Mutant of burkholderia cepacia lipase, and recombinant expression vector thereof and use thereof

Abstract

Provided in the present invention are a mutant of Burkholderia cepacia lipase, and a recombinant expression vector thereof and the use thereof. The mutant comprises P131S, T132A, T251A, T251S, P131A, P131Q, T132G, Q88S or P113A single mutation, Q88S and P113A double mutation, and combinations of multi-site mutations including combination 1 of Q88S, P113A, T132G and L287S, combination 2 of Q88S, P113A, P131S and L287S, and combination 3 of Q88S, P113A, P131S and T251A.

Description

Mutants of Burkholderia lipase, their recombinant expression vectors, and their applications Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to mutant Burkholderia lipase, its recombinant expression vector, and its applications. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] Eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, plays a vital role in human health, but the human body cannot synthesize it on its own. Studies have confirmed that EPA has multiple positive effects in maintaining cardiovascular health, anti-tumor activity, regulation of glucose and lipid metabolism, and anti-inflammation. Currently, EPA is mainly derived from marine fish and is widely used in food, health supplements, and disease treatment. However, fish oil contains relatively low levels of polyunsaturated fatty acids (PUFAs) such as EPA and DHA (5%–26%), which cannot meet market demand for health supplements. Therefore, how to effectively and selectively enrich EPA from fish oil presents a challenge and has become a hot topic of research.

[0004] The key issue of the easy degradation and oxidation of unsaturated fatty acids in fish oil during molecular distillation remains to be addressed. Enzymatic enrichment of EPA is considered a promising approach due to its mild reaction conditions, high efficiency, and strong specificity, effectively reducing oxidation and isomerization. Lipases, belonging to the class of hydrolases, can hydrolyze triglycerides into diglycerides, monoglycosides, and fatty acids. These enzymes also exhibit esterification, transesterification, and alcoholysis. However, lipases typically show different selectivity towards medium-chain free fatty acids (FFA) or long-chain free fatty acids, as well as saturated or unsaturated free fatty acids. Currently, researchers mainly focus on the total amount of DHA and EPA. Some researchers have used Geotrichum sp. lipase to hydrolyze n-3PUFA, selectively enriching the total content of EPA (1.53%–1.85%) and DHA (24.1%–30.9%) in fish oil. However, these lipases show low selectivity and catalytic stability in enriching EPA in fish oil.

[0005] Studies have shown that Burkholderia cepacia lipase (BCL) has good selectivity for EPA, increasing the EPA content in fish oil by 22.4%. However, this level is still far from meeting market demand. Summary of the Invention

[0006] The purpose of this invention is to address the limited effectiveness of wild-type Burkholderia cepacia lipase in enriching EPA in fish oil. This invention provides a mutant of Burkholderia cepacia lipase, its recombinant expression vector, and its applications, which can significantly improve the enrichment of EPA in fish oil, increasing the EPA content in fish oil by 33%.

[0007] The technical solution of the present invention is as follows:

[0008] This invention provides a Burkholderia lipase mutant, wherein the mutant contains at least one or more of the following mutations:

[0009] P131S: The 131st amino acid of LipA in wild-type Burkholderia lipase is mutated from proline to serine.

[0010] T132A: The threonine of the 132nd amino acid in LipA of wild-type Burkholderia lipase is mutated to alanine.

[0011] T251A: The 251st amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from threonine to alanine.

[0012] T251S: The 251st amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from threonine to serine.

[0013] P131A: The 131st amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from proline to alanine.

[0014] P131Q: The 131st amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from proline to glutamine;

[0015] T132G: The threonine in the 132nd amino acid sequence of LipA in wild-type Burkholderia lipase is mutated to glycine;

[0016] Q88S: The glutamine-serine mutation at amino acid position 88 of the LipA sequence of wild-type Burkholderia lipase.

[0017] P113A: The proline sequence at position 113 of the LipA lipase of wild-type Burkholderia lipase is mutated to alanine.

[0018] In another aspect, the present invention provides a Burkholderia lipase mutant, wherein the mutant exhibits the following two mutations:

[0019] Q88S: The 88th amino acid sequence of wild-type Burkholderia lipase contains a glutamine-serine mutation.

[0020] And P113A: The 113th amino acid sequence of wild-type Burkholderia lipase has been mutated from proline to alanine.

[0021] Another aspect of the present invention provides a Burkholderia lipase mutant, said mutant having any of the following combinations of mutations:

[0022] Combination 1:

[0023] Q88S: The 88th amino acid sequence of wild-type Burkholderia lipase contains a glutamine-serine mutation.

[0024] P113A: The amino acid sequence at position 113 of the wild-type Burkholderia lipase is mutated from proline to alanine.

[0025] T132G: The threonine in the 132nd amino acid sequence of the wild-type Burkholderia lipase is mutated to glycine;

[0026] L287S: The 287th amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from lysine to leucine.

[0027] Combination 2:

[0028] Q88S: The 88th amino acid sequence of wild-type Burkholderia lipase contains a glutamine-serine mutation.

[0029] P113A: The amino acid sequence at position 113 of the wild-type Burkholderia lipase is mutated from proline to alanine.

[0030] P131S: The 131st amino acid of wild-type Burkholderia lipase is mutated from proline to serine.

[0031] L287S: The 287th amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from lysine to leucine.

[0032] Combination 3:

[0033] Q88S: The 88th amino acid sequence of wild-type Burkholderia lipase contains a glutamine-serine mutation.

[0034] P113A: The amino acid sequence at position 113 of the wild-type Burkholderia lipase is mutated from proline to alanine.

[0035] P131S: The 131st amino acid of wild-type Burkholderia lipase is mutated from proline to serine.

[0036] T251A: The threonine in the 251st amino acid sequence of the wild-type Burkholderia lipase is mutated to alanine.

[0037] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, the mutation primers for P131S including P131S-F as shown in SEQ ID NO.3; and P131S-R as shown in SEQ ID NO.4.

[0038] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, the mutation primers for T132A including T132A-F as shown in SEQ ID NO.13; and T132A-R as shown in SEQ ID NO.14.

[0039] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, the mutation primers for T251A including T251A-F as shown in SEQ ID NO.9; and T251A-R as shown in SEQ ID NO.10.

[0040] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, the mutation primers for T251S including T251S-F as shown in SEQ ID NO.11; and T251S-R as shown in SEQ ID NO.12.

[0041] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, wherein the mutation primers for P131A include P131A-F as shown in SEQ ID NO.1; and P131A-R as shown in SEQ ID NO.2.

[0042] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, wherein the mutation primers for P131Q include P131N-F as shown in SEQ ID NO.17; and P131M-R as shown in SEQ ID NO.18.

[0043] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, wherein the mutation primers for T132G include T132N-F as shown in SEQ ID NO.19; and T132N-R as shown in SEQ ID NO.20.

[0044] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, the mutation primers for Q88S including Q88S-F as shown in SEQ ID NO.5; and Q88S-R as shown in SEQ ID NO.6.

[0045] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, wherein the mutation primers for P113A include P113A-F as shown in SEQ ID NO.7; and P113A-R as shown in SEQ ID NO.8.

[0046] According to a preferred embodiment, the mutation is prepared by induction with mutation primers, the mutation primers for L287S including L287S-F as shown in SEQ ID NO.15; and L287S-R as shown in SEQ ID NO.16.

[0047] Another aspect of the present invention provides the application of a Burkholderia lipase mutant as described above in the purification and enrichment of EPA.

[0048] Another aspect of the present invention provides a Burkholderia lipase recombinant expression vector, comprising a plasmid backbone and a nucleotide sequence encoding a Burkholderia lipase mutant as described above, inserted into the plasmid backbone.

[0049] According to a preferred embodiment, the plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a, or pET-25.

[0050] According to a preferred embodiment, the nucleotide sequence encoding a Burkholderia lipase mutant as described above is inserted upstream of the f1 ori site of the pETDuet plasmid.

[0051] According to a preferred embodiment, the nucleotide sequence encoding a Burkholderia lipase mutant as described above includes lipA and lipB genes, wherein lipB, lipA, and f1 ori on the pETDuet plasmid are tandemly linked, and the lipA gene sequence has the mutation described above.

[0052] The wild-type Burkholderia lipase described is a lipase derived from Burkholderia sp. ZYB002, with NCBI accession number 573762. This lipase contains two genes, lipA (NCBI Gene ID: 56661727) and its corresponding chaperone protein lipB (NCBI Gene ID: 56662904). lipA has 993 nucleotides and 330 amino acids.

[0053] According to a preferred embodiment, the engineered bacteria are obtained by transforming them into host bacteria using the recombinant expression vector as described above.

[0054] According to a preferred embodiment, the engineered bacteria described above are used in the purification and enrichment of EPA.

[0055] Another aspect of the present invention provides a method for purifying and enriching EPA, using ethyl ester-type fish oil as raw material and adding a Burkholderia lipase mutant as described above for reaction.

[0056] In another aspect, the present invention provides a gene that encodes a Burkholderia lipase mutant as described above.

[0057] Another aspect of the present invention provides the use of the Burkholderia lipase mutant as described above in the preparation of foods, pharmaceuticals, cosmetics, health products or feeds with high EPA content.

[0058] Compared with existing technologies, the advantages of this invention are:

[0059] 1. Burkholderia lipase mutants: Several Burkholderia lipase mutants with significant effects on EPA purification and enrichment were discovered through induced mutation. These mutants involve single or combined mutations at multiple sites. The mutants increased the protein yield of Burkholderia lipase and all showed better enzyme activity (up to 57%) and catalytic efficiency in the hydrolysis of ethyl ester-type fish oil; they also demonstrated good EPA purification and enrichment effects.

[0060] 2. After constructing a heterologous expression vector using a mutant of Burkholderia lipase, the mutant can be heterologously expressed in other fields, achieving a wider range of effects and production sources, and reducing the production cost of the Burkholderia lipase mutant. Attached Figure Description

[0061] Figure 1 shows the recombinant expression vector of the lipase-encoding gene in this invention;

[0062] Figure 2 is a standard curve of protein solution concentration in this invention. Detailed Implementation

[0063] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of this invention.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production. Unless otherwise specified, the methods used in this invention are conventional methods in the art; unless otherwise specified, all reagents used in this invention are commercially available.

[0065] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0066] Example 1

[0067] I. Construction of mutants

[0068] Burkholderia lipA and lipB (synthesized by Beijing Qingke Biotechnology Co., Ltd.) were constructed in the order shown in Figure 1 into the pETDuet plasmid (purchased from EMD Biosciences (Novagen)). The mutant plasmid was constructed using the Novizan Mut Express II Fast Mutagenesis Kit V2. Site-directed mutagenesis primers were designed using the Novizan website. The designed primers are shown in Table 1 below. The target plasmid was amplified using Phanta Max Super-Fidelity DNA Polymerase. After recombination and circularization of the amplification product, it was transformed into E. coli DE3 (BL21) competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.). 70 μL of bacterial culture was spread onto LB agar plates containing Ampicillin (Amp, 50 mg / L) using glass beads and incubated overnight at 37°C. Identification of recombinant products: Randomly select 5 single colonies and add them to 1.5 mL or 2 mL centrifuge tubes containing 600 μL of LB liquid medium containing Amp antibiotic. Incubate at 37℃ and 220 rpm for 5-6 h. When the bacterial solution becomes turbid, aliquot 100 μL and send for sequencing. Store the remaining bacterial solution at 4℃. If the sequencing results are correct, preserve the mutant strain using 60% glycerol (LB medium formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl; for solid medium, add 15 g / L agar powder).

[0069] Table 1. Site-directed mutagenesis primers

[0070] II. Expression and Collection of Mutant Proteins

[0071] Mother liquor preparation: Inoculate with a single colony and incubate overnight;

[0072] Inoculate the stock solution into TB medium (2% by volume);

[0073] (TB medium formulation: peptone 20 g / L, yeast extract 24 g / L, glycerol 4 g / L, dipotassium hydrogen phosphate 12.25 g / L, potassium dihydrogen phosphate 2.3 g / L.)

[0074] Incubate for approximately 4 hours until OD 600 Once the concentration reaches 0.7–0.9, add IPTG (0.1 mmol / L) and express for 48 h (180 rpm, 20 °C), then collect the bacterial cells.

[0075] Collect bacterial cells by centrifugation at 4℃, 4000 rpm, and 10 min; wash the bacterial cells once with approximately 100 mL of pure water and once with PBS.

[0076] Add PBS buffer (1:10 with bacterial cells) and mix well. Sonicate (15%, 4 seconds sonication, 3 seconds pause, 5 min), then centrifuge twice (10000 rpm, 8 min). The supernatant is the protein solution containing the mutant enzyme, i.e., the enzyme solution. PBS buffer formulation: NaCl (sodium chloride): 8 g / L, KCl (potassium chloride): 0.2 g / L, Na₂HPO₄ (disodium hydrogen phosphate): 1.44 g / L, KH₂PO₄ (potassium dihydrogen phosphate): 0.24 g / L, adjust pH to 7.4.

[0077] III. Protein Content Detection

[0078] To prepare standard protein solutions of different concentrations: Take 7 clean test tubes. Put 0.1 mL of deionized water into tube 1 as a blank group. Add 0.02, 0.04, 0.06, 0.08 and 0.10 mL of 1.0 mg / mL BSA solution to tubes 2-6 respectively using a micropipette. Then, add deionized water to each tube to make up to 0.1 mL. Put 0.1 mL of the protein solution to be tested into tube 7. Mix all the test tubes thoroughly with a vortex mixer.

[0079] Add staining reagent: Add 3.0 mL of Coomassie Brilliant Blue G250 reagent to each of the above test tubes using a pipette or pipette, and mix gently and quickly with a vortex mixer.

[0080] Colorimetric analysis: Coomassie Brilliant Blue reagent binds rapidly to proteins, completing the binding process in 2–5 minutes and remaining stable for up to one hour. Therefore, the absorbance (A) of each test tube solution at a wavelength of 595 nm can be measured on a spectrophotometer after the solution has stood for 5 minutes. 595 value;

[0081] Construct a standard curve: Plot the concentration of the standard protein solution (mg / mL) on the x-axis, and the absorbance value A 595 By plotting the vertical axis, a straight line can be fitted to obtain a standard curve; the result is shown in Figure 2.

[0082] Calculate the concentration of the protein to be tested: Measure the absorbance A of the protein solution to be tested using a spectrophotometer. 595 By comparing the values ​​with the standard curve, the concentration of the protein to be tested can be calculated, and the results are shown in Table 2.

[0083] Table 2 Protein Concentration

[0084] As shown in Table 2, these mutants effectively increased protein yield and could significantly reduce the production cost of Burkholderia lipase in industrial production.

[0085] IV. Protein Purification

[0086] First, the HisTrap™ HP affinity chromatography column was equilibrated with 5 column volumes of nickel column equilibration buffer at a flow rate of 0.5 mL / min. Then, 50 mL of crude enzyme solution, filtered through a 0.22 μm filter, was loaded into the HisTrap™ HP affinity chromatography column at a flow rate of 0.3 mL / min. After loading, the column was washed with more than 10 column volumes of HisTrap™ HP affinity chromatography column equilibration buffer at a flow rate of 0.5 mL / min to remove unbound proteins. Finally, the HisTrap™ HP affinity chromatography column was eluted with a gradient of HisTrap™ HP affinity chromatography column equilibration buffer and HisTrap™ HP affinity chromatography column elution buffer. The elution conditions were: total elution volume 20 mL, flow rate 0.3 mL / min, and 2 mL eluent collected in fractions per tube.

[0087] V. Enzyme Activity Assay

[0088] The specific method for measuring lipase activity using olive oil titration is as follows: 4g of polyvinyl alcohol was added to 180mL of deionized water and slowly stirred at 90℃ until completely dissolved. The solution was then cooled and brought to a final volume of 200mL. 150mL of the solution was taken, and 50mL of olive oil was added. The mixture was emulsified on a tissue mixer for 3–5 minutes to obtain an olive oil emulsion. 4mL of the olive oil emulsion was taken, and 5mL of 50mM pH 7.5 PB buffer solution was added. The blank control group was pre-treated with 15mL of ethanol. The mixture was then incubated in a 30℃ water bath for 15 minutes. 1mL of enzyme solution was quickly added, and the mixture was magnetically stirred at 800r / min and timed for 15 minutes. 15mL of ethanol was added to the sample group to terminate the reaction. Finally, two drops of 1% phenolphthalein indicator were added, and the enzyme activity was measured using a 100mM sodium hydroxide solution. The results are shown in Table 3.

[0089] Table 3 Enzyme Activity

[0090] As shown in Table 3, the enzyme activities of Q88S, P113A, P131S, and T251A were increased by 57% compared to WT; Q88S, P113A, T132G, and L287S by 34%; and Q88S, P113A, P131S, and L287S by 34.5%. The increased enzyme activity of the mutants suggests that these mutants have a stronger ability to catalyze subsequent reactions.

[0091] VI. Test for hydrolysis reaction of ethyl ester fish oil

[0092] Ethyl ester fish oil with an initial EPA content of 56% was used as the reaction raw material: 15 mL of fish oil was added to the prepared enzyme solution, and then water was added to bring the volume to 22.5 mL (1.5 times the volume of the fish oil). The reaction was carried out at 400 rpm under vacuum for 2–3 hours, and then the acid value was measured.

[0093] Acid value test: Take about 5g of upper layer oil, add 20mL of acid value reagent (ethanol and ether mixed at 1:1 (V / V)), add 3 drops of phenolphthalein, and titrate with 0.1mol / L sodium hydroxide standard titration solution until the sample solution turns light pink and does not fade after shaking for 30s. Record the volume of sodium hydroxide titration solution consumed.

[0094] Acid value (NaOH mg / g) = 56.11 * V * C / W, where:

[0095] W -- Sample weight, g;

[0096] V--V1-VO (volume of titrant consumed by the sample minus the volume of titrant consumed by the blank), mL.

[0097] C -- Concentration of sodium hydroxide standard titrant solution, mol / L.

[0098] Acid value is a measure of the number of free carboxylic acid groups in a compound (e.g., fatty acids) or mixture, which reflects the enzyme's ability to hydrolyze ethyl ester fish oil into ethanol and free fatty acids in the reaction.

[0099] The acid value test results are shown in Table 4 below:

[0100] Table 4. Acid Value Test Results

[0101] As shown in Table 4, the acid values ​​of the constructed mutants were all higher than those of the wild-type WT of the starting strain, indicating that these mutants had a stronger ability to catalyze the hydrolysis reaction.

[0102] When the acid value is greater than 20, take more than 10g of oil for alkali refining:

[0103] NaOH addition calculation: (0.714 * oil mass (g) * acid value) / 1000.

[0104] Add NaOH to the oil, stir in a homogenizer until fully reacted, collect the oil by centrifugation, and wash three times with hot water until the oil is clear and transparent.

[0105] Component content detection:

[0106] Take 30 mg of the sample to be tested into a 10 mL volumetric flask, dissolve and dilute to the mark with internal standard solution. Accurately transfer 2.0 mL into a glass test tube and slowly remove the solvent by nitrogen purging. Add 1.5 mL of 2% (w / v) sodium hydroxide-methanol solution, seal tightly with a PTFE-lined cap, mix well, and heat in a boiling water bath for 7 min. Cool, add 2 mL of boron trichloride-methanol solution, purge with nitrogen, seal tightly, mix well, heat in a boiling water bath for 30 min, cool to 40–50 °C, add 1 mL of isooctane, seal tightly, vortex or shake for at least 30 s, and immediately add 5 mL of saturated sodium chloride solution (containing 1 volume of sodium chloride and 2 volumes of water). Purge with nitrogen, seal tightly, and vortex or shake for at least 15 s. After standing and separating, the supernatant was transferred to a test tube and shaken again with 1 mL of isooctane to separate the layers. The supernatant was then combined with the previous one and washed twice with 1 mL of water each time. The extract was dried with anhydrous sodium sulfate and the supernatant was transferred to a 2 mL brown sample vial for GC detection of the product.

[0107] The chromatographic column material was a quartz capillary column with a size of 0.25 mm × 25 m. The stationary phase was polyethylene glycol stationary phase. The detector temperature was 270 °C, the injection port temperature was 250 °C, the column temperature was initially set at 170 °C for 2 min, and then increased to 240 °C at an increase rate of 3 °C per minute and held for 15 min. The carrier gas was nitrogen / helium with a split ratio of 1:200 and a flow rate of 1 mL / min.

[0108] The GC results are shown in Table 5 below. Compared with the starting strain WT, the EPA content of each mutant strain was significantly increased. Among them, the EPA content of the best mutant strain P131S reached 73.18%, which was 33% higher than the initial EPA content and 11.7% higher than that of WT.

[0109] Table 5 EPA Content Table

[0110] Therefore, it can be seen that the Burkholderia lipase mutant provided by this invention exhibits higher selectivity for EPA, can be enriched to provide more possibilities for the industrial production of EPA, and also provides some insights for the engineering modification of other multi-domain proteins.

[0111] The amino acid and nucleotide sequence of LipA in the wild-type Burkholderia lipase is as follows:

[0112] LipA ​​amino acid sequence - SEQ ID No. 21:

[0113] LipA ​​nucleotide sequence - SEQ ID NO.22:

[0114] The amino acid sequences of lipA in each mutant are as follows:

[0115] P131S: SEQ ID NO.23:

[0116] T132A: SEQ ID NO. 24:

[0117] T251A: SEQ ID NO. 25:

[0118] T251S: SEQ ID NO. 26:

[0119] P131A: SEQ ID NO. 27:

[0120] P131Q: SEQ ID NO. 28:

[0121] T132G: SEQ ID NO. 29:

[0122] P113A: SEQ ID NO. 30:

[0123] Q88S: SEQ ID NO.31:

[0124] Q88S, P113A dual protrusions: SEQ ID NO.32:

[0125] The four protrusions of Q88S, P113A, T132G, and L287S are: SEQ ID NO. 33.

[0126] The four protuberances of Q88S, P113A, P131S, and L287S: SEQ ID NO. 34

[0127] The four protrusions of Q88S, P113A, P131S, and T251A: SEQ ID NO. 35

[0128] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A Burkholderia lipase mutant, characterized in that, The mutant contains at least one or more of the following mutations: P131S: The 131st amino acid of LipA in wild-type Burkholderia lipase is mutated from proline to serine. T132A: The threonine of the 132nd amino acid in LipA of wild-type Burkholderia lipase is mutated to alanine. T251A: The 251st amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from threonine to alanine. T251S: The 251st amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from threonine to serine. P131A: The 131st amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from proline to alanine. P131Q: The 131st amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from proline to glutamine; T132G: The threonine sequence of the 132nd amino acid in the LipA of wild-type Burkholderia lipase is mutated to glycine; Q88S: The glutamine-serine mutation at amino acid position 88 of the LipA sequence of wild-type Burkholderia lipase. P113A: The proline at position 113 of the LipA amino acid sequence of wild-type Burkholderia lipase is mutated to alanine. The amino acid sequence of LipA of the wild-type Burkholderia lipase is shown in SEQ ID No.

21.

2. The Burkholderia lipase mutant according to claim 1, characterized in that, The mutation is prepared by induction with mutation primers, including P131S-F as shown in SEQ ID NO.3 and P131S-R as shown in SEQ ID NO.

4.

3. The Burkholderia lipase mutant according to claim 1, characterized in that, The mutation is prepared by induction with mutation primers, including T132A-F as shown in SEQ ID NO.13 and T132A-R as shown in SEQ ID NO.

14.

4. A Burkholderia lipase mutant according to claim 1, characterized in that, The mutation is prepared by induction with mutation primers, including T251A-F as shown in SEQ ID NO.9 and T251A-R as shown in SEQ ID NO.

10.

5. A Burkholderia lipase mutant according to claim 1, characterized in that, The mutation is prepared by induction with mutation primers, including T251S-F as shown in SEQ ID NO.11 and T251S-R as shown in SEQ ID NO.

12.

6. A Burkholderia lipase mutant according to claim 1, characterized in that, The mutation is prepared by induction with mutation primers, including P131A-F as shown in SEQ ID NO.1 and P131A-R as shown in SEQ ID NO.

2.

7. A Burkholderia lipase mutant according to claim 1, characterized in that, The mutation is prepared by induction with mutation primers, including P131N-F as shown in SEQ ID NO.17 and P131M-R as shown in SEQ ID NO.

18.

8. A Burkholderia lipase mutant according to claim 1, characterized in that, The mutation is prepared by induction with mutation primers, including T132N-F as shown in SEQ ID NO.19 and T132N-R as shown in SEQ ID NO.

20.

9. A Burkholderia lipase mutant according to claim 1, characterized in that, The mutation is prepared by induction with mutation primers, including Q88S-F as shown in SEQ ID NO.5 and Q88S-R as shown in SEQ ID NO.

6.

10. A Burkholderia lipase mutant according to claim 1, characterized in that, The mutation is prepared by induction with mutation primers, including P113A-F as shown in SEQ ID NO.7 and P113A-R as shown in SEQ ID NO.

8.

11. A Burkholderia lipase mutant, characterized in that, The mutant exhibits the following two mutations: Q88S: The 88th amino acid sequence of wild-type Burkholderia lipase contains a glutamine-serine mutation. And P113A: The amino acid sequence at position 113 of the wild-type Burkholderia lipase is mutated from proline to alanine; The amino acid sequence of LipA of the wild-type Burkholderia lipase is shown in SEQ ID No.

24.

12. A Burkholderia lipase mutant, characterized in that, The mutant can exist in any of the following combinations of mutations: Combination 1: Q88S: The 88th amino acid sequence of wild-type Burkholderia lipase contains a glutamine-serine mutation. P113A: The amino acid sequence at position 113 of the wild-type Burkholderia lipase is mutated from proline to alanine. T132G: The threonine in the 132nd amino acid sequence of the wild-type Burkholderia lipase is mutated to glycine; L287S: The leucine at amino acid position 287 is mutated to serine; Combination 2: Q88S: The 88th amino acid sequence of wild-type Burkholderia lipase contains a glutamine-serine mutation. P113A: The amino acid sequence at position 113 of the wild-type Burkholderia lipase is mutated from proline to alanine. P131S: The 131st amino acid of wild-type Burkholderia lipase is mutated from proline to serine. L287S: The lysine at amino acid position 287 is mutated to leucine; Combination 3: Q88S: The 88th amino acid sequence of wild-type Burkholderia lipase contains a glutamine-serine mutation. P113A: The amino acid sequence at position 113 of the wild-type Burkholderia lipase is mutated from proline to alanine. P131S: The 131st amino acid of wild-type Burkholderia lipase is mutated from proline to serine. T251A: The 251st amino acid sequence of LipA in wild-type Burkholderia lipase is mutated from threonine to alanine. The amino acid sequence of LipA of the wild-type Burkholderia lipase is shown in SEQ ID No.

21.

13. The application of a Burkholderia lipase mutant as described in any one of claims 1-12 in the purification and enrichment of EPA.

14. A Burkholderia lipase recombinant expression vector, characterized in that, The plasmid backbone includes a nucleotide sequence encoding a Burkholderia lipase mutant as described in any one of claims 1-12.

15. The Burkholderia lipase recombinant expression vector according to claim 14, characterized in that, The plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a, or pET-25.

16. The Burkholderia lipase recombinant expression vector according to claim 15, characterized in that, The nucleotide sequence encoding a Burkholderia lipase mutant as described in any one of claims 1-12 is inserted upstream of the f1 ori site in the plasmid backbone.

17. The Burkholderia lipase recombinant expression vector according to claim 16, characterized in that, The nucleotide sequence encoding a Burkholderia lipase mutant as described in any one of claims 1-12 includes the lipA and lipB genes, wherein the lipB, lipA and f1 ori on the pETDuet plasmid are tandemly linked, and the lipA gene sequence contains the mutation as described in any one of claims 1-12.

18. An engineered bacterium expressing a mutant as described in any one of claims 1-12, characterized in that, The engineered bacteria were obtained by transforming them into host bacteria using the recombinant expression vector as described in any one of claims 14-17.

19. The application of the engineered bacteria as described in claim 18 in the purification and enrichment of EPA.

20. A method for purifying and enriching EPA, characterized in that, Using ethyl ester-type fish oil as raw material, a Burkholderia lipase mutant as described in any one of claims 1-12 is added for reaction.

21. A gene characterized by, The gene encodes the Burkholderia lipase mutant as described in claim 1, claim 11, or claim 12.

22. The use of the Burkholderia lipase mutant as described in claim 1, claim 11 or claim 12 in the preparation of food, pharmaceutical, cosmetic, health product or feed containing EPA.

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