Recombinant vector for expressing LPL, recombinant bacterium and LPL production method

Abstract

The invention provides a recombinant vector for expressing LPL, a recombinant bacterium and an LPL production method, and belongs to the technical field of gene engineering. The invention provides a recombinant expression vector of Pseudomonas aeruginosa lipoprotein lipase LPL, wherein an LPL coding gene is derived from Pseudomonas aeruginosa. The recombinant expression vector is introduced into a pseudomonas aeruginosa PAO1 cell, an expression strain is successfully constructed, the expression quantity of target protein is improved, efficient expression of LPL in PAO1 is achieved, and the produced LPL is high in enzyme activity and wider in application range.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant vector for expressing LPL, a recombinant bacterium and a method for producing LPL. Background Art

[0002] Lipases are important industrial hydrolases that catalyze the hydrolysis of various forms of long-chain triacylglycerides to form fatty acids and glycerol. Lipases can catalyze the hydrolysis of oils and fats at the oil-water interface and can also undergo complex catalytic reactions such as esterification, transesterification, transesterification, and acidolysis in both microaqueous and non-aqueous phases. Therefore, lipases are considered one of the most important catalysts in the biological field and have great commercial value in food, detergents, feed, and biodiesel.

[0003] Unlike common lipases, lipoprotein lipase, derived from Pseudomonas aeruginosa, is a specialized triglyceride proteolytic enzyme that hydrolyzes triglycerides in chylomicrons and other low-density lipoproteins. In clinical diagnostic medicine, lipoprotein lipase is widely used to quantify triglycerides in serum and other samples.

[0004] However, the expression of large amounts of active lipoprotein lipase has always been a problem that has plagued the industry. Conventional breeding, fermentation condition optimization, heterologous expression and other strategies have not been able to fundamentally improve its production. There are two main reasons for this: First, common model host cells often do not have a homologous secretion system corresponding to lipoprotein lipase. Large amounts of expressed enzymes are likely to be highly toxic to the host cells and die prematurely, or they are mostly expressed as inclusion bodies (to avoid toxicity to the host cells), requiring complex and tedious renaturation steps to obtain enzyme proteins with poor folding and low enzyme activity; second, the lipoprotein lipase gene has a homologous folding enzyme gene, which requires the action of the homologous folding enzyme to form an active protein with the correct conformation, but heterologous host cells often do not have the corresponding folding enzyme. Summary of the Invention

[0005] The present invention provides a recombinant vector for expressing LPL, a recombinant bacterium and an LPL production method. Pseudomonas aeruginosa is used as an expression cell, which can significantly improve the expression amount and enzyme activity of LPL.

[0006] The present invention provides a recombinant vector for expressing Pseudomonas aeruginosa lipoprotein lipase. The amino acid sequence of the Pseudomonas aeruginosa lipoprotein lipase is shown in SEQ ID No. 1.

[0007] In a preferred embodiment of the present invention, the base vector of the recombinant vector comprises a lac promoter.

[0008] In a preferred embodiment of the present invention, the basic vector of the recombinant vector comprises the shuttle vector pBBR1MCS-5.

[0009] In a preferred embodiment of the present invention, in the recombinant vector, the gene encoding Pseudomonas aeruginosa lipoprotein lipase is inserted between HindIII and BamHI of the basic vector.

[0010] The present invention also provides a recombinant Pseudomonas aeruginosa comprising the above recombinant vector and expressing Pseudomonas aeruginosa lipoprotein lipase.

[0011] The present invention also provides a method for constructing the above-mentioned recombinant Pseudomonas aeruginosa, comprising the following steps: inserting the coding gene of Pseudomonas aeruginosa lipoprotein lipase into the shuttle vector pBBR1MCS-5 to construct a recombinant vector, and transferring the recombinant vector into the Pseudomonas aeruginosa host bacteria to construct the recombinant Pseudomonas aeruginosa.

[0012] In a preferred embodiment of the present invention, the coding gene is amplified using Pseudomonas aeruginosa PAO1 as a template and an upstream primer having a nucleotide sequence as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No. 4.

[0013] In a preferred embodiment of the present invention, the Pseudomonas aeruginosa host bacteria includes Pseudomonas aeruginosa PAO1.

[0014] The present invention also provides a method for producing Pseudomonas aeruginosa lipoprotein lipase, comprising culturing the recombinant Pseudomonas aeruginosa, wherein the culture solution contains the Pseudomonas aeruginosa lipoprotein lipase;

[0015] The recombinant Pseudomonas aeruginosa includes the above-mentioned recombinant Pseudomonas aeruginosa or the recombinant Pseudomonas aeruginosa constructed using the above-mentioned construction method.

[0016] In a preferred embodiment of the present invention, after obtaining the culture fluid, it further comprises ion exchange chromatography purification.

[0017] Beneficial Effects: The present invention provides a recombinant expression vector for Pseudomonas aeruginosa lipoprotein lipase (LPL), wherein the LPL encoding gene is derived from Pseudomonas aeruginosa. The present invention successfully constructs an expression strain by introducing the recombinant expression vector into Pseudomonas aeruginosa PAO1 cells, thereby increasing the expression level of the target protein and achieving efficient expression of LPL in PAO1 cells.

[0018] In the embodiment of the present invention, Pseudomonas aeruginosa PAO1 was selected as the host bacteria for homologous expression, and the LPL gene sequence was constructed into the expression vector pBBR1MCS-5, thereby achieving efficient expression of LPL in Pseudomonas aeruginosa PAO1. The produced LPL has high enzyme activity and a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the results of colony PCR verification of PAO1-pBBR1MCS-5-LPL. In the figure, M: DNA Marker; 1-3: colony PCR verification results of three PAO1-pBBR1MCS-5-LPL single colonies; CK: negative control, using water as the colony PCR template;

[0020] Figure 2 This is the result of colony PCR verification of Ecoli.BL21(DE3)-pBBR1MCS-5-LPL, where M is DNA Marker; 1-3 are the results of colony PCR verification of three Ecoli.BL21(DE3)-pBBR1MCS-5-LPL single colonies; CK is the negative control, using water as the template for colony PCR.

[0021] Figure 3 This is the SDS-PAGE identification result of PAO1-pBBR1MCS-5-LPL, in which M: Protein Marker; Lane 1: LPL supernatant produced by fermentation of the PAO1-pBBR1MCS-5-LPL expression strain;

[0022] Figure 4 This is a diagram showing the SDS-PAGE identification results of Ecoli.BL21(DE3)-pBBR1MCS-5-LPL, where M: Protein Marker; Lane 1: LPL supernatant produced by fermentation of the Ecoli.BL21(DE3)-pBBR1MCS-5-LPL expression strain;

[0023] Figure 5 This is a diagram showing the SDS-PAGE purification results of PAO1-pBBR1MCS-5-LPL, where M: Protein Marker; Lane 1: purification results of LPL produced by fermentation of the PAO1-pBBR1MCS-5-LPL expression strain. DETAILED DESCRIPTION

[0024] The present invention provides a recombinant vector for expressing Pseudomonas aeruginosa lipoprotein lipase. The amino acid sequence of the Pseudomonas aeruginosa lipoprotein lipase is shown in SEQ ID No. 1.

[0025] The amino acid sequence of LPL of the present invention is shown in SEQ ID No. 1:

[0026] MKKKSLLPLGLAIGLASLAASPLIQASTYTQTKYPIALAHGALGFDNILGVDYWFGIPSALRRDGAQVYVTEVSQLDTSEVRGEQLLQQVEEIVALSGQPKVNLIGHSHGGPTIRYVAAVRPDLIASATSVGAPHKGSDTADFLRQIPPGSAEAAI LSGLVNSLGALISFLSSSGSTGTQNALGSLESLSSEGAARFNAKYPHGVPTSACGEGAYKVNGVSYYSWSGSSPLTNFLDPSDAFLGASSLTFKNGTANDGLVGTCSSHLGMVIRDNYKMNHLDEVNQVFGLTSLFEASPVSVYRQHANRLKNASL*;

[0027] The nucleotide sequence encoding the LPL is shown in SEQ ID No. 2:

[0028] ATGAAGAAGAAGAGCCTGCTGCCGCTGGGCCTGGCCATCGGCCTGGCCAGCCTGGCCGCCAGCCCGCTGATCCAGGCCAGCACCTACACCCAGACCAAGTACCCGATCGCCCTGGCCCACGGCGCCCTGGGCTTCGACAACATCCTGGGCGTGGACTACTGGTTCGGCATCCCGAGCGCCCTGCGCCGCGACGGCGCCCAGGTGTACGTGACCGAGGTGAGCCAGCTGGACACCAGCGAGGTGCGCGGCGAGCAGCTGCTGCAGCAGGTGGAGGAGATCGTGGCCCTGAGCGGCCAGCCGAAGGTGAACCTGATCGGCCACAGCCACGGCGGCCCGACCATCCGCTACGTGGCCGCCGTGCGCCCGGACCTGATCGCCAGCGCCACCAGCGTGGGCGCCCCGCACAAGGGCAGCGACACCGCCGACTTCCTGCGCCAGATCCCGCCGGGCAGCGCCGAGGCCGCCATCCTGAGCGGCCTGGTGAACAGCCTGGGCGCCCTGATCAGCTTCCTGAGCAGCGGCAGCACCGGCACCCAGAACGCCCTGGGCAGCCTGGAGAGCCTGAGCAGCGAGGGCGCCGCCCGCTTCAACGCCAAGTACCCGCACGGCGTGCCGACCAGCGCCTGCGGCGAGGGCGCCTACAAGGTGAACGGCGTGAGCTACTACAGCTGGAGCGGCAGCAGCCCGCTGACCAACTTCCTGGACCCGAGCGACGCCTTCCTGGGCGCCAGCAGCCTGACCTTCAAGAACGGCACCGCCAACGACGGCCTGGTGGGCACCTGCAGCAGCCACCTGGGCATGGTGATCCGCGACAACTACAAGATGAACCACCTGGACGAGGTGAACCAGGTGTTCGGCCTGACCAGCCTGTTCGAGGCCAGCCCGGTGAGCGTGTACCGCCAGCACGCCAACCGCCTGAAGAACGCCAGCCTGTAG。

[0029] The LPL described in the present invention is derived from Pseudomonas aeruginosa. When obtaining the coding gene of the LPL, the genomic DNA of Pseudomonas aeruginosa PAO1 was used as a template in the embodiment to design primers for amplification. The primers used for amplification are as follows:

[0030] Upstream primer (SEQ ID No. 3): cgacggtatcgataagcttATGAAGAAGAAGAGCCTGCT;

[0031] Downstream primer (SEQ ID No. 4): gctctagaactagtggatcccTACAGGCTGGCGTTCTTC.

[0032] When the amplification is performed in the present invention, the PCR program used includes: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 62°C for 15 seconds, extension at 72°C for 1 minute, 32 cycles; and further extension at 72°C for 5 minutes.

[0033] The present invention inserts the amplified LPL encoding gene LPL into a base vector containing the lac promoter. In one embodiment of the present invention, the shuttle vector pBBR1MCS-5 is used as the base vector, and pBBR1MCS-5 homologous sequences are added to both ends of the amplified LPL sequence for seamless cloning, with insertion sites at HindIII and BamHI.

[0034] The present invention also provides a recombinant Pseudomonas aeruginosa comprising the recombinant vector and expressing Pseudomonas aeruginosa lipoprotein lipase.

[0035] The present invention also provides a method for constructing the above-mentioned recombinant Pseudomonas aeruginosa, comprising the following steps: inserting the coding gene of Pseudomonas aeruginosa lipoprotein lipase into the shuttle vector pBBR1MCS-5 to construct a recombinant vector, and transferring the recombinant vector into the Pseudomonas aeruginosa host bacteria to construct the recombinant Pseudomonas aeruginosa.

[0036] The recombinant Pseudomonas aeruginosa described herein is obtained by transforming the PAO1 strain using the recombinant vector, using Pseudomonas aeruginosa as the expression host. The present invention does not specifically limit the transformation method; conventional transformation methods in the art may be used for transformation, such as electroporation in the examples.

[0037] The present invention also provides a method for producing Pseudomonas aeruginosa lipoprotein lipase, comprising culturing the recombinant Pseudomonas aeruginosa, wherein the culture solution contains the Pseudomonas aeruginosa lipoprotein lipase;

[0038] The recombinant Pseudomonas aeruginosa includes the above-mentioned recombinant Pseudomonas aeruginosa or the recombinant Pseudomonas aeruginosa constructed using the above-mentioned construction method.

[0039] The culture described herein includes a liquid culture medium based on LB liquid culture medium and containing gentamicin at a final concentration of 40 μg / mL. The culture is performed with an inoculum size of 1%. The culture described herein is accompanied by shaking at a frequency of 220 rpm, and the culture is performed under the shaking conditions, such as at 37° C. for 48 hours.

[0040] The present invention centrifuges the bacterial solution obtained after fermentation and collects the supernatant, which is the crude LPL enzyme solution. The centrifugation speed is 8000 rpm in the embodiment and the time is 5 minutes. After obtaining the crude LPL enzyme solution, the present invention further includes ion exchange chromatography purification.

[0041] To further illustrate the present invention, a recombinant vector expressing LPL, a recombinant bacterium and an LPL production method provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1 Extraction of Pseudomonas aeruginosa PAO1 genome

[0043] The genomic DNA of Pseudomonas aeruginosa PAO1 was extracted using a bacterial DNA extraction kit (Yisheng Biotechnology (Shanghai) Co., Ltd.), and its concentration and purity were detected by Nanodrop after 1% agarose gel electrophoresis. The DNA was stored at -20°C for future use.

[0044] Example 2 Construction of recombinant expression vector

[0045] The present invention designs primers to amplify the LPL gene based on the genomic DNA of Pseudomonas aeruginosa PAO1 (Taxonomy ID: 208964). The primers are the upstream primers and downstream primers shown in SEQ ID No. 3 to 4, and are commissioned to Wuxi Aorui Dongyuan Biotechnology Co., Ltd. for synthesis.

[0046] The amplified fragment containing the pBBR1MCS-5 homologous sequence was seamlessly cloned into the expression vector pBBR1MCS-5.

[0047] Take 10 μL of the seamlessly cloned product and add it to 100 μL of DH5α competent cells (Shanghai Weidi Biotechnology Co., Ltd.). Gently tap the tube to mix thoroughly. Incubate on ice for 25 minutes, then in a 42°C water bath for 45 seconds. Immediately transfer to ice for 2 minutes. Add 700 μL of antibiotic-free LB liquid medium, incubate at 37°C, 220 rpm for 1 hour, and centrifuge at 5000 rpm for 1 minute. Aspirate 600 μL of the remaining medium and mix thoroughly by pipetting. Then spread the remaining medium onto a gentamicin plate (40 μg / mL) and incubate inverted at 37°C for 12 hours. The next day, single colonies were picked from the plate for colony PCR to verify successful transformation with DH5α-pBBR1MCS-5-LPL. Simultaneously, shake the cells to extract the plasmid pBBR1MCS-5-LPL, which is the recombinant expression vector.

[0048] Example 3 Construction of LPL production strain

[0049] 3.1 Preparation of competent Pseudomonas aeruginosa PAO1

[0050] (1) The strain was inoculated into 2 mL of LB liquid medium and cultured overnight at 37°C and 220 rpm;

[0051] (2) Inoculate 50 mL of LB liquid medium at a 1% inoculum volume and culture at 37°C and 220 rpm for about 2 to 3 hours until the OD 600 About 0.5;

[0052] (3) Centrifuge the bacterial solution at 8000 rpm for 10 min at 4°C and discard the supernatant.

[0053] (4) Resuspend the pellet in 30 mL of pre-cooled 300 mM (10%) sucrose solution and wash 2 to 3 times;

[0054] (5) Finally, add 500 μL of 300 mM sucrose solution and resuspend. This completes the preparation of Pseudomonas aeruginosa competent cells. Aliquot the competent cells into 100 μL / tubes and store in a -80°C refrigerator.

[0055] 3.2 Electroporation of Pseudomonas aeruginosa PAO1

[0056] (1) Mix approximately 500 ng of the recombinant expression vector with 100 μL of Pseudomonas aeruginosa competent cells, gently tap the tube wall to mix, and let stand on ice for 10 min;

[0057] (2) Transfer the electroporation mixture into an electroporation cuvette under the electroporation conditions of 25 μF, 200 Ω, 2.5 kW, and electric shock;

[0058] (3) After electroporation, add 1 mL of LB liquid medium, transfer to a centrifuge tube, and incubate at 30°C and 100 rpm for 2 h;

[0059] (4) Spread the culture medium onto an LB plate containing gentamicin resistance and culture at 37°C overnight to obtain clones; pick the clones and perform colony PCR to verify whether they are PAO1-pBBR1MCS-5-LPL that has been successfully transformed. The results are as follows Figure 1 As shown, it proved that PAO1-pBBR1MCS-5-LPL was successfully transformed.

[0060] 3.3 Transformation of Ecoli.BL21(DE3)

[0061] (1) Take 2 μL of the recombinant expression vector and add it to 100 μL of Ecoli.BL21 (DE3) competent cells (Shanghai Weidi Biotechnology Co., Ltd.), flick the tube wall to mix, and incubate on ice for 25 min;

[0062] (2) 42°C water bath for 45 seconds, then immediately transfer to ice bath for 2 minutes;

[0063] (3) Add 700 μL of LB medium without antibiotics and incubate at 37°C and 220 rpm for 1 h;

[0064] (4) Centrifuge at 5000 rpm for 1 min, aspirate 600 μL of culture medium, mix the remaining medium by pipetting, and spread it onto a gentamicin plate (40 μg / mL). Incubate the plate in an inverted manner at 37°C for 12 h.

[0065] The next day, single colonies were picked from the plate for colony PCR to verify whether the transformation was successful with Ecoli.BL21(DE3)-pBBR1MCS-5-LPL. Figure 2 As shown, it was proved that Ecoli.BL21(DE3)-pBBR1MCS-5-LPL was successfully transformed.

[0066] Example 4 Fermentation of LPL expression strain

[0067] The PAO1-pBBR1MCS-5-LPL and Ecoli.BL21(DE3)-pBBR1MCS-5-LPL strains constructed in Example 3 were inoculated with 1% inoculum into LB liquid culture medium with a final concentration of 40 μg / mL gentamicin, cultured at 37°C, 220 rpm for 12 h, and then inoculated with 1% inoculum into 1 L of LB liquid culture medium and fermented at 37°C for 48 h. The fermented bacterial solution was centrifuged at 8000 rpm for 5 min, and the supernatant was collected to obtain the crude LPL enzyme solution. The crude LPL enzyme solution was subjected to SDS-PAGE detection. The SDS-PAGE results are shown in FIG. Figure 3 、 Figure 4 shown.

[0068] Example 5 LPL activity detection

[0069] 1. Detection principle:

[0070] 4-Nitrophenylbutyrate (PNPB) is a widely used substrate in lipase hydrolysis activity assays. LPL hydrolyzes PNPB to form p-nitrophenol (PNP). PNP is yellow, has a maximum absorbance at 400 nm, and is highly sensitive. Released p-nitrophenol is continuously measured by monitoring the increase in absorbance at 400 nm.

[0071] 2. Definition of enzyme activity unit:

[0072] Using p-nitrophenylbutyrate as substrate, it can release 1.0 micromol (10 -6 mol) of p-nitrophenol.

[0073] 3. Main reagents:

[0074] Reagent 1: 100 mmol / L NaH2PO4, 150 mmol / L NaCl, 0.5% Triton X-100, pH 7.2.

[0075] Reagent 2: 50 mmol / L PNPB (4-nitrophenyl butyrate) was dissolved in 1 mL of acetonitrile.

[0076] Reagent 3: LPL enzyme solution.

[0077] 4. Sample determination:

[0078] Mix 0.9 mL of Reagent 1 with 0.1 mL of Reagent 3, preheat at 37°C for 2 minutes, then add 0.01 mL of Reagent 2. Immediately after mixing, measure the absorbance change at 400 nm over a 5-minute period to obtain ΔA400 nm / min. Substitute water for the enzyme solution as a blank and perform the same assay.

[0079] 5. Calculation formula:

[0080]

[0081] 1.01 = assay volume (mL);

[0082] df = dilution factor;

[0083] 0.0148 = molar extinction coefficient of p-nitrophenol at 400 nm;

[0084] 0.1 = volume of enzyme used (mL).

[0085] 6. LPL enzyme activity of Ecoli.BL21 and PAO1

[0086] The enzyme activity of the crude enzyme produced by Ecoli.BL21(DE3)-pBBR1MCS-5-LPL was determined to be 120 U / L, while the enzyme activity of the crude enzyme produced by PAO1-pBBR1MCS-5-LPL was 5616 U / L.

[0087] Example 6 Purification of LPL

[0088] (1) At 4°C, slowly add ammonium sulfate solid to the LPL supernatant to a final concentration of 65%. Let stand for 1 hour, centrifuge, and precipitate for later use.

[0089] (2) Redissolve the precipitate in Tris-HCl buffer (0.05 mol / L, pH = 7.5) containing 0.5 mM EDTA;

[0090] (3) Dialysis with Tris-HCl buffer (0.02 mol / L, pH = 8.0) containing 0.5 mM EDTA at 4°C to remove ammonium sulfate;

[0091] (4) Packing a DEAE Sepharose ion exchange chromatography column;

[0092] (5) Equilibrate with Tris-HCl equilibration buffer (0.05 mol / L, pH = 7.5) containing 0.5 mM EDTA;

[0093] (6) Filter through a 0.22 μm water-based microporous membrane, load the filtrate onto a DEAE Sepharose ion exchange chromatography column, and collect the sample;

[0094] (7) Wash with Tris-HCl buffer (0.05 mol / L, pH = 7.5) containing 0.5 mM EDTA and collect the sample;

[0095] (8) Elution was performed on DEAE Sepharose using a gradient of 0.15, 0.25, 0.5, and 1.0 M NaCl at a flow rate of 0.5 mL / min, and the samples were collected;

[0096] (9) Pipette 20 μL of the collected sample and run SDS-PAGE.

[0097] The results are as follows Figure 5 As shown in the figure, M: protein marker, Lane 1: protein eluted with 0.25M NaCl eluent. The purified protein concentration was 0.4 mg / mL; the purified enzyme activity was verified to be 8950 U / L.

[0098] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A recombinant vector expressing Pseudomonas aeruginosa lipoprotein lipase, characterized in that: The amino acid sequence of the Pseudomonas aeruginosa lipoprotein lipase is shown in SEQ ID No.

1.

2. The recombinant vector according to claim 1, characterized in that The basic vector of the recombinant vector comprises a lac promoter.

3. The recombinant vector according to claim 1 or 2, characterized in that The basic vector of the recombinant vector comprises the shuttle vector pBBR1MCS-5.

4. The recombinant vector according to claim 3, characterized in that: In the recombinant vector, the coding gene of Pseudomonas aeruginosa lipoprotein lipase is inserted between HindIII and BamHI of the basic vector.

5. A recombinant Pseudomonas aeruginosa comprising the recombinant vector according to any one of claims 1 to 4 and expressing Pseudomonas aeruginosa lipoprotein lipase.

6. The method for constructing the recombinant Pseudomonas aeruginosa according to claim 5, characterized in that: The following steps are involved: The coding gene of Pseudomonas aeruginosa lipoprotein lipase is inserted into the shuttle vector pBBR1MCS-5 to construct a recombinant vector, and the recombinant vector is transferred into a host bacterium of Pseudomonas aeruginosa to obtain the recombinant Pseudomonas aeruginosa.

7. The construction method according to claim 6, characterized in that: When the coding gene is amplified, Pseudomonas aeruginosa PAO1 is used as a template and the upstream primer with the nucleotide sequence shown in SEQ ID No. 3 and the downstream primer with the nucleotide sequence shown in SEQ ID No. 4 is used for amplification.

8. The construction method according to claim 6, characterized in that: The Pseudomonas aeruginosa host bacteria include Pseudomonas aeruginosa PAO1.

9. A method for producing Pseudomonas aeruginosa lipoprotein lipase, characterized in that: The method comprises culturing the recombinant Pseudomonas aeruginosa, wherein the culture solution contains the Pseudomonas aeruginosa lipoprotein lipase; The recombinant Pseudomonas aeruginosa includes the recombinant Pseudomonas aeruginosa described in claim 5 or the recombinant Pseudomonas aeruginosa constructed by the construction method described in any one of claims 6 to 8.

10. The production method according to claim 9, characterized in that: After obtaining the culture solution, the method further includes ion exchange chromatography purification.

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