombinant E. coli BL21(DE3)-LDC1, and the protein size is about 70-80 kDa. This indicates that LDC1 exists in the supernatant of the cell lysate with a high concentration.
[0036] Example 2
[0037] Determination of the enzyme activity of L-lysine decarboxylase LDC1:
[0038] 1. Preparation of reaction mother solution: Prepare L-lysine solution with a concentration of 200 g / L, pyridoxal phosphate (PLP) solution with a concentration of 5 mM, adjust the pH of both to 6.5 with hydrochloric acid, and buffers of different pH values. The buffer of pH 7.0 is 100 mM citric acid-200 mM Na2HPO4 buffer, the buffer of pH 8.0 is 100 mM citric acid-200 mM Na2HPO4 buffer, and the buffer of pH 9.0 is 50 mM Tris-HCl buffer.
[0039] 2. Purification of L-lysine decarboxylase LDC1:
[0040] The protein purification method using nickel column, first of all, centrifuge the cell lysate at 8,000rpm for 10 minutes at 4℃ to remove the crude enzyme solution fragments, and take the supernatant for subsequent steps. At the same time, prepare the nickel column, and equilibrate the nickel NTA affinity chromatography column with 20mM Tris-HCl (pH 8.0) and 0.5M NaCl equilibration buffer in advance. Make sure the column remains moist during operation and avoids drying. Then mix the supernatant and nickel column, stir at low speed in an ice bath for 1-2h, so that the His-tagged protein can fully bind to the nickel ion. Then wash the column 1-2 times with pure water to remove unbound proteins and impurities. Subsequently, further wash with a washing buffer containing 20mM imidazole to remove non-specifically bound proteins. Finally, elute the target protein with elution buffer containing 50, 100, 200, 300, 400 and 500mM imidazole in sequence. Collect the eluate and use SDS-PAGE to analyze the purification effect. Finally, the nickel column was washed three times with pure water and stored in a 20% ethanol solution at low temperature.
[0041] 3. Reaction system: Enzyme activity detection was carried out in a 500 μL catalytic system, using 10 μg / mL LDC 1 to catalyze 50 g / L L-lysine added with 0.1 mM PLP, and reacted at 200 rpm for 10 min at pH 7.0, 8.0 and 9.0, temperature 37°C, and after the reaction, the reaction was terminated by boiling in a boiling water bath for 5 min. The remaining amount of L-lysine was detected and the enzyme activity was calculated, indicating that the enzyme activity of LDC1 was 20.5 U.
[0042] The enzyme activity was defined as: 1 U of enzyme was required to consume 1.0 μmol of L-lysine in 1 min under the assay conditions.
[0043] Example 3
[0044] Characterization of enzymatic properties of L-lysine decarboxylase LDC1:
[0045] 1. Study on the optimal pH and pH stability of L-lysine decarboxylase LDC1
[0046] (1) Optimum pH: The catalytic activity of LDC1 was tested in the pH range of 4.0-9.0. Figure 3 As shown in (a), when the pH is below 5.0, LDC1 has almost no catalytic activity. When the pH is 6.0, the relative enzyme activity increases rapidly to 25%, and then continues to increase with the increase of pH value. When it reaches 7.0, the enzyme activity reaches the maximum. Subsequently, as the pH further increases, the enzyme activity gradually decreases. From the results, it can be seen that the optimal catalytic pH of LDC1 is 7.0, and in the pH range of 7.0-9.0, the relative enzyme activity remains above 35%.
[0047] (2) pH stability: LDC1 was incubated at 37°C in a pH 4-9 buffer and the enzyme activity was measured after 12 h. Figure 3 (b) As shown in the figure, the residual enzyme activity of LDC1 was maintained above 60% in the pH range of 6.0-8.0.
[0048] 2. Study on the optimal temperature and temperature stability of L-lysine decarboxylase LDC1
[0049] (1) Optimum catalytic temperature: The optimum temperature of LDC1 was investigated at 37°C-62°C. The results are as follows Figure 4 As shown in (a), the catalytic activity increases with the increase of temperature. When the temperature reaches 42°C, the catalytic activity of LDC1 is the highest. However, as the temperature continues to rise, the enzyme activity of LDC1 gradually decreases. When the catalytic temperature is 47°C, the relative enzyme activity is only 40%.
[0050] (2) Temperature stability: The temperature stability of LDC1 is as follows Figure 4 As shown in (b), after incubation at 37-47℃ for 12h, the catalytic activity of the enzyme still remained above 80%; after incubation at 52℃, the catalytic activity remained at around 60%. In previous studies, the catalytic activity of L-lysine decarboxylase from E. coli MG1655 remained at 50% after incubation at 50℃ for 10h. In comparison, LDC1 has better temperature stability.
[0051] Example 4
[0052] Optimization of induction conditions for L-lysine decarboxylase LDC1:
[0053] In order to obtain the optimal expression conditions of LDC1, the induction temperature, induction time and induction agent IPTG concentration were optimized. The strain culture method was the same as in Example 1.
[0054] The temperature optimization results are as follows: Figure 5As shown in (a), when the induction temperature is 25°C, the enzyme activity reaches the highest level, and when the temperature is ≥30°C, the enzyme activity and total enzyme activity are relatively low. This may be because lowering the induction temperature can slow down the transcription and translation rates, which is conducive to the correct folding of proteins and the maintenance of activity.
[0055] The induction time has a direct impact on the amount of protein accumulation. The test results of different induction times are as follows Figure 5 As shown in (b), after adding IPTG, the induction time was 12h to 24h, and the enzyme activity gradually increased with the increase of induction time. When the induction time reached 16h, the LDC1 enzyme activity reached the highest (20.5U), which may be because at this time point, the cell growth and the expression of the target protein reached the best balance.
[0056] The results of optimizing the concentration of inducer IPTG are as follows Figure 5 As shown in (c), when the IPTG concentration is 0.5 mM, the catalytic activity of LDC1 reaches the maximum value, but as the inducer concentration increases, the enzyme activity level shows a gradual downward trend. Excessive IPTG concentration leads to a gradual increase in the induction intensity, and the protein cannot be properly folded after excessive translation to form inclusion bodies; it may also cause cytotoxicity, affecting cell growth and the expression of the target protein; while too low a concentration may not be able to fully induce the expression of the target gene.
[0057] Example 5
[0058] L-lysine decarboxylase LDC1 as catalyst for the preparation of 1,5-pentanediamine:
[0059] 1. Reaction system: Cultivate the strain under the optimal induction conditions (temperature 25°C, induction time 16h, inducer concentration 0.5mMIPTG) and add L-lysine to synthesize pentamethylenediamine. After obtaining the strain, add it to LB medium adjusted to pH 7.0 with dilute hydrochloric acid and containing appropriate amount of antibiotics, and incubate at 37°C until OD 600 After reaching 0.5, 0.5 mM IPTG was added for induction, and 100 g / L of L-lysine and a final concentration of 0.1 mM coenzyme pyridoxal phosphate (PLP) were added. After induction with IPTG, the culture was incubated at 30 ° C and 200 rpm for 24 h. Samples were taken to measure cell growth and analyze the concentration of substrates and products.
[0060] To investigate the catalytic performance of LDC1, the catalytic ability of whole-cell LDC1 was investigated using 100 g / L L-lysine as substrate. Under the initial conditions (temperature 25°C, induction time 16 h, IPTG concentration 0.5 mM), the yield of pentamethylenediamine was only 12.3 g / L. By optimizing the whole-cell induction conditions, under the optimal conditions (induction time 16 h, temperature 37°C, inoculation OD 6004) The production of pentamethylenediamine increased to 62.2 g / L.
[0061] The HPLC detection method of pentamethylenediamine is as follows: pentamethylenediamine is treated by pre-column derivatization. Take 1 mL of fermentation broth, add 300 μL of NaHCO3-NaOH buffer at pH 9.5, then add 1 mL of 5 mg / mL dansyl chloride solution prepared with acetone, mix well and react the mixed solution at 40°C and 150 rpm for 45 minutes to obtain pentamethylenediamine derivatives. Then add 200 μL of 25% ammonia water to terminate the derivatization reaction. Finally, add 2.7 mL of methanol to make up the total volume to 5 mL. Finally, filter through a 0.45 μm filter membrane for testing.
[0062] The HPLC detection conditions are as follows: column model: Shimadzu C18 column (4.6×250mm, 5μm); mobile phase: methanol: water = 8:2 (volume ratio); flow rate: 1mL / min; column temperature: 40℃; detection wavelength: 254nm; injection volume: 10μL; peak elution time: about 5.5min; detector: UV detector.
[0063] The results are as follows Figure 6 As shown in the figure, the optimal catalytic conditions for LDC1 whole cells can be seen. Figure 6 As shown in (a) and (b), the highest yield of 1,5-pentanediamine is 12.5 g / L at an induction time of 16 h; while the 1,5-pentanediamine synthesized at other induction times is less than 11 g / L. Temperature is a key factor affecting enzyme activity and stability. At induction temperatures of 16, 20, 24, 30, 37, and 42 °C, the whole cell catalytic production of 1,5-pentanediamine showed different effects. Figure 6 As shown in (c), 37°C was determined to be the optimal induction temperature, and the yield of 1,5-pentanediamine was significantly increased to 50.1 g / L. 600 Reflects the density of the bacteria and is proportional to the number of cells. 600 4 is one of the best conditions determined in this embodiment, such as Figure 6 (d) as shown.
[0064] The above results show that the L-lysine decarboxylase LDC1 of the present invention has high catalytic ability, fast reaction speed, and is suitable for reaction at high lysine concentration. Under the above conditions, the yield of pentamethylenediamine under the optimal catalytic conditions of the whole cell is increased from 12.3 g / L to 62.2 g / L. The recombinant Escherichia coli containing L-lysine decarboxylase LDC1 of the present invention can be used for industrial large-scale production of 1,5-pentamethylenediamine.
[0065] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A nucleotide sequence encoding the LDC1 gene of L-lysine decarboxylase derived from Pantoea 1.19, characterized in that: As shown in SEQ ID No.
2.
2. A recombinant expression vector, characterized in that: The invention relates to a recombinant expression vector pET28a-LDC1 containing the nucleotide sequence of claim 1.
3. The method for constructing a recombinant expression vector according to claim 2, characterized in that: The specific steps are: performing high-fidelity PCR amplification on the nucleotide sequence described in claim 1, inserting the target fragment after gel recovery and purification into the EcoRI and XhoI restriction sites of the pET28a vector, and obtaining the recombinant expression vector pET28a-LDC1 after verification by colony PCR and sequencing.
4. A recombinant Escherichia coli producing L-lysine decarboxylase LDC1, characterized in that: It contains the recombinant expression vector according to claim 2.
5. The method for constructing a recombinant Escherichia coli according to claim 4, characterized in that: The specific steps are as follows: The recombinant expression vector pET28a-LDC1 according to claim 2 is linearized by restriction enzyme digestion and then transformed into competent Escherichia coli BL21 (DE3). After resistance screening, the correct transformants are selected by colony PCR to obtain recombinant Escherichia coli BL21 (DE3)-LDC1.
6. Use of the recombinant Escherichia coli according to claim 4 in producing L-lysine decarboxylase LDC1, characterized in that: The specific steps are as follows: The recombinant Escherichia coli of claim 4 is inoculated into the culture medium and cultured until the bacterial OD 600 After the pH value is 0.6-0.8, IPTG is added to a final concentration of 0.5-0.7 mM for induction. The induction temperature is 25-30°C and the induction time is 16-18 hours. After the induction, the wet cells are collected by centrifugation, the cell pellet is resuspended and ultrasonically broken, the supernatant is collected by centrifugation and purified to obtain L-lysine decarboxylase LDC1.
7. Use of the recombinant Escherichia coli according to claim 4 in the synthesis of pentamethylenediamine, characterized in that: The specific steps are as follows: The recombinant Escherichia coli of claim 4 is inoculated into the culture medium and cultured until the bacterial OD 600 After the concentration of IPTG reached 0.5-0.8, IPTG was added to a final concentration of 0.5-0.7 mM for induction. L-lysine and coenzyme pyridoxal phosphate were added at the same time, and the culture was continued to synthesize pentamethylenediamine.
8. The use according to claim 7, characterized in that: The induction temperature was 37°C and the induction time was 16 h.
9. The use according to claim 7, characterized in that: The total concentration of L-lysine was 100 g / L, and the final concentration of coenzyme pyridoxal phosphate was 0.1 mM.