Burkholderia lipase with T251A mutation as well as recombinant expression vector and application thereof

Abstract

The invention relates to the technical field of bioengineering, and discloses Burkholderia lipase with T251A mutation and a recombinant expression vector and application thereof, threonine of the 251 amino acid sequence of LipA of wild Burkholderia lipase is mutated into alanine. The expression quantity of the mutant enzyme is increased by 32%, meanwhile, the hydrolysis capacity on ethyl ester type fish oil is remarkably improved, the selectivity on EPA is improved, and the excellent EPA purification and enrichment effect is achieved. According to the mutant, the preparation cost is reduced, the hydrolysis capability and selectivity are improved, and the mutant has great popularization and application values.

Description

[0001] The present invention is a divisional application with application number 2025101891393, application date 2025-02-20, application type invention, and invention name “Mutant of Burkholderia lipase and its recombinant expression vector and application”. Technical Field

[0002] The present invention relates to the technical field of bioengineering, and in particular to a Burkholderia lipase with a T251A mutation, a recombinant expression vector thereof, and an application thereof. Background Art

[0003] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0004] Eicosapentaenoic acid (EPA), a polyunsaturated fatty acid in the omega-3 family, plays a vital role in human health, yet the body cannot synthesize it. Studies have confirmed that EPA has multiple beneficial effects in maintaining cardiovascular health, including anti-tumor activity, regulating glucose and lipid metabolism, and anti-inflammatory properties. Currently, EPA is primarily derived from marine fish and has been widely used in food, health supplements, and disease treatment. However, the content of EPA and polyunsaturated fatty acids (PUFAs) such as docosahexanoic acid (DHA) in fish oil is relatively low (5%-26%), failing to meet market demand for health supplements. Therefore, the challenge of effectively and selectively enriching EPA from fish oil remains a hot topic for researchers.

[0005] The key issue of unsaturated fatty acids in fish oil being susceptible to degradation and oxidation 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 belong to the class of hydrolases and can hydrolyze triglycerides into diglycerides, monoglycerides, and fatty acids. These enzymes also perform esterification, transesterification, and alcoholysis. However, lipases generally exhibit varying selectivity for medium-chain free fatty acids (FFA) or long-chain free fatty acids, as well as for saturated and unsaturated free fatty acids. Currently, researchers primarily focus on the total amount of DHA and EPA. Some researchers have used Geotrichum sp. lipase to hydrolyze n-3 PUFAs, selectively enriching the total amount of EPA (1.53%–1.85%) and DHA (24.1%–30.9%) in fish oil. However, these lipases exhibit low selectivity and catalytic stability for EPA enrichment in fish oil.

[0006] Research has shown that Burkholderia cepacia lipase (BCL) has good selectivity for EPA, increasing the EPA content in fish oil by 22.4%. However, this content is still far from meeting market demand. Summary of the Invention

[0007] The present invention aims to address the problem that the current wild-type Burkholderia cepacia lipase has limited effect in enriching EPA in fish oil. The present invention provides a mutant of Burkholderia cepacia lipase and a recombinant expression vector and application thereof, which can significantly improve the effect of enriching EPA in fish oil and increase the EPA content in fish oil by 33%.

[0008] The technical solutions of the present invention are as follows: In one aspect, the present invention provides a Burkholderia lipase mutant, wherein the mutant has at least one or more of the following mutations: P131S: The proline at amino acid 131 of the wild-type Burkholderia lipase LipA is mutated to serine; T132A: The threonine at amino acid 132 of the wild-type Burkholderia lipase LipA is mutated to alanine; T251A: The threonine in the 251st amino acid sequence of the wild-type Burkholderia lipase LipA is mutated to alanine; T251S: The 251st amino acid sequence of the wild-type Burkholderia lipase LipA is mutated from threonine to serine; P131A: The proline at amino acid 131 of the wild-type Burkholderia lipase LipA was mutated to alanine; P131Q: The proline at amino acid 131 of the wild-type Burkholderia lipase LipA was mutated to glutamine; T132G: The threonine at amino acid 132 of the wild-type Burkholderia lipase LipA was mutated to glycine; Q88S: glutamine at amino acid position 88 of the wild-type Burkholderia lipase LipA is mutated to serine; P113A: The proline at amino acid position 113 of the wild-type Burkholderia lipase LipA is mutated to alanine.

[0009] Another aspect of the present invention provides a Burkholderia lipase mutant, wherein the mutant has two mutations as shown below: Q88S: glutamine at amino acid position 88 of the wild-type Burkholderia lipase LipA is mutated to serine; and P113A: the proline at amino acid position 113 of the wild-type Burkholderia lipase LipA is mutated to alanine.

[0010] Another aspect of the present invention provides a Burkholderia lipase mutant, wherein the mutant has any one of the following mutation combinations: Combination 1: Q88S: glutamine at amino acid position 88 of the wild-type Burkholderia lipase LipA is mutated to serine; P113A: The proline at amino acid position 113 of the wild-type Burkholderia lipase LipA was mutated to alanine; T132G: The threonine at amino acid 132 of the wild-type Burkholderia lipase LipA was mutated to glycine; L287S: Leucine at amino acid position 287 of the wild-type Burkholderia lipase LipA is mutated to serine; Combination 2: Q88S: glutamine at amino acid position 88 of the wild-type Burkholderia lipase LipA is mutated to serine; P113A: The proline at amino acid position 113 of the wild-type Burkholderia lipase LipA was mutated to alanine; P131S: The proline at amino acid 131 of the wild-type Burkholderia lipase LipA is mutated to serine; L287S: Leucine at amino acid position 287 of the wild-type Burkholderia lipase LipA is mutated to serine; Combination three: Q88S: glutamine at amino acid position 88 of the wild-type Burkholderia lipase LipA is mutated to serine; P113A: The proline at amino acid position 113 of the wild-type Burkholderia lipase LipA was mutated to alanine; P131S: The proline at amino acid 131 of the wild-type Burkholderia lipase LipA is mutated to serine; T251A: The threonine at the 251st amino acid sequence of the wild-type Burkholderia lipase LipA is mutated to alanine.

[0011] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the mutation primers of P131S include P131S-F as shown in SEQ ID NO.3; and P131S-R as shown in SEQ ID NO.4.

[0012] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the T132A mutation primer includes T132A-F as shown in SEQ ID NO.13; and T132A-R as shown in SEQ ID NO.14.

[0013] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the T251A mutation primer includes T251A-F as shown in SEQ ID NO.9; and T251A-R as shown in SEQ ID NO.10.

[0014] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the T251S mutation primer includes T251S-F as shown in SEQ ID NO.11; and T251S-R as shown in SEQ ID NO.12.

[0015] According to a preferred embodiment, the mutation is prepared by inducing mutation primers, and the mutation primers of P131A include P131A-F as shown in SEQ ID NO.1; and P131A-R as shown in SEQ ID NO.2.

[0016] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and 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.

[0017] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the T132G mutation primer includes T132N-F as shown in SEQ ID NO.19; and T132N-R as shown in SEQ ID NO.20.

[0018] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the Q88S mutation primers include Q88S-F as shown in SEQ ID NO.5; and Q88S-R as shown in SEQ ID NO.6.

[0019] According to a preferred embodiment, the mutation is prepared by inducing mutation primers, and the mutation primers of P113A include P113A-F as shown in SEQ ID NO.7; and P113A-R as shown in SEQ ID NO.8.

[0020] According to a preferred embodiment, the mutation is prepared by inducing a mutation primer, and the L287S mutation primer includes L287S-F as shown in SEQ ID NO.15; and L287S-R as shown in SEQ ID NO.16.

[0021] Another aspect of the present invention provides the use of the aforementioned Burkholderia lipase mutant in the purification and enrichment of EPA.

[0022] 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, which is inserted into the plasmid backbone.

[0023] According to a preferred embodiment, the plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a or pET-25.

[0024] 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.

[0025] According to a preferred embodiment, the nucleotide sequence encoding a Burkholderia lipase mutant as described above includes lipA and lipB genes, the lipB, lipA and the f1 ori on the pETDuet plasmid are connected in series, and the lipA gene sequence has the mutation as described above.

[0026] The wild-type Burkholderia lipase is derived from Burkholderia sp. ZYB002 and has NCBI accession number 573762. The lipase comprises two genes: lipA (NCBI Gene ID: 56661727) and its corresponding chaperone protein, lipB (NCBI Gene ID: 56662904). LipA has a total of 993 nucleotides and 330 amino acids.

[0027] Compared with the existing technology, the beneficial effects of the present invention are: 1. Mutants of Burkholderia lipase. Several mutants with significant effects on EPA purification and enrichment have been discovered through induced mutagenesis. These mutants involve single or combined mutations at multiple sites. These mutants increase protein production, exhibit superior enzymatic activity (up to 57%), and catalyze the hydrolysis of ethyl ester fish oils, demonstrating superior EPA purification and enrichment. 2. After constructing a heterologous expression vector using the Burkholderia lipase mutant, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a recombinant expression vector of the lipase encoding gene of the present invention; Figure 2 This is a standard curve diagram of protein solution concentration in the present invention. DETAILED DESCRIPTION

[0029] The specific embodiments listed in the present invention are only examples of the present invention, and the present 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 the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without the manufacturer specified are conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present invention can also be implemented 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 purpose of the present invention.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are International Standard Units, and the numerical values ​​and numerical ranges appearing in this invention should be understood to include the inevitable systematic errors in industrial production. Unless otherwise specified, the methods used in this invention are conventional methods in the art; unless otherwise specified, the reagents used in this invention are commercially available.

[0031] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0032] Example 1 1. Construction of mutants The lipA and lipB of Burkholderia (synthesized by Beijing Qingke Biotechnology Co., Ltd.) were used to Figure 1The sequence shown was constructed in the pETDuet plasmid (purchased from EMD Biosciences (Novagen)). Mutation plasmids were constructed using the Novagen MutExpress II Fast Mutagenesis Kit V2. Site-directed mutagenesis primers were designed by visiting the Novagen website. The designed primers are shown in Table 1 below. The target plasmid was amplified using Phanta Max Super-Fidelity DNA Polymerase. The amplified product was recombinantly circularized and transformed into competent E. coli DE3 (BL21) cells (purchased from Beijing Qingke Biotechnology Co., Ltd.). 70 μL of the bacterial suspension was spread using glass beads onto solid LB plates containing 50 mg / L of ampicillin (Amp) and incubated in an inverted position overnight at 37°C. Identification of recombinant products: Five single colonies were randomly picked and added to a 1.5-mL or 2-mL centrifuge tube containing 600 μL of LB liquid medium containing the Amp antibiotic. The culture was shaken at 37°C and 220 rpm for 5-6 h. When the bacterial solution became turbid, 100 μL was aliquoted and sent for sequencing. The remaining bacterial solution was stored at 4°C. If the sequencing results were correct, the mutant strain was preserved in 60% glycerol (LB medium formula: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 15 g / L agar powder for solid medium).

[0033] Table 1 Site-directed mutagenesis primers

[0034] 2. Expression and Collection of Mutant Proteins Preparation of stock solution: Inoculate a single colony from overnight culture; The mother liquid was inoculated into TB medium (2% volume ratio); (TB medium formula: 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.) Culture for about 4 h until OD 600 When the concentration reached 0.7-0.9, IPTG (0.1 mmol / L) was added and the cells were collected after expression for 48 h (180 rpm, 20°C); Collect the cells by centrifugation at 4°C, 4000 rpm, for 10 min; wash the cells once with approximately 100 mL of pure water and once with PBS; Add PBS buffer (1:10 mass ratio to bacterial cells) and mix thoroughly. Ultrasonicate (15% CO, 4 seconds on, 3 seconds off, 5 minutes). Centrifuge twice (10,000 rpm, 8 minutes). The supernatant is the protein solution containing the mutant enzyme, also known as the enzyme solution. PBS buffer recipe: 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 the pH to 7.4.

[0035] 3. Protein content detection Prepare standard protein solutions of different concentrations: Take 7 ordinary clean test tubes, put 0.1 mL of deionized water in tube No. 1 as a blank group, use a micropipette to add 0.02, 0.04, 0.06, 0.08, and 0.10 mL of 1.0 mg / mL BSA solution to tubes No. 2 to 6, respectively, and then fill each tube with deionized water to 0.1 mL. In addition, put 0.1 mL of the protein solution to be tested in test tube No. 7. Mix all test tubes thoroughly with a vortex mixer; Add staining reagent: Use a pipette or pipette to add 3.0 mL of Coomassie Brilliant Blue G250 reagent to each of the above test tubes and mix gently and quickly using a vortex mixer; Colorimetry: Coomassie Brilliant Blue reagent binds to proteins quickly, completing the process in 2 to 5 minutes and remaining stable for 1 hour. Therefore, the absorbance value A of each test tube solution at a wavelength of 595 nm can be measured on a spectrophotometer after the solution has been allowed to stand for 5 minutes. 595 value; Make a standard curve: take the concentration of standard protein solution (mg / mL) as the horizontal axis and the absorbance value A as the horizontal axis. 595 As the vertical axis, a straight line fitting can be performed to obtain a standard curve; the result is as follows Figure 2 As shown; Calculate the concentration of the protein to be tested: The light absorbance A of the protein solution to be tested measured by the spectrophotometer 595 The protein concentration to be tested can be calculated by comparing the values ​​with the standard curve. The results are shown in Table 2.

[0036] Table 2 Protein concentration

[0037] According to the results shown in Table 2, these mutants effectively increased protein yield and can greatly reduce the production cost of Burkholderia lipase in industrial production.

[0038] 4. Protein Purification First, a HisTrap™ HP affinity column was equilibrated with 5 column volumes of nickel 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 onto the HisTrap™ HP affinity column at a flow rate of 0.3 mL / min. After loading, the column was flushed with more than 10 column volumes of HisTrap™ HP affinity column equilibration buffer at a flow rate of 0.5 mL / min to remove unbound proteins. Finally, the HisTrap™ HP affinity column was eluted using a gradient of HisTrap™ HP affinity column equilibration buffer and HisTrap™ HP affinity column elution buffer. Elution conditions were: a total elution volume of 20 mL, a flow rate of 0.3 mL / min, and 2 mL / tube fractions of eluate.

[0039] 5. Enzyme activity determination The olive oil titration method for measuring lipase activity is as follows: 4 g of polyvinyl alcohol is added to 180 mL of deionized water and slowly stirred at 90°C until completely dissolved. The solution is then cooled and brought to a volume of 200 mL. To 150 mL of the solution, 50 mL of olive oil is added and emulsified on a tissue mixer for 3–5 minutes to obtain an olive oil emulsion. To 4 mL of the olive oil emulsion, 5 mL of 50 mM PB buffer (pH 7.5) is added. For the blank control, 15 mL of ethanol is added beforehand. The sample is then incubated in a 30°C water bath for 15 minutes. Then, 1 mL of enzyme solution is quickly added. The sample is magnetically stirred at 800 rpm and a timer is started for 15 minutes. The reaction is terminated by adding 15 mL of ethanol to the sample. Finally, two drops of 1% phenolphthalein indicator are added. The enzyme activity is titrated with 100 mM sodium hydroxide solution. The results are shown in Table 3.

[0040] Table 3 Enzyme activity

[0041] The results in Table 3 show that compared to the WT, the enzyme activity of the mutants Q88S, P113A, P131S, and T251A increased by 57%, the Q88S, P113A, T132G, and L287S increased by 34%, and the Q88S, P113A, P131S, and L287S increased by 34.5%. The increased enzyme activity of these mutants suggests that these mutants are more capable of catalyzing subsequent reactions.

[0042] 6. Ethyl ester fish oil hydrolysis reaction test Use ethyl ester fish oil with an initial EPA content of 56% as the reaction medium: Take 15 mL of fish oil, add the prepared enzyme solution, and then add water to make up to 22.5 mL (1.5 times the volume of the fish oil). Rotate at 400 rpm while simultaneously applying vacuum. After 2–3 hours of reaction, test the acid value.

[0043] Acid value test: Take about 5 g of the upper layer of oil, add 20 mL of acid value reagent (ethanol and ether mixed in a ratio of 1:1 (V / V)), then add 3 drops of phenolphthalein, and titrate with 0.1 mol / L sodium hydroxide standard solution until the sample solution turns light pink and does not fade after shaking for 30 seconds. Record the volume of sodium hydroxide solution consumed.

[0044] Acid value (NaOH mg / g) = 56.11*V*C / W, where: W--the weight of the sample, g; V--V1-VO (the volume of titrant consumed by the sample minus the volume of titrant consumed by the blank), mL.

[0045] C--Concentration of sodium hydroxide standard titrant solution, mol / L.

[0046] Acid number is a measure of the number of free carboxylic acid groups in a compound (e.g., fatty acid) or mixture and, in this reaction, reflects the ability of the enzyme to hydrolyze ethyl ester fish oil into ethanol and free fatty acids.

[0047] The acid value test results are shown in Table 4 below: Table 4 Acid value test results

[0048] According to the results shown in Table 4, the acid values ​​of the constructed mutants were all higher than that of the starting strain wild type WT, indicating that these mutants had stronger ability to catalyze the hydrolysis reaction.

[0049] When the acid value is greater than 20, take more than 10g of oil for alkali refining operation: Calculation of NaOH addition amount: (0.714 * oil mass (g) * acid value) / 1000.

[0050] Add NaOH to the oil, stir with a homogenizer until fully reacted, collect the oil by centrifugation, and wash with hot water three times until the oil is clear and transparent.

[0051] Detection of content of each component: Place 30 mg of the sample to be tested in a 10 mL volumetric flask, dissolve it with the internal standard solution, and dilute to the mark. Accurately pipette 2.0 mL into a glass test tube and slowly remove the solvent with a nitrogen purge. Add 1.5 mL of 2% (w / v) sodium hydroxide-methanol solution, cap tightly with a tetrafluoroethylene-lined cap, mix thoroughly, and heat in a boiling water bath for 7 minutes. Cool, add 2 mL of boron trichloride-methanol solution, purge with nitrogen, cap tightly, mix thoroughly, and heat in a boiling water bath for 30 minutes. Cool to 40-50°C, add 1 mL of isooctane, cap tightly, and vortex or shake for at least 30 seconds. Immediately add 5 mL of saturated sodium chloride solution (containing 1 volume sodium chloride and 2 volumes water). Pour nitrogen, cap tightly, and vortex or shake for at least 15 seconds. After standing and stratification, the supernatant was transferred to a test tube, and the layers were shaken again with 1 mL of isooctane. The supernatant was combined with the previous one, and then washed twice with water, 1 mL each time. The extract was dried over anhydrous sodium sulfate, and the supernatant was transferred to a 2 mL brown injection bottle and the product was detected by GC.

[0052] The chromatographic column material is a quartz capillary column with a size of 0.25 mm×25 m. The stationary phase is polyethylene glycol stationary liquid. The detector temperature is 270°C, the injection port temperature is 250°C, the column temperature is 170°C for the first 2 minutes, then increased to 240°C at an increase of 3°C per minute and maintained for 15 minutes. The carrier gas is nitrogen / helium with a split ratio of 1:200 and a flow rate of 1 mL / min.

[0053] 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 optimal mutant strain P131S reached 73.18%, an increase of 33% over the initial EPA content and an increase of 11.7% compared with the WT.

[0054] Table 5 EPA content

[0055] Therefore, it can be seen that the Burkholderia lipase mutant provided by the present invention exhibits higher selectivity for EPA, can enrich EPA, provide more possibilities for industrial production, and also provide some insights for the engineering transformation of other multi-domain proteins.

[0056] The amino acid and nucleotide sequences of the wild-type Burkholderia lipase LipA are as follows: LipA ​​amino acid sequence - SEQ ID No. 21: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. LipA nucleotide sequence - SEQ ID NO.22: GCCGATGGCTACGCGGCGACGCATTACCCGATCATCCTCGTTCACGGGCTCTCGGGTACCGACAAGTACGCGGGCGTGCTCGAGTATTGGTACGGCATCCAGGAGGACCTGCAACAGAACGGTGCGACCGTCTACGTCGCGAACCTGTCGGGTTTCCAGAGCGACGACGGCCCGAACGGGCGCGGCGAACAGTTGCTCGCTTACGTGAAGACGGTGCTCGCGGCGACGGGGGCGACCAAGGTGAATCTCGTCGGCCACAGCCAGGGCGGCCTCTCGTCGCGCTATGTTGCTGCCGTCGCGCCCGATCTCGTTGCGTCGGTGACGACGATCGGCACGCCGCATCGCGGCTCGGAATTCGCCGACTTCGTGCAGAACGTGCTGGCGTACGATCCGACCGGGCTTTCGTCATCGGTGATCGCCGCGTTCGTCAATGTGTTCGGAATCCTGACGAGCAGCAGCCACAACACCAACCAGGACGCGCTCGCCGCACTGCAGACGCTGACCACCGCACGGGCCGCCACGTACAACCAGAACTATCCGAGCGCGGGCCTGGGTGCGCCGGGCAGTTGCCAGACCGGCGCGCCGACCGAAACCGTCGGCGGCAACACGCACCTGCTGTATTCGTGGGCCGGCACGGCGGTCCAGCCGACGCTCTCCGTGTTCGGCGTCACGGGTGCGACGGACACGAGCACCCTTCCGCTCGTCGATCCGGCGAACGTGCTCGACCTGTCGACGCTCGCGCTGTTCGGCACCGGAACGGTGATGATCAACCGCGGCTCCGGACAGAACGACGGGCTCGTGTCGAAGTGCAGTGCGCTGCACGGCAAGGTGCTGAGCACGAGCTACAAGTGGAACCACCTCGACGAGATCAACCAGCTGCTCGGCGTGCGCGGCGCGTATGCTGAAGATCCGGTCGCGGTGGTCCGCACGCATGTGAACCGGCTGAAGCTGGCGGGCGTGTAA. The amino acid sequences of lipA of each mutant are as follows: P131S:SEQ ID NO.23: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDSTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T132A:SEQ ID NO.24: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPAGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T251A:SEQ ID NO.25: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGAGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T251S:SEQ ID NO.26: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGSGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. P131A:SEQ ID NO.27: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDATGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. P131Q:SEQ ID NO.28: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDQTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. T132G:SEQ ID NO.29: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPGGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. P113A:SEQ ID NO.30: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSQGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Q88S:SEQ ID NO.31: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTPHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Double mutant Q88S, P113A: SEQ ID NO.32: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Quadruple mutant Q88S, P113A, T132G, L287S: SEQ ID NO.33: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDPGGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHSDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Quadruple mutants Q88S, P113A, P131S, L287S: SEQ ID NO.34: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDsTGLSSSVIAAFVNVFGILTSSSHNTNQDALAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGTGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHSDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. Quadruple mutants Q88S, P113A, P131S, T251A: SEQ ID NO.35: ADGYAATHYPIILVHGLSGTDKYAGVLEYWYGIQEDLQQNGATVYVANLSGFQSDDGPNGRGEQLLAYVKTVLAATGATKVNLVGHSSGGLSSRYVAAVAPDLVASVTTIGTAHRGSEFADFVQNVLAYDsTGLSSSVIAAFVNVFGILTSSSHNTNQDA LAALQTLTTARAATYNQNYPSAGLGAPGSCQTGAPTETVGGNTHLLYSWAGTAVQPTLSVFGVTGATDTSTLPLVDPANVLDLSTLALFGAGTVMINRGSGQNDGLVSKCSALHGKVLSTSYKWNHLDEINQLLGVRGAYAEDPVAVVRTHVNRLKLAGV. The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A Burkholderia lipase having a T251A mutation, characterized in that The Burkholderia lipase has the following mutations: T251A: The threonine in the 251st amino acid sequence of LipA of the wild-type Burkholderia lipase is mutated to alanine; the sequence of LipA of the wild-type Burkholderia lipase is shown in SEQ ID NO.

21.

2. Use of the Burkholderia lipase having the T251A mutation as claimed in claim 1 in purifying or enriching EPA.

3. A recombinant expression vector for Burkholderia lipase, characterized in that: The invention comprises a plasmid backbone and a nucleotide sequence encoding the Burkholderia lipase with T251A mutation according to claim 1 and inserted into the plasmid backbone.

4. The Burkholderia lipase recombinant expression vector according to claim 3, characterized in that: The plasmid backbone is pETDuet, pET-28a, pET-22b, pET-32a or pET-25.

5. The Burkholderia lipase recombinant expression vector according to claim 4, characterized in that: The nucleotide sequence encoding the Burkholderia lipase with the T251A mutation as claimed in claim 1 is inserted upstream of the f1 ori site of the pETDuet plasmid.

6. The Burkholderia lipase recombinant expression vector according to claim 5, characterized in that: The nucleotide sequence encoding the Burkholderia lipase with T251A mutation as claimed in claim 1 includes the lipB gene and the mutated lipA gene, and the lipB, lipA and the f1 ori on the pETDuet plasmid are sequentially connected in series.

7. Use of a Burkholderia lipase recombinant expression vector according to any one of claims 3 to 6 in the preparation of Burkholderia lipase.

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