Recombinant lipase with self-purification function and high stability and application thereof

Abstract

This invention discloses a recombinant lipase with self-purification function and high stability, and its applications. The recombinant lipase is formed by linking a lipase with an arthropod-like polypeptide via a linker. The arthropod-like polypeptide is an octapeptide repeat sequence, (QYPSDGRG)n, where n is 20-60 repeats. The recombinant lipase of this invention can be efficiently expressed in an *E. coli* prokaryotic expression system and exhibits strong self-purification ability and high stability. It has broad application prospects in food processing, biofuel production, papermaking, and leather industry, and is of great significance for improving production efficiency and reducing production costs, demonstrating significant practical value.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant lipase with self-purification function and high stability and its applications. Background Technology

[0002] Lipases are important hydrolases that catalyze various reactions involving the hydrolysis, esterification, and transesterification of lipid substrates such as triglycerides. Due to their high efficiency, specificity, and catalytic ability in non-aqueous systems, lipases have been widely applied in many key areas of industrial and agricultural production. In the food industry, lipases are used to improve food flavor, synthesize edible flavorings, and modify oils and fats; in the detergent industry, they are a core component of environmentally friendly enzyme-added detergents; in the bioenergy field, lipases are a core biocatalyst for the production of biodiesel; furthermore, lipases play an irreplaceable role in the pharmaceutical, leather, paper, and fine chemical synthesis industries.

[0003] With the increasing demand for lipase applications, obtaining high-purity, high-activity lipases efficiently and at low cost has become a key technological bottleneck restricting their large-scale application. Currently, commonly used industrial lipase separation and purification methods (such as salting out and chromatography) often suffer from cumbersome procedures, high costs, significant enzyme activity loss, or difficulty in scaling up. In particular, to facilitate purification, recombinant lipases often have histidine tags fused to their amino or carboxyl ends and are purified in one step using immobilized metal ion affinity chromatography (IMAC). Although immobilized metal ion affinity chromatography is simple to operate, it still has significant limitations in practical applications: firstly, the chromatographic media are expensive, resulting in high purification costs and hindering industrial scale-up; secondly, metal ions are prone to detachment, leading to decreased purification efficiency and potential contamination of the target product; and thirdly, the elution process usually requires the use of high concentrations of competing reagents such as imidazole, which may cause irreversible denaturation of the enzyme protein, affecting enzyme activity recovery.

[0004] Resilin, a member of the elastin family, is an insoluble, cross-linked protein with excellent elasticity, playing a crucial role in the jumping movements of arthropods. Resilin exhibits remarkable heat resistance, remaining stable and undegraded up to 100°C; it is resistant to chemical denaturation, tolerating high concentrations of urea and guanidine hydrochloride, and possesses structural stability; it is also resistant to proteolytic degradation, exhibiting strong resistance to various proteases.

[0005] Resilin-like polypeptides (RLPs) are short peptides that are artificially designed and synthesized by humans to mimic arthropod elastin. RLPs have a wide range of potential applications, particularly in biomedicine, materials science, and protein delivery. However, no research has been reported on combining RLPs with lipases to improve lipase performance. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant lipase with self-purification function and high stability and its application. This recombinant lipase can be expressed efficiently in the Escherichia coli prokaryotic expression system and has the characteristics of strong self-purification ability and high stability. It has broad application prospects in food processing, biofuel production, papermaking and leather industry, and is of great significance for improving production efficiency and reducing production costs, with significant practical value.

[0007] The technical solution adopted by this invention to solve its technical problem is: A recombinant lipase with self-purification function and high stability, wherein the recombinant lipase is formed by linking a lipase with an arthropod-like polypeptide through a linker, wherein the arthropod-like polypeptide is an octapeptide repeat sequence, the octapeptide repeat sequence being (QYPSDGRG)n, where n is 20-60 repeats.

[0008] This invention is based on the inventors' first artificial synthesis of an octapeptide repeat sequence arthropod-like polypeptide (RLP), which possesses unique thermosensitive properties. Furthermore, this invention connects a lipase to this arthropod-like polypeptide via a linker (rigid linker AEAAAAKEAAAKA) to construct a recombinant lipase. The recombinant lipase inherits the thermosensitive properties of RLPs, which are characterized by: low-temperature precipitation; when the temperature is below the highest critical dissolution temperature, hydrogen bonds between RLP chains dominate, intermolecular association is enhanced, and cold-induced aggregation occurs, forming a protein-rich condensed phase; redissolution upon heating, the process is reversible, and this property appears to be unique to arthropod elastin and arthropod-like elastin. This property can be used to purify RLPs and their fusion proteins. This invention achieves precipitation and resolution of the recombinant lipase through simple temperature cycling, thereby efficiently and rapidly separating and purifying the lipase from complex lysates. More importantly, the native catalytic activity of the lipase is not significantly affected after fusion with RLPs.

[0009] The recombinant lipase of the present invention is formed by linking lipase and arthropod-like polypeptides (RLPs) through a linker, and is referred to as Lipase-Linker-RLPs.

[0010] The amino acid sequence of the lipase is shown in SEQ ID No. 1.

[0011] The linker is a linker peptide, and the amino acid sequence of the linker peptide is AEAAAAKEAAAKA.

[0012] A polynucleotide encoding the recombinant lipase described above.

[0013] A recombinant vector comprising the aforementioned polynucleotide sequence.

[0014] The recombinant vector is a recombinant prokaryotic vector, and the prokaryotic vector is a pET(+) plasmid or a pGEX vector.

[0015] A recombinant genetically engineered bacterium comprising the polynucleotide or the recombinant vector.

[0016] The host bacteria of the recombinant genetically engineered bacteria include Escherichia coli.

[0017] A purification method for the recombinant lipase, employing cooling agglutination purification, specifically involves: precipitating the recombinant lipase with ammonium sulfate, then resuspending the precipitate in Tris-HCl buffer, heating to 25-60℃ and redissolving, centrifuging to collect the supernatant, and obtaining the purified recombinant lipase.

[0018] The recombinant lipase is used in food processing, paper deinking, leather degreasing, detergent production, biodiesel preparation, or pharmaceutical intermediate synthesis. The food processing includes noodle processing, dairy processing, and oil processing.

[0019] The beneficial effects of this invention are: 1. In this invention, lipase is linked to RLPs via a linker, giving the lipase thermal stability and cooling agglutination properties. This characteristic allows the target protein to be obtained through a simple purification process of cooling agglutination, followed by reheating and resolution. Compared to traditional IMAC technology, the purification method provided by this invention has significant advantages such as simple operation, no need for expensive chromatographic media, no use of chemical elution reagents, mild conditions, high enzyme recovery rate, and ease of large-scale production. It provides a promising new technical route for the low-cost, high-efficiency industrial production of lipase.

[0020] 2. The recombinant lipase of the present invention is expressed using an Escherichia coli expression system. Escherichia coli cells have strong metabolic capabilities and rich cellular mechanisms, enabling them to efficiently synthesize and fold proteins. Compared with other expression systems, the Escherichia coli expression system is simple to operate and low in cost. The culture and protein expression cycle of the recombinant engineered bacteria described in the present invention is relatively short, making it suitable for rapid large-scale production. Attached Figure Description

[0021] Figure 1This is a schematic diagram of the construction of the recombinant expression plasmid pET28a(+)-Lipase-Linker-RLPs; Lipase-Linker-RLPs represents the recombinant lipase of this invention.

[0022] Figure 2 SDS-PAGE images of recombinant lipase expression at different induction temperatures (16, 25, 30, 37℃). M: Standard protein molecular weight; Lane Ctrl: Empty vector-induced fragmented expression. E. coli Whole bacteria; Lane Uni: Uninduced strain containing recombinant lipase E. coli Broken whole bacteria; lane Lys: induction at different temperatures containing recombinant lipase E. coli Broken whole bacteria; lane Sup: supernatant containing recombinant lipase; lane Pel: precipitate containing recombinant lipase.

[0023] Figure 3 The following is a graph showing the effect of temperature on the separation of recombinant lipase. (A) SDS-PAGE of the effect of different temperatures on the separation of recombinant lipase. Lane M: standard protein molecular weight; Lane Ctrl: E. coli transformed with empty plasmid; Lane Lys: whole-cell lysate of recombinant lipase; Lanes S1, S2, and S3 show the separation effect of supernatant containing recombinant lipase at 25, 40, and 60℃, respectively; Lanes P1, P2, and P3 show the separation effect of precipitate containing recombinant lipase at 25, 40, and 60℃, respectively; (B) Graph showing the effect of different temperatures on supernatant and precipitate containing recombinant lipase.

[0024] Figure 4 This is a diagram showing the thermal stability of the recombinant lipase in Example 5.

[0025] Figure 5 This is a diagram showing the storage stability of the recombinant lipase in Example 5. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0027] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0028] Example 1: Construction of recombinant expression plasmid pET28a-Lipase-Linker-RLPs Nanjing GenScript Biotech Co., Ltd. synthesizes whole-genome lipase ( Ideonella sakaiensisThe nucleotide sequence of the lipase (NCBI accession number GAP38373.1) is shown in SEQ ID NO:2, and the amino acid sequence of the lipase is shown in SEQ ID NO:1. The 5' and 3' ends of the above lipase sequence were respectively ligated with Spe I and Nhe I enzyme (Takara Bio Engineering (Dalian) Co., Ltd.) restriction sites to obtain the lipase nucleotide sequence with Spe I and Nhe I restriction sites. Then, the lipase nucleotide sequence with Spe I and Nhe I restriction sites was constructed into the pET28a(+) plasmid (purchased from Novagen), denoted as pET28a(+)-Lipase.

[0029] Suzhou Hongxun Biotechnology Co., Ltd. synthesized the Linker-RLPs nucleotide sequence in its entirety. The linker has an Nhe I restriction site at 5' and the RLPs have a Hind III restriction site at 3'. The above sequence was then constructed on the PUC18 plasmid (purchased from Addgene) and designated as PUC18-Linker-RLPs.

[0030] The linker is a linker peptide AEAAAAKEAAAKA (SEQ ID NO:3), whose nucleic acid sequence is shown in SEQ ID NO:4; the RLPs are arthropod-like polypeptides with repeating octapeptide (QYPSDGRG, SEQ ID NO:5) sequences, and in this embodiment, the RLPs are (QYPSDGRG). 40 40 is a repeating number.

[0031] Figure 1 The restriction enzyme sites for the recombinant plasmid pET28a(+)-Lipase-Linker-RLPs are specified. pET28a(+)-Lipase and PUC18-Linker-RLPs were double-digested with Nhe I and HindIII. The double digestion system was as follows: Nhe I: 1µL (10U / µL); Hind III: 1µL (15U / µL); 10× buffer (10×M Buffer, purchased from TaKaRa): 2µL; pET28a(+)-Lipase or PUC18-Linker-RLPs: 5µL (100 ng / µL); Sterile water: Add to a total of 20 μL.

[0032] Add the above double enzyme digestion system to a centrifuge tube and mix well. Digest at 37℃ for 2-3 h. Then, recover the Linker-RLP gene fragment and linearized pET28a(+)-Lipase according to the instructions of the gel extraction kit (Shanghai Sangon Biotech, EZ-10 column DNA gel extraction kit). Next, ligate the gel-extracted Linker-RLP gene fragment and pET28a(+)-Lipase using T4 DNA ligase (Takara Bio Engineering (Dalian) Co., Ltd.). The ligation system is as follows: 10×T4 ligase buffer (Takara Bio Engineering (Dalian) Co., Ltd.): 2 μL; pET28a(+)-Lipase (50 ng / µL): 1µL; Linker-RLPs (50 ng / µL): 1µL; T4 DNA ligase (10 U / μL): 1 μL; Sterile water: Add to a total of 20 μL.

[0033] The reaction was carried out in an incubator at 16℃ for approximately 18 hours. After ligation, the recombinant plasmid pET28a(+)-Lipase-Linker-RLPs was obtained. The recombinant plasmid pET28a(+)-Lipase-Linker-RLPs was sent to GenScript Biotech in Nanjing for sequencing. Plasmids with correct sequencing results were then used for subsequent expression.

[0034] Example 2: Large-scale culture of Escherichia coli expressing Lipase-Linker-RLPs (1) Transform the recombinant plasmid pET28a(+)-Lipase-Linker-RLPs into E. coli BL21(DE3) competent cells: Take out the BL21(DE3) competent cells and immediately place them on ice to thaw slowly. After thawing, in a clean bench, add 2 μL of recombinant plasmid to 100 μL of thawed competent cells and mix well. Incubate on ice for 30 min. Heat shock in a 42℃ water bath for 45-50 s, then immediately place on ice and incubate for 3-5 min. Add 800 μL of culture medium (antibiotic-free sterile LB liquid medium) to the clean bench and incubate at 37℃ with shaking at 190 rpm for 40 min. Take out the EP tube, centrifuge at 10,000 rpm for 30 s, remove 600 μL of culture medium from the EP tube in the clean bench, leaving about 200 μL of culture medium to resuspend the cells.

[0035] (2) Resuspend 200 μL of culture medium in solid LB medium containing 50 μg / mL kanamycin (15 g / L agar powder, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.4) and incubate at 37°C. Then, transfer the single colonies after overnight culture to 5 ml of liquid LB medium containing 50 μg / mL kanamycin (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.4) and incubate overnight at 37°C and 200 rpm in an orbital shaker. Then, inoculate 3 mL of the overnight cultured bacteria into 300 mL of liquid LB medium containing 50 μg / mL kanamycin and grow at 37°C and 200 rpm for 3 hours or until the bacterial OD600 reaches between 0.4 and 0.6.

[0036] (3) Place the culture obtained in step (2) on ice for 20 minutes, add IPTG to it to a final concentration of 0.4 mM, and then incubate at 16℃, 25℃, 30℃ and 37℃ with shaking at 180 rpm for 16-20 h (overnight) to induce the expression of recombinant lipase Lipase-Linker-RLPs. Finally, centrifuge the liquid culture medium at 3000 rpm and 4℃ for 20 minutes and discard the supernatant to obtain cell pellets cultured at different temperatures, and store them at -80℃ for later use.

[0037] (4) Thaw the cell pellets obtained at different culture temperatures in step (3), resuspend them in 10 mL of Tris-HCl buffer (50 mM, pH 8.0, the same below), and centrifuge at 3000 rpm and 4°C for 20 minutes. Discard the supernatant and collect the bacterial cells. Wash the bacterial cells twice with Tris-HCl buffer. Then resuspend the cells in 20 mL of Tris-HCl buffer containing 1 mM PMSF and sonicate them on ice using an ultrasonic cell disruptor: work for 6 seconds, pause for 6 seconds, and sonicate for a total of 30 minutes to obtain a lysis buffer containing recombinant lipase. Centrifuge the lysis buffer at 4°C and 14,000 rpm for 30 minutes to obtain the supernatant and pellet. Transfer the supernatant to a new EP tube and preserve the supernatant and pellet.

[0038] To determine the nature of the target protein expressed in soluble or inclusion body form, the lysis buffer, supernatant, and precipitate obtained in step (4) (resuspended in Tris-HCl buffer) were used for SDS-PAGE analysis. The results showed that under induction conditions at 16°C and 25°C, a significant band was added at the 66.4 kDa target location in the supernatant. Figure 2 The results showed a high degree of agreement with the predicted theoretical molecular weight of the recombinant protein (approximately 68 kDa), indicating successful induction and the acquisition of soluble recombinant lipases, Lipase-Linker-RLPs.

[0039] Example 3: Cooling and agglutination purification of recombinant lipase In this embodiment, cooling agglutination is used to purify recombinant lipase, thereby verifying the self-purification performance and purification efficiency of the recombinant lipase prepared in Example 2.

[0040] 3.1 Add (NH4)2SO4 to 500 μL of the supernatant obtained in step (4) of Example 2, with a final concentration of (NH4)2SO4 of 1 M. After mixing, place on ice for 30 min, then centrifuge at 12000 rpm for 15 min at 4 °C to separate the supernatant and precipitate. Remove the supernatant and resuspend the precipitate in Tris-HCl buffer. Recombinant lipase undergoes a phase transition at temperatures above the phase transition temperature, transforming into a soluble state. To study the effect of different temperatures on the separation effect of recombinant lipase, the solutions obtained after resuspending the above precipitate (named crude enzyme solution) were placed in water baths at 25 °C, 40 °C, and 60 °C for 12 h, respectively. Afterward, centrifuge at 5000 rpm for 30 seconds at 25 °C, and transfer the supernatant (purified recombinant lipase) to a clean 1.5 mL EP tube for later use. The precipitate was also retained for later use.

[0041] 3.2 The supernatant and precipitate obtained in 3.1 were subjected to SDS-PAGE. Figure 3 The results showed that the protein (recombinant lipase) was present in the supernatant after treatment at 25℃, 40℃, and 60℃. At 60℃, the supernatant contained fewer impurities, indicating the best separation and purification effect. Figure 3 (A) in the middle. Figure 3 As can be seen from (B) in the figure, as the temperature increases, more and more impurities form precipitates, with the most precipitation occurring at 60℃. Figure 3 The results show that more impurities can be removed at 60℃, achieving the best separation effect.

[0042] 3.3 The enzyme activity of the supernatant and precipitate obtained in 3.1 was measured, and the total enzyme activity, specific enzyme activity, and purification fold were calculated. The results are shown in Table 1. The results show that the enzyme activity in the precipitate is much lower than that in the supernatant. In addition, the purification folds in the supernatant were 2.72, 3.47, and 7.23 times when treated at 25℃, 40℃, and 60℃, respectively, further indicating that the purification effect was best at 60℃.

[0043] Table 1. Effect of temperature on the purification efficiency of recombinant lipase .

[0044] Example 4: Enzyme activity detection method Lipases catalyze the hydrolysis of p-nitrophenylacetic acid (p-NPA) to produce p-nitrophenol (pNP) and acetic acid. Under alkaline or neutral conditions, the product pNP is yellow and exhibits a characteristic absorption peak at 410 nm. The rate of pNP formation can be calculated by real-time monitoring with a spectrophotometer or by determining the rate of increase in absorbance at 410 nm using the endpoint method, thus determining the catalytic activity of the lipase.

[0045] Set the spectrophotometer sample chamber temperature to 40°C and preheat the required volume of PBS buffer (pH 7.4) and substrate solution in a 40°C water bath for at least 10 minutes. Add 970 μL of preheated PBS buffer (pH 7.4) and 20 μL of room temperature PBS buffer to the cuvette. Add 970 μL of preheated PBS buffer and 20 μL (1.2 mg / mL) of active enzyme solution to the cuvette. Quickly add 10 μL of preheated 10 mM p-NPA substrate solution to each of the above two cuvettes, and immediately mix with a pipette (avoiding air bubbles), while simultaneously starting the timing. After reacting precisely for 10 minutes, immediately add 100 μL of 1 M NaOH solution to the cuvette to terminate the reaction. After mixing, zero the instrument using a blank tube and read the absorbance of the measurement tube at a wavelength of 410 m.

[0046] The lipase activity unit is defined as: under the above conditions, the amount of enzyme that releases 1 μmol of p-nitrophenol per minute is defined as 1 lipase activity unit (U).

[0047] The calculation formula is: X = cV / tV', Where X is the lipase activity (U / ml), c is the p-nitrophenol concentration (μmol / L), V is the final volume of the reaction solution after acid-base adjustment (mL), V' is the amount of enzyme solution used (mL), and t is the reaction time (min).

[0048] Example 5: Thermal and storage stability of recombinant lipase The plasmid pET28a(+)-Lipase from Example 1 was transformed into competent cells, induced to express, and subjected to ultrasonic centrifugation using the same method as in Example 2. The supernatant was then purified by Ni-NTA affinity column chromatography to obtain wild-type lipase (Lipase), which served as a control.

[0049] Wild-type lipase purified from equal amounts of Ni-NTA and recombinant lipase purified from the supernatant treated at 60°C in Example 3 were placed at different temperatures (20-80°C) for 30 minutes each without reaction solution. The enzymes were then transferred to ice to adjust the enzyme temperature and centrifuged at 12,000 rpm for 10 minutes to remove denatured and precipitated proteins. The enzyme activity was measured according to the method described in Example 4. The highest enzyme activity was taken as 100%, and other enzyme activity values ​​were compared with the highest enzyme activity value to plot the curves of relative activity at different temperatures.

[0050] Figure 4 The graph shows the thermostability of wild-type lipase and recombinant lipase at different temperatures. As can be seen from the graph, the relative enzyme activities of wild-type lipase and recombinant lipase are as follows: When the temperature rises to 60℃, the enzyme activity of wild-type lipase is only 30.03%, while the recombinant lipase still retains 74.81% activity; at 70℃, the enzyme activity of wild-type lipase is only 10.22%, while the recombinant lipase still retains 41.92% activity; at 80℃, the activity of wild-type lipase is only 1.33%, while the recombinant lipase retains 11.19% activity. Therefore, it can be concluded that the recombinant lipase prepared in this invention has good thermostability.

[0051] In this embodiment, the storage stability of wild-type lipase and recombinant lipase was also investigated. The specific method is as follows: two portions of wild-type lipase and recombinant lipase were prepared. One portion was stored at 4°C and the other portion was stored at 25°C. After storage for 0, 5, 10, 15, 20, 25 and 30 days, the enzyme activity was detected according to the method described in Example 4. The highest enzyme activity was taken as 100%, and the other enzyme activities were compared with the highest enzyme activity. At the same time, the time versus relative enzyme activity curve was plotted.

[0052] Storage stability results are as follows Figure 5 Purified wild-type and recombinant lipases were stored at 4°C and 25°C for 30 days, with residual enzyme activity measured every 5 days. The activities of both enzymes decreased with prolonged storage, but the decrease was more pronounced in the wild-type lipase. Furthermore, the enzymes showed higher stability at 4°C than at 25°C. Under both temperature conditions, the recombinant lipase exhibited greater stability than the wild-type lipase. After 30 days of treatment at 4°C, the recombinant lipase retained 88.28% of its activity, while the wild-type lipase retained 72.75%. After 30 days of treatment at 25°C, the recombinant lipase retained 76.88% of its activity, while the wild-type lipase retained only 52.43%, indicating that the recombinant lipase showed better storage stability than the wild-type lipase.

[0053] In summary, the recombinant lipase exhibits significantly higher thermal and storage stability than the wild-type lipase. Therefore, the recombinant lipase prepared by this invention demonstrates significantly improved thermal and storage stability, and can be easily, rapidly, and efficiently self-isolated and purified.

[0054] Example 6: The difference between this embodiment and Embodiment 1 is that RLPs are (QYPSDGRG). 20 20 represents the number of replicates. Recombinant lipases were then prepared and their performance tested according to the methods described in Examples 2-5. The results were compared with those of RLPs (QYPSDGRG). 40 Similarity is achieved when 40 is the number of repetitions.

[0055] Example 7: The difference between this embodiment and Embodiment 1 is that RLPs are (QYPSDGRG). 60 60 represents the number of replicates. Recombinant lipases were then prepared and their performance tested according to the methods described in Examples 2-5. The results were compared with those of RLPs (QYPSDGRG). 40 Similarity is achieved when 40 is the number of repetitions.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

[0057] SEQ ID NO:1: MNFPRASRLMQAAVLGGLMAVSAAATAQTNPYARGPNPTAASLEASAGPFTVRSFTVSRPSGYGAGTVYYPTNAGGTVGAIAIVPGYTARQSSIKWWGPRLASHGFVVITIDTNSTLDQPSSRSSQQMAALRQVASLNGTSSSPI YGKVDTARMGVMGWSMGGGGSLISAANNPSLKAAAPQAPWDSSTNFSSVTVPTLIFACENDSIAPVNSSALPIYDSMSRNAKQFLEINGGSHSCANSGNSNQALIGKKGVAWMKRFMDNDTRYSTFACENPNSTRVSDFRTANCS; SEQ ID NO:2: ATGAACTTCCCGCGCGCGAGCCGTCTGATGCAGGCAGCGGTTCTGGGCGGCTTGATGGCGGTGAGCGCCGCTGCGACGGCGCAGACCAACCCGTACGCGCGTGGTCCGAACCCGACCGCCGCCAGCCTGGAAGCCAGCGCGGGTCCGTTCACCGTGCGTAGCTTCACCGTGAGCCGTCCGAGCGGTTACGGTGCGGGTACCGTGTACTACCCGACCAACGCCGGTGGTACCGTGGGTGCCATCGCCATCGTGCCGGGTTACACCGCGCGTCAGAGCAGCATCAAATGGTGGGGTCCGCGTCTGGCGAGCCACGGTTTCGTTGTTATCACCATCGACACCAACAGCACCCTGGACCAGCCGAGCAGCCGTAGCAGCCAGCAGATGGCGGCGCTGCGTCAGGTGGCCAGCCTGAACGGTACCAGCAGCAGCCCGATCTACGGTAAAGTTGACACCGCGCGTATGGGTGTTATGGGTTGGAGCATGGGTGGTGGTGGTAGCCTGATCAGCGCCGCCAACAACCCGAGCCTGAAAGCCGCCGCCCCGCAGGCCCCGTGGGACAGCAGCACCAACTTCAGCAGCGTGACCGTGCCGACCCTGATCTTCGCCTGCGAAAACAGCAGCATCGCCCCGGTTAACAGCAGCGCGCTGCCGATCTACGACAGCATGAGCCGTAACGCCAAACAGTTTCTGAAAATCAACGGTGGTAGCCACAGCTGCGCCAACAGCGGTAACAGCAACCAGGCGCTGATCGGTAAAAAAGGTGTTGCCTGGATGAAACGTTTCATGGACAACGACACCCGTTACAGCACCTTCGCCTGCGAAAACCCGAACAGCACCCGTGTGAGCGACTTCCGTACCGCCAACTGCAGC; SEQ ID NO 3:AEAAAKEAAAKA; SEQ ID NO 4:GCTGAAGCTGCTGCTAAAGAAGCTGCTGCTAAAGCT; SEQ ID NO 5:QYPSDGRG。

Claims

1. A recombinant lipase with self-purification function and high stability, characterized in that, The recombinant lipase is formed by linking a lipase with an arthropod-like polypeptide via a linker. The arthropod-like polypeptide is an octapeptide repeat sequence, which is (QYPSDGRG)n, where n is 20-60 repeats.

2. The recombinant lipase according to claim 1, characterized in that, The amino acid sequence of the lipase is shown in SEQ ID No.

1.

3. The recombinant lipase according to claim 1, characterized in that, The linker is a linker peptide, and the amino acid sequence of the linker peptide is AEAAAAKEAAAKA.

4. A polynucleotide, characterized in that, It encodes the recombinant lipase as described in claim 1.

5. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 4.

6. The recombinant vector according to claim 5, characterized in that, The recombinant vector is a recombinant prokaryotic vector, and the prokaryotic vector is a pET(+) plasmid or a pGEX vector.

7. A recombinant genetically engineered bacterium, characterized in that, It comprises the polynucleotide of claim 4 or the recombinant vector of any one of claims 5-6.

8. The recombinant genetically engineered bacteria according to claim 7, characterized in that, The host bacteria of the recombinant genetically engineered bacteria include Escherichia coli.

9. A method for purifying the recombinant lipase as described in claim 1, characterized in that, The recombinant lipase was purified by cooling agglutination, specifically by precipitating the recombinant lipase with ammonium sulfate, then resuspending the precipitate in Tris-HCl buffer, heating to 25-60℃ and redissolving, centrifuging to collect the supernatant, and obtaining the purified recombinant lipase.

10. The application of the recombinant lipase as described in claim 1, 2, or 3 in food processing, paper deinking, leather degreasing, detergent production, biodiesel preparation, or pharmaceutical intermediate synthesis, characterized in that, The food processing includes noodle processing, dairy processing, and oil processing.

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